PREVALENCE AND CHARACTERIZATION OF ENTEROCOCCI IN CLINICAL SAMPLES FROM PUBLIC HOSPITALS IN UYO, NIGERIA
ABSTRACT
In this study, the prevalence of Enterococci from 3 clinical samples (wound, urine and stool) of patients attending University of Uyo Teaching Hospital and Anua General Hospital and their virulence factors were investigated. A descriptive cross-sectional study design was adopted and data obtained were analyzed statistically. Two hundred samples, including Urine, Wound and Stool were obtained and cultured by standard methods. Isolates obtained were tested for their antimicrobial susceptibilities and thepresence of virulence factors. Isolates obtained were Enterococcus faecalis (56.7%), Enterococcus faecium (26.5%), Enterococcus durans (10.5%) and Enterococcus gallinarium (5.3%). The prevalence of Enterococci species in the clinical samples from the two hospitals was 9.5% of which more isolates were obtained from University of Uyo Teaching Hospital (11.4%) than Anua General Hospital (6.5%). The difference in the prevalence of Enterococci isolates among the subjects in the two hospitals was statistically significant(9.5%). The isolates showed high resistance to Erythromycin (89.5%), Gentamycin (84.2%) and Vancomycin (68.4%) while the susceptibility to Ampicillin and Aztreonam was seen with increasing frequency. The virulence factors detected among Enterococci species were haemolysin and biofilm formation.The production of biofilm in Enterococcus faecalis (63.6%) were comparatively higher than Enterococcusfaecium (60.0%),also the production of hemolysin were comparatively higher in E.faecalis(81.7%) than E.faecium(60.0%). The presence of these pathogens in patients and elaboration of virulence factors indicates a possible outbreak of nosocomial infections by these pathogens in hospital settings; hence, there is need for regular monitoring of these pathogens in hospital laboratories to curb the emergence of these infections.
TABLE OF CONTENTS
CHAPTER TITLE PAGE
Cover Page
Title Page
Declaration
Certification
Dedication
Acknowledgements
Abstract
Table of Contents
List of Tables
List of Figures
List of Plates
Abbreviations/symbols
CHAPTER ONE: INTRODUCTION
1.1 Background of the Study
1.2 Statement of the Problem
1.3 Aim and objectives of the Study
1.4 Justification of the Study
1.5 Significance of the Study
CHAPTER TWO: REVIEW OF RELATED LITERATURE
2.0 Definition of Enterococci
2.1 Historical perspectives of the Study
2.2 Habitat and Environmental Significance of Enterococci
2.3 Morphological Features of Enterococci
2.4 Growth requirements and Cultural Characteristics
2.5 Identification and Typing
2.6 Species Identification
2.7 Virulence factors
2.7.1 Cytolysin
2.7.1.1Alpha Hemolysis
2.7.1.2Beta Hemolysis
2.7.1.3Gamma Hemolysis
2.7.2Enterococcal Surface Protein
2.7.3 Enterococcus Faecalis Antigen
2.7.4 Angiotensin Binding Protein
2.7.5 Aggregation Surface
2.7.6Hyluronidase
2.7.7Extracellular Polysaccharides
2.7.8 Extracellular Superoxide
2.7.9 Lipoteichoic Acid
2.7.10 Gelatinase
2.7.11 Biofilm
2.8 Environmental Parameters Affecting Growth
2.8.1 Influence of PHon Enterococci
2.8.2 Sodium Chloride tolerance
2.8.3 Influence of temperature
2.8.4 Interaction with other bacteria
2.9 Disinfectant Resistance
2.10 Heavy metal resistance in Enterococci
2.11 Antibiotics resistance in Enterococci
2.11.1 Betalactam
2.11.2 Clindamycin
2.11.3 Aminoglycosides
2.11.4 Trimethoprim/Sulphametoxazole
2.11.5 Flouroquinolones
2.11.6 Chloramphenicol
2.11.7 Erythromycin
2.11.8 Tetracycline
2.11.9 Bacitracin
2.11.10 Linezolid
2.11.11 Tigecycline
2.11.12 Quinupristin-Dalfopristin
2.11.14 Glycopeptide resistance
2.11.14.1 Teicoplanin and others
2.11.14.2 Vancomycin
2.12 Enterococcal Resistance genes
2.12.1 Plasmid
2.12.2 Transposons
2.13 Treatment of Vancomylin Resistant Enterococci infection
2.14 Enterococcal infections
2.14.1 Endocarditis
2.14.2 Enterococcal Bacteremis
2.14.3 Urinary Tract infections
2.14.4 Central Nervous System infection
2.14.5 Intra-abdominal and Pelvic infection
2.14.6 Skin and Soft Tissue infection
2.14.7 Biomaterial Associated infection
2.14.8 Nosocomial infections
2.14.9 Community Acquired infections
2.14.10 Food Borne infection
2.15 Epidermiology of Enterococcal Infection
2.15.1 Environmental Reservoir
2.15.2 Human Beings
2.15.3 Drinking water Resources
2.15.4 Animal Reservoir
2.16 Epidemiological Typing Methods
2.16.1 Phenotyping method
2.16.2 Genotyping Method
2.17 Pathogenesis
2.18 Clinical Manifestation of Enterococci
2.19 Treatment and Management of Enterococci infection
2.20 Prevention and control of Enterococcal infections
CHAPTER THREE: MATERIALS AND METHODS
3.1 Research Design
3.2 The Study Area
3.3 Study Population
3.4 Sample Size
3.5 Sample Collection
3.6 Data Collection
3.7 Laboratory Analysis
3.7.1 Isolation and Identification of Enterococci isolates
3.7.2 Gram’s staining
3.7.3 Catalase
3.8 Salt Tolerance test
3.9 Bile Esculin test for identification of Enterococci species
3.10 Carbohydrate fermentation test for the identification of Enterococci species
3.11 Antimicrobial susceptibility test
3.12 Detection of virulence factor
3.12.1 Hemolysin Detection
3.12 Biofilm Detection using Tube Method
CHAPTER FOUR: RESULT AND DISCUSSION
4.1 Results
4.1.1 The Age and Sex Distribution of subject
4.1.2 Distribution of Clinical Samples based on gender
4.1.3 Duration of Admission of patients in days
4.1.4 Prevalence of Enterococci from patience in UUTH and AGH
4.1.5 Prevalence of Enterococci isolates from patients in different
Hospital wards
4.1.6 Distribution of isolates in the two study areas based on Clinical Samples
4.1.7 Distribution of Enterococci isolates according to Clinical Samples
4.1.8 Antibiotics Susceptibility Test
4.1.9 Virulence factor production by various isolates
4.2 Discussion
CHAPTER FIVE: SUMMARY, CONCLUSIONS AND RECOMMENDATIONS
5.1 Summary
5.2 Conclusions
5.3 Recommendations
REFERENCES
CHAPTER 1
INTRODUCTION
1.1 Background of the Study
Enterococci are gram positive cocci bacteria that are normally present in the human intestine and in the female genital tract and are often found in the environment. Though they were believed to be harmless commensals for many years with no medical significance,they have emerged recently as one of the most common nosocomial pathogen (Lopes et al., 2006).
Enterococci are part of the normal flora or organisms found in the intestine of humans and animals. They have been long recognized as important human pathogen and are becoming increasingly so. The genus Enterococcus includes more than seventeen (17) species (Patel et al., 2000), although only a few cause clinical infection in humans (Lopes et al., 2006).Enterococci exist as commensals, in harmony with gut flora, and the dynamics of the host commensals relationship like antibiotics treatment, host injury, diminished immunity; allow these intestinal bacteria to gain access to extra intestinal host sites to cause infection. The nosocomial Enterococci might have extra capacities to colonize, overgrow, and invade host tissue. This poses an emerging threat to patient’s safety. Since the beginning of the antibiotic era, they have posed major therapeutic challenges, including the need for synergistic combination of antibiotics to successfully treatEnterococcal infective endocarditis (IE).They have emerged recently as one of the most common nosocomial pathogens. To emphasize the intestinal origin of gram positive diplococcus, Thiercelin in 1899 described the group as Enterococcus. Subsequent members of this genus such as Streptococcus faecalis was described by Andrew and Horders in 1906, while Streptococcus durans was described by Sherman in 1937.
The name “enterocogue” was first used by Thiercelin in a paper from France published in 1899, the name was proposed to emphasize the intestinal origin of this new Gram positive diplococci. In the same year (1899), MacCallum and Hasting reported a case of endocarditis caused by an organism they called Micrococcus zymogenes. It was later suggested that this organism was actually a hemolyticenterococcus. The name Streptococcusfaecalis (faecalis relating to faeces) was first coined in 1906 by Andrew and Horder, who isolated this organism from a patient with endocarditis.
In an excellent review in 1937, Sherman emphasized that the term enterococcus had been used to mean different things ranging from the broad definition of any faecal Streptococcus to a restricted definition of any organism that appeared to be identical to Streptococcus faecalis. Sherman proposed a classification scheme which separated Streptococci in to four divisions: Pyogenic, Viridans, Lactic and Enterococcus. The later term (Enterococcus) was used for facultative anaerobes and organism that can survive temperature of 600C for short periods and grew in high salts concentrations.
In the laboratory, many characteristicssuch as the abilityto:( 1) Hydrolyze esculin, (2) their growth in 6.5% sodium chloride (3) their hydrolysis of perrolidonyl arylamidase and leucine aminopeptidase, and (4) their reaction with group D antiserum has been widely used to differentiate between enterococci and non-enterococci, Streptococci such as Streptococci bovis, (Murray, 2000). Before they were assigned their own genus they were classified as group D Streptococcus. Enterorcoccus faecalis and Enterococcus faecium are the most prevalent species cultured from humans, which account for more than 90% of clinical isolates. Other Enterococcal species known to cause human infection includeEnterococcus avium, Enterococcus durans, Enterococcus raffinose, Enterococcus gallinarium and Enterococcus mundtii, Enterococcusfaecium. They are responsible for most Vancomycin-resistant Enterococci (VRE) infection (Murray;2000). Isolation of Enterococci resistant to multiple antibiotics has become increasingly common in the hospital setting. According to National nosocomial infection surveillance (NNIS) data from January 2003 through December 2003, it was obvious that, out of the 300 participating hospitals in international conference of United States (ICUS), 28% 0f Enterococci isolates from thesehospitalswere vancomycin-resistant. Clonal spread is the dominant factor responsible forthe dissemination of multidrug-resistant Enterococci in North America and Europe. Virulence and pathogenicity factors of Enterococci have been described using molecular technique and several genes isolated from resistant Enterococcisuch as(Agg, gel, E,ace, Cyllls, esp, cpd, fsrB) is responsible for encoding the virulence factors of enterococci, which include the production of gelatinase and hemolysin, adherence to the caco-2 and hep-2 cells of the urinary bladder and capacity for biofilm formation.
Enterococci have both an intrinsic and acquired resistance to antibiotics, making them important nosocomial pathogens. Intrinsically, Enterococci tolerate or resist beta–lactam antibiotics because they contain penicillin- binding protein (PBPs); therefore, they are still able to synthesize some cell wall components (Willemse et al., 2009). They are intrinsically resistant to penicillinase susceptibilityPenicillin (low level), penicillinase-resistant Penicillins, Cephalosporins, Nalidixic acid, Aztreonam, Macrolides and low levels of Clindamycin and Aminoglycosides. They used already formed folic acid, which allows them to bypass the inhibition of folate synthesis, resulting in resistance to sulfamethoxazole.
Unlike Streptococcal species, Enterococci are relatively resistant to Penicillin, with minimum inhibitory concentrations (MICS) that generally range from 1-8mcg/ml forEnterococcusfaecalis and 6-64mcg/ml for Enterococcus faecium. Therefore, acquisition of Vancomycin resistance by Enterococci has seriously affected the treatment and infection control of this organism.Vancomycin resistance Enterococci (VRE), particularly E.faecium strain are frequently resistant to all antibiotics that are effective against vancomycin-resistantEnterococci, which leaves clinicians with limited therapeutic options. Newer antibiotics such as (Quinupristin-Dalfopristin, Linezolid, Daptomycin, Tigecycline) with activity against many VRE strains have improved this situation. A mutationin the domain V of the 23s rRNA of Enterococci is responsible for linezolid resistance, whereas resistance to quinupristin-dalfopristin may be the result of several mechanism which include:- modification of enzymes, active efflux, and target modification. Resistance of Enterococci faecalisand Enterococci faeciumto daptomycin, newer cycli lipopeptide antibiotics that act on the bacterial cell membrane, has also been reported (Brender and Jander, 2002).
Six phenotypes of Vancomycin resistanceEnterococci, termed vanA, vanB, vanC, vanD, vanE and VanG have been described. The VanA and VanB phenotypes are clinically significant and mediated by 1-2 acquired transferable operons that consist of 7 genes in 2 clusters termed VAN A and VAN B operon. In 1988, these genes clusters were first reported in Enterococcus strain (Kuhnen et al., 1987). Van A is carried on a transposon Tn1546 that is almost always plasmid-mediated. In the United States, and Europe, the 3 major phenotypes include van A, van B and van D. Van A is the most common and the Enterococcal isolates exhibit high–level resistance to both Vancomycin and Teicoplanin, While van B isolate have variable resistance to Vancomycin and remain susceptible to Teicoplanin. The van C phenotype is mediated by the chromosomal VANC 1 and VANC 2 genes, which are constitutively present in Enterococci gallinariumand Enterococcuscasseliflavus respectively. These genes confer relatively low resistance levels to Vancomycin and are not transferable. To date, the Van D, Van E and Van G phenotypes have been described in only a few strains of Enterococci. Three patients infected with vancomycin-resistant Staphylococcus aureus (VRSA) have been reported in the United States. This report shows that the infection was due to the invitro conjugative transfer potential of the Van A resistant gene from Vancomycin resistant Enterococci faecalis to methicillin resistant Staph aureus(MRSA). An in vitro study by Guzman et al.,(1989) provided evidence for the role of adherence in the pathogenesis of Enterococcusfaecalis in urinary tract infection and endocarditis.
It has been revealed that the beta-lactam antibiotics Ceftaroline, Ertapenem, ampicillin, cefepime, and ceftriaxone can increase the invitro activity of daptomcin against vancomycin-resistant E.faecium and E.faecalis. Ceftaroline and daptomycin appeared to be the most effective combination (Shankar et al., 2002). In a study of synergistic combinations against isolates resistant to Daptomycin, a combination of daptomycin and ampicillin appeared to be the most synergistic. The unavailability of clinical synergistic data like the combination oflinezolid and daptomycin to a specific isolates limits treatments of the therapy against Vancomycin resistantEnterococci.
The global emergence of vancomycin resistantEnterococcifaecium isolates in hospital and community settings in 5 continents over just the past two decades from 2004 till date, (Jones et al., 2004), have been characterized as the clonal spread of Complex 17 (CC17) Enterococcus faecium which is defined upon multilocus sequence typing and is characterised by resistance to Quinolones and Ampicillin. This continued global spread of resistant organism and the creation of new highly virulent pathogen from transfer of resistance genes underscore the importance of infection control and prevention, active surveillance and use of appropriate antibiotics.
Therefore, this study was carried out to determine the prevalence and virulence factors of Enterococci species isolates from urine, wound and stool samples of patients attending Secondary and Tertiary Hospitals in Uyo, South-South Nigeria, as suchrecords of prevalence of Enterococci species are not available in the state in other to compare with the 23% prevalence rate of Enterococci species from urine, wounds and stool samples as obtained from Oshogbo, South -Western Nigeria,(Agarwal et al 2009). And also access its antimicrobial profile, so as to suggest possibleantibiotic for its treatment in order to reduce the risk of nosocomial infections, such as urinary tract infection, Endocarditis and bacteremia caused by enterococci species.
1.2 Statement of Problem:
In 2003, fifteen years after the first public report in 1988 of clinical strains of Enterococci in Nigeria as one of the major cause of hospital acquired infections.The organism in the last three years has become very resistant to many antimicrobial agents and is one of the major causes of hospital acquired infections. Recently, the National Nosocomial Surveillance System (NNSS) predicted that the percentage of Enterococci isolates exhibiting resistance in Nigeria will be on the increase. A study of the prevalence rate of Enterococci and the virulence expressed by this organism in hospital settings in Uyo, South-South Nigeria, is required especially as records of such studies are not available. This pathogen can cause important nosocomial epidemics and can increase morbidity, mortality and cost of management of the disease.
1.3 Aim and Objectives of the Study
1.3.1 Aim:
The aim of the study is to determine the Prevalence and Characterization of Enterococci in Clinical Samples from Public Hospitals in Uyo, Nigeria
1.3.2 Objectives:
The study was carried out to:
i. Determine the prevalence of Enterococci species in urine, wound and stool samples of patients in secondary and tertiary hospital in Uyo, South-South, Nigeria.
ii. Determine the antimicrobial susceptibility profile of these isolates.
iii. Determine the virulence factors expressed by strains of Enterococci isolated from these clinical samples.
1.4 Justification of Study
Enterococci are one of the leading cause of nosocomial infections and are thus a persisting clinical problem globally, particularly Enterococcus faecium and Enterococcus faecalis. Also, the emergence of multi-drug resistant strains that are currently responsible for approximately 23% of all nosocomial infections in urine, wounds and Stool samples in Oshogbo, South Western Nigeria is worrisome and such a study need be replicated in Uyo environment.
1.6 Significance of Study
This significance of the study is to isolatepathogensand analyse the antimicrobial profile and detection of the virulence factors of Enterococci, in other to provide data and consequently reduce the nosocomial infection caused by them.
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