Doi:10.1016/j.vetmic.2007.01.027

Veterinary Microbiology 122 (2007) 366–372 High prevalence of methicillin resistant Staphylococcus A.J. de Neeling , M.J.M. van den Broek , E.C. Spalburg , M.G. van Santen-Verheuvel W.D.C. Dam-Deisz , H.C. Boshuizen A.W. van de Giessen , E. van Duijkeren X.W. Huijsdens a National Institute for Public Health and the Environment, Bilthoven, The Netherlands b Food and Consumer Product Safety Authority, Zutphen, The Netherlands c Veterinary Faculty, Utrecht University, Utrecht, The Netherlands Received 17 November 2006; received in revised form 25 January 2007; accepted 26 January 2007 Recently methicillin resistant Staphylococcus aureus (MRSA) was isolated from pigs and pig farmers in The Netherlands. In order to assess the dissemination of MRSA in the Dutch pig population, we screened 540 pigs in 9 slaughterhouses, where arepresentative portion of Dutch pigs (63%) was slaughtered in 2005. We found 209 (39%) of the pigs to carry MRSA in their nares.
Forty-four of 54 groups of 10 consecutive pigs (81%), each group from a different farm, and all slaughterhouses were affected.
All MRSA isolates belonged to 1 clonal group, showing Multi-Locus Sequence Type 398 and closely related spa types (mainly t011, t108 and t1254). Three types of the Staphylococcal Chromosome Cassette (SCCmec) were found: III (3%), IVa(39%) and V (57%). All 44 tested isolates (1 isolate per group) were resistant to tetracycline, reflecting the high and predominantuse of tetracyclines in pig husbandry. Twenty-three percent of the isolates were resistant to both erythromycin and clindamycinand 36% to kanamycin, gentamicin and tobramycin but only a single isolate was resistant to co-trimoxazole and none tociprofloxacin and several other antibiotics.
The percentage of MRSA positive pigs was significantly different among slaughterhouses and among groups within slaughterhouses, indicating a high prevalence of MRSA in pigs delivered from the farms as well as cross contamination in theslaughterhouses.
# 2007 Elsevier B.V. All rights reserved.
Keywords: Swine microbiology; Staphylococcus aureus; Methicillin resistance; Tetracycline resistance; Genetics; The Netherlands * Corresponding author at: National Institute for Public Health and the Environment (RIVM), Diagnostic Laboratory for InfectiousDiseases and Perinatal Screening, pb 22, Antonie van Leeuwen- Methicillin resistant Staphylococcus aureus (MRSA) hoeklaan 9, 3721 MA Bilthoven, The Netherlands.
in humans is still rare in The Netherlands. Last year 2% Tel.: +31 30 2742729; fax: +31 30 2744418.
of the S. aureus isolates from hospitals were resistant to 0378-1135/$ – see front matter # 2007 Elsevier B.V. All rights reserved.
doi: A.J. de Neeling et al. / Veterinary Microbiology 122 (2007) 366–372 were transferred into tubes containing 5 mL Phenol Red (0.03%) of patients admitted to hospitals carried MRSA Mannitol Broth (Brunschwig Chemie, Amsterdam) with 4 mg/L oxacillin (Sigma) and 75 mg/L aztreonam cultured from dogs, cats and diseased horses, but no (ICN). After 18 h incubation at 35 8C, the bacteria from MRSA was found in a survey of 200 healthy horses in each tube were plated onto sheep blood agar and three selective agar media: MRSA Select Agar (BioRad, Veenendaal), Oxacillin Resistance Screening Agar and three patients who had contact with pigs. These Chromogenic MRSA Agar (Oxoid, Haarlem).
authors also tested 26 pig farmers. Six of them (23%) After 18 h incubation at 35 8C, suspected colonies carried MRSA. Subsequently MRSA was isolated were plated onto sheep blood agar and incubated for from several members of a family living on a pig farm 18 h. Colonies suspect of being MRSA were tested by and 8 out of 10 pigs at the same farm carried MRSA PCR for the S. aureus specific DNA-fragment human and porcine origin in these investigations were non-typeable by standard Pulsed-Field Gel Electro- phoresis (PFGE) using the SmaI restriction enzyme(NT). The NT MRSA contain a restriction modifica- tion enzyme, which methylates the SmaI-recognitionsequence ( MRSA-isolates were typed by PFGE using SmaI as These observations prompted us to determine the restriction enzyme according to the Harmony protocol prevalence of MRSA in healthy pigs in nine Dutch slaughterhouses. We further analyzed the porcine from pigs (1–3 per group) were typed by spa-typing MRSA by molecular typing and susceptibility testing.
) and 1 isolate per group wassubjected to Multi-Locus Sequence Typing (MLST)Typing of the Staphylococcal Chromosome Cassette (SCCmec) was performed byPCR ( From November 2005 to January 2006 in each of nine slaughterhouses all over The Netherlands 6 groups, The susceptibility to antimicrobials of one isolate 10 pigs per group, 540 pigs in total, were screened. In per group was tested by agar dilution using Mueller 2005, 63% of the pigs raised in The Netherlands were Hinton Agar (BBL) and multipoint inoculation slaughtered in the nine investigated slaughterhouses. In The Netherlands each slaughterhouse buys pigs from a The antibiotics tested were clindamycin (Pharmacia), broad range of farms, with few exclusive contracts teicoplanin (Aventis Pharma), mupirocin (Glaxo- between a slaughterhouse and the farms supplying pigs.
SmithKline), linezolid (Pfizer), chloramphenicol, So we are confident that we have screened a repre- ciprofloxacin, doxycycline, erythromycin, fusidic sentative sample of the pigs in the Dutch slaughterlines.
acid, gentamicin, kanamycin, neomycin, oxacillin, A group consisted of 10 consecutive pigs in the rifampicin, tetracycline, tobramycin, trimethoprim- slaughterline, each group from a different farm, except sulfamethoxazole (co-trimoxazole) and vancomycin 1 group, which was composed of pigs from 3 farms and (MP Biomedicals). S. aureus ATCC 43300 and S.
2 groups, which were both from 1 farm.
aureus ATCC 29213 were used as reference strains.
A swab (Medical Wire & Equipment Co. (Bath) Ltd. Corsham, Wiltshire, no. MW102) was taken from the nares of the pigs just after stunning, by officials ofthe Dutch Food and Consumer Product Safety Statistical analyses were performed in GAUSS Authority (VWA). Within 5 h after sampling, swabs (Aptech Systems, Inc. Black Diamond, WA, USA).
A.J. de Neeling et al. / Veterinary Microbiology 122 (2007) 366–372 A two level logistic-normal model was used, assuming fermenting organisms. However, the subsequent a normal distribution both of the log(odds) among MRSA-selective agar media and the PCR showed groups within slaughterhouses, and of the log(odds) only a minority of these bacteria to be MRSA. We did among slaughterhouses. Fitting was by maximum not systematically subculture or identify the other likelihood and profile likelihood was used to obtain bacteria. Some were oxacillin resistant Staphylococ- confidence bounds of the variance parameters.
cus lentus, Staphylococcus sciuri and Enterococcusfaecalis and oxacillin sensitive S. aureus, Staphylo-coccus chromogenes and Staphylococcus simulans.
The predominant spa types of the NT MRSA from MRSA was found in 209 (39%) of the 540 screened the pigs were t011, t108 and t1254, whereas spa types pigs. At least 1 of the 10 sampled pigs carried MRSA t1255, t567, t034 and t943 were found sporadically in 44 (81%) of the 54 investigated groups. The number (All spa types were closely related. Type of MRSA-carrying animals per group of 10 pigs is t1254 was found only in slaughterhouse 5, where 13 of the 14 selected MRSA in 5 of the 6 groups belonged to The log(odds) of MRSA carrying pigs differed this spa type. Spa type t1254 differs by only 1 base significantly among groups ( p < 0.0001) and among substitution (G to C) in the first repeat from spa type slaughterhouses ( p < 0.0001). The normal logistic model showed a variance of the log(odds) among All isolates showed ST 398. SCCmec types IVa groups of 2.2 (95% CI 1.0–4.5), and an almost equally (n = 41) and V (n = 59) were most prevalent, whereas large variance among slaughterhouses of 2.1 (95% CI type III was present in only four isolates. We did not 0.65–7.5). The geographic location of the farmers who detect the genes of Panton-Valentine Leucocidin in supplied the groups of pigs appeared representative for the distribution of pigs over The Netherlands. Groupswith a high number of MRSA carrying pigs did not Swabs taken from the nares of pigs in the The oxacillin MICs of the NT MRSA from pigs slaughterhouses were incubated in an enrichment broth containing mannitol and the pH indicator phenol intermediate or resistant to doxycycline and resistant red. The enrichment broth turned yellow in nearly all to tetracycline. The 10 isolates (23%) resistant to tubes during incubation indicating growth of mannitol erythromycin were cross-resistant to clindamycin.
Table 1Number of NT MRSA carrying pigs in 6 groups of 10 pigs in each of 9 slaughterhouses A.J. de Neeling et al. / Veterinary Microbiology 122 (2007) 366–372 All 16 strains (36%) resistant to kanamycin were dominant spa type in slaughterhouse no. 5 was cross-resistant to gentamicin and all but 1 were cross- probably due to transmission of this particular MRSA resistant to tobramycin. Nearly all tested isolates were strain among groups of pigs from different farms in susceptible to ciprofloxacin, co-trimoxazole, rifampi- cin, teicoplanin, vancomycin, linezolid, amikacin, The origin of the NT MRSA in the pigs remains chloramphenicol, fusidic acid and mupirocin.
unclear. In The Netherlands, there are three types ofpig farms: breeding farms, rearing or reproductionfarms and fattening farms. Farmers may rear piglets at the same farm or they buy piglets for fattening(finishing pigs) at rearing farms. To date, we do not We found an unexpected high prevalence of know if the pigs get infected at the fattening farms or if MRSA in healthy pigs originating from more than they have already been infected when they arrive on 50 different farms in The Netherlands. In our country these farms. If the pigs on the breeding farms or the prevalence of MRSA in companion animals and rearing farms are colonized with MRSA, finishing horses is low. However, we detected MRSA in 39% of the 540 pigs, in 81% of the 54 groups of 10 pigs and in Possible sources of the mecA gene are coagulase all 9 slaughterhouses. All of the MRSA isolated from negative staphylococci belonging to the normal the pigs were non-typeable by PFGE using SmaI microflora of the pig, which may have transmitted macrorestriction. We conclude that NT MRSA has the mecA gene to a methicillin susceptible S. aureus widely spread in the Dutch pig population. These strain. Alternatively, the MRSA strain as such may results are in line with the earlier isolation of NT have been transmitted to pigs from another source.
MRSA from pigs and humans on the same farm Perhaps pigs are just a good host for this special MRSA strain, which originated from another host or However, it is likely that the number of positive possibly feed. In household contacts humans may groups was raised considerably by transmission of the infect companion animals with MRSA and vice versa.
NT MRSA in the lairages of the slaughterhouses. We A similar phenomenon may have been the initial cause found a significant difference in the prevalence of of the emergence of the NT MRSA in pigs.
MRSA-positive pigs among slaughterhouses. Thirteen MRSA may also be disseminated from contami- of 14 tested pigs delivered to slaughterhouse no. 5 had nated feed and dust. Tetracycline resistant S. aureus the same spa type, which differed from the main spa might survive or even thrive in feed medicated with type t011 in one nucleotide. Two delivering farms did tetracyclines. detected resistant S.
not obtain pigs from elsewhere and four had received aureus inside and downwind of swine confinement pigs from several rearing farms. So the aberrant facilities in levels, which they considered a potential A.J. de Neeling et al. / Veterinary Microbiology 122 (2007) 366–372 Table 3Minimum inhibitory concentrations of antibiotics (mg/L) for NT MRSA from pigs (44 groups tested, 1 isolate per group) (-) no growth; (*) no CLSI breakpoints; vertical lines are breakpoints.
human health hazard. They found S. aureus to be the barn to prevent exposure of vulnerable populations predominant bacterium in the air within a swine barn, (children, elderly, immunocompromized individuals) being present at 104 CFU/m3, and concluded that to bacteria from the swine barn adhering to their swine facilities should be placed at least 200 m from residential areas to avoid detrimental effects on human The significant difference in the prevalence of MRSA-positive pigs among groups may have been due pig farmers should wear particle respirators and to transmission among pigs within groups and to should change clothes and shower prior to leaving the differences in risk factors among the farms or farm A.J. de Neeling et al. / Veterinary Microbiology 122 (2007) 366–372 compartments where the pigs were raised, particularly method and show a wide variety of different PFGE-, the use of tetracyclines which may select bacteria which spa- and MLST types which are also common in are resistant to tetracycline. In 2004, the use of tetracyclines as group medication in breeding and In conclusion, we observed a high prevalence of fattening facilities for pigs was 10 resp. 9 daily doses per MRSA in pigs in Dutch slaughterhouses. Further animal year, which may have been an underestimation research into causes and effects is needed.
(Other antibiotics were used in atleast 10-fold lower amounts. In contrast sows andpiglets in breeding facilities received much more penicillins and aminoglycosides as compared to pigs forfattening. So the resistance to the former antibiotics We thank M.E.O.C. Heck, G.N. Pluister and L. de may have been due to selection in breeding facilities.
Heer for PFGE analyses and Dr. P.J. van der Wolf and The selection effect of the considerable and Ir. H. Rang for their comments on the manuscript.
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