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PROJECT TOPICS ON PHENOTYPIC AND GENOTYPIC CHARACTERIZATION OF VANCOMYCIN RESISTANT STAPHYLOCOCCUS AUREUS ISOLATED FROM IN-PATIENTS IN SELECTED HOSPITALS IN KADUNA METROPOLIS, KADUNA STATE NIGERIA

PROJECT TOPICS ON PHENOTYPIC AND GENOTYPIC CHARACTERIZATION OF VANCOMYCIN RESISTANT STAPHYLOCOCCUS AUREUS ISOLATED FROM IN-PATIENTS IN SELECTED HOSPITALS IN KADUNA METROPOLIS, KADUNA STATE NIGERIA

ABSTRACT

Staphylococcus aureus is an important pathogen associated with various hospital-and community-acquired infections as such it is regarded as a public health problem. Vancomycin was an approved and highly recommended antibiotic worldwide for the treatment of infections caused by S. aureus especially methicillin resistant S. aureus. This study was carried out in order to phenotypically and genotypically characterize vancomycin resistant S.

aureus isolated from patients on admission in some selected hospitals in Kaduna metropolis, Kaduna state Nigeria. A total of 300 skin swab samples were collected from patients on admission. The swab sticks were inoculated into brain-heart infusion broth, transported to the laboratory and then inoculated on to mannitol salt agar and blood agar. Isolates with the characteristic colonial morphology of the S.

aureus were then characterized microscopically and identified biochemically. The susceptibility of S. aureus isolates to seven antibiotics was determined using Kirby-Bauer method. Vancomycin minimum inhibitory concentration for the isolates was determined by broth dilution method. The VRSA were confirmed by the detection of vanA gene in the isolates by polymerase chain reaction. A total of 52 (17.33%) S.

aureus isolates were obtained from skin swabs of in-patients in selected hospitals in Kaduna metropolis based on microscopic and biochemical identification. Multiple antibiotic resistance index analysis showed that the MAR indices of the isolates were between 0.14 and 0.71. Most of the isolates had MARI values greater than 0.2 suggesting that the isolates were from high risk environment where antibiotics are frequently used.

Among the 52 isolates, 2 were vancomycin intermediate S. aureus (MIC value 4-8μg/ml) another 2 were vancomycin resistant S. aureus (MIC values ≥ 16μg/ml) while the remaining 48 isolates were vancomycin susceptible S. aureus (MIC value ≤2μg/ml). PCR result for the detection of vanA gene showed that all the 2 VRSA had the vanA gene. It then means the resistance of the VRSA isolates was mediated by vanA gene. VRSA was characterized both phenotypically (by determination of vancomycin susceptibility) and genotypically (by detection of Van A gene) in this study. The occurrence of S. aureus and VRSA in this study was found to be 17.33% and 3.85% respectively.

CHAPTER ONE

1.0 INTRODUCTION

 

1.1 Background of the Study

Staphylococcus aureus is one of the most common causes of serious infections in community and hospital settings (Lowry et al., 1998). Staphylococcus aureus, is a major human pathogen causing large variety of infections worldwide and predominates in surgical wound infections with prevalence rate ranging from 4.6% – 54.4% (Bannerman, 2003).

It is one of the species of the genus Staphylococcus. It is a Gram positive, non-motile, catalase positive, mostly coagulase positive, facultative anaerobe, involved in causing a number of diseases including boils, pustules, impetigo, osteomyelitis, mastitis, septicemia, meningitis, pneumonia and toxic shock syndrome,post operation wound infection, food borne intoxication and nosocomial bacteremia (Talaro and Talaro, 2002;Jun et al., 2004;Cheesbrough, 2006).

It is considered the most resistant of all non-spore forming pathogens, with well-developed capacities to withstand high salt concentrations (7.5 – 10%), extremes in pH and high temperatures up to 60oC for 60 minutes(Jun et al., 2004).

Staphylococcus aureus colonizes the skin and nasal carvities, it occurs in about 25-30% of healthy people. It is a versatile human pathogen responsible for nosocomial (HA) and community acquired (CA) infection, with clinical manifestation of superficial and systemic diseases, associated with high morbidity and mortality rates (Jun et al., 2004).

The unique characteristic of S. aureus is the production of virulence factors responsible for the establishment of staphylococcal diseases and propensity to develop resistance to multiple antibiotics (Jun et al., 2004).Patients subjected to broad-spectrum antibiotics and immunosuppressive therapies have higher risk of infection by this microorganism (Prasad, 2014).

Methicillin-resistant Staphylococcus aureus (MRSA) is now endemic in health care facilities, with rates up to 15.0% in some health care settings (Fridkin et al., 1999). Also, recent reports describe MRSA carriage in persons in the community who do not have health care–associated risks. The increased incidence of MRSA has led to more frequent use of vancomycin, the drug commonly relied on for treating MRSA infections (Salgado et al., 2003).

Vancomycin is a glycopeptide antibiotic effective against majority of Gram positive bacteria, particularly against multiple drug resistant enterococci and staphylococci which are resistant to β-lactum antibiotics (Reynolds, 1998). These pathogens have acquired resistance to this compound by virtue of their intrinsic property especially in clinical isolates. This leads to severe complications in immunocompromised as well as surgical patients. Vancomycin resistant enterococci (VRE) were first reported in 1988 in Europe and USA (Prasad, 2014).

Vancomycin acts by binding with the C-terminal D-alanyl-D-alanine (D-Ala-D-Ala) residue of pentapeptide, thereby blocking the addition of precursors by transglycosylation to peptidoglycan chain and hence inhibits cross linking of cell wall by transpeptidation (Courvalin, 2006). Resistance to vancomycin is caused by synthesis of precursors with low affinity for these antibiotics conferred by operons present on Van gene clusters that encode enzymes which synthesize low affinity precursors wherein C-terminal D-Ala residue is replaced by D-lactate (D-Lac) or D-serine (D-Ser) that modify vancomycin binding site. These genes confer high or low level resistance to vancomycin and teicoplanin which may be inducible or constitutive (Prasad, 2014).

In vitro conjugative transfer of the vanA gene from enterococci to Staphylococcus aureus was demonstrated in 1992 (Noble et al., 1992). However, it was not until 1996, when the first case of vancomycin- intermediate Staphylococcus aureus (VISA; MIC, 8–16 mg/mL) was detected, and since then decreased susceptibility to vancomycin became a clinical reality (CDC,1997). None of the VISA strains identified contained the vanA gene or any of the other vancomycin-resistant genes found in VRE (Oliveira et al., 2001). In vitro studies suggest that, with prolonged vancomycin exposure, VISA organisms produce a thickened cell wall matrix, limiting drug penetration (Hanaki et al., 1998).

For many years, vancomycin has been considered the drug of choice for the treatment of Staphylococcus aureus infections due to strains that had become resistant to methicillin (Oliveira et al., 2001). However, in July 2002, things changed when the Centers for Disease Control (CDC) in the USA published the first documented report of Staphylococcus aureus that was resistant to vancomycin as well as being resistant to methicillin. The infection occurred in a diabetic patient with chronic kidney (renal) failure who was undergoing peritoneal dialysis in a hospital in Michigan. Approximately 5 years earlier, the Japanese had reported the first strain of Staphylococcus aureus with reduced (or intermediate) susceptibility to vancomycin followed by two additional cases from the USA. These earlier isolates were termed Vancomycin Intermediate Staphylococcus aureus (VISA) (CDC, 2003).

The definitions of VRSA and VISA are based on the results of laboratory testing which determine the minimum concentration of vancomycin that is required to inhibit the growth of Staphylococcus aureus in an in vitro study. It should be noted that these definitions are not universal and some countries lump all strains of Staphylococcus aureus that require increased concentrations of vancomycin to inhibit their growth into a single VRSA category. Regardless of the specific categorization of these isolates, the potential clinical impact of these strains on the management of patients is enormous (Cosgrove et al., 2004).

The mechanisms of resistance that have been identified for VISA and VRSA strains are quite different and are not fully understood. For VISA strains, the proposed mechanism resulting in reduced susceptibility to vancomycin is believed to be a thickening of the bacterial cell wall such that vancomycin is trapped within the bacterial cell wall and is thus unable to reach its target on the surface of the bacterial cytoplasmic membrane (Cosgrove et al., 2004). The VRSA strain that was isolated from the patient in Michigan as well as a second isolate from a patient in Pennsylvania contained the vanA gene which codes for an altered target such that the binding of vancomycin to the target is significantly reduced and thus, it cannot carry out its normal function of inhibiting bacterial cell wall synthesis. The source of the van A gene isolated in VRSA appears to have come from co-infection with vancomycin resistant Enterococcus (VRE) (Applebaum, 2006).

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The future of VISA and VRSA strains is not clear and much research is needed to help further understand all aspects of these organisms including their epidemiology, Microbiology, clinical and infection control implications and optimal treatment. As well, in addition to VISA and VRSA strains, there appear to be strains of Staphylococcus aureus that are referred to as ―heteroresistant.‖ These strains appear to be susceptible to vancomycin based on standard laboratory testing, but contain subpopulations of Staphylococcus aureus that have intermediate susceptibility to vancomycin. The clinical significance of these strains requires further investigation (Chang et al., 2003).

PROJECT TOPICS ON PHENOTYPIC AND GENOTYPIC CHARACTERIZATION OF VANCOMYCIN RESISTANT STAPHYLOCOCCUS AUREUS ISOLATED FROM IN-PATIENTS IN SELECTED HOSPITALS IN KADUNA METROPOLIS, KADUNA STATE NIGERIA

1.2 Statement of Problem

Vancomycin is commonly used for patients with Methicillin-resistantStaphylococcus aureus infections, and reduced susceptibility to vancomycin is becoming increasingly common. Therefore, the treatment of Staphylococcus aureus infections is becoming increasingly more complicated due to the emergence of various types of antibiotic resistance (Prasad, 2014).

The high prevalence rate of Vancomycin resistant Staphylococcus aureus can be attributed to the high rate of indiscriminate abuse of antibiotics. This lead to antibiotic resistant strains such as MRSA, VISA and VRSA

Vancomycin was considered to be the best alternative for the treatment of multi-drug resistant MRSA. However, there are increasing numbers of reports indicating the emergence of VRSA strains exhibiting two different resistant mechanisms namely modification of Vancomycin-binding target and removal of Vancomycin-binding target. Multi-drug resistant VRSA is a major cause of nosocomial and community acquired infections and is on the rise in humans (Jun et al., 2004).

The number of cases of VISA and VRSA reported so far has remained relatively small and thus the epidemiology and risk factors associated with infection with these organisms is not completely known. However, there is evidence that prior exposure to vancomycin, particularly repeated or prolonged courses. For patients infected with VISA strains, mortality appears to increase significantly compared to patients who are infected with vancomycin susceptible Staphylococcus aureus (Chang et al., 2003).

A prevalence of 5.3% of VRSA was observed in a study carried out at Abakaliki. This high prevalence rate was attributed to the high rate of indiscriminate abuse of antibiotics (Moses etal., 2013). So also a significant rise in the number of strains of Staphylococcus aureus with reduced susceptibility to vancomycin, teicoplanin and oxacillin was found by Tiwari and Sen (2006).

Presence of vancomycin resistant S. aureus among in-patients and hospital environment posses serious danger, because in such environment there is easy of transmission of the VRSA among patients and health care personnel through contact.

1.3 Justification

  1. aureus causes a wide range of infections, from a variety of skin, wound and deep tissue infections to more life-threatening conditions such as pneumonia, endocarditis, septic arthritis and septicemia. This bacterium is also one of the most common species in nosocomial infections. In addition, S. aureus may also cause food poisoning, scalded-skin syndrome and toxic shock syndrome, through production of different toxins (Winn Washington 2006).

Staphylococcus aureus is an important pathogen that causes health care and community acquired infection in every region of the world. Although S. aureus is a common commensal of the skin, there is need to investigate the prevalence of the organism on the skin of patients on admission especially MRSA and VRSA because most infections, both community and nosocomial, occur as a result of auto-inoculation even though nosocomial infections may also result from person-person transmission. Cross-contamination by Staphylococcus aureus among patients, professionals and medical supplies in health facilities is a constant concern (Breves et al., 2015).

Staphylococcus aureus, especially methicillin-resistant S. aureus is a major cause of nosocomial infections worldwide. Patients subjected to broad-spectrum antibiotics and immunosuppressive

therapies have higher risk of infection by this microorganism. S. aureus infections are often extremely difficult to treat due to the large population heterogeneity, phenotypic switching, intra-strain diversity, hypermutability and most importantly the small colony variants. It is very important to emphasise that host immune responses against persistent infections by S. aureus is insufficient resulting normally into chronic infections, which in turn can lead to life threatening situations (Prasad, 2014).

It has also been reported that S. aureus strains have a wide variety of multi-drug resistance genes on plasmids, which can be exchanged and spread among different species of Staphylococcus and can be transferred to new bacterial hosts by transduction, conjugation or transformation method (Lujan et al., 2007). S. aureus is known to be notorious in its acquisition of resistance to new drugs and continues to defy control measures (Talaro and Talaro, 2002). Consequential effects of S. aureus multidrug resistance include prolonged hospitalization of patients, difficulty in patient management and treatment, and problem in infection control (Kleven 2007).

The treatment of infection caused by Staphylococcus aureus is a problem due to the emergence and spread of methicillin resistance gene. Vancomycin is commonly used for treatment of methicillin resistant Staphylococcus aureus (MRSA) (Prakash et al., 2006). But the incidence of vancomycin intermediate S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA) has been increasing in various parts of the world (Tiwari et al., 2008).

There is dearth of information on the prevalence of VRSA in Kaduna metropolis. It is therefore important to determine the susceptibility pattern of Staphylococcus aureus to vancomycin to be investigated.

1.4 Aim and Objectives

1.4.1 Aim

The study was aimed at phenotypic and genotypic characterization of vancomycin resistant Staphylococcus aureus (VRSA) isolated from in-patients in some selected hospitals in Kaduna Metropolis, Kaduna State Nigeria.

1.4.2 Objectives

The objectives were to:

  1. Isolate and characterize Staphylococcus aureus from skin swabs using standard microbiological techniques.
  2. Determine the antibiotic susceptibility pattern of the isolates using Kirby-Bauer method and the Minimum Inhibitory Concentration of Vancomycin.
  3. Screen the Vancomycin Resistant Staphylococcus aureus (VRSA) isolates for VanA gene using Polymerase Chain Reaction.

See Also : NUTRITIONAL STATUS, DIETARY PATTERN AND PREVALENCE OF SOME CHRONIC DISEASES AMONG ADULTS IN BIDA LOCAL GOVERNMENT AREA, NIGER STATE, NIGERIA

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