1. Molecular characterisation of
Panton-Valentine Leukocidin (PVL)-positive
Staphylococcus aureus from Wales
N. Bome-Mannathoko1, M. Wooton3, L. G. Harris1, R. Howe3, D. Mack1,2
1Medical Microbiology and Infectious Diseases, Institute of Life Science, School of Medicine, Swansea University NPHS Microbiology Swansea, Singleton Hospital, Abertawe-Bro Morgannwg University NHS Health Board, Swansea
3Specialist Antimicrobial Chemotherapy Unit, NPHS Microbiology Cardiff
Introduction
PVL-positive S.aureus typically cause primary skin and soft-tissue abscesses but have also been implicated in life threatening infections i.e. haemorrhagic pneumonia and necrotising fasciitis. Recent widespread dissemination of PVL-positive community-acquired MRSA clones, i.e. USA300 and USA400 in the USA, has heightened the question of the clinical relevance of PVL. Although the prevalence of PVL-positive S. aureus in the UK is low (<2%), the enhanced virulence and transmissibility of USA300 in the USA accentuates the need for continuous surveillance of PVL-positive strains in the UK.
Of 61 SACU isolates, 16 MRSA had the USA pulsotype, ACME and the MRSAIVa-t008 genotype. Most of the remaining MRSA belonged to MRSAIVc-t044 and MRSAIVc-t002 genotypes (Table 3).
Table 3. Molecular epidemiology of SACU S. aureus isolates
spa type
No.
SCCmec
ACME
PFGE
MRSA
t008 (t024, t1578, t1624, t2743)
16 (26.2%)
IVa
POS
USA
t008
1 (1.6%)
IVc
NEG
Unique
t044 (t4725)
5 (8.2%) B1
t002
3 (4.9%) C1
t127
2 (3.2%)
USA400 (MW2)
Miscellaneous*
8 (13.1%)
IVMISC
Miscellaneous
USA300 reference strains
FPR3857
n/a
SF8300
MSSA
t159 H1
t211 K1
t2271 G1
Miscellaneous**
18 (29.6%)
Total 61
Methods
560 consecutive S. aureus wound isolates from NPHS Microbiology Swansea, Singleton Hospital (ABMU) were screened for PVL by real-time PCR and 19 (3.4%) were positive. This non-selected PVL-positive ABMU cohort (n=19) and a PVL-positive cohort (n=61) from the Specialist Antimicrobial Chemotherapy Unit, NPHS Microbiology Cardiff (SACU) were evaluated. The 80 isolates were characterised by: detection of mecA and the arginine catabolic mobile element (ACME) by PCR; pulsed-field gel electrophoresis (PFGE); spa and SCCmec typing. Susceptibility testing was performed with BD Phoenix PMIC/ ID-67 panels.
Results – genotypic characterisation
Table 1. Prevalence of MRSA and MSSA per location
Location
MRSA
MSSA
Total
SACU, Cardiff
35 (57.4%)
26 (42.6%) 61
ABMU, Swansea
2 (10.5%)
17 (89.5%) 19
** t019, t105, t138, t160, t162, t177, t189, t314, t345, t1425, t1635, t1869, t2393, t3204, 2xnew(c), 2xnew(d)
*t005, t202, t223, t275, t311, 2xnew (a&b),1-untypeable
Results- antibiotic resistance
All 80 PVL-positive S. aureus were susceptible to: vancomycin, daptomycin, mupirocin, rifampin, teicoplanin and had varying resistance to other antibiotics (Table 4).
p =0.0004:Fisher’s 2-tailed exact test
17/19 of the ABMU isolates were MSSAs and only 2 were MRSAs. spa types t021 and t314 were exhibited by 3 strains, and the remaining isolates had various spa types (Table 2).
Table 4. Antibiotic resistance of PVL-positive S. aureus
All (n=80)
SACU (n=61)
ABMU (n=19)
Penicillin
98.8% (79)
100% (61)
94.7% (18)
Oxacillin
45.0% (36)
57.4% (35)
5.3% (1)*
Gentamicin
5.0% (4)
1.6% (1)
15.8% (3)*
Tobramycin
36.3% (29)
41.0% (25)
21.1% (4)
Erythromycin
28.8% (23)
31.1% (19)
Clindamycin
1.3% (1)
0.0% (0)
5.3% (1)
Tetracycline
17.5% (14)
9.8% (6)
42.1% (8)*
Trimethoprim
63.8% (51)
67.2% (41)
52.6% (10)
TMP-SMZ
14.8% (9)
26.3% (5)
Ciprofloxacin
4.9% (3)
Fusidic acid
8.8% (7)
Table 2. Molecular epidemiology of ABMU S. aureus isolates
spa type
No.
SCCmec
ACME
PFGE
MRSA
t019
1 (5.3%)
IVd
NEG
Unique
t437 V
MSSA
t021
3 (15.8%)
n/a M1
t314 L1
Miscellaneous*
11 (57.8%)
Miscellaneous
Total 19
*t002, t005, t084, t159, t645, t891, t903, t917, t4363, t4791, t3011
*Oxacillin p=0.0001; Gentamicin p=0.04; Tetracycline p=0.003: Fisher’s 2-tailed exact test
89.5% of the ABMU cohort comprised extensively diverse MSSA, 57.4% of the SACU isolates were MRSA and PFGE revealed that these isolates belonged to several clones (Figure 1).
Conclusion
3.4% of unselected S. aureus from ABMU were PVL-positive.
Distinct differences in MRSA prevalence and clonal composition of the PVL-positive cohorts from ABMU and the SACU referral unit.
USA300 was predominant and strains affiliated to other clones i.e. European clone (MRSAIVc-t044), USA800 (MRSAIVc-t002) were present in the SACU cohort but not in the ABMU cohort.
PVL genes are present in MSSA strains of diverse genotypes and not limited to specific MRSA clones.
Molecular epidemiology of S. aureus strains submitted to referral units is valuable but is not necessarily representative of strains of a geographical location.
Verification by evaluation of unselected isolates from the same area is vital.
USA B1 C1
USA400
Unique
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References
Ellington et al Clin Microbiol Infect . 28 :1113–1121
Ellington et al J Antimicrob Chem . 61:73- 77
Holmes et al J Clin Microbiol. 43:
Strommenger et al J Clin Microbiol. 44: 2533–2540
Deurenberg et al FEMS Microbiol Let. 240:
Acknowledgements
Thanks to Binh A. Diep for provision of the USA300 reference strains. Our gratitude to the staff at NPHS Microbiology Swansea, Singleton Hospital for their support and collaboration
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Figure 1. PFGE of 20 PVL-positive MRSA from SACU