María López

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

Contribution of Efflux Pumps, Porins, and β-Lactamases to Multidrug Resistance in Clinical Isolates of Acinetobacter baumannii

ABSTRACT We investigated the mechanisms of resistance to carbapenems, aminoglycosides, glycylcyclines, tetracyclines, and quinolones in 90 multiresistant clinical strains of Acinetobacter baumannii isolated from two genetically unrelated A. baumannii clones: clone PFGE-ROC-1 (53 strains producing the OXA-58 β-lactamase enzyme and 18 strains with the OXA-24 β-lactamase) and clone PFGE-HUI-1 (19 strains susceptible to carbapenems). We used real-time reverse transcriptase PCR to correlate antimicrobial resistance (MICs) with expression of genes encoding chromosomal β-lactamases (AmpC and OXA-51), porins (OmpA, CarO, Omp33, Dcap-like, OprB, Omp25, OprC, OprD, and OmpW), and proteins integral to six efflux systems (AdeABC, AdeIJK, AdeFGH, CraA, AbeM, and AmvA). Overexpression of the AdeABC system (level of expression relative to that by A. baumannii ATCC 17978, 30- to 45-fold) was significantly associated with resistance to tigecycline, minocycline, and gentamicin and other biological functions. However, hyperexpression of the AdeIJK efflux pump (level of expression relative to that by A. baumannii ATCC 17978, 8- to 10-fold) was significantly associated only with resistance to tigecycline and minocycline (to which the TetB efflux system also contributed). TetB and TetA(39) efflux pumps were detected in clinical strains and were associated with resistance to tetracyclines and doxycycline. The absence of the AdeABC system and the lack of expression of other mechanisms suggest that tigecycline-resistant strains of the PFGE-HUI-1 clone may be associated with a novel resistance-nodulation-cell efflux pump (decreased MICs in the presence of the inhibitor Phe-Arg β-naphthylamide dihydrochloride) and the TetA(39) system.

Detection of vanA and vanB2-containing enterococci from food samples in Spain, including Enterococcus faecium strains of CC17 and the new singleton ST425.

Two-hundred-twenty-nine food samples of animal origin were tested to know the prevalence of vancomycin-resistant enterococci (VRE) after a decade of avoparcin ban as animal growth promoter in Spain. VRE with acquired mechanism of resistance were detected in 9 of these 229 samples (3.9%, obtained from chicken, veal and rabbit), and one VRE per food sample was further characterized. The vanA gene was identified in seven isolates (2 E. faecium, 3 E. durans, and 2 E. hirae), and the vanB2 gene in the remaining 2 isolates (identified as E. faecium). The two vanB2 isolates showed a phenotype of multiresistance that included, in addition to vancomycin, also ampicillin, erythromycin, tetracycline, streptomycin, kanamycin, ciprofloxacin, chloramphenicol and trimethoprim-sulfamethoxazole and contained, among others, erm(B), tet(M), ant(6), and aph(3)-III genes. Most of vanA enterococci showed erythromycin and tetracycline resistance and contained the erm(B) and tet(M) genes. One vanA- and both vanB2-positive E. faecium isolates were classified by MLST analysis into the CC17 clonal complex (ST17 and ST78), and one additional vanA isolate was included in a new sequence type named ST425 (singleton). Co-transference by conjugation of erm(B) and vanA genes was demonstrated in one vanA-positive E. faecium isolate. The inclusion of vanB2 cluster into Tn5382 structure was demonstrated in the two vanB2 isolates, as well as the linkage pbp5-Tn5382, and beta-haemolysis and gelatinase production was identified in one of them. Food sample of animal origin could be a vehicle of transference of VRE of vanA and vanB2 type that could be transferred to humans.

Proteomic characterization of vanA-containing Enterococcus recovered from Seagulls at the Berlengas Natural Reserve, W Portugal

BackgroundEnterococci have emerged as the third most common cause of nosocomial infections, requiring bactericidal antimicrobial therapy. Although vancomycin resistance is a major problem in clinics and has emerged in an important extend in farm animals, few studies have examined it in wild animals. To determine the prevalence of van A-containing Enterococcus strains among faecal samples of Seagulls (Larus cachinnans) of Berlengas Natural Reserve of Portugal, we developed a proteomic approach integrated with genomic data. The purpose was to detect the maximum number of proteins that vary in different enterococci species which are thought to be connected in some, as yet unknown, way to antibiotic resistance.ResultsFrom the 57 seagull samples, 54 faecal samples showed the presence of Enterococcus isolates (94.7%). For the enterococci, E. faecium was the most prevalent species in seagulls (50%), followed by E. faecalis and E. durans (10.4%), and E. hirae (6.3%). VanA-containing enterococcal strains were detected in 10.5% of the 57 seagull faecal samples studied. Four of the vanA-containing enterococci were identified as E. faecium and two as E. durans. The tet(M) gene was found in all five tetracycline-resistant vanA strains. The erm(B) gene was demonstrated in all six erythromycin-resistant vanA strains. The hyl virulence gene was detected in all four van A-containing E. faecium isolates in this study, and two of them harboured the pur K1 allele. In addition these strains also showed ampicillin and ciprofoxacin resistance. The whole-cell proteomic profile of van A-containing Enterococcus strains was applied to evaluate the discriminatory power of this technique for their identification. The major differences among species-specific profiles were found in the positions corresponding to 97-45 kDa. Sixty individualized protein spots for each vanA isolate was identified and suitable for peptide mass fingerprinting measures by spectrometry measuring (MALDI/TOF MS) and their identification through bioinformatic databases query. The proteins were classified in different groups according to their biological function: protein biosynthesis, ATP synthesis, glycolysis, conjugation and antibiotic resistance. Taking into account the origin of these strains and its relation to infectious processes in humans and animals, it is important to explore the proteome of new strains which might serve as protein biomarkers for biological activity.ConclusionsThe comprehensive description of proteins isolated from vancomycin-resistant Enterococcus faecium and E. durans may provide new targets for development of antimicrobial agents. This knowledge may help to identify new biomarkers of antibiotic resistance and virulence factors.

Analysis of excess enthalpies of ethyl formate + n-alkane or 1-alkanol with two group contribution models

Abstract We present experimental values of enthalpies of mixing at 298.15 K for mixtures composed of ethyl formate with n-alkanes (from n-hexane to n-decane) or 1-alkanols (from 1-propanol to 1-decanol). Then hE data for the latter mixtures have been used to determine characteristic formate-alkanol interaction parameters for three group contribution models: the UNIFAC model with and without consideration of 1-alkanol association effects, and the Nitta-Chao model. The experimental data are reproduced within mean errors of less than 6% by all the used models.

Antibiotic resistance, virulence determinants and production of biogenic amines among enterococci from ovine, feline, canine, porcine and human milk

BackgroundRecent studies have shown that mammalian milk represents a continuous supply of commensal bacteria, including enterococci. The objectives of this study were to evaluate the presence of enterococci in milk of different species and to screen them for several genetic and phenotypic traits of clinical significance among enterococci.ResultsSamples were obtained from, at least, nine porcine, canine, ovine, feline and human healthy hosts. Enterococci could be isolated, at a concentration of 1.00u2009×u2009102 -1.16u2009×u2009103xa0CFU/ml, from all the porcine samples and, also from 85, 50, 25 and 25% of the human, canine, feline and ovine ones, respectively. They were identified as Enterococcus faecalis, Enterococcus faecium, Enterococcus hirae, Enterococcus casseliflavus and Enterococcus durans. Among the 120 initial enterococcal isolates, 36 were selected on the basis of their different PFGE profiles and further characterized. MLST analysis revealed a wide diversity of STs among the E. faecalis and E. faecium strains, including some frequently associated to hospital infections and novel STs. All the E. faecalis strains possessed some of the potential virulence determinants (cad, ccf, cob, cpd, efaAfs, agg2, gelE, cylA, espfs) assayed while the E. faecium ones only harboured the efaAfm gene. All the tested strains were susceptible to tigecycline, linezolid and vancomycin, and produced tyramine. Their susceptibility to the rest of the antimicrobials and their ability to produce other biogenic amines varied depending on the strain. Enterococci strains isolated from porcine samples showed the widest spectrum of antibiotic resistance.ConclusionsEnterococci isolated from milk of different mammals showed a great genetic diversity. The wide distribution of virulence genes and/or antibiotic resistance among the E. faecalis and E. faecium isolates indicates that they can constitute a reservoir of such traits and a risk to animal and human health.

MLST and a genetic study of antibiotic resistance and virulence factors in vanA-containing Enterococcus from buzzards (Buteo buteo)

Aims:u2002 To analyse the occurrence of faecal carriage of vancomycin‐resistant enterococci (VRE) in Buteo buteo and to study the associated resistance and virulence genes.

Tn1546 structures and multilocus sequence typing of vanA-containing enterococci of animal, human and food origin

OBJECTIVESnTo characterize the Tn1546 structure and to perform the genetic typing of 51 PFGE-unrelated vanA-containing enterococci of different origin (clinical, food and faecal samples of healthy humans and healthy poultry).nnnMETHODSnTn1546 structure was characterized by a PCR primer walking strategy and sequencing. Multilocus sequence typing (MLST) was performed for Enterococcus faecium and Enterococcus faecalis strains, and esp and hyl genes were detected by PCR.nnnRESULTSnNine different Tn1546 structures were identified in the studied strains. Type I was the most prevalent structure (75%) (identical to GenBank M97297). Two new Tn1546 structures were identified (in three clinical and animal strains), containing two new insertion sequences (ISs; ISEfa11 disrupting vanS and ISEfa10 disrupting orf1). An additional new Tn1546 structure was found in one animal strain, containing ISEf1 interrupting vanY and IS1542 in the orf2-vanR region. A high diversity of sequence types (STs) was detected among clinical (6 ST/18 strains) and non-clinical E. faecium strains (18 ST/24 strains). STs associated with clonal complexes CC17 and CC9 were mainly detected among clinical and non-clinical E. faecium strains, respectively. Seven new STs were identified in non-clinical strains. The esp and hyl genes were only found among clinical E. faecium strains.nnnCONCLUSIONSnA moderate variability in Tn1546 structure has been detected among unrelated vanA-containing enterococci of different origins, showing three new structures including two new ISs. A high diversity of STs was detected among E. faecium strains, especially among non-clinical strains, and new STs have been identified.

Species distribution, antibiotic resistance and virulence traits in enterococci from meat in Tunisia

Antimicrobial resistance and the mechanisms implicated were studied in 119 enterococci from 105 meat samples from Tunisian markets. Almost 24.5% of recovered enterococci showed resistance against four or more antimicrobial agents and these isolates were identified to the species level. Enterococcus faecalis was the most prevalent species (41%). High percentages of erythromycin and tetracycline resistances were found among our isolates, and lower percentages were identified to aminoglycosides, ciprofloxacin and chloramphenicol. All tetracycline-resistant isolates carried the tet(M) and/or tet(L) genes. The erm(B) gene was detected in 78.5% of erythromycin-resistant isolates, ant(6)-Ia gene in 58.8% of streptomycin-resistant isolates, and cat(A) gene in one chloramphenicol-resistant isolate. Forty-eight isolates carried the gelE gene and exhibited gelatinase activity. The hyl and esp genes were detected in one and three Enterococcus faecium isolates, respectively. Streptomycin-resistant isolates showed a high genetic diversity by PFGE and MLST. Meat might play a role in the spread through the food chain of enterococci with these virulence and resistance characteristics to humans.

First Detection of the Staphylococcal Trimethoprim Resistance Gene dfrK and the dfrK-Carrying Transposon Tn559 in Enterococci

The trimethoprim resistance gene dfrK has been recently described in Staphylococcus aureus, but so far has not been found in other bacteria. A total of 166 enterococci of different species (E. faecium, E. faecalis, E. hirae, E. durans, E. gallinarum, and E. casseliflavus) and origins (food, clinical diseases in humans, healthy humans or animals, and sewage) were studied for their susceptibility to trimethoprim as determined by agar dilution (European Committee on Antimicrobial Susceptibility Testing) and the presence of (a) the dfrK gene and its genetic environment and (b) other dfr genes. The dfrK gene was detected in 49% of the enterococci (64% and 42% of isolates with minimum inhibitory concentrations of ≥2u2009mg/L or ≤1u2009mg/L, respectively). The tet(L)-dfrK linkage was detected in 21% of dfrK-positive enterococci. The chromosomal location of the dfrK gene was identified in one E. faecium isolate in which the dfrK was not linked to tet(L) gene but was part of a Tn559 element, which was integrated in the chromosomal radC gene. This Tn559 element was also found in 14 additional isolates. All combinations of dfr genes were detected among the isolates tested (dfrK, dfrG, dfrF, dfrK+dfrG, dfrK+dfrF, dfrF+dfrG, and dfrF+dfrG+dfrK). The gene dfrK gene was found together with other dfr genes in 58% of the tested enterococci. This study suggested an exchange of the trimethoprim resistance gene dfrK between enterococci and staphylococci, as previously observed for the trimethoprim resistance gene dfrG.