Abstract

In New Zealand, it is estimated that greater than half of the methicillin-resistant Staphylococcus aureus (MRSA) strains recovered from patients belong to what has been termed Western Samoan phage pattern types 1 and 2 (WSPP1, WSPP2). These strains differ from classical MRSA isolates in terms of their lack of multiresistance and community occurrence, suggesting that such strains possess properties and/or characteristics different from those of other MRSA. To address this hypothesis, 10 WSPP1 and WSPP2 isolates from Western Samoa, New Zealand and Australia were compared with common hospital MRSA isolates. All WSPP isolates were identical with regard to pulsed-field gel electrophoretic pattern of SmaI-digested DNA, coagulase gene restriction fragment length polymorphism pattern and localization of mecA to a 194 kb SmaI digestion fragment. The WSPP strains were no more resistant/sensitive to various environmental stresses (e.g. skin fatty acids, UV light, desiccation) compared with hospital epidemic MRSA strains, except for their higher tolerance to salt. In terms of virulence, the WSPP MRSA were quantitatively better at attaching to the epithelial cell line HEp2, were uniformly egg-yolk opacity factor negative and produced higher levels of haemolytic toxins compared with non-WSPP MRSA isolates.

Received 30 May 2002; returned 15 July 2002; revised 10 September 2002; accepted 11 September 2002

Introduction

Methicillin-resistant Staphylococcus aureus (MRSA) have become an increasing problem worldwide. MRSA was first isolated in New Zealand in the mid-1970s, but remained uncommon until 1986, when a marked increase occurred in the number of isolates referred to the Communicable Disease Group, Institute of Environmental Science and Research (ESR) at Porirua. Based on data collected from hospital and community diagnostic laboratories, the prevalence of methicillin resistance in clinical S. aureus in New Zealand has steadily risen from 1.1% in 1994 to 6.8% in 2001. In 1995, around half of the MRSA received by ESR belonged to what was termed Western Samoan phage pattern types 1 and 2 (29/52/52A/80/55/54/77/84/95/96+, WSPP1; 29/81/54/77/84+, WSPP2). In 1997, a complete surveillance involving all hospital and community laboratories in New Zealand revealed that of 2085 MRSA isolated, 76% were of the WSPP type. These were regarded as community based with a predilection for Pacific Island races.15 By 2000, it was estimated that almost 4500 isolates of MRSA were recovered in New Zealand.2 At this time, WSPP strains comprised 61.7% of the isolates, with the next most common (15.4% isolates) strain being EMRSA-15. Most (73.4%) of the WSPP isolates were recovered from community patients, and 73.7% of the EMRSA-15 isolates were from hospital patients. It is worth noting that during the 1990s, New Zealand had an excellent reporting system with almost all diagnostic laboratories referring possible MRSA to ESR.

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.

Materials and methods

Strains and culture conditions

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 (0.5–15%) 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 0.025–0.4% for linoleic acid and 1–7% 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 × 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 α- and β-haemolysin toxins, and adherence to HEp2 cells

The quantitative measurement of α- and β-haemolysintoxin production was carried out in terms of haemolytic titre as described previously.25 To confirm the presence of the genes encoding α-haemolysin (hla) and β-haemolysin (hlb), PCR and Southern hybridization were carried out as described previously.17,18 The following primers were used: α-haemolysin HLA-1 5′-CTGATTACTATCCAAGAAATTCGATTG-3′ and HLA-2 5′-CTTTCCAGCCTACTTTTTTATCAGT-3′, and β-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 α- and β-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 × 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 × 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.

Results

Characterization of the WSPP isolates

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.

The oxacillin MIC for the 10 isolates studied ranged from 8 to 128 mg/L (Table 1). All isolates were susceptible to gentamicin, erythromycin, ciprofloxacin and co-trimoxazole, except ST94/1208 (resistant to these four antimicrobials) and MR99/692 (resistant to erythromycin). This latter isolate was also mupirocin resistant. All isolates were β-lactamase producers. Clindamycin susceptibility was not tested.

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).

The chromosomal location and vicinity of the mecA gene after SmaI digestion and PFGE of genomic DNA from the different isolates was investigated with a DNA probe that was internal to the mecA gene (Figure 1). In all WSPP strains, mecA was localized to a 194 kb SmaI band.

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 0.0001–0.002% 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 6–10% for other MRSA (Table 2).

At relative humidities of 3–84%, all isolates studied showed a similar pattern of cell survival with no viable cells detected after 35 days of incubation, and therefore WSPP strains did not appear better adapted to drying than other strains of MRSA (data not shown).

All isolates of WSPP studied were found to produce consistently high levels of α- and β-haemolysin toxins in the ranges 256–4096 and 128–512 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 α-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.

Discussion

The WSPP1 and WSPP2 MRSA were first isolated from Western Samoan patients in the Auckland region of New Zealand during 1992–1993. Whereas Pacific Island groups originally appeared most at risk from WSPP strains, these strains are now widespread throughout New Zealand and occur in all racial populations (although still strongly associated with Pacific Island people), accounting for >60% (around 2700 isolates from a total of 4500) of all MRSA isolated in New Zealand in 2000.2 They are consistently associated with overt skin lesions (abscess or cellulitis) in younger age groups. In the 2001 New Zealand census, 200 301 (5.4%) of the total population of 3 737 490 identified themselves as Polynesian.

Our sample of 10 WSPP isolates from New Zealand, Samoa and Australia displayed characteristics consistent with those found for all other isolates so far examined—identical macrorestriction patterns of SmaI-digested DNA, oxacillin MICs in the 8–128 mg/L range, with all susceptible to vancomycin and most susceptible to a variety of non-β-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 β-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. β-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 α- and β-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 α-Haemolysin toxin is a pore-forming haemolytic and membrane-damaging toxin,30 whereas β-haemolysin toxin is produced by a large number of S. aureus strains, especially those of animal origin.31 Our results indicate that α-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 β-haemolysin toxin, all were egg-yolk opacity factor negative, suggesting that the β-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.

Acknowledgements

The financial assistance of the Otago Medical Research Foundation and the Deans Fund, Otago School of Medical Sciences is gratefully acknowledged. R.P.A. was the recipient of a New Zealand Official Development Assistance (NZODA) Study Award.

*

Corresponding author. Tel: +64-3-479-7718; Fax: +64-3-479-8540; E-mail: sandy.smith@stonebow.otago.ac.nz

Figure 1. (a) PFGE patterns of representative MRSA isolates after SmaI digestion. Lane 1, low range PFG marker (New England Biolabs); lane 2, 98M7611; lane 3, 98M7675; lane 4, MA0116; lane 5, ST92/398; lane 6, M1126; lane 7, MR98/2554. (b) Hybridization of SmaI DNA digests in (a) with mecA gene. The size of the fragments is shown in kb.

Figure 1. (a) PFGE patterns of representative MRSA isolates after SmaI digestion. Lane 1, low range PFG marker (New England Biolabs); lane 2, 98M7611; lane 3, 98M7675; lane 4, MA0116; lane 5, ST92/398; lane 6, M1126; lane 7, MR98/2554. (b) Hybridization of SmaI DNA digests in (a) with mecA gene. The size of the fragments is shown in kb.

Table 1.

 Bacterial strains used in the study and some of their properties

Isolate number ID32 numbera Oxacillin MIC (mg/L) β-Lactamase Descriptionb PFGE pattern Coagulase gene RFLP type mecA position (kb) Isolated 
MA0126 367317610   8 WSPP1 194 Western Samoa 
MA0116 367316610 12 WSPP1 Ac 194 Western Samoa 
98M7611 367336610 12 WSPP1 194 Australia 
98M7675 367337610 12 WSPP2 194 Australia 
98M22661 367336610 16 WSPP1 194 Australia 
MA0111 367332730 16 WSPP1 194 Western Samoa 
MR99/692 367337710 16 WSPP1 194 New Zealand 
ST92/398 367332711 32 WSPP1 194 New Zealand 
ST94/24 367336611 64 WSPP1 194 New Zealand 
ST94/1208 367337610  128 WSPP1 194 New Zealand 
MR95/658   32d EMRSA-15 NDe New Zealandf 
MR98/1675  ≥256 EMRSA-16 ND New Zealandf 
M1126  ≥256 MRSA  97 New Zealand 
MR99/854  ≥256 WNWH1  97 New Zealand 
MR98/2554  ≥256 AKAH2 NPg 194 New Zealand 
Isolate number ID32 numbera Oxacillin MIC (mg/L) β-Lactamase Descriptionb PFGE pattern Coagulase gene RFLP type mecA position (kb) Isolated 
MA0126 367317610   8 WSPP1 194 Western Samoa 
MA0116 367316610 12 WSPP1 Ac 194 Western Samoa 
98M7611 367336610 12 WSPP1 194 Australia 
98M7675 367337610 12 WSPP2 194 Australia 
98M22661 367336610 16 WSPP1 194 Australia 
MA0111 367332730 16 WSPP1 194 Western Samoa 
MR99/692 367337710 16 WSPP1 194 New Zealand 
ST92/398 367332711 32 WSPP1 194 New Zealand 
ST94/24 367336611 64 WSPP1 194 New Zealand 
ST94/1208 367337610  128 WSPP1 194 New Zealand 
MR95/658   32d EMRSA-15 NDe New Zealandf 
MR98/1675  ≥256 EMRSA-16 ND New Zealandf 
M1126  ≥256 MRSA  97 New Zealand 
MR99/854  ≥256 WNWH1  97 New Zealand 
MR98/2554  ≥256 AKAH2 NPg 194 New Zealand 

aID32 STAPH generated identification number.

bBased largely on phage pattern.

cOne band different.

dConsidered unusually low for EMRSA-15.

eND, not determined.

fFrom a health care worker who had worked in the UK.

gNP, no PCR product.

Table 2.

 Toxin production, salt tolerance and adherence studies

 Haemolytic titrea     
Strain no. α-toxin β-toxin  Egg-yolk factor MIC NaCl (%) Adherenceb % ± s.d
MA0126 1024 512  – 14.0 8.88 ± 1.42 
MA0116  512 256  – 14.0 6.87 ± 0.56 
98M7611  256 256  – 13.0 13.76 ± 1.69 
98M7675 2048 256  – 14.0 11.49 ± 2.10 
98M22661 4096 256  – 14.0 8.45 ± 1.25 
MA0111  512 128  – 14.0 6.13 ± 0.76 
MR99/692 2048 128  – 14.0 4.62 ± 0.80 
ST92/398 1024 128  – 14.0 5.83 ± 0.80 
ST94/24 4096 256  – 14.0 4.76 ± 0.95 
ST94/1208 2048 128  – 14.0 7.32 ± 1.59 
MR95/658 2048 256   9.5 4.73 ± 0.40 
MR98/1675   0  4   6.0 4.59 ± 0.45 
M1126 2048 128   7.0 1.36 ± 0.57 
MR99/854 1024 128   9.5 2.45 ± 0.37 
MR98/2554   0  4  10.0 5.86 ± 0.87 
NCTC 10345 NTc NT  NT NT 1.32 ± 0.31 
 Haemolytic titrea     
Strain no. α-toxin β-toxin  Egg-yolk factor MIC NaCl (%) Adherenceb % ± s.d
MA0126 1024 512  – 14.0 8.88 ± 1.42 
MA0116  512 256  – 14.0 6.87 ± 0.56 
98M7611  256 256  – 13.0 13.76 ± 1.69 
98M7675 2048 256  – 14.0 11.49 ± 2.10 
98M22661 4096 256  – 14.0 8.45 ± 1.25 
MA0111  512 128  – 14.0 6.13 ± 0.76 
MR99/692 2048 128  – 14.0 4.62 ± 0.80 
ST92/398 1024 128  – 14.0 5.83 ± 0.80 
ST94/24 4096 256  – 14.0 4.76 ± 0.95 
ST94/1208 2048 128  – 14.0 7.32 ± 1.59 
MR95/658 2048 256   9.5 4.73 ± 0.40 
MR98/1675   0  4   6.0 4.59 ± 0.45 
M1126 2048 128   7.0 1.36 ± 0.57 
MR99/854 1024 128   9.5 2.45 ± 0.37 
MR98/2554   0  4  10.0 5.86 ± 0.87 
NCTC 10345 NTc NT  NT NT 1.32 ± 0.31 

aHighest titre giving erythrocyte lysis.

bMean of four experiments each done in triplicate.

cNT, not tested.

References

1.
Smith, J. M. B. (
1998
). Emerging problems of antibiotic resistant bacteria.
New Zealand Medical Journal
 
111
,
441
–4.
2.
Lang, S., Taylor, S. & Morris, A. (
2001
). Community-acquired methicillin-resistant Staphylococcus aureus.
Antibiotics and Chemotherapy
 
5
,
12
–3.
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,
S74
–9.
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
,
593
–8.
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
,
273
–81.
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
,
797
–800.
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
,
935
–6.
11.
Kak, V. & Levine, D. P. (
1999
). Editorial response: community-acquired methicillin-resistant Staphylococcus aureus infections—where do we go from here?
Clinical Infectious Diseases
 
29
,
801
–2.
12.
Nimmo, G. R., Schooneveldt, J., O’Kane, G., McCall, B. & Vickery, A. (
2000
). Community acquisition of gentamicin-sensitive methicillin-resistant Staphylococcus aureus in southeast Queensland, Australia.
Journal of Clinical Microbiology
 
38
,
3926
–31.
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
,
627
–30.
14.
Chambers, H. F. (
2001
). The changing epidemiology of Staphylococcus aureus?
Emerging Infectious Diseases
 
7
,
178
–82.
15.
National Committee for Clinical Laboratory Standards. (
2000
). Performance Standards for Antimicrobial Disk Susceptibility Tests—Seventh 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
,
64
–73.
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
,
1768
–72.
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
,
1642
–5.
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
,
2598
–600.
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
,
2233
–9.
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
,
59
–62.
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
,
1125
–32.
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
,
41
–9.
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
,
1045
–9.
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
,
631
–41.
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
,
27
–32.
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
,
1674
–5.
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
,
195
–200.
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
,
14978
–84.
31.
Dinges, M. M., Orwin, P. M. & Schlievert, P. M. (
2000
). Exotoxins of Staphylococcus aureus.
Clinical Microbiology Reviews
 
13
,
16
–34.

Author notes

Departments of 1Microbiology and 2Biochemistry, Otago School of Medical Sciences, University of Otago, Dunedin; 3Middlemore Hospital and Diagnostic Medlab, Auckland; 4Institute of Environmental Science and Research, Porirua, New Zealand