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.

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.

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.

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 beta-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 bla (CTX-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.

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:-.

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.

Late detection of a shigellosis outbreak in a school in Madrid.

Even though shigellosis in Spain is rare, an indigenous outbreak is occasionally detected. We describe an outbreak in a school in Madrid caused by person-to-person transmission of Shigella sonnei. After the detection of Shigella sonnei in a stool sample from a 3 year old girl, an investigation at her school was initiated. Questionnaires were distributed to the parents of 520 pupils attending the school. A case was defined as a school case if it was the first case in a child's household, and as a household case if other members of the household had fallen ill first. We identified 88 cases (60 pupils and 28 of their family members). The attack rate (AR) was 12% in the school and 32% in the families. There was a significant association between higher AR and lower age. The outbreak lasted for two months. The length and the shape of the epidemic curve of the 60 cases in pupils suggests person-to-person transmission. Shigella sonnei isolated from 5 different cases were typed by pulsed field gel electrophoresis (PFGE) and was found to be an identical strain. The prolonged duration of the outbreak was probably due to delayed detection, and stopped as soon as control measures were introduced.

Comparison of phenotypic and genotypic markers for characterization of an outbreak of Salmonella serotype Havana in captive raptors.

AIMS: To establish a typing method for tracing the epidemic relationship of 16 strains of Salmonella serotype Havana isolated from captive raptors showing no symptomatology and residing in a wildlife hospital in Spain. METHODS AND RESULTS: Antimicrobial susceptibility testing, ribotyping, pulsed field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP) methodology were applied. Ten unrelated strains of serotype Havana were included as a control group to provide a basis of for the efficiency of the different markers used. All outbreak-related strains were resistant to nalidixic acid and streptomycin and showed the same ripotype, pulsotype and AFLP pattern. CONCLUSIONS: This is the first time that AFLP analysis has been tested with serotype Havana isolates and it has demonstrated to be the most useful epidemiological tool for discriminating between unrelated and outbreak-related strains of this serotype. The results obtained suggest that all the Salmonella serotype Havana isolates represented a common outbreak strain whose origin of contamination could not be established although it is thought that it was the poultry meat used for raptors'diet. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study suggests the importance of microbiological analysis of these products in order to prevent contamination and dissemination of Salmonellae in this kind of Hospital.

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.

Use of AFLP, plasmid typing and phenotyping in a comparative study to assess genetic diversity of Shigella flexneri strains.

Shigella flexneri infections are one of the main causes of acute diarrhoea in Cuba. Twenty strains isolated from sporadic cases in nine different Cuban provinces were characterized. Serotyping, antibiotic-resistance typing, plasmid-typing and AFLP-typing were used to determine their suitability for use in epidemiological studies of S. flexneri. The predominant serotypes were serotype 6 (35%) and serotype 2 (35%). Eleven different plasmid profiles were detected (Diversity Index = 0.92). AFLP-typing discriminated 12 different patterns (DI = 0.95), these patterns were not coincident with plasmid-typing patterns. Both techniques combined distinguished 14 patterns among the 20 studied strains (DI = 0.99). There was no consistent relationship between plasmid-typing and AFLP-typing patterns or antibiotic-resistance typing patterns. Ninety-five percent of S. flexneri strains were multiresistant.

Atypical, fljB-negative Salmonella enterica subsp. enterica strain of serovar 4,5,12:i:- appears to be a monophasic variant of serovar Typhimurium.

An fljB-negative, multidrug-resistant Salmonella enterica serovar 4,5,12:i:- phage type DT U302 strain (resistant to ampicillin, chloramphenicol, sulfonamide, gentamicin, streptomycin, tetracycline, and sulfamethoxazole-trimethoprim) emerged and spread in Spain in 1997. Sequences specific for Salmonella serovar Typhimurium and phage type DT 104 and U302 were present in this atypical Salmonella strain, suggesting that it is a monophasic Salmonella serovar Typhimurium variant.

[Distribution of Salmonella spp. serotypes isolated in Spain during a 4-year period (1993-1996)]. / Distribución de serotipos de Salmonella spp. aislados en España durante un período de 4 años (1993-1996).

BACKGROUND: Knowledge of the epidemiology of salmonellosis over time is the main tool for public health care control of this disease which is the main cause of alimentary toxic infections in Spain. The epidemiologic marker of choice in this genus is the stable, simple technique of serotyping, which, given its wide use allows the follow up of the main serotypes over the years. This study analyzes the seasonal trends of the main serotypes of Salmonella received in the National Reference Laboratory of Salmonella and Shigella in Spain (LNRSSE) from 1993-1996. MATERIALS AND METHODS: Serotyping of the strains of Salmonella received in the LNRSSE during the years of the study with sera induced in our laboratory and commercial laboratories was undertaken. The strains of human origin the trends of the main serotypes were evaluated with the Mantel-Haenszel chi 2 test. RESULTS AND DISCUSSION: Nineteen thousand seven hundred forty-one strains of Salmonella from most of the autonomous communities of Spain were analyzed. Ninety-two point one two percent of the strains of human origin were received from laboratories that sent strains throughout at least three of the four years of the study, thereby allowing evaluation of the serotype trends for this group of strains. The enteritidis and typhimurium serotypes were the most frequently observed. In these serotypes the strains of human origin were typed with double the frequency of that found among those from food and three-fold greater than that found among the strains of environmental origin. Typhimurium was the more frequently found than enteriditis in the group of strains from diseased animals. Finally, the significant statistical increase of the hadar serotype, which surpassed the virchow serotype in recent years, is of note.

Chromosomal rearrangements in Salmonella enterica serotype typhi affecting molecular typing in outbreak investigations.

Salmonella enterica serotype Typhi strains belonging to eight different outbreaks of typhoid fever that occurred in Spain between 1989 and 1994 were analyzed by ribotyping and pulsed-field gel electrophoresis. For three outbreaks, two different patterns were detected for each outbreak. The partial digestion analysis by the intron-encoded endonuclease I-CeuI of the two different strains from each outbreak provided an excellent tool for examining the organization of the genomes of epidemiologically related strains. S. enterica serotype Typhi seems to be more susceptible than other serotypes to genetic rearrangements produced by homologous recombinations between rrn operons; these rearrangements do not substantially alter the stability or survival of the bacterium. We conclude that genetic rearrangements can occur during the emergence of an outbreak.

Molecular typing of Salmonella enterica serovar typhi.

The efficiencies of different tests for epidemiological markers--phage typing, ribotyping, IS200 typing, and pulsed-field gel electrophoresis (PFGE)--were evaluated for strains from sporadic cases of typhoid fever and a well-defined outbreak. Ribotyping and PFGE proved to be the most discriminating. Both detected two different patterns among outbreak-associated strains.

Prevalence of Salmonella serotypes isolated in Spain from human and non human sources (1983-1987).

Salmonella serotypes over a five year period were studied in order to know their prevalence in Spain. The Salmonella Reference Centre received a total of 17,612 strains from 1983-1987. The majority (16,133) were of human origin and only 1,479 strains were isolated from non-human sources. The serotyping yielded 100 different serotypes, Salmonella enterica serotype Enteritidis (8) being the commonest in both groups, 61.18% of human origin and 31.91% of non-human origin. Salmonella enterica serotype Typhimurium the commonest serotype in many countries, occupies second place in our results with the following percentages 11.87% and 9.67% respectively. Among the strains of human origin Salmonella enterica serotype Typhi occupies fourth place (3.24%). This is very low compared with the high number of clinically diagnosed typhoid fever cases declared in the country: over 5,000 cases per year.