Characteristics of methicillin-resistant Staphylococcus aureus from broiler farms in Germany are rather lineage- than source-specific.

Methicillin-resistant Staphylococcus aureus (MRSA) are a major concern for public health, and broiler farms are a potential source of MRSA isolates. In this study, a total of 56 MRSA isolates from 15 broiler farms from 4 different counties in Germany were characterised phenotypically and genotypically. Spa types, dru types, SCCmec types, and virulence genes as well as resistance genes were determined by using a DNA microarray or specific PCR assays. In addition, PFGE profiles of isolates were used for analysis of their epidemiological relatedness. While half of the isolates belonged to spa type t011, the other half was of spa types t1430 and t034. On 3 farms, more than 1 spa type was found. The most common dru type was dt10a (n = 19), followed by dt11a (n = 17). Susceptibility testing of all isolates by broth microdilution revealed 21 different resistance phenotypes and a wide range of resistance genes was present among the isolates. Up to 10 different resistance phenotypes were found on individual farms. Resistance to tetracyclines (n = 53), MLSB antibiotics (n = 49), trimethoprim (n = 38), and elevated MICs of tiamulin (n = 29) were most commonly observed. Microarray analysis detected genes for leucocidin (lukF/S), haemolysin gamma (hlgA), and other haemolysines in all isolates. In all t1430 isolates, the egc cluster comprising of genes encoding enterotoxin G, I, M, N, O, U, and/or Y was found. The splitstree analysis based on microarray and PCR gene profiles revealed that all CC9/SCCmec IV/t1430/dt10a isolates clustered apart from the other isolates. These findings confirm that genotypic patterns were specific for clonal lineages rather than for the origin of isolates from individual farms.

Variability of SCCmec elements in livestock-associated CC398 MRSA.

The most common livestock-associated lineage of methicillin-resistant Staphylococcus aureus (MRSA) in Western Europe is currently clonal complex (CC) 398. CC398-MRSA spread extensively across livestock populations in several Western European countries, and livestock-derived CC398-MRSA strains can also be detected in humans. Based on their SCCmec elements, different CC398 strains can be distinguished. SCCmec elements of 100 veterinary and human CC398-MRSA isolates from Germany and Austria were examined using DNA microarray-based assays. In addition, 589 published SCC and/or genome sequences of CC398-MRSA (including both, fully finished and partially assembled sequences) were analysed by mapping them to the probe sequences of the microarrays. Several isolates and sequences showed an insertion of a large fragment of CC9 genomic DNA into the CC398 chromosome. Fifteen subtypes of SCCmec elements were detected among the 100 CC398 isolates and 41 subtypes could be discerned among the published CC398 sequences. Eleven of these were also experimentally detected within our strain collection, while four subtypes identified in the isolates where not found among the sequences. A high prevalence of heavy metal resistance genes, especially of czrC, was observed among CC398-MRSA. A possible co-selection of resistances to antibiotics and zinc/copper supplements in animal feed as well as a spill-over of SCCmec elements that have evolved in CC398-MRSA to other, possibly more virulent and/or medically relevant S. aureus lineages might pose public health problems in future.

Recombination-Mediated Host Adaptation by Avian Staphylococcus aureus.

Staphylococcus aureus are globally disseminated among farmed chickens causing skeletal muscle infections, dermatitis, and septicaemia. The emergence of poultry-associated lineages has involved zoonotic transmission from humans to chickens but questions remain about the specific adaptations that promote proliferation of chicken pathogens. We characterized genetic variation in a population of genome-sequenced S. aureus isolates of poultry and human origin. Genealogical analysis identified a dominant poultry-associated sequence cluster within the CC5 clonal complex. Poultry and human CC5 isolates were significantly distinct from each other and more recombination events were detected in the poultry isolates. We identified 44 recombination events in 33 genes along the branch extending to the poultry-specific CC5 cluster, and 47 genes were found more often in CC5 poultry isolates compared with those from humans. Many of these gene sequences were common in chicken isolates from other clonal complexes suggesting horizontal gene transfer among poultry associated lineages. Consistent with functional predictions for putative poultry-associated genes, poultry isolates showed enhanced growth at 42 °C and greater erythrocyte lysis on chicken blood agar in comparison with human isolates. By combining phenotype information with evolutionary analyses of staphylococcal genomes, we provide evidence of adaptation, following a human-to-poultry host transition. This has important implications for the emergence and dissemination of new pathogenic clones associated with modern agriculture.

Characterization of canine and feline methicillin-resistant Staphylococcus pseudintermedius (MRSP) from Thailand.

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) in small animal practice are very difficult to treat due to multi-resistance. In contrast to other countries, little is known about MRSP from Thailand. In particular, information on feline MRSP isolates in general is rare. In total, 39 MRSP isolates from dogs (n=28) and cats (n=11) from Thailand collected from independent clinical cases were used. Oxacillin resistance and presence of the mecA gene was confirmed. Susceptibility to additional 29 antimicrobial agents was tested according to CLSI recommendations. Antimicrobial resistance genes were detected by PCR assays. Molecular typing comprised spa typing, dru typing and macrorestriction analysis with subsequent pulsed-field gel electrophoresis (PFGE). For selected isolates, multi-locus sequence typing (MLST) was performed. All isolates were multi-resistant with resistance to at least six classes of antimicrobial agents. In all cases corresponding resistance genes were detected. In addition to mecA, the genes blaZ, catpC221, aacA/aphD, erm(B), dfrG, tet(M) and tet(K) were identified. Six spa types (t02, t05, t09, t10, t23, t72), eleven dru types (dt8ak, dt10ao, dt10cp, dt10cq, dt11a, dt11bo, dt11cb, dt11cj, dt11v, dt11y, dt11z) and 27 PFGE types (designated as A1-A10, B1-B8, C1-C2, D, E, F, G, H, I, J) were identified. MLST for one isolate of each main PFGE pattern A-J revealed seven types [ST45 (n=3), ST112, ST155, ST282 and the novel types ST432, ST433 (n=2) and ST434]. This study showed that MRSP isolates from clinical cases in individual dogs and cats in Thailand are multi-resistant with similar resistance genes and characteristics as isolates from Europe and North America.

Detection of the macrolide-lincosamide-streptogramin B resistance gene erm(44) and a novel erm(44) variant in staphylococci from aquatic environments.

Resistance to macrolides, lincosamides and streptogramin B antibiotics (MLSB) is not restricted to staphylococci from clinical samples but can also be present in staphylococci from the aquatic environment. Two coagulase-negative staphylococci-Staphylococcus xylosus and S. saprophyticus were obtained from sewage and receiving river water samples and were investigated for the genetic basis of inducible MLSB resistance by whole-genome sequencing. Two rRNA methylases encoded by erm(44) and a novel erm(44) variant were identified, which had only 84% amino acid identity. While fragments of phage DNA were found in the vicinity of the erm(44) gene of S. xylosus, no relics of mobile genetic elements were detected in the sequences flanking the erm(44) variant gene in the S. saprophyticus strain. The functionality of the erm genes was confirmed by cloning and transformation experiments. Based on the obtained sequences, specific PCR assays for both erm genes were developed and used to identify erm(44) in another 7 S. xylosus and 17 S. saprophyticus isolates from aquatic environments.

Identification of ABC transporter genes conferring combined pleuromutilin-lincosamide-streptogramin A resistance in bovine methicillin-resistant Staphylococcus aureus and coagulase-negative staphylococci.

The aim of this study was to investigate the genetic basis of combined pleuromutilin-lincosamide-streptogramin A resistance in 26 unrelated methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative staphylococci (CoNS) from dairy cows suffering from mastitis. The 26 pleuromutilin-resistant staphylococcal isolates were screened for the presence of the genes vga(A), vga(B), vga(C), vga(E), vga(E) variant, sal(A), vmlR, cfr, lsa(A), lsa(B), lsa(C), and lsa(E) by PCR. None of the 26 isolates carried the genes vga(B), vga(C), vga(E), vga(E) variant, vmlR, cfr, lsa(A), lsa(B), or lsa(C). Two Staphylococcus haemolyticus and single Staphylococcus xylosus, Staphylococcus lentus, and Staphylococcus hominis were vga(A)-positive. Twelve S. aureus, two Staphylococcus warneri, as well as single S. lentus and S. xylosus carried the lsa(E) gene. Moreover, single S. aureus, S. haemolyticus, S. xylosus, and Staphylococcus epidermidis were positive for both genes, vga(A) and lsa(E). The sal(A) gene was found in a single Staphylococcus sciuri. All ABC transporter genes were located in the chromosomal DNA, except for a plasmid-borne vga(A) gene in the S. epidermidis isolate. The genetic environment of the lsa(E)-positive isolates was analyzed using previously described PCR assays. Except for the S. warneri and S. xylosus, all lsa(E)-positive isolates harbored a part of the previously described enterococcal multiresistance gene cluster. This is the first report of the novel lsa(E) gene in the aforementioned bovine CoNS species. This is also the first identification of the sal(A) gene in a S. sciuri from a case of bovine mastitis. Moreover, the sal(A) gene was shown to also confer pleuromutilin resistance.

Emerging issues in antimicrobial resistance of bacteria from food-producing animals.

During the last decade, antimicrobial resistance in bacteria from food-producing animals has become a major research topic. In this review, different emerging resistance properties related to bacteria of food-producing animals are highlighted. These include: extended-spectrum ß-lactamase-producing Enterobacteriaceae; carbapenemase-producing bacteria; bovine respiratory tract pathogens, such as Pasteurella multocida and Mannheimia haemolytica, which harbor the multiresistance mediating integrative and conjugative element ICEPmu1; Gram-positive and Gram-negative bacteria that carry the multiresistance gene cfr; and the occurrence of numerous novel antimicrobial resistance genes in livestock-associated methicillin-resistant Staphylococcus aureus. The emergence of the aforementioned resistance properties is mainly based on the exchange of mobile genetic elements that carry the respective resistance genes.

Multidrug resistance genes in staphylococci from animals that confer resistance to critically and highly important antimicrobial agents in human medicine.

Most antimicrobial resistance genes known so far to occur in staphylococci of animal origin confer resistance to a specific class of antimicrobial agents or to selected members within such a class. However, there are also a few examples of multidrug resistance (MDR) genes that confer resistance to antimicrobial agents of different classes by either target site methylation or active efflux via ATP-binding cassette (ABC) transporters. The present review provides an overview of these MDR genes with particular reference to those genes involved in resistance to critically or highly important antimicrobial agents used in human and veterinary medicine. Moreover, their location on mobile genetic elements and colocated resistance genes, which may play a role in coselection and persistence of the MDR genes, are addressed.

Two different erm(C)-carrying plasmids in the same methicillin-resistant Staphylococcus aureus CC398 isolate from a broiler farm.

During a study on plasmid-borne antimicrobial resistance among methicillin-resistant Staphylococcus aureus (MRSA) isolates from broiler farms, an MRSA isolate was identified which carried multiple plasmids. This MRSA isolate belonged to CC398 and exhibited spa type t3015 and dru type dt11a. Plasmid profiling revealed the presence of one large and two small plasmids. The resistance genes tet(L) (tetracycline resistance), dfrK (trimethoprim resistance) and aadD (kanamycin/neomycin resistance) were located on the large plasmid. Both small plasmids, designated pSWS371 and pSWS372, carried only an erm(C) gene for macrolide/lincosamide resistance. Sequence analysis revealed that the 2458-bp plasmid pSWS371 carried only a repL gene for plasmid replication in addition to the erm(C) gene. In contrast, the 3882-bp plasmid pSWS372 harbored - in addition to the erm(C) gene - three more genes: a repF gene for plasmid replication, a cop-6 gene for a small protein potentially involved in copy number control of the plasmid and a novel pre/mob gene for a protein involved in plasmid recombination and mobilization. The erm(C) genes of both small plasmids exhibited constitutive erm(C) gene expression and analysis of the respective translational attenuators identified deletions of 16 bp and 74 bp which explain the constitutive expression. The simultaneous presence of two small plasmids that carry the same resistance gene in the same MRSA isolate is a rare observation. The fact that both plasmids belong to different incompatibility groups as specified by the different rep genes, repL and repF, explains why they can stably coexist in the same bacterial cell.

Identification of a novel vga(E) gene variant that confers resistance to pleuromutilins, lincosamides and streptogramin A antibiotics in staphylococci of porcine origin.

OBJECTIVES: To investigate the genetic basis of pleuromutilin resistance in coagulase-negative staphylococci of porcine origin that do not carry known pleuromutilin resistance genes and to determine the localization and genetic environment of the identified resistance gene. METHODS: Plasmid DNA of two pleuromutilin-resistant Staphylococcus cohnii and Staphylococcus simulans isolates was transformed into Staphylococcus aureus RN4220. The identified resistance plasmids were sequenced completely. The candidate gene for pleuromutilin resistance was cloned into shuttle vector pAM401. S. aureus RN4220 transformants carrying this recombinant shuttle vector were tested for their MICs. RESULTS: S. cohnii isolate SA-7 and S. simulans isolate SSI1 carried the same plasmid of 5584 bp, designated pSA-7. A variant of the vga(E) gene was detected, which encodes a 524 amino acid ATP-binding cassette protein. The variant gene shared 85.7% nucleotide sequence identity and the variant protein 85.3% amino acid sequence identity with the original vga(E) gene and Vga(E) protein, respectively. The Vga(E) variant conferred cross-resistance to pleuromutilins, lincosamides and streptogramin A antibiotics. Plasmid pSA-7 showed an organization similar to that of the apmA-carrying plasmid pKKS49 from methicillin-resistant S. aureus and the dfrK-carrying plasmid pKKS966 from Staphylococcus hyicus. Sequence comparisons suggested that recombination events may have played a role in the acquisition of this vga(E) variant. CONCLUSIONS: A novel vga(E) gene variant was identified, which was located on a small plasmid and was not associated with the transposon Tn6133 [in contrast to the original vga(E) gene]. The plasmid location may enable its further dissemination to other staphylococci and possibly also to other bacteria.

Identification of the novel lincosamide resistance gene lnu(E) truncated by ISEnfa5-cfr-ISEnfa5 insertion in Streptococcus suis: de novo synthesis and confirmation of functional activity in Staphylococcus aureus.

The novel lincosamide resistance gene lnu(E), truncated by insertion of an ISEnfa5-cfr-ISEnfa5 segment, was identified in Streptococcus suis. The gene lnu(E) encodes a 173-amino-acid protein with ≤69.4% identity to other lincosamide nucleotidyltransferases. The lnu(E) gene and its promoter region were de novo synthesized, and Staphylococcus aureus RN4220 carrying a shuttle vector with the cloned lnu(E) gene showed a 16-fold increase in the lincomycin MIC. Mass spectrometry experiments demonstrated that Lnu(E) catalyzed the nucleotidylation of lincomycin.

Plasmid-Mediated Antimicrobial Resistance in Staphylococci and Other Firmicutes.

In staphylococci and other Firmicutes, resistance to numerous classes of antimicrobial agents, which are commonly used in human and veterinary medicine, is mediated by genes that are associated with mobile genetic elements. The gene products of some of these antimicrobial resistance genes confer resistance to only specific members of a certain class of antimicrobial agents, whereas others confer resistance to the entire class or even to members of different classes of antimicrobial agents. The resistance mechanisms specified by the resistance genes fall into any of three major categories: active efflux, enzymatic inactivation, and modification/replacement/protection of the target sites of the antimicrobial agents. Among the mobile genetic elements that carry such resistance genes, plasmids play an important role as carriers of primarily plasmid-borne resistance genes, but also as vectors for nonconjugative and conjugative transposons that harbor resistance genes. Plasmids can be exchanged by horizontal gene transfer between members of the same species but also between bacteria belonging to different species and genera. Plasmids are highly flexible elements, and various mechanisms exist by which plasmids can recombine, form cointegrates, or become integrated in part or in toto into the chromosomal DNA or into other plasmids. As such, plasmids play a key role in the dissemination of antimicrobial resistance genes within the gene pool to which staphylococci and other Firmicutes have access. This chapter is intended to provide an overview of the current knowledge of plasmid-mediated antimicrobial resistance in staphylococci and other Firmicutes.

[Antimicrobial resistance, ESBL and MRSA--definitions and laboratory diagnostics]. / Antimikrobielle Resistenz, ESBL und MRSA--Definitionen und Labordiagnose.

In the light of frequent discussions about the correct performance of in vitro susceptibility testing and the interpretation of the results obtained, the aim of the present report is to summarize basic facts that may facilitate the understanding of this complex topic. For this, the terms "antimicrobial resistance", "ESBL", and "MRSA" are defined. Besides the statements on antimicrobial resistance, information on intrinsic and acquired resistance properties as well as basic rules for the correct performance of antimicrobial susceptibility testing in routine diagnostics are presented. Moreover, the two groups of interpretive criteria--clinical breakpoints and epidemiological cut-off values--including their applications are explained in detail. Furthermore, currently valid diagnostic procedures--as published by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Clinical and Laboratory Standards Institute (CLSI)--for the screening of ESBL-producing Enterobacteriaceae and MRSA as well as for the confirmation of suspicious isolates are presented and compared. Based on the information given, it becomes obvious that the correct performance of the diagnostic tests, which includes strict following the performance standards and the detailed information given therein, is an indispensable prerequisite for a standardized and harmonized in vitro susceptibility testing and--as a consequence--for the determination of valid and reliable susceptibility data in routine diagnostics. This is of utmost importance since the susceptibility data based on the use of clinical breakpoints often represent the basis for therapeutic interventions.

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.

Complete sequence of the multi-resistance plasmid pV7037 from a porcine methicillin-resistant Staphylococcus aureus.

The aim of this study was to determine the complete sequence of the multi-resistance plasmid pV7037 to gain insight into the structure and organization of this plasmid. Of the four XbaI clones of pV7037, one clone of 17,577 bp has already been sequenced and shown to carry a multi-resistance gene cluster. The remaining three clones of approximately 12.5, 6.5 and 4.5 kb were sequenced, the entire plasmid sequence correctly assembled and investigated for reading frames. In addition, two reading frames one coding for an ABC transporter and the other coding for an rRNA methylase were cloned and expressed in a S. aureus host to see whether they confer antimicrobial resistance properties. Plasmid pV7037 proved to be 40,971 bp in size. Besides the previously determined resistance gene cluster, it carried a functionally active tet(L) gene for tetracycline resistance, a complete cadDX operon for cadmium resistance and also a variant of the ß-lactamase transposon Tn552. Two single bp deletions, which resulted in frame shifts, functionally deleted the genes for the BlaZ ß-lactamase and the signal transducer protein BlaR1 in this Tn552 variant of pV7037. Plasmid pV7037 seems to be composed of various parts previously known from plasmids and transposons of staphylococci and other Gram-positive bacteria. However, there are also parts of the plasmid which do not show any homology to so far known sequences deposited in the databases. The novel ABC transporter and rRNA methylase genes identified on pV7037 do not seem to play a role in antimicrobial resistance. The co-location of numerous antimicrobial resistance genes bears the risk of co-transfer and co-selection of resistance genes, but also persistence of resistance genes even if no direct selective pressure by the use of the respective antimicrobial agents is applied.

Resistance phenotypes and genotypes of methicillin-resistant Staphylococcus aureus isolates from broiler chickens at slaughter and abattoir workers.

OBJECTIVES: To comparatively investigate the resistance phenotypes and genotypes of various methicillin-resistant Staphylococcus aureus (MRSA) isolates from broilers at slaughter and workers at the respective poultry slaughterhouses. METHODS: Forty-six MRSA isolates (28 from broilers and 18 from humans) obtained at four different slaughterhouses were included. In addition to previously determined sequence types (STs) and spa types, the isolates were characterized by dru typing, SCCmec typing and PFGE. Resistance phenotypes were determined by broth microdilution. Resistance genes and clonal complexes (CCs) were detected by DNA microarray or specific PCR assays. RESULTS: MRSA of CC398, spa type t011 and varying dru types represented 23/28 broiler isolates and 12/18 human isolates. Three ST9/t1430/dt10a isolates were each seen among the isolates from the abattoir workers and the broilers. In addition, two human CC398/ST1453/t4652/dt3c isolates, a single human CC398/t034/dt6j isolate and two chicken CC398/t108/dt11a isolates were detected. All CC398 isolates (including ST1453) and some of the ST9 isolates from chickens and humans showed resistance to four to nine classes of antimicrobial agents and carried a wide range of resistance genes. While the resistance phenotypes and genotypes of the chicken isolates of the same flock were closely related, they usually differed from the resistance phenotypes and genotypes of the isolates from the workers at the respective slaughterhouse. CONCLUSIONS: The apparent homogeneity of MRSA isolates from the same flock suggests exchange of isolates between the respective animals. The apparent heterogeneity of MRSA isolates from abattoir workers might reflect their occupational contact with animals from numerous chicken flocks.

The diversity of antimicrobial resistance genes among staphylococci of animal origin.

Staphylococci of animal origin harbor a wide variety of resistance genes. So far, more than 40 different resistance genes have been identified in staphylococci from animals. This includes genes that confer resistance to virtually all classes of antimicrobial agents approved for use in animals, such as penicillins, cephalosporins, tetracyclines, macrolides, lincosamides, phenicols, aminoglycosides, aminocyclitols, pleuromutilins, and diaminopyrimidines. The gene products of some of these resistance genes confer resistance to only specific members of a class of antimicrobial agents, whereas others confer resistance to the entire class or even to members of different classes of antimicrobial agents. The resistance mechanisms specified by the resistance genes fall into three major categories: (i) enzymatic inactivation, (ii) active efflux, or (iii) protection/modification/replacement of the cellular target sites of the antimicrobial agents. Mobile genetic elements, in particular plasmids and transposons, play a major role as carriers of antimicrobial resistance genes in animal staphylococci. They facilitate the exchange of resistance genes with staphylococci of human origin but also with other Gram-positive bacteria.