The Emergence and Spread of Multiple Livestock-Associated Clonal Complex 398 Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Strains among Animals and Humans in the Republic of Ireland, 2010–2014

Clonal complex (CC) 398 methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) are associated with carriage and infection among animals and humans but only a single case of CC398 MRSA has been reported in the Republic of Ireland (ROI). The present study investigated the molecular epidemiology of CC398 MRSA (n = 22) and MSSA (n = 10) from animals and humans in the ROI from 2010–2014. Isolates underwent antimicrobial susceptibility testing, spa typing, DNA microarray profiling and PCR for CC398-associated resistance genes. All MRSA underwent SCCmec IV or V subtyping. Four distinct CC398-MRSA incidents were identified from (i) a man in a nursing home (spa type t011-SCCmec IVa, immune evasion complex (IEC) negative), (ii) a horse and veterinarian who had recently travelled to Belgium (t011-IVa, IEC positive), (iii) pigs (n = 9) and farm workers (n = 9) on two farms, one which had been restocked with German gilts and the other which was a finisher farm (t034-V_T, IEC negative, 3/9 pigs; t011- V_T, IEC negative, 6/9 pigs & 9/9 farm workers), and (iv) a child who had worked on a pig farm in the UK (t034-V_T, IEC negative). Isolates also carried different combinations of multiple resistance genes including erm(A), erm(B), tet(K), tet(M) & tet(L), fexA, spc, dfrG, dfrK aacA-aphD and aadD further highlighting the presence of multiple CC398-MRSA strains. CC398 MSSA were recovered from pigs (n = 8) and humans (n = 2). CC398 MSSA transmission was identified among pigs but zoonotic transmission was not detected with animal and human isolates exhibiting clade-specific traits. This study highlights the importation and zoonotic spread of CC398 MRSA in the ROI and the spread of CC398 MSSA among pigs. Increased surveillance is warranted to prevent further CC398 MRSA importation and spread in a country that was considered CC398 MRSA free.     

Clonal Complex 398 Methicillin Susceptible Staphylococcus aureus: A Frequent Unspecialized Human Pathogen with Specific Phenotypic and Genotypic Characteristics

Clonal complex 398 livestok-associated-MRSA (CC398 LA-MRSA) clone is described as a major animal pathogen that can also colonize and infect humans. CC398 methicillin susceptible Staphylococcus aureus (CC398 MSSA) is less described. We identified 126 CC398 MSSA strains of human origin within 6380 S. aureus isolates gathered between 2009 and 2011, from the French National Reference Centre for Staphylococci. They were characterized using antimicrobial susceptibility testing, spa typing, DNA microarrays (Identibac S. aureus Genotyping ®, Alere), CC398-specific sequence PCR, ermT (encoding macrolides résistance) PCR. Fifty-three CC398 LA-MRSA collected from French pigs and veal were used as comparators, and phylogenetic relations between human CC398 MSSA and animal CC398 MRSA populations were explored on the basis of spa-typing and DNA microarrays. CC398 MSSA were able to induce a large spectrum of infections (especially skin, bloodstream, and pneumonias). The prevalence rate of this clone was high in MSSA population, i.e., 24.7% in a local prospective study on nasal colonization, and 7.5% in a national prospective study on infective endocarditis. CC398 MSSA isolates were frequently (89%) erythromycin resistant, due to the presence of the ermT gene, a gene not detected in erythromycin resistant CC398 LA-MRSA strains. Expression of staphylococcal complement inhibitor (scn) and the chemotaxis inhibitory protein (chp), was also specific to this population. The CC398 MRSA signature included also a panel of antibiotic resistance genes, especially a type IV or V cassette mec and tetM. CC398 MSSA and CC398 LA-MRSA populations were closely related based on spa-typing and DNA microarrays, with the MRSA strains forming the most derived lineage in phylogenic trees. Both MSSA and MRSA populations may come from common ancestors, which would have evolved in the settings of different selective pressures, explaining the acquisition of ermT, chp and scn for MSSA, and antibiotic resistance genes for MRSA.     

Transmission of Methicillin-Resistant Staphylococcus aureus CC398 from Livestock Veterinarians to Their Household Members

There are indications that livestock-associated MRSA CC398 has a reduced human-to-human transmissibility, limiting its impact on public health and justifying modified control measures. This study determined the transmissibility of MRSA CC398 from livestock veterinarians to their household members in the community as compared to MRSA non-CC398 strains. A one-year prospective cohort study was performed to determine the presence of MRSA CC398 in four-monthly nasal and oropharyngeal samples of livestock veterinarians (n  =  137) and their household members (n  =  389). In addition, a cross-sectional survey was performed to detect the presence of MRSA non-CC398 in hospital derived control patients (n  =  20) and their household members (n  =  41). Staphylococcus aureus isolates were genotyped by staphylococcal protein A (spa) typing and multiple-locus variable-number tandem repeat analysis (MLVA). Mean MRSA CC398 prevalence over the study period was 44% (range 41.6–46.0%) in veterinarians and 4.0% (range 2.8–4.7%) in their household members. The MRSA CC398 prevalence in household members of veterinarians was significantly lower than the MRSA non-CC398 prevalence in household members of control patients (PRR 6.0; 95% CI 2.4–15.5), indicating the reduced transmissibility of MRSA CC398. The impact of MRSA CC398 appears to be low at the moment. However, careful monitoring of the human-to-human transmissibility of MRSA CC398 remains important.     

Comparative genotypic and phenotypic characterisation of methicillin-resistant Staphylococcus aureus ST398 isolated from animals and humans

The high prevalence of methicillin-resistant Staphylococcus aureus (MRSA) ST398 among pigs in certain European countries and North America and its occurrence in other animal species raises a question concerning the molecular mechanisms mediating the success of this lineage. In this study a panel of S. aureus strains belonging to sequence type (ST) 5 (n = 4), ST8 (n = 5), ST15 (n = 5), ST22 (n = 8), clonal complex (CC) 30 (n = 8), CC97 (n = 8), CC130 (n = 4), CC151 (n = 4) and ST398 (n = 18) were screened by DNA microarray and PCR for the carriage of virulence and antimicrobial resistance genes. Isolates belonging to the same sequence type/clonal complex (ST/CC) were found to share similar virulence gene profiles. The ST398 lineage displayed the lowest content of virulence genes, which consisted mainly of genes detected among the majority or all of the analysed lineages. All MRSA ST398 isolates lacked accessory virulence genes that were detected in other ST/CC. In contrast to virulence genotype, the antimicrobial resistance genes profiles varied between isolates belonging to the same ST/CC and profile similarities could be observed for isolates from different lineages. MRSA ST398 isolates in particular displayed significant diversity and high content of antimicrobial resistance genes. This was comparable with certain MRSA belonging to other sequence types particularly the equine MRSA ST8. The apparent lack of significant virulence genes among MRSA ST398 strains, demonstrates that the lineage features a unique genetic background but no ST398-specific virulence markers could be identified.     

Molecular Epidemiology of Methicillin-Resistant Staphylococcus aureus Isolated from Australian Veterinarians

This work investigated the molecular epidemiology and antimicrobial resistance of methicillin-resistant Staphylococcus aureus (MRSA) isolated from veterinarians in Australia in 2009. The collection (n = 44) was subjected to extensive molecular typing (MLST, spa, SCCmec, dru, PFGE, virulence and antimicrobial resistance genotyping) and antimicrobial resistance phenotyping by disk diffusion. MRSA was isolated from Australian veterinarians representing various occupational emphases. The isolate collection was dominated by MRSA strains belonging to clonal complex (CC) 8 and multilocus sequence type (ST) 22. CC8 MRSA (ST8-IV [2B], spa t064; and ST612-IV [2B], spa variable,) were strongly associated with equine practice veterinarians (OR = 17.5, 95% CI = 3.3–92.5, P < 0.001) and were often resistant to gentamicin and rifampicin. ST22-IV [2B], spa variable, were strongly associated with companion animal practice veterinarians (OR = 52.5, 95% CI = 5.2–532.7, P < 0.001) and were resistant to ciprofloxacin. A single pig practice veterinarian carried ST398-V [5C2], spa t1451. Equine practice and companion animal practice veterinarians frequently carried multiresistant-CC8 and ST22 MRSA, respectively, whereas only a single swine specialist carried MRSA ST398. The presence of these strains in veterinarians may be associated with specific antimicrobial administration practices in each animal species.     

A Livestock-Associated,Multidrug-Resistant,Methicillin-Resistant Staphylococcus aureus Clonal Complex 97 Lineage Spreading in Dairy Cattle and Pigs in Italy

Pandemic methicillin-resistant Staphylococcus aureus (MRSA) clonal complex 97 (CC97) lineages originated from livestock-to-human host jumps. In recent years, CC97 has become one of the major MRSA lineages detected in Italian farmed animals. The aim of this study was to characterize and analyze differences in MRSA and methicillin-susceptible S. aureus (MSSA) mainly of swine and bovine origins. Forty-seven CC97 isolates, 35 MRSA isolates, and 6 MSSA isolates from different Italian pig and cattle holdings; 5 pig MRSA isolates from Germany; and 1 human MSSA isolate from Spain were characterized by macrorestriction pulsed-field gel electrophoresis (PFGE) analysis, multilocus sequence typing (MLST), spa typing, staphylococcal cassette chromosome mec (SCCmec) typing, and antimicrobial resistance pattern analysis. Virulence and resistance genes were investigated by PCR and microarray analysis. Most of the isolates were of SCCmec type V (SCCmec V), except for two German MRSA isolates (SCCmec III). Five main clusters were identified by PFGE, with the German isolates (clusters I and II) showing 60.5% similarity with the Italian isolates, most of which (68.1%) grouped into cluster V. All CC97 isolates were Panton-Valentine leukocidin (PVL) negative, and a few (n = 7) tested positive for sak or scn. All MRSA isolates were multidrug resistant (MDR), and the main features were erm(B)- or erm(C)-mediated (n = 18) macrolide-lincosamide-streptogramin B resistance, vga(A)-mediated (n = 37) pleuromutilin resistance, fluoroquinolone resistance (n = 33), tet(K) in 32/37 tet(M)-positive isolates, and blaZ in almost all MRSA isolates. Few host-associated differences were detected among CC97 MRSA isolates: their extensive MDR nature in both pigs and dairy cattle may be a consequence of a spillback from pigs of a MRSA lineage that originated in cattle as MSSA and needs further investigation. Measures should be implemented at the farm level to prevent spillover to humans in intensive farming areas.     

Recent Emergence of Staphylococcus aureus Clonal Complex 398 in Human Blood Cultures

Background

Recently, a clone of MRSA with clonal complex 398 (CC398) has emerged that is related to an extensive reservoir in animals, especially pigs and veal calves. It has been reported previously that methicillin-susceptible variants of CC398 circulate among humans at low frequency, and these have been isolated in a few cases of bloodstream infections (BSI). The purpose of this study was to determine the prevalence of S. aureus CC398 in blood cultures taken from patients in a geographic area with a high density of pigs.

Methodology/Principal Findings

In total, 612 consecutive episodes of S. aureus BSI diagnosed before and during the emergence of CC398 were included. Three strains (2 MSSA and 1 MRSA) that were isolated from bacteremic patients between 2010–2011 were positive in a CC398 specific PCR. There was a marked increase in prevalence of S. aureus CC398 BSI isolated between 2010–2011 compared to the combined collections that were isolated between 1996–1998 and 2002–2005 (3/157, 1.9% vs. 0/455, 0.0%; p = 0.017).

Conclusions/Significance

In conclusion, in an area with a relative high density of pigs, S. aureus CC398 was found as a cause of BSI in humans only recently. This indicates that S. aureus CC398 is able to cause invasive infections in humans and that the prevalence is rising. Careful monitoring of the evolution and epidemiology of S. aureus CC398 in animals and humans is therefore important.     

Molecular Analysis of Human and Canine Staphylococcus aureus Strains Reveals Distinct Extended-Host-Spectrum Genotypes Independent of Their Methicillin Resistance

Staphylococcus aureus causes a wide range of infectious diseases in humans and various animal species. Although presumptive host-specific factors have been reported, certain genetic lineages seem to lack specific host tropism, infecting a broad range of hosts. Such Extended-Host-Spectrum Genotypes (EHSGs) have been described in canine infections, caused by common regional human methicillin-resistant S. aureus (MRSA) lineages. However, information is scarce about the occurrence of methicillin-susceptible S. aureus (MSSA) EHSGs. To gain deeper insight into EHSG MSSA and EHSG MRSA of human and canine origin, a comparative molecular study was carried out, including a convenience sample of 120 current S. aureus (70 MRSA and 50 MSSA) isolates obtained from infected dogs. spa typing revealed 48 different spa types belonging to 16 different multilocus sequence typing clonal complexes (MLST-CCs). Based on these results, we further compared a subset of canine (n = 48) and human (n = 14) strains, including isolates of clonal complexes CC5, CC22, CC8, CC398, CC15, CC45, and CC30 by macrorestriction (pulsed-field gel electrophoresis [PFGE]) and DNA-microarray analysis. None of the methods employed was able to differentiate between clusters of human and canine strains independently of their methicillin resistance. In contrast, DNA-microarray analysis revealed 79% of the 48 canine isolates as carriers of the bacteriophage-encoded human-specific immune evasion cluster (IEC). In conclusion, the high degree of similarity between human and canine S. aureus strains regardless of whether they are MRSA or MSSA envisions the existence of common genetic traits that enable these strains as EHSGs, challenging the concept of resistance-driven spillover of MRSA.     

Prevalence and Characterization of Staphylococcus aureus in Growing Pigs in the USA

A decade of research of methicillin-resistant S. aureus (MRSA) in pigs shows that the prevalence and predominant genotypes (i.e., ST398, ST9, ST5) of MRSA vary widely geographically, yet knowledge of the epidemiology of S. aureus generally in swine remains rudimentary. To characterize S. aureus, including MRSA, in the US swine industry, we sampled 38 swine herds in 11 states in major swine producing regions. The herds sampled included pigs sourced from 9 different breeding stock companies, and the sample was likely biased towards larger herds that use regular veterinary services. Twenty nasal swabs were collected from 36 groups of growing pigs by 36 swine veterinarians, 2 more herds were sampled opportunistically, and a historically MRSA-positive herd was included as a positive control. S. aureus was detected on 37 of the 38 herds, and in 77% of pigs sampled. Other than the positive control herd, no MRSA were detected in the study sample, yielding a 95% upper confidence limit of 9.3% for MRSA herd prevalence. All but two (ST1-t127; ST2007-t8314) of 1200 isolates belonged to three MLST lineages (ST9, ST398, and ST5) that have been prominent in studies of MRSA in pigs globally. A total of 35 spa types were detected, with the most prevalent being t337 (ST9), t034 (ST398), and t002 (ST5). A purposively diverse subset of 128 isolates was uniformly negative on PCR testing for major enterotoxin genes. The findings support previous studies suggesting a relatively low herd prevalence of MRSA in the US swine industry, but confirm that methicillin susceptible variants of the most common MRSA genotypes found in swine globally are endemic in the US. The absence of enterotoxin genes suggests that the source of toxigenic S. aureus capable of causing foodborne enterotoxicosis from pork products is most likely post-harvest contamination.     

An Ex Vivo Porcine Nasal Mucosa Explants Model to Study MRSA Colonization

Staphylococcus aureus is an opportunistic pathogen able to colonize the upper respiratory tract and skin surfaces in mammals. Methicillin-resistant S. aureus ST398 is prevalent in pigs in Europe and North America. However, the mechanism of successful pig colonization by MRSA ST398 is poorly understood. To study MRSA colonization in pigs, an ex vivo model consisting of porcine nasal mucosa explants cultured at an air-liquid interface was evaluated. In cultured mucosa explants from the surfaces of the ventral turbinates and septum of the pig nose no changes in cell morphology and viability were observed up to 72 h. MRSA colonization on the explants was evaluated followed for three MRSA ST398 isolates for 180 minutes. The explants were incubated with 3×10⁸ CFU/ml in PBS for 2 h to allow bacteria to adhere to the explants surface. Next the explants were washed and in the first 30 minutes post adhering time, a decline in the number of CFU was observed for all MRSA. Subsequently, the isolates showed either: bacterial growth, no growth, or a further reduction in bacterial numbers. The MRSA were either localized as clusters between the cilia or as single bacteria on the cilia surface. No morphological changes in the epithelium layer were observed during the incubation with MRSA. We conclude that porcine nasal mucosa explants are a valuable ex vivo model to unravel the interaction of MRSA with nasal tissue.     

Alarming Proportions of Methicillin-Resistant Staphylococcus aureus (MRSA) in Wound Samples from Companion Animals,Germany 2010–2012

Staphylococcus (S.) aureus is an important cause of wound infections in companion animals, and infections with methicillin-resistant S. aureus (MRSA) are of particular concern due to limited treatment options and their zoonotic potential. However, comparable epidemiological data on MRSA infections in dogs, cats and horses is scarce, also limiting the knowledge about possible links to MRSA isolates from human populations. To gain more knowledge about the occurrence and genotypic variation of MRSA among wound swabs of companion animal origin in Germany we performed a survey (2010–2012) including 5,229 samples from 1,170 veterinary practices. S. aureus was identified in 201 (5.8%) canine, 140 (12.2%) feline and 138 (22.8%) equine swabs from a total of 3,479 canine, 1,146 feline and 604 equine wounds, respectively. High MRSA rates were identified with 62.7%, 46.4% and 41.3% in S. aureus of canine, feline and equine origin, respectively. Further genotyping including spa typing and multilocus sequence typing (MLST) revealed a comparable distribution of spa types among canine and feline MRSA with CC22 (47.6%; 49.2%) and CC5 (30.2%; 29.2%) as predominant lineages followed by CC398 (13.5%; 7.7%) and CC8 (4.0%; 9.2%). In contrast, the majority of equine MRSA belonged to CC398 (87.7%). Our data highlight the importance of S. aureus and MRSA as a cause of wound infections, particularly in cats and horses in Germany. While “human-associated” MRSA lineages were most common in dogs and cats, a remarkable number of CC398-MRSA was detected in horses, indicating a replacement of CC8-MRSA as the predominant lineage within horses in Germany. These data enforce further longitudinal epidemiological approaches to examine the diversity and temporal relatedness of MRSA populations in humans and animals to assess probable sources of MRSA infections. This would enable a sound risk assessment and establishment of intervention strategies to limit the additional spread of MRSA.     

Nasal Colonization of Humans with Methicillin-Resistant Staphylococcus aureus (MRSA) CC398 with and without Exposure to Pigs

Background

Studies in several European countries and in North America revealed a frequent nasal colonization of livestock with MRSA CC398 and also in humans with direct professional exposure to colonized animals. The study presented here addresses the question of further transmission to non exposed humans.

Methods

After selecting 47 farms with colonized pigs in different regions of Germany we sampled the nares of 113 humans working daily with pigs and of their 116 non exposed family members. The same was performed in 18 veterinarians attending pig farms and in 44 of their non exposed family members. For investigating transmission beyond families we samples the nares of 462 pupils attending a secondary school in a high density pig farming area. MRSA were detected by direct culture on selective agar. The isolates were typed by means of spa-sequence typing and classification of SCCmec elements. For attribution of spa sequence types to clonal lineages as defined by multi locus sequence typing we used the BURP algorithm. Antibiotic susceptibility testing was performed by microbroth dilution assay.

Results

At the farms investigated 86% of humans exposed and only 4.3% of their family members were found to carry MRSA exhibiting spa-types corresponding to clonal complex CC398. Nasal colonization was also found in 45% of veterinarians caring for pig farms and in 9% of their non exposed family members. Multivariate analysis revealed that antibiotic usage prior to sampling beard no risk with respect to colonization. From 462 pupils only 3 were found colonized, all 3 were living on pig farms.

Conclusion

These results indicate that so far the dissemination of MRSA CC398 to non exposed humans is infrequent and probably does not reach beyond familial communities.     

Dissemination of Methicillin-Susceptible CC398 Staphylococcus aureus Strains in a Rural Greek Area

A large collection of Staphylococcus aureus including a. 745 clinically significant isolates that were consecutively recovered from human infections during 2012–2013, b. 19 methicillin-susceptible (MSSA), randomly selected between 2006–2011 from our Staphylococcal Collection, c. 16 human colonizing isolates, and d. 10 strains from colonized animals was investigated for the presence and the molecular characteristics of CC398. The study was conducted in Thessaly, a rural region in Greece. The differentiation of livestock-associated clade from the human clade was based on canSNPs combined with the presence of the φ3 bacteriophage and the tetM, scn, sak, and chp genes. Among the 745 isolates, two MRSA (0.8% of total MRSA) and thirteen MSSA (2.65% of total MSSA) were found to belong to CC398, while, between MSSA of our Staphylococcal Collection, one CC398, isolated in 2010, was detected. One human individual, without prior contact with animals, was found to be colonized by a MSSA CC398. No CC398 was identified among the 10 S. aureus isolated from animals. Based on the molecular markers, the 17 CC398 strains were equally placed in the livestock-associated and in the human clades. This is the first report for the dissemination of S. aureus CC398 among humans in Greece.     

Livestock-Associated Methicillin Resistant and Methicillin Susceptible Staphylococcus aureus Sequence Type (CC)1 in European Farmed Animals: High Genetic Relatedness of Isolates from Italian Cattle Herds and Humans

Methicillin-resistant Staphylococcus aureus (MRSA) Sequence Type (ST)1, Clonal Complex(CC)1, SCCmec V is one of the major Livestock-Associated (LA-) lineages in pig farming industry in Italy and is associated with pigs in other European countries. Recently, it has been increasingly detected in Italian dairy cattle herds. The aim of this study was to analyse the differences between ST1 MRSA and methicillin-susceptible S. aureus (MSSA) from cattle and pig herds in Italy and Europe and human isolates. Sixty-tree animal isolates from different holdings and 20 human isolates were characterized by pulsed-field gel electrophoresis (PFGE), spa-typing, SCCmec typing, and by micro-array analysis for several virulence, antimicrobial resistance, and strain/host-specific marker genes. Three major PFGE clusters were detected. The bovine isolates shared a high (≥90% to 100%) similarity with human isolates and carried the same SCCmec type IVa. They often showed genetic features typical of human adaptation or present in human-associated CC1: Immune evasion cluster (IEC) genes sak and scn, or sea; sat and aphA3-mediated aminoglycoside resistance. Contrary, typical markers of porcine origin in Italy and Spain, like erm(A) mediated macrolide-lincosamide-streptograminB, and of vga(A)-mediated pleuromutilin resistance were always absent in human and bovine isolates. Most of ST(CC)1 MRSA from dairy cattle were multidrug-resistant and contained virulence and immunomodulatory genes associated with full capability of colonizing humans. As such, these strains may represent a greater human hazard than the porcine strains. The zoonotic capacity of CC1 LA-MRSA from livestock must be taken seriously and measures should be implemented at farm-level to prevent spill-over.     

Time-Scaled Evolutionary Analysis of the Transmission and Antibiotic Resistance Dynamics of Staphylococcus aureus Clonal Complex 398

Staphylococcus aureus clonal complex 398 (CC398) is associated with disease in humans and livestock, and its origins and transmission have generated considerable interest. We performed a time-scaled phylogenetic analysis of CC398, including sequenced isolates from the United Kingdom (Scotland), along with publicly available genomes. Using state-of-the-art methods for mapping traits onto phylogenies, we quantified transitions between host species to identify sink and source populations for CC398 and employed a novel approach to investigate the gain and loss of antibiotic resistance in CC398 over time. We identified distinct human- and livestock-associated CC398 clades and observed multiple transmissions of CC398 from livestock to humans and between countries, lending quantitative support to previous reports. Of note, we identified a subclade within the livestock-associated clade comprised of isolates from hospital environments and newborn babies, suggesting that livestock-associated CC398 is capable of onward transmission in hospitals. In addition, our analysis revealed significant differences in the dynamics of resistance to methicillin and tetracycline related to contrasting historical patterns of antibiotic usage between the livestock industry and human medicine. We also identified significant differences in patterns of gain and loss of different tetracycline resistance determinants, which we ascribe to epistatic interactions between the resistance genes and/or differences in the modes of inheritance of the resistance determinants.     

Livestock-Associated Methicillin-Resistant Staphylococcus aureus (MRSA) as Causes of Human Infection and Colonization in Germany

Pigs, cattle and poultry are colonized with MRSA and the zoonotic transmission of such MRSA to humans via direct animal contact, environmental contaminations or meat are a matter of concern. Livestock-associated (LA) MRSA are mostly belonging to clonal complex (CC) 398 as defined by multilocus sequence typing. However, MRSA of other clonal lineages including CC5, CC9 and CC97 have also been detected in livestock animals in Germany. Within the framework of a Dutch-German network project (EUREGIO), 14,036 MRSA isolated from clinical and screening specimens (January 2008 - June 2012) derived from human patients in hospitals as well as general or specialized practices in a German region characterized by a high density of livestock production, were subjected to S. aureus protein A (spa) sequence typing. The prevalence of putative LA-MRSA among the human MRSA isolates was determined by analyzing the detection of livestock-indicator (LI) spa types which had already been reported in German livestock. Overall, 578 spa types were detected among the MRSA isolates. LI spa types t011, t034, t108, t1451, t2011, t571, t1456, t1250, t1255, t1580, t2970, t2346, t1344, t2576, t2330 and t2510 (all of which are indicative for LA-MRSA CC398) accounted for 18.6% of all human isolates. The LI spa types t1430 (CC9), t3992 (CC97), t002 (CC5) and t007 (CC30) were found in 0.14%, 0.01%, 1.01% and 0.04% of all human MRSA isolates, respectively. LI spa types associated with CC398 represented 23% of all MRSA from screening samples and a varying proportion among isolates from clinical specimens ranging between 0% in cerebrospinal fluid, 8% in blood cultures and 14% in deep respiratory fluids. Our findings indicate that LA-MRSA are a major cause for human infection and stress the need for close surveillance. Although LA-MRSA CC398 predominates, the occurrence of putative LA-MRSA from other clonal lineages should be monitored.     

Rapid Differentiation between Livestock-Associated and Livestock-Independent Staphylococcus aureus CC398 Clades

Staphylococcus aureus clonal complex 398 (CC398) isolates cluster into two distinct phylogenetic clades based on single-nucleotide polymorphisms (SNPs) revealing a basal human clade and a more derived livestock clade. The scn and tet(M) genes are strongly associated with the human and the livestock clade, respectively, due to loss and acquisition of mobile genetic elements. We present canonical single-nucleotide polymorphism (canSNP) assays that differentiate the two major host-associated S. aureus CC398 clades and a duplex PCR assay for detection of scn and tet(M). The canSNP assays correctly placed 88 S. aureus CC398 isolates from a reference collection into the human and livestock clades and the duplex PCR assay correctly identified scn and tet(M). The assays were successfully applied to a geographically diverse collection of 272 human S. aureus CC398 isolates. The simple assays described here generate signals comparable to a whole-genome phylogeny for major clade assignment and are easily integrated into S. aureus CC398 surveillance programs and epidemiological studies.     

苯唑西林敏感、mecA基因阳性的猪源金黄色葡萄球菌的流行、分子分型及耐药性研究

[目的] 为了解我国猪源苯唑西林敏感-mecA阳性金黄色葡萄球菌（Oxacillin-susceptible,mecA-positive Staphylococcus aureus,OS-MRSA）的流行情况、菌株分子特征及耐药性,本研究对我国中西部4个省份（甘肃、陕西、河南和广西）的9个规模化养猪场进行鼻腔拭子样本采集。[方法] 运用PCR扩增nuc和mecA基因及苯唑西林耐药性检测对OS-MRSA菌株进行分离鉴定。然后对分离所得的OS-MRSA菌株进行26种毒素编码基因、16种抗生素耐药性以及spa、MLST和SCCmec分型检测。[结果] 结果表明,采集的884份样本中,67份样本7.6%（67/884）分离到金黄色葡萄球菌,包括50株甲氧西林敏感菌株（Methicillin-sensitive Staphylococcus aureus,MSSA）、8株苯唑西林耐受-mecA阳性金黄色葡萄球菌（Oxacillin-resistant mecA-positive,OR-MRSA）和9株OS-MRSA菌株。26种被检毒素编码基因中有9种毒素编码基因被检出,其中hla基因检出率最高,其次为hld、hlb、hlg、sei、sem、seg、sen和seo。此外,67株分离株中仅有16株携带肠毒素编码基因,其中OR-MRSA和OS-MRSA菌株分别占37.5%（6/16）和50.0%（8/16）,且携带毒素编码基因的菌株克隆型均为ST9-t899。16种所测试抗生素中,菌株对12种抗生素表现为耐药,其中MSSA、OR-MRSA和OS-MRSA分离株分别主要对1-8、10-12和7-11种抗生素耐药。所有分离株共有4种克隆型ST398-t571、ST9-t899、ST398-t034和t11241,其中ST9-t899为MRSA菌株唯一克隆型和ST398-t571为MSSA优势克隆型。除4株分离株未检测到SCCmec分型外,IV_b（76.5%,13/17）是MRSA分离株的唯一分型。[结论] 结果表明,我国猪源MRSA分离株对苯唑西林药物敏感性发生了改变,出现了较多的苯唑西林敏感菌株。此外,MSSA和MRSA分离株优势克隆型分别为ST398-t571和ST9-IVb-t899。研究还发现,克隆型与毒素编码基因有显著相关性,携带毒素编码基因的菌株克隆型均为ST9-t899。通过了解我国猪源MSSA、OR-MRSA和OS-MRSA的流行、分子特征和耐药性,可以为我国猪源金黄色葡萄球菌的防控提供数据支持。     

Analysis of Transmission of MRSA and ESBL-E among Pigs and Farm Personnel

Livestock-associated bacteria with resistance to two or more antibiotic drug classes have heightened our awareness for the consequences of antibiotic consumption and spread of resistant bacterial strains in the veterinary field. In this study we assessed the prevalence of concomitant colonization with livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) and enterobacteriaceae expressing extended-spectrum betalactamases (ESBL-E) in farms at the German-Dutch border region. Nasal colonization of pigs with MRSA (113/547 (20.7%)) was less frequent than rectal colonization with ESBL-E (163/540 (30.2%)). On the individual farm level MRSA correlated with ESBL-E recovery. The data further provide information on prevalence at different stages of pig production, including abattoirs, as well as in air samples and humans living and working on the farms. Notably, MRSA was detected in stable air samples of 34 out of 35 pig farms, highlighting air as an important MRSA transmission reservoir. The majority of MRSA isolates, including those from humans, displayed tetracycline resistance and spa types t011 and t034 characteristic for LA-MRSA, demonstrating transmission from pigs to humans. ESBL-E positive air samples were detected on 6 out of 35 farms but no pig-to-human transmission was found. Detection of ESBL-E, e.g. mostly Escherichia coli with CTX-M-type ESBL, was limited to these six farms. Molecular typing revealed transmission of ESBL-E within the pig compartments; however, related strains were also found on unrelated farms. Although our data suggest that acquisition of MRSA and ESBL-E might occur among pigs in the abattoirs, MRSA and ESBL-E were not detected on the carcasses. Altogether, our data define stable air (MRSA), pig compartments (ESBL-E) and abattoir waiting areas (MRSA and ESBL-E) as major hot spots for transmission of MRSA and/or ESBL-E along the pig production chain.