The first detection of methicillin-resistant Staphylococcus aureus ST398 in pigs in Korea

In order to determine the presence of methicillin-resistant Staphylococcus aureus (MRSA) in pigs in Korea, a total of 657 nasal swabs were collected from pigs on 66 different pig farms nationwide during February 2008-May 2009. The prevalences of MRSA positive samples in pigs and farms were 3.2% (21/657) and 22.7% (15/66), respectively. Two different types were found among the 21 MRSA isolates: 17 strains of livestock-associated type (LA; ST398 or ST541/spa t034) and 4 strains of human-associated type (HA; ST72/spa t664 or t2461). The most prevalent type of MRSA strain was ST398/t034 (12/21, 57%), followed by ST541/t034 (5/21, 23.8%). The rest of the isolates were ST72/t664 (n=2) and ST72/t2461 (n=2), respectively. Our data provide evidence for the existence of not only LA types (ST398 and ST541) but also HA type (ST72) MRSA in pigs in Korea. This survey provides the first evidence of LA type MRSA in animals in Korea. In addition, the presence of human MRSA clones in pigs observed in this study suggests an additional reservoir for human MRSA infection, and vice versa.     

Swine as reservoirs of zoonotic borderline oxacillin-resistant Staphylococcus aureus ST398

Methicillin resistance mediated by the mecA gene in Staphylococcus aureus, also known as “true MRSA”, is typically associated with high oxacillin MIC values (≥8 mg/L). Because non-mecA-mediated oxacillin resistant S. aureus phenotypes can also cause hard-to-treat diseases in humans, their misidentification as methicillin-susceptible S. aureus strains (MSSA) can compromise the efficiency of the antimicrobial therapy. These strains have been refereed as Borderline Oxacillin-Resistant S. aureus (BORSA) but their characterization and role in clinical microbiology have been neglected. Considering the increasing importance of livestock-associated methicillin-resistant S. aureus ST398 (LA-MRSA) as an emerging zoonotic pathogen worldwide, this study aimed to report the genomic context of oxacillin resistance in porcine S. aureus ST398 strains. S. aureus isolates were recovered from asymptomatic pigs from three herds. Oxacillin MIC values ranged from 4 to 32 mg/L. MALDI-TOF-confirmed isolates were screened for mecA and mecC by PCR and genotyped by means of PFGE and Rep-PCR. Seven isolates were whole genome sequenced. None of the isolates harbored the mecA gene or its variants. Although all seven sequenced isolates belonged to one sequence type (ST398), two different spa types (t571 and t1471) were identified. All isolates harbored conserved blaZ gene operon and no mutations on genes encoding for penicillin-binding-proteins were detected. Genes conferring resistance against other drugs such as aminoglycosides, chloramphenicol, macrolide, lincosamide and streptogramin (MLS), tetracycline and trimethoprim were also detected. Isolates also harbored virulence genes encoding for adhesins (icaA; icaB; icaC; icaD; icaR), toxins (hlgA; hlgB; hlgC; luk-PV) and protease (aur). Pigs can serve as reservoirs of non-mecA-mediated oxacillin-resistant ST398 strains potentially pathogenic to humans. Considering that mecA has been the main target to screen methicillin-resistant staphylococci, the occurrence of BORSA phenotypes is probably underestimated in livestock.     

Retrospective detection of methicillin resistant and susceptible Staphylococcus aureus ST398 in Danish slaughter pigs

This retrospective study documents the first detection in Danish pigs of methicillin resistant and susceptible variants of the Staphylococcus aureus lineage ST398, which was previously described among pigs, pig farmers and veterinary staff in The Netherlands as well as in human patients and infected animals in Europe. Ten out of 100 pigs tested were shown to carry ST398 in the nasal cavity and two out of three farms tested were positive. The 10 porcine ST398 isolates had spa type t034 (n=9) and t1793 (n=1), and were resistant to erythromycin, clindamycin and tetracycline in addition to penicillin. The detection of ST398 in Danish pigs suggests that this new emerging zoonotic bacterium is rapidly spreading in the pig population in Europe.     

Characterization of methicillin-resistant Staphylococcus aureus CC398 obtained from humans and animals on dairy farms

In this study MRSA isolates from dairy farms were investigated for their genetic relationships and antimicrobial susceptibility. In total, 125 MRSA isolates from 26 dairy farms were studied, including isolates from milk samples (n=46), dairy cattle (n=24), calves (n=6), dust samples from pig (n=16) and veal calf sheds (n=1), dogs (n=2), a horse, a sheep and humans (n=28). CC398-specific PCRs, spa typing, SCCmec typing and ApaI macrorestriction analysis were conducted. Susceptibility testing was performed by broth microdilution. All 125 isolates belonged to CC398. Eight spa types (t011, t108, t034, t567, t1184, t1451, t2287 and t3934) were detected. SCCmec elements of types IV (n=48) and V (n=67) were identified with 10 isolates being non-typeable. Six main macrorestriction patterns - with up to 23 sub-patterns - and twelve resistance patterns were identified. Sixty-eight isolates showed a multiresistance phenotype. Farm-by-farm analysis revealed different scenarios: in some farms, the MRSA CC398 isolates from dairy cattle, humans, pig sheds and/or sheep were indistinguishable suggesting an interspecies exchange of the same MRSA CC398 subtype. In other farms, several MRSA CC398 subtypes were detected in different host species/sources with occasionally even more than one MRSA CC398 subtype from the same host species/source. These latter results may suggest that either different MRSA subtypes associated with humans or animals have been imported into the respective farm or that one MRSA CC398 strain has undergone diversification, reflected by more or less expanded changes in PFGE patterns, spa type or resistance pattern, during colonization of different hosts on the same farm.     

Methicillin‐Resistant Staphylococcus aureus (MRSA) ST398 in Pig Farms and Multispecies Farms

During the last few years, methicillin‐resistant Staphylococcus aureus (MRSA) ST398 has been isolated frequently from livestock, especially from pigs and to a lesser extent from cattle and poultry. To gain insight into the distribution of this bacterium in pig farms versus multispecies farms, 30 Belgian farms (10 pig, 10 pig/poultry and 10 pig/cattle farms) were screened for the presence of MRSA. On each farm, 10 nasal swabs were taken from pigs. When present, cattle (n = 10) were sampled in the nares and poultry (n = 10) in the nares, earlobes and cloaca. A selection of the obtained isolates were further characterized using multilocus sequence typing (MLST), spa typing, SCCmec typing, pulsed field gel electrophoresis (PFGE), multiple‐locus variable‐number tandem repeat analysis (MLVA) and antimicrobial susceptibility testing. On 26 of 30 farms, MRSA was isolated from pigs. Furthermore, MRSA was also isolated from poultry and cattle on one pig/poultry and five pig/cattle farms, respectively. All tested MRSA isolates belonged to ST398. Eight spa types (t011, t034, t567, t571, t1451, t2974, t3423 and t5943) were detected, among which t011 predominated. SCCmec cassettes type IVa and V were present in 20% and 72% of the isolates, respectively. When combining the results of the two remaining typing methods, PFGE and MLVA, eighteen genotypes were obtained of which one genotype predominated (56% of the positive farms). All MRSA isolates were resistant to tetracycline. Resistance to trimethoprim, aminoglycosides, macrolides, lincosamides, fluoroquinolones and chloramphenicol was also observed. In conclusion, there was no effect of the farm type on the MRSA status of the pigs. A statistically significant difference was observed when comparing the pig/poultry or the pig/cattle MRSA status on the multispecies farms. Additionally, a wide variety of MRSA ST398 strains was found within certain farms when combining different typing methods.     

Effects of tetracycline and zinc on selection of methicillin-resistant Staphylococcus aureus (MRSA) sequence type 398 in pigs

An in vivo experiment was conducted to evaluate the effects of tetracycline and zinc on pig colonization and transmission of methicillin-resistant Staphylococcus aureus (MRSA) sequence type (ST) 398. Eight piglets naturally colonized with MRSA ST398 and 8 MRSA-negative piglets of the same age and breed were assigned to three groups treated with tetracycline and zinc (Group 1), zinc (Group 2) or tetracycline alone (Group 3) and one non-treated group (Group 4), each containing two MRSA-positive and two MRSA-negative animals. Two additional non-treated control groups composed of only MRSA-positive (Group 5) and MRSA-negative (Group 6) animals were used to check for stability of MRSA carriage status. Nasal swabs and environmental wipes were collected on Days 0, 7, 14, and 21, and the occurrence of MRSA in each sample was quantified by bacteriological counts on Brilliance? MRSA agar. Significantly higher nasal MRSA counts were observed in the zinc-treated (p=0.015) and tetracycline-treated (p=0.008) animals compared to the non-treated animals. Environmental MRSA counts appeared to increase over time in Groups 1 and 2 but such an increase was not statistically significant. MRSA-negative animals housed with MRSA-positive animals became positive in all groups, whereas the carriage status of the animals in Groups 5 and 6 did not change. This study demonstrates that feed supplemented with tetracycline or zinc increases the numbers of MRSA ST398 in the nasal cavity of pigs. Transmission of MRSA from positive to negative animals housed within the same pen was not influenced by exposure to these agents.     

Dynamic of nasal colonization by methicillin-resistant Staphylococcus aureus ST398 and ST1 after mupirocin treatment in a family in close contact with pigs

Nasal colonization by methicillin-resistant Staphylococcus aureus (MRSA) was evaluated after a mupirocin treatment in a family previously colonized by MRSA sequence type ST398 and ST1, who lived close to a pig farm. Eight nasal samples were swabbed from each of the four family members on different moments after mupirocin treatment. The efficacy of treatment was low in those family members who worked in the farm, and higher in the remaining two family members with sporadic contact with pigs. In addition, nasal and skin swabs from randomly selected pigs of the farm were taken. MRSA were detected in 33% of pigs tested. All MRSA isolates obtained were characterized by Staphylococcal-Cassette-Chromosome mec (SCCmec) determination, Multilocus-Sequence-Typing (MLST), spa- and agr-typing, Pulsed-field-gel-electrophoresis (PFGE), antimicrobial susceptibility, detection of antimicrobial resistance genes, and toxin gene profiling. Spa-types t011, t1255 and t1197 were detected in humans and animals, with indistinguishable PFGE patterns, suggesting animal to human MRSA transmission. Each spa-type was ascribed to a specific pulsotype. Spa-types t127 and t108 were only detected in MRSA isolates obtained from humans, and t012 only in those from animals. MRSA ST1-t127 isolates and some ST398-t011 and ST398-t1197 isolates presented a multiantimicrobial-resistance phenotype. None of them harbored lukF/lukS, tst, eta and etb virulence genes. This study showed that the efficacy of nasal MRSA decolonization in healthy people with very close contact with pigs is especially low.     

Clindamycin-resistance in methicillin-resistant Staphylococcus aureus isolated from animals

Clindamycin is widely used in veterinary medicine to treat a variety of bacterial infections. Resistance to clindamycin amongst staphylococci may be due to modification of the ribosomal target site. Resistance due to this mechanism is encoded by the erm genes, which may be either constitutively or inducibly expressed. Inducible expression of resistance is not usually demonstrable by routine laboratory susceptibility testing, however, a relatively simple double disc agar diffusion test (D-test) may be used to detect the presence of inducible resistance. Two hundred and eighty-five isolates of methicillin-resistant Staphylococcus aureus (MRSA) isolated from infections in animals were tested using the D-test for the presence of inducible clindamycin resistance. One hundred and seven (71%) of the strains tested demonstrated inducible resistance to clindamycin, which were not detected on initial susceptibility testing. It has been shown that there is potential for staphylococci to develop resistance to clindamycin whilst on treatment when inducible resistance mechanisms are present. Therefore, routine screening for inducible resistance may be prudent.     

Carriage of methicillin-resistant Staphylococcus aureus by veterinarians in Australia

Objective To estimate the prevalence of carriage of methicillin‐resistant Staphylococcus aureus (MRSA) among Australian veterinarians. Methods Individuals attending veterinary conferences in Australia in 2009 were recruited to provide nasal swabs and complete a questionnaire about their professional activities. Swabs were processed by standard methods for detecting MRSA and questionnaire responses were used to group veterinarians according to their areas of major work emphasis (species and practice type). Prevalence was estimated for each of these grouping and contingency tables and regression tree analysis used to explain the variation in MRSA carriage. Results Among the 771 respondents ‘industry and government veterinarians’ (controls) had the lowest prevalence of MRSA carriage at 0.9%. Veterinarians with horses as a major area of work emphasis had a prevalence of 11.8% (13‐fold that of controls) and those whose only major emphasis was horses had a prevalence of 21.4% (23‐fold that of controls). Veterinarians with dogs and cats as a major activity had a 4.9% prevalence (5‐fold that of controls). Prevalence rates for other major activities (pigs, dairy and beef cattle, avian and wildlife) were also increased, but were estimated from smaller numbers of respondents. Regression tree analysis clearly isolated equine veterinarians and dog and cat practitioners as groups at increased risk of carriage of MRSA. Conclusion Carriage of MRSA is a notable occupational health issue for veterinarians in clinical practice in Australia, particularly those who work with horses.     

Epidemiology of methicillin-resistant Staphylococcus aureus in dairy herds.

Sixty-eight methicillin-resistant Staphylococcus aureus strains were isolated from mastitis milk samples originating from 20 Belgian dairyherds. All these strains appeared to be representatives of one single strain which was probably of human origin. Evidence is presented indicating a rapid in vivo evolutionary change in this strain. The following characteristics were found to be variable: the production of beta haemolysin inversely connected with fibrinolysin (staphylokinase) activity; the production of lipase, enterotoxin B and delta haemolysin; the resistance to neomycin, chloramphenicol, tetracyclines, methicillin and spectinomycin associated with constitutive or inducible macrolide resistance.     

Staphylococcus aureus (MSSA) and MRSA (CC398) isolated from post-mortem samples from pigs

There are many reports on the occurrence of Livestock Associated Methicilline resistant Staphylococcus aureus (LA-MRSA, CC398) in healthy pigs. There are however, very few reports of LA-MRSA being associated with pathological lesions in pigs. With this study we try to find the answers to the questions: (1) how often is S. aureus found in post-mortem material from pigs, (2) how many of these isolates are methicillin resistant, (3) are these equally distributed over the years? Here we report the isolation of MRSA and of methicillin sensitive S. aureus (MSSA) from samples derived from post-mortem examinations at the Animal Health Service in The Netherlands in the period from 2003 through October 2008. The MSSA and MRSA described here were isolated from 159 pathological lesions and from 7 submissions of aborted foetuses derived from a total of 116 animals, representing 103 submissions coming from 92 different herds. This is approximately 0.5% of all pigs submitted for post mortem examination in those years. The proportion of pigs from which S. aureus (both MSSA and MRSA) was isolated from, did not increase over the years. MSSA (N=97) and LA-MRSA CC398 (N=18) were present mainly in (peri)arthritis in over 30% of all cases, but were also isolated from internal organs such as lung, brain, spleen, kidneys, heart, indicating septicaemia. Remarkably, one non-CC398 MRSA (ST1) was isolated in a joint and a kidney of one pig. This isolate was resistant to 5 out of 6 antimicrobials tested. There was no significant difference in the type of lesions in which LA-MRSA was found compared to MSSA. The number of antimicrobials these isolates were resistant to, increased rapidly after 2004. LA-MRSA was isolated for the first time in 2005 and then again in 2007 and 2008, suggesting that this is an emerging pathogen. However, due to changes in the panel of antimicrobials used to test S. aureus for antimicrobial susceptibility in 2005 and 2007, the possibility exists that we may have missed some MRSA isolates. LA-MRSA isolates are resistant to at least three but sometimes five out of six antimicrobials tested. All isolates were susceptible to the combination of Trimethoprim/Sulfamethaxol.     

Dissimilarity of ccrAB gene sequences between methicillin-resistant Staphylococcus epidermidis and methicillin-resistant Staphylococcus aureus among bovine isolates in Korea

The sequences of the ccrAB genes from bovine-, canine- and chicken-originating methicillin-resistant Staphylococcus (S.) epidermidis (MRSE) and bovine methicillin-resistant Staphylococcus (S.) aureus (MRSA) were compared to investigate the frequency of intra-species horizontal transfer of the staphylococcal cassette chromosome mec (SCCmec) complex. Nineteen MRSE strains were isolated from bovine milk, chickens, and dogs, and their genetic characteristics were investigated by multilocus sequence typing and SCCmec typing. Among the animal MRSE strains, the most frequent SCCmec type was type IV, which consisted of the type B mec complex and ccrAB type 2. The ccrA2 and ccrB2 genes were sequenced from the bovine, chicken and canine MRSE strains and compared with those of the bovine MRSA strains. The sequences generally clustered as MRSA and MRSE groups, regardless of the animal source. Additionally, no bovine MRSE sequence was associated with the bovine MRSA groups. Although most of the bovine MRSE and MRSA isolates possessed SCCmec type IV sequences, our results suggest that the intra-species gene transfer of the SCCmec complex between bovine S. aureus and bovine S. epidermidis strains is not a frequent event.     

The prevalence of methicillin-resistant Staphylococcus aureus colonization in feedlot cattle

Methicillin-resistant Staphylococcus aureus (MRSA) is an important human pathogen and recent evidence has implicated food animals in the epidemiology of human infections in some regions. While the role of food in MRSA transmission and human health relevance are unclear, MRSA can be found in retail meat products internationally, including beef, yet there has been minimal investigation of MRSA in beef cattle. This study involved screening feedlot cattle for nasal and gastrointestinal colonization with MRSA shortly before the time of slaughter. Methicillin-resistant S. aureus was not isolated from any of 491 nasal swabs and 488 faecal samples. This finding is in contrast to studies that have isolated MRSA from retail beef in Canada, performed in the same laboratory using comparable culture techniques. The reason for this discrepancy is unclear but it demonstrates that further study of MRSA in livestock as well as slaughter, processing and retail environments is needed to elucidate the epidemiology of MRSA contamination of meat.     

Evaluation of prevalence and risk factors for methicillin-resistant Staphylococcus aureus colonization in veterinary personnel attending an international equine veterinary conference

Infection and colonization with methicillin-resistant Staphylococcus aureus (MRSA) are now increasingly being reported in animals and people who work with animals, including horses and horse personnel. The objectives of this study were to evaluate the prevalence of MRSA colonization in veterinary personnel attending an international equine veterinary conference, and to identify risk factors for MRSA colonization in this group. Volunteers were recruited at the conference to provide a single nasal swab and complete a questionnaire regarding contact with the human health care system, contact with horses, and personal hand hygiene. Colonization with MRSA was found in 26/257 (10.1%) of study participants. Multivariable analysis showed an increased risk of MRSA colonization associated with having been diagnosed with or having treated a patient diagnosed with MRSA colonization or infection in the last year (odds ratio [OR] 8.41, 95% confidence interval [CI] 1.51–49.92, P = 0.015; OR 3.27, 95% CI 1.05–10.11, P = 0.039, respectively), whereas hand washing between infectious cases (OR 0.27, 95% CI 0.11–0.72, P = 0.009) and hand washing between farms (OR 0.35, 95% CI 0.14–0.98, P = 0.047) were protective. The prevalence of MRSA colonization among equine veterinary personnel found in this study was high compared to that of other studies of the general population. These data support previous suggestions that equine veterinary personnel are at increased risk of colonization with MRSA. This is the first study to demonstrate a statistically significant association between hand hygiene practices and a measurable clinical outcome in veterinary medicine.     

Characterization of methicillin-resistant Staphylococcus aureus isolated from dogs in Korea.

Twelve strains of the methicillin-resistant Staphylococcus aureus (MRSA) recovered from hospitalized dogs were analyzed for in vitro antimicrobial susceptibility and virulence, and were genetically characterized by pulsed-field gel electrophoresis (PFGE). Antibiotic susceptibility test showed that nearly all isolates were resistant to beta-lactam antibiotics tested and all the strains were fully susceptible to glycopeptides. There were no inhibitory activities among the aminoglycosides. The 50% lethal dose (LD50) was determined by intraperitoneal injection of cell suspensions and estimated by the Spearman-K?rber method. The mouse lethality of MRSA and methicillin-susceptible S. aureus (MSSA) was not significantly different in both normal and cyclophosphamide-treated mice (p>0.05), indicating that they were equally virulent. There was a great difference in the incidence of toxin production between the MRSA and MSSA group; 83.3% (10 of 12) of the MRSA and 14.3% (1 of 7) of the MSSA were toxin producers. The predominant types produced by MRSA was B. All the MRSA strains were capsular type 5 producers, while of 7 MSSA strains, four were type 5, one for type 8, and two were nontypeable. Based on the PFGE analysis, the 12 MRSA isolates generated 9 to 11 fragments in the size range of <48.5 to 630.5 kb, and yielded 6 different patterns. The results indicated that production of toxin and capsule type do not play a role in the pathogenicity to mouse and PFGE is a valuable tool for the characterization of MRSA. This report is the first such cases in the veterinary literature in Korea and may indicate the frequent emergence of MRSA in veterinary clinic hereafter.     

The first nosocomial outbreak of methicillin-resistant Staphylococcus aureus in horses in Sweden

Background

Methicillin-resistant Staphylococcus aureus (MRSA) in animals is a rare finding in Sweden. In horses, MRSA was first detected in a screening survey in 2007. In 2008, six clinical cases occurred in an equine hospital, indicating an outbreak.

Method

All MRSA isolates detected, 11 spa-type t011 and one t064 (n = 12), in infected horses (n = 10) and screening of horses (n = 2) in Sweden from December 2007 to March 2010 were retrospectively analysed with pulsed-field gel electrophoresis (PFGE) using Cfr9I and ApaI restriction enzymes, to study relationship between the isolates. Medical records of infected horses and outbreak investigation notes were scrutinised to monitor the clinical outcome and other aspects of the outbreak.

Results

Eight of the 10 infected horses were linked to one equine hospital and two to another hospital in the same region. The six horses infected with MRSA in 2008 underwent surgery during the period 22 May-7 July in one of the hospitals. Four more infections linked to the two hospitals were notified between 2009 and March 2010.Nine of the 11 spa-type t011 isolates had identical Cfr9I and ApaI PFGE pattern. All six infected horses from 2008 presented with this MRSA. Two t011 isolates differed in one and two bands, respectively, in PFGE.Nine horses suffered from surgical site infections (SSI). No antimicrobials were used following the MRSA diagnosis and the infections cleared. The time from surgery to MRSA diagnosis differed greatly between the horses (range 15-52 days).

Conclusions

Association in time and space of six horses infected with an identical MRSA strain of spa-type t011 confirmed an outbreak. Two isolates found in 2009 and 2010 in the outbreak hospital were closely related to the outbreak strain, indicating one circulating strain. Both spa-type t011 and t064 have been reported in horses in Europe prior to these findings. The observation that the infections cleared although antimicrobials were not used is encouraging for future prudent use of antimicrobials. The time from surgery to bacteriological diagnosis was not acceptable in most cases, as contagious spread was a risk. Sampling when symptoms of infection are noticed and accurate analysis are thus important.