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Keywords:

  • Arginine catabolic mobile element;
  • dru ;
  • methicillin-resistant Staphylococcus aureus;
  • pulsed-field gel electrophoresis;
  • Panton–Valentine leukocidin;
  • Qatar;
  • SCC mec ;
  • Spa

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgements
  8. Transparency Declaration
  9. References

Global spread of epidemic methicillin-resistant Staphylococcus aureus (MRSA) is an issue of increasing clinical concern especially problematic community-associated (CA) -MRSA. However, data regarding MRSA epidemiology in regions of the Middle East, including Qatar, are still insufficient. A representative subset of 61 MRSA isolates from multinational patients from hospital in Qatar during a 2-year period (2009/2010) was examined. Molecular characterization for MRSA isolates was performed by pulsed-field gel electrophoresis (PFGE), SCCmec, spa and dru typing, and PCR for the presence of the arginine catabolic mobile element and genes for the Panton–Valentine leukocidin (PVL). Prevalence of MRSA among S. aureus isolated was 176/840 (21%). Of the 61 MRSA isolates examined, three (5%) represented hospital-acquired infection. By PFGE, 32 isolates (52%) were CA-MRSA USA300 (n = 4), USA400 (n = 3), USA1100/Southwest (SW) Pacific (n = 17) and ST80-MRSA-IV (n = 8) strains. The remaining isolates were well-known healthcare-associated EMRSA-15 (n = 8) and USA800 (n = 13). Three isolates were USA900, one was USA1200 and four were unrelated to any known strains in the international database. Unexpectedly, the USA900 isolates were all positive for PVL and USA400 isolates were PVL negative. Five of the eight EMRSA-15 isolates were PVL positive. ST80-MRSA-IV and USA300 strains exhibited typical dru types (dt10a and dt9g, respectively). Eleven different spa types were observed in this study. All USA300 isolates were arginine catabolic mobile element positive. The high prevalence of CA-MRSA, especially including USA300, in this setting underscores the importance of global epidemiological monitoring to better understand and hopefully help prevent the emergence and spread of these problem pathogens in patient populations.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgements
  8. Transparency Declaration
  9. References

Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogen of worldwide concern, a cause of morbidity and mortality in both hospital and community populations. Global spread of epidemic MRSA is an issue of increasing clinical concern especially regarding problematic community-associated MRSA. Several molecular typing techniques have been developed to provide knowledge of both transmission and epidemiology of MRSA strains and to support infection control measures [1]. These techniques include pulsed-field gel electrophoresis (PFGE) as a reference standard for MRSA strain typing [2, 3], SCCmec typing [4], as well as DNA sequence-based typing such as multilocus sequence typing [5], sequencing of the SCCmec-associated direct repeat unit (dru) [6] and single-locus DNA sequencing of repeat regions of the spa gene (protein A) [7]. Nucleic acid sequence typing has become more popular than PFGE because of the easy data transfer and excellent comparability of results through online databases [8].

Panton–Valentine leukocidin (PVL) is an S. aureus-specific exotoxin pore-forming protein encoded by two co-transcribed genes, lukF-PV and lukS-PV, and is associated with skin and soft-tissue infections and severe necrotizing pneumonia [9]. Healthcare-associated MRSA strains seldom carry PVL, in contrast community-associated MRSA frequently carries this gene [10].

The arginine catabolic mobile element (ACME) was shown to contribute to bacterial growth, survival, transmission and colonization within the host. Although it is frequently found in coagulase-negative staphylococcal species, a variety of strains such as USA300 (ST8-MRSA-IVa) also carry this element [11]. ACME is integrated into orfx, the same site on the S. aureus chromosome into which SCCmec integrates, and contains the structural gene arcA with a distinctive nucleotide sequence, encoding arginine deiminase [12].

Epidemiological data on MRSA in regions of the Middle East, including Qatar, are still insufficient. The present study was undertaken to characterize MRSA strains during a 2-year period (2009/2010) in a private 250-bed tertiary-care general hospital in Doha, Qatar.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgements
  8. Transparency Declaration
  9. References

Bacterial isolates

A representative subset of 61 MRSA were isolated from multinational patients in a tertiary-care hospital in Doha, Qatar over a 2-year period (2009/2010). Only isolates from patients resident in Doha for > 6 months and who had a recent history, 3 months before the study, of antibiotic treatment were selected. Duplicate isolates and isolates with incomplete clinical data were excluded from the study. All the isolates were recovered from different patients and different pathological samples (Table 1). The study was approved by the hospital ethics and research committee. MRSA isolates were detected in the clinical laboratory using VITEK 2 AST-P580 cards (bioMérieux, Marcy l'Etoile, France), cefoxitin disc diffusion, and PBP2 latex agglutination (Denka Seiken Co., Niigata, Japan) according to the manufacturers' instructions. Methicillin sensitive S. aureus ATCC 25923 and MRSA ATCC 43300 were used as negative and positive controls, respectively in each run. Further confirmation for suspected MRSA isolates was performed by PCR amplification of the mecA gene [13]. All MRSA isolates were also tested for mupirocin and inducible clindamycin susceptibility using VITEK 2 AST-P580 cards according to the manufacturer's instructions.

Table 1. Clinical and molecular characteristicsa of isolates
SexAgeSpecimenNationalityPVLACMEPFGE pattern; SCCmec; dru; spa; Predicted MLST (ST)
  1. F, female; M, male; MLST, multilocus sequence typing; ND, not determined; PFGE, pulsed-field gel electrophoresis; UNK, unknown; HVS, high vaginal swab.

  2. a

    In some instances, molecular characteristics were inferred from the strain type indicated by the PFGE pattern.

  3. b

    spa, dru, SCCmec typing were performed on a representative subset of isolates.

F5 yearsThroatEgyptianNegativeNegativeEMRSA 15; SCCmec IV; ND; t852; 22b
F24 yearsAbscessIndianPositiveNegative
M44 yearsAbscessCanadianPositiveNegative
M14 yearsAbscessQatariPositiveNegative
M40 yearsAbscessBritishPositiveNegative
M5 yearsAbscessBritishNegativeNegative
M46 yearsAbscessIndianPositiveNegative
F25 yearsAbscessLebaneseNegativeNegative
M15 yearsAbscessTunisianPositiveNegativeEUST80; SCCmec IV; dt10a; t044; 80b
M2 yearsSkin vesiclesLebanesePositiveNegative
M40 yearsWoundCanadianPositiveNegative
F13 yearsAbscessQatariPositiveNegative
M31 yearsNasal swabSyrianPositiveNegative
M 1 yearSkin vesiclesEgyptianPositiveNegative
M35 yearsAbscessEgyptianPositiveNegative
F2 yearsWoundEgyptianPositiveNegative
F25 yearsHVSQatariNegativeNegative(Pediatric clone) USA 800; SCCmec IV; ND; t002; 5b
F 7 monthsSkin vesiclesQatariNegativeNegative
M39 yearsWoundSudaneseNegativeNegative
F1 yearsAbscess (groin)PakistaniNegativeNegative
F32 yearsSkinPakistaniNegativeNegative
F22 yearsUrineQatariNegativeNegative
M3 yearsPus, AbscessQatariNegativeNegative
F20 yearsAbscessQatariNegativeNegative
M3 yearsAbscessQatariNegativeNegative
M29 yearsAbscessQatariNegativeNegative
M44 yearsEye swabEgyptianNegativeNegative
F32 yearsAbscessSri LankanNegativeNegative
M34 yearsAbscessQatariNegativeNegative
F10 daysPusEgyptianPositiveNegative(Southwest- Pacific clone) USA 1100; SCCmec IV; ND; t019; 30b
F31 yearsAbscessQatariPositiveNegative
F34 yearsAbscessQatariPositiveNegative
M54 yearsAbscessAmericanPositiveNegative
M37 yearsAbscessQatariPositiveNegative
F29 yearsAbscess (arm)IranianPositiveNegative
F33 yearsWoundBritishPositiveNegative
F3 yearsAbscessIndianPositiveNegative
F33 yearsAbscessBritishPositiveNegative
F3 yearsWoundEgyptianPositiveNegative
M41 yearsWoundFilipinoPositiveNegative
M62 yearsAbscessFilipinoPositiveNegative
M2 yearsAbscessQatariPositiveNegative
F33 yearsNasal swabFilipinoPositiveNegative
M35 yearsAbscessFilipinoPositiveNegative
F35 yearsAbscessQatariPositiveNegative
F38 yearsAbscessQatariPositiveNegative
M54 yearsAbscessEgyptianPositiveNegativeUSA 1200VNDt314121
M52 yearsAbscessAmericanPositivePositiveUSA 300IVdt9gt0088
F32 yearsAbscessCanadianPositivePositiveUSA 300IVdt9gt1218
M35 yearsAbscessAmericanPositivePositiveUSA 300IVdt9gt0088
F32 yearsHVSCanadianPositivePositiveUSA 300IVdt9gt1218
F37 yearsWoundQatariNegativeNegativeUSA 400IVNDND8
F54 yearsAbscessBangladeshiNegativeNegativeUSA 400IVNDND8
M44 yearsWoundEgyptianNegativeNegativeUSA 400IVNDt2678
M41 yearsAbscessBahrainiPositiveNegativeUSA 900VNDND15
M45 yearsAbscessQatariPositiveNegativeUSA 900VNDt65715
F48 yearsAbscessIndianPositiveNegativeUSA 900VNDt735815
M 8 daysSkin vesiclesIndianPositiveNegativeUnknownIVNDt69088
M34 yearsWoundQatariPositiveNegativeUnknownIVNDt69088
M42 yearsSkin blisterFilipinoNegativePositiveUnknownVNDUNKUNK
F30 yearsHVSQatariNegativeNegativeUnknownVNDUNKUNK

Clinical and epidemiological information for patients was obtained from clinic documents to determine whether these isolates were recovered from hospital-acquired MRSA or community-acquired MRSA infections based on patient history according to the criteria published by the CDC (Atlanta, GA, USA; http://www.cdc.gov/mrsa/diagnosis/index.html). MRSA strains were considered to be community-acquired MRSA if isolates were recovered from outpatients, within 48 h of hospitalization or if the patient lacked the following hospital-acquired MRSA risk factors: haemodialysis, surgery, residence in a long-term care facility or hospitalization during the previous year, the presence of an indwelling catheter or a percutaneous device at the time of culture, or previous isolation of MRSA from the patient.

Molecular characterization

Genomic DNA was extracted by a 10-min incubation with lysostaphin (0.5 mg/mL) at 37°C followed by boiling for 10 min. After centrifugation, the supernatant was used as a template for amplification. PCR for the presence of the gene encoding PVL was performed as previously described [14]. ACME-arcA gene was detected by PCR using ACME-specific primers [12]. PFGE analysis with SmaI was carried out according to Goering [15]. Macro-chromosomal restriction fragments were separated for 23 h at 6 V/cm, using a 120° included angle at 14°C using a CHEF-DR II System (Bio-Rad, Hercules, CA, USA), with initial and final pulse times of 1 and 35 s, respectively. Staphylococcus aureus 8325 was used as a reference strain. Banding patterns were analysed with BioNumerics V6.5 (Applied Maths, St-Martens-Latem, Belgium) with unweighted pair-group arithmetic averages (UPGMA) and Dice coefficients. Band patterns that were >80% identical were considered related as per the recommendation of Tenover et al. [3]. PFGE banding patterns were interpreted to different epidemic-type clones following the published guidelines and database [16].

SCCmec typing was performed using six multiplex PCR assays [17, 18]. For dru typing, the dru region was amplified and sequenced as described previously [19]. The BioNumerics tandem repeat sequence typing plug-in tool was used for dru sequence analysis and assignment of dru types used an alphanumeric nomenclature [19]. For spa typing, the primers and thermal cycling conditions of the European Network of Laboratories for Sequenced Based Typing of Microbial Pathogens (SeqNet [http://www.seqnet.org]) were used. Analysis of spa sequences and assignment of spa types were performed using the Spa typing plug-in tool of the BioNumerics software package. Confirmation of sequenced dru and spa types was also performed via the freely available dru server (http://www.dru-typing.org/search.php) and Ridom Spa Server (http://spa.ridom.de/index.shtml), respectively.

Results and Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgements
  8. Transparency Declaration
  9. References

A total of 840 S. aureus isolates were recovered from a tertiary-care hospital in Doha, Qatar over a 2-year period (2009/2010). The MRSA isolates represent 21% (176 isolates) of the total S. aureus isolated. A subset of 61 MRSA isolates from different multinational patients with a recent history of antibiotic treatment was chosen for the current study. According to the CDC criteria, 58 of these were considered to be community-acquired MRSA based on patient history whereas the remaining three were hospital-acquired MRSA from skin infections. Results of molecular characterization of the isolates by PFGE, and PCR for SCCmec, PVL, ACME, dru and spa typing are shown in Table 1 and Fig. 1. There were no significant associations between nationality, age of the patient, or nature of the specimen and MRSA strain type.

image

Figure 1. Frequency distribution of methicillin-resistant Staphylococcus aureus isolate strain types (%).

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Analysis by PFGE demonstrated patterns in 32 isolates (52%) consistent with a community-associated MRSA designation (i.e. strain types previously described as community-associated MRSA) [3, 16]. The most prevalent clone was the PVL-positive USA1100 (SW Pacific Clone) which represented 28% (17 isolates) of the total number of isolates (Fig. 1). Well known community-associated MRSA strains (ST80-MRSA-IV, USA300 and USA400) were 13% (eight isolates), 7%, (four isolates) and 5% (three isolates) of the total, respectively, cumulatively representing 25% of all isolates. PFGE patterns for the remaining isolates were consistent with a healthcare-associated MRSA designation (strain types previously described as healthcare-associated MRSA) [3, 16]. USA800 (Paediatric Clone) isolates were 21% (13 isolates) of the total while 13% (eight isolates) were a mixture of PVL-positive and PVL-negative EMRSA-15.

In contrast to previous reports [20, 21], in which USA900 and USA1200 were detected only as methicillin-sensitive S. aureus, three MRSA isolates in the current study were USA900, one was USA1200, and four were unrelated to any known strains in the international database (two of them were inferred to be ST88-MRSA-IV). Unexpectedly, the USA900 isolates were all positive for PVL and USA400 isolates were PVL negative. USA400 isolates were cultured in the current study from non-North American patients (i.e. patients from Qatar, Bangladesh and Egypt).

In addition, five of the eight EMRSA-15 isolates were PVL positive. This finding is surprising because previous studies detected that all EMRSA-15 clone isolates were PVL negative [22, 23]. Sequence analysis of the mec-associated direct repeat (dru) region in USA300 and ST80-MRSA-IV isolates yielded dru types 9g and 10a, respectively, confirming the clonal spread of these strains as seen in other geographic locations [24-26]. As expected, the ACME arcA gene was only found in USA300 isolates [12] and also in one additional strain that lysed poorly and is the subject of further investigation. Eleven different spa types were observed in this study. Two spa types (t008 ‘2’ and t121 ‘2’) were associated with USA300 isolates. A USA300 clone was previously demonstrated to move from the community to the hospital setting where it underwent an adaptive process combining both resistance and virulence characteristics [26, 27].

Inducible clindamycin resistance was detected in 12 (20%) isolates that were USA800 (five isolates), EMRSA-15 (three isolates), ST80-MRSA-IV (two isolates), and individual isolates of USA300 and the inferred ST88-MRSA-IV clone. These data are in agreement with a previous report [28] where 20% of 60 MRSA isolated from clinical samples were found to have inducible resistance to clindamycin detected by D-test.

Only five (8%) of our isolates were resistant to mupirocin. According to the hospital's infection control policy, mupirocin nasal ointment was used routinely for MRSA decolonization either in nasal carriers or for treatment of patients. In a previous study, among 4980 MRSA isolates from 32 Canadian hospitals, high-level mupirocin resistance increased from 1.6% in 1995–99 to 7.0% in 2000–04 [29].

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgements
  8. Transparency Declaration
  9. References

The predominant MRSA strain types found were USA1100 (SW Pacific Clone), USA800 (Paediatric Clone) and EMRSA-15. The global spread of community-associated MRSA strains continues unabated and clearly extends to the Middle East. This not only includes ST80-MRSA-IV, which is well known in Europe, but also USA300 and USA400 strains typically associated with North America. Finding the USA300 clone in the Middle East region stresses the need for active and continuous monitoring of the incidence of this threatening clone.

The current study underscores the value of molecular typing to monitor global trends in the emergence, spread and persistence of epidemic MRSA strains. Our findings also highlight the need for continued vigilance and surveillance.

According to our knowledge, this is the first published report concerning MRSA typing in Qatar; however, further studies are still required in this area and in the Middle East as a whole to compare the spread of different clones with findings in other geographical regions in the world.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgements
  8. Transparency Declaration
  9. References

Part of this study was presented to the 51st ICAAC (Interscience Conference of Antimicrobial Agents and Chemotherapy) 2011, Chicago, USA.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgements
  8. Transparency Declaration
  9. References
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