Departments of 1 Microbiology and 2 Biochemistry, Otago School of Medical Sciences, University of Otago, Dunedin; 3 Middlemore Hospital and Diagnostic Medlab, Auckland; 4 Institute of Environmental Science and Research, Porirua, New Zealand
Received 30 May 2002; returned 15 July 2002; revised 10 September 2002; accepted 11 September 2002
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Abstract |
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Keywords: community MRSA, phenotypic and molecular characteristics
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Introduction |
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Although there are difficulties in demonstrating that so-called community MRSA (CMRSA) are not acquired as the result of a visit to a health care facility, it does seem that CMRSA are of increasing importance in a number of countries.614 In general, such strains have not been multiresistant and often have low methicillin/oxacillin MICs, typically 64 mg/L. The predilection of such strains for children and/or minority communities has also been a recorded feature in some surveys.3,5,6,8,9,12,13 In many respects, these CMRSA appear to behave in a similar manner to methicillin-susceptible S. aureus (MSSA), and can be brought into hospitals and serve as an important source of nosocomial infection. For some reason, clindamycin9,10 or gentamicin12 susceptibility appears to have been taken as an indicator of possible community acquisition.
In New Zealand, the rapid spread of the WSPP clone of CMRSA throughout the country, and occurrence in patients devoid of the risk factors commonly reported for MRSA acquisition,1 suggest that such strains possess properties and/or characteristics different from those of other MRSA. Despite the increasing problems of WSPP in New Zealand, features of the physiology and molecular genetics of this clone are still largely unknown. The present study was therefore undertaken to compare various characteristics (e.g. toxin production, environmental survival) of WSPP strains from New Zealand, Australia and Western Samoa with those of other MRSA strains.
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Materials and methods |
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All isolates used in this study were from the culture collections of the Department of Microbiology, University of Otago, and ESR. In addition to control strains, 15 MRSA isolates were included in the comparative investigations 10 WSPP1 or WSPP2 chosen on the basis of geographical isolation and oxacillin MICs, and five multiresistant hospital MRSA. MRSA were defined as S. aureus with an oxacillin MIC of 4 mg/L. All MICs were determined using Etests (AB Biodisk, Solna, Sweden) as recommended by the manufacturer. Where required, disc diffusion sensitivity tests were carried out as described by the NCCLS.15 Other bacterial strains used in this study were S. aureus strain ATCC 25923 as an antimicrobial sensitivity test control, S. aureus strain 417 as a mecA-positive control, S. aureus strain 8325-4 (Hla+, Hlb+) and attachment-negative strain S. aureus NCTC 10345 (CN56 Wood 46). All strains were maintained on sheep blood agar plates at 4°C with regular subculturing. For long-term storage, strains were stored in skimmed milk (Difco) at 70°C.
PCR, DNA hybridization, pulsed-field gel electrophoresis (PFGE) and restriction fragment length polymorphism (RFLP) of the coagulase gene
All MRSA strains were tested for the presence of mecA by PCR and Southern blot hybridization using standard molecular biology protocols and primers described previously.1618 The RFLP of the coagulase gene was carried out using the method described by Goh et al.19
PFGE was carried out as described previously20 using the contour-clamped homogeneous electric field (CHEF-DRIII) electrophoresis system (Bio-Rad Laboratories, Richmond, CA, USA) at 6 V/cm and 14°C. The Low Range PFG Marker (New England Biolabs, Inc., Beverly, MA, USA) containing lambda concatemers and lambda-digested HindIII fragments was used as a size standard. Separated DNA fragments were stained with ethidium bromide and visualized with a UV transilluminator. The clonality of isolates was judged using previously described criteria from visual comparisons of banding patterns of samples run together in the same gel.21
Salt tolerance, and resistance to desiccation, skin fatty acids and ultraviolet radiation
Salt tolerance of the MRSA strains was determined using an agar dilution technique employing different concentrations (0.515%) of NaCl incorporated into nutrient agar. The lowest concentration of NaCl that inhibited colony formation was interpreted as the MIC of NaCl, as described previously.22
Survival of MRSA strains under different humidities was carried out using desiccators and a range of relative humidities.23 Samples were taken at 7 day intervals and the number of viable cells remaining on the filter determined by dilution and plating techniques. Cell viability (survival) was assessed as the number of bacteria remaining as a percentage of the starting count.
Sensitivity to two free fatty acids was determined by incorporating the acids into blood agar plates [5% blood in tryptic soy agar (TSA)] to create final fatty acid concentrations in the range 0.0250.4% for linoleic acid and 17% for oleic acid. MICs were determined by identifying the lowest concentration of the fatty acid that inhibited growth after incubation at 37°C for 24 h.24
Resistance of each test strain to UV light was observed by spotting 10 µL of an overnight culture (1 x 108 cfu/mL) on to the surface of sheep blood agar plates followed by exposure (0, 2, 5, 8, 15, 30, 45, 60, 90 and 150 s) to UV light (254 nm wavelength). Plates were then incubated overnight at 37°C and the presence or absence of growth recorded.
Production of - and ß-haemolysin toxins, and adherence to HEp2 cells
The quantitative measurement of - and ß-haemolysin toxin production was carried out in terms of haemolytic titre as described previously.25 To confirm the presence of the genes encoding
-haemolysin (hla) and ß-haemolysin (hlb), PCR and Southern hybridization were carried out as described previously.17,18 The following primers were used:
-haemolysin HLA-1 5'-CTGATTACTATCCAAGAAATTCGATTG-3' and HLA-2 5'-CTTTCCAGCCTACTTTTTTATCAGT-3', and ß-haemolysin HLB-1 5'-GTGCACTTACTGACAATAGTGC-3' and HBL-2 5'-GTTGATGAGTAGCTACCTTCAGT-3'.26 Strain 8325-4 (Hla+ Hlb+) was used as a positive control for
- and ß-haemolysin.25 All PCR products were sequenced to confirm that the correct gene had been amplified. Positive PCR products for hla and hlb were used as DNA probes for Southern hybridization, as described above.
The adherence assay employed a continuous human epithelial cell line, HEp2, and was carried out as described by Aathithan et al.27 Tryptic soy broth (TSB) cultures of each strain were radiolabelled by mixing with 0.925 MBq [3H]thymidine in an orbital shaker (200 rpm) for 18 h at 37°C. The radiolabelled bacteria were then centrifuged and washed twice in 20 mL of PBS, and the pellet resuspended to an OD540 of 0.4 (1 x 108 cells/mL). The specific activity of the 3H-labelled bacteria was determined by transferring 100 µL of labelled bacteria into a scintillation vial containing 1 mL of scintillant, and the radioactivity determined using an LKB Wallac Scintillation Counter. Adherence assays were carried out using fresh HEp2 monolayers in 24-well tissue culture plates inoculated with 0.5 mL of culture, followed by incubation at 37°C for 2 h. After incubation, the monolayer was gently washed twice with 1 mL of PBS and then lysed by adding 250 µL of pre-warmed trypsin with incubation at 37°C for 15 min. After complete detachment and solubilization of the monolayer, the content of each well was transferred to a scintillation vial containing 1 mL of scintillation fluid and the radioactivity determined as above. Percentage adherence was calculated by applying the following formula: % of adherence = (mean cpm of the lysed monolayer x 100)/(mean cpm of the original bacterial suspension).
Egg-yolk opacity factor
Each isolate was inoculated on to plates of mannitol egg-yolk medium [0.1% beef extract (BBL), 1% peptone 140 (Difco), 1% mannitol, 1% NaCl, 2% solution of Phenol Red (12.5 mL/L), 50% saline solution of egg yolk (11 mL/L) and 1.5% agar] and incubated for 48 h at 37°C. Any obvious zone of opacity occurring around the subsequent growth was then recorded.
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Results |
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Ten WSPP MRSA isolates with varying methicillin MICs from New Zealand, Western Samoa and Australia were included in this study (Table 1). All WSPP isolates revealed a similar biochemical profile using the ID32 STAPH system (Table 1). Variations were observed in the fermentation of ribose and arabinose, acetoin production, the formation of arginine arylamidase and novobiocin resistance.
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Nine of the WSPP isolates revealed identical SmaI DNA fingerprint patterns following PFGE (Figure 1, lanes 2 and 3 showing representative pattern). One WSPP isolate (MA0116) showed absence of a band at 146 kb (Figure 1, lane 4). Each PFGE profile was found to be stable during numerous passages of the bacterium in vitro and unrelated to the profiles generated for other MRSA (i.e. non-WSPP) (Figure 1, lanes 6 and 7). Coagulase gene RFLP patterns generated from the WSPP isolates were of one type and unrelated to that of the non-WSPP isolates (Table 1).
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All WSPP were tested for their resistance/sensitivity to various environmental stresses and compared with the more classical hospital MRSA strains. The MIC of oleic acid for all MRSA isolates examined was >7%. The MIC of linoleic acid ranged from 0.3% to 0.4% for all strains and there were no significant differences between the WSPP and other MRSA isolates (data not shown). Similarly, percentage survival after 15 s exposure to UV light was in the range 0.00010.002% for all isolates (data not shown). In the case of salt tolerance, the WSPP isolates appeared consistently more tolerant to salt than the other MRSA investigated. Inhibitory levels of NaCl were 14% for WSPP compared with 610% for other MRSA (Table 2).
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All isolates of WSPP studied were found to produce consistently high levels of - and ß-haemolysin toxins in the ranges 2564096 and 128512 haemolytic titre, respectively (Table 2). High-level production of these toxins was not a consistent feature of the other MRSA studied. All WSPP MRSA were positive for the presence of the hla gene coding for
-haemolysin toxin production, but were negative for the hlb gene as confirmed by PCR. However, Southern hybridization of genomic DNA with an hlb probe from strain 8325-4 (Hla+, Hlb+) demonstrated that all strains were positive for a putative hlb gene (data not shown).
The percentage adherence results and the mean values for each isolate are shown in Table 2. Mean adherence values ranged from 1.36% to 13.76% for MRSA isolates. The control strain Wood 46 (a known low-adherer) had a value of 1.32%. Analysis of the data using the t-test indicated that as a group the WSPP isolates had significantly (P < 0.005) increased adherence compared with other MRSA.
Of the 15 S. aureus isolates included in the present study, all 10 WSPP were egg-yolk opacity factor negative. All five non-WSPP MRSA were egg-yolk positive.
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Discussion |
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Our sample of 10 WSPP isolates from New Zealand, Samoa and Australia displayed characteristics consistent with those found for all other isolates so far examinedidentical macrorestriction patterns of SmaI-digested DNA, oxacillin MICs in the 8128 mg/L range, with all susceptible to vancomycin and most susceptible to a variety of non-ß-lactam antimicrobials such as gentamicin, co-trimoxazole and ciprofloxacin. The most recent (July 2000) local survey in New Zealand revealed that 1.3% of 229 WSPP isolates were resistant to two or more classes of antimicrobials in addition to ß-lactams, with >99% still susceptible to chloramphenicol, ciprofloxacin, clindamycin, co-trimoxazole, fusidic acid, gentamicin and tetracycline. Mupirocin resistance was at 3.5%.
Apart from New Zealand and Western Samoa, the WSPP clone has been found in Australia;12,13 these isolates were referred to as gentamicin-sensitive, methicillin-resistant S. aureus (GS-MRSA) in one publication.12 The Australian isolates clearly have identical characteristics to the New Zealand and Western Samoan isolates (Table 1), and as in New Zealand were first isolated from Polynesians. In the Australian studies, it was assumed that the strain was introduced into Australia from Polynesia via New Zealand,12 or occurred more commonly (compared with multiresistant MRSA or MSSA) in patients born in New Zealand, Samoa or Tonga.13
The possible origins of CMRSA like the WSPP clone is open to debate. It seems unlikely that they are feral descendants of hospital isolates, but more likely represent a community MSSA strain that has acquired the mec DNA element from some other cutaneous staphylococcal species.28 Although it is conceivable that the WSPP clone is of animal origin (e.g. ß-haemolysin toxin producers, egg-yolk opacity factor negative), the phage patterns of isolates suggest that this is unlikely. To some degree, the WSPP clone behaves like the old phage group I S. aureus, with resistance only to penicillin-type antimicrobials and a predilection for skin abscess/boil formation.
Presumably the WSPP clone originated in Samoa and was readily disseminated amongst individuals because of the living conditions found in that country. Thirty-two of 110 (29%) adults passing through the front door of a hospital in Western Samoa were colonized with S. aureus, including 2.7% with WSPP MRSA.2 Clearly there is no barrier to its colonization and spread amongst other people of any race should the opportunity for spread occur. The high salt tolerance of the WSPP clone probably helps in this respect, as would the ability to adhere to cell surfaces. In general, studies27,29 attempting to correlate adherence with epidemicity and spread amongst MRSA strains have reported no significant differences in the abilities of epidemic MRSA, other MRSA and MSSA to adhere to human nasal epithelial cells, HEp-2 cells or to other cultured cell lines. In our investigations, the WSPP MRSA as a group appeared to adhere significantly better to HEp-2 cells than did all other MRSA studied. This finding is possibly related in some way to the unique mec DNA element (Staphylococcus cassette chromosome mec) found in WSPP MRSA (data not shown).
The WSPP isolates were found to be high level - and ß-haemolysin toxin producers (as determined by assays used here and the presence of hla and hlb) in comparison with the other S. aureus isolates tested; this may positively influence the ability of such isolates to initiate joint/bone infections.25
-Haemolysin toxin is a pore-forming haemolytic and membrane-damaging toxin,30 whereas ß-haemolysin toxin is produced by a large number of S. aureus strains, especially those of animal origin.31 Our results indicate that
-haemolysin toxin production is a consistent feature of WSPP isolates, and may in part be responsible for their association with overt cutaneous lesions. All WSPP MRSA were positive for the presence of the hla gene, but the hlb gene could only be detected by Southern hybridization. This result suggested that the primers used here to amplify hlb were not homologous to the hlb gene of the WSPP MRSA. Moreover, despite the WSPP isolates being able to produce ß-haemolysin toxin, all were egg-yolk opacity factor negative, suggesting that the ß-haemolysin toxin produced by the WSPP isolates may differ from that of other S. aureus isolates. Further work will be required to determine how this toxin differs in WSPP MRSA strains.
In conclusion, WSPP1 and WSPP2 strains are now the most common type of MRSA found in New Zealand populations. In the main, these non-multiresistant strains appear to be associated with community-acquired cutaneous lesions, rather than being hospital acquired as is the case for most MRSA in other countries. WSPP strains are especially common in Polynesian populations and younger age groups. We have shown WSPP MRSA from Western Samoa and New Zealand 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-negative strains are clearly clonally related. The WSPP strains as a group show increased adherence to tissue culture cells compared with other MRSA, and this may relate to their increased fitness and the relative success of this clone.
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Acknowledgements |
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Footnotes |
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References |
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2 . Lang, S., Taylor, S. & Morris, A. (2001). Community-acquired methicillin-resistant Staphylococcus aureus. Antibiotics and Chemotherapy 5, 123.
3 . Mitchell, J. M., MacCulloch, D. & Morris, A. J. (1996). MRSA in the community. New Zealand Medical Journal 109, 411.
4 . Riley, D., MacCulloch, D. & Morris, A. J. (1998). Methicillin-resistant S. aureus in the suburbs. New Zealand Medical Journal 111, 59.
5 . Rings, T., Findlay, R. & Lang, S. (1998). Ethnicity and methicillin-resistant S. aureus in South Auckland. New Zealand Medical Journal 111, 151.
6 . Ayliffe, G. A. (1997). The progressive intercontinental spread of methicillin-resistant Staphylococcus aureus. Clinical Infectious Diseases 24, Suppl. 1, S749.[ISI][Medline]
7
.
Herold, B. C., Immergluck, L. C., Maranan, M. C., Lauderdale, D. S., Gaskin, R. E., Boyle-Vavra, S. et al. (1998). Community-acquired methicillin-resistant Staphylococcus aureus in children with no identified predisposing risk. Journal of the American Medical Association 279, 5938.
8 . Maguire, G. P., Arthur, A. D., Boustead, P. J., Dwyer, B. & Currie, B. J. (1998). Clinical experience and outcomes of community-acquired and nosocomial methicillin-resistant Staphylococcus aureus in a northern Australian hospital. Journal of Hospital Infection 38, 27381.[ISI][Medline]
9 . Gorak, E. J., Yamada, S. M. & Brown, J. D. (1999). Community-acquired methicillin-resistant Staphylococcus aureus in hospitalized adults and children without known risk factors. Clinical Infectious Diseases 29, 797800.[ISI][Medline]
10 . Frank, A. L., Marcinak, J. F., Daisy Mangat, P. & Schreckenberger, P. C. (1999). Increase in community-acquired methicillin-resistant Staphylococcus aureus in children. Clinical Infectious Diseases 29, 9356.[ISI][Medline]
11 . Kak, V. & Levine, D. P. (1999). Editorial response: community-acquired methicillin-resistant Staphylococcus aureus infectionswhere do we go from here? Clinical Infectious Diseases 29, 8012.[ISI][Medline]
12
.
Nimmo, G. R., Schooneveldt, J., OKane, G., McCall, B. & Vickery, A. (2000). Community acquisition of gentamicin-sensitive methicillin-resistant Staphylococcus aureus in southeast Queensland, Australia. Journal of Clinical Microbiology 38, 392631.
13 . Gosbell, I. B., Mercer, J. L., Neville, S. A., Crone, S. A., Chant, K. G., Jalaludin Bin, B. et al. (2001). Non-multiresistant and multiresistant methicillin-resistant Staphylococcus aureus in community-acquired infections. Medical Journal of Australia 174, 62730.[ISI][Medline]
14 . Chambers, H. F. (2001). The changing epidemiology of Staphylococcus aureus? Emerging Infectious Diseases 7, 17882.[ISI][Medline]
15 . National Committee for Clinical Laboratory Standards. (2000). Performance Standards for Antimicrobial Disk Susceptibility TestsSeventh Edition: Approved Standard M2-A7. NCCLS, Villanova, PA, USA.
16 . de Lencastre, H., Couto, I., Santos, I., Melo-Cristino, J., Torres-Pereira, A. & Tomasz, A. (1994). Methicillin-resistant Staphylococcus aureus disease in a Portuguese hospital: characterization of clonal types by a combination of DNA typing methods. European Journal of Clinical Microbiology and Infectious Diseases 13, 6473.[ISI][Medline]
17 . Geha, D. J., Uhl, J. R., Gustaferro, C. A. & Persing, D. H. (1994). Multiplex PCR for identification of methicillin-resistant Staphylococcus aureus in the clinical laboratory. Journal of Clinical Microbiology 32, 176872.[Abstract]
18 . Sambrook, J., Fritsch, E. F. & Maniatis, T. (1998). Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.
19 . Goh, S. H., Byrne, S. K., Zhang, J. L. & Chow, A. W. (1992). Molecular typing of Staphylococcus aureus on the basis of coagulase gene polymorphisms. Journal of Clinical Microbiology 30, 16425.[Abstract]
20 . Matushek, M. G., Bonten, M. J. & Hayden, M. K. (1996). Rapid preparation of bacterial DNA for pulsed-field gel electrophoresis. Journal of Clinical Microbiology 34, 2598600.[Abstract]
21
.
Tenover, F. C., Arbeit, R. D., Goering, R. V., Mickelsen, P. A., Murray, B. E., Persing, D. H. et al. (1995). Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. Journal of Clinical Microbiology 33, 22339.
22 . Jones, E. M., Bowker, K. E., Cooke, R., Marshall, R. J., Reeves, D. S. & MacGowan, A. P. (1997). Salt tolerance of EMRSA-16 and its effect on the sensitivity of screening cultures. Journal of Hospital Infection 35, 5962.[ISI][Medline]
23 . Mary, P., Dupuy, N., Dolhem-Biremon, C., Defives, C. & Tailliez, R. (1994). Differences among Rhizobium meliloti and Bradyrhizobium japonicum strains in tolerance to desiccation and storage at different humidities. Soil Biology and Biochemistry 26, 112532.
24 . Lacey, R. W. & Lord, V. L. (1981). Sensitivity of staphylococci to fatty acids: novel inactivation of linolenic acid by serum. Journal of Medical Microbiology 14, 419.[Abstract]
25
.
Nilsson, I.-M., Hartford, O., Foster, T. & Tarkowski, A. (1999). Alpha-toxin and gamma-toxin jointly promote Staphylococcus aureus virulence in murine septic arthritis. Infection and Immunity 67, 10459.
26
.
Jarraud, S., Mougel, C., Thioulouse, J., Lina, G., Meugnier, H., Forey, F. et al. (2002). Relationships between Staphylococcus aureus genetic background, virulence factors, agr groups (alleles), and human disease. Infection and Immunity 70, 63141.
27 . Aathithan, S., Dybowski, R. & French, G. L. (2001). Highly epidemic strains of methicillin-resistant Staphylococcus aureus not distinguished by capsule formation, protein A content or adherence to HEp-2 cells. European Journal of Clinical Microbiology and Infectious Diseases 20, 2732.[ISI][Medline]
28 . Wielders, C. L. C., Vriens, M. R., Brisse, S., de Graaf-Miltenbury, L. A. M., Troelstra, A., Fleer, A. et al. (2001). Evidence of in-vivo transfer of mecA DNA between strains of Staphylococcus aureus. Lancet 357, 16745.[ISI][Medline]
29 . Duckworth, G. J. & Jordens, J. Z. (1990). Adherence and survival properties of an epidemic methicillin-resistant strain of Staphylococcus aureus compared with those of methicillin-sensitive strains. Journal of Medical Microbiology 32, 195200.[Abstract]
30
.
Harshman, S., Boquet, P., Duflot, E., Alouf, J. E., Montecucco, C. & Papini, E. (1989). Staphylococcal alpha-toxin: a study of membrane penetration and pore formation. Journal of Biological Chemistry 264, 1497884.
31
.
Dinges, M. M., Orwin, P. M. & Schlievert, P. M. (2000). Exotoxins of Staphylococcus aureus. Clinical Microbiology Reviews 13, 1634.