ant(6)-I Genes Encoding Aminoglycoside O-Nucleotidyltransferases Are Widely Spread Among Streptomycin Resistant Strains of Campylobacter jejuni and Campylobacter coli.

Thermotolerant Campylobacter species C. jejuni and C. coli are actually recognized as the major bacterial agent responsible for food-transmitted gastroenteritis. The most effective antimicrobials against Campylobacter are macrolides and some, but not all aminoglycosides. Among these, susceptibility to streptomycin is reduced by mutations in the ribosomal RPSL protein or by expression of ANT(6)-I aminoglycoside O-nucleotidyltransferases. The presence of streptomycin resistance genes was evaluated among streptomycin-resistant Campylobacter isolated from humans and animals by using PCR with degenerated primers devised to distinguish ant(6)-Ia, ant(6)-Ib and other ant-like genes. Genes encoding ANT(6)-I enzymes were found in all possible combinations with a major fraction of the isolates carrying a previously described ant-like gene, distantly related and belonging to the new ant(6)-I sub-family ant(6)-Ie. Among Campylobacter isolates, ant(6)-Ie was uniquely found functional in C. coli, as shown by gene transfer and phenotype expression in Escherichia coli, unlike detected coding sequences in C. jejuni that were truncated by an internal frame shift associated to RPSL mutations in streptomycin resistant strains. The genetic relationships of C. coli isolates with ANT(6)-Ie revealed one cluster of strains presented in bovine and humans, suggesting a circulation pathway of Campylobacter strains by consuming contaminated calf meat by bacteria expressing this streptomycin resistance element.

Co-occurrence of colistin-resistance genes mcr-1 and mcr-3 among multidrug-resistant Escherichia coli isolated from cattle, Spain, September 2015.

Colistin resistance genes mcr-3 and mcr-1 have been detected in an Escherichia coli isolate from cattle faeces in a Spanish slaughterhouse in 2015. The sequences of both genes hybridised to same plasmid band of ca 250 kb, although colistin resistance was non-mobilisable. The isolate was producing extended-spectrum beta-lactamases and belonged to serotype O9:H10 and sequence type ST533. Here we report an mcr-3 gene detected in Europe following earlier reports from Asia and the United States.

Detection of plasmid mediated colistin resistance (MCR-1) in Escherichia coli and Salmonella enterica isolated from poultry and swine in Spain.

Recent findings suggest that use of colistin as a last resort antibiotic is seriously threatened by the rise of a new plasmid mediated mechanism of resistance (MCR-1). This work identifies, for the first time in Southern Europe, the gene mcr-1 in nine strains from farm animals (poultry and swine) corresponding to five Escherichia coli and four Salmonella enterica, among which three belong to serovar Typhimurium and one to Rissen. The MCR-1 was found encoded by a plasmid highly mobilizable by conjugation to the E. coli J53 strain. Two E. coli strains carried two determinants, mcr-1 plus pmrA or pmrB mutations, known to confer colistin resistance.

Identification of the main quinolone resistance determinant in Campylobacter jejuni and Campylobacter coli by MAMA-DEG PCR.

Among zoonotic diseases, campylobacteriosis stands out as the major bacterial infection producing human gastroenteritis. Antimicrobial therapy, only recommended in critical cases, is challenged by resistance mechanisms that should be unambiguously detected for achievement of effective treatments. Quinolone (ciprofloxacin) resistance of Campylobacter jejuni and Campylobacter coli, the 2 main Campylobacter detected in humans, is conferred by the mutation gyrA C-257-T, which can be genotyped by several methods that require a previous identification of the pathogen species to circumvent the sequence polymorphism of the gene. A multiplex PCR, based on degenerated oligonucleotides, has been designed for unambiguous identification of the quinolone resistance determinant in Campylobacter spp. isolates. The method was verified with 249 Campylobacter strains isolated from humans (141 isolates) and from the 3 most important animal sources for this zoonosis: poultry (34 isolates), swine (38 isolates), and cattle (36 isolates). High resistance to ciprofloxacin, MIC above 4µg/mL, linked to the mutated genotype predicted by MAMA-DEG PCR (mismatch amplification mutation assay PCR with degenerated primers) was found frequently among isolates from the different hosts.

Prevalence of quinolone resistance determinants in non-typhoidal Salmonella isolates from human origin in Extremadura, Spain.

Resistance to the quinolones nalidixic acid (NAL) and ciprofloxacin (CIP) and the occurrence of quinolone resistance determinants have been investigated in 300 non-typhoidal Salmonella from human origin, isolated in the years between 2004 and 2008, in 6 hospitals within Extremadura (Spain). Salmonella Enteritidis was the major serotype found among quinolone-resistant isolates, most of which were clustered by clonal analysis to a single clone, which presented D87 or S83 substitutions in GyrA. Eleven isolates presented the non-classical quinolone resistance phenotype (resistance to CIP and susceptibility to NAL), lacking mutations in the quinolone resistance determinant region of topoisomerase genes. Among them, one Salmonella Typhimurium isolate carried a qnrS1 gene in a low-molecular-weight plasmid, pQnrS1-HLR25, identical to plasmids previously found in the UK, Taiwan, and USA. The occurrence of this genetic element could represent a risk for the horizontal transmission of quinolone resistance among Enterobacteriaceae in the Iberian Peninsula.

Co-occurrence of ACSSuT and cephalosporin resistance phenotypes is mediated by int1-associated elements in nontyphoidal Salmonella enterica from human infections in Spain.

A screening of antimicrobial resistance and its genetic determinants has been performed on 300 Salmonella enterica isolates collected during 2004-2008 from human infections in Spain. Salmonella Typhimurium and Salmonella Enteritidis were the major serotypes, which were found with similar frequencies covering 80% of the bacterial collection. Salmonella Typhimurium isolates frequently shared low susceptibility to antimicrobials of the penta-resistance phenotype (ACSSuT) and/or cephalosporin resistance. The ACSSuT profile was found closely linked to int1-associated gene cassettes, with major elements carrying DNA fragments of 1.0 Kb (aadA2 gene) plus 1.2 Kb (blaPSE-1 gene) or 2.0 Kb (aadA1 and blaOXA-1 genes). Among these, ACSSuT and cephalosporin resistances were associated in Salmonella Typhimurium isolates expressing the blaOXA gene. ß-lactamase activities were also detected from isolates carrying blaTEM, blaCMY, or blaSHV, although only the two last genes expressed extended-spectrum ß-lactamases. The clonal analysis of S. enterica strains suggests that both horizontal and vertical transfer mechanisms are involved in the wide dissemination of their antimicrobial resistance.

Dissemination of antimicrobial-resistant clones of Salmonella enterica among domestic animals, wild animals, and humans.

Non-typhoidal salmonellosis is an important zoonotic disease caused by Salmonella enterica. This work focuses on the identification of Salmonella enterica clonal strains which, presenting a wide distribution potential, express resistance determinants that compromise effectiveness of the antimicrobial therapy. The screening was performed on 506 Salmonella enterica isolates from animals and humans, which were characterized by serovar and phage typing, genome macrorestriction and pulsed-field gel electrophoresis, and detection of phenotypic and genotypic traits for antimicrobial resistance. A Salmonella Enteritidis strain with strong quinolone resistance is spread on three host environments carrying one of the four variants found for the GyrA protein: (1) Asp87Tyr, the major polymorphism found in 39 Salmonella isolates from human origin and six from poultry; (2) Ser83Phe, with four isolates from human origin and one from white stork (Ciconia ciconia); and (3) Asp87Asn or (4) Asp87Gly, with two isolates each from human origins. Several Salmonella Typhimurium strains that presented int1 elements and the classically associated pentaresistance (ACSSuT) phenotype were found distributed between two host environments: domestic animals and humans, domestics and wild animals, or wild fauna plus humans. This study points out the importance of monitoring gut microbiota and its antimicrobial resistance from wildlife, in parallel to livestock animals and humans, especially for animal species that are in close contact with people.

Antimicrobial resistance determinants among anaerobic bacteria isolated from footrot.

Antibiotic resistance has been evaluated among 36 Gram negative and anaerobic bacilli (10 Bacteroides, 11 Prevotella, 7 Porphyromonas and 8 Fusobacterium strains) isolated from clinical cases of caprine and ovine footrot (necrotic pododermatitis). The initial analysis on this bacterial consortium evaluates the relationships existing among antimicrobial resistance determinants, phenotype expression and mobilization potential. The Bacteroides strains were generally resistant to penicillins, first-generation cephalosporins, tetracycline and erythromycin, and expressed low level of ß-lactamase activity. The main determinants found among the Bacteroides strains were cepA and tetQ genes, conferring resistance to ß-lactams and tetracycline, respectively. A general susceptibility to ß-lactams was shown for most Prevotella, Porphyromonas and Fusobacterium strains, where none of the ß-lactamase genes described in Bacteroides was detected. Resistance to tetracycline and/or erythromycin was found among the three bacterial groups. Although tetQ genes were detected for several Prevotella and Porphyromonas strains, a unique ermF positive was revealed among Prevotella strains. The expression of resistance markers was not related with the polymorphism of their coding sequences. However, the finding of sequence signatures for conjugative transposons in the vicinities of tetQ and ermF suggests a mobilization potential that might have contributed to the spread of antimicrobial resistance genes.

Discrimination between Bacteroides, Dichelobacter, Fusobacterium, Porphyromonas and Prevotella isolated from caprine footrot by PCR-RFLP - short communication.

Footrot is widely considered the most severe and most common foot pathology in small ruminants. This study tested the ability of a molecular typing system based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay of the 16S rRNA gene to discriminate between the strict anaerobe genera most commonly isolated from footrot ( Bacteroides, Dichelobacter, Fusobacterium, Porphyromonas and Prevotella ) in goats in Extremadura (Spain), with a view to facilitating identification for diagnostic purposes and thus providing a useful tool for future epidemiological studies. Although the electrophoretic patterns obtained with the enzyme Tru 1I were more readily interpreted, and may thus be the best initial option, results may be confirmed by a second enzyme ( RsaI). The PCR-RFLP assay of the 16S rRNA gene may therefore prove a useful addition to conventional biochemical identification techniques, providing taxonomic information at genus level.

Gene Context and DNA rearrangements in the carbapenemase locus of division II strains of Bacteroides fragilis.

The cfiA gene is clustered in a bicistronic operon encoding an N-acetyltransferase and an O-acetyltransferase related to resistance markers. This genetic context, exclusively found in strains of Bacteroides fragilis division II, has been highly rearranged by the successive integration of two new mobile sequences, a miniature element and ISBf9. Besides that, among the DNA polymorphisms detected in the cfiA locus, only the integration of IS942 at its promoter was a determinant for expression of carbapenemase activity.

Genetic determinants for cfxA expression in Bacteroides strains isolated from human infections.

OBJECTIVES: To identify genetic determinants that determine beta-lactamase expression in Bacteroides strains isolated from human infections. METHODS: Beta-lactam susceptibility and beta-lactamase enzyme expression were characterized in selected strains. Beta-lactamase genes and surrounding regions were analysed by PCR, inverse PCR and Southern hybridization. RESULTS: High resistance to penicillins and cephalosporins was found among most isolated strains, in which all known beta-lactamase genes from Bacteroides are represented, but differences were found in their expression of enzyme activity. In contrast to the cepA gene, ubiquitously found but frequently inactive, or cfiA, which only confers carbapenem resistance in two strains, the detection of high beta-lactamase expression correlates closely with the presence of cfxA genes. This genetic determinant shares variability of upstream regulatory elements, including sequence tags from Tn4555, Tn4351 and IS614B, and polymorphisms of encoded amino acid sequences at positions G(57)C and Y(259)C, which might determine enzyme expression characteristics. CONCLUSIONS: The main determinant for beta-lactamase expression in Bacteroides strains is the cfxA gene, in which IS614B integration upstream of the coding sequence represents a molecular marker for higher levels of enzyme activity.

Penicillin-binding proteins of Bacteroides fragilis and their role in the resistance to imipenem of clinical isolates.

In this study penicillin-binding proteins (PBPs) of Bacteroides fragilis and the resistance mechanisms of this micro-organism to 11 beta-lactam antibiotics were analysed. The study focused on the role of PBP2Bfr and metallo-beta-lactamase in the mechanism of resistance to imipenem. The mechanism of beta-lactam resistance in B. fragilis was strain dependent. The gene encoding the orthologue of Escherichia coli PBP3 gene (pbpBBfr, which encodes the protein PBP2Bfr) was sequenced in five of the eight strains studied, along with the ccrA (cfiA) gene in strain 119, and their implications for resistance were examined. Differences were found in the amino-acid sequence of PBP2Bfr in strains AK-2 and 119, and the production of beta-lactamases indicated that these differences may be involved in the mechanism of resistance to imipenem. In vitro binding competition assays with membrane extracts using imipenem indicated that the PBP that bound imipenem with the highest affinity was PBP2Bfr, and that increased affinity in strain 7160 may be responsible for the moderate susceptibility of this strain to imipenem. In the same way, the importance of the chromosomal class A beta-lactamase CepA in the resistance mechanism of the B. fragilis strains NCTC 9344, 7160, 2013E, AK-4, 0423 and R-212 was studied. In these strains this is the principal resistance mechanism to antimicrobial agents studied other than imipenem.

The use of melatonin as a vaccine agent.

Molecules with immunomodulatory properties determine the magnitude and quality of immune responses specific for the coadministered antigen. Melatonin is considered a biological-response modifier of the immune system with broad application in veterinary medicine. In seasonally-breeding animals, the indolamine is able to improve reproductive performance. With the purpose of expanding new advantageous roles for melatonin, we investigated the effect of subcutaneous slow-release melatonin implants in the humoral response after a vaccination. We reported here a new feature of melatonin as an adjuvant-like system towards Dichelobacter nodosus (A1 and C serotypes)--the bacterium which cause ovine footrot--the most important cause of lameness in sheep. Antibody titres determined by both agglutination and ELISA techniques were substantially higher and were sustained for a longer duration than non-implanted animals. Remarkably, the effect of melatonin was completely dependent on the presence of aluminium hydroxide. The finding that melatonin enhances a defined immune response in vivo opens new perspectives for the improvement of Th2-biased immune responses by alum adjuvants.

Relationship between penicillin-binding protein patterns and beta-lactamases in clinical isolates of Bacteroides fragilis with different susceptibility to beta-lactam antibiotics.

This study examines the role of the penicillin-binding proteins (PBPs) of Bacteroides fragilis in the mechanism of resistance to different beta-lactam antibiotics. Six of the eight strains used were beta-lactamase-positive by the nitrocefin assay. These strains displayed reduced susceptibility to imipenem (MIC, 2-16 mg l(-1)) and some of them were resistant to the actions of ampicillin, cefuroxime, cephalexin, cefoxitin and piperacillin. When studying specific enzymic activity, the capacity to degrade cefuroxime was only detected in strains AK-4, R212 and 0423 and the capacity to degrade cephalexin was only detected in strains R212 and 2013E; no specific activity was detected on imipenem. Metallo-beta-lactamase activity was only detected in strains AK-2 and 119, despite the fact that the cfiA gene was identified in four strains (AK-2, 2013E, 119 and 7160). The cepA gene was detected in six of the eight strains studied. Three high-molecular-mass PBPs were detected in all strains; however, in some cases, PBP2Bfr and/or PBP3Bfr appeared as a faint band. PBP4Bfr and PBP5Bfr were detected in six strains. PBP6Bfr only was detected in B. fragilis strains AK-2, 0423, 119 and 7160. By analysis of the sequence of B. fragilis chromosomal DNA and comparison with genes that are known to encode PBPs in Escherichia coli, six genes that encode PBP-like proteins were detected in the former organism. The gene that encodes the PBP2 orthologue of E. coli (pbpABfr, PBP3Bfr) was sequenced in six of the eight strains and its implications for resistance were examined. Differences in the PBP3Bfr amino acid sequences of strains AK-2 and 119 and their production of beta-lactamases indicate that these differences are not involved in the mechanism of resistance to imipenem and/or cephalexin.