Phenotypic and molecular characterization of community occurring ...

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Phenotypic and molecular characterization of community occurring ...

WSPP community MRSATable 2. Toxin production, salt tolerance and adherence studiesHaemolytic titre aStrain no. α-toxin β-toxin Egg-yolk factor MIC NaCl (%) Adherence b % ± S.D.MA0126 1024 512 – 14.0 8.88 ± 1.42MA0116 512 256 – 14.0 6.87 ± 0.5698M7611 256 256 – 13.0 13.76 ± 1.6998M7675 2048 256 – 14.0 11.49 ± 2.1098M22661 4096 256 – 14.0 8.45 ± 1.25MA0111 512 128 – 14.0 6.13 ± 0.76MR99/692 2048 128 – 14.0 4.62 ± 0.80ST92/398 1024 128 – 14.0 5.83 ± 0.80ST94/24 4096 256 – 14.0 4.76 ± 0.95ST94/1208 2048 128 – 14.0 7.32 ± 1.59MR95/658 2048 256 + 9.5 4.73 ± 0.40MR98/1675 0 4 + 6.0 4.59 ± 0.45M1126 2048 128 + 7.0 1.36 ± 0.57MR99/854 1024 128 + 9.5 2.45 ± 0.37MR98/2554 0 4 + 10.0 5.86 ± 0.87NCTC 10345 NT c NT NT NT 1.32 ± 0.31a Highest titre giving erythrocyte lysis.b Mean of four experiments each done in triplicate.c NT, not tested.Zealand during 1992–1993. Whereas Pacific Island groupsoriginally appeared most at risk from WSPP strains, thesestrains are now widespread throughout New Zealand andoccur in all racial populations (although still strongly associatedwith Pacific Island people), accounting for >60%(around 2700 isolates from a total of 4500) of all MRSA isolatedin New Zealand in 2000. 2 They are consistently associatedwith overt skin lesions (abscess or cellulitis) in youngerage groups. In the 2001 New Zealand census, 200 301 (5.4%)of the total population of 3737490 identified themselves asPolynesian.Our sample of 10 WSPP isolates from New Zealand,Samoa and Australia displayed characteristics consistent withthose found for all other isolates so far examined—identicalmacrorestriction patterns of SmaI-digested DNA, oxacillinMICs in the 8–128 mg/L range, with all susceptible to vancomycinand most susceptible to a variety of non-β-lactamantimicrobials such as gentamicin, co-trimoxazole and ciprofloxacin.The most recent (July 2000) local survey in NewZealand revealed that 1.3% of 229 WSPP isolates were resistantto two or more classes of antimicrobials in addition toβ-lactams, with >99% still susceptible to chloramphenicol,ciprofloxacin, clindamycin, co-trimoxazole, fusidic acid,gentamicin and tetracycline. Mupirocin resistance was at3.5%.Apart from New Zealand and Western Samoa, the WSPPclone has been found in Australia; 12,13 these isolates werereferred to as gentamicin-sensitive, methicillin-resistantS. aureus (GS-MRSA) in one publication. 12 The Australianisolates clearly have identical characteristics to the NewZealand and Western Samoan isolates (Table 1), and as inNew Zealand were first isolated from Polynesians. In theAustralian studies, it was assumed that the strain was introducedinto Australia from Polynesia via New Zealand, 12 oroccurred more commonly (compared with multiresistantMRSA or MSSA) in patients born in New Zealand, Samoa orTonga. 13The possible origins of CMRSA like the WSPP clone isopen to debate. It seems unlikely that they are feral descendantsof hospital isolates, but more likely represent acommunity MSSA strain that has acquired the mec DNA elementfrom some other cutaneous staphylococcal species. 28Although it is conceivable that the WSPP clone is of animalorigin (e.g. β-haemolysin toxin producers, egg-yolk opacityfactor negative), the phage patterns of isolates suggest thatthis is unlikely. To some degree, the WSPP clone behaves likethe old phage group I S. aureus, with resistance only topenicillin-type antimicrobials and a predilection for skinabscess/boil formation.Presumably the WSPP clone originated in Samoa andwas readily disseminated amongst individuals because of theliving conditions found in that country. Thirty-two of 110(29%) adults passing through the front door of a hospital inWestern Samoa were colonized with S. aureus, including829


R. P. Adhikari et al.2.7% with WSPP MRSA. 2 Clearly there is no barrier to itscolonization and spread amongst other people of any raceshould the opportunity for spread occur. The high salttolerance of the WSPP clone probably helps in this respect,as would the ability to adhere to cell surfaces. In general,studies 27,29 attempting to correlate adherence with epidemicityand spread amongst MRSA strains have reported nosignificant differences in the abilities of epidemic MRSA,other MRSA and MSSA to adhere to human nasal epithelialcells, HEp-2 cells or to other cultured cell lines. In ourinvestigations, the WSPP MRSA as a group appeared toadhere significantly better to HEp-2 cells than did all otherMRSA studied. This finding is possibly related in some wayto the unique mec DNA element (Staphylococcus cassettechromosome mec) found in WSPP MRSA (data not shown).The WSPP isolates were found to be high level α- andβ-haemolysin toxin producers (as determined by assays usedhere and the presence of hla and hlb) in comparison with theother S. aureus isolates tested; this may positively influencethe ability of such isolates to initiate joint/bone infections. 25α-Haemolysin toxin is a pore-forming haemolytic andmembrane-damaging toxin, 30 whereas β-haemolysin toxin isproduced by a large number of S. aureus strains, especiallythose of animal origin. 31 Our results indicate that α-haemolysintoxin production is a consistent feature of WSPP isolates,and may in part be responsible for their association withovert cutaneous lesions. All WSPP MRSA were positive forthe presence of the hla gene, but the hlb gene could only bedetected by Southern hybridization. This result suggested thatthe primers used here to amplify hlb were not homologous tothe hlb gene of the WSPP MRSA. Moreover, despite theWSPP isolates being able to produce β-haemolysin toxin, allwere egg-yolk opacity factor negative, suggesting that theβ-haemolysin toxin produced by the WSPP isolates maydiffer from that of other S. aureus isolates. Further work willbe required to determine how this toxin differs in WSPPMRSA strains.In conclusion, WSPP1 and WSPP2 strains are now themost common type of MRSA found in New Zealand populations.In the main, these non-multiresistant strains appear tobe associated with community-acquired cutaneous lesions,rather than being hospital acquired as is the case for mostMRSA in other countries. WSPP strains are especiallycommon in Polynesian populations and younger age groups.We have shown WSPP MRSA from Western Samoa and NewZealand to be identical to Australian isolates (GR-MRSA)and all of the isolates are likely to represent a single clone.These highly toxigenic, salt tolerant and egg-yolk-negativestrains are clearly clonally related. The WSPP strains as agroup show increased adherence to tissue culture cells comparedwith other MRSA, and this may relate to their increasedfitness and the relative success of this clone.AcknowledgementsThe financial assistance of the Otago Medical ResearchFoundation and the Deans Fund, Otago School of MedicalSciences is gratefully acknowledged. R.P.A. was the recipientof a New Zealand Official Development Assistance (NZODA)Study Award.References1. Smith, J. M. B. (1998). Emerging problems of antibiotic resistantbacteria. New Zealand Medical Journal 111, 441–4.2. Lang, S., Taylor, S. & Morris, A. (2001). 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