Antimicrobial resistance and phage and molecular typing of Salmonella strains isolated from food for human consumption in Spain.

The aims of this study were to ascertain the population structure and antimicrobial susceptibility of Salmonella enterica serovars isolated in 2002 from food in 16 Spanish regions. Serovars were characterized by serotyping, phage typing, antimicrobial susceptibility, and pulsed-field gel electrophoresis (PFGE) typing, and 264 nonrelated strains were selected for further analysis. The main sources were eggs and their derivatives (21.6% of strains), poultry and related products (16.6%), and seafood (16.3%). High serotype diversity was detected (51 serotypes); the most common were Enteritidis (n = 96, 36.3%) and Typhimurium (n = 53, 20.1%), followed by a miscellaneous group of 49 different serotypes (n = 115, 43.5%). A 15% increase in Salmonella Enteritidis isolation was observed. Common phage types for Salmonella Enteritidis were PT1 (41.6% of isolates), PT4 (9.4%), PT6 (9.4%), and PT6a (9.4%), and common types for Salmonella Typhimurium were DTU302 (18.8%), DT104 (15.1%), and DT104B (13.2%). Salmonella Enteritidis strains were categorized into eight PFGE types with a similarity of 81 to 96%, and 73.9% of the strains were grouped into just one cluster. Salmonella Typhimurium isolates were divided into 13 PFGE types with a similarity of 64 to 86%, and one predominant clone contained 41.5% of the strains. Resistance rates for Salmonella Enteritidis, Salmonella Typhimurium, and the miscellaneous group were, respectively, 8.3, 69.8, and 13.9% for ampicillin, 3.1, 52.8, and 59% for streptomycin, 40.6, 22.6, and 10.4% for nalidixic acid, 15.6, 71.7, and 31.1% for tetracycline, 7.3, 18.8, and 9.5% for trimethoprim-sulfamethoxazole, 0, 50.9, and 4.3% for chloramphenicol, and 6.2, 71.7, and 17.4% for multiple (at least four) antimicrobials. All the strains remained susceptible to other beta-lactams and fluoroquinolones. Surveillance of S. enterica isolated from food is strongly recommended to reduce community exposure to antimicrobial resistant strains.

Epidemiological tracing of Salmonella enterica serotype Abortusovis from Spanish ovine flocks by PFGE fingerprinting.

Salmonella enterica serotype Abortusovis, an ovine host-specific serotype, rare in most countries, is responsible for epidemic abortion episodes in Spain. With the aim of surveillance and detection of the spread of specific clones, 55 Abortusovis isolates collected during 1996-2001 from flocks in 11 provinces, were studied using XbaI-PFGE. Despite the fact that the strains were geographically and spatially related, PFGE demonstrated an epidemiologically acceptable discriminating power, identifying 20 clones (similarity, 52-96%). Clones Sabv6, 1, 5 were disseminated in seven, five and two areas respectively, while another 17 clones appeared in single places. Clones from nearby geographic regions showed a high relatedness (one band of difference in the PFGE profile) Sabv1-2-3, Sabv5-6, Sabv7-8, and Sabv13-14, suggesting a common ancestor. Co-isolation in the same flock (Sabv5-6, Sabv1-3, Sabv1-6) was detected. PFGE surveillance detected the predominance and widespread distribution of clone Sabv6 in 21 out of the 55 Abortusovis serotype episodes studied in Spain.

A foodborne outbreak of Salmonella infection due to overproduction of egg-containing foods for a festival.

A large outbreak of gastroenteritis occurred in Catalonia in June 2002 with 1435 cases and 117 hospitalizations. Consumption of a hard pastry with vanilla cream was strongly associated with illness. Stool samples from cases and food-handlers were analysed. The premises of the food manufacturer were inspected and food samples were taken for microbiological analysis. Salmonella serotype Enteriditis was isolated from 154 cases, three food-handlers and nine food samples. Outbreak-associated strains showed a coincident phage type, antibiotype and pulse-field gel electrophoresis pattern. Inadequate handling of foods containing eggs occurred because the establishment exceeded its safe food production capacity to meet demand for the pastry, which was consumed on the day of a traditional festival. Excessive production of foods for holidays or special events represents a potential public health threat.

Fecal shedding of Escherichia coli O157, Salmonella, and Campylobacter in Swiss cattle at slaughter.

Fecal samples from 2,930 slaughtered healthy cattle were examined with the following goals: (i) to monitor the shedding of Escherichia coli O157, Salmonella, and Campylobacter in cattle; and (ii) to further characterize the isolated strains. The percentage of the 2,930 samples that tested positive for E. coli O157 by PCR was 1.6%. Thirty-eight strains from different animals that agglutinated with Wellcolex E. coli O157 were isolated. Of the six sorbitol-negative strains, five tested positive for stx2 genes (two times for stx2c and three times for stx2), and one strain tested positive for stx1 and stx2c genes. All sorbitol-negative strains belonged to the serotypes O157:H7- and O157:H7 and harbored the eae type gamma 1 and ehxA genes. The 32 sorbitol-positive strains tested negative for stx genes and belonged to the serotypes O157:H2, O157:H7, O157:H8, O157:H12, O157:H19, O157:H25, O157:H27, O157:H38, O157:H43, O157:H45, and O157:H-. All O157:H45 strains harbored the eae subtype alpha 1 and therefore seem to be atypical enteropathogenic E. coli strains. Whereas none of 1,000 examined samples was positive for Salmonella, 95 of 935 (10.2%) samples were positive for Campylobacter, and all strains were identified as C. jejuni. Sixteen Campylobacter strains were resistant to tetracycline, five were resistant to nalidixic acid/ciprofloxacin, four were resistant to streptomycin, and one was resistant to nalidixic acid/ciprofloxacin and streptomycin. Fecal shedding of zoonotic pathogens in slaughter animals is strongly correlated with the hazard of carcass contamination. Therefore, the maintenance of slaughter hygiene is of crucial importance.

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.

Antimicrobial resistance in salmonellae from humans, food and animals in Spain in 1998.

We studied 1710 Salmonella: spp. isolates from human (1051), food (421) and animal (238) sources. They were tested by the disc diffusion method for susceptibility to 12 different antimicrobial agents. The incidence of resistance and multiple resistance (MR) among the salmonella strains of different origins, the relationship between their most frequent serotypes and phage types (PTs) and their antimicrobial resistance patterns were determined. In general, the incidence of resistance and MR was significantly higher in animal isolates than in human and food isolates (P < 0.05). Resistance to each individual drug among the human isolates and food isolates was very similar, with resistance to ampicillin, tetracycline, streptomycin and sulphonamides most frequently observed. MR has remained uncommon in Salmonella enteritidis. Nevertheless, 90% of PT6A of the human isolates and 100% of the food isolates were ampicillin resistant and 80 and 60%, respectively, of the PT1 isolates were nalidixic acid resistant. Salmonella typhimurium was the most multiresistant serotype in the three sample populations and ten different patterns of MR were seen. Almost 100% of the Salmonella hadar isolates, from human and food sources, were resistant. We recommend restriction of the use of antibiotics in veterinary medicine in order to reduce the selection and spread of multiresistant strains.

[Food poisoning outbreak due to the consumption of spaghetti a la carbonara caused by Salmonella enteritidis]. / Brote de toxiinfección alimentaria por consumo de espaguetis a la carbonara causado por Salmonella enteritidis.

INTRODUCTION: This paper reports a clinico-epidemiological and microbiological investigation conducted into an outbreak of gastrointestinal infection due to Salmonella enteritidis, where the most likely food vehicle was spaghetti a la carbonara. METHODS: An historic cohort study was conducted out among persons exposed to menus at a school canteen. Data were gathered on age, sex, foods consumed and clinical symptoms. School premises and menus were inspected, food samples obtained (spaghetti and meat balls), and stool samples taken from 30 affected subjects and 8 food handlers. Isolated strains were studied using pulsed-field electrophoresis. Attack rates were computed, and the odds ratio adjusted for the remaining foodstuffs (ORa) used to calculate the independent contribution made by the respective foods to risk of infection. RESULTS: Study coverage was 75.7% (140/185). The overall attack rate was 72.1% (101/140), with 12.9% of those affected requiring hospitalisation. The multivariate analysis showed that, while the spaghetti maintained its association (ORa = 8.4; 95% CI 1.4-51.8), the meat balls registered a reduction in risk (ORa = 1.8; 95% CI 0.4-7.5). S. enteritidis was isolated in stool cultures from 28 affected subjects, and in 2 blood and 6 stool cultures from food handlers (5 of whom were classed as cases). Moreover, S. enteritidis was also isolated in the food samples. On pulsed-field electrophoresis, the strains registered the same electrophoresis pattern. CONCLUSIONS: This outbreak serves to underscore the gravity of Salmonella spp. food poisoning, the danger of using inadequately cooked eggs, and the importance of interviewing food handlers to ensure proper classification (i.e., as patients or carriers). Existing recommendations as to the use of pasteurised egg products ought to be extended in scope.

Evaluation of phenotypic and genotypic markers for characterisation of the emerging gastroenteritis pathogen Salmonella hadar.

Over the last 4 years Salmonella hadar has increasingly been isolated in Europe in conjunction with food-borne gastroenteritis. The aim of this study was to evaluate epidemiological methods (phage typing, antimicrobial susceptibility testing, DNA plasmid analysis, ribotyping and pulsed-field gel electrophoresis analysis) for characterising Salmonella hadar isolates. The 100% phage typeability of isolates and the high discriminatory index of 0.8856 suggest that phage typing is the method of choice. In order to obtain subdivisions of the most frequent Salmonella hadar phage types, a combination of molecular methods, such as ribotyping performed with Bg/I and EcoRI or pulsed-field gel electrophoresis using XbaI and XhoI, would be desirable as the usefulness of each technique varies with the phage type being analysed. Of note was the high (86%) rate of resistance to tetracycline and nalidixic acid but full susceptibility to ciprofloxacin in the strains studied.

[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.

[Antimicrobial susceptibility of a selection of Salmonella enterica strains of various origins isolated in Spain]. / Estudio de la sensibilidad a antimicrobianos de cepas de Salmonella enterica de distintos orígenes aisladas en España.

The widespread use of antimicrobials in human and veterinary practice is increasingly causing the emergence of different multidrug-resistant human pathogens. This situation makes treating infections caused by these microorganisms difficult. Salmonella enterica is an ubiquitous organism and may be a good indicator of the influence of the use and abuse of antimicrobials on the appearance of multiresistant strains. One hundred and ninety S. enterica strains of different origins isolated in Spain in 1996 were randomly selected. The minimal inhibitory concentration (MIC) was studied using the agar dilution method according to NCCLS criteria in the following antimicrobials: ampicillin, ticarcillin, amoxicillin-clavulanic acid, cefazolin, cefuroxime, cefotaxime, imipenem, gentamicin, apramycin, ciprofloxacin, streptomycin, chloramphenicol, tetracycline, sulfamethoxazole and co-trimoxazole. Sixty-three percent of the S. enterica tested were resistant and 24% were multiresistant. The percentage of resistant and multiresistant strains of S. enterica of human origin was slightly higher than those of nonhuman origin. Statistically, ampicillin, ticarcillin and amoxicillin-clavulanic acid were significantly more resistant in strains of human origin. Ninety-one percent of the strains of Typhimurium serotype and phagotype 104 were multiresistant. The Salmonella Typhimurium serotype and phagotype 104 ACSTSu-resistant clone, which is widespread in various Western countries, was also isolated in this study. The use of different antimicrobials in human and veterinary practice needs to be rationalized.

Multiple analysis of a foodborne outbreak caused by infant formula contaminated by an atypical Salmonella virchow strain.

An outbreak of food poisoning involving most autonomous Spanish communities was detected in the first half of 1994. The causative food was infant formula milk contaminated by lactose-fermenting Salmonella virchow. It was not possible to isolate the causative strain from the manufacturer's facilities. During the same period of time, there was a significant increase in lactose-non-fermenting Salmonella virchow strains compared with the same period in previous years. Simultaneously, lactose-non-fermenting strains were recovered from clinical samples from children and from some milk samples that were involved in the outbreak. Therefore, it was speculated that the outbreak might be more extensive than initially thought. The following epidemiological markers were used for typing the Salmonella virchow strains involved in the outbreak: (i) phage typing: (ii) ribotyping, using a set of 20 different endonucleases: and (iii) pulsed-field gel electrophoresis, using three different endonucleases. The most useful markers for this serotype were phage typing and pulsed-field gel electrophoresis, since ribotyping was not able to distinguish all strains tested. The results obtained revealed that the outbreak was caused by at least two strains: one presenting phage type 4-4a and pulsed-field patterns A1 or A2 and L+ or L-, and another presenting phage type 2 and pulsed-field patterns A1 or A2 and L+ or L-. The results indicate that the outbreak was more extensive than initially thought and that the Virchow serotype is very clonal in Spain.

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.

[Epidemic outbreaks of salmonellosis caused by eating eggs]. / Brotes epidémicos de salmonelosis por consumo de huevos.

BACKGROUND: Investigation of outbreaks of foodborne infections (OFI) by Salmonella associated with the consumption of hens' eggs from a same classification center of Castellón, and determination of origin, extension and to take measures of control and prevention. METHODS: Descriptive and case-control studies, microbiologic analysis of feces, suspicious foods, and eggs. Sampling of eggs in the classification center for estimation the prevalence of Salmonlla. RESULTS: In 1992, 5 OFI were detected, 4 collective (1 school, 2 restaurants, and 1 residence) and 1 at home, by the consumption of food prepared with eggs: fried or boiled eggs, omelette, soufflé, 2 times, and home-made russian salad. Five hundred and forty-five persons were exposed and 364 were studied, with 100 case patients and 16 hospitalized. The range of attack rates was 10.5-87.0%. Samonella enteritidis (3 OFI) and S. typhimurium (3 OFI) were the infectious agents. In February 1993, the prevalence of Salmonella in eggs from the center was 0.26% (4/1.524) (S. enteritidis, 2 isolates, and S. typhimurium, 2 isolate), three on the shell, and one in the yolk. Two farmhouses of the six supplied were infected. CONCLUSIONS: These outbreaks indicate that salmonellosis by egg's consumption are frequent here and investigation of infection sources is necessary.