A persistent antimicrobial resistance pattern and methicillin-resistance associated genotypes in a short term Staphylococcus aureus carriage of a student population

Background: Staphylococcus aureus is an opportunistic commensal of human anterior nares. Under favorable conditions, colonization may persist and pose significant threat in healthy individuals leading to infections. Antimicrobial treatment may be limited due to the emergence of methicillin-resistant Staphylococcus aureus (MRSA) which is also frequently resistant to a wide range of antibiotics. Objective: This study aims to assess and compare the antimicrobial sensitivity pattern and methicillin resistance-associated genotypes of carriage S. aureus previously isolated from a student population at two isolations of one-month interval. Materials and Methods: In a previous study, S. aureus was isolated from 31.3% (60/192) and 33% (60/180) of a student population during two isolations in October and November 2013 respectively (Mat Azis et al., 2014). Thirty-nine (65%) students were detected for S. aureus at both isolation events and referred as persistent carriers. All isolates were screened for MRSA by PCR detecting the mecA gene. mecA positive isolates were subjected to staphylococcal cassette chromosome (SCC) mec typing. In this current study, all 120 isolates from both isolation episodes were subjected to antibiotic susceptibility test (AST) by Kirby-Bauer disc-diffusion method against cefoxitin (30 µg), erythromycin (15 µg), vancomycin (30 µg) gentamicin (10 µg), ciprofloxacin (5 µg), rifampin (5 µg), penicillin (10 units), and tetracycline (30 µg). Isolates that showed resistant towards cefoxitin were further validated by Etest. Isolates from the 39 persistent carriers were further subjected to spa typing. Results and Conclusion: Overall, all 120 S. aureus isolates from both isolation events were susceptible toward vancomycin, ciprofloxacin and gentamycin. A highest frequency of resistance was observed for penicillin at both isolations (70% and 65% respectively). This was followed by tetracycline with a similar resistance rate (11.67%) in both isolation events. While low level of resistance was observed against erythromycin at both events. This indicates the persistence of the antimicrobial resistance pattern in the population over the short study period. As for methicillin resistance, out of the 120 isolates of S. aureus, 10 (8.33%) were positive for mecA gene with 4 and 6 isolates from first and second isolation events respectively; 2 isolates were from a same individual. However, among the mecA positive isolates, only 8 isolates showed resistance towards cefoxitin (4 isolates from each isolation event) while the other two mecA positive isolates (from second event) were cefoxitin-susceptible by both disc and Etest methods. The mecA-positive isolates belonged to SCCmec types I (n = 9) and V (n = 1). This indicates the tendency of MRSA to persist although at a low rate with limited genotypes. As for the persistent S. aureus carriers, it was found that for 19 (48.72%) of them, respective individual carried S. aureus of a similar spa types in the respective individuals over the short term period. The limitation of this study is that it only represents a short term carriage in a student population. Whether the observed findings reflect the population at large requires more studies with a longer study period and a wider population size. The incidence of mecA carrying isolates susceptible to cefoxitin requires more validation on potential heterogeneous characteristics of MRSA found in this student population

Genotypic and phenotypic characterization of methicillin resistance determinants and β-lactamase in Staphylococcus species

Aims: To characterize the genotypic distribution of mec complex, bla complex, methicillin-resistance level (cefoxitinMIC) and β-lactamase activity in carriage methicillin-resistant Staphylococcus species for a potential correlation. Methodology and results: Biochemical test, 30 µg cefoxitin diffusion disc test, cefoxitin E-test, mec and bla complexes distributions, Pbp2a and β-lactamase assays were conducted to characterize phenotypic and genotypic of MRSA and MRCoNS in our collection. Phylogenetic tree was constructed using MEGA6 software to trace the diversity of blaZ gene of MRSA and MRCoNS. Sixteen MRSA and nineteen MRCoNS were identified by biochemical tests followed by 30 µg cefoxitin antibiotic disc susceptibility test and mecA gene screening. Twenty nine isolates carry complete mecA genes (2.1 kb), incomplete mec regulator (negative or truncated) and positive Pbp2a assay for both MRSA and MRCoNS. Only MRCoNS SC177 isolate with cefoxitin MIC of 32 µg/mL carries complete mec complex. Thirty-one of thirty-five isolates carry complete bla complex (blaZ, blaRI, blaI) with 10 MRSA produce strong β-lactamase and cefoxitin MIC of ≥12 µg/mL. Only 4 MRCoNS with cefoxitin MIC of ≤8 µg/mL produce strong β-lactamase. The diversity of blaZ gene was demonstrated by phylogenetic analysis and unusual amino acid mutation at position 145 for MRSA SA60 isolate may compromise its β-lactamase activity with low cefoxitin MIC level (2 µg/mL). Conclusions, significance and impact of the study: Isolates that carry complete complete mecA gene were largely consistent with the expression of Pbp2a. Nevertheless, there is no clear correlation of mec regulator genes in relation to cefoxitin-MIC in both methicillin resistant (MR) Isolates that carry Staphylococcus species. On the other hand, various expression level of β-lactamase may correlate with cefoxitin-MIC level in MRSA as compared to MRCoNS

A persistent antimicrobial resistance pattern and limited methicillin-resistance-associated genotype in a short-term Staphylococcus aureus carriage isolated from a student population

The aim of the present study was to assess and compare the antimicrobial susceptibility pattern against a panel of antibiotics and molecular and methicillin resistance-associated genotypes of 120 carriage S. aureus isolates previously isolated from a student population at two isolation events within a one-month interval. The antibiotic susceptibility of isolates was determined using the Kirby-Bauer disc-diffusion method (cefoxitin by Etest). The MRSA was screened using polymerase chain reaction for the presence of the mecA gene. The mecA-positive isolates were subjected to staphylococcal cassette chromosome (SCC) mec typing, multilocus sequence typing (MLST) and eBURST analysis. All isolates were characterized for the presence of the Panton–Valentine leukocidin (PVL) gene, an enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) pattern and the spa type. For the two occasions where S. aureus was isolated, the highest frequency of resistance was observed for penicillin (70% and 65%, respectively), with a lower rate against erythromycin and tetracycline (<12%). All isolates were susceptible to ciprofloxacin and gentamycin. As for methicillin resistance, eight isolates had minimum inhibitory concentrations (MIC) of resistant categories, but 10 isolates (8.33%) were positive for the mecA gene. The mecA-positive isolates belonged to SCCmec types I (n = 9) and V (n = 1). MLST was resolved for only three MRSAs, ST508 (n = 1), ST88 (n = 1) and ST96 (n = 1). The results of the eBURST analysis showed that the MRSA isolates analyzed in the present study were potentially related to MRSA identified in other countries. Approximately half of the persistent S. aureus carriers harbored S. aureus of a similar spa type in the respective individuals during both isolation events. A persistent antimicrobial pattern and limited distinct MRSAs were observed over the short study period. The latter frequently exhibited SCCmec type I, commonly associated with hospital-acquired (HA) characteristics, but further delineation is needed to justify the origins of these bacteria

Presence of methicillin resistance and heteroresistance among Coagulase Negative Staphylococci (CoNS) isolates obtained from health sciences students at a Public University

This study was carried out to investigate the Coagulase Negative Staphylococci (CoNS) nasal carriage and the presence of methicillin resistant Coagulase Negative Staphylococci (MR-CoNS) among health sciences students at Faculty of Medicine and Health Sciences, Universiti Putra Malaysia. A total of 120 isolates of CoNS (62.5%) was isolated from 192 student volunteers. The mecA gene was detected in 15 isolates of CoNS (12.5%). Eight out of the 15 isolates of mecA positive CoNS were resistant to cefoxitin in disc diffusion test whereas the remaining seven isolates of mecA positive CoNS were susceptible to cefoxitin. Analysis of questionnaires showed no significant association between CoNS nasal carriage and the socio-demographic and risk factors except for the genders and history of cold (P < 0.050). Generally, this finding showed a relatively low level of methicillin resistance among CoNS nasal carriage from student volunteers

Phenotypic and molecular analysis of staphylococcus aureus for carriage and transmission in a student population in a Malaysian public university

Staphylococcus aureus is a human commensal bacteria that colonize the skin and mucosal surfaces of healthy individuals. This opportunistic pathogen is the most common nosocomial pathogen responsible for life-threatening diseases in humans worldwide. The anterior nares are the most frequent colonization site for S. aureus and nasal carriage is a major risk factor for infections and a source of transmission of this pathogen. The incidence of S. aureus and MRSA are growing at an alarming rate not only in the healthcare settings but also in the community. In this regards, this study aims to assess the S. aureus carrier rate and persistence, risk factors for nasal carriage, antimicrobial resistance and epidemiological molecular characteristics among the university student population. A set of self-administered questionnaires on socio-demographics, hygienic practices, medical and medication history together with a consent form were distributed prior to nasal swab collection. The collection was done twice in a one month interval during October and November 2013 from 192 and 180 health sciences students, respectively, at the Faculty of Medicine and Health Sciences, Universiti Putra Malaysia. Identification of bacteria isolated was done based on basic phenotypic methods. All S. aureus isolates were subjected to antibiotic susceptibility test (AST) by Kirby-Bauer disc diffusion method against eight antibiotics and screened for MRSA by PCR detecting the mecA gene. All mecA positive isolates were subjected to staphylococcal cassette chromosome (SCC) mec typing, multilocus sequence typing (MLST) and eBURST analysis. All isolates were further characterized by spa typing, screening of PVL genes and enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). In this study, the colonization rates of S. aureus was 31.3% (60/192) and 33.3% (60/180) of the student population during the first and second sampling respectively. Thirty-nine (65%) students were detected for S. aureus at both isolation events and referred as persistent carriers. There is no significant correlation between the carriage and the tested risk factors except for the habit of touching the nose and chronic illnesses (P<0.05). All 120 S. aureus isolates from both isolation events were susceptible towards vancomycin, ciprofloxacin and gentamycin. The highest frequency of resistance was observed for penicillin at both isolations (70% and 65% respectively). This was followed by tetracycline with a similar resistance rate (11.67%) in both isolation events. Low level of resistance was observed against erythromycin at both events. This indicates the persistence of the antimicrobial resistance pattern in the population over the short study period. As for methicillin resistance, out of the 120 isolates of S. aureus, 10 (8.33%) were positive for the mecA gene with four and six isolates from first and second isolation events respectively; four isolates were from two individuals. However, among the mecA positive isolates, only eight isolates showed resistance towards cefoxitin (four isolates from each isolation event) while the other two mecA positive isolates (from second event) were cefoxitin-susceptible by both dics and Etest methods. The mecA-positive isolates belonged to SCCmec types I (n=9) and V (n=1). MLST analysis of MRSA isolates revealed three STs: ST508 (n=1), ST88 (n=1) and ST96 (n=1) while other seven of MRSA isolates showed non typeable sequences type. This indicates the tendency of MRSA to persist, although at a low rate with limited genotypes. eBURST analysis showed that MRSA isolates found in this study were potentially related to those MRSA found in Asian countries and might be disseminated regionally. Based on ERIC analysis, the majority of isolates were largely genetically distinct. As for the persistent S. aureus carriers, it was found that for 19 (48.72%) of them, respective individual carried S. aureus of a similar spa type during both isolation events. This indicates the persistence of certain spa types in the respective individuals over the short term period. Although the prevalence of S. aureus carriage and MRSA in this study cannot be generalized to entire population due to limitations of the study, but this indicates the need of periodic screening to monitor S. aureus and MRSA status among community

Assessment of pathogenicity of community-acquired MRSA isolates in mice-induced peritonitis

Introduction: Methicillin-Resistance Staphylococcus aureus (MRSA) is known as a major nosocomial pathogen in healthcare. However, it has now spread in the community known as community-acquired MRSA (CA-MRSA). Thus, the survival and pathogenicity of CA-MRSA isolates were assessed using in vivo peritonitis model with comparison to ATCC-MRSA. Two CA-MRSA isolates; CA-MRSA1 and CA-MRSA2 that were isolated from healthy population, were studied and compared. Methods: Mice were assigned into 4 groups and injected intraperitoneally with ATCC-MRSA, CA-MRSA1 or CA-MRSA2, respectively. Sterile Dulbecco’s Phosphate-Buffered Saline (DPBS) represents negative control. Mice were observed twice daily, 0-72 hours of post-infection. Any signs of distress were recorded for severity score and survival analyses. Mice were euthanised at 72 hours post-inoculation or by referring to the Peritonitis Severity Scoring (PSS) system. Organs of interest, peritoneal lavage and abscess were processed for bacterial counts. Tissue samples were analysed for histopathological scores. Results: All mice inoculated with MRSA showed clear signs of illness with peritonitis symptoms of p<0.001 and comparable PSS scores were recorded in all infected mice groups. Intraperitoneal injection of lethal dose of MRSA resulted in significant death of ATCC-MRSA (p<0.05) and CA-MRSA-infected mice (p<0.01), compared to the un-infected. Bacterial burden was significantly high in all samples harvested from mice challenged with CA-MRSA2 compared to ATCC-MRSA except in abscess and lung. Significant liver necrosis and spleen inflammation were observed in CA-MRSA1, and lung inflammation in ATCC-MRSA-infected mice. Conclusion: Nasal carriage CA-MRSA isolates from a healthy population has the potential to cause peritonitis with comparable severity as ATCC-MRSA

Staphylococcus aureus infection risk in a population of health sciences students at a public university

Background: This study was undertaken to evaluate Staphylococcus aureus carriage and persistence in health sciences students at the Faculty of Medicine and Health Sciences, Universiti Putra Malaysia. Methods: Consent form and self-administered questionnaires on socio-demographics, hygienic practices, medical and medication history were distributed followed by nasal swab collection; sampling was done twice in one month interval in October and November 2013. Bacterial identification followed the standard phenotypic methods. Antibiotic susceptibility of isolates against oxacillin and cefoxitin was tested by disc diffusion method. Methicillin resistance determinant gene (mecA) was detected through polymerase chain reaction-assay. Results: S. aureus was isolated from 31.3% (60/192) and 33.3% (60/180) of the student population during the first and second sampling respectively. Among the S. aureus-positive participants, about 65% of them were persistent carrier (S. aureus was detected during both sampling exercises). Six methicillin-resistant S. aureus (MRSA) were detected (four and two isolates in the two sampling events respectively) as inferred by decreased susceptibility to oxacillin and cefoxitin and presence of mecA gene; two of the strains were from a single individual. Fisher’s exact test showed no significant correlation between carriage and the tested risk factors except for the habit of touching nose and chronic illnesses (P < 0.05), with a higher incidence of S. aureus among those associated with the two risk factors. Conclusions: As far as the limited sampling period is concerned, these findings indicate that a proportion of the student population may be at infection risk. Avoiding frequent nose-touching could be one of the preventive measures