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General enquiries on this form should be made to:Defra, Science Directorate, Management Support and Finance Team,Telephone No. 020 7238 1612E-mail: [email protected]
SID 5 Research Project Final Report
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NoteIn line with the Freedom of Information Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. The SID 5 (Research Project Final Report) is designed to capture the information on the results and outputs of Defra-funded research in a format that is easily publishable through the Defra website. A SID 5 must be completed for all projects.
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Project identification
1. Defra Project code OD2020
2. Project title
Staphylococcus aureus in Cattle - an Investigation into Selected Properties of Isolates Recovered from Clinical Veterinary Diagnostic Samples.
3. Contractororganisation(s)
Veterinary Laboratories Agency,Woodham Lane,New Haw,Weybridge,Surrey,KT15 3NB.
54. Total Defra project costs £ 186,558(agreed fixed price)
5. Project: start date................ 02 January 2006
end date................. 31 March 2008
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6. It is Defra’s intention to publish this form. Please confirm your agreement to do so...................................................................................YES NO (a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They
should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow.Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer.In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.
(b) If you have answered NO, please explain why the Final report should not be released into public domain
Executive Summary7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the
intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.
The main objective of this research project was to determine whether or not methicillin-resistant Staphylococcus aureus (MRSA) was present in dairy cattle in England and Wales and to study some aspects of the population biology of S. aureus isolates recovered from cattle.
The population of organisms studied comprised S. aureus recovered from bovine clinical mastitis samples submitted to fourteen VLA regional laboratories strategically located throughout England and Wales.
989 isolates presumptively identified as S. aureus on phenotypic criteria were collected from VLA regional laboratories over the 18-month period April 2006 to September 2007. 940 isolates were confirmed as S. aureus by genetic methods.
S. aureus isolates were examined by PCR for the presence of the mecA gene; this gene was not detected in any bovine S. aureus isolates. The prevalence of MRSA in S. aureus from clinical bovine mastitis samples was therefore 0/940 (0%, 95% confidence interval 0 – 0.32%).
The S. aureus isolates originated from 465 different cattle herds; 4 isolates were submitted from unnamed herds of origin. The prevalence of infected cattle herds, (assuming that an infected herd would submit a sample from an infected cow and that MRSA would be recovered from that sample), was therefore approximately 0/465 (0%).
Breed data was available for 747 isolates confirmed genotypically as S. aureus; 669 (90%) of these were from Holstein, Friesian or Holstein-Friesian cattle. Only 8 isolates (1%) originated from pure beef breeds of cattle. This was an expected finding, as most mastitis samples submitted to VLA originate from dairy cows.
The isolates were submitted from 202 different veterinary practices (branch practices
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were counted as a separate practice).
Resistance to penicillin was relatively high at 43% and the high prevalence of resistance probably relates to the widespread usage of penicillin for the treatment of mastitis. Most isolates remained relatively susceptible to the other antimicrobials tested and 55% of bovine S. aureus isolates were fully-susceptible to all antimicrobials tested. Resistance figures to selected other antimicrobials were as follows: amoxicillin/ clavulanate 1%; ciprofloxacin 6%; erythromycin 2%: tetracycline 2%; gentamicin 0.3%; trimethoprim/ sulphonamides 0%.
Glycopeptide intermediate S. aureus and heterogeneous glycopepetide intermediate S. aureus (GISA and hGISA) were not detected (i.e. isolates with resistance to the important antimicrobial vancomycin, which is used in humans, were not present).
205 strains were selected for molecular characterisation by PFGE using the restriction endonuclease Sma1 on the basis of antibiogram and geographical data (i.e. isolates were selected from different geographical regions from within the study area). Of these, 100 isolates with distinct PFGE profile and/or antibiogram were examined by multiplex PCR for the presence of 14 genes encoding staphylococcal toxins (namely, enterotoxin A-E and G-J, exfoliative toxins A, B and D, toxic shock syndrome toxin-1 and Panton Valentine Leukocidin toxin).
PFGE analysis demonstrated 63.3% identity amongst the 205 strains subjected to PFGE. 50 different PFGE profile types were identified and there were 5 main cluster groups (designated types 14, 15, 30 31 and 40). Each of the main cluster groups contained ≥7 subtypes.
Toxin characterisation of 100 selected S. aureus isolates revealed that 51.5% of isolates possessed at least one toxin. Staphylococcal enterotoxins and TSST-1 occurred in the strains examined as follows; sea (1%), seb (1%), 13% (sec), 1% (sed), 18% (seg), 1% (seh), 18% (sei), 1% (sej), 12% (tst) and 1% (etd). All isolates were negative for PVL and for the exfoliative genes eta and etb. Seg/sei are usually co-selected and carried on the same pathogenicity island in S. aureus; it was therefore perhaps to be expected that they occurred at the same frequency in the study population. Other veterinary studies have detected similar enterotoxins in some S. aureus isolates from animals elsewhere in Europe. The Panton-Valentine leukocidin has been particularly associated with some strains of community-acquired MRSA and was not detected in bovine S. aureus isolates.
There was no obvious correlation between PFGE type, geographical location, toxin carriage and antibiogram. There were no isolates of S. aureus which were untypable using PFGE. This is an important finding as the strain of MRSA that has been detected in pigs and cattle in continental Europe (MLST ST398) is untypable by PFGE.
Further analysis of the results is necessary before definitive conclusions can be made regarding the possible relatedness of any human and bovine methicillin-susceptible S. aureus isolates. Whilst the current data for human methicillin-susceptible (MSSA) isolates is limited, any future comparisons with UK and international isolates would be assisted by the use of spa-typing.
Cattle in England and Wales, based on the findings in this extensive survey of S. aureus isolates from diagnostic samples, do not currently appear to be a reservoir for MRSA.
In view of that finding, the current public health safeguards regarding milk produced from dairy cattle are considered to be adequate.
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Bovine S. aureus strains do not currently appear to be a reservoir for the Panton-Valentin Leukocidin. Certain enterotoxins can be detected in S. aureus from cattle and there are some potential food safety implications relating to that finding. However, mastitic milk is excluded from human consumption and refrigeration and pasteurisation should limit any possible potential to cause human disease.
Project Report to Defra8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with
details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include: the scientific objectives as set out in the contract; the extent to which the objectives set out in the contract have been met; details of methods used and the results obtained, including statistical analysis (if appropriate); a discussion of the results and their reliability; the main implications of the findings; possible future work; and any action resulting from the research (e.g. IP, Knowledge Transfer).
Final Report to Defra for Project OD2020.
MRSA in Cattle – an investigation into selected properties of isolates recovered from clinical veterinary diagnostic samples.
Scientific Objectives.
1) Determination of the prevalence of MRSA in bovine mastitis samples.2) Develop and implement standardised procedures for detection and identification of MRSA in food-producing animals that are fully-harmonised and directly comparable to current medical procedures.3) Develop a typing scheme for bovine S. aureus isolates based mainly on PFGE that will enable comparison of bovine and human isolates and thus help to provide information regarding the likely origin of such strains.4) Genotype any MRSA isolates recovered from animals and provide information on their relatedness to human isolates.5) Provide a framework within which ongoing monitoring for MRSA in food-producing animals could be maintained in Great Britain.6) Provide veterinary facilities capable of typing MRSA isolates according to current medical procedures, which can be deployed at short notice, should the need arise.
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Details of the Methods Used, Results Obtained and the Extent to which Objectives have been met. Objective 1) Determination of the prevalence of MRSA in bovine mastitis samples.
The main objective of this research project was to determine whether or not methicillin-resistant Staphylococcus aureus (MRSA) was present in dairy cattle in England and Wales and to study some aspects of the population biology of S. aureus isolates recovered primarily from the udder of cattle. The primary aim was to investigate the MRSA status of dairy cattle in England and Wales.
The population of organisms studied comprised S. aureus recovered from bovine clinical mastitis samples submitted to fourteen VLA regional laboratories strategically located throughout England and Wales.
989 isolates presumptively identified as S. aureus on phenotypic criteria were collected from VLA regional laboratories over the 18-month period April 2006 to September 2007. Criteria used for presumptive identification of S.aureus isolates included Gram-stain reactions, catalase, oxidase and coagulase tests as appropriate. 940 (95%) of these isolates were subsequently confirmed as S. aureus using a PCR to confirm the presence of a species specific sequence in the thermonuclease (nuc) gene (Brakstad et al. 1992). Staphylococcal thermonuclease is an extracellular phosphodiesterase produced in large quantities by S. aureus (Hazen and Cotton, 1978). There are regions within the gene, which are species specific (Shortle, 1983) and these have been successfully and frequently used to detect and identify S. aureus by PCR amplification (Brakstad et al, 1992)
S. aureus isolates were examined by PCR for the presence of the mecA gene (Bignardi et al. 1996); this gene was not detected in any bovine S. aureus isolates. The prevalence of MRSA in S. aureus from clinical bovine mastitis samples was therefore 0/940 (0%, 95% confidence interval 0 – 0.32%).
The S. aureus isolates originated from 465 different cattle herds; 4 isolates were submitted from unnamed herds of origin. The prevalence of infected cattle herds, (assuming that an infected herd would submit a sample from an infected cow and that MRSA would be recovered from that sample), was therefore approximately 0/465 (0%).
The total number of cattle eligible for being sampled for mastitis in herds submitting samples to VLA Regional Laboratories and from which S. aureus was isolated could not be estimated accurately because herd size details were missing from a significant number of submissions. This information is routinely requested from the submitting veterinarian, but is not always supplied.
Breed data was available for 747 isolates confirmed genotypically as S. aureus; 669 (90%) of these were from Holstein, Friesian or Holstein-Friesian cattle (figure 1). Only 8 isolates (1%) originated from pure beef breeds of cattle. This was an expected finding, as most mastitis samples submitted to VLA originate from dairy cows.
The isolates were submitted from 202 different veterinary practices (branch practices were counted as a separate practice).
Figure 1: Cattle Breeds represented in the Study.
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Data relating to the county of origin of the cattle from which the S. aureus isolates were recovered, was available for 929 isolates. These isolates were recovered from 45 different counties in England and Wales; an additional 5 isolates originated from the Channel Islands of Jersey or Guernsey and one isolate originated from Dumfrieshire. Numbers of isolates examined according to county of origin are shown at figure 2.
Figure 2: S. aureus Isolates Included in the Study by County of Origin.
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Epidemiological Considerations.
The study aimed to examine approximately 1000 bovine S. aureus isolates from clinical veterinary diagnostic samples. This figure was chosen to ensure that if the true prevalence is 1%, it would have been determined within 1% confidence intervals. The study examined 989 isolates presumptively identified as S. aureus and of these, 940 were genotypically confirmed as S. aureus. 95% confidence intervals have already been given above.
[If the prevalence of MRSA in dairy cattle was 1%, then 459 isolates would have to be examined to give a 99% probability of detecting a single MRSA isolate. If MRSA was present
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in dairy cattle at a prevalence of 1%, then the study would have given more than a 99% probability of detecting it, because the sample size greatly exceeded 459 isolates].
The study was designed with the aim of examining a minimum of 459 cattle herds. This sample size provided a 99% probability of detecting an MRSA - infected herd if the herd-level prevalence of infection was 1%. This probability was dependent on the assumption that an infected herd, would submit at least one infected sample. This objective was met as isolates from at least 465 herds were examined (the total range was from 465 to 469 herds, as 4 samples came from unnamed herds).
Antimicrobial Susceptibility Testing Results.
All isolates were examined for their antimicrobial susceptibility using the disc diffusion susceptibility testing recommendations and interpretation guidelines of the British Society for Antimicrobial Chemotherapy (BSAC 2007).
Antimicrobial (Disc concentration)
Antimicrobial Abbreviation
Number Resistant/ Total Tested
Percentage Resistant.
95% Confidence Interval
Penicillin (1i.u.) P 404/940 43 39.8-46.2%
Amoxicillin/ clavulanate (2/1ug)
AMC 12/940 1 0.7-2.2%
Ciprofloxacin (1ug) Cip 55/940 6 4.4-7.5%
Erythromycin (5ug) E 18/940 2 1.1-3.0%
Tetracycline (10ug) T 18/940 2 1.1-3.0%
Gentamicin (10ug) CN 3/940 0.3 0.07-0.93%
Trimethoprim/ sulphonamide (25ug)
SXT 0/940 0 0 - 0.32%
Table 1: Susceptibility of Bovine S. aureus Isolates to Selected Antimicrobials.
All isolates were also examined for beta-lactamase production using commercial nitrocefin test strips or discs; there were 534 beta-lactamase negative isolates; of these 533 were penicillin susceptible and one was penicillin resistant. There were 406 beta-lactamase positive isolates and of these 403 were penicillin resistant; therefore 3 isolates that were beta-lactamase positive were susceptible to penicillin. Table 2 shows the resistance to beta-lactams and the nitrocefin disc reactions of the S. aureus isolates which were resistant to either cefoxitin or oxacillin.
All 12 isolates resistant to amoxicillin / clavulanate were also resistant to penicillin as expected.
Of the eighteen isolates resistant to erythromycin, 16 were also resistant to penicillin and three were resistant to amoxicillin / clavulanate. None of the erythromycin-resistant isolates were resistant to tetracyclines or gentamicin.
Of the eighteen isolates resistant to tetracyclines, 17 were also resistant to penicillin and two were resistant to amoxicillin / clavulanate. Two isolates resistant to tetracyclines were also resistant to ciprofloxacin.
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Resistance Patterns of S. aureus isolates recovered during the Project.
T (n=1)E (n=2)P, T, Cip (n=2)P,T,AMC (n=2)P,T (n=13)P, E, AMC (n=3)P, E (n=13)P, CN (n=2)CN (n=1)P (n=322)P, AMC (n=5)P, Cip, AMC (n=2)P, Cip (n=40)Cip (n=11)Fully-susceptible (n=521)
Results of Disc Diffusion Testing Using Cefoxitin 10μg Discs.
All S. aureus isolates were examined for susceptibility to cefoxitin 10μg discs on Iso-Sensitest agar as described by BSAC (BSAC 2007). 10/940 isolates were resistant to cefoxitin with zones of inhibition less than 21mm in diameter. These isolates were all resistant to penicillin and all produced beta-lactamase in the nitrocefin test; three were also resistant to amoxicillin/ clavulanate. The oxacillin MICs for these isolates ranged from 1 to 32. All of the isolates were negative for the mecA gene by PCR. The resistance of cefoxitin-resistant isolates to other beta-lactams is given in table 2. The ten isolates resistant to cefoxitin were all susceptible to tetracyclines, trimethoprim/ sulphonamides and gentamicin. 2/10 were resistant to erythromycin and 5/10 were resistant to ciprofloxacin.
Oxacillin and Vancomycin MIC Determinations.
In addition to determination of the minimum inhibitory concentration (MIC) of oxacillin, the MIC of vancomycin was also determined for all S. aureus isolates in view of the recent emergence of vancomycin resistant S. aureus in human clinical isolates.
Figure 3: Oxacillin MIC Distribution of 940 Bovine S. aureus Isolates.
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The oxacillin MIC was determined according to the agar dilution method recommended by BSAC (Brown et al. 2005). The BSAC breakpoint for resistance to oxacillin is > 2 μg/ml and 24/940 (2.6%) isolates had an oxacillin MIC exceeding this breakpoint. No isolates had an MIC > 32 μg/ml; a control mecA positive EMRSA-15 isolate had an oxacillin MIC of >128 μg/ml. S. aureus isolates which have borderline resistance to oxacillin and which are not mecA positive may hyper-produce β-lactamase or possess altered PBPs (Barg et al, 1991).
S.aureus isolates resistant to oxacillin were all susceptible to tetracyclines, trimethoprim/ sulphonamides and gentamicin; 2/24 were resistant to erythromycin and 10/24 were resistant to ciprofloxacin.
Table 2: Resistance to other Beta-Lactams in Isolates Resistant to either Oxacillin or Cefoxitin, according to BSAC Interpretive Criteria.
Beta-lactamase production
Resistant to penicillin
Resistant to amoxicillin/ clavulanate
Resistant to cefoxitin
Resistant to oxacillin
Isolates resistant to oxacillin (MIC > 2 μg/ml) n=24
21/24* 21/24* 1/24 7/24 -
Isolates resistant to cefoxitin (zone diameter < 21mm) n=10
10/10 10/10 3/10 - 7/10
Control EMRSA-15 isolate n=1
1/1 1/1 1/1 1/1 1/1 (MIC >128 μg/ml)
*3/24 isolates were sensitive to penicillin and did not produce beta-lactamase in the nitrocefin test.
Figure 4: Vancomycin MIC Distribution of 940 Bovine S. aureus Isolates.
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27 bovine S. aureus isolates had an MIC of 4 and 1 isolate an MIC of 16 against vancomycin in an agar dilution MIC on brain heart infusion (BHI) agar, performed according to the method recommended by Brown et al. 2005. Brown et al. recommended a breakpoint for resistance to vancomycin in MRSA of > 4 μg/ml, though noted that BHI supplemented with 4 μg/ml vancomycin often gave high false-positive rates of resistance. The 28 isolates from this study with a vancomycin MIC of > 4 μg/ml on BHI were subsequently examined by etest against vancomycin and teicoplanin according to the standard method (Mueller-Hinton agar, an inoculum with a density of 0.5 McFarland standard and 24 hours incubation) and according to the “macro etest” method (BHI, inoculum with a density of a 2 McFarland standard and 48 hours incubation) (Brown et al. 2005). The recommended interpretative criteria for the ‘macro etest’ were > 8 μg/ml for both vancomycin and teicoplanin or >12 μg/ml for teicoplanin alone. In this study, all 28 isolates examined with a vancomycin MIC of > 4 μg/ml on BHI in the agar dilution test, had a vancomycin MIC of < 3 μg/ml in the standard etest and a vancomycin “MIC reading” of < 4 μg/ml in the macro etest. All 28 isolates had a teicoplanin “MIC reading” of < 8 μg/ml in the macro etest. Glycopeptide intermediate S. aureus and heterogeneous glycopepetide intermediate S. aureus (GISA and hGISA) were therefore not detected.
Of the 28 isolates in this study with a vancomycin MIC > 4 μg/ml, one was resistant to tetracyclines and two (different isolates) were resistant to ciprofloxacin. 18 of these isolates were resistant to penicillin, though none were resistant to amoxicillin/ clavulanate.
Objective 1 was met and no MRSA isolates were detected.
Objective 2) Develop and implement standardised procedures for detection and identification of MRSA in food-producing animals that are fully-harmonised and directly comparable to current medical procedures.
It is highly desirable that procedures in the medical and veterinary fields are standardised and harmonised so that results of this and future studies are directly comparable. This study incorporated the current medical approaches to the analysis of S. aureus to ensure complete harmonisation between the main medical and veterinary bacteriological reference laboratories in England and Wales.
Prior to DNA extraction isolates were sub-cultured onto 5% Sheep Blood agar and incubated at 37oC for 18-24 hrs as a purity check. For each isolate, DNA was extracted from the growth plates using reagent Prepman Ultra (Applied Biosytems). The remaining culture was harvested using commercially available glycerol beads and then stored at -80oC to ensure viability of strains for a minimum of 5 years.
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Literature reviews and collaboration within the project identified that simultaneous detection of the mecA gene together with one of several S. aureus species-specific genes (coa, femA/B, 16S rDNA, clfA, nuc) is possible by PCR (Unal et al, 1992. Barski et al, 1996 and Stepan et al, 2004). A number of genes have been used to identify S. aureus or MRSA, for example:
coa – an extracellular protein produced by several strains of S. aureus which forms a complex, named staphylothrombinfemA/B - cytoplasmic proteins which can influence the level of expression of resistance to methicillin.16S rRNA - ribosomal RNA, contains regions which are species specificclfA - clumping factor fibrinogen, surface-associated binding protein of S. aureus nuc – extracellular thermostable nuclease (TNase) protein
Following recommendations from collaborators in this project at the HPA and colleagues at the Scottish MRSA Reference Laboratories (SMRSARL), a multiplex PCR method based on the detection of the S. aureus thermonuclease (nuc) gene in combination with methicillin resistance gene (mecA) was selected to identify MRSA isolates. The PCR procedure used in this study was based on that used at the SMRSARL and is given at appendix 1. The method describes a multiplex PCR for the simultaneous detection of the mecA gene and the nuc gene of S. aureus. To resolve reproducibility problems the PCR cycle was optimised. Improvements included alteration of the denaturation time (5mins to 3mins), annealing time (30secs to 60 secs) and extension time (5mins to 10mins). Control strains EMRSA -15 and -16 were used to check the performance of the multiplex PCR.
Objective 2 was fully met.
Objective 3) Develop a typing scheme for bovine S. aureus isolates based mainly on PFGE that will enable comparison of bovine and human isolates and thus help to provide information regarding the likely origin of such strains.
This objective was to develop laboratory procedures suitable for use in discriminating between S. aureus strains of bovine or human origin. These procedures would have been used to attempt to determine the likely origin of any MRSA isolates that are recovered, though no MRSA isolates were recovered from cattle. Pulsed field gel electrophoresis (PFGE) has formerly been established as a gold standard for strain discrimination and investigation of the molecular epidemiology of methicillin and other resistant S. aureus. Pulsed-field gel electrophoresis separates large genomic DNA fragments after digestion with a restriction endonuclease. DNA fragments migrate and separate through agarose gels as a result of alternating the direction of the electrical field during electrophoresis. Representatives of all PFGE subtypes were examined according to the methods used by the HPA to determine their toxin gene complement and the use of different PFGE patterns ensured that a genetically diverse range of strains were examined for toxins. PFGE was utilised as a tool for the identification of the main cluster types of S. aureus. These clusters were then targeted for further investigation using techniques such as toxins possessed, spa typing and MLST. PFGE and toxin results, together with the antimicrobial resistance phenotype and county of origin are given at appendix 2.
PFGE blocks were prepared according to a standard protocol supplied by the HPA. DNA fragments digested with SmaI were separated on a CHEF-DR II apparatus (Bio-Rad, UK) using the following parameters: ramp of 1s-80s for 30h at field strength of 6 V/cm, with buffer chilled at 14oC. Images were photographed and imported into BioNumerics (Applied Maths) software for analysis.
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Data analysis was conducted using BioNumerics software. Clustering was performed by the unweighted pair grouping method with arithmetic average (UPGMA), with a 2% tolerance window using the Dice similarity coefficient.
The dendogram produced using BioNumerics was inspected visually to assign profile types. Isolates that differed by one to four bands were assigned a numbered subtype. The PFGE patterns were interpreted according to Tenover et al. (1995) where three or more band differences between two strains defined different genotypes. The dendrogram is shown at appendix 2.
Relatively few methicillin-susceptible S. aureus isolates are routinely examined by PFGE in human medicine and this limited the comparisons which could be made between human and veterinary isolates. During the course of the project, it became evident that in many studies spa-typing is often replacing PFGE for the characterisation of S. aureus isolates.
205 strains were selected for PFGE characterisation using the restriction endonuclease Sma1 on the basis of antibiogram and geographical data (i.e. isolates were selected from different geographical regions from within the study area). Of these, 100 isolates with distinct PFGE profile and/or antibiogram were examined by multiplex PCR for the presence of 14 genes encoding staphylococcal toxins (namely, enterotoxin A-E and G-J, exfoliative toxins A, B and D, toxic shock syndrome toxin-1 and Panton Valentine Leukocidin toxin) at the Staphylococcus Reference Laboratory, HPA Colindale (Monday and Bohach 1999, Becker et al. 1998, Yamaguchi et al. 2002, Lina et al. 1999, Oliveira and de Lencastre 2002). These 100 isolates represented 50 PFGE types (83 types including the sub-types) and 18 different antibiogram profiles and results are shown in appendix 2.
PFGE analysis demonstrated 63.3% identity amongst the 205 strains. 50 different PFGE profile types were identified and there were 5 main cluster groups (designated types 14, 15, 30 31 and 40). Each of the main cluster groups contained ≥7 subtypes. Appendix 2 lists the designated profile types, including the sub-types. The results (based on the sub-set of isolates examined) suggest that particular strains of S. aureus are prevalent in cattle and that these strains are widely dispersed geographically within England and Wales.
The diversity between the 100 strains selected for toxin characterisation, using a 2% tolerance was at 63.7%.
Toxin characterisation of 100 selected S. aureus isolates revealed that 51.5% of isolates possessed at least one toxin. Staphylococcal enterotoxins and TSST-1 were detected as follows (figure 5); sea (1%), seb (1%), 13% (sec), 1% (sed), 18% (seg), 1% (seh), 18% (sei), 1% (sej), 12% (tst) and 1% (etd). All isolates were negative for PVL and exfoliative genes (eta and etb). Seg/sei are usually co-selected and carried on the same pathogenicity island in S. aureus; it was therefore perhaps to be expected that they occurred at the same frequency in the study population. The Panton-Valentine leukocidin has been particularly associated with some strains of community-acquired MRSA and was not detected in bovine S. aureus isolates.
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Figure 5: Toxins Identified in Bovine S. aureus Isolates.
Information relating to geographical location, antimicrobial susceptibility and toxin carriage has also been included in the dendogram, to assist in identifying any geographical or antimicrobial phenotypic similarities between isolates with similar PFGE profiles.
There was no obvious correlation between PFGE type, geographical location, toxin carriage and antibiogram. There were no isolates of S. aureus which were untypable using PFGE. This is an important finding as the strain of MRSA that has been detected in pigs and cattle in continental Europe (MLST ST398) is untypable by PFGE.
A robust typing scheme, discriminating between human and bovine strains would be very useful for tracing the origin of any opportunistic infection of domestic animals with human MRSA strain(s). When the project proposal was written, PFGE was established as the gold standard technique for the investigation of the molecular epidemiology of methicillin and other resistant S. aureus. However spa-typing is emerging as a more convenient method of comparing such isolates, particularly if spa-typing is routinely applied to typing methicillin-susceptible S. aureus.
The molecular epidemiology of MSSA both in the UK and internationally, is less well characterised than is the case with MRSA. Notwithstanding that, the panel of bovine isolates for which PFGE and toxin-typing data were available were compared to data on human methicillin-susceptible S. aureus isolates available at the HPA. Isolates representing the main PFGE types (types 14, 15, 30, 31 and 40) which had been screened for toxins were selected for comparison to human types. Toxin profiling was also used as an additional marker to maximise discriminatory power for comparison to human isolates. Whilst the current data for human methicillin-susceptible (MSSA) isolates is limited any future comparisons with UK and international isolates would be assisted by the use of spa-typing.
The objective to examine 100 bovine isolates of S. aureus by PFGE was exceeded and in fact 205 isolates were examined by PFGE and from these, 100 isolates were examined for possession of toxins. All 205 isolates examined by PFGE were typable, indicating that S. aureus MLST ST398 was not present, as this strain is non-typable using standard PFGE protocols. PFGE data was only available for relatively low numbers of human methicillin-susceptible S. aureus isolates and further analysis of the results is necessary before definitive conclusions can be made regarding the possible relatedness of any human and bovine methicillin-susceptible S. aureus isolates.
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seb1% sec
13%
sed1%
seg18%
seh1%
sei18%
sej1%tst
12%
etd1%
see, eta, etb, pvl0%
not detected33%
sea1%
Toxin characterisation revealed 51.5% of isolates tested possess at least one toxin.
4) Genotype any MRSA isolates recovered from animals and provide information on their relatedness to human isolates.
Genetic analyses to determine staphylococcal cassette chromosome mec (SCCmec) subtype and epidemic strain type were to be performed on any MRSA isolates that were recovered. MLST (Multi-locus Sequence Typing) in addition to Single Locus Sequence Typing of the spa gene were also to be used on all MRSA isolates recovered to further identify them to the clonal complex level. Multi-locus Sequence Typing (MLST) and spa typing were selected to provide additional information on the relatedness of any MRSA isolates recovered from bovine milk samples and the relatedness of the isolates to human MRSA isolates.
Although no MRSA isolates were recovered from cattle samples, MLST typing methods provided by the HPA have been successfully implemented at the VLA to fulfil objective 5 using two positive MRSA control strains (EMRSA15 and EMRSA 16) and two methicillin-susceptible bovine S. aureus isolates. Of the bovine methicillin-susceptible S. aureus isolates, one was ST133 and the other contained sequences not previously recognised.
The S. aureus MLST scheme uses internal fragments of the following seven house-keeping genes:-
arc (Carbamate kinase) aro (Shikimate dehydrogenase) glp (Glycerol kinase) gmk (Guanylate kinase) pta (Phosphate acetyltransferase) tpi (Triosephosphate isomerase) yqi (Acetyle coenzyme A acetyltransferase)
Sequences are analysed in DNAStar (EditSeq) and sequences obtained at each of the seven loci are used to define a sequence type (ST) (Maiden et al. 1998). MLST type can be determined using BioNumerics software or at www.mlst.net (Enright et al. 2000) where sequences are compared to previously typed strains. Strains belonging to the same ST are defined as being related and collectively form a clonal complex.
Spa typing is a discriminatory single-locus sequence typing technique used for epidemiological typing of S. aureus. It is based on sequencing the so-called region X of the protein A (spa) gene. The X-region of the spa gene is a polymorphic region containing short sequences of tandem repeats. These repeats are assigned unique codes; the combination of repeats result in a specific spa type (www.ridom.de). Spa typing is becoming widely and routinely used for typing Staphylococcus strains.
Objective 4 was met as far as possible in that the molecular techniques which would have been used had MRSA been isolated from cattle, were successfully transferred from HPA Colindale and implemented at VLA. Spa-typing was used to examine a number of methicillin-susceptible bovine S. aureus isolates and results are given in Appendix 3.
5) Provide a framework within which ongoing monitoring for MRSA in cattle could be maintained in England and Wales and develop a standardised approach to the detection and molecular investigation of suspect MRSA isolates recovered from food animals in England and Wales.
SID 5 (Rev. 3/06) Page 16 of 30
A robust typing scheme for bovine S. aureus molecular characterisation of MRSA has been established at the VLA. Developed technologies have been successfully transferred to the VLA from the MRSA Reference Laboratory at the Health Protection Agency (HPA), Colindale. Standardised methods have been followed to allow harmonised molecular identification and comparison of isolates recovered from food animals and humans.
S. aureus strain typing methods transferred to the VLA are as follows:
Multiplex PCR for simultaneous detection of species specific thermonuclease (nuc) gene and methicillin resistance (mecA) gene.Pulsed Field Electrophoresis (PFGE).Toxin Typing.MLST.Spa Typing.
An Access database was developed for gathering relevant epidemiological information from background details submitted with the samples to VLA.
A further database was created using BioNumerics software (Applied Maths) to record and analyse epidemiological strain data. This database contained genotypic (PFGE and toxin profile), phenotypic (antibiogram) and relevant epidemiological information relating to samples which were selected for further investigation.
Objective 5 was fully met in that a framework now exists within which ongoing monitoring for MRSA in food-producing animals could be maintained in Great Britain.
Objective 6) Provide veterinary facilities capable of typing MRSA isolates according to current medical procedures, which can be deployed at short notice, should the need arise.
Trained staff and requisite facilities are now available at VLA for this purpose; the recent EFSA MRSA survey in pigs has meant that expertise can be maintained in this area.
Outputs
A poster has been accepted at ECCMID 2008 describing many of the findings from this work (Sharma et al. 2008).
It is likely that the research will lead to the publication of one or more peer-reviewed papers.
This project presented an excellent opportunity for a multidisciplinary group of scientists from HPA and VLA to examine cattle as one of the major food-producing animals from England and Wales for the presence of MRSA and provided a basis for the harmonisation and enhancement of their routine diagnostic and surveillance activities in this area. The project has provided a firm collaborative foundation on which further work may be based, should the MRSA status of food-producing animals warrant it in future.
Discussion of Results and their Reliability.
MRSA is a major cause of healthcare-associated infection and an increasing problem in human medicine, causing serious illness, including death and significant economic loss. Most human infections are thought to be nosocomial, though community-acquired infection is now being increasingly observed. In humans, a number of epidemic strains of MRSA (EMRSA) have been identified in the UK since the early 1980’s and these strains have been identified
SID 5 (Rev. 3/06) Page 17 of 30
using bacteriophage typing and pulsed field gel electrophoresis (PFGE). In the last few years, infections have also been increasingly reported in companion animals, particularly dogs and cats. More recently a particular strain of MRSA (ST398) has been particularly associated with pigs in some European countries.
The epidemiology of MRSA is dynamic and the relatively recent finding of MRSA in companion animals, horses and pigs provides a good example of how MRSA may be evolving or adapting to exploit other reservoirs or environmental niches. Prior to this study, there was a need to clarify whether or not there have been any similar changes in cattle and to determine if MRSA occurred in this species.
Prior to this study, it was not known whether MRSA is currently present in cattle in England and Wales, since no recent surveys have been performed to address this question; the last survey of bovine mastitis S. aureus isolates was published in 1986. Devriese and others reported bovine infections with MRSA in the early 1970s in cattle herds in Belgium, where it caused clinical mastitis (Devriese et al 1972, Devriese and Hommez 1975). The origin of infection was considered to be human contacts and this study demonstrated the potential for infection of cattle. However, recent studies in Korea, where 12 bovine MRSA isolates were detected in a collection of 265 bovine S. aureus isolates, showed that the bovine MRSA isolates were different at the molecular level from a collection of Korean human MRSA isolates. The Korean studies detected no MRSA in S. aureus isolates from pigs or beef cattle. The potential for farmed livestock other than pigs and cattle, to act as significant carriers of MRSA is largely unknown. Recent studies in Germany/ Switzerland identified the presence of two MRSA isolates in cattle (Monecke et al. 2007) and MRSA have also been identified in cattle from other EU Member States (van Loo et al. 2007, also personal communications). Clearly, the European position relating to MRSA in animals is rapidly evolving.
Analysis of S. aureus isolates causing mastitis in cattle in Denmark from the 1950’s, 1992 and 2000 by phage typing showed that the staphylococcal population in Denmark has remained relatively unchanged for more than 50 years (Vintov et al). These data suggest that typing schemes for bovine S. aureus are likely to be suitable for use in longitudinal studies for many years . These authors also used the international set of S. aureus phages to compare human and bovine strains in Denmark and concluded that there was no interaction between the bovine and human population and that the two populations had evolved independently. More recently, PFGE rather than phage typing has been regarded as the gold standard for strain differentiation within S. aureus since it provides manageable restriction profiles representing the entire bacterial genome and has a high discriminatory power. In turn, PFGE is now being replaced by spa-typing.
The findings from this study suggest that cattle in England and Wales are not reservoirs of MRSA or the PVL toxin. The study included S. aureus isolates from farms in diverse geographical locations in England and Wales, from a range of cattle breeds and from number of different veterinary practices. The large number of strains examined and diverse origin suggest that the results are likely to be representative of the current situation in England and Wales. The large number of veterinary practices involved is also significant, because carriage of MRSA by veterinary surgeons has previously been recognised (Wulf et al. 2008). However, dairy cattle could be a potential reservoir of TSST-1, enterotoxins and to a lesser extent, exfoliative toxins that were identified in the isolates tested. One isolate tested positive for an exfoliative toxin (etd). Other veterinary studies have also detected similar toxins in S. aureus isolates from animals (Jorgensen et al 2005).
Resistance to penicillin was relatively high and the high prevalence of resistance probably relates to the widespread usage of penicillin. Most isolates remained relatively susceptible to
SID 5 (Rev. 3/06) Page 18 of 30
the other antimicrobials tested and 55% of bovine S. aureus isolates were fully-susceptible to all antimicrobials tested.
No correlation between PFGE types, geographical location, antimicrobial susceptibility and toxin carriage was observed in this study. PFGE revealed 50 clonal groups with 5 main cluster groups- however no strong relationship was seen in relation to the antibiogram/ geographical distribution and toxin carriage. It is not clear how far the occurrence of 5 main cluster groups is linked to the structure and associated movements of the cattle industry, which could allow dissemination of particular strains, or the extent to which other factors such as antimicrobial usage or intrinsic strain properties influence the distribution and prevalence of these groups.
Further analysis of the results is necessary before definitive conclusions can be made regarding the possible relatedness of any human and bovine methicillin-susceptible S. aureus isolates.
Main Implications of the Findings.
Cattle in England and Wales, based on the findings in this extensive survey of S. aureus isolates from diagnostic samples, do not currently appear to be a reservoir for MRSA.
In view of that finding, the current public health safeguards regarding milk produced from dairy cattle are considered to be adequate.
The Panton-Valentin Leukocidin was not detected in 100 bovine S. aureus isolates (one-sided 95% confidence interval 0-3.0).
Certain enterotoxins can be detected in S. aureus from cattle and there are some food safety implications of that finding. However, mastitic milk is excluded from human consumption and refrigeration and pasteurisation should limit the potential to cause human disease.
The results also indicate that cefoxitin is likely to be useful as a screening antimicrobial for MRSA in disc diffusion tests performed on veterinary S. aureus isolates. Screening of isolates resistant to amoxicillin/ clavulanate for methicillin resistance is considered to be a possible alternative approach, if ongoing surveillance for MRSA is considered.
Recommendations and Possible Future Work.
Recent studies in Europe have detected MRSA in cattle, including the ST398 strain which has recently emerged in pigs in some European countries (Monecke et al. 2007). The position relating to MRSA in cattle in Europe is clearly evolving and in the light of this ongoing surveillance for MRSA in cattle should be considered. This could take the form of repeated periodic surveys or ongoing surveillance achieved through slight modification of current procedures performed at VLA Regional Laboratories. Practically, such ongoing surveillance for MRSA could be done by either:
1) Including cefoxitin in routine susceptibility tests.
Or,
2) Performing supplementary testing on the relatively low number of S. aureus isolates resistant to amoxillin / clavulanate.
SID 5 (Rev. 3/06) Page 19 of 30
Three isolates were resistant to oxacillin but susceptible to penicillin and did not produce beta-lactamase; the resistance to oxacillin could be further investigated for example to look for novel alterations to the penicillin binding protein.
Spa typing is recommended as the current optimum technique for rapid comparison of human and veterinary S. aureus isolates. A major disadvantage for PFGE in this role is the low number of methicillin-susceptible S. aureus isolates currently typed in human medicine. It is strongly recommended that a proportion of medical and veterinary S. aureus and MRSA isolates are subjected to spa-typing on an ongoing basis. The objectives are two-fold:- comparison of medical and veterinary S. aureus isolates within the UK.- identification of S. aureus types recognised to be epidemic in humans or animals in other countries.
Action Resulting from the Research (IP and Knowledge Transfer).
Knowledge transfer to veterinarians engaged in cattle practice will be achieved through dissemination of results to VLA Regional Laboratories.
Knowledge transfer to dairy farms and the agricultural community will be achieved through communications to the farming press.
The study has provided a baseline against which future trends may be measured and updates the study performed in 1986 on lower numbers of isolates.
Knowledge Transfer.
Two members of staff from VLA visited the Staphylococcal Reference Laboratory at HPA Colindale and received training on multi-locus sequence typing (MLST) and spa-typing and MLST typing during the course of the project.
References.
Barg N, Chambers H, Kernodle D. 1991. Borderline susceptibility to antistaphylococcal penicillins is not conferred exclusively by the hyperproduction of beta-lactamase. Antimicrob Agents Chemother. 35: 1975-9.
Barski P, Pieckowicz P, Galinski J and Kur Jozef (1996) Rapid assay for the detection of methicillin-resistant Staphylococcus aureus using multiplex PCR. Molecular and Cellular Probes. 10, 471-475.
Becker K., Roth R. and Peters G. (1998). Rapid and specific detection of toxigenic Staphylococcus aureus: use of two multiplex PCR enzyme immunoassays for amplification and hybridisation of staphylococcal enterotoxin genes, exfoliative toxin genes and toxic shock syndrome toxin 1 gene. J Clin Microbiol, 36, 2548-2553.
Bignardi GE, Woodford N, Chapman A, Johnson AP and Speller DCE (1996) Detection of the mec-A gene and phenotypic detection of resistance in Staphylococcus aureus isolates with borderline or low-level methicillin resistance. J Antimicrob Chemotherapy 37, 53-63
Brakstad OG, Aasbakk K, and Maeland JA. (1992). Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene. J Clinical Microbiology 30, 1654–60
SID 5 (Rev. 3/06) Page 20 of 30
Brown DFG, Edwards DI, Hawkey PM, Morrison D, Ridgway GL, Towner KJ and Wren MWD (2005) Guidelines for the laboratory diagnosis and susceptibility testing of methicillin-resistant Staphylococcus aureus (MRSA). J Antimicrobial Chemotherapy 56, 1000-1018.
BSAC (2007). British Society for Antimicrobial Chemotherapy Standardized Disc Susceptibility Method. Available at http://www.bsac.org.uk/susceptibility_testing/bsac_standardized_disc_susceptibility_method.cfm (accessed 28/03/2008).
Devriese LA, Van Damme LR and Fameree L. 1972. Methicillin (cloxacillin)-resistant Stahpylococcus aureus strains isolated from bovine mastitis. Zentralblatt fur Veterinarmedizin. Reihe B. 17: 598-605.
Devriese LA and Hommez J. 1975 Epidemiology of methicillin-resistant Staphylococcus aureus in dairy herds. Research in Veterinary Science. 19:23-27.
Enright MC, Day NPJ, Davies CE, Peacock SJ and Spratt BG (2000) Multilocus Sequence Typing for Characterization of Methicillin-Resistant and Methicilin-Susceptible Clones of Staphylococcus aureus. Journal of Clinical Microbiology 38, 1008-1015
Hazen EE Jr and Cotton A. 1978. Staphylococcal nuclease reviewed: A prototypic study in contemporary enzymology. I. isolation; physical and enzymatic properties. Molecular and Cellular Biochemistry. 22:67-77.
Jorgensen HJ, Mork T, Caugant DA, Kearns A and Rorvik LM. 2005. Genetice Variation among Staphylococcus aureus Strains from Norwegian Bulk Milk. Appl and Envirn Micrbiol. 71 8352-8361.
Lina G, Piemont Y, Dodail-Gamot F, Bes M, Peter M-O, Gauduchon V, Vandenesch F and Etienne J. (1999) Involvement of Panton-Valentine-Leucocidin-producing Staphylococcus aureus in primary skin infections and pneumonia. Clin Infect Dis 29, 1128-32.
Maiden MCJ, Bygraves JA, Feil E, Morelli G, Russell JE, Urwin R, Zhang Q, Zhou J, Zurth K, Caugant DA, Feavers IM, Achtman M and Spratt BG. 1998. Multilocus sequence typing: Aportable approach to the identification of clones within populations of pathogenic microorganisms. Proc Natl Acad Sci USA. 95: 3140-3145.
Monday S.R. and Bohach G.A. (1999). Use of multiplex PCR to detect classical and newly described pyrogenic toxin genes in Staphylococcal isolates. J Clin Microbiol, 37, 3411-3414.
Monecke S, Kuhnert P, Hotzel H, Slickers P and Ehricht P 2007. Microarray based study on virulence-associated genes and resistance determinants of Staphylococcus aureus isolates from cattle. Vet Microbiol 125, 128-140.
Oliveira DC and de Lencastre H (2002). Multiplex PCR strategy for rapid identification of structural types and variants of the mec element in methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 46, 2155-61.
Sharma M, Rogers J, Kearns A, Ganner M, Coldham NG and Teale C. (2008) Absence of MRSA in clinical mastitis samples recovered from dairy cattle across England and Wales.18 th
ECCMID, Barcelona, Spain, 19-22 April 2008.
Shortle D. 1983. A genetic system for analysis of staphylococcal nuclease. Gene. 22: 181-189.
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Stepan J, Pantucek J and Doskar J. Molecular diagnostics of clinically important staphylococci. Folia Microbiol 49, 353-386
Tenover FC, Arbeit RD, Goering RV, Mickelsen PA,Murray BE, Persing DH and Swaminathan B. 1995. Interpreting Chromosomal DNA Restriction Patterns Produced by Pulsed-Field Gel Electrophoresis: Criteria for Bacterial Strain Typing. Journal of Clinical Microbiology. 33: 2233-2239.
Unal S, Hoskins J, Flokowitsch JE, Ernie Wu CY, Preston DA and Skatrud PL (1992) Detection of Methicillin-Resistnace Staphylococci by Using the Polymerase Chain Reaction. J Clin Microb 1685-1691
Van Loo I, Huijsdens X, Tiemersma E, de NeelingA, van de Sande-Bruinsma N, Beaujean D, Voss A and Kluytmans J. (2007) Emergence of MRSA of animal origin in humans. Emerging and Infectious Disease 13, 1834 - 1839.
Vintov J, Aarestrup FM, Elsberg Zinn C and Olsen JE (2003) Phage types and antimicrobial Resistance among Danish bovine Staphylococcus aureus isolates since the 1950s. Vet Microbiol. 97, 63-72.
Wulf MW, Sørum M, van Nes A, Skov R, Melchers WJ, Klaassen CH and Voss A. (2008) Prevalence of methicillin-resistant Staphylococcus aureus among veterinarians: An international study. Clin Microbiol Infection 14, 29-34.
Yamaguchi T, Nishifuji K, Sasaki M, Fudaba Y, Aepfelbacher M, Takata T, Ohara M, Komatsuzawa H, Amagai M and Sugai M. (2002). Identification of the Staphylococcus aureus etd pathogenicity island which encodes a novel exfoliative toxin, ETD, and EDIN-B. Infect Immun 70, 5835-5845.
Appendix 1.
Multiplex PCR for the species-specific nuc gene of S. aureus and the methicillin resistance gene, mecA.
This protocol describes a duplex PCR method for detection of the methicillin resistance via penicillin binding protein 2a (PBP2a), encoded by mecA gene and the species specific nuc gene of S. aureus.
The method is based on the standard operating procedure (SOP) used at the Scottish MRSA reference laboratory. Optimisation of the PCR cycle was required due to reproducibility problems.
Primers
Primer Primer sequence 5’-3’ Product size (bp)
mecA-1 CTC AGG TAC TGC TAT CCA CC 449bpmecA-2 CAC TTG GTA TAT CTT CAC Cnuc-1 GCG ATT GAT GGT GAT ACG GTT 280bpnuc-2 AGC CAA GCC TTG ACG AAC TAA AGC
PCR set up (reaction volume 50μl)
PCR Controls:
SID 5 (Rev. 3/06) Page 22 of 30
Negative control – waterPositive control – for nuc and mecA EMRSA 15 and/or EMRSA 16
PCR cycle:
95oC 3 min95oC 30 sec55oC 60 sec 30 cycles 72oC 60 sec72oC 10 min4 oC Hold
Analyse PCR product by agarose gel electrophoresis to determine presence/absence of genes. 1-2% agarose prepared in x1TAE buffer (1mM EDTA, 40mM Tris acetate, pH8).Gel running conditions 100 V for 1.5hrsStain gel with ethidium bromide. View and save image to disk.
Appendix 2.Dendrogram of bovine S. aureus isolates examined by PFGE.
SID 5 (Rev. 3/06) Page 23 of 30
Dice (Opt:2.00%) (Tol 2.0%-2.0%) (H>0.0% S>0.0%) [0.0%-100.0%]PFGE - Sma
100
908070PFGE - Sma
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VLA ref no
16-C0132-06-06
16-C0216-09-06
17-C0209-12-06
16-C0047-05-07
23-C0364-07-06
14-C0473-01-07
27-C0283-05-06
27-C0133-07-06
27-C0133-07-06
24-C0069-08-06
12-C0250-09-06
16-C0199-09-06
24-C0204-10-06
27-C0163-03-07
27-C0343-03-07
26-C0423-09-06
23-C0234-01-07
16-C0140-01-07
28-C0270-01-07
14-C0090-08-06
24-C0120-01-07
17-C0266-09-06
24-C0186-01-07
24-C0368-05-07
23-C0018-03-07
28-C0190-03-07
26-C0044-05-06
24-C0251-07-06
23-C0377-07-06
24-C0415-07-06
23-C0417-08-06
22-C0074-08-06
22-C0026-09-06
17-C0274-09-06
28-C0010-01-07
24-C0310-04-07
17-C0004-10-06
12-C0249-01-07
17-C0186-10-06
26-C0705-04-06
21-C0384-05-06
15-C0115-07-06
17-C0289-08-06
26-C0214-09-06
26-C0410-09-06
15-C0108-10-06
26-C0190-01-07
16-C0155-01-07
16-C0244-03-07
16-C0187-08-06
17-C0134-09-06
29-C0089-09-06
bead ref
C02747
C03363
C03828
C04637
C02964
C04060
C02551
C02924
C02925
C03029
C03279
C03353
C03406
C04346
C04441
C03316
C03927
C03979
C04020
C03003
C03908
C03306
C03968
C04761
C04280
C04344
C02522
C02953
C02957
C02995
C03091
C03097
C03221
C03312
C03868
C04576
C03385
C04011
C03403
C02496
C02665
C02918
C03163
C03317
C03326
C03388
C03987
C04062
C04434
C03175
C03257
C03283
sample ref
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22
32
25
32
11
13
46
03
03
42
08
44
42
14
14
56
16
22
57
05
15
48
16
11
45
60
35
15
11
36
07
07
21
55
15
48
08
37
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21
52
53
48
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09
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06
55
notes
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nuc gene
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Toxin Profile
seg, sei
Not Detected
seg, sei
sec, tst
Not Detected
Not Detected
tst
sec, tst
sec, tst
sec, tst
sec, tst
sec, tst
Not Detected
Not Detected
seg, sei
seg, sei
seg, sei
seg, sei
seg, sei
seg, sei
seg, sei
seg, sei
seg, sei
PFGE Type
1
2
3
4
5
6
7
8
8a
9
9
9
9
10
11
12
13
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13
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14a
14b
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14c
14a
14a
14
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14
14
14
14
14c
14a
14e
14e
14e
14e
14e
14f
14e
14d
14e
14e
14e
14e
14e
14e
14e
Antibiogram
P
P-Cip-Fox-Ox
P
$
$
$
$
$
$
$
$
P-Te
$
$
$
$
P
$
$
$
$
$
$
$
$
$
$
$
$
$
$
$
$
$
$
$
$
$
$
$
$
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$
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$
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$
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$
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Cn
SID 5 (Rev. 3/06) Page 24 of 30
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24-C0314-05-06
23-C0037-07-06
16-C0011-01-07
15-C0168-04-07
17-C0043-08-06
17-C0236-03-07
15-C0257-06-06
28-C0002-09-06
15-C0103-05-07
15-C0098-02-07
17-C0292-01-07
17-C0292-01-07
26-C0279-07-06
23-C0147-09-06
17-C0018-10-06
24-C0378-10-06
17-C0251-09-06
17-C0118-10-06
23-C0234-01-07
16-C0306-02-07
24-C0238-05-07
24-C0238-05-07
17-C0251-09-06
24-C0028-04-07
23-C0501-06-07
15-C0241-12-06
15-C0075-02-07
23-C0020-08-06
23-C0020-08-06
16-C0199-09-06
16-C0267-02-07
23-C0071-06-07
23-C0452-02-07
23-C0051-07-06
16-C0018-07-06
23-C0282-07-06
12-C0003-08-06
28-C0001-09-06
23-C0254-09-06
28-C0182-10-06
21-C0194-11-06
26-C0638-04-06
24-C0480-06-06
23-C0374-07-06
23-C0376-11-06
17-C0190-02-07
17-C0125-07-06
17-C0196-09-06
17-C0196-09-06
12-C0197-06-07
12-C0197-06-07
23-C0071-09-06
14-C0301-02-07
16-C0157-02-07
16-C0045-12-06
C02535
C02866
C03886
C04489
C03021
C04369
C02758
C03186
C04633
C04115
C03996
C03999
C02940
C03238
C03377
C03461
C03288
C03378
C03926
C04285
C04668
C04669
C03286
C04472
C04878
C03800
C04104
C03053
C03056
C03352
C04249
C04776
C04225
C02868
C02877
C02936
C03010
C03185
C03254
C03427
C03622
C02491
C02843
C03088
C03610
C04206
C02904
C03609
C03267
C04853
C04857
C03187
C04181
C04238
C03810
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48
55
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21
21
Gwyn.
36
21
15
21
21
16
22
16
16
21
42
16
48
48
36
36
44
49
45
36
36
37
11
08
55
34
55
15
35
02
36
34
06
11
06
06
08
14
31
28
43
09
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Not Detected
Not Detected
Not Detected
Not Detected
Not Detected
etd, seg, sei
Not Detected
Not Detected
Not Detected
sec
Not Detected
Not Detected
sec
Not Detected
Not Detected
Not Detected
Not Detected
Not Detected
Not Detected
Not Detected
Not Detected
Not Detected
Not Detected
Not Detected
Not Detected
Not Detected
sec, tst
sec, tst
sec, tst
25
26
27
27a
27b
27c
28
28
28a
28b
29
29
29a
30
30a
30b
30b
30a
30a
30b
30b
30b
30c
30a
30b
30d
30d
30e
30e
30e
30e
30e
31
31
31
31
31
31
31
31
31
31a
31
31
31
31a
31a
31b
31b
31c
31c
31d
31d
31c
31e
P
P
$
$
$
$
P
P
P
P
P
P
P
Cip
P-Te
P
P
P-Cip-Te
P
P
P
P
P-Cn
P
P
P
P
P
P-Cip
P
P
P
$
$
$
P-Ox
P
P-Te
P
P-Te
P
P
P
$
$
P
$
$
$
P-Amc-E
P-E
P
P
$
P
SID 5 (Rev. 3/06) Page 25 of 30
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14-C0301-02-07
16-C0157-02-07
16-C0045-12-06
27-C0098-06-07
26-C0176-08-06
24-C0161-03-07
17-C0009-09-06
24-C0245-04-07
28-C0293-09-06
23-C0471-11-06
24-C0221-12-06
12-C0162-07-06
21-C0016-08-06
16-C0134-09-06
15-C0236-03-07
23-C0387-05-06
22-C0102-03-07
22-C0170-03-07
17-C056-02-07
26-C0210-02-07
17-C0013-09-06
28-C0221-10-06
23-C0039-03-07
28-C0165-11-06
24-C0081-08-06
16-C0162-08-06
12-C0197-06-07
26-C0138-06-06
12-C0270-09-06
13-C0078-08-06
26-C0052-02-07
17-C0253-10-06
12-C0330-11-06
21-C0233-08-06
24-C0073-09-06
28-C0134-12-06
12-C0489-03-07
16-C0102-06-06
16-C0060-07-06
17-C0279-07-06
26-C0153-05-06
16-C002-08-07
22-C0149-05-06
24-C0125-12-06
15-C0071-09-06
16-C0216-09-06
14-C0475-08-06
16-C0216-09-06
16-C0216-09-06
26-C0338-07-06
C04181
C04238
C03810
C04817
C03066
C04330
C03214
C04533
C03313
C03640
C03850
C02946
C03047
C03274
C04348
C02543
C04352
C04421
C04120
C04166
C03217
C03294
C04274
C03611
C03028
C03169
C04859
C03067
C03307
C03158
C04164
C03423
C03708
C03130
C03232
C03791
C04449
C02744
C02888
C03000
C02523
C05139
C02569
C03808
C03224
C03371
C03125
C03367
C03368
C02972
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28
43
09
Warwi.
25
15
44
11
55
11
42
08
10
09
48
45
07
07
19
37
21
55
34
55
15
37
08
35
08
31
35
48
21
21
Jersey
57
75
09
37
21
53
09
07
42
48
32
38
32
32
37
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Not Detected
Not Detected
Not Detected
sec, tst
Not Detected
Not Detected
sea
Not Detected
Not Detected
sec, tst
Not Detected
sec, tst
sec, tst
Not Detected
sec, tst
Not Detected
seg, sei
sea
Not Detected
Not Detected
Not Detected
Not Detected
Not Detected
Not Detected
Not Detected
31d
31c
31e
31e
31
31
31
31a
31
31
32
33
33a
34
34
35
36
37
38
38a
39
39
40
40a
40
40a
40b
40
40b
40
40
41
42
43
43a
43b
43c
44
44
44
44a
45
46
47
48
48
48a
48a
48b
49
P
$
P
$
$
P
P
Cip
P
$
P
Cip
$
$
P
P
$
P
P-Cip
P
P-Cip
$
P
P-Cip-Ox
P
P
P-E-Ox
P
P-E
P-Te
P-Amc-E-Fox
P
$
P
P
P
P-Te
$
$
P
P
P-Amc-Cip
P-Cip
P
P
P-Cip
P
P-Cip-Fox-Ox
P-Cip-Fox-Ox
$
SID 5 (Rev. 3/06) Page 26 of 30
Appendix 3.
Spa typing of selected isolates chosen on the basis of antibiotic sensitivity, PFGE, toxin and geographical information
Bead Reference
County of Origin (Defra county code) Antibiogram
Toxins Present
Spa Type
Spa type global frequency*
C04421 07 P sea t024 1.31%C04911 07 P-Amc-Te seb t189 0.23%C04912 07 P-Amc-Te seb t189 0.23%C03378 21 P-Cip-Te sec t521 0.02%C04530 Guernsey P sec, seh unknown C04857 08 P-E sec, tst t359 0.09%C04853 08 P-Te sec, tst t359 0.09%C04859 08 P-E-Ox sec, tst t359 0.09%C03158 31 P-Te sec, tst t224 0.03%C02986 48 Sensitive sec, tst unknown C03611 55 P-Cip-Ox sec, tst t224 0.03%C03163 21 Sensitive seg, sei t529 0.02%
C03098 48 Sensitivesec, seg, sei, tst t529 0.02%
C02978 07 Psed, seg, sei, sej t002 6.01%
C04369 14 Sensitiveetd, seg, sei t140 0.02%
C02936 11 P-OxNot Detected t521 0.02%
C03461 15 PNot Detected t044 2.59%
C03286 21 P-CnNot Detected t044 2.59%
C04164 35P-Amc-E-Fox
Not Detected t359 0.09%
C03187 36 PNot Detected t1234 0.01%
C03800 48 PNot Detected t131 0.19%
* information from www.ridom.de
.
SID 5 (Rev. 3/06) Page 27 of 30 0.19%t131Not DetectedP 48C03800
0.09%t359Not DetectedP-Amc-E-Fox
35C04164
2.59%t044Not DetectedP-Cn 21C03286
2.59%t044Not DetectedP 15C03461
0.02%t521Not DetectedP-Ox 11C02936
0.02%t140etd, seg, seiSensitive14C04369
6.01%t002sed, seg, sei, sej
P 07C02978
0.02%t529sec, seg, sei, tst
Sensitive48C03098
0.02%t529seg, seiSensitive21C03163
0.03%t224sec, tstP-Cip-Ox55C03611
unknownsec, tstSensitive48C02986
0.03%t224sec, tstP-Te 31C03158
0.09%t359sec, tst P-E-Ox 08C04859
0.09%t359sec, tstP-E 08C04857
unknownsec, sehP GUC04530
0.02%t521secP-Cip-Te21C03378
0.23%t189sebP-Amc-Te07C04911
1.31%t024seaP 07C04421
Spa Type Global
Frequency
Spa Type
Toxin§
carriageAntibiogra
mCount
ySample Ref
References to published material9. This section should be used to record links (hypertext links where possible) or references to other
published material generated by, or relating to this project.
SID 5 (Rev. 3/06) Page 28 of 29
References.
Barg N, Chambers H, Kernodle D. 1991. Borderline susceptibility to antistaphylococcal penicillins is not conferred exclusively by the hyperproduction of beta-lactamase. Antimicrob Agents Chemother. 35: 1975-9.
Barski P, Pieckowicz P, Galinski J and Kur Jozef (1996) Rapid assay for the detection of methicillin-resistant Staphylococcus aureus using multiplex PCR. Molecular and Cellular Probes. 10, 471-475.
Becker K., Roth R. and Peters G. (1998). Rapid and specific detection of toxigenic Staphylococcus aureus: use of two multiplex PCR enzyme immunoassays for amplification and hybridisation of staphylococcal enterotoxin genes, exfoliative toxin genes and toxic shock syndrome toxin 1 gene. J Clin Microbiol, 36, 2548-2553.
Bignardi GE, Woodford N, Chapman A, Johnson AP and Speller DCE (1996) Detection of the mec-A gene and phenotypic detection of resistance in Staphylococcus aureus isolates with borderline or low-level methicillin resistance. J Antimicrob Chemotherapy 37, 53-63
Brakstad OG, Aasbakk K, and Maeland JA. (1992). Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene. J Clinical Microbiology 30, 1654–60
Brown DFG, Edwards DI, Hawkey PM, Morrison D, Ridgway GL, Towner KJ and Wren MWD (2005) Guidelines for the laboratory diagnosis and susceptibility testing of methicillin-resistant Staphylococcus aureus (MRSA). J Antimicrobial Chemotherapy 56, 1000-1018.
BSAC (2007). British Society for Antimicrobial Chemotherapy Standardized Disc Susceptibility Method. Available at http://www.bsac.org.uk/susceptibility_testing/bsac_standardized_disc_susceptibility_method.cfm (accessed 28/03/2008).
Devriese LA, Van Damme LR and Fameree L. 1972. Methicillin (cloxacillin)-resistant Stahpylococcus aureus strains isolated from bovine mastitis. Zentralblatt fur Veterinarmedizin. Reihe B. 17: 598-605.
Devriese LA and Hommez J. 1975 Epidemiology of methicillin-resistant Staphylococcus aureus in dairy herds. Research in Veterinary Science. 19:23-27.
Enright MC, Day NPJ, Davies CE, Peacock SJ and Spratt BG (2000) Multilocus Sequence Typing for Characterization of Methicillin-Resistant and Methicilin-Susceptible Clones of Staphylococcus aureus. Journal of Clinical Microbiology 38, 1008-1015
Hazen EE Jr and Cotton A. 1978. Staphylococcal nuclease reviewed: A prototypic study in contemporary enzymology. I. isolation; physical and enzymatic properties. Molecular and Cellular Biochemistry. 22:67-77.
SID 5 (Rev. 3/06) Page 29 of 29
