THE PSEUDOMONAD GROUP
The pseudomonads are gram-negative, motile, aerobic rods some of which produce water-soluble pigments. Pseudomonads occur widely in soil, water, plants, and animals. Pseudomonas aeruginosa is frequently present in small numbers in the normal intestinal flora and on the skin of humans and is the major pathogen of the group. Other pseudomonads infrequently cause disease. The classification of pseudomonads is based on rRNA/DNA homology and common culture characteristics.
P aeruginosa is widely distributed in nature and is commonly present in moist environments in hospitals. It can colonize normal humans, in whom it is a saprophyte. It causes disease in humans with abnormal host defenses.
MORPHOLOGY AND IDENTIFICATION
- Typical organisms
P aeruginosa is motile and rod-shaped, measuring about 0.6 x 2µm. it is gram-negative and occurs as single bacteria, in paris, and occasionally in short chains.
P aeruginosa is an obligate aerobe that grows readily on many types of culture media, sometimes producing a sweet or grape-like of corn taco-like odor. Some strains hemolyze blood. P aeruginosa from smooth round colonies with a florescent greenish color. It often produces the nonfluorescnet bluish pigment pyocyanin, which diffuses into the agar. Other pseudomonas species do not produce the fluorescent pigment pyoverdin, which gives a greenish color to the agar. Some strains produce the dark red pigment pyorubin or the black pigment pyomelanin.
P aeruginosa in a culture can produce multiple colony types. P aeruginosa from different colony types may also have different biochemical and enzymatic activities and different antimicrobial susceptibility patterns. Sometimes it is not clear if the colony types represent different strains of P aeruginosa or are variants of the same strain.
Cultures from patients with cystic fibrosis (CF) often yield P aeruginosa organisms that form muciod colonies as a result of overproduction of alginate, an exopolysaccharde. In CF patients, the exopolysaccharide appears to provide the matrix for the organisms to live in a bioflim.
- GROWTH CHARACTERISTICS
P aeruginosa grows well at 37-420C; its growth at 420C helps differentiate it from other pseudomonas species in the fluorescent group. It is oxidase-positive. It does not ferment carbohydrates, but many strains oxidize glucose. Identification is usually based on colonial morphology, oxidase positively, the presence of characteristics pigments, and growth at 420C. Differentiation of P aeruginosa from other psedomonads on the basis of biochemical activity requires testing with a large battery of substrates.
ANTIGEN STRUCTURE AND TOXINS
Pili (fimbriae) extend from the cell surface and promote attachment to host epithelial cells. The exopolysaccharide is responsible for the mucoid colines seen in cultures from patients with CF. the lipopolysaccharide, which exists in multiple immunotypes, is responsible for many of the end toxic properties of the organism. P aeruginosa isolates from clinical infections produce extracellular enzymes, including elastases, proteases, and two hemolysins; a heat-labile phospholipase C and a heat – stable glycolipid.
Many strains of P aeruginosa produce exotoxin A, which causes tissue necrosis and is lethal for animals when injected in purified form. The toxin blocks protein synthesis by a mechanism of action identical to that of diphtheriatoxin, though the structures of the two toxins are not identical. Antitoxins to exotoxin A are found in some human sera, including those of patients who have recovered from serious P aeruginosa infection.
P aeruginosa is pathogenic only when introduced into areas devoid of normal defenses, e.g., when mucous membranes and skin are disrupted by direct tissue damage; when intravenous or urinary catheters are used; or when neutropenisa is present, as in cancer chemotherapy. The bacterium attaches to and colonizes the mucos membrane or skin, invades locally, and produces systemic disease. These processes are promoted by the pili, enzymes, and toxins described above, lipopolysaccharide plays a direct role in causing fever, shock, oliguria, leukocytosis and leucopenia, disseminated intravascular coagulation, and adult respiratory distress syndrome.
P aeruginosa and other pseudomonads are resistant to many antimicrobial agents and therefore become dominant and important when more susceptible bacteria of the normal flora are suppressed.
DIAGNOSTIC LABORATORY TESTS
Specimens from skin lesions, pus, urine blood, spinal fluid, sputum, and other material should be obtained as indicated by the type of infection.
Gram-negative rods are often seen in smears. There are no specific morphologic characteristics that differentiate pseudomonads in specimens from enteric or other gram-negative rods.
Specimens are plated on blood agar and the differential media commonly used to grow the enteric gram-negative rods. Pseudomonads grow readily on most of these media, but they may grow more slowly than the enteric. P aeruginosa does not ferment lactose and is easily differentiated from the lactose-fermenting bacteria. Culture is the specific test for diagnosis of P aeruginosa infection.
Clinically significant infections with P aeruginosa should not be treated with single-drug therapy because the success rate is low with such therapy and because the bacteria can rapidly develop resistance when single drugs are employed. A penicillin such as piperacillin such as piperacillinactive against P aeruginosa is used in combination with an aminoglycoside, usually tobramycin. Other drugs active against P aeruginosa include aztreonam, carbapenems such as imipenem or meropenem, and the newer quinolones, including ciprofloxacin. Of the newer cephalosporeins, ceftazidime and cefoperazone are active against P aeruginosa; ceftazidime is used with an aminoglycoside in primary therapy of P aeruginosa infections. The susceptibility patterns of P aeruginosa vary geographically, and susceptibility tests should be done as an adjunct to selection of antimicrobial therapy.
EPIDEMIOLOGY AND CONTROL
P aeruginosa is primarily a nosocomial pathogen, and the methods for control of infection are similar to those for other nosocomial pathogens. Since pseudomonas thrives in moist environments, special attention should be paid to sinks, water baths, showers, hot tubs, and other wet areas. For epidemiologic purposes, strains can be typed using molecular typing techniques.
Vibrios are among the most common bacteria in surface waters worldwide. They are curved aerobic rods and are motile, possessing a polar flagellum. V cholera serogruops O1 and O139 cause cholera in humans, while other vibrios may cause sepsis or enteritis.
The epidemiology of cholera closely parallels the recognition of V cholerae transmission in water and the development of sanitary water system.
MORPHOLOGY AND IDENTIFICATION
- Typical organisms
Upon first isolation, V cholerae is a comma-shaped, curved rod-2-4µm long. It is actively motile by means of a polar flagellum. On prolonged cultivation, vibrios may become straight rods that resemble the gram-negative enteric bacteria.
V cholerae produces convex, smooth, round colonies that are opaque and granular in transmitted light. V cholerae and most other vibros grow well at 370C on many kinds of media, including defined media containing mineral salts and asparagines as sources of carbon and nitrogen. V cholerae grows well on thiosulfate-citrate-bile-sucrose (TCBS) agar, on which it produces yellow colonies that are readily visible against the dark-green background of the agar. Vibrios are oxidase-positive, which differentiates them from enteric gram-negative, bacteria. Characteristically, vibrios grow at a very high pH (8.5-9.5) and arc rapidly killed by acid. Cultures containing fermentable carbohydrates therefore quickly become sterile.
In areas where cholera is endemic, direct cultures of stool on selective media, such as TCBS, and enrichment cultures in alkaline peptone water are appropriate. However, routine stool cultures on special media such as TCBS generally are not necessary or cost-effective in areas whore cholera is rare.
- Growth Characteristics
V cholerae regularly ferments sucrose and mannose but not arabinose. A positive oxidase test is a key step in the preliminary identification of V cholerae and other vibrios. Vibrio species are susceptible to the compound O/129 (2,4-dhimmo-6,7-diisopropylpteridine phosphate), which differentiates them from Aeromonas species, which arc resistant to O/129. Most Vibrio species arc halotolerant, and NaCl often stimulates their growth. Some vibrios are halophilic, requiring the presence of NaCl to grow. Another difference between vibrios and aeromonas is that vibrios grow on media containing 6% NaCl, whereas aeromonas does not.
ANTIGENIC STRUCTURE & BIOLOGIC CLASSIFICATION
Many vibrios share a single heat-labile flagellar II antigen. Antibodies to the H antigen arc probably not involved in the protection of susceptible hosts.
V cholerae has O lipopolysaccharides that confer sero-logic specificity; There are at least 139 O antigen groups. V cholerae strains of O group 1 and O group 139 cause classic cholera; occasionally, non-Ol/non-O139 V cholerae causes cholera-like disease. Antibodies to the O antigens tend to protect laboratory animals against infections with V cholerae.
The V cholerae serogroup OL antigen has determinants that make possible further typing; the serolypes are Ogawa, Inaba, and Hikojhna. Two biotypes of epidemic V cholerae have been defined, classic and El Tor. The El Tor biotype produces a bemolysin, gives positive results on the Voges-Pioskauer test, and is resistant to polymyxin B. Molecular techniques can also be used to type V cholerae. Typing is used for epidcmiologic studies, and tests generally are done only in reference laboratories.
V cholerae O139 is very similar to V cholerae Ol El Tor biotype. V cholerae O139 does not produce the Oi lipopoly-saccharide and does not have all the genes necessary to make this antigen. V cholerae O139 makes a polysaccharide capsule-like other non-Ol V cholerae strains, while V chol-erae Ol does not make a capsule.
VIBRIO CHOLERAE ENTEROTOXIN
V cholerae produce a heat-labile enterotoxin with a molecular weight of about 84,000, consisting of subunits A (MW 28,000) and B (see Chapter 9). Ganglioside GM} serves as the mucosal receptor for subunit B, which promotes entry of subunit A into the cell. Activation of subunit A; yields increased levels of intracellular cAMP and results in prolonged hypersecretion of water and electrolytes. There is increased sodium-dependent chloride secretion, and absorption of sodium and chloride is inhibited. Diarrhea occurs—as much as 20-30 L/day— with resulting dehydration, shock, acidosis, and death. The genes for V cholerae enterotoxin are on the bacterial chromosome. Cholera enterotoxin is antigenicaUy related to LT of Escherichia coli and can stimulate the production of neutralizing antibodies. However, the precise role of antitoxic and antibacterial antibodies in protection against cholera is not clear.
Under natural conditions, V cholerae is pathogenic only for humans. A person with normal gastric acidity may have to ingest as many as 10’° or more V cholerae to become infected when the vehicle is water, because the organisms are susceptible to acid. When the vehicle is food, as few as 10;-104 organisms are necessary because of the buffering capacity of food. Any medication or condition that decreases stomach acidity makes a person more susceptible to infection with V cholerae.
Cholera is not an invasive infection. The organisms do not reach the bloodstream but remain within the intestinal tract. Virulent V cholerae organisms attach to the microvilli of the brush border of epithelial cells. There they multiply and liberate cholera toxin and perhaps mucinases and endotoxin.
DIAGNOSTIC LABORATORY TESTS
Specimens tor culture consist of mucus flecks from stools.
The microscopic appearance of smears made from stool samples is not distinctive. Dark-field or phase contrast microscopy may show the rapidly motile vibrios.
Growth is rapid in peptone agar, on blood agar with a pH near 9.0 or on TCBS agar, and typical colonies can be picked in 18 hours. For enrichment, a few drops of stool can be incubated for 6-8 hours in taurocholatepeptone broth (pH 8.0-9.0); organisms from this culture can be stained or subcultured.
- Specific Tests
V cholerae organisms are further identified by slide agglutination tests using anti-O group 1 or group 139 antisera and by biochemical reaction patterns.
The most important part of therapy consists of water and electrolyte replacement to correct the severe dehydration and salt depletion. Many antimicrobial agents are effective against V cholerae. Oral tetracycline tends to reduce stool output in cholera and shortens the period of excretion of vibrios. In some endemic areas, tetracycline resistance of V cholerae has emerged; the genes are carried by transmissible plasmids.
EPIDEMIOLOGY, PREVENTION, & CONTROL
Six pandemics (worldwide epidemics} of cholera occurred between 18) 7 and 1923, caused most likely by V cholerae O1 of the classic biotype and largely originating in Asia, usually the Indian subcontinent. The seventh pandemic began in 1961 in the Celebes Islands, Indonesia, with spread to Asia, the Middle East, and Africa. This pandemic has been caused by V cholerae biotype El Tor. Starting in 1991, the seventh pandemic spread to Peru and then to other countries of South America and Central America. Cases also occurred in Africa. Millions of people have had cholera in this pandemic. Some consider the cholera caused by the serotype O139 strain to be the eighth pandemic that began in the Indian subcontinent in 1992-1993, with spread to Asia. The disease has been rare in North America since the mid-lSOOs, but an endemic focus exists on the Gulf Coast of Louisiana and Texas.
Cholera is endemic in India and Southeast Asia. From these centers, it is carried along shipping lanes, trade routes, and pilgrim migration routes. The disease is spread by contact involving individuals with mild or early illness and by water, food, and flies. In many instances, only 1-5% of exposed susceptible persons develop disease. The carrier state seldom exceeds 3-4 weeks, and the importance of carriers in transmission is unclear. Vibrios survive in water for up to 3 weeks.
V cholerae lives in aquatic environments. And such environments are the vibrios natural reservoir. V cholerae lives attached to algae, copepods, and crustacean shells. It can survive for years and grow, but when conditions are not suitable for growth it can become dormant.
Control rests on education and on improvement of sanitation, particularly of food and water. Patients should be isolated, their excreta disinfected, and contacts followed up. Chemoprophylaxis with antimicrobial drugs may have a place. Repeated injection of a vaccine containing either lipopolysaccharides extracted from vibrios or dense Vibrio suspensions can confer limited protection to heavily exposed persons (e.g. family contacts) but is not effective as an epidemic control measure.
Neisseria meningitides, often called meningococcus, is a gram-negaitve, nonsporulating, obligately aerobic, oxidase-positive, encapsulated diplococcus about 0.6-1.0µm in diameter: at least 13 pathogenic strains of N. meningtidis are recognized, based on antigenic differences in their capsular polysaccharides.
Al least 13 serogroups of meningococci have been identified by immunologic specificity of capsiilar polysaccharides. The most important serogroups associated with disease in humans are A, R, C, X, Y, and W-135. The group A polys accharide is a polymer of N-acetyimannosamine phosphate, and that of group C is a polymer of Af-acetyl-O-acetylneuraminic acid. Meningococcal antigens are found in blood and cerebrospi-nal fluid of patients with active disease. Outbreaks and sporadic cases in the Western Hemisphere in the last decade have been caused mainly by groups B, C, W-135, and Y; outbreaks in southern Finland and Sao Paulo, Brazil, were due to groups A and C; outbreaks in New Zealand have been due to a par ticular.B strain; those in Africa were due mainly to group Group C and, especially, group A are associated with demic disease.
The outer membrane proteins of meningococci have been divided into classes on the basis of molecular weight. All strains have either class 1, class 2, or class 3 proteins; these are analogous to the For proteins of gonococci and are responsible for the serotype specificity of meningococci. They help form pores in the meningococcal ceil wall. As many as 20 serotypes have been denned; serotypes 2 and 15 have been associated with epidemic disease. The Opa (class 5) protein is comparable to Opa of the gonococci. Meningococci are piliated, but unlike gonococci, they do not form distinctive colony types indicating piliated bacteria. Meningococcal I, PS is responsible for many of the toxic effects found in meningococcal disease. The highest levels of endotoxin measured in sepsis have been found in patients with meningococce-mia (50- to 100-fold greater than with other gram-negative infections).
Humans are the only natural hosts for whom meningococci are pathogenic. The nasopharynx is the portal of entry. There, the organisms attach to epithelial cells with the aid of pili; they may form part of the transient flora without producing symptoms. From the nasopharynx, organisms may reach the bloodstream, producing bacleremia; the symptoms may be like those of an upper respiratory tract infection. Fulminant meningococcemia is more severe, with high fever and hemorrhagic rash; there may be disseminated intra-vascuhr coagulation and circulatory collapse (Waterhouse-Friderichsen syndrome).
Meningitis is the most common complication of meningococcemia. It usually begins suddenly, with intense headache, vomiting, and stiff neck, and progresses to coma wifhin a few hours.
During meningococcemia, there is thrombosis of many smali blood vessels in many organs, with perivascular infiltration and petechffll hemorrhages. There may be interstitial myocarditis, arthritis, and skin lesions. In meningitis, the meninges are acutely inflamed, with thrombosis of blood vessels and exudation of polymorphonuclear leukocytes, so thai the surface of the brain is covered with a thick purulent exudaie.
It is no (known what transforms an asymptomatic infection of the nasopharynx into meningococcemia and meningitis, but this can be prevented by specific bactericidal serum antibodies against the infecting serotype. Nehseria bacter-emia is favored by the absence of bactericidal antibody (IgM and IgG), inhibition of serum bactericidal action by a blocking IgA antibody, or a complement component deficiency (C5, C6, C7, or C8). Meningococci are readily phagocytosed jn the presence of a specific opsonin.
Diagnostic Laboratory Tests
Specimens of blood are taken for culture, and specimens of sftinai fluid are taken for smear, culture, and chemical determinations. Nasopharyngeal swab cultures are suitable for carrier surveys. Puncture material from pelechiae may be taken for smear ant! culture.
Gram-stained smears of the sediment of centrifuged spinal fluid or of petechial aspirate often show typical neisseriae within polymorphonuclear leukocytes or extracellulariy.
Culture media without sodium polyanethol sulfonate are helpful in culturing blood specimens. Cerebrospinal fluid specimens are plated on “chocolate” agar and incubated at 37°C in an atmosphere of 5% CO, (candle jar). Freshly drawn spinal fluid can be directly incubated at 37″C if agar culture media are not immediately available. A modified Thayer-Martin medium with antibiotics (vancomycin, colis-tin, amphotericin) favors the growth of neisseriae, inhibits many other bacteria, and is used for nasopharyngeal cultures. Presumptive colonies of neisseriae on solid media, particularly in mixed culture, can be identified by Gram’s stain and the oxidase test. Spinal fluid and blood generally yield pure cultures that can be further identified by carbohydrate oxi-dative reactions and agglutination with type-specific or polyvalent serum.
Antibodies to meningococcal polysaccharides can be measured by iatex agglutination or hemagglutination tests or by their bactericidal activity. These tests are done only in reference laboratories.
Penicillin G is the drug of choice for treating meningococcal . ‘ disease. Either chlorampheiiicol or a third-generation ccpha-osporin such as cefotaxime or ceftriaxone is used in persons allergic to penicillins.
Epidemiology, Prevention, & Control
Meningococcal meningitis occurs in epidemic waves ‘eg, in military encampments, in religious pilgrims, and in sub-S3 ha ran Africa; in Brazil, there were more than 15,000 cases in 1974) and a smaller number of sporadic interepidemic cases. Five to 30% of the normal population may harbor meningococci (often nontypeable isolates) in the nasopharynx during interepidemic periods. During epidemics, the carrier rale goes up to 70-80%. A rise in the number of cases is preceded by an increased number of respiratory carriers. Treatment with oral penicillin does not eradicate the carrier state. Rifampin, 600 mg orally twice daily for 2 days (or ciprofloxacin in adults, 500 mg as a single dose), can often eradicate the carrier state and serve as chemoprophylaxis for household and other close contacts. Since the appearance of many sulfonamide-resistant meningococci, chemoprophylaxis with sulfonamides is no longer reliable.
Clinical cases of meningitis present only a negligible source of infection, and isolation therefore has only limited usefulness. More important is the reduction of personal contacts in a population with a high carrier rate. This is accomplished by avoidance of crowding or administration of vaccines as discussed above. As mentioned, such vaccines are currently used in selected populations (eg, the military and in civilian epidemics).
e natural habitat of shigellae is limited to the intestinal tracts of humans and other primates, where they produce bacillary dysentery.
The genus Proteus is characterized by rapid motility and by production of the en/.yme ureasv. By the extent of genomic DNA hybridization, it shows only a distant relationship to E. coli. Proteus is a frequent cause ol urinary tract infections in humans and probably benefits in this regard from its ready ability to degrade urea. Because of the rapid molility ol ‘Proteus cells, colonies growing on agar plates often exhibit a characteristic swarming phcnotype Cells at the edge of the growing colony are more rapidly motile than those in the center of the colony. The former move a short distance away from the colony in a mass and then undergo a reduction in mobility, settle down, and divide, forming a new population of motile cells that again swarm. As a result, the mature colony appears as a series of concentric rings, with higher concentrations oi cells alternating with lower concentrations.
Morphology & Identification
- Typical Organisms
Shigellae are slender gram-negative rods; coccobacillary forms occur in young cultures.
Shigellae are facultative anaerobes but grow best aerobically. Convex, circular, transparent colonies with intact edges reach a diameter of about 2 mm in 24 hours.
- Growth Characteristics
All shigellae ferment glucose. With the exception of Shigella sonnet, they do not ferment lactose. The inability to ferment lactose distinguishes shigellae on differential media. Shigellae form acid from carbohydrates but rarely produce gas. They may also be divided into those Chat ferment mannitol and those that do not (Table 15-4).
Shigellae have a complex antigenic pattern. There is great overlapping in the serologic behavior of different species, and most of them share O antigens with other enteric bacilli.
The somatic O antigens of shigellae are lipopolysaccha-rides. Their serologic specificity depends on the polysaccha-dde. “There are more than 40 serotypes. The classification of shigellae relies on biochemical and antigenic characteristics.
Pathogensis & pathology
Shigclla infections are almost always limited to the gastrointestinal tract; bloodstream invasion is quite rare. Shigellae are highly communicable; the infective dose is on the order of 10J organisms (whereas it usually is 10;-103 for salmonellae and vibrios). The essential pathologic process is invasion of the mucosal epithelial cells (eg, M cells) by induced phagocytosis, escape from the phagocytic vacuole, multiplication and spread within the epithelial cell cytoplasm, and passage to adjacent cells. Microabscesses in the wall of the large intestine and terminal ileum lead to necrosis of the mucous membrane, superficial ulceration, bleeding, and formation of a “pseudoniembrane” on the ulcerated area. This consists of fibrin, leukocytes, cell debris, a necrotic mucous membrane, and bacteria. As the process subsides, granulation tissue fills the ulcers and scar tissue forms.
Diagnostic Laboratory Tests
Specimens include fresh stool, mucus flecks, and rectal swabs. for culture. Large numbers of fecal leukocytes and some red blood cells often are seen microscopically. Serum specimens, if desired, must be taken 10 days apart to demonstrate a rise in tiler of agglutinating antibodies.
The materials are streaked on differential media (eg, MacConkey or EMB agar) and on selective media (Hektoen enteric agar or SalmoneUa-Shigella agar), which suppress other Enterobacteriaceae and gram-positive organisms. Colorless (lactose-negative) colonies are inoculated into triple sugar iron agar. Organisms that fail to produce H2S, that produce acid but not gas in the butt and an alkaline slant in triple sugar iron agar medium, and that are nonmotile should be subjected to slide agglutination by specific Shigella antisera.
Normal persons often have agglutinins against several Shigelia species. However, serial determinations of antibody tilers may show a rise in specific antibody. Serology is not used to diagnose Shigella infections.
Ciprofloxacin, amplillin, doxycycline, and trimethoprimsulfamethoxazole are most commonly inhibitor for Shigella isolates and can suppress acute clinical attacks of dysentery and shorten the duration of symptoms. They may fail to eradiate the organism from the intestinal tract. Multiple drug resistance can be transmitted by plasmids, and resistant infections are widespread. Many cases are self-limited. Opioids should be avoided in Shigella dysentery.
Epidemiology, Prevention, & Control
Shigellae are transmitted by “food, finger, feces, and flies” from person to person. Most cases of Shigella infection occur in children under 10 years of age. Shigellosis has become an important problem in day care centers in the United States. S dysenteriae can spread widely. Mass chemoprophylaxis for limited periods of time (e.g, in military personnel) has been tried, but resistant strains of shigellae tend to emerge rapidly. Since humans are the main recognized host of pathogenic shigellae, control efforts must be directed at eliminating the organisms from this reservoir by (1) sanitary control of water, food, and milk; sewage disposal; and fly control; (2) isolation of patients and disinfection of excreta; (3) detection of sub-clinical cases and carriers, particularly food handlers; and (4). antibiotic treatment of infected individuals.
Morphology & Identification
Corynebacteria are 0.5-1 µm in diameter and several micrometers long. Characteristically, they possess irregular swellings at one end that give them the “club-shaped” appearance. Irregularly distributed within the rod (often near the poles) are granules staining deeply with aniline dyes (metachromatic granules) that gives the rod a beaded appearance. Individual corynebacteria in stained smears tend to lie parallel or at acute angles to one another. True branching is rarely observed in cultures.
On blood agar, the C diphtheriae colonies are small, granular, and gray, with irregular edges, and may have small ones of homolysis. On agar containing potassium tellurite, the colonies are brown to black with a brown-black halo because the tellurite is reduced intracellularly (staphylococci and streptococci can also produce black colonies). Four bio-types of C diphtheriae have been widely recognized: gravis, mitis, interrnedius, and belfanti. These variants have been classified on the basis of growth characteristics such as colony morphology, biochemical reactions, and severity of disease produced by infection, Very few reference laboratories provide the biotype characterization; the incidence of diphtheria has greatly decreased and the association of severity of disease with biovar is not important to clinical or public health management of cases or outbreaks. If necessary in the setting of an outbreak, immunochemical and molecular methods can be used to type the C diphtheriae isolates.
diphtheriae and other corynebacteria grow aerobically on most ordinary laboratory media. On Loeffler serum medium, corynebacteria grow much more readily than other respiratory organisms, and the morphology of organisms is typical in smears made from these colonies,
Corynebacteria tend to pleomorphism in microscopic and colonial morphology. When some nontoxigenic diphtheria organisms are infected with bacteriophage from certain toxigenic diphtheria bacilli, the offspring of the exposed bacteria are lysogenic and toxigenic, and this trait is subsequently hereditary. When toxigenic diphtheria bacilli are serially sub-cultured in specific antiserum against the temperate phage that they carry, they tend to become nontoxigenic. Thus, acquisition of phage leads to toxigenicity (lysogenic conversion). The actual production of toxin occurs perhaps only when the prophage of the lysogenic C diphtheriae becomes induced andlyses the cell. Whereas toxigenicity is under control of the phage gene, invasiveness is under control of bacterial genes.
The principal human pathogen of the genus Corynebacterium is C diphtheriae, the causative agent of respiratory or cutaneous diphtheria. In nature, C diphtheriae occurs in the respiratory tract, in wounds, or on the skin of infected persons or normal carriers. It is spread by droplets or by contact to susceptible individuals; the bacilli then grow on mucous membranes or in skin abrasions, and those that are toxigenic start producing toxin.
All toxigenic C diphtheriae are capable of elaborating the same disease-producing exotoxm. In vitro production of this toxin depends largely on the concentration of iron. Toxin production is optimal at 0.14 |ig of iron per milliliter of medium but is virtually suppressed at 0.5 ug/mL. Other factors influencing the yield of toxin in vitro are osmotic pressure, arnino acid concentration, pH, and availability of suitable carbon and nitrogen sources. The factors that control toxin production in vivo are not well understood,
Diphtheria toxin is a heat-labile polypeptide (MW 62,000) that can be lethal in a dose of 0.1 ug/kg. If disul-fide bonds are broken, the molecule can be split into two fragments. Fragment B (MW=38,000), which has no independent activity, is functionally divided into a receptor domain and a translation domain. The binding of the receptor domain to host cell membrane proteins CD-9 and heparin-binding epidermal growth factor (HBEGF)-like precursor, triggers the entry of” the toxin into the cell through receptor-mediated endocytosis. Acidification of the translocation domain within a developing endosome leads to creation of a protein channel that facilitates movement of Fragment A into the host cell cytoplasm. Fragment A inhibits polypeptide chain elongation—provided nicoti-namide adenine dinucleotide (NAD) is present—by inactivating the elongation factor EF-2. This factor is required for translocation of polypeptidyl-transfer RNA from the acceptor to the donor site on the eukaryotic ribosome. Toxin fragment A inactivates EF-2 by catalyzing a reaction that yields free nicotinamide plus an inactive adenosine diphosphate-ribose-EF-2 complex (ADP-ribosylation). It is assumed that the abrupt arrest of protein synthesis is responsible for the necrotizing and neurotoxic effects of diphtheria toxin. An exotoxin with a similar mode of action can be produced by strains of Pseitdotnonas aeruginosa.
Diagnostic Laboratory Tests
These serve to confirm the clinical impression and are of epidemiologic significance. Note: Specific treatment must never be delayed for laboratory reports if the clinical picture is strongly suggestive of diphtheria. Physicians should notify the clinical laboratory before collecting or submitting samples for culture.
Dacron swabs from the nose, throat, or oilier suspected lesions must be obtained before antimicrobial drugs are administered. Swabs should be collected from beneath any visible membrane. The swab should then be placed in semi-solid transport media such as Amies. Smears stained with alkaline methylene blue or Gram stain show beaded rods in typical arrangement.
Specimens should be inoculated to a blood agar plate (to rule out hemolytic streptococci), a Loeffler slant and a tellurite plate (eg, cystine-tellurite agar or modified Tinsdale’s medium)- and incubated at 37°C. In 12-l8 hours, the Loeffler slant may yield organisms of typical “diphtheria-like” morphology. In 36-48 hours, the colonies on tellurite medium are sufficiently definite for recognition of C diphtheria.
- A filter paper disk containing antitoxin (10 lU/disk) is placed on an agar plate, The cultures to be iested for toxigenicity are spot innoculatcd 7-9 mm away from the disk. After 48 hours of incubation, the antitoxin diffusing from the paper disk has precipitated the toxin diffusing from toxigenic cultures and has resulted in precipitin bands between- the disk and the bacterial growth. This is the modified Elek method described by the WHO Diphtheria Reference Unit.
- Polymerase chain reaction-based methods have been described for detection of the diphtheria toxin gene (tox). PCR assays for tax can also be used directly on, patient specimens before culture results are available, positive culture confirms a positive PCR assay. A negative culture following antibiotic therapy along with a positive PCR assay suggests that the patient probam-y has diphtheria.
- Enzyme-linked immunosorbent assays can be used to detect diphtheria toxin from clinical C diphtheria
- An immunochromographic strip assay allows detection of diphtheria toxin in a matter of hours. This assay is highly sensitive.
The latter two assays are not widely available. Historically, toxigenicity of a C diphtheriae isolate has been demonstrated by injecting two guinea pigs with the emulsified isolate. If the guinea pig protected with diphtheria antitoxin survives while the unprotected one dies, the isolate is considered to be toxigenic. This test has largely been replaced by more modern technology.
The treatment of diphtheria rests largely on rapid suppression of toxin-producing bacteria by antimicrobial drugs and the early administration of specific antitoxin against the toxin formed by the organisms at their site of entry and multiplication. Diphtheria antitoxin is produced in various animals (horses, sheep, goals, and rabbits) by the repeated injection of purified and concentrated toxoid. Treatment with antitoxin is mandatory when there is strong clinical suspicion of diphtheria. From 20,000-100,000 units are injected intramuscularly or intravenously after suitable precautions have been taken fskin or conjunctival test) to rule out hypersen-sitivity to the animal serum. The antitoxin should be given on the day the clinical diagnosis of diphtheria is made and need not be repeated. Intramuscular injection may be used in mild cases.
Antimicrobial drugs (penicillin, erythromycin) inhibit the growth of diphtheria bacilli. Although these drugs have virtually no effect on the disease process, they arrest toxin production. They also help to eliminate coexistent streptococci and C diphtheriae from the respiratory tracts of patients or carriers.
Epidemiology. Prevention, & Control
Before artificial immunization, diphtheria was mainly a disease of small children. The infection occurred either clinically or subclinically at an early age and resulted in the widespread production of antitoxin in the population. An asymptomatic infection during adolescence and adult life served as a stimulus for maintenance of high antitoxin levels. Thus, most members of the population, except children, were immune. By age 6-8 years, approximately 75% of children in developing countries where skin infections with C diphtheriae. are common have protective serum antitoxin levels. Absorption of small amounts of diphtheria toxin from the skin infection presumably provides the antigenic stimulus for the immune response; the amount of absorbed toxin does not produce disease.
Active immunization in childhood with diphtheria tox-oid yields antitoxin levels that are generally adequate until adulthood. Young adults should be given boosters of toxoid, because toxigenic diphtheria bacilli are not sufficiently prevalent in the population of many developed countries to provide the stimulus of subclinical infection with stimulation of resistance. Levels of antitoxin decline with time, and many older persons have insufficient amounts of circulating antitoxin to protect them against diphtheria.
The principal aims of prevention are to limit the distribution of toxigenic diphtheria bacilli in the population and to maintain as high a level of active immunization as possible.
To limit contact with diphtheria bacilli to a minimum, patients with diphtheria should be isolated. Without treatment, a large percentage of infected persons continue to shed diphtheria bacilli for weeks or months after recovery (convalescent carriers). This danger may be greatly reduced by active early treatment with antibiotics.
A filtrate of broth culture of a toxigenic strain is treated with 0.3% formalin and incubated at 37°C until toxicity has disappeared. This fluid toxoid is purified and standardized in flocculating units (Lf doses). Fluid toxoids prepared as above are adsorbed onto aluminum hydroxide or aluminum phosphate. This material remains longer in a depot after injection and is a better antigen. Such toxoids are commonly combined with tetanus toxoid (Td) and sometimes with pertussis vaccine (DPT or DaPT) as a single injection to be used in initial immunization of children. For booster injection of adults, only Td toxoids or Td toxoids combined with acellular pertussis vaccine (for a one-time injection for those individuals who received whole cell pertussis vaccine as children) are used; these combine a full dose of tetanus toxoid with a tenfold smaller dose of diphtheria toxoid in order to diminish the likelihood of adverse reactions.
All children must receive an initial course of immunizations and boosters. Regular boosters with Td are particularly important for adults who travel to developing countries, where the incidence of clinical diphtheria may be 1000-fold higher than in developed countries, where immunization is universal.
There are several species in the genus Listeria. Of these, L nionocytogenes is important as a cause of a wide spectrum of disease in animals and humans. L monocytogenes is capable of growing and surviving over a wide range of environmental conditions. It can survive at refrigerator temperatures (4°C), under conditions of low pH and high salt conditions. Therefore, it is able to overcome food preservation and safety barriers making it an important food-borne pathogen.
Morphology and identification
L monocytogenes is a short, gram-positive, nonspore-forming rod (Figure 12-2). It is catalase-positive and has a tumbling end-over-end motility at 22-280C but not at 370c; the motility test rapidly differentiate liseria from diphtherodis that are members of the normal flora of the skin.
Gram stain of the gram-positive bacillus Listeria monocytogenes in a blood culture. Original magnification x 1000. Red blood cells are present in the backgt-ound. Listeria isolated from clinical specimens frequently show variation in length and often in shape as welt. Typically they are 0.4-0.5 pm in diameter and 0.5-2 urn long.
Culture & Growth Characteristics
Listeria grows well on media such as 5% sheep blood agar on which it exhibits the characteristic small zone of hemolysis around and under colonies. The organism is a facultative anaerobe and is catalase-positive, esculin hydrolysis positive, and motile. Listeria produces acid but not gas from utilization of a variety of carbohydrates.
The motility at room temperature and hemolysin production are primary findings that help differentiate listeria from coryneform bacteria.
Serologic classification is done only in reference laboratories and is primarily used for epidemiologic studies. Serotypes l/2a, l/2b, and 4b make up more than 95% of the isolates from humans. Serotype 4b causes most of the food-borne outbreaks.
Pathogenesis & Immunity
L monocytogenes enters the body through the gastrointestinal tract after ingestion of contaminated foods such as cheese or vegetables. The organism has several adhesin proteins (Ami, Fbp A, and flagellin proteins) that facilitate bacterial binding to the host cells and that contribute to virulence. It has a cell wall surface protein called intcrnalin A that interacts with E-cadherin, a receptor on epithelial cells, promoting phagocytosis into the epithelial cells. After phagocytosis, the bacterium is enclosed in a phagolysosome, where the low pH activates the bacterium to produce listeri-olysin O, This enzyme lyses the membrane of the phagolysosome and allows the hsteriae to escape into the cytoplasm of the epithelial cell. The organisms proliferate and ActA, another listerial surface protein, induces host cell actin polymerization, which propels them to the cell membrane. Pushing against the host cell membrane, they cause formation of elongated protrusions called fiiopods. These filopods are ingested by adjacent epithelial cells, macrophages, and hepatocytes, the listeriae are released, and the cycle begins again. L monocytogenes can move from cell to cell without being exposed to antibodies, complement, or polymorpho-nuclear cells. Shigella fiexneri and rickettsiae also usurp the host cells’ actin and contractile system to spread their infections.
Iron is an important virulence factor. Listeriae produce siderophores and are able to obtain iron from transferring.
Immunity to L monocytogenes is primarily cell-mediated, as demonstrated by the intracellular location of infection and by the marked association of infection and conditions of impaired cell-mediated immunity such as pregnancy, AIDS, lymphoma, and organ transplantation. Immunity can be transferred by sensitized lymphocytes but not by antibodies.
- monocytogenes is found widely in soil and water; virtually no food source is sale from possible L. nionocytogene contamination. Food can become contaminated at any stage during food growth or processing. Food preservation by refrigeration, which ordinarily slows microbial growth, is ineffective in limiting growth of this psyehrotolerant organism. Ready-to-eat meats, fresh soft cheeses, unpasteurized dairy products, and inadequately pasteurized milk are the major food vehicles for this pathogen, even when foods are properly stored at refrigerator temperature (4CC).
- monocytogenes is an intracellular pathogen. It enters the body through the gastrointestinal tract with ingcstion of contaminated food. Uptake of the pathogen by phagocytes results in growth and proliferation of the bacterium, lysis of the phagocyte, and spread to surrounding cells such a.s libn bla.sts. Immunity to L. monocytogenes i.s mainly TI(l cell-mediated. Particularly susceptible populations include the elderly, pregnant women, neonalcs, and immunosuppressed individuals (for example, transplant patients undergoing steroid therapy and AIDS patients). Although exposure to L monocylogenes is undoubtedly very common, there arc only about 2,501) estimated cases of clinical listeriosis each year, and fewer than 1,000 are reported. Nearly all diagnosed cases require hospitalization. Acute lisleriosis is rare and is characterized by sepliccmia, often leading (o meningitis, with a mortality rate of 20% or higher. About 32 listeriosis deaths are reported annually.
Diagnosis, Treatment, and Prevention
Listeriosis is diagnosed by eulluriiig L. monocytogeiicb from the blood or spinal lluid. L. monocytogenes can be identified in food by direct culture or by molecular methods such as ribo-lyping and the polymerase chain reaction (PCR). Ail clinical isolates are analyzed by pulscd-field ge] eleclrophoresis to determine molecular subtypes. The subtype patterns are reported to PulseNel at CDC. Intravenous antibiotic treatment with penicillin, ampicillin, or Irimethoprim plus sulfamethox-azole is recommended for invasive disease.
Prevention measures include recalling contaminated food and taking steps to limit L. monocyogi’iies contamination at the food-processing site. Because L. monocytogenes is susceptible to heat and radiation, raw food and food-handling equipment can be readily decontaminated. However, without pasteurizing the Hnishcd food product, the risk of contamination cannot be eliminated because of the widespread distribution of the pathogen.
Individuals who are irnmunocompromised should avoid unpasteurized dairy products and ready-to-eat processed meats. Spontaneous abortion is a frequent outcome of liste-riosis. Therefore, to protect the fetus, pregnant women should also avoid foods that may transmit L. monocytogenes.
Most streptococci that contain the group A antigen are S pyogenes. It is a prototypical human pathogen. It is used here to illustrate general characteristics of streptococci and specific characteristics of the species. S pyogenes is the main human pathogen associated with local or systemic invasion and poststreptococcal immunologic disorders. 5 pyogenes typically produces large (1 cm in diameter) zones of p hemolysis around colonies greater than 0.5 mm in diameter. They are PYR-positive (hydrolysis of l-pyrrolidonyl-2-naphthylamide) and usually are susceptible to bacitracin.
Morphology & Identification
- Typical Organisms
Individual cocci are spherical or ovoid and are arranged in chains. The cocci divide in a plane perpendicular to the long axis of the chain. The members of the chain often have a striking diplococcal appearance, and rod-like forms are occasionally seen. The lengths of the chains vary widely and are conditioned by environmental factors. Streptococci are gram-positive; however, as a culture ages and the bacteria die, they lose their gram-positivity and can appear to be gram-negative; for some streptococci, this can occur after overnight incubation.
Most group A strains produce capsules composed of hyaluronic acid. The capsules are most noticeable in very young cultures. They impede phagocytosis. The hyaluronic acid capsule likely plays a greater role in virulence than is generally appreciated and together with M protein is felt to be an important factor in the resurgence of rheumatic fever in the United States in the 1980s and 1990s. The capsule binds to hyaluronic-acid-binding protein, CD44, present on human epithelial cell,1;. Binding induces disruption of intercellular junctions allowing microorganisms to remain extracellular as they penetrate the epithelium (Sroll,erman GH and Dale fB). Capsules of other streptococci (eg, 5 agalactiae and S pneumoniae) are different. The Spyogenes eel wall contains proteins (M, T, R antigens), carbohydrates (group-specific), and pcptidoglycans. Hair-like pili project through the capsule of group A streptococci. The PH consist partly of M protein and are covered with lipoteichoic acid. The latter is important in the attachment of streptococci to epithelial cells.
Most streptococci grow in solid media as discoid colonies, usually 1 -2 mrn in diameter. Spyogenes is other species have variable hemolytic characteristics.
- Growth Characteristics
Energy is obtained principally from the utilization of glucose with lactic acid as the end product. Growth of streptococci tends to be poor on solid media or in broth unless enriched with blood or tissue fluids. Nutritive requirements vary widely among different species. The human pathogens are most exacting, requiring a variety of growth factors. Growth and hemolysis are aided by incubation in 10% COr Most pathogenic hemolytic streptococci grow best at 37°C. Most streptococci are facultative anaerobes and grow under aerobic and anaerobic conditions. Peptostreptococci are obligate anaerobes.
Variants of the same streptococcus strain may show different colony forms. This is particularly marked among S pyogenes strains, giving rise to either matte or glossy colonies. Matte colonies consist of organisms that produce much M protein and generally are virulent. The Spyogenes in glossy colonies tend to produce little M protein and arc often not virulent.
A variety of distinct disease processes are associated with S pyogenes infections. The infection can be divided into several categories.
- Diseases Attributable to Invasion byS pyogenes, p-Hemblytic Group A Streptococci
The portal of entry determines the principal clinical picture. In each case, however, there is a diffuse and rapidly spreading infection that involves the tissues and extends along lymphatic pathways with only minimal local suppuration. From the lymphatics, the infection can extend to the bloodstream.
- Erysipelas—If the portal of entry is the skin, erysipelas results, with assive brawny edema and a rapidly advancing margin of infection.
- Celfulitis—Streptococcal cellulitis is an acute, rapidly spreading infection of the skin and subcutaneous tissues. It follows infection associated with mild trauma, burns, wounds, or surgical incisions. Pain, tenderness, swelling, and erythema occur. Cellulitis is differentiated from erysipelas by two clinical findings: In cellulitis, the lesion is not raised, and the line between the involved and uninvolved tissue is
- Necrotizing fasciitis (streptococcal gangrene)—This is infection of the subcutaneous tissues and fascia. There is extensive and very rapidly spreading necrosis of the skin and subcutaneous tissues. Bacteria other than S pyogenes can also cause necrotizing fasciitis. The group A streptococci that cause necrotizing fasciitis have sometimes been termed “flesh-eating bacteria.”
- Puerperal fever—If the streptococci enter the uterus
after delivery, puerperal fever develops, which is essentially a
septicemia originating in the infected wound (endometritis).
- Batteremia/sepsis—Infection of traumatic or surgical wounds with streptococci results in bacteremia, which rap idly can be fatal. S pyogenes bacteremia can also follow skin infections, such as cellulitis and rarely pharyngitis.
- Diseases Attributable to Local Infection with S pyogenes and Their By-Products
- Streptococcal sore throat—The most common infection due to [3-hernolytic S pyogenes is streptococcaj sore throat or pharyngitis. S pyogenes adhere to the pharyngeal epithelium by means of lipoteichoic acid-covered surface pili and alfjo by means of hyaluronic acid in encapsulated strains. The glycoprotein fibronectin (MW 440,000) on epithelial cells probably serves as lipoteichoic acid Jigand. In infants and small children, the sore throat occurs as a subacute nasopharyngitis with a thin serous discharge and little fever but with a tendency of the infection to extend to the middle ear and the mastoid The cervical lymph nodes are usually enlarged. The, illness may persist for weeks. In older children and adults, the disease is more acute and is characterized by intense nasopharyngitis, tonsillitis, and intense redness and edema of the mucous membranes, with purulent exudate, enlarged, tender cervical lymph nodes, and (usually) a high fever. Twenty percent of infections are asymptomatic. A similar clinical picture can occur with infectious mononucleosis, diphtheria, gonococcal infection, and adenovirus infection.
S pyogenes infection of the upper respiratory tract does not usually involve the lungs. Pneumonia, when it does occur, is rapidly progressive and severe and is most commonly a sequela to viral infections, eg, influenza or measles, which seem to enhance susceptibility greatly.
- Streptococcal pyoderma—Local infection of super– ficial layers of skin, especially in children, is called impetigo. It consists of superficial vesicles that break down and eroded areas whose denuded surface is covered with pus and later is encrusted. It spreads by continuity and is highly communicable, especially in hot, humid climates. More widespread infection occurs in eczematous or wounded skin or in burns and may progress to cellulitis. Group A Streptococcal skin infections are often attributable to M types 49, 57, and 59-61 and may precede glomerulonephritis but do not often lead to rheumatic fever.
A clinically identical infection can be caused by S aureus and sometimes both S pyogenes and S aureus arc present.
- Invasive group a streptococcal infections, streptococcal toxic shocks syndrome, and scarlet fever
Fulminant, invasive S pyogenes infections with Streptococcal toxic shock syndrome are characterized by shock, bac-teremia, respiratory failure, and multiorgan failure. Death occurs in about 30% of patients. The infections tend to follow minor trauma in otherwise healthy persons with several presentations of soft tissue infection. These include necrotlzing fasciitis, myositis, and infections at other soft tissue sites; bac-teremia occurs frequently. In some patients, particularly those infected with group A streptococci of M types 1 or 3, the disease presents with focal soft tissue infection accompanied by fever and rapidly progressive shock with multiorgan failure.
Death occurs in about 30% of patients. The infections tend to follow minor trauma in otherwise healthy persons with several presentations of soft tissue infection. These include necrotlzing fasciitis, myositis, and infections at other soft tissue sites; bac-teremia occurs frequently. In some patients, particularly those infected with group A streptococci of M types 1 or 3, the disease presents with focal soft tissue infection accompanied by fever and rapidly progressive shock with multiorgan failure. Erythema and desquamation may occur. The S pyogenes of the M types 1 and 3 (and types 12 and 28) that make pyrogenic exotoxin A or B are associated with the severe infections.
Pyrogenic exotoxins A-C also cause scarlet fever in association with S pyogenes pharyngitis or with skin or soft tissue infection- The pharyngitis maybe severe. The rash appears on the trunk after 24 hours of illness and spreads to involve the extremities. Streptococcal toxic shock syndrome and scarlet fever are clinically overlapping diseases.
- Poststreptococcal Diseases (Rheumatic Fever, lomerulonephritis)
Following an acute S pyogenes infection, there is a latent period of 1-4 weeks, after which nephritis or rheumatic fever occasionally develops. The latent period suggests that these poststreptococcal diseases are not attributable to the direct effect of disseminated bacteria but represent instead a hyper-sensitivity response. Nephritis is more commonly preceded by infection of the skin; rheumatic fever is more commonly preceded by infection of the respiratory tract.
- Acute glomerulonephritis—This sometimes develops 1-4 weeks after S pyogenes skin infection (pyoderma, impetigo). Some strains are particularly nephritogenic, principally with M types 2, 42,49, 56, 57, and 60 (skin). Other nephritogenic M types associated with throat infections and glomerulonephritis are 1, 4, 12, and 25. After random Streptococcal skin infections, the incidence of nephritis is less than 0.5%.
Glomerulonephritis maybe initiated by antigen-antibody complexes on the glomerular basement membrane. The most important antigen is probably in the Streptococcal protoplast membrane. In acute nephritis, there is blood and protein in the urine, edema, high blood pressure, and urea nitrogen retention; serum complement levels are also low. A few patients die; some develop chronic glomerulonephritis with ultimate kidney failure; and the majority recover completely.
- Rheumatic fever—This is the most serious sequela of S pyogenes because it results in damage lo heart muscle and valves. Certain strains of group A streptococci contain cell membrane antigens that cross-react with human heart tissue antigens. Sera from patients with rheumatic fever contain antibodies to these antigens.
The onset of rheumatic fever is often preceded by S pyogenes infection 1-4 weeks earlier, although the infection may be mild and may not be detected. In general, however, patients with more severe Streptococcal sore throats have a greater chance of developing rheumatic fever. In the 1950s, untreated Streptococcal infections were followed by rheumatic fever in up to 3% of military personnel and 0.3% of civilian children. Rheumatic fever is now relatively rare in the United States (<0.05% of streptococcal infections), but it occurs up to 100 times more frequently in tropical countries and is the most important cause of heart disease in young people in developing countries-Typical symptoms and signs of rheumatic fever include fever, malaise, a migratory nonsuppurative polyarthritis, and evidence of inflammation of all parts of the heart (endocardium, myocardium, and pericardium). The carditis characteristically leads to thickened and deformed valves and to small perivascular granulomas in the myocardium (AschoSf bodies) that are finally replaced by scar tissue. Erythrocyte sedimentation rates, serum transaminase levels, electrocardiograms, and other tests are used to estimate rheumatic activity.
Rheumatic fever has a marked tendency to be reactivated by recurrent streptococcal infection whereas nephritis does not. The first attack of rheumatic fever usually produces only slight cardiac damage, which, however, increases with each subsequent attack. It is therefore important to protect such patients from recurrent S pyogenes infections by prophylactic penicillin administration.
Specimens to be obtair tococcal infection. A for culture. Serum is c
Smears from pus oftei definite chains. Cocci the organisms are no to retain the blue. If smears of pus shov anaerobic organisms swabs are rarely cont are always present an> streptococci on stained smears.
Specimens suspected of containing streptococci are cultured on blood agar plates. If anaerobes are suspected, suitable anaerobic media 10% CO2 often speeds hemolysis. Slicing the inoculums into the blood agar has a similar effect, because oxen does not readily diffuse through the organisms, and it is oxygen that inactivates streptolysin O.
Blood cultures will grow hemolytic group A streptococci (e.g, in sepsis) within hours or a few days. Certain a-hemolytic streptococci and enterococci may grow slowly, so blood cultures in cases of suspected endocarditis occasionally do not turn positive for a few days.
The degree and kind of hemolysis (and colonial appearance) may help place an organism in a definition group. S pryogenes can be identified by rapid tests specific for the presence of belonging to group inhibition of growth only when more definitive tests are not available.
- Antigen Detection test
Several commercial kits arte available for rapid detection of group a streptococalal antigen from throat swabs. These kits use enzymatic or chemical methods to extract the antigen from the swab, then use EIA or agglutination tests to demonstrate the presence of the antigen. The tests can be complted minutes to hours after the specimen is obtained. They are 60-90% sensitive, depending upon the prevalence of the disease on the population, and 98-99% specific when compared to culture methods.
A rise in the titer of antigens to many group A streptococcal antigens can be estimed. Such antibodies include ASO, particularly in respiratory disease; anti – Dnase and anthyaluronidase, particularly in skin infections; antistreptokinase; anti-M type specific antibodies; and others of these, the anti-ASO titer is most widely used.
All spyogenes are susceptible to penicillin G, and most are susceptible to erythromycin. Antimicrobial drugs have no effect on established glomerulonephritis and rheumatic fever. In acute streptococcal infections, however, every effort must be made to rapidly eradicate streptococci from the patient, eliminate the anti-genie stimulus (before day 8), and thus prevent poststrep-tococcal disease. Doses of penicillin or erythromycin that result in effective tissue levels for 10 days usually accomplish this. Antimicrobial drugs are also very useful in preventing reinfection with |}-hemolytic group A streptococci in rheumatic fever patients.
Epidemiology, prevention, and control
Although humans can be asymptomatic nasopharyngeal or perineal carriers of Spyogenes, the organism should be considered significant if it is detected by culture or other means. The ultimate source of group. A streptococci is a person harboring these organisms. The individual may have a clinical or from the swab, then use EIA or agglutination tests to demon- subclinical infection or may be a carrier distributing streptococci directly to other persons via droplets from the respiratory tract or skin. The nasal discharges of a person harboring Spyogenef are the most dangerous source for spread of these organisms.
Many other streptococci (viridans streptococci, enterococci, etc) are members of the normal flora of the human body. They produce disease only when established in parts of the body where they do not normally occur (eg, heart valves). To prevent such accidents, particularly in the course of surgical procedures on the respiratory, gastrointestinal, and urinary tracts that result in temporary bacteremia, antimicrobial 1. Detection and early antimicrobial therapy of respiratory and skin infections with group A streptococci. Prompt eradication of streptococci from early infections can effectively prevent the development of poststreptococcal .disease. This requires maintenance of adequate penicillin levels in tissues for 10 days (eg, benzathine penicillin G given once intramuscularly). Erythromycin is an alternative drug, although some S pyogciies are resistant.
- Antistreptococcal chemoprophylaxis in persons who have suffered an attack of rheumatic fever. This involves giving one injection of benzalhine penicillin G intramuscularly, every 3-4 weeks, or daily oral penicillin or oral sulfonamide. The first attack of rheumatic fever infrequently causes major heart damage; however, such persons are particularly susceptible to reinfections with streptococci that precipitate relapses of rheumatic activity and give rise to cardiac damage. Chemoprophylaxis in such individuals, children, must be continued for years. Chemoprophylaxis is not used in glomeru-lonephritis because of the small number of ncphritogenic types of streptococci. An exception may be family groups with a high rate of poslstreptococcal nephritis.
- Eradication of Spyogcnes from carriers. This is especially important when carriers are in areas such as obstetric delivery rooms, operating rooms, classrooms, or nurseries. Unfortunately, it is often difficult to eradicate (5-hemolytic streptococci from permanent carriers, and individuals may occasionally have to be shifted away from “sensitive” areas for some time.
The pneurnococci (S pneumonias) are gram-positive diplo-cocci, often lancet-shaped or arranged in chains, possessing a capsule of polysaccharide that permits typing with specific antisera. Pneurnococci are readily lysed by surface-active agents, which probably remove or inactivate the inhibitors of cell wall autolysins. Pneurnococci are normal inhabitants of the upper respiratory tract of 5-40% of humans and can cause pneumonia, sinusitis, otitis, bronchitis, bacteremia, meningitis, and other infectious processes.
Morphology & Identification
- Typical Organisms
The typical gram-positive, lancet-shaped diplococi are often seen in areoften seen in specimens of young cultures. In sputum or pus, single cocci or chains are also seen. With age, the organisms rapidly become grarn-negative and (ipnd o lyse spontaneously. Autolysis of pneumococci is gseatly
enhanced by surface-active agents. Lysis of pneumococct occurs in a few minutes when ox bile (10%) or sodium deoxcholate (2%) is added to a broth culture or suspension organisms at neutral pH. Viridans streptococci do not J
and are thus easily differentiated from pneumococci, On solid media, the growth of pneumococci is inhibited around a disk of Optochin; viridans streptococci are not inhibited by Optochin. Other identifying points include almost uniform %iru-lence for mice when injected intraperitoneally and tha “capsule swelling test,” or quellung reaction.
Pneumococci form small, round colonies, at first some shaped and later developing a central plateau with an deviated rim. Pneumococci are a-hemolytic on blood agar. Growth is enhanced by 5-10% CO2.
- Growth Characteristics
Most energy is obtained from fermentation of glucose this is accompanied by the rapid production of lactic acid, which limits growth. Neutralization of broth cultures with alkali at intervals results in massive growth.
Pneumococcal isolates that produce large amount of capsules produce large mucoid colonies. Capsule production is not essential for growth on agar medium, and capsular production is, therefore, lost after a small number of subcultures. The pneumococci will, however, again produce capsules and have enhanced virulence if injected into mice.
- Production of Disease
Pneumococci produce disease through their ability to multiply in the tissues. They produce no toxins of significance. The virulence of the organism is a function of its capsule, which prevents or delays ingestion by phagocytes. A serum that contains antibodies against the type-specific polysaccharide protects against infection. If such a serum is absorbed with the type-specific polysaccharide, it loses its protective power. Animals or humans immunized with a given type of pneumococcal polysaccharide arc subsequently Immune to that type of pneumococcus and possess precipitating and opsonizing antibodies for that type of polysaccharide.
- Loss of natural resistance
Since 40-70% of humans are at some time carriers of virulent pneumococci, the normal respiratory mucosa must possess great natural resistance to the pneumococcus. Among the factors that probably lower this resistance and thus predispose to pneumococca infection are the following:
- Viral and other respiratory tract infections that damage surface cells; abnormal accumulations of mucus (eg, allergy), which protect pneumococci from phagocytosis; bronchial obstruction (eg, atelectasis); and respiratory tract injury due to irritants disturbing its rnucociliary function.
- Alcohol or drug intoxication, which depresses phagocytic activity, depresses the cough reflex, and facilitates aspiration of foreign material.
- Abnormal circulatory dynamics (eg, pulmonary congestion, heart failure), Other mechanisms, e.g, malnutrition, general debility, sickle cell anemia, hyposplenism, nephrosis, or complement deficiency.
Diagnostic Laboratory Tests
Blood is drawn for culture; CSF and sputum are collected for /’demonstration of pneumococci by smear and culture. Serum antibody tests are impractical. Sputum may be examined in several ways.
- Stained Smears
A Gram-stained film of rusty-red sputum shows typical organisms, many polymorphonuclear neutrophils, and many red cells.
- Capsule Swelling Tests
Fresh emulsified sputum mixed with antiserum causes capsule swelling (the quellung reaction) for identification of pneumococci.
The culture is created by sputum cultured on blood agar and incubated in CO2 or a candle jar. A blood culture is also taken.
Immunity to infection with pneumococci is type-specific and depends both on antibodies to capsular polysacchari.de and on intact phagocytic function. Vaccines can induce production of antibodies to capsular polysaccharides.
Since pneumococci are sensitive to many antimicrobial drugs, early treatment usually results in rapid recovery, and antibody response seems to play a much diminished role. Penicillin G is the drug of choice, but in the United States 15% of pneumococci are penicillin-resistant (MIC >2 ug/mL) and about 18% are moderately resistant (MIC 0.1-1 ug/mL). High-dose penicillin G with MICs of 0.1-2 ug/ml, appears to be effective in treating pneumonia caused by pneumococci but would not be effective in treatment of meningitis due to the same strains. Some penicillin-resistant strains are resistant to cefo-taxime. Resistance to tetracycline and erythromycin occurs also. Pneumococci remain susceptible to vancomycin.
Epidemiology/ Prevention, & Control
PneUinococcal pneumonia accounts for about 60% of all bac-lefia! pneumonias. In the development of illness, predisposing factors (see above) are more important than exposure to the infectious agent, and the healthy carrier is more important in disseminating pneumococci than the sick patient.
It is possible to immunize individuals with type-specific polysaccharides. Such vaccines can probably provide 90% protection against bacteremic pneumonia. A polysaccharide vaccine containing 23 types is licensed in the United States. This vaccine is appropriate for elderly, debilitated, or immu-nosuppressed individuals. A pneumococcal conjugate vaccine contains capsular polysaccharides conjugated to diphtheria CRM|97 protein. This seven-valent vaccine is recommended for all children aged 2-23 months, to help prevent invasive infections, and for selected children aged 24-59 months.
The enterococci have the group D group-specific substance and were previously classified as group D streptococci. Because the group D cell wall specific antigen is a teichoic acid, it is not an antigenically good marker; enterococci are usually identified by characteristics other than immuno-logic reaction with group-specific antisera. They are part of the normal enteric flora. They are usually nonhemolytic, but occasionally a-hemolytic. Enterococci are PYR-positive. They grow in the presence of bile and hydrolyze esculin (bile esculin-positive). They grow in 6.5% NaCl. They grow well at between 10°C and 45°C whereas streptococci generally grow at a much narrower temperature range. They arc more resistant to penicillin G than the streptococci, and rare isolates have plasmids that encode for pMactamase. Many isolates are vancomycin-resistant. There are at least 12 species of enterococci. Enterococcus faecalis is the most common and causes 85-90% of entero-coccal infections, while Enterococcus faecium causes 5~LO%. The enterococci are among the most frequent causes of noso-comia! infections, particularly in intensive care units, and are selected by therapy with cephalosporins and other antibiotics to which they are resistant. Enterococci are transmitted from one patient to another primarily on the hands of hospital personnel, some of whom may carry the enterococci in their gastrointestinal tracts. Enterococci occasionally are transmitted on medical devices. In patients, the most common sites of infection are the urinary tract, wounds, biliary tract.
Tetanus is a serious, often life-threatening disease. Although tetanus is preventable through immunization, about 500 individuals have acquired tetanus in the United Stales within the last decade and about 75 of these have died. Worldwide, tetanus causes over 200,000 deaths per year, even though it is a preventable infectious disease.
Telanus is caused by an cxotoxin produced by Clostndium tetani, an obligalcly anaerobic, end of pore- forming rod (‘ » ‘Section 16.2). The natural reservoir of C. tetani is soil, where it is a ubiquitous resident, although it is occasionally found in the gut of mammals, as are other Clostridium species.
Cells of C. teictni normally gain access to the body through a soil-contaminated wound, typically a deep puncture. In the wound, anoxic conditions allow germination of endospores, growth of the organism, and produclion of a potent exoloxin, the tetanus toxin. The organism is n on invasive; its sole method of causing disease is through the action of tetanus toxin on host cells. The incubation time is variable and may take from 4 days to several weeks, depending on the number of endospores inoculated at the time of injury. Tetanus is not transmitted from person to person.
Diagnosis, Control, Prevention, and Treatment
Diagnosis of tetanus is based on exposure, clinical symptoms, and, rarely, identification of the toxin in the blood or tissues of the patient. The organism may also be cultured from the wound, but success is highly variable.
The natural reservoir of C. lelaui is the suil. Because C. tetani is. an accidental pathogen in humans and is not dependent on humans or other animals for its propagation, there is no possibility for eradication. Therefore, control measures must focus on prevention.
Tetanus is a preventable disease. The existing loxoid vaccine is completely effective for disease prevention. Virtually all tetanus cases occur in individuals: who were inadequately immunized. Individuals from 25-59 years of age are the fastest growing age group for contracting tetanus, presumably because public health immunization programs target infants, school-age individuals, and seniors 60 years of age and older.
Appropriate treatment ol” serious cuts, lacerations, and punctures includes administration of a “booster” tetanus loxoid immunization. If the wound is severe and is contaminated by soil, treatment should also include administration of an antitoxin preparation, especially if the patient’s immunization stalus is unknown or is out of date. The tetanus antitoxin is typically pooled human ami-tetanus immunoglobulin (approved for human use worldwide) or a preparation of antibodies to tetanus made in horses (approved for use in many developing countries). Both of these preparations work by binding and neutralizing the tetanus exoloxin. The anli-loxin is generally given intramuscularly, but intrathccal injection (injection into ihc sheath surrounding the spinal cord) is superior because the antitoxin can then get to the affected nerve root much more efficiently ( Section 28.10). These measures prevent active tetanus irom occurring.
Acute symptomatic tetanus is treated with antibiotics, usually penicillin, to stop growth and toxin production by C. tetani and antitoxin to prevent binding of newly released toxin to cells. Supportive therapy such as sedation, administration of muscle relaxants, and mechanical respiration may be necessary to control the effects of paralysis. Treatment cannot provide a reversal of symptoms, because toxin that is already bound to tissues cannot be neutralized. Even with; antitoxin, antibiotics, and support therapy, tetanus patients have significant morbidity and morality.
Clostridial Food Poisoning
Clostriditini pefringens and Clostridium botulinum cause serious food poisoning. Members of the genus Clostridium are anaerobic endospore forming rods. Canning and cooking procedures kill living organisms but do not necessarily kill endospores. Under appropriate anaerobic condition, the endospores can then germinate and toxin is produced.
Clostridium perfringnes food poisoning
- Perfringens is an anaerobic, gram-positive endospore forming rod commonly found in soil. It also lives in small numbers in the intestinal tract of many animals and humans and is therefore found in sewage. C. perfringens is the most prevalent reported cause of food poisoning in the united state, with an estimated 248,000 annual cases.
Perfringers food poisoning requires the ingestion of a large does of C. perfringens (>108 cells) in contaimined cooked or uncooked foods, especially high-protein foods such as meat, dishes cooked in bulk. In such food preparations, heat penetration is often insufficient, and surviving
- perfringenes endospores germinate under anoxic conditions, such as in a sealed container. The C. perfringens grows quickly in the meat, especially if the food is left to cool at 20-400C for short time periods. However, the toxin is not yet present.
After consumption of the contamined food, the living C. perfringens begin to sporulate in the intestine, which coincides with production of the perfingenes entertoxin. When ingested, perfringenes enterotoxin alters the permeability of the intestinal epithelium, leading to nausea, diarrhea, and intestinal cramps, usually with no fever. The onset of perfringens food poisoning begins about 7-15 hours after consumption of the contamined food but usually resolves within 24 hours, and fatalities are rare.
Diagnosis, treatment, and prevention
Diagnosis of perfringens food poisoning is made by isolation of C. perfringens from the feces or, more reliably, by a direct enzyme-linked immunosorbent assay (ELISA) to detect C. perfringens entertoxin in feces. Because C. perfringenes food poisoning is self-limiting, antibiotic treatment is not indicated. Supportive therapy can be used in serious cases. Prevention oi: perfringens food poisoning requires measures to prevent contamination of raw and cooked foods and control of cooking and canning procedures to ensure proper heat treatment of ail foods. Cooked foods should be refrigerated as soon as possible to rapidly lower temperatures and inhibit C. perfriagcns growth,
Botulism is a severe, often fatal, food poisoning that occurs / following the consumption of food containing the exotoxin produced by C. botulinurn. This bacterium normally inhabits soil or water, but its endospores may contaminate raw foods before harvest or slaughter. If the foods are properly processed so that the C. botulinum endospores are removed or killed, no problem arises; but if viable endospores are present, they may germinate and produce toxin. Even a small amount of the resultant neurotoxin can be dangerovis.
We discussed the nature and activity of bolulinum toxin in Section 28.10 Botulinum toxin is a neurotoxin that causes flaccid paralysis, usually affecting the autonomic nerves that control body functions such as respiration and heartbeat. At least seven distinct botulinum toxins are known. However, because the toxins are destroyed by heat (80°C for 10 minutes), thoroughly cooked food, even if contaminated with toxin, can be totally harmless.
Most cases of foodborne botulism are caused by eating foods that are not cooked after processing. For example, nonacid, home-canned vegetables (e.g., home-canned corn and beans) are often used without cooking when food poisoning. An average of 24 cases of foodborne.. botulism have occurred per year in the United Slates from 2000-2005.
The majority of botulism cases occur following infection with C. botulinum. For example, infant botulism occurs when neonates ingest endospores of C. botulinum In some cases, raw honey is the vehicle, but more often the source cannot be identified. If the infant’s normal flora is not developed or if the infant is undergoing antibiotic therapy, endospores can germinate in the infant’s intestine, triggering C. bolulinum growth and toxin production. Most cases of infant botulism occur between the first week of life and 2 months of age, rarely occurring in children older than 6 months when the normal intestinal flora is more developed. Over 60% of all botulism cases in the United States are in infants. An average of 85 cases of infant botulism have occurred per year in the United Slates from 2000 to 2005. Wound botulism can also occur from infection, presumably from endospores introduced by introduction of contaminating material by a parenteral route. Wound botulism is most commonly associated with illicit injectable drug use and in the United States has averaged 26 cases per year from 2000 to 2005.
All forms of botulism arc quite rare, with at most six cases occurring per 10 million individuals per year in the United States in recent years. Botulism, however, is a very serious disease because of the high mortality associated with the disease. Over the last decade there have been fewer than 155 cases each year, but about 25% of all cases were fatal. Death occurs from respiratory paralysis or cardiac arrest due to the paralyzing action of the botilium neurotoxin.
Diagnosis, treatment, and prevention
Diagnosis of botulism is by demonstrating botulism toxin in patient serum or by finding toxin or live C. botulinum in suspected food products. Laboratory findings are coupled with clinical observations, including neurological signs of localr/ed paralysis (impaired vision and speech) beginning 18-24 hours after ingestion of contaminated food. Treatment involves administration of botulinum antitoxin if the diagnosis is early, and mechanical ventilation for flaccid respiratory paralysis. In infant botulism. C. bolulinum and toxin are often found in bowel contents. Infant botulism is usually self-limiting, and most infants recover wilh only supportive therapy, such as assisted ventilation. Antitoxin administration is not recommended. Respiratory failure causes occasional deaths.
Prevention of botulism requires maintaining careful controls over canning and preservation methods. Susceptible foods should be heated to destroy endospores; boiling for 20 minutes destroys the toxin. Home-prepared foods are the most common source of foodborne botulism outbreaks. In addition, feeding honey to children under 2 years of age is not recommended because honey is an occasional source of C. botulinum edospores.
The spores bf Clostridium tetani_are ubiquitous. They occur in the gastrointestinal tracts of man and animals. They are also present in the soil especially in manured soil. Tetanus develops following the contamination of wound with Clostridium tetani spores. The source of infection may be soil, dirty clothing or faeces. Spores of Clostridium tetani may be embedded in surgical catgut (prepared from cattle and sheep gut). Germination of spores is dependent upon the reduced oxygen tension occurring in devitalized tissue.
Infection strictly remains localized in the wound and the disease is due to the effect of a potent diffusible exotoxin (tetanospasmin). Conditions that favour the germinatioiji of spores and the multiplication of the organisms in the tissues are similar to those of Clostridiwn perfringens. However, tetanus may also develop following (superficial abrasion, septic abortion, thorn prick, and cleansing of auditory meatus with a small slick. Tetanus neonatorum follows the infection of umbilical wound of newborn infants. Postoperative tetanus may be due to imperfectly; sterilized catgut, intestinal contents during abdominal surgery and air of the operation theatre containing spore-bearing dust.
Clostridium tetani remains localized at the site of initial infection and produces tetanus toxin. It is absorbed from the site of its production and ascends to the central neryous system via motor nerves. However, some toxin may be delivered from the siie of infection via the blood to all nerves in the body and the subsequent transmission to the central nervous system depends upon uptake through neuromusqular nerve endings and intra-axbnal transport. Therefore, the first symptoms Jin human tetanus appear in head and neck because of the shorter length.
of the cranial nerves.
Tetanospalmhi resembles strychnine in its effect. It appears to act by interfering with the normal inhibition of motor impulses exerpised by the upper motor neuron over the lower. This results in sustained muscle spasm and the characteristic signs spasm pi jaw (lockjaw, trismus) and facial muscles (rims sardonicus), and arching of the body (opisthotonicus).
The incubation period of tetanus varies from 2 days to several weeks but commonly. It is 6-12 days. Tetanus is a serious disease with a high mortality rate of 80-90% without proper treatment and even with proper treatment is 15-50%. Tetanus nonatorum and uterine tetanus have very high fatality rates (70-100%). In rural India, it in fourth commonest cause of death.
CLOSTRIDIUM PSEUDOMEMBRANOUS COLITIS
Pseudomembranous colitis, a cause of antibiotic-associated diarrhea (AAD), is an infection of the colon. It is often, but not always, caused by the bacterium Closlridium difficile. Because of this, the informal name C. difficile colitis is also commonly used. The illness is characterized by offensive-smelHng diarrhea, fever, and abdominal pain. In severe cases, life-threatening complications develop, such as toxic megacolon.
Mechanism of disease
The use of clindamycin, broad-spectrum antibiotics such as cephalosporins, or any penicillin-based antibiotic such as amoxicilljn causes the normal bacterial flora of the bowel to be altered. In particular, when the antibiotic kills off other competing bacteria in the intestine, any bacteria remaining will have less competition for space and nutrients. The net effect is to’ permit more extensive growth than normal of certain bacteria. Clostridium difficile is one such type of bacterium. In addition to proliferating in the bowel, C, difficile also produces-toxins. Without either Toxin A or Toxin B, C. difficile may colonize the gut but is unlikely to cause pseudomembranous colitis.
Risk factors and epidemiology
In most cases, a patient presenting with pseudomembranous colitis has recently been on antibiotics. Antibiotics disturb the normal bowel bacterial flora. Certain antibiotics such as ampicillin have a higher propensity to create an environment where pseudomembranous colitis can outcoinpete the normal gut flora. Clindamycin is the antibiotic classically associated with this disorder, but any antibiotic can cause the condition. Even though they are not particularly likely to cause pseudomembranous colitis, due to their very frequent use, cephalosporin antibiotics (such as cefazolin and cephalexin) account for a large percentage of cases. Diabetics and the elderly are also at increased risk, although half of cases are not associated with risk factors.
Other risk factors include increasing age and recent major surgery. There is some evidence that jproton pump inhibitors are a risk factor for C. difficile infection and pseudomembranous colitis, but others question whether this is a false association or statistical artifact (increased PPI use is itself a marker of increased age and co-morbid illness).; indeed, one large case-control study showed that PPIs are not a risk factor.
Pseudomembranous colitis in compuiertomography.
As noted above, pseudomembranous colitis is characterized by diarrhea, abdominal pain, and fever. Usually, the diarrhea is non-bloody, although blood may be present if the affected individual is taking blood thinners or has an underlying lower bowel condition such as hemorrhoids. Abdominal pain is almost always present and may be severe. So-called “peritoneal” signs (e.g., rebound temlcrncss) may be present “Constitutional” signs such as fever, fatigue, and loss of appetite are prominent. In fact, one of the main ways of
distinguishing pseudomembranous colitis .from other antibiotic-associated diarrheal states is that patients with the former are sick. That is, they are often prostrate, lethargic, and in general look unwell. Their “sick” appearance tends to be paralleled by the results of their blood tests, which often show anemia, an elevated white blood cell count, and low serum albumin.
Endoscopic image of pseudomembranous colitis, the yellow pseudomembranes seen on the wail of the sigmoid colon Pathological specimen showing pseudomembranous colitis.
In order to make the diagnosis, it is, of course, essential that the treating physician be aware of any recent antibiotic usage. The disease may occur as late as six months after Antibiotic use. Although there is some relationship between dose/duration of antibiotic and the likelihood of developing pseudomembranous colitis, it may occur even after a single dose of antibiotic. In fact, the of a single-dose prophylactic antibiotic is a common practice in surgical and dental patients to prevent infections associated with a procedure.
Hence, even though unlikely to cause pseudomembranous colitis on aper-case basis, single-dose antibiotic treatment, by virtue of the large number of patients receiving such, is an important cause of pseudomembranous colitis. Use of’ proton pump inhibitor’ drugs such as omcprazolc for gastric-reflux, or some forms of asthma inhaler, in fact, all drugs with anlicholinergjc effects that slow the digestive transit time lead to retention of toxins and exacerbate the effects of broad-spectrum antibiotics.
Prior to the advent of tests to detect Clostndium difficile toxins, the iagnosis was most often made by colonoscopy or sigmoidoseopy. The appearance of “pseudomembranes” on the mucosa of the colon or rectum is diagnostic of the condition. The pseudomembranes arc composed of an exudate made of inflammatory debris, white blood cells, etc.
Although colonoscopy and sigmoidoseopy are Mill employed, stool testing for the presence of Clostridium difficile toxins is now often the first-line diagnostic approach. Usually, only two toxins arc tested for – Toxin A and Toxin B – but the organism produces several others. This test is not 100% accurate, and there is a considerable false negative rate even with repeat testing.
Another, more recent two-step approach involves testing for the presence of C. in the stool and then testing for toxin production. The first step is performed by testing for the presence of the C. Diff GDH antigen. If the first step is positive, a second test, a PCR assay targeting the toxin genes, is performed.
The disease is treated either with oral vancomycin or with intravenous melronidazole. Choice of drug depends on severity of disease and the ability to tolerate and absorb oral medications. Vancomycin treatment docs present the risk of the development of vancomycin-resistant cnterococcus, though it is only minimally absorbed into the blood
stream from the gastrointestinal tract Mclronidazolc has on occasion? been associated with the development of pscudomcmbranouz colitis. In these cases, metronidazole is still an effective treatment, since tiic cause of the colitis is not the antibiotic, but rather the change in bacterial flora from a previous round of antibiotics. Clostridium difficile infections thai do not respond lo vancomycin or mclronidazole arc sometimes treated with oral rifaximin. Fidaxomicin is a new alternative that has been approved for treatment in mid-201 1.
Cholcslyraminc and other bile acid scquestrants should not be used as adjunctive therapy because, though they may bind the C..difficile toxin, they can also inhibit the effects of the primary antibiotic.
Several probiolic therapies have bcSRscd as adjunct therapies for pseudomembranous colitis. Saccharomyce.? bonlqrdii (JjB’s yeast) has been shown in one small study of 124 patients to reduce the recurrence rate of pseud omembranous colitis. A number of mechanisms have been proposed to explain this effect. Fecal bacteriotherapy in a medical treatment, which involves restoration of colon homcostasi; f reintroducing normal bacterial flora from stool obtained from a healthy donor, has been successfully used to treat acute pseudomembranous colitis.
If antibiotics do not control the infection, the patient may require a olectomy (removal of the colon) for treatment of the colitis.
A randomized controlled trial using a probiotic drink containing Laclobacillus casei, L bulgaricux, and Streptococcus thermophilus was reported to have some efficacy. This study was, however, sponsored by the company that produces the drink. Although intriguing, several other studies have been unable to demonstrate any benefit of oral supplements of similar bacteria at preventing C. difficile-associated diarrhea (CDAD).
Sarah A. kuehen T, Stephen T. Cartman, John T. Heap, Michelle L. Kelly, Alan
rockaync & Nigel P. Minion (2010). “The role of toxin A and toxin B in Clostridium
difficile infection””. 467 (7316): 711-3.
Kalzung, Bertram G. (2G* 17). Basic and ‘finical Pharmacology.
New York, NY: McGraw Mill Medical, pp. 733.
Deslipandc A, Pant C, Pasupulcli V (201 1). “Association between Proton Pump
Injjjtmor therapy and Clostridium difficile infection in a Meta-Analysis”. Clin.
Jawetz, Melnicks and Adelberg, ed. (2010) medical microbiology (25th ed)
Mc Graw Hill pp. 176-350.
Ha-rfis L.G, Foster S.J, Richards S. G. (2002). “An introduction to Staphylococcus laureus, and techniques for identifying and quantifying S. aureus adhesins in relation to adhesion to biomaterials: review”. European cells and materials 4: 39-60.
Madiagn M, Martinko J, ed. (2009). Brock biology of microorganisms (12th ed).
Prentice Hall. ISBN 0131443291.
Takahashi T, Satoh I, Kikuchi N. (1999). “Phylogenetic relationships of 38 taxa
of the genus staphylococcus based on 16S rRNA gene sequence analysis”.
Int. J. syst. Bacterial. 49 (2): 725-728.
Kloos WE, Ballard DN, George CG, Webster JA, Hubner RJ, Ludwig W, Schleifer KH, Fiedler F, Schubert K (1998). “Delimiting the genus Staphylococcus through Inscription of Macrococcits caseolyticits gen. nov.. comb. rtov. and Macrococcus eqiiipercicus sp. nov., and Macrococcus bovicus sp. nov. and Macrococcus carouselicus sp. nov”. Int J Syst Bacteriol 48 (3): 859-877..
Svec P., Vancanneyt M., Sedlaek L, Engelbeen K., Stetina V., Swings, J. & Petra, P. (2004). “Reclassifiealion of Staphylococcus pulvereri Zakrzewska-Czerwiska et al. 1995 as a later synonym of Staphylococcus vitulinus Webster et al 1994″. Int. J. Syst. Evoi MicrQbiot 54 (6): 2213-2215.
Ghebremedhin B, Layer F, Konig W, Konig B (2008). “Genetic classification and distinguishing of Staphylococcus species based on different partial gap, 16S rRNA, hsp60,rpoB,sodA, and tufgpne sequences”. J. din. Microbiol. 46 (3): 1019-1025..
Beuchat, L. R_; E. K.. Heaton (2003). “Salmonella Survival on Pecans as Influenced b\ Processing and …Storage Conditions”. Applied and Environmental Microbiology 29 (6): 795-801. PMC !8~OS2. “\XX PMID 1098573. Retrieved 2010-08-19. “Little decrease in viable population of the three species was noted on inoculated pecan halves stored at -18. -7, and 5 °C for 32 weeks.
Cornelis P (2000). “expressing genes in different eschericha coli compartments”. Curr. Opin. Biotechnol. 11 (5): 450-454.
Eckburg PB, Bik EM, Bernstein CN, Purdon E, Dethlesfen l et al. (2005). “Diversity of the human intestinal microbial flora”. Science 308 (5728): 1635-1638.
Fotadar U, zaveloff P, Terracio L (2005). “Growth of escherichai coli at elevated temperatures”. J. Basic Microbiol 45. (5): 403-4.
George M Gsrfrity, ed. (2005) [1984(Wiliiams & Wilkins)J. The Gammaproteobacteria, I3erge>’s [anual of Systematic Bacteriology. 2B (2nd ed.)- Ne\v York: Springer, pp. 1 108. ISBN 978-0-387-24144-9. British Library no. International Bulletin of Bacteriological Nomenclature and Taxonomy 8:73-74 (1958) .
Ishii S and Sjidowsky MJ. 2008. Escherlchia coli in the Environment: Implications for Water Quality and furnan Health. Microbes andEnvironments 23: 101-108.
Lledo W^Hernandez M, Lopez. E, (2009). Guidance for Control of Infections with Carbapenem-Resistant or Carbapenemase-Producing “Enterobacteriaceae”” in Acute Care Facilities. 58( 10);256-26() https://www. cdc.gov/mmwr/preview/mmwrhtml/mm5 810a4.htm.
MadigaJ-rlM, Martinko J, ed. (2005). Brock Biology of Microorganisms (1 1th ed.). Prentice Hall. ISBN 0-3-144329-1.
Ryan KJ, Rt^CG (editors) (2004). Sherris Medical Microbiology (4th ed.). McGraw Hill. pp. 362-8. !BN 0-8385-8529-9.
Todar, K. Pathogenic £. cofi”. Online Tex/book of Bacteriology. University of Wisconsin Madison Department,of Bacteriology. Retrieved 2007-11-30.
Whitt, Dixie D:. Salvers, Abigail A. (2002) . “14”. Baclerial Paihogenew: A Molecular Approach (2nded.). USA: ASM Press. ISBN 1-55581-1 71-X.
Ice cream is a frozen blend of sweetened cream mixture and air, with added flavouring. A wide variety of ingredients are allowed in ice cream, but the minimum amount of milk fat, milk solids (protein + lactose + minerals) and air are defined by standard of identify in the united state ice cream must contain at least 10% milk fat and at least 20% total milk solids and may contain safe and suitable sweeteners, emulsifiers and stabilizers and flavouring materials. Ice cream is sold as hard or soft curve. After freezing process only a portion of the water is actually in a frozen state. Soft ice cream is served directly from the freezer where only a small amount of water has been frozen. Hard ice cream is packaged from the freezer and then goes through a hardening process that freezes more of the water in the mix.
There are wide range of ingredient and formulations (recipes) that can be used in ice cream. The basic types of ingredients and their function are described below:
Milk fat provides creaminess and richness to ice cream and contributes to its melting characteristics. The minimum fat content is 10% and the premium ice cream can contain as much as 16% milk fat. Source of milk fat include milk, cream and butter.
The total solids component of ice cream includes both the fat and other solids. The other solids milk consists of protein and lactose. The nonfat solid plays an important role in the body and texture of ice cream by stabilizing the air is incorporated during the freezing process. Some of solid not fat include milk, cream, condensed milk, evaporated milk, dry milk and whey.
Sweeteners are used to provide the characteristics sweetness of ice cream, it also lower the freezing point of the mix to allow some water to remain unfrozen at serving temperature. A lower freezing point uncikes ice cream easier to scope and eat, though the addition of too much sugar can make the product too soft. Sweeteners used include sugar and corn syrup. Corn syrup improves and help to maintain smoothness when ice cream encounters temperature fluctuation of heat-shock. It gives a desirable chewiness to the body of the product due to the presence of dixtrins, high molecular-weight polysaccharides that remain after acid hydrolysis of the cornstarch to produce corn syrup. When the flavoring of the syrup itself cannot be detected, there is a masking effect on ice cream flavour particularly for vanilla.
Stabilizers are proteins or carbohydrates used in ice cream to add viscosity and control ice crystallization. Over time during frozen storage small ice crystals naturally migrate together and from larger ice crystals stabilizers help to keep the small crystals isolated and prevent the growth of larger crystals, which causes ice cream to be coarse, icy and unpleasant to eat. Stabilizers used include alginates (carageenan) gums (locust bean, guar) and gelatins. Hums of various kind are useful as a stabilizers in ice cream because of their characteristics property of imbibing or absorbing large amount of water. Some gums are especially effective in increasing viscosity; other produces a heavier body or provide better resistance to heat short.
Emulsifiers are used to help keep the milk fat evenly dispersed in the ice cream during freezing and storage. A good distribution of fat helps stabilize the air incorporated into ice cream and provide smooth product. Emulsifiers used in ice cream become more inform, more versatile and almost essential to modern ice cream manufacturing.
Different range of flavouring are used in ice cream production which include both natural and artificial flavours such as fruits, nuts, and bulky inclusions like chocolate chunks and candies for better quality products.
General manufacturing procedure of ice cream
- blend ingredients
- pasteurize mix
- age mix
- add liquid flavour and colours
- add fruits, nuts, and bulky flavourings
- BLEND THE ICE CREAM MIXTURE
The milk fat source, nonfat solid, stabilize and emulsifiers are blended to ensure complete mixing of liquid and dry ingredients.
- Pasteurize mix
Ice cream mix is pasteurize at 68.30C for 30 minutes. The condition used to pasteurizes ice cream mix are grater than those use for fluid milk because of increased viscosity front higher fat solid and sweetener content and condition of egg yolks in custard products.
Ice cream is homogenize to decrease the milk fat globule size to form a better emulsion and contribute to a smoother, creamier ice cream. It also ensures that emulsifiers and stabilizer are well blended and evenly distributed in the ice cream mix before it is frozen.
- Age mix
Ice cream mixed is aged to 50C for at last 4 hours or overnight aging the mix cools it down before freezing. Aging improves the whipping properties of the mix.
- Add liquids flavours and colours
Liquid flavours and colour may be added to the mix before freezing.
- Freeze: The process involves freezing the mix and incorporating air. Ice cream mix can be frozen in batch or continuous freezer and the condition used will depend on the type of freezer. The addition of air is called overrun and contributes to the lightness of ice cream. The overrun level can be set as desired to adjust the denseness of the finished product.
- Add fruit, nut and bulky flavouring: All these may be added at this point. These ingredient cannot be added before freezing or the would interfere with smooth flow of the mix through the freezer.
- Package: as desired, depending on the product.
- Harden: The ice cream is cooled as quickly as possible down to a holding temperature of less than 250 The temperatures and time of cooling will depend in the type of storage freezer. Storage at 250C will help to stabilize the ice crystals and inclination products quality.
Chocolate is made from the cocoa bean, found in pods, growing from the trunk and lower braches of the cocoa tree (theobroma cacao) which means food for the gods.
Chocolate as we know today was largely made possible by three events:-
- In 1828, Dutch Chemist Johannes Van Houten, invested a methods of extracting the fat or cocoa butter, from ground cocoa beans. The resulting cocoa power was much less bitter testing and when combined with sugar or honey, made a drink much more palatable to our test.
- This process known as van Houten process made it possible for try and sons of Bristol, England to manufacture and sell the first solid chocolate bar in 1847.
- In 1875 a Swiss manufacturer, Daniel peters used van Houten process to successfully combine chocolate with powered milk to produce the first milk chocolate.
CHOCOLATE MILK FOR MUTATION
- Dutched cocoa
- Marine colloids
- Dry blended cocoa, sucrose and marine colloids add to milk
- If liquid sugar or corn syrup is used, meter into batch
- Add dry blended ingredients.
- Pasteurize by batch UHT heat processing techniques.
- Cool to 40C and fill. If liquid sugar or corn syrup is used meter into batch.
Chocolate milk is manufactured in dairies primarily with hot process dairy power base mixes. Hot process dairy syrups are also available but their use is limited. At the dairy, sweetener is added to the base mix combined with raw milk to process the finished chocolate milk under appropriate pasteurization and homogenization parameters. The hot process base mixes contain carrageen an which fruition to:-
- suspend the cocoa
- Contribute body and mouth feel
- Stabilize the fat and inhibit creaming.
Carrageen an is a unique hydrocolloid stabilizer for dairy application because of its ability to react predictably with milk proteins to permanently suspend cocoa at appropriate use levels and storage conditions. Products such as starch and guar gum may be included in the base mixes as auxiliary thickeners but the cocoa suspension contributed by these components is only viscosity related and thus temporary.
Chocolate milk is typically processed under pasteurization conditions UHT pasteurized products are also available and will become more significant as the trend towards greater convenience continues. Batch pasteurization is observed infrequently in the field and occur generally in dairies with older processing equipment.
Production of chocolate milk can be affected by many variables which differs from dairy to dairy. A generally uniform finished product is produced over a wide geographical market segment by minimizing the various parameters which inherently differ between diaries.
Factors affecting finished chocolate milk quality includes:-
- Source of milk
- Butterfat level in milk
- Variation in protein content of the milk
- Cocoa formulation
- Total heat exposure during pasteurization
- Cooling methods and temperature of the cooling unit.
- Stabilizer source
- Shear stress due to processing equipment.
The mechanisms by which carrageenan impart it’s unique functionality to chocolate milk is through to be due to both the water gel and milk get potential of the stabilizer. Since milk is composed of about 87% water, there is ample opportunity for carrageenan-carrageenan helix formation after pasteurization and during the cooling phase of production. Additionally, the 3.3% protein in milk as well as protein contributed by the coca allow for carrageenan protein interaction. Data indicates that cocoa suspension is due to the water gel potential or carrageenan- carrageenan interaction. The suspension is due to rapid formation of a weak gel matrix which entraps the cocoa particles during the cooling phase of production. Protein carrageenan interactions account for viscosity development, mouth feel and body of the finished chocolate milk. This interaction progresses with time and maximum viscosity development is attained with a day.
Finally, the international best practice of chocolate milk production account for improve quality milk in order to be more completive globally; milky safely, milk quality and must meat the minimum safely standards also ensure that good bacteria court are not destroyed and with no antibiotics or chemical residues.
So Swiss (Switzerland) is known to produce the world highest quality milk because they produce safely regulated milk and also quality milk.
- Gram-positive bacteria: are those bacteria that retain the stain or that are resistance to decolonization by alcohol during gram method of staining.
- Gram-negative bacteria: Is a common class of bacteria normally found in the gastro intestinal treat that can be responsible for disease in man.
- Homo fermentative: a fermentative resulting wholly in a single end-product-used, especially of economically important lactic-acid bacteria that ferment CHO to lactic-acid
- Hetero fermentative: a fermentative resulting in a number of end product –used especially of lactic-acid bacteria and produce volatile and corbondioxide as well as la tic acid.
- Facultative-micro-organism that can grow in presence or in absence of oxygen
- Agar- Is a medium for facilitating the growth of micro-organism.
The increasing rate of bacterial resistance to antimicrobial agents for the therapeutic treatment of diseases in animals has been a major source of concern to scientists for some years now. Antimicrobial resistance takes place when bacteria adjust or adapt in a way that permits them to stay alive in the presence of antibiotics designed to kill them; bacteria evolve resistance to these drugs, typically by acquiring chromosomal mutations and multi drug resistance plasmid and tranponson etc et al; 2003; Lautenbach et al; 1987; Levine et al; 2002; Nichol et al; 2005; Pena, 1995; Sherg, 2002.
Some other researchers, state that there is not enough evidence that might point towards such potential risk or even consider potential human health benefits derived from antibiotic use in food animals (Witte, 1998); Doyle, 2006 .
The worldwide mergence of antibiotic-resistant bacteria threatens to undo the dramatic advances in human health that were ushered in with the discovery of these drugs in the mid-1900s. Today, resistance has rendered most of the original antibiotics obsolete for many infections, mandating an increased reliance on synthetic drugs (Cirz et al., 2003). In poultry production antibiotics are widely used as growth promoter and treatment of infectious disease (Wolfgang, 1998). The use of antibiotics in the poultry production industries for the promotion of growth largely contributes to the high resistance to antimicrobial agents in normal flora of poultry (Allan et al., 1993; Aronson et al., 1975) and pathogenic microorganisms (Amara, et al., 1995). These resistance microorganisms may act as a possible source for the transfer of antimicrobial resistance to human pathogens (van den et al., 2001). Plasmid and transposon-mediated resistance is widely transmitted between different bacterial species and genera including human pathogens (Davies, 1994; Wise, 1985). Multi-drug resistant strains of E. coli are prevalent in both human and animal isolates in different parts of the world (Amara et al., 1995; Bebora et al., 1994; mahipal et al., 1992). E.coil is a common normal flora organism in the gastrointestinal tract of animals and man but may become pathogenic to both (Jacob et al. 1970; Levine et al. 1987) serious outbreak of gastrointestinal illness caused by food born pathogenic E.coli, have occurred during the past two decades (Armstong et al, 1996). Thus resistant strain of E.coli arising from the exposure of animals to antimicrobials may possibly become infectious organism in humans.
Allan B. J Van-den-Hurk W. and Potter A. A. 1993. Characterization of Escherichia coli isolated from cases of avian colibacilosis. Canadian. Jounal of Veterinary Research. 57: 45-151.
Allen D. G. Pringle X. Smith D. Conjon P.D. and Burgmann P.M. 1993. Handbook of veterinary Drugs. Lippincott, Philadephia, Page 257- 258.
Bauer A. W. Kirby W. M. M. Sheris J.C and Turk M. 1999. Antibiotic susceptibility testing by a standard single diskmethod. American Joournal of Clinical pathology. 4 5:493-496.
Bebora L. C. Oundo J O and Yamamoto H. 1994. Resistance
of E.coli strains. Recovered from chickens, to antibiotics with particular references to trimethprim sulfamethoxazole (Septrin). East African medical journal. 71: 624-627.
Cirz RT, Chin JK, Andes DR, de Crecy-Lagard V. and Craig
W.A 2005. Inhibition of mutation and combating the evolution of antibiotic resistance. PLos boil. 3 (6): e17.
Davies J. 1994. Inactivation of antibiotics ad the
dissemination of resistance genes. Science 264-375-382
Finch R.G., Greenwood D., NorrbyS.R and Whittley R.J. 2003.
Antibiotic and chemotherapy anti-infective agents and their use in therapy. 8 ed. Edinbugh: Churchill Livingstone. 964 p.
Islam M. J., Sultana S., Das K. K. Sharimin N. and Hasan M. N.
- Isolation of plasmid-mediated multidrug resistant Escherchia coli from poultry. Int. J. Sustain. Crop prod. 3 (5): 46 – 50.
Jacobs S.I., Halizel A and wolman B. 1970. Outbreak of infantile
gastoentieritis caused by E. coli 0114. Archives of desease in chikdhood. 45:243
Johnson, J.R kuskowski M.A Menard M., Gajewski A., Xercavins
- and Garau J. 2006. Similarity between human and chicken Escherichia coli isolated in realtion to ciprofloxacin resistance status. J Infect Dis. 194 (1) 8-10.
Kariuki S., Gilks C., Kimari J., Obanda A., Muyodi J., Waiyaki P.
and Hart C.A. 1999. Genotype analysis of Escherichia coli strains isolated from children and chickens living in close contant .Appl environ microbial. 65 (2): 472-6.
Lindergren P.K., Karlsson A. and Hughes D. 2003. Mutation rate
and evolution of fluroquinolone resistance in Escherichia coli isolates fropm patients with urinary tract infections. Antimicro agents chemother. 47:3222-3232.
Lautenbach E., Fishman N.O., Bilker W.B., Castigloni, A. and
Metley J.P 2002. Risk factors for fluroroquinolone resistance in nosocomial Escherichia coli and klebsiella pneumonia infections. Arch intern med. 162-2469-2477.
Marier R., Wells J.G., Swanson R.C., collahan W. and Mehlaman
I.J. 1973. An outbreak of enteropathogenic E. coli food borne disease traced to imported French cheese. Lancet.1376.
Matsuo L. ma, Ishii H. Y. and Yamaguchi K. 2002. Characterization of cefotaxime-resistant
Escherichia coli isolates from a nosocomial outbreak at three geriatric hospital. Journal of infection and chemotherapy publisher: Springer Japan. 8 (2): 155-162.
Mulamattathil S.G, Esterhutsen H.A., Pretorius P.J. 2000.
Antibiotic-resistant Gram-negative bacteria in a virtually closed water reticulation system. Journal of applied microbiology. 88 (6): 930-937.
Nichol K., Zhanel G.G and Hoban D.J. 2003. Molecular epidemiology of penicillin-resistant and ciprofloaxin-resistant streptoccous pneumonia in Canada. Antimicrb agents chemother. 47: 804-808.
Pena C., Albareda J. M., Pallares R., Pujol M. and Tubau F. 1995. Relationship between quinolone use and emergence of ciprofloxacin-resistant Escherichia coli in bloodstream infections. Anitmicrob Agents Chemother. 39:520-524.
Sheng W., Chen Y., Wang J., Chang S. and Luh K. 2002. Emerging fluoroqunolone-resistance for common clinically important gram- negative bacteria in Taiwan. Diagn Microb Infect Dis 43:141-147
|BANK||ACCOUNT NAME||ACCOUNT NUMBER|
|DIAMOND BANK||FREEMANBIZ COMMUNICATION||007 031 2905|
|FIDELITY BANK||FREEMANBIZ COMMUNICATION||560 028 4107|
|GTB||FREEMANBIZ COMMUNICATION||013 772 5121|
|ZENITH BANK||FREEMANBIZ COMMUNICATION||101 326 3297|