Myriam Zarazaga

Mechanisms of Resistance in Multiple-Antibiotic-Resistant Escherichia coli Strains of Human, Animal, and Food Origins

ABSTRACT Seventeen multiple-antibiotic-resistant nonpathogenic Escherichia coli strains of human, animal, and food origins showed a wide variety of antibiotic resistance genes, many of them carried by class 1 and class 2 integrons. Amino acid changes in MarR and mutations in marO were identified for 15 and 14 E. coli strains, respectively.

Antibiotic Resistance in Campylobacter Strains Isolated from Animals, Foods, and Humans in Spain in 1997–1998

ABSTRACT Colonization by Campylobacter strains was investigated in human, broiler, and pig fecal samples from 1997- 1998, as well as in foods of animal origin, and antibiotic susceptibility testing was carried out for these strains. Campylobacter strains were isolated in the foods of animal origin (55 of 101 samples; 54.4%), intestinal samples from broilers (85 of 105; 81%), and pigs (40 of 45; 88.9%). A total of 641 Campylobacter strains were isolated from 8,636 human fecal samples of clinical origin (7.4%).Campylobacter jejuni was the most frequently isolated species from broilers (81%) and humans (84%), and Campylobacter coli was most frequently isolated from pigs (100%). An extremely high frequency of ciprofloxacin resistance was detected amongCampylobacter strains, particularly those isolated from broilers and pigs (99%), with a slightly lower result for humans (72%); cross-resistance with nalidixic acid was almost always observed. A higher frequency of resistance to erythromycin (81.1%), ampicillin (65.7%), gentamicin (22.2%), and amikacin (21.6%) was detected in C. coli strains isolated from pigs compared to those isolated from humans (34.5, 29.3, 8.6, and 0%, respectively). A low frequency of erythromycin resistance was found in C. jejuni or C. coli isolated from broilers. A greater resistance to ampicillin and gentamicin (47.4 and 11.9%, respectively) was detected in C. jejuni isolated from broilers than in human strains (38 and 0.4%, respectively). β-Lactamase production was found in 81% of the Campylobacter strains tested, although 44% of them were characterized as ampicillin susceptible. The increasing rates of Campylobacter resistance make advisable a more conservative policy for the use of antibiotics in farm animals.

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.

Detection of CMY-2, CTX-M-14, and SHV-12 β-Lactamases in Escherichia coli Fecal-Sample Isolates from Healthy Chickens

ABSTRACT Genes encoding the CMY-2, CTX-M-14, and SHV-12 β-lactamases were detected in three of five Escherichia coli isolates from fecal samples from healthy chickens which showed resistance or diminished susceptibility to extended-spectrum cephalosporins. A −42 mutation at the promoter region of the ampC gene was detected in the other two isolates.

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.

Prevalence of extended-spectrum beta-lactamase-producing Escherichia coli isolates in faecal samples of broilers

Seventy-six faecal samples were obtained from broilers at slaughterhouse level in Portugal. Samples were inoculated on cefotaxime-supplemented Levine agar plates. Cefotaxime-resistant Escherichia coli isolates were recovered from 32 samples (42.1%), obtaining a total of 34 E. coli isolates (one or two isolates per sample). Susceptibility to 16 antibiotics was studied by disk diffusion method, and 85% of the isolates presented a phenotype of multi-resistance that included antimicrobial agents of at least four different families. Extended-spectrum-beta-lactamases (ESBL) of the TEM and CTX-M groups were detected in 31 ESBL-positive E. coli isolates. Twenty-six isolates harboured the bla(TEM-52) gene and two of them also harboured bla(TEM-1b). The bla(CTX-M-14) gene was identified in three isolates (in association with bla(TEM-1b) in one of them), and bla(CTX-M-32) was demonstrated in two additional isolates. Three of the 34 cefotaxime-resistant isolates (9%) did not produce ESBLs, and two of them presented mutations at positions -42 (C-->T), -18 (G-->A), -1 (C-->T), and +58(C-->T) of the promoter/attenuator region of ampC gene. tet(A) and/or tet(B) genes were detected in all 34 tetracycline-resistant isolates, aadA in all 26 streptomycin-resistant isolates; cmlA in 3 of 6 chloramphenicol-resistant isolates, and aac(3)-II or aac(3)-I + aac(3)-IV genes in all 4 gentamicin-resistant isolates. Different combinations of sul1, sul2 and sul3 genes were demonstrated among the 22 trimethoprim-sulfamethoxazole-resistant isolates. Amino acid changes in GyrA and ParC proteins were identified in all 18 ciprofloxacin-resistant isolates. The results of this study indicate that the intestinal tract of healthy poultry is a reservoir of ESBL-positive E. coli isolates.

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.