S Nordin, Z Wahab, S Hussin, M Rahman
antibiogram, antibiotic resistance, molecular characterization, mrsa, rep-pcr
S Nordin, Z Wahab, S Hussin, M Rahman. Molecular Characterization of Nosocomial Methicillin Resistant Staphylococci aureus by Rep-PCR. The Internet Journal of Infectious Diseases. 2010 Volume 9 Number 2.
Infection with nosocomial methicillin-resistance
Detection and characterization MRSA strains during outbreaks are important to monitor trace and control the intrahospital and interhospital spread or evolution of bacterial strains. Molecular methods have been proven to be cost-effective for the study of nosocomial outbreaks and give genomic nature of infected organisms. However, PFGE is time consuming, tedious and the reagents are expensive, hence may not be practical in resource limited settings. Rep-PCR seems to be a promising technique in view of its adequate discriminatory power, good reproducibility and at a low cost in detecting MRSA.5,6 The present communication deals with use of rep-PCR to characterize genetic relatedness among the MRSA strains isolated and identified from clinical samples.
Materials And Methods
The present research was conducted on the patients admitted to Hospital Kuala Lumpur (HKL), Malaysia from December 2005 to April, 2007.
Blood, tracheal aspirate, nasal swab and pus specimens obtained from the suspected patients were processed in the Microbiology laboratory for the isolation and identification of Methicillin Resistant Staphylococcus aureus(MRSA) organisms.
Isolation and Identification
Methicillin Resiatant Staphylococcus aureus(MRSA) organisms were isolated and identified based on colonial morphology, Gram’s stain, Coagulase test, Antibiotic resistance to methicillin by oxacillin 1 µg, disk susceptibility test following the guidelines of the Clinical and Laboratory Standards Institute.7
The MRSA identified bacteria were kept in microbank bead and stored at -70°C. These were sub-cultured on blood agar before use.
Antibiotic sensitivity test
Antibiotic susceptibility tests were performed by disc diffusion method using the Mueller-Hinton agar which was inoculated with a suspension adjusted to 0.5 McFarland turbidity standards. Plates were incubated overnight at 35°C. CLSI guidelines were used as reference for determination of susceptibility. In addition to oxacillin (1 µg), the antimicrobial panel include fusidic acid (10 µg), mupirocin (5 µg), clindamycin (2 µg), vancomycin (30 µg) gentamicin (10 µg), erythromycin (15 µg), cotrimoxazole (1.25 / 23.75 µg), rifampicin (5 µg), linezolid (30 µg) and teicoplanin (30 µg). Reading of the inhibition zone and interpreting susceptibility were performed using the Biomic Vision Microbiology Analyser.
Quality control strains methicillin-sensitive Staphylococcus aureus ATCC 25923 was included in the testing. Confirmatory test for MRSA detection was confirmed by performing PCR to detect mecA gene, adapted from study done by Delvecchio et al.8
Patients’ profiles were retrieved from the case files of patients with the isolated MRSA, from the medical record office.
DNA extraction was performed by High Pure PCR Template Preparation Kit (Roche Diagnostics, Germany). The nucleotide sequences of the primers to amplify targets in the rep-PCR of MRSA were obtained from previously published article by Delvecchio et al.8 Briefly 25µL of Master Mix (Roche Diagnostics, Germany) in which there was 1µL of primer, 23µL of sterile double distilled water and 1µL of DNA. The thermo-cycling conditions were: initial denaturation at 95 °C for 3 minutes, denaturation at 94 °C for 1 minute, annealing at 54 °C for 1 minute and extension at 72 °C for 2 minutes. Thirty cycles were performed followed by a second extension at 72 °C for 5 minutes. In every PCR run, a negative control (nuclease free water) was included. Methicillin-sensitive
The study was approved by the Research and Ethical Committee of Medical Faculty, UKM
Isolation and Identification of bacteria
It was observed that twenty seven samples were positive for MRSA obtained from 23 patients (4 patients had two MRSA isolates), who were admitted to Hospital Kuala Lumpur. 14 (51.9%) of the MRSA isolates from blood samples, 9 (33.3%) from tracheal aspirate, 3 (11.1%) from swab samples, 2 of which were nasal swabs and 1 (3.7%) was pus.
Fingerprinting patterns of MRSA
Antibiogram analysis of the MRSA collection resolved these strains into six groups designated (I-VI) (Table 1). Group II, which was resistant to cotrimoxazole, gentamicin, clindamycin and erythromycin, was the commonest accounting for 40.7% (11/27) of the isolates collected. Group I accounts for 33.3% (9/27) of the isolates and this group was resistant to cotrimoxazole, gentamicin and erythromycin. Group III accounts for 3.7% (1/27) of the isolates and this group was resistant to gentamicin, clindamycin and erythromycin. Group IV accounts for 3.7% (1) of the isolates and this group was resistant to fusidic acid, cotrimoxazole, rifampicin, gentamicin and erythromycin. Group V accounts for 11.1% (3/27) of the isolates and this group was resistant to fusidic acid, cotrimoxazole, rifampicin, gentamicin, clindamycin and erythromycin. Group VI accounts for 7.4% (2/27) of the isolates and this group was resistant to rifampicin, gentamicin, clindamycin and erythromycin.
Band size: 210 to 1150 bp
Lane 1: Negative control, Lanes 2 and 3: methicillin-sensitive
MRSA :lanes 4, 7 and 9 pattern ‘a’; lanes 5 and 10: pattern ‘b’; lane 6: pattern ‘c’; lane 8: pattern ‘d’; and lane 11: pattern ‘e’)
FU fusidic acid; SXT cotrimoxazole; GM gentamicin; RIF rifampicin; CLI clindamycin; ER erythromycin;
* Repeated samples of MRSA isolation.
This study was undertaken with a view to identification of MRSA and to do their molecular characterization which is important to monitor, trace and control of the intrahospital and interhospital spread of MRSA strains. In the present study rep- PCR was used as a tool to perform molecular study with the advantages of being simpler and less time consuming.9
In the present study 17 positive MRSA positive isolates obtained from 23 patients (4 patients had two MRSA isolates), from the patients who were admitted to Hospital Kuala Lumpur. 14 (51.9%) of the MRSA isolates from blood samples, 9 (33.3%) from tracheal aspirate, 3 (11.1%) from swab samples, 2 of which were nasal swabs and 1 (3.7%) was pus.
In the study designed published primer of Delvecchio’s study8 was used to perform rep-PCR to determine the molecular nature of the above MRSA isolates.
The patterns of bands from the MRSA isolates varied with 4 to 5 bands visible, ranging in size from 210 bp to 1150 bp (Fig 1). By comparing the banding patterns between MSSA and MRSA, distinction between these organisms is easily determined. The MSSA isolates lack the 650 bp band that was observed in all the MRSA isolates. The MRSA strain belonging to PCR pattern ‘e’ is the predominant strain. This strain was present throughout the study period, in December 2005, then November 2006 till April 2007. Norazah et al.10 also determined a predominant MRSA strain in their study. It is also interesting to note in this study that patient ‘B’, whom had repeated blood samples sent 3 days apart, and MRSA were isolated in both samples; genotypically they were not identical, hence belonged to the different strains. Therefore, patient ‘B’ was infected by two strains of MRSA. Likewise, two blood samples sent 2 days apart from patient ‘G’ did not isolate identical MRSA strains. Patient ‘V’ had two tracheal aspirate samples sent on the same day and showed two identical MRSA strains. This makes sense since the sample was taken from the same site on the same day. Patient ‘P’‘s nasal was colonized by MRSA in January 2007. MRSA isolated from patient ‘P’s blood sample in April 2007 and this strain was identical to the colonizer isolated in January 2007. This highlights the importance of screening for MRSA carrier as an infection control measure and the role of rep-PCR in determining and verifying the MRSA strains that cause infection in a colonized patient.
There were clusters of MRSA infections observed in this study. The predominant MRSA strain in this study belonged to pattern ‘e’. This information can be used by an Infectious Disease Physician or a Clinical Microbiologist to trace the source of the MRSA strain. Screening the environment and personnel in the ICU may show the relatedness of the MRSA between them and isolates from patients. Thus, infection control measures can then be applied to contain further spread. Van der Zee et al9 compared repetitive element sequence-based PCR (rep-PCR), with other genotypic methods and found that rep-PCR was better than others in terns of its efficacy. They also mentioned that rep-PCR was easier and faster with good reproducibility. Delvecchio et al.8 also employed rep-PCR in their study and found similar discriminatory power.
It is also verified in this study that MRSA isolates with the same rep-PCR pattern exhibited different antibiotic susceptibility patterns, while strains with different rep-PCR patterns had the same antibiotic susceptibility. Therefore, there was no relationship between antibiotic susceptibility and rep-PCR patterns. This is in agreement with other studies that reported antibiotic susceptibility testing has a relatively limited use in molecular studies due to phenotypic variation of the isolates.9
In conclusion, genotypic method is more reliable method while performing molecular characterization and molecular epidemiology study of nosocomial MRSA pathogens. In resource-limited settings, rep-PCR has the potential implication in molecular epidemiology investigation of nosocomial pathogens to monitor, tracing and control the spread or evolution of bacterial strains.
Authors acknowledge the authority of University Kebangsaan Malaysia for providing fund (FF-200-2006) to conduct the present research. The authors also acknowledge the authority of Hospital Kuala Lumpur, Malaysia for cordial assistance during the study.