Application of Antibiogram, Ribosome Spacer PCR (RS-PCR), and Arbitrarily Primed PCR (AP-PCR) for Typing of Methicillin-Resistant Staphylococcus Aureus (MRSA)
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Date
2001
Authors
Ahmad Mahmoud Hasan Saleh
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Abstract
Nosocomial infections caused by methicillin-resistant Staphylococcus aurous (MRSA) represent an increasing problem in hospitals worldwide. Quick and reliable typing methods are required to obtain information about the relatedness of MRSA isolates and to allow faster implementation of appropriate infection control measures. The current study describes the distribution of forty four MRSA isolates collected between February and May 1998 from 3 hospitals in Palestine, and the ability of antibiogram and two different genotyping techniques - Ribosome Spacer PCR (RS-PCR) and Arbitrarily Primed PCR (AP-PCR), to detect the relatedness among the MRSA isolates.
The ability of these techniques to detect different types among the MRSA isolates was found to be as follows; seventeen antibiogram types were noted by using 15 different antimicrobial agents, designated 1 through 17. MRSA isolates were resistant to the majority of antimicrobial agents tested; oxacillin (100%), ampicillin (100%), clindamycin (95.5%), erythromycin (86.4%), tetracytlin (72.7%), and trimethoprime (61.4%). However, all isolates were susceptible to both vancomycin and refampicin. Around 78% of the isolates were found to be resistant to more than 3 different antimicrobial agents. RS-PCR generated eleven different genotypes among the 40 examined isolates and 4 of the isolates were non-typeable, designated I through XI; each spacer pattern was represented by 1-6 fragments ranging from 300-800bp. AP-PCR identified fifteen different genotypes among the 38 MRSA examined and 6 isolates were non type able using the BG2 arbitrarily primer (GGT TGG GTG AGA ATT GCA CG; 5' to 3'), designated A through 0; each BG2 pattern was represented by 1-7 fragments ranging from 300-800 bp. XI AP-PCR using P7 arbitrarily primer (GTG GAT GCG A; 5' to 3') revealed fourteen distinct genotypes among the 42 MRSA isolates and two isolates were non-type able, designated a through n. The revealed fragments size (1-11) ranged from 300-900 bp. In our study, genotyping was more powerful tool than antibiogram in differentiating between unrelated isolates of MRSA, and strongly indicates that the source of MRSA was the environment in both the neonatal unit (NU) and intensive care unit (ICU), since most of the environmental and colonized MRSA isolates of both units revealed identical genetic patterns. Combination of RS-PCR and AP-PCR (BG2 and P7 primers) resulted in five major clones, while six clones were shown among MRSA isolates using BG2 and P7 arbitrarily primers. Analysis of isolates distribution in the most prevalent clones indicated that the environment of the NU and ICU was the predicted source of MRSA because most of isolates from the environment and colonized patients in both units showed the same clone.
Thus, our results confirm the usefulness of combined usage of both genotypic methods (RS-PCR and AP-PCR) and AP-PCR with different arbitrarily primers for epidemiological studies of MRSA.
Nosocomial infections caused by methicillin-resistant Staphylococcus aurous (MRSA) represent an increasing problem in hospitals worldwide. Quick and reliable typing methods are required to obtain information about the relatedness of MRSA isolates and to allow faster implementation of appropriate infection control measures. The current study describes the distribution of forty four MRSA isolates collected between February and May 1998 from 3 hospitals in Palestine, and the ability of antibiogram and two different genotyping techniques - Ribosome Spacer PCR (RS-PCR) and Arbitrarily Primed PCR (AP-PCR), to detect the relatedness among the MRSA isolates. The ability of these techniques to detect different types among the MRSA isolates was found to be as follows; seventeen antibiogram types were noted by using 15 different antimicrobial agents, designated 1 through 17. MRSA isolates were resistant to the majority of antimicrobial agents tested; oxacillin (100%), ampicillin (100%), clindamycin (95.5%), erythromycin (86.4%), tetracytlin (72.7%), and trimethoprime (61.4%). However, all isolates were susceptible to both vancomycin and refampicin. Around 78% of the isolates were found to be resistant to more than 3 different antimicrobial agents. RS-PCR generated eleven different genotypes among the 40 examined isolates and 4 of the isolates were non-typeable, designated I through XI; each spacer pattern was represented by 1-6 fragments ranging from 300-800bp. AP-PCR identified fifteen different genotypes among the 38 MRSA examined and 6 isolates were non type able using the BG2 arbitrarily primer (GGT TGG GTG AGA ATT GCA CG; 5' to 3'), designated A through 0; each BG2 pattern was represented by 1-7 fragments ranging from 300-800 bp. XI AP-PCR using P7 arbitrarily primer (GTG GAT GCG A; 5' to 3') revealed fourteen distinct genotypes among the 42 MRSA isolates and two isolates were non-type able, designated a through n. The revealed fragments size (1-11) ranged from 300-900 bp. In our study, genotyping was more powerful tool than antibiogram in differentiating between unrelated isolates of MRSA, and strongly indicates that the source of MRSA was the environment in both the neonatal unit (NU) and intensive care unit (ICU), since most of the environmental and colonized MRSA isolates of both units revealed identical genetic patterns. Combination of RS-PCR and AP-PCR (BG2 and P7 primers) resulted in five major clones, while six clones were shown among MRSA isolates using BG2 and P7 arbitrarily primers. Analysis of isolates distribution in the most prevalent clones indicated that the environment of the NU and ICU was the predicted source of MRSA because most of isolates from the environment and colonized patients in both units showed the same clone. Thus, our results confirm the usefulness of combined usage of both genotypic methods (RS-PCR and AP-PCR) and AP-PCR with different arbitrarily primers for epidemiological studies of MRSA.
Nosocomial infections caused by methicillin-resistant Staphylococcus aurous (MRSA) represent an increasing problem in hospitals worldwide. Quick and reliable typing methods are required to obtain information about the relatedness of MRSA isolates and to allow faster implementation of appropriate infection control measures. The current study describes the distribution of forty four MRSA isolates collected between February and May 1998 from 3 hospitals in Palestine, and the ability of antibiogram and two different genotyping techniques - Ribosome Spacer PCR (RS-PCR) and Arbitrarily Primed PCR (AP-PCR), to detect the relatedness among the MRSA isolates. The ability of these techniques to detect different types among the MRSA isolates was found to be as follows; seventeen antibiogram types were noted by using 15 different antimicrobial agents, designated 1 through 17. MRSA isolates were resistant to the majority of antimicrobial agents tested; oxacillin (100%), ampicillin (100%), clindamycin (95.5%), erythromycin (86.4%), tetracytlin (72.7%), and trimethoprime (61.4%). However, all isolates were susceptible to both vancomycin and refampicin. Around 78% of the isolates were found to be resistant to more than 3 different antimicrobial agents. RS-PCR generated eleven different genotypes among the 40 examined isolates and 4 of the isolates were non-typeable, designated I through XI; each spacer pattern was represented by 1-6 fragments ranging from 300-800bp. AP-PCR identified fifteen different genotypes among the 38 MRSA examined and 6 isolates were non type able using the BG2 arbitrarily primer (GGT TGG GTG AGA ATT GCA CG; 5' to 3'), designated A through 0; each BG2 pattern was represented by 1-7 fragments ranging from 300-800 bp. XI AP-PCR using P7 arbitrarily primer (GTG GAT GCG A; 5' to 3') revealed fourteen distinct genotypes among the 42 MRSA isolates and two isolates were non-type able, designated a through n. The revealed fragments size (1-11) ranged from 300-900 bp. In our study, genotyping was more powerful tool than antibiogram in differentiating between unrelated isolates of MRSA, and strongly indicates that the source of MRSA was the environment in both the neonatal unit (NU) and intensive care unit (ICU), since most of the environmental and colonized MRSA isolates of both units revealed identical genetic patterns. Combination of RS-PCR and AP-PCR (BG2 and P7 primers) resulted in five major clones, while six clones were shown among MRSA isolates using BG2 and P7 arbitrarily primers. Analysis of isolates distribution in the most prevalent clones indicated that the environment of the NU and ICU was the predicted source of MRSA because most of isolates from the environment and colonized patients in both units showed the same clone. Thus, our results confirm the usefulness of combined usage of both genotypic methods (RS-PCR and AP-PCR) and AP-PCR with different arbitrarily primers for epidemiological studies of MRSA.