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.

Isolation of a point mutation associated with altered expression of the CmeABC efflux pump in a multidrug-resistant Campylobacter jejuni population of poultry origin.

The objective of this study was to investigate the antibiotic resistance phenotype of Campylobacter jejuni isolates from a poultry flock of broiler production in Spain. Isolates were characterised by RFLP-PCR of the flaA gene and multilocus sequence typing. Minimum inhibitory concentrations of quinolones, aminoglycosides, ß-lactams, tetracyclines, phenicols, macrolides and lincosamides were determined by Etest. Determinants of resistance and the regulatory region of the cmeABC operon were investigated in all isolates by PCR detection and sequencing. Expression of the CmeABC efflux pump was investigated by quantitative RT-PCR and accumulation assay. Based on their molecular markers, two different populations of C. jejuni were identified: one resistant to quinolones, ß-lactams and tetracyclines, considered multidrug-resistant (MDR); and another resistant only to tetracyclines. Both populations possessed the tetO gene, previously associated with tetracycline resistance. The blaOXA-61 gene was also present in both populations, although only the MDR population showed ß-lactamase activity. In addition, MDR isolates possessed the Thr86Ile mutation in the gyrA gene responsible for quinolone resistance. Moreover, sequencing of the regulatory region of the cmeABC operon revealed the presence of the C-32→T mutation in the MDR isolates, which was accompanied by an increase in cmeA mRNA levels compared with the non-mutant population. In conclusion, this is the first report of the mutation C-32→T in the cmeABC operon in C. jejuni isolates of veterinary origin. This mutation is associated with overexpression of the CmeABC efflux pump in a MDR population and is possibly related to enhanced tolerance to antimicrobials that favours the development of resistance.

Aplicación del análisis de los polimorfismos de los tamaños de los fragmentos de restricción del gen flaA amplificado por reacción en cadena de la polimerasa y resistotipo para identificar potenciales brotes de campilobacteriosis no diagnosticados en España / Application of restriction fragment length polymorphism-polymerase chain reaction- flaA and resistotype to identify potential undiagnosed outbreaks of campylobacteriosis in Spain

INTRODUCCIÓN: Los brotes detectados de campilobacteriosis son poco frecuentes y, por lo general, cursan con un bajo número de pacientes, aunque se estima que muchos más permanecerían sin diagnosticar. Las técnicas de investigación de brotes más exitosas en Campylobacter spp. (PFGE, MLST) tienen el inconveniente de ser laboriosas y no estar disponibles en muchos laboratorios. MÉTODOS: Durante el año 2008 se recibieron 352 aislados de C. jejuni y C. coli procedentes de 16 hospitales. Todas las cepas fueron tipificadas genotípicamente mediante RFLP-PCR-flaA (tipo flaA) y fenotípicamente con su resistotipo. Se estableció que aquellas cepas de la misma especie procedentes del mismo hospital, aisladas en un periodo de hasta 11 días, con valores de CMI de±1 dilución y con el mismo tipo flaA, podrían pertenecer a un brote. Las cepas que cumpliesen con estos criterios serían posteriormente tipificadas mediante KpnI-PFGE y MLST. RESULTADOS: Veintitrés de los 352 aislados, en 10 grupos, cumplían con los criterios de pertenencia a posibles brotes no diagnosticados. En 8 grupos los pulsotipos (PFGE) de los aislados de cada grupo tenían una semejanza entre ellos mayor del 95%. En 7 de ellos los secuenciotipos (MLST) eran coincidentes. CONCLUSIONES: El uso de 2 marcadores sencillos (resistotipo y RFLP-PCR-flaA) puede detectar aislados que probablemente formen parte de un brote de campilobacteriosis no diagnosticado. Para su confirmación se requieren otros marcadores moleculares y los datos epidemiológicos de cada aislado. El estudio apunta a que, como en otros países, el número de brotes de campilobacteriosis en España está probablemente infraestimado

INTRODUCTION: Outbreaks of campylobacteriosis are infrequent and usually involve a low number of patients, although it is estimated that many more remain undiagnosed. The most successful techniques for outbreak investigation in Campylobacter spp. (PFGE, MLST) have the drawback of being laborious and not available in many laboratories. METHODS: During the year 2008, 352 isolates of C. jejuni and C. coli from 16 hospitals were received in our laboratory. All strains were genotyped by RFLP-PCR-flaA (flaA type) and phenotyped with their resistotype. It was established that the strains of the same species from the same hospital, isolated over a period of up to 11 days, with MIC values of±1 dilution with the same flaA type could belong to an outbreak. Strains that met these criteria would be later subtyped by KpnI-PFGE and MLST.RESULTS:A total of 23 out of 352 isolates, distributed in 10 groups, met the criteria for being associated with putative undiagnosed outbreaks. The similarity of the PFGE-profiles in 8 groups was greater than 95% among the isolates from each group. In 7 of the groups, the sequence types (MLST) were coincident. CONCLUSIONS: The use of 2 easy markers (resistotype and RFLP-PCR-flaA) may detect isolates probably belonging to an undiagnosed outbreak of campylobacteriosis. Accurate diagnosis requires other molecular markers and epidemiological data of each isolate. The study suggests that, as in other countries, the number of outbreaks of campylobacteriosis in Spain is probably underestimated

[Application of restriction fragment length polymorphism-polymerase chain reaction-flaA and resistotype to identify potential undiagnosed outbreaks of campylobacteriosis in Spain]. / Aplicación del análisis de los polimorfismos de los tamaños de los fragmentos de restricción del gen flaA amplificado por reacción en cadena de la polimerasa y resistotipo para identificar potenciales brotes de campilobacteriosis no diagnosticados en España.

INTRODUCTION: Outbreaks of campylobacteriosis are infrequent and usually involve a low number of patients, although it is estimated that many more remain undiagnosed. The most successful techniques for outbreak investigation in Campylobacter spp. (PFGE, MLST) have the drawback of being laborious and not available in many laboratories. METHODS: During the year 2008, 352 isolates of C. jejuni and C. coli from 16 hospitals were received in our laboratory. All strains were genotyped by RFLP-PCR-flaA (flaA type) and phenotyped with their resistotype. It was established that the strains of the same species from the same hospital, isolated over a period of up to 11 days, with MIC values of±1 dilution with the same flaA type could belong to an outbreak. Strains that met these criteria would be later subtyped by KpnI-PFGE and MLST. RESULTS: A total of 23 out of 352 isolates, distributed in 10 groups, met the criteria for being associated with putative undiagnosed outbreaks. The similarity of the PFGE-profiles in 8 groups was greater than 95% among the isolates from each group. In 7 of the groups, the sequence types (MLST) were coincident. CONCLUSIONS: The use of 2 easy markers (resistotype and RFLP-PCR-flaA) may detect isolates probably belonging to an undiagnosed outbreak of campylobacteriosis. Accurate diagnosis requires other molecular markers and epidemiological data of each isolate. The study suggests that, as in other countries, the number of outbreaks of campylobacteriosis in Spain is probably underestimated.

Salmonella spp. and Shiga toxin-producing Escherichia coli prevalence in an ocellated lizard (Timon lepidus) research center in Spain.

The aim of this work was to study the epidemiological status of Salmonella spp. and Shiga toxin-producing Escherichia coli (STEC) in an ocellated lizard research center focusing on the risk and hygiene aspects. Fecal and environmental samples were collected and examined for Salmonella spp. and STEC. Isolates were detected using real-time polymerase chain reaction (RT-PCR) and characterized using serotyping and pulsed-field gel electrophoresis (PFGE). Overall, 52% of samples were positive for Salmonella spp. using RT-PCR and seven isolates were obtained from samples from ocellated lizards and their environment, whereas no samples were positive for STEC. Salmonella isolates belonged to S. enterica subsp. enterica serovar Kibusi and S. enterica subsp. salamae serovars 41:z10:z6 and 18:z10:z6, some of which have previously been isolated from human sources. Indistinguishable and closely related PFGE types were found, which supported the existence of horizontal transmission between animals due to crowding of animals and the persistence of Salmonella in the environment. The results of the current study emphasize the need for improved prevention efforts and good hygiene practices in research centers, recuperation centers, and zoos with reptiles to minimize the exposure of personnel and visitors to this pathogen.

Caracterización molecular de la cepa responsable de un brote de shigelosis en un centro escolar de Madrid / Molecular characterization of the strain causing a shigellosis outbreak in a Madrid school

Introducción. El objetivo de este estudio es describir un brote de gastroenteritis por Shigella sonnei en un colegio. Métodos. Se realizó una encuesta epidemiológica. En 5 pacientes se obtuvieron coprocultivos. La tipificación molecular de los aislamientos se realizó mediante electroforesis en gel de campos pulsantes (PFGE), secuenciación de genes metabólicos (MLST) y análisis plasmídico. Resultados. La tasa de ataque fue del 14,3% (67 alumnos). Se aisló S. sonnei en los 5 enfermos. Se tipificaron 4 cepas que resultaron indistinguibles. Conclusión. Estos resultados sugieren una identidad común de la cepa causal (AU)

Introduction. The aim of this study is to describe a Shigella sonnei outbreak in a school. Method. An epidemiological inquiry was performed. Stool samples from 5 patients were cultured. Molecular typing of the isolates was carried out by PFGE, MLST and plasmid analysis. Results. The attack rate was 14.3% (67 students). Shigella sonnei was isolated from all 5 patients. The four strains available for typing were indistinguishable. Conclusion. These results suggest a common identity of the outbreak strain (AU)

[Molecular characterization of the strain causing a shigellosis outbreak in a Madrid school]. / Caracterización molecular de la cepa responsable de un brote de shigelosis en un centro escolar de Madrid.

INTRODUCTION: The aim of this study is to describe a Shigella sonnei outbreak in a school. METHOD: An epidemiological inquiry was performed. Stool samples from 5 patients were cultured. Molecular typing of the isolates was carried out by PFGE, MLST and plasmid analysis. RESULTS: The attack rate was 14.3% (67 students). Shigella sonnei was isolated from all 5 patients. The four strains available for typing were indistinguishable. CONCLUSION: These results suggest a common identity of the outbreak strain.

Blind comparison of traditional serotyping with three multiplex PCRs for the identification of Salmonella serotypes.

Salmonella serotypes are defined on the basis of somatic (O) antigens which define the serogroup and flagellar (H) factor antigens, both of which are present in the cell wall of Salmonella. Most Salmonella organisms alternatively express phase-1 or phase-2 flagellar antigens encoded by fliC and fljB genes, respectively. Our group previously published two multiplex PCRs for distinguishing the most common first- and second-phase antigens. In this paper we describe a third multiplex PCR to identify the most common serogroups (O:B; O:C1; O:C2; O:D and O:E). The combination of these three PCRs enabled us to completely serotype organisms belonging to the Salmonella species. This multiplex PCR includes 10 primers. A total of 67 Salmonella strains belonging to 32 different serotypes were tested. Each strain generated one serogroup-specific fragment ranging between 162 and 615bp. Twenty-eight strains belonging to 21 serotypes, with a serogroup different from those tested in this work, did not generate any fragments. To compare molecular serotyping with traditional serotyping, 500 strains, received according to the order of arrival in the laboratory, were serotyped using both methods. The three multiplex PCRs were able to serotype 84.6% of the tested strains. This method was found to be very helpful in our laboratory as an alternative method for typing strains causing outbreaks, and it can be used to supplement conventional serotyping, since it is also applicable to motionless and rough strains.

[Description of an outbreak of Campylobacter jejuni gastroenteritis and molecular characterization of the implicated strain]. / Descripción de un brote de gastroenteritis por y caracterización molecular de la cepa implicada.

INTRODUCTION: The aim of this study is to describe an outbreak of diarrhea caused by Campylobacter jejuni in a primary school. METHODS: Stool samples from five patients were cultured. Molecular typing of the isolated strains was performed using PCR-RFLP-flaA, PFGE and MLST. RESULTS: Campylobacter jejuni was isolated from all five patients. Two of the five strains were available for typing. The DNA patterns of the two isolates were indistinguishable. CONCLUSION: The results suggest that the causal strain had a common identity.

Descripción de un brote de gastroenteritis por y caracterización molecular de la cepa implicada / Description of an outbreak of Campylobacter jejuni gastroenteritis and molecular characterization of the implicated strain

Introducción. El objetivo de este trabajo es describir el estudio de un brote de diarrea por Campylobacter jejuni en una escuela infantil. Métodos. Se obtuvieron coprocultivos en 5 pacientes. La tipificación molecular se realizó mediante (RFLP-PCR flaA), electroforesis en campo pulsante (PFGE) y secuenciación de genes metabólicos (MLST). Resultados. Se aisló Campylobacter jejuni en los 5 enfermos. Sólo pudieron recuperarse para la tipificación 2 cepas. La tipificación mediante los 3 métodos resultó indistinguible en ambos aislamientos. Conclusión. Estos resultados sugieren una identidad común de la cepa causal (AU)

Introduction. The aim of this study is to describe an outbreak of diarrhea caused by Campylobacter jejuni in a primary school. Methods. Stool samples from five patients were cultured. Molecular typing of the isolated strains was performed using PCR-RFLP-flaA, PFGE and MLST. Results. Campylobacter jejuni was isolated from all five patients. Two of the five strains were available for typing. The DNA patterns of the two isolates were indistinguishable. Conclusion. The results suggest that the causal strain had a common identity (AU)

[Serotype and phage type distribution of human Salmonella strains isolated in Spain, 1997-2001]. / Distribución de los serotipos y fagotipos de Salmonella de origen humano aislados en España en 1997-2001.

INTRODUCTION: Salmonellosis is one of the most frequent causes of gastroenteritis in Spain. Serotyping is the gold standard epidemiological marker for subdividing Salmonella spp. strains. A small number of serotypes are very frequently isolated, reducing the discriminatory power of serotyping. Thus, to increase our knowledge of Salmonella spp. epidemiology, additional epidemiological markers, such as phage typing, should be used for this purpose. METHODS: Salmonella spp. strains of human origin sent to the Laboratorio Nacional de Referencia de Salmonella y Shigella (LNRSSE, Spanish Reference Laboratory for Salmonella and Shigella) between 1997 and 2001 were serotyped using conventional agglutination methods, and Enteritidis, Typhimurium, Hadar, Virchow and Typhi serotypes were additionally phage typed according to internationally-developed schemes. RESULTS: A total of 30,856 Salmonella spp. strains, isolated in the majority of Spanish Autonomous Communities, were analyzed. Enteritidis (51%) and Typhimurium (24%) were the most frequently isolated serotypes. The following were the most frequent serotype/phage type combinations: Enteritidis/PT1 (18%), Enteritidis/PT4 (15%), Enteritidis/PT6a (5%), Typhimurium/DT104 (5%) and Enteritidis/PT6 (3%). The serotype Enteritidis/PT1 showed the greatest increase over the period studied, from 11.61% in 1997 to 24.74% in 2001. CONCLUSIONS: A hierarchical typing approach for Salmonella spp., using serotyping coupled with phage typing allowed a higher level of discrimination among Salmonella serotypes. Application of this approach in epidemiological studies could be highly useful for early characterization of related strains.

Distribución de los serotipos y fagotipos de Salmonella de origen humano aislados en España en 1997-2001 / Serotype and phage type distribution of human Salmonella strains isolated in Spain, 1997-2001

INTRODUCCIÓN. La salmonelosis continúa siendo una de las causas principales de gastroenteritis en España, siendo la serotipificación el marcador epidemiológico universalmente utilizado para la caracterización de los aislamientos de Salmonella spp. Algunos serotipos se identifican muy frecuentemente, reduciendo el poder de discriminación de esta técnica. Por ello, para el estudio epidemiológico de las salmonelosis producidas por estos serotipos es necesario utilizar marcadores complementarios como la fagotipificación. MÉTODOS. Se serotipificaron, por aglutinación directa, las cepas de Salmonella spp. de origen humano recibidas en el Laboratorio Nacional de Referencia de Salmonella y Shigella (LNRSSE) entre los años 1997 y 2001 y se fagotipificaron, según 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 mayoría de las Comunidades Autónomas. Los serotipos Enteritidis (51%) y Typhimurium (24%) fueron los mayoritarios. Las combinaciones serotipo/fagotipo más 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 período de tiempo, pasando del 11,61% en 1997al 24,74% en 2001. CONCLUSIONES. La utilización jerárquica de la serotipificación y posteriormente de la fagotipificación en cepas de Salmonella spp. de los serotipos más frecuentes aumentó enormemente el poder de discriminación de la serotipificación. Su aplicación en estudios epidemiológicoses de gran utilidad en la caracterización temprana de cepas relacionadas (AU)

INTRODUCTION. Salmonellosis is one of the most frequent causes of gastroenteritis in Spain. Serotyping is the gold standard epidemiological marker for subdividing Salmonella spp. strains. A small number of serotypes are very frequently isolated, reducing the discriminatory power of serotyping. Thus, to increase our knowledge of Salmonella spp. epidemiology, additional epidemiological markers, such as phage typing, should be used for this purpose. METHODS. Salmonella spp. strains of human origin sent to the Laboratorio Nacional de Referencia de Salmonellay Shigella (LNRSSE, Spanish Reference Laboratory for Salmonella and Shigella) between 1997 and 2001 were serotyped using conventional agglutination methods, and Enteritidis, Typhimurium, Hadar, Virchow and Typhiserotypes were additionally phage typed according to internationally-developed schemes. RESULTS. A total of 30,856 Salmonella spp. strains, isolatedin the majority of Spanish Autonomous Communities,were analyzed. Enteritidis (51%) and Typhimurium (24%)were the most frequently isolated serotypes. The following were the most frequent serotype/phage type combinations: Enteritidis/PT1 (18%), Enteritidis/PT4 (15%),Enteritidis/PT6a (5%), Typhimurium/DT104 (5%) and Enteritidis/PT6 (3%). The serotype Enteritidis/PT1 showed the greatest increase over the period studied, from 11.61%in 1997 to 24.74% in 2001. CONCLUSIONS. A hierarchical typing approach for Salmonellaspp., using serotyping coupled with phage typing allowed a higher level of discrimination among Salmonella serotypes. Application of this approach in epidemiological studies could be highly useful for early characterization of related strains (AU)