Methicillin-resistant Staphylococcus Aureus CC398 Research Articles

Transmission of methicillin-resistant Staphylococcus aureus isolates on broiler farms

The aim of the present study was to investigate the resistance pheno- and genotypes and the molecular typing characteristics of methicillin-resistant Staphylococcus aureus (MRSA) isolates from broiler farms in order to explore transmission between the different reservoirs. Thirty-seven MRSA CC398 isolates (11 from broilers, 15 from the broiler houses, 5 from farm residences and 6 from humans living and/or working on the farms) cultured from samples at four different farms during a previous study, were included. In addition to the previously determined spa types, the isolates were characterized by dru typing, SCCmec typing, pulsed-field gel electrophoresis and DNA microarray. Resistance phenotypes were determined by broth microdilution. Resistance genes were detected by DNA microarray or specific PCR assays. Selected isolates from broilers and humans (n=7) were analysed by whole genome mapping. On the same farm, isolates from chickens, broiler houses, the farm residences and humans were often closely related or indistinguishable. On three of the four farms, however, MRSA isolates with different characteristics were present. On the one hand, the apparent similarity of MRSA isolates from the same farm indicates transmission between broilers, humans and their environment. On the other hand, different MRSA isolates were present on the same farm, indicating introduction from different sources or diversification over time. This study shows that different typing methods should be used to investigate epidemiological links between isolates and that whole genome mapping can be a useful tool to establish these links.

Livestock-Associated Methicillin and Multidrug Resistant Staphylococcus aureus Is Present among Industrial, Not Antibiotic-Free Livestock Operation Workers in North Carolina

ObjectivesAdministration of antibiotics to food animals may select for drug-resistant pathogens of clinical significance, such as methicillin-resistant Staphylococcus aureus (MRSA). In the United States, studies have examined prevalence of MRSA carriage among individuals exposed to livestock, but prevalence of multidrug-resistant S. aureus (MDRSA) carriage and the association with livestock raised with versus without antibiotic selective pressure remains unclear. We aimed to examine prevalence, antibiotic susceptibility, and molecular characteristics of S. aureus among industrial livestock operation (ILO) and antibiotic-free livestock operation (AFLO) workers and household members in North Carolina.MethodsParticipants in this cross-sectional study were interviewed and provided a nasal swab for S. aureus analysis. Resulting S. aureus isolates were assessed for antibiotic susceptibility, multi-locus sequence type, and absence of the scn gene (a marker of livestock association).ResultsAmong 99 ILO and 105 AFLO participants, S. aureus nasal carriage prevalence was 41% and 40%, respectively. Among ILO and AFLO S. aureus carriers, MRSA was detected in 7% (3/41) and 7% (3/42), respectively. Thirty seven percent of 41 ILO versus 19% of 42 AFLO S. aureus-positive participants carried MDRSA. S. aureus clonal complex (CC) 398 was observed only among workers and predominated among ILO (13/34) compared with AFLO (1/35) S. aureus-positive workers. Only ILO workers carried scn-negative MRSA CC398 (2/34) and scn-negative MDRSA CC398 (6/34), and all of these isolates were tetracycline resistant.ConclusionsDespite similar S. aureus and MRSA prevalence among ILO and AFLO-exposed individuals, livestock-associated MRSA and MDRSA (tetracycline-resistant, CC398, scn-negative) were only present among ILO-exposed individuals. These findings support growing concern about antibiotics use and confinement in livestock production, raising questions about the potential for occupational exposure to an opportunistic and drug-resistant pathogen, which in other settings including hospitals and the community is of broad public health importance.

Dynamics and Determinants of Staphylococcus aureus Carriage in Livestock Veterinarians: A Prospective Cohort Study

Since 2003, a new clade of methicillin-resistant Staphylococcus aureus (MRSA) belonging to clonal complex (CC) 398 and associated with animal husbandry has emerged in the Netherlands. The purpose of this study was to determine the dynamics of carriage in persons with direct contact to livestock. A 2-year prospective cohort study was performed in which the anterior nares and oropharynx of 137 livestock veterinarians were sampled for the presence of S. aureus every 4 months during the first year and again 1 year later. All S. aureus isolates were genotyped by staphylococcal protein A (spa) typing and with multilocus variable-number tandem repeat analysis (MLVA). The mean prevalence of MRSA CC398 carriage was 44% (range, 42%-46%), and for S. aureus the prevalence was 72% (range, 69%-75%). Thirty-two veterinarians (23%) were always carrying MRSA CC398 and 18 of those (56%, 13% of all veterinarians) had identical MLVA types at all sampling moments. A high proportion of veterinarians had persistent MRSA CC398 carriage during the 2-year study period, indicating that this variant may colonize humans for prolonged periods. Furthermore, prevalence of S. aureus carriage was extremely high, indicating that MRSA CC398 is not replacing the susceptible strains, but comes on top of it.

Identification of the novel spectinomycin resistance gene spw in methicillin-resistant and methicillin-susceptible Staphylococcus aureus of human and animal origin

Sir, Spectinomycin resistance in staphylococci is mostly mediated by spectinomycin 9-O-adenyltransferase encoded by the spc gene. This gene is located together with the macrolide–lincosamide– streptogramin B resistance gene erm(A) on transposon Tn554. The analysis of methicillin-resistant Staphylococcus aureus (MRSA) CC398 from cases of bovine mastitis showed that all spectinomycin-resistant isolates carried the gene spc along with erm(A), whereas MRSA CC398 isolates from turkey meat and turkey meat products occasionally exhibited spectinomycin resistance, but lacked the spc gene. This observation pointed towards the existence of other as yet unknown spectinomycin resistance genes in staphylococci. Most recently, the analysis of MRSA ST398 and methicillin-susceptible S. aureus (MSSA) ST9 isolates of human origin from Spain, but also MRSA ST9 isolates from swine in China, revealed the presence of multiresistance gene clusters that were most likely of enterococcal origin. – 7 So far, two different types of these multiresistance gene clusters have been identified, both of which carry the resistance genes aadE (streptomycin resistance), lnu(B) (lincosamide resistance) and lsa(E) (pleuromutilin– lincosamide–streptogramin A resistance). A closer look at these clusters revealed the presence of a reading frame for a putative spectinomycin resistance gene between aadE and lsa(E). This reading frame is 810 bp in size, has an unusual start codon (TTG) and codes for an adenyltransferase protein of 269 amino acids. Database searches identified identical or closely related proteins in Enterococcus faecium (e.g. GenBank accession number EFF20873), Enterococcus faecalis (e.g. GenBank accession number EEU82264) and Lactobacillus johnsonii (e.g. GenBank accession number EGP12870) (Figure 1). The corresponding proteins were referred to as streptomycin 3′′-adenylyltransferase, spectinomycin 9-O-adenylyltransferase, putative toxin-antitoxin system or a hypothetical protein in the respective database entries. Moreover, three database entries (GenBank accession numbers AAL05551, AFM38045 and AFU35063) reported a protein that was 27 amino acids shorter. This was due to the annotation of a wrong start codon (ATG) upstream of which no ribosome binding site was found. Analysis of the corresponding nucleotide sequences (GenBank accession numbers AF408195, JQ861959 and JX560992, respectively) confirmed the presence of the larger correct reading frame of 810 bp. To determine whether or not this protein confers resistance to streptomycin or spectinomycin, we decided to clone the gene and express it in an S. aureus host. For this, the recently developed PCR5 assay (forward: 5′-TTGGATTGCAGCATTATTGG-3′, reverse: 5′-ATTTGGTCGAAGCCTT GTTG-3′; annealing temperature 568C) was used to generate an amplicon of 1985 bp from the cloned 17.5 kb segment of plasmid pV7037 (GenBank accession number JX560992) from the porcine MRSA ST9 isolate SA7037. This amplicon included the entire reading frame for the putative resistance gene and 239 bp in the upstream and 936 bp in the downstream parts. The amplicon was initially cloned into pCR-Blunt II-TOPO (Life Technologies GmbH, Darmstadt, Germany), and the recombinant vector was transformed into Escherichia coli recipient strain TOP10. The amplicon was then cut off from this vector by EcoRI digestion and inserted into the single EcoRI site of the E. coli–S. aureus shuttle vector pLI50. This recombinant shuttle vector was first introduced by electrotransformation into E. coli TOP10 and subsequently into S. aureus RN4220. Antimicrobial susceptibility testing was performed by broth macrodilution for spectinomycin and streptomycin according to CLSI document M31-A3 with S. aureus ATCC 29213 as the quality control strain. The MICs of streptomycin were 4 mg/L for S. aureus RN4220, S. aureus RN4220 carrying pLI50 and S. aureus RN4220 carrying the recombinant pLI50, suggesting that the cloned gene does not confer streptomycin resistance. In contrast, the MICs of spectinomycin were 32 mg/L for S. aureus RN4220 and S. aureus RN4220 carrying pLI50, but were distinctly higher at 4096 mg/L for S. aureus RN4220 carrying the recombinant pLI50 with the cloned gene. This observation confirmed that the gene in question was a spectinomycin resistance gene, which was then designated spw. Besides the occurrence of spw in E. faecium, E. faecalis and L. johnsonii, a gene was detected in Streptococcus suis that encoded a protein of 262 amino acids (GenBank accession number AER19630) that showed 93.9% amino acid identity to Spw. In contrast, the Spw protein exhibited only 64.7% identity to the Tn554-associated Spc protein (Figure 1). To see whether spw was present in other staphylococci, a PCR assay (forward: 5′-CGGCAGTAATGGGTGGTTTA-3′, reverse: 5′-CAGCCACC TCAGATTCCATT-3′; annealing temperature 548C) was developed

Livestock-associated Staphylococcus aureus CC398: Animal reservoirs and human infections

Over the past 15years the epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) has changed significantly. Being initially a nosocomial pathogen, other clones have been detected in the community, leading to infections in relatively young and healthy individuals lacking contact with healthcare. More recently, a specific clone of MRSA CC398 emerged, which has spread extensively in livestock animals and is also found in retail meat. People in contact with food production animals are at high risk of colonization. The ways in which MRSA CC398 can be transmitted to humans are direct contact with animals, environmental contamination, and eating or handling contaminated meat. The role of MRSA CC398 as a food pathogen needs further research. Recently, whole genome sequencing and other genetic analyses have shown that livestock-associated strains are distinct from human-derived strains. However, there is also an exchange of strains between the reservoirs. Livestock-associated and human-associated strains of CC398 share some virulence factors, but there are also distinct virulence factors that appear to be important in host adaptation. Exchange of genes encoding these virulence factors between strains may expand the host range and thereby threaten public health. Since the emergence of MRSA CC398 in humans, approximately 10years ago, this clone has shown a remarkable evolution, which is described in this review.

Transmission Dynamics of Methicillin-Resistant Staphylococcus aureus in Pigs.

From the mid-2000s on, numerous studies have shown that methicillin-resistant Staphylococcus aureus (MRSA), renowned as human pathogen, has a reservoir in pigs and other livestock. In Europe and North America, clonal complex (CC) 398 appears to be the predominant lineage involved. Especially worrisome is its capacity to contaminate humans in close contact with affected animals. Indeed, the typical multi-resistant phenotype of MRSA CC398 and its observed ability of easily acquiring genetic material suggests that MRSA CC398 strains with an increased virulence potential may emerge, for which few therapeutic options would remain. This questions the need to implement interventions to control the presence and spread of MRSA CC398 among pigs. MRSA CC398 shows a high but not fully understood transmission potential in the pig population and is able to persist within that population. Although direct contact is probably the main route for MRSA transmission between pigs, also environmental contamination, the presence of other livestock, the herd size, and farm management are factors that may be involved in the dissemination of MRSA CC398. The current review aims at summarizing the research that has so far been done on the transmission dynamics and risk factors for introduction and persistence of MRSA CC398 in farms.

Low MRSA prevalence in horses at farm level

BackgroundIn Europe, methicillin-resistant Staphylococcus aureus (MRSA) belonging to the clonal complex (CC) 398 has become an important pathogen in horses, circulating in equine clinics and causing both colonization and infection. Whether equine MRSA is bound to hospitals or can also circulate in the general horse population is currently unknown. This study, therefore, reports the nasal and perianal MRSA screening of 189 horses on 10 farms in a suspected high prevalence region (East- and West-Flanders, Belgium).ResultsOnly one horse (0.53%) from one farm (10%) tested positive in the nose. It carried a spa type t011-SCCmecV isolate, resistant to β-lactams and tetracycline, which is typical for livestock-associated MRSA CC398.ConclusionIn the region tested here, horses on horse farms seem unlikely to substantially contribute to the large animal associated ST398 MRSA reservoir present at intensive animal production units.

High prevalence of spa types associated with the clonal lineage CC398 among tetracycline-resistant methicillin-resistant Staphylococcus aureus strains in a Spanish hospital

The clonal lineages, resistance mechanisms and virulence traits of tetracycline-resistant (Tet(R)) methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in a Spanish hospital during 2009 and 2010 were investigated. Fifty-two Tet(R) MRSA strains from unrelated patients were included in this study. Susceptibility to 26 antimicrobial agents was determined and 24 resistance genes were tested for by PCR. The sequences of the genes grlA and gyrA were analysed in all ciprofloxacin-resistant MRSA isolates. For all strains, spa, SCCmec and agr typing was implemented. Multilocus sequence typing was performed for 16 representative strains of the different spa types. The presence of the genes tst, lukF/lukS-PV, eta, etb, etd and cna was investigated by PCR. Fifteen different spa types, four of them new ones, were detected among the 52 strains, being associated with the following clonal complexes (CCs): CC398 (67.3%), CC1 (11.5%), CC5 (11.5%) and CC8 (9.6%). A novel sequence type (ST), ST2077, belonging to CC398 was identified. Most MRSA CC398 strains were typed as SCCmecV-agrI. In addition to β-lactam resistance, isolates showed resistance to (gene/number of strains): tetracycline [tet(K)/36, tet(L)/8 and tet(M)/48], macrolides and lincosamides [erm(B)/6, erm(C)/25, erm(T)/2, msr(A)/msr(B)/4 and mph(C)/4], aminoglycosides [aac(6')-Ie-aph(2')-Ia/8, ant(4')-Ia/13 and aph(3')-IIIa/15], trimethoprim [dfrS1/2 and dfrK/3] and mupirocin (mupA/3). Strains investigated for mutations mediating quinolone resistance revealed an S80F exchange in GrlA and different changes in GyrA. Three strains were Panton-Valentine leucocidin-positive (ST8 and ST94) and 41 strains were cna-positive. All MRSA isolates were negative for the genes tst, eta, etb and etd. Tetracycline resistance could be a good marker for MRSA CC398, although this resistance can also be found in other lineages.

Farm-specific lineages of methicillin-resistant Staphylococcus aureus clonal complex 398 in Danish pig farms

The objective of this study was to investigate the genetic diversity of methicillin-resistant Staphylococcus aureus (MRSA) clonal complex (CC) 398 using pulsed-field gel electrophoresis (PFGE). Dust and pigs at five age groups were sampled in six Danish MRSA-positive pig farms. MRSA CC398 was isolated from 284 of the 391 samples tested, including 230 (74%) animal and 54 (68%) environmental samples. PFGE analysis of a subset of 48 isolates, including the six strains previously isolated from farm workers, revealed the existence of farm-specific pulsotypes. With a single exception, human, environmental and porcine isolates originating from the same farm clustered together in the PFGE cluster analysis, indicating that spread of MRSA CC398 in Danish pig farms is mainly due to clonal dissemination of farm-specific lineages that can be discriminated by PFGE. This finding has important implications for planning future epidemiological studies investigating the spread of CC398 in pig farming.

Detection of the novel vga(E) gene in methicillin-resistant Staphylococcus aureus CC398 isolates from cattle and poultry

Detection of the novel vga(E) gene in methicillin-resistant Staphylococcus aureus CC398 isolates from cattle and poultry

Zinc resistance of Staphylococcus aureus of animal origin is strongly associated with methicillin resistance

This study was conducted to determine the occurrence of zinc and copper resistances in methicillin-resistant Staphylococcus aureus (MRSA) from swine and veal calves in a global strain collection.The test population consisted of 476 porcine MRSA isolates from ten European countries, 18 porcine MRSA isolates from Canada and seven MRSA from China, 92 MRSA and 60 methicillin-susceptible S. aureus (MSSA) isolates from veal calves in the Netherlands and 88 porcine MSSA isolates from four European countries. Most porcine MRSA (n=454) and all bovine MRSA belonged to clonal complex (CC) 398 whereas 37 of the pig MRSA from Europe and the seven Chinese isolates belonged to other CCs and 3 isolates were not classified into a CC.All isolates were tested for susceptibility to zinc chloride and copper sulphate using agar dilution and tested by PCR for the czrC gene encoding zinc resistance.Phenotypic zinc resistance (MIC>2mM) was observed in 74% (n=324) and 42% (n=39) of European MRSA CC398 from pigs and veal calves, respectively, and in 44% of the Canadian isolates (n=8), but not among the Chinese isolates. Almost all (99%) zinc-resistant MRSA carried czrC. Of the 37 European non-CC398 MRSA, 62% were resistant to zinc, but only 46% of them carried czrC. The MICs of the MSSA isolates to zinc chloride ranged from 1 to 4mM and none carried czrC. The MICs of copper sulphate were associated neither with methicillin resistance nor with the detection of czrC.This study showed that zinc resistance and the czrC gene are widespread among CC398 MRSA isolates. This suggests that the use of zinc in feed might have contributed to the emergence of MRSA.

Detection, Molecular Characterization, and Clonal Diversity of Methicillin-ResistantStaphylococcus aureusCC398 and CC97 in Spanish Slaughter Pigs of Different Age Groups

The objective of this study was to determine the frequency of nasal carriage of methicillin-resistant Staphylococcus aureus (MRSA) in slaughter pigs, to characterize the recovered isolates, and to investigate their genomic relatedness. Nasal swabs were collected from 53 finishing-pigs (F-pigs) and 53 suckling-piglets (S-piglets) at two different abattoirs in La Rioja (Northern Spain) coming from six production holdings. MRSA isolates were characterized by spa−, agr−, SCCmec−, and multilocus sequence typing, pulsed-field gel electrophoresis (PFGE)-ApaI, toxin gene profiling, antimicrobial susceptibility, and determination of antimicrobial resistance genes. MRSA isolates were recovered from 11 F-pigs (14 isolates) and 26 S-piglets (30 isolates). Forty of the 44MRSA presented the spa-types t011, t108, t1197, and t2346, which corresponded to the sequence type ST398 and to the clonal complex CC398. Interestingly, the remaining four isolates from F-pigs presented the spa-type t3992, and they were ascribed to a new sequence type named ST1379 (a single-locus variant of ST97), which was included in clonal complex CC97. Five PFGE-ApaI clusters with up to nine individual patterns detected among our MRSA and low genomic relatedness was observed between F-pig and S-piglet isolates. All MRSA were positive for hla, hld, and hlg hemolysin genes. ST1379 isolates harbored eta, lukE/D, and hlg-2 toxin genes, whereas ST398 isolates were positive for hlb. A great variety of distinct resistance gene patterns were observed, most of them coming from F-pig isolates. MRSA virulence properties seem to be dependent of the isolate clonal lineage. This study showed that slaughter pigs are frequently colonized by MRSA CC398; moreover, the detection of strains belonging to CC97 underlines that other lineages are also able to spread in livestock. Further studies should assess the risk of CC398 and non-CC398 MRSA to enter the food chain as well as the human health implications.

Methicillin-resistantStaphylococcus aureus(MRSA) in food production animals

Until recently, reports on methicillin-resistant Staphylococcus aureus (MRSA) in food production animals were mainly limited to occasional detections in dairy cattle mastitis. However, since 2005 a MRSA clone, CC398, has been reported colonizing pigs, veal calves and broiler chickens and infecting dairy cows. Many aspects of its prevalence in pigs remain unclear. In other livestock, colonizing capacity and reservoir status still require elucidation. MRSA CC398 has also been detected in meat, but, as for other MRSA, the risk this poses is somewhat unclear. Currently, the most worrying aspect of MRSA CC398 appears to be its capacity to spread to humans. This might complicate MRSA control measures in human healthcare, urging research into risk factors and transmission routes. Although infections with MRSA CC398 are much less reported than carriage, more investigation into its pathogenic potential is required. Moreover, the origin and evolution of this clone remain unknown.

Single-nucleotide polymorphism in the SCCmec-orfX junction distinguishes between livestock-associated MRSA CC398 and human epidemic MRSA strains

A number of real-time PCR assays for direct detection of methicillinresistant (MRSA) in clinical specimens are targeting staphylococcal cassette chromosome mec (SCCmec) right extremity sequences and the S. aureus chromosomal orfX gene sequences located to the right of the SCCmec integration site. When testing 184 MRSA strains of human and animal origin from geographically distinct locations, we identified several characteristic single-nucleotide polymorphisms (SNPs) within the SCCmec-orfX junction of livestock-associated (LA) MRSA CC398 which serve as suitable strain markers for screening purposes. Within an assay time of 60 minutes and an additional 10 minutes for the melting curve analysis, all MRSA CC398 isolates were correctly identified by their characteristic T(m) value in the commercial LightCycler MRSA Advanced test. Studies to confirm the diagnostic accuracy of the SNP-based strain identification assay with a larger collection of clinical and LA-MRSA strains are ongoing.