Fernanda Ruiz-Larrea

β-Lactamases in Ampicillin-Resistant Escherichia coli Isolates from Foods, Humans, and Healthy Animals

ABSTRACT TEM-, SHV-, and OXA-type β-lactamases were studied by PCR with 124 ampicillin-resistant (AMPr) Escherichia coli isolates recovered from foods of animal origin (n = 20) and feces of humans (n = 49) and healthy animals (n = 55). PCR showed that 103 isolates were positive for TEM and negative for SHV and OXA. Three E. coli isolates showed a positive reaction for OXA, and one showed a positive reaction for SHV. The remaining 17 E. coli isolates were negative for the three enzymes by PCR. Fifty-seven of the 103 blaTEM amplicons were sequenced. Different molecular variants of blaTEM-1 were found in 52 isolates: blaTEM-1a (n = 9), blaTEM-1b (n = 36), blaTEM-1c (n = 6), and blaTEM-1f (n = 1). Four inhibitor-resistant TEM (IRT) β-lactamase-encoding genes were also detected: blaTEM-30c (IRT-2), blaTEM-34b (IRT-6), blaTEM-40b (IRT-11), and blaTEM-51a (IRT-15). A new blaTEM gene, named blaTEM-95b, which showed a mutation in amino acid 145 (P→A) was detected. It was found in a food isolate of chicken origin (AMPr, amoxicillin-clavulanic acid susceptible). The promoter region in 24 blaTEM amplicons was analyzed, and the weak P3 promoter was found in 23 of them (blaTEM-1 in 20 amplicons and blaTEM-51a, blaTEM-30c, and blaTEM-95b in 1 amplicon each). The strong Pa/Pb promoter was found only in the blaTEM-34b gene. No extended-spectrum β-lactamases were detected. Mutations at position −42 or −32 in the ampC gene promoter were demonstrated in 4 of 10 E. coli isolates for which the cefoxitin MIC was ≥16 μg/ml. Different variants of blaTEM-1 and IRT blaTEM genes were found among the AMPrE. coli isolates from foods and the feces of humans and healthy animals, and a new gene, blaTEM-95b (P3), was detected.

Design and Evaluation of PCR Primers for Analysis of Bacterial Populations in Wine by Denaturing Gradient Gel Electrophoresis

ABSTRACT Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified ribosomal DNA (rDNA) is routinely used to compare levels of diversity of microbial communities and to monitor population dynamics. While using PCR-DGGE to examine the bacteria in wine fermentations, we noted that several commonly used PCR primers for amplifying bacterial 16S rDNA also coamplified yeast, fungal, or plant DNA present in samples. Unfortunately, amplification of nonbacterial DNA can result in a masking of bacterial populations in DGGE profiles. To surmount this problem, we developed two new primer sets for specific amplification of bacterial 16S rDNA in wine fermentation samples without amplification of eukaryotic DNA. One primer set, termed WLAB1 and WLAB2, amplified lactic acid bacteria, while another, termed WBAC1 and WBAC2, amplified both lactic acid bacterial and acetic acid bacterial populations found in wine. Primer specificity and efficacy were examined with DNA isolated from numerous bacterial, yeast, and fungal species commonly found in wine and must samples. Importantly, both primer sets effectively distinguished bacterial species in wine containing mixtures of yeast and bacteria.

Macrolide Resistance Genes in Enterococcus spp.

ABSTRACT Seventy-eight isolates of different Enterococcusspecies (E. faecalis, n = 27; E. faecium, n = 23; E. durans,n = 8; E. avium, n = 6;E. hirae, n = 9; E. gallinarum, n = 3; and E. casseliflavus, n = 2) with a variety of erythromycin resistance phenotypes were examined for the presence of macrolide resistance genes (ermA, ermB,ermC, ermTR, mefA/E, andmsrA). Positive PCR amplifications of ermB were obtained for 39 of 40 highly erythromycin-resistantEnterococcus isolates (MICs, >128 μg/ml) of different species; the remaining highly resistant E. faecium isolate was positive for PCR amplification of ermA but was negative for PCR amplification of the ermB and ermCgenes. For all enterococcal strains for which erythromycin MICs were ≤32 μg/ml PCRs were negative for erm methylase genes. For all E. faecium isolates PCR amplified products of the expected size of 400 bp were obtained when msrA primers were used, with the results being independent of the erythromycin resistance phenotype. All the other enterococcal species gave negative results by msrA PCRs. Sequencing of the msrAPCR products from either erythromycin-susceptible, low-level-resistant, or highly resistant E. faecium strains showed that the amplicons did not correspond to the msrA gene described forStaphylococcus epidermidis but corresponded to a new putative efflux determinant, which showed 62% identity with themsrA gene at the DNA level and 72% similarity at the amino acid level. This new gene was named msrC.

Antibiotic resistance in Escherichia coli isolates obtained from animals, foods and humans in Spain

Antibiotic resistance was investigated in 474 Escherichia coli isolates recovered from animal faeces (broilers, pigs, pets, bulls and horses), human faeces (patients and healthy volunteers) and food products of animal origin. E. coli isolates (3260) recovered from human significant infectious samples were also included. There was a high frequency of nalidixic acid, ciprofloxacin and gentamicin resistance in E. coli isolates from broilers (88, 38 and 40%, respectively), and from foods (53, 13 and 17%). High levels of resistance to trimethoprim-sulphamethoxazole and tetracycline have been found in E. coli isolates from broilers, pigs and foods. These data raise important questions about the potential impact of antibiotic use in animals and the possible entry of resistant pathogens into the food chain.

High tolerance of wild Lactobacillus plantarum and Oenococcus oeni strains to lyophilisation and stress environmental conditions of acid pH and ethanol

A total of 76 Lactobacillus plantarum and Oenococcus oeni wild strains were recovered from traditionally elaborated Spanish red wines and were investigated with respect to their response to acid pH, lyophilisation, temperature and ethanol concentrations which are normally lethal to lactic acid bacteria. Both L. plantarum and O. oeni strains were able to grow at pH 3.2, were highly resistant to lyophilisation treatment and proliferated in the presence of up to 13% ethanol at 18 degrees C. Therefore, it is shown that both species are highly tolerant to stress conditions and that similarly to O. oeni strains, L. plantarum strains are of interest in beverage biotechnology.

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.

Bacteriocin production by lactic acid bacteria isolated from Rioja red wines

L. NAVARRO, M. ZARAZAGA, J. SÁENZ, F. RUIZ‐LARREA & C. TORRES.2000. Forty‐two lactic acid bacteria (LAB) of the genera Lactobacillus (32), Leuconostoc (6), Pediococcus (3) and Lactococcus (1), isolated from Rioja red wines, were tested for antimicrobial activity. All these strains, as well as 18 Leuconostoc oenos and 19 yeast strains were used as indicators. Only nine strains showed antimicrobial activity, and all were of the species Lactobacillus plantarum, which constitutes the predominant microflora in Rioja red wines after alcoholic fermentation. Lact. plantarum strain J‐51 showed the widest range of action, inhibiting the growth of 31 strains of the four studied LAB genera. Lact. plantarum J‐51 antimicrobial activity was lost after treatment with proteases, suggesting a proteinaceous nature for this activity. It was found to be stable between pH 3 and 9 and under strong heating conditions (100 °C for 60 min). Polymerase chain reaction (PCR) analysis of Lact. plantarum J‐51 genome revealed the presence of the plnA gene that encodes the plantaricin precursor PlnA. A 366‐bp fragment was sequenced and showed 95% identity with pln locus of Lact. plantarum C‐11. The deduced precursor peptide sequence showed one mutation (Gly7 to Ser7) at the double glycine leader peptide, and the three putative 26‐, 23‐ and 22‐residue active peptides remain identical to those of Lact. plantarum C‐11. Therefore, antimicrobial peptides constitute a potent adaptation advantage for those strains that dominate in a medium such as wine, and can play an important role in the ecology of wine microflora.

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

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.

Genetic diversity of the pln locus among oenological Lactobacillus plantarum strains.

A total of 33 Lactobacillus plantarum strains obtained from grape musts and wines during alcoholic and malolactic fermentations were submitted to PCR analysis with specific primers directed to 27 genes of the plantaricin (pln) locus previously described for L. plantarum strains. The number of genes that were detected varied depending on the strain, and cluster analysis of results rendered seven groups, named plantaritypes (similarity within each group >89%) that included all the 33 oenological strains, four L. plantarum type strains (C11, NC8, J23 and J51) that had been previously described, and strain WCFS1 whose genome had been fully sequenced. The common features for most strains (74%) were the presence of the plnABCD regulatory system (which includes genes of the inducing peptide, its coupled membrane-located histidine protein kinase and two response regulators), the two-peptide bacteriocin plnEF genes and the genes of a membrane-bound ABC transport system. The pln locus is shown to be widespread among oenological strains (94% of appearance), as well as to possess a remarkable plasticity and variable regions related to its regulation and bacteriocin production.