Carmen Lozano

Detection, molecular characterization, and clonal diversity of methicillin-resistant Staphylococcus aureus CC398 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.

Detection of methicillin-resistant Staphylococcus aureus ST398 in food samples of animal origin in Spain

Sir, Methicillin-resistant Staphylococcus aureus (MRSA) strains belonging to clonal lineage sequence type (ST) 398 are being reported at an increasing frequency in Europe. This new MRSA type has been isolated from colonized and infected animals and humans, and also from meat in some countries, representing a risk to human health; nevertheless, so far, no data about detection of MRSA ST398 in food in Spain have been published. A total of 318 raw food samples of food-producing animals (148 chicken, 55 pork, 46 veal, 19 lamb, 10 turkey, 8 rabbit and 12 minced-meat samples) and of wild animals (8 game bird, 4 wild boar, 4 deer and 4 hare samples) were collected from November 2007 to March 2009 in La Rioja (Spain). Samples were suspended in saline solution and 100 mL was inoculated in brain heart infusion (BHI) broth containing 6.5% NaCl, and incubated at 358C for 24 h. Then, 300 mL was seeded on ORSAB plates (Oxoid) with oxacillin (2 mg/L), and incubated at 358C for 36 h. One blue presumptive MRSA colony per sample was selected and identified by DNase assay and by PCRs of the mecA and nuc genes. MICs of 10 antibiotics were determined by the agar dilution method; the disc diffusion method was used for 7 additional antibiotics (Table 1). The presence of resistance genes was studied by PCR. Mutations in quinolone targets were determined by sequence analysis of grlA and gyrA genes. Recovered MRSA isolates were characterized by multilocus sequence typing (MLST) (saureus.mlst.net), staphylococcal cassette chromosome mec (SCCmec) typing, spa typing (www.ridom. com) and agr typing. The presence of Panton–Valentine leucocidin (PVL) genes was investigated by PCR. MRSA was detected in 5 of the 318 (1.6%) food samples (from pork, chicken, rabbit, veal and wild boar). MRSA strains were characterized and the results are summarized in Table 1. The two strains from pork and veal corresponded to ST398-SCCmecV (spa types t011 and t1197, respectively), the two strains from chicken and rabbit were typed as ST125-SCCmecIVa-t067, and the strain from a wild boar was ST217-SCCmecIVa-t032. All MRSA were PVL negative. MRSA has also been isolated from meat in other countries, and the percentages detected varied widely (0%–11%). The rate found in our study (1.6%) is in the lower range of the reported data, and higher percentages have recently been reported in the Netherlands and the USA. A correlation between meat type and rate of MRSA contamination cannot be established as the numbers of samples of different origins in our study differ significantly. The detection of the MRSA ST217 strain in one of the four tested wild boar samples is noteworthy. A previous study had also reported non-ST398 MRSA in food derived from game in a low percentage of samples (2.2%). Although MRSA prevalence in raw food is low, the risk of its transmission through the food chain cannot be disregarded, especially in uncooked meat. Indeed, foodborne disease outbreaks caused by MRSA have been reported. Moreover, contaminated foods may also constitute a health risk for food handlers. Carcasses obtained from animals colonized by MRSA may become contaminated during slaughter and foods can also become contaminated during processing by food handlers colonized by MRSA. Molecular typing is a useful tool for understanding the origin of these strains. In our study two MRSA strains were ST398, suggesting that animals could be the source of contamination. Both ST398 strains presented resistance to tetracycline, a characteristic of animal-related MRSA. Tetracycline is the most widely used antibiotic in the pig industry; macrolides and aminoglycosides are also used, but less frequently. The origin of ST398 is unclear, but the excessive use of certain antibiotics in animal production might be implicated in its emergence. A reservoir of MRSA ST398 seems to be present in food-producing animals and a high rate of nasal carriers has been found in humans in contact with these animals, which may have consequences for human health. ST398 has been detected in several European countries, America and Asia. Due to the significant spread of this variant, it was expected to also be present in Spain, but, to our knowledge, this is the first report of ST398 in foods in a Mediterranean country. The other non-ST398 strains in our study were ST125-t067 and ST217-t032, types associated with human infections, suggesting a possible human origin, and might have been transmitted by colonized food handlers. According to a recent publication, MRSA ST125-t067 was implicated in .50% of the invasive infections in Spanish hospitals. Moreover, those authors described that simultaneous resistance to ciprofloxacin, tobramycin and erythromycin is frequently found in isolates belonging to spa-t067, a variant carrying ant(40)-Ia and msrA genes. One of our ST125-t067 strains presented all these characteristics. On the other hand, ST217 is a variant of epidemic EMRSA-15 and was reported to present the characteristics of nosocomial MRSA with a high level of ciprofloxacin resistance, which is in accordance with our results. In conclusion, MRSA was detected in 1.6% of meat samples in our study. Strain characterization suggests that they could be from both animal and human origin. Although the presence of MRSA in food is low, it has to be monitored because it can contribute to the spread of MRSA. To our knowledge, this is the first study concerning the prevalence of MRSA in food in Spain. Journal of Antimicrobial Chemotherapy (2009) 64, 1325–1346 doi:10.1093/jac/dkp378 Advance Access publication 21 October 2009

Staphylococcus aureus nasal carriage, virulence traits, antibiotic resistance mechanisms, and genetic lineages in healthy humans in Spain, with detection of CC398 and CC97 strains.

S. aureus nasal carriage was investigated in 278 healthy humans, determining the antibiotic resistance mechanisms, virulence traits, and genetic lineages of recovered isolates. Nasal samples were cultured in specific media for S. aureus and methicillin-resistant S. aureus (MRSA) recovery. S. aureus was detected in 53 of 278 nasal samples (19.1%): MRSA was found in one sample (0.4%) and methicillin-susceptible S. aureus (MSSA) in the remaining 52 samples. The MRSA isolate was typed as ST1649-t701-agrI-SCCmec-IVc and only exhibited resistance to beta-lactams. A high diversity of spa types (n=37) was identified among the 52 MSSA, identifying 5 new spa-types. Multi-locus sequence typing (MLST) typing was performed in 30 selected MSSA, detecting 16 different sequence types, 2 of them being new. MSSA strains presented agr types I (30.2%), II (30.2%), III (34%), and IV (5.6%). Eleven strains showed erythromycin resistance and harbored different combinations of erm(A), erm(B), erm(C), erm(T), and msr(A) genes. Two strains exhibited ciprofloxacin resistance, and one of them presented amino acid changes in GyrA and GrlA proteins. The presence of 28 genes encoding staphylococcal toxins was investigated by PCR in all 53 S. aureus isolates. The toxic shock syndrome toxin 1 (TSST-1) gene was detected in 15 MSSA isolates (11 of them typed within the clonal complex CC30) and the gene of exfoliative toxin A in 2 strains. Different combinations of enterotoxin genes were identified among S. aureus strains. None of the S. aureus isolates harbored the Panton-Valentine leukocidin gene. Two MSSA presented the sequence-type ST398 [harboring erm(T) gene], and 2 additional isolates were typed as ST97. Interestingly, MSSA CC398 and CC97 isolates were detected. These clonal complexes are associated with food-producing animals.

Genetic environment and location of the lnu(A) and lnu(B) genes in methicillin-resistant Staphylococcus aureus and other staphylococci of animal and human origin

OBJECTIVES To detect the presence of lnu genes in staphylococcal strains with the unusual phenotype lincosamide resistance/macrolide susceptibility (L(R)/M(S)), and to determine their locations and genetic environments. METHODS Six staphylococcal strains of human and animal origin with the phenotype L(R)/M(S) were studied. The presence of 15 resistance genes was tested by PCR. SCCmec typing was performed for all methicillin-resistant strains. agr typing, spa typing and multilocus sequence typing were carried out for Staphylococcus aureus strains. Transformation experiments were carried out by electrotransformation. Plasmid or chromosomal gene location was determined by Southern blot analysis and the genetic environments of the lnu genes were studied in all strains. RESULTS Three methicillin-resistant staphylococcal strains contained the lnu(A) gene. The presence of the pLNU1 plasmid carrying lnu(A) was confirmed in one methicillin-resistant S. aureus (MRSA) ST398-t108 and one methicillin-resistant Staphylococcus sciuri. A novel lnu(A)-carrying plasmid (pUR5425) was identified in one MRSA ST125-t067 strain. Transformants of the three lnu(A)-positive strains presented increased lincomycin MIC values. The remaining three studied staphylococcal strains harboured the lnu(B) gene: two methicillin-susceptible S. aureus (MSSA) ST9-t337 and one MRSA ST398-t011. The lnu(B) gene was embedded in the chromosome in the two MSSA strains and in a large-sized plasmid in the MRSA strain. The same lnu(B) genetic environment was detected in these three strains. CONCLUSIONS The resistance phenotype L(R)/M(S) seems to be related to S. aureus animal-associated clonal lineages (ST398 and ST9). A novel lnu(A)-carrying plasmid was identified and this is the first detection of the lnu(B) gene in MRSA ST398.

High diversity of Staphylococcus aureus and Staphylococcus pseudintermedius lineages and toxigenic traits in healthy pet-owning household members. Underestimating normal household contact?

Forty-three unrelated pet-owning households were screened in Spain to study the Staphylococcus aureus and Staphylococcus pseudintermedius nasal carriage, their genetic lineages and virulence traits. Sixty-seven healthy owners and 66 healthy pets were investigated. Isolates characterization was performed and potential interspecies transmission was assessed. S. aureus was present in 51.2% of households studied while S. pseudintermedius in 30.2%. Twenty-eight owners (41.8%) carried S. aureus: one methicillin-resistant S. aureus (MRSA) [t5173-ST8-SCCmecIVa] and 27 methicillin-susceptible S. aureus (MSSA). Three owners (4.5%) were colonized by methicillin-susceptible S. pseudintermedius (MSSP). Fifteen pets (22.7%) carried S. pseudintermedius: two methicillin-resistant S. pseudintermedius (MRSP) [ST71-SCCmecII/III; ST92-SCCmecV] and 13 MSSP; in addition, 8 pets (12.1%) presented MSSA. High diversity of spa and sequence types (STs) was detected. Typical livestock-associated S. aureus lineages (CC398, CC9) were observed in humans and/or companion animals and hospital and/or community-acquired S. aureus lineages (CC45, CC121, CC5, CC8) were detected among pets. Almost 40% of S. pseudintermedius were multidrug-resistant. S. aureus isolates harboured a remarkable high number of virulence genes. The expA gene was detected in 3 S. pseudintermedius isolates. Identical strains from both owners and their pets were identified in 5 households (11.6%): (a) four MSSA (t073-ST45/CC45, t159-ST121/CC121, t209-ST109/CC9, t021-ST1654([new])/singleton) and (b) one multidrug-resistant MSSP (ST142([new])). Highly clonally diverse and toxigenic S. aureus and S. pseudintermedius are common colonizers of healthy humans and pets. The presence of these bacterial species, virulence genes, and interspecies transmission detected, points out to consider pet ownership as a risk factor to acquire, maintain and spread, potential pathogenic bacteria.

Detection and characterization of methicillin-resistant Staphylococcus pseudintermedius in healthy dogs in La Rioja, Spain.

The objective was to identify the methicillin-resistant coagulase-positive staphylococci (MRCoPS) nasal carriage rate of healthy dogs in La Rioja (Spain) and to characterize the recovered isolates by different molecular techniques. Nasal samples from 196 dogs were obtained (98 household-dogs, 98 pound-dogs). Isolates were identified and characterized by spa-, SCCmec- and MLST-typing, SmaI-PFGE, antimicrobial susceptibility, determination of antimicrobial resistance and toxin genes profiling. S. pseudintermedius was the only species recovered. Nine methicillin-resistant S. pseudintermedius (MRSP) were obtained from 9 of 196 sampled dogs (8% pound-dogs, 1% household-dogs). MRSP isolates were typed (MLST/PFGE/spa/SCCmec) as: ST71/A/t02/II-III (7 isolates), ST92/C/t06/V (1 isolate), and ST26/B/non-typable/non-typable (1 isolate). All MRSP were resistant to [resistance gene/number isolates]: β-lactams [mecA+blaZ/9], tetracycline [tet(K)/7, tet(M)/2], macrolides and lincosamides [erm(B)/9], aminoglycosides [aacA-aphD+aadE+aphA-3/9], and co-trimoxazol [dfr(G)/9]. Eight MRSP isolates showed also resistance to fluoroquinolones and amino acid changes in GyrA [Ser84Leu+Glu714Lys, 7 isolates; Ser84Leu, 1 isolate] and GrlA [Ser80Ile, 8 isolates] proteins were detected. The remaining isolate was chloramphenicol resistant and harboured cat(pC221) gene. All MRSP isolates harboured the aadE-sat4-aphA-3 multiresistance-gene-cluster linked to erm(B) gene as well as the siet, si-ent and lukS/F-I toxin genes. MRSP is a moderately common (4.6%) colonizer of healthy dogs in Spain. A major MRSP lineage (ST71) was detected and its future evolution should be tracked.

GelJ – a tool for analyzing DNA fingerprint gel images

BackgroundDNA fingerprinting is a technique for comparing DNA patterns that has applications in a wide variety of contexts. Several commercial and freely-available tools can be used to analyze DNA fingerprint gel images; however, commercial tools are expensive and usually difficult to use; and, free tools support the basic functionality for DNA fingerprint analysis, but lack some instrumental features to obtain accurate results.ResultsIn this paper, we present GelJ, a feather-weight, user-friendly, platform-independent, open-source and free tool for analyzing DNA fingerprint gel images. Some of the outstanding features of GelJ are mechanisms for accurate lane- and band-detection, several options for computing migration models, a number of band- and curve-based similarity methods, different techniques for generating dendrograms, comparison of banding patterns from different experiments, and database support.ConclusionsGelJ is an easy to use tool for analyzing DNA fingerprint gel images. It combines the best characteristics of both free and commercial tools: GelJ is light and simple to use (as free programs), but it also includes the necessary features to obtain precise results (as commercial programs). In addition, GelJ incorporates new functionality that is not supported by any other tool.

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

Identification of novel vga(A)-carrying plasmids and a Tn5406-like transposon in meticillin-resistant Staphylococcus aureus and Staphylococcus epidermidis of human and animal origin.

Nine staphylococcal strains of human and animal origin with a lincomycin-resistant/erythromycin-susceptible phenotype and carrying vga genes were characterised to determine the genetic elements involved in the dissemination of these uncommon resistance genes. These strains were typed by multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec) and/or spa typing. Antimicrobial susceptibility was studied by disk diffusion and agar dilution methods. Presence of the genes lnu(A), lnu(B), vga(A), vga(A)v, vga(B), vga(C), vga(E), lsa(B) and cfr was studied by PCR. Transformation experiments were carried out in all strains, and the plasmid or chromosomal gene location was determined by Southern blot analysis. Genetic environments of the vga genes were analysed by PCR mapping or inverse PCR and sequencing. Five meticillin-resistant Staphylococcus aureus (MRSA) ST398 strains and three Staphylococcus epidermidis strains harboured the gene vga(A), and one MRSA-ST8 strain contained the gene vga(A)v. One MRSA-ST398 strain, which also contained the gene lnu(A), showed the highest minimum inhibitory concentration (MIC) to lincomycin. The vga(A)v-positive strain presented lower MIC values than the vga(A)-positive strains. Presence of the pVGA plasmid was confirmed in two MRSA-ST398 strains. Four novel vga(A)-carrying plasmids were detected: pUR2355 (in two MRSA and one meticillin-susceptible S. epidermidis); pUR4128 (one MRSA); pUR3036 [one meticillin-resistant S. epidermidis (MRSE)]; and pUR3937 (one MRSE). The plasmid pUR4128 was very similar to pUR2355. Plasmids pUR3036 and pUR3937 were related and were very similar to plasmid pSE-12228-06. The gene vga(A)v was located in a transposon analogous to Tn5406. Therefore, four novel vga(A)-carrying plasmids and a variant of Tn5406 were identified in this study.

High diversity of genetic lineages and virulence genes in nasal Staphylococcus aureus isolates from donkeys destined to food consumption in Tunisia with predominance of the ruminant associated CC133 lineage

BackgroundThe objective of this study was to determine the genetic lineages and the incidence of antibiotic resistance and virulence determinants of nasal Staphylococcus aureus isolates of healthy donkeys destined to food consumption in Tunisia.ResultsNasal swabs of 100 donkeys obtained in a large slaughterhouse in 2010 were inoculated in specific media for S. aureus and methicillin-resistant S. aureus (MRSA) recovery. S. aureus was obtained in 50% of the samples, being all of isolates methicillin-susceptible (MSSA). Genetic lineages, toxin gene profile, and antibiotic resistance mechanisms were determined in recovered isolates. Twenty-five different spa-types were detected among the 50 MSSA with 9 novel spa-types. S. aureus isolates were ascribed to agr type I (37 isolates), III (7), II (4), and IV (2). Sixteen different sequence-types (STs) were revealed by MLST, with seven new ones. STs belonging to clonal clomplex CC133 were majority. The gene tst was detected in 6 isolates and the gene etb in one isolate. Different combinations of enterotoxin, leukocidin and haemolysin genes were identified among S. aureus isolates. The egc-cluster-like and an incomplete egc-cluster-like were detected. Isolates resistant to penicillin, erythromycin, fusidic acid, streptomycin, ciprofloxacin, clindamycin, tetracycline, or chloramphenicol were found and the genes blaZ, erm(A), erm(C), tet(M), fusC were identified.ConclusionsThe nares of donkeys frequently harbor MSSA. They could be reservoirs of the ruminant-associated CC133 lineage and of toxin genes encoding TSST-1 and other virulence traits with potential implications in public health. CC133 seems to have a broader host distribution than expected.