María Aurora Echeita

Antibiotic Resistance of Salmonella spp. from Animal Sources in Spain in 1996 and 2000

Emergence of resistant and multiresistant bacteria has become an important worldwide sanitary problem. International agencies recommend improving resistance surveillance studies in not only human but also animal origin strains. Because of its ubiquitous characteristics and zoonotic agent consideration, Salmonella spp. can be used as a good indicator microorganism for resistance surveillance studies. Salmonella spp. strains from animal sources isolated in 1996 (107) and 2000 (474) in Spain were tested against 12 different antimicrobials agents, using the disc diffusion method. Results were interpreted following the NCCLS criteria. Data showed that Salmonella spp. strains (61.7% in 1996 and 81.5% in 2000) were resistant to at least one antibiotic. Pig-related strains were considerably more resistant than strains from other sources. Enteritidis serotype was less resistant than other serotypes, except for ampicillin in 1996 (50% resistant) and nalidixic acid in 2000 (65.1% resistant). An emergent monophasic serotype, 4,5,12:i:-, first detected in 1997 in Spain was 100% resistant and 90% multiresistant. Typhimurium serotype was the most common Salmonella serotype from animal sources in both years. It was widely distributed among animals and was among the serotypes with a higher degree of resistance. The ampicillin, chloramphenicol, sulfonamides, streptomycin, and tetracycline resistance pattern, commonly associated with Salmonella serotype Typhimurium DT 104, had spread among other Typhimurium phage types and other Salmonella serotypes. Salmonella spp. strains isolated from feeding stuffs were considerably more susceptible than animal source strains, suggesting that the high Salmonella spp. resistance percentage was probably due to the use of antibiotics in animal farms rather than the consumption of contaminated feeding stuffs.

Salmonella enterica Infections in Spanish Swine Fattening Units

The present study is the first conducted in Spain to estimate the bacteriological herd prevalence of Salmonella enterica in fattening units and to describe the Salmonella serovar diversity on these farms using a sample representative of the entire swine population. For this purpose, 10 faecal samples were collected from 10 different pens containing pigs close to market weight in a total of 232 fattening units. Total sample size was proportionally distributed according to the fattener census in each of the regions of the country and all the samples were examined by culture of 25 g of faecal material. One hundred (43.1%) farms had at least one Salmonella‐positive sample (95% CI: 37–49.1%). Salmonella enterica was detected in 290 (12.5%) pooled faecal floor samples (95% CI: 11.2–13.8%). The apparent herd prevalence of salmonellosis was similar among multi‐site, finishing and farrow to finish farms. Overall, 24 different serovars were identified, with S. Typhimurium, S. Rissen and S. Derby being the most common both at herd and sample level. Results of phage typing were available for the 91 isolates of S. Typhimurium. A total number of 10 different phage types were identified, with DT 193 being the most frequent. Phage types DT 104, DT 104b and DT U302, which have been associated with several multi‐resistant patterns, accounted for 23% and 29% of the Typhimurium total isolates or Typhimurium infected farms respectively.

Study of the molecular mechanisms involved in high-level macrolide resistance of Spanish Campylobacter jejuni and Campylobacter coli strains

OBJECTIVES To investigate the molecular mechanisms involved in the high-level erythromycin resistance of clinical Spanish Campylobacter jejuni and Campylobacter coli strains. METHODS Overall susceptibilities of 678 C. jejuni and 119 C. coli strains, collected from 10 Spanish provinces during 2006 and 2007, were determined by Etest. In high-level erythromycin-resistant strains, molecular determinants were studied. The analysis was focused on region V of the 23S rRNA gene, the rplD and rplV ribosomal genes, and the regulatory region of the CmeABC efflux pump. RESULTS The global resistance rate to erythromycin was 3.8%. Among the resistant strains, 93% were C. coli and 7% were C. jejuni. The A2075G mutation in the 23S rRNA gene was detected in all of the resistant strains except for two, which carried the A2074G mutation. None of the ribosomal rplD and rplV genes harboured the described mutations that confer resistance to macrolides. Different mutations affecting the regulatory region of the CmeABC efflux pump were also found. CONCLUSIONS C. coli strains are clearly more resistant to erythromycin than C. jejuni. The mutation A2075G in the 23S rRNA gene was responsible for the resistance in most of the strains; A2074G was only found in two strains. Further studies are required to ascertain the effect of mutations in the regulatory region of cmeABC. Our data indicate that the rate of resistance was similar to that of other European countries.

Antimicrobial resistance of Salmonella enterica isolates from apparently healthy and clinically ill finishing pigs in Spain.

This study was the first conducted in Spain to evaluate the occurrence of antimicrobial resistance and multi‐resistance in Salmonella isolates recovered from finishing pigs from Spanish swine farms distributed over the whole country. For this purpose, 290 Salmonella isolates recovered from apparently healthy finishing pigs in a farm‐based cross‐sectional study and 192 Salmonella isolates recovered from faecal samples of finishing pigs suffering from diarrhoea were investigated. Resistance to a panel of 17 antimicrobials was determined using a broth microdilution technique. Resistance was a common finding and was detected in 90.3% of the Salmonella isolates from apparently healthy finishing pigs and 95.3% of the Salmonella isolates from clinically diseased finishing pigs. Resistance was particularly high among isolates of serogroup B and serovars Typhimurium and its monophasic variant S. 4,5,12:i:‐. Higher frequencies of resistance were found to tetracycline, sulphamethoxazole, streptomycin, spectinomycin, ampicillin, chloramphenicol and trimethoprim‐sulphamethoxazole. Less than 10% of the isolates were resistant to amoxicillin/clavulanic acid, neomycin, cephalotin, apramycin and gentamicin. Resistance to ciprofloxacin, colistin and ceftiofur was rare (under 1%). Multi‐resistance, defined as resistance to four or more drugs, was detected in more than 50% of the isolates. Although multi‐resistance was particularly frequent among isolates of S. Typhimurium, it was also high among other serovars as Bredeney and the S. Typhimurium monophasic variant. 4,5,12:i:‐.

Distribución de los serotipos y fagotipos de Salmonella de origen humano aislados en España en 1997-20

Introduccion. La salmonelosis continua siendo una de las causas principales de gastroenteritis en Espana, siendo la serotipificacion el marcador epidemiologico universalmente utilizado para la caracterizacion de los aislamientos de Salmonella spp. Algunos serotipos se identifican muy frecuentemente, reduciendo el poder de discriminacion de esta tecnica. Por ello, para el estudio epidemiologico de las salmonelosis producidas por estos serotipos es necesario utilizar marcadores complementarios como la fagotipificacion. Metodos. Se serotipificaron, por aglutinacion directa, las cepas de Salmonella spp. de origen humano recibidas en el Laboratorio Nacional de Referencia de Salmonella y Shigella (LNRSSE) entre los anos 1997 y 2001 y se fagotipificaron, segun esquemas internacionales, las cepas de los serotipos Enteritidis, Typhimurium, Hadar, Virchow y Typhi. Resultados. Se analizaron 30.856 cepas de Salmonella spp. procedentes de la mayoria de las Comunidades Autonomas. Los serotipos Enteritidis (51%) y Typhimurium (24%) fueron los mayoritarios. Las combinaciones serotipo/fagotipo mas frecuentes fueron: Enteritidis/FT1 (18%), Enteritidis/FT4 (15%), Enteritidis/FT6a (5%), Typhimurium/FT104 (5%) y Enteritidis/FT6 (3%). Las cepas del serotipo Enteritidis/FT1 tuvieron el mayor aumento en este periodo de tiempo, pasando del 11,61% en 1997 al 24,74% en 2001. Conclusiones. La utilizacion jerarquica de la serotipificacion y posteriormente de la fagotipificacion en cepas de Salmonella spp. de los serotipos mas frecuentes aumento enormemente el poder de discriminacion de la serotipificacion. Su aplicacion en estudios epidemiologicos es de gran utilidad en la caracterizacion temprana de cepas relacionadas.

Novel genetic environment of qnrB2 associated with TEM-1 and SHV-12 on pB1004, an IncHI2 plasmid, in Salmonella Bredeney BB1047 from Spain.

The presence of the qnrB2 gene in animal isolates and zoo-notic pathogens opens the possibility that genetic exchange and plasmid acquisition of the qnrB2 gene could occur in the faecal flora of the animals. Interestingly, p137.25 belongs to the IncN plasmid family that is able to replicate in different enterobacter-ial strains, but also seems prevalent in faecal flora from animals. In fact, a study performed on a large collection of E. coli from the USA demonstrated that the prevalence of IncN plasmids is high in avian E. coli (10% – 16%) but negative in E. coli from faeces of healthy humans. 15 This evidence supports the hypothesis that the Salmonella 137.25 strain acquired the qnrB2 gene on an IncN plasmid circulating in avian bacterial flora. This strain could cause infections in humans through the food chain and the resistance plasmid contributes to the dissemination of the qnrB2 gene in other Enterobacteriaceae. None to declare.

Dynamics of populations of Campylobacter jejuni in two grandparent broiler breeder farms: Persistent vs. transient strains

The objectives of the study were to characterize and investigate the populations of Campylobacter jejuni in two grandparent broiler breeder farms over four years. Caecal as well as farm environmental samples were obtained. Campylobacter isolates were characterized by macrorestriction profile (SmaI and KpnI-PFGE) and PCR-RFLP of the flaA gene. Susceptibility tests against seven antimicrobials were also performed. Birds were negative for Campylobacter spp. when they came to these two production farms (20 weeks), and most of the flocks remained uncolonized until they were 23 weeks old. Eighteen genotypes were characterized, with one of them (genotype 2) appearing and persisting over the study period in the two farms. In general, the strains exhibited high genetic stability, and most of them could be seen as transient in the farms, being substituted by other strains when their flock was substituted. Only one environmental sampling was positive for C. jejuni. Two different genotypes were characterized; one of them was isolated from the birds of that farm two years before. The susceptibility data point to the idea of an environmental source or reservoir of this genotype. Regarding the susceptibility of the populations, as other studies have shown, quinolone resistance (alone or combined with other resistances) was the most frequent: 68.5%. Quinolone- and multidrug-resistant strains are a matter of concern in public health. In conclusion, this survey shows the complexity of the study of the colonization of farms by C. jejuni.

Development of Three Multiplex PCR Assays Targeting the 21 Most Clinically Relevant Serogroups Associated with Shiga Toxin-Producing E. coli Infection in Humans

Escherichia coli serogroups O5, O15, O26, O45, O55, O76, O91, O103, O104, O111, O113, O118, O121, O123, O128, O145, O146, O157, O165, O172, and O177 are the O-antigen forms of the most clinically relevant Shiga toxin-producing E. coli (STEC) serotypes. In this study, three multiplex PCR assays able to specifically detect these 21 serogroups were developed and validated. For this purpose, the O-antigen gene clusters of E. coli O5 and O76 were fully sequenced, their associated genes were identified on the basis of homology, and serogroup-specific primers were designed. After preliminary evaluation, these two primer pairs were proven to be highly specific and suitable for the development of PCR assays for O5 and O76 serogroup identification. Specific primers were also designed for serogroups O15, O45, O55, O91, O104, O113, O118, O123, O128, O146, O157, O165, O172, and O177 based on previously published sequences, and previously published specific primers for serogroups O26, O103, O111, O121, and O145 were also included. These 21 primer pairs were shown to be specific for their target serogroup when tested against E. coli type strains representing 169 known O-antigen forms of E. coli and Shigella and therefore suitable for being used in PCR assays for serogroup identification. In order to validate the three multiplex PCR assays, 22 E. coli strains belonging to the 21 covered serogroups and 18 E. coli strains belonging to other serogroups were screened in a double-blind test and their sensitivity was determined as 1 ng chromosomal DNA. The PCR assays developed in this study could be a faster, simpler, and less expensive strategy for serotyping of the most clinically relevant STEC strains in both clinical microbiology and public health laboratories, and so their development could benefit for clinical diagnosis, epidemiological investigations, surveillance, and control of STEC infections.

Molecular mechanisms of quinolone, macrolide, and tetracycline resistance among Campylobacter isolates from initial stages of broiler production

The aim of this study was to investigate the resistance mechanisms of quinolones, macrolides and tetracycline in campylobacter isolates from grandparent and parent broiler breeders in Spain. Twenty-six isolates were investigated for quinolone resistance, three isolates for macrolide resistance and 39 for tetracycline resistance. All of the quinolone-resistant isolates possessed the mutation Thr86Ile in the quinolone resistance-determining region of gyrA and one isolate possessed the mutation Pro104Ser. Only one Campylobacter coli population (defined by restriction fragment length polymorphism–polymerase chain reaction of flaA and pulsed field gel electrophoresis) was resistant to erythromycin, and the mutation A2075G (23S rDNA) was responsible for macrolide resistance. The tetO gene was found in all of the tetracycline-resistant isolates. Twenty-two out of the 39 isolates investigated by Southern blot possessed chromosomic location of tetO and 17 were located on plasmids. Most of the plasmids with tetO were of around 60 kb and conjugation was demonstrated in a selection of them. In conclusion, we showed that Thr86Ile is highly prevalent in quinolone-resistant isolates as well as mutation A2075G in macrolide-resistant isolates of poultry origin. More variability was found for tetO. The possibility of horizontal transmission of tetO among campylobacter isolates is also an issue of concern in public health.

Spread of a multiresistant CTX-M-9-producing Salmonella enterica serotype Virchow phage type 19 in Spain.

The purpose of this study was to survey Salmonella enterica serotype Virchow phage type 19 (S. Virchow PT19) strains submitted to the Spanish National Reference Laboratory for Salmonella (SNRLS) from 2002 to 2006 in order to determine the rate type and genetic background of β-lactam resistance and to further identify the associated resistances. Ninety-nine S. Virchow PT19 strains were analysed. Antimicrobial susceptibility was determined by the disk diffusion method using Mueller–Hinton agar medium. Polymerase chain reaction (PCR) assays and, later, sequencing of the obtained fragments were performed for the molecular characterisation of the resistances. Pulsed-field gel electrophoresis (PFGE) and plasmid analysis (using conjugation, Southern blot hybridisation and replicon typing) were used for characterisation. The characterisation of S. Virchow PT19 strains allowed the identification of a clonal multiresistant S. Virchow PT19 harbouring an IncH12 plasmid with the blaCTX-M-9 gene within the complex integron In60 distributed across Spain. An IncH12 plasmid widely reported and studied in Enterobacteria is described in a clonal multiresistant S. Virchow PT19 which has successfully spread throughout Spain