Genomics of the Argentinian cholera epidemic elucidate the contrasting dynamics of epidemic and endemic Vibrio cholerae.

In order to control and eradicate epidemic cholera, we need to understand how epidemics begin, how they spread, and how they decline and eventually end. This requires extensive sampling of epidemic disease over time, alongside the background of endemic disease that may exist concurrently with the epidemic. The unique circumstances surrounding the Argentinian cholera epidemic of 1992-1998 presented an opportunity to do this. Here, we use 490 Argentinian V. cholerae genome sequences to characterise the variation within, and between, epidemic and endemic V. cholerae. We show that, during the 1992-1998 cholera epidemic, the invariant epidemic clone co-existed alongside highly diverse members of the Vibrio cholerae species in Argentina, and we contrast the clonality of epidemic V. cholerae with the background diversity of local endemic bacteria. Our findings refine and add nuance to our genomic definitions of epidemic and endemic cholera, and are of direct relevance to controlling current and future cholera epidemics.

Development and validation of a PulseNet standardized protocol for subtyping isolates of Cronobacter species.

Cronobacter (formerly known as Enterobacter sakazakii) is a genus comprising seven species regarded as opportunistic pathogens that can be found in a wide variety of environments and foods, including powdered infant formula (PIF). Cronobacter sakazakii, the major species of this genus, has been epidemiologically linked to cases of bacteremia, meningitis in neonates, and necrotizing enterocolitis, and contaminated PIF has been identified as an important source of infection. Robust and reproducible subtyping methods are required to aid in the detection and investigation, of foodborne outbreaks. In this study, a pulsed-field gel electrophoresis (PFGE) protocol was developed and validated for subtyping Cronobacter species. It was derived from an existing modified PulseNet protocol, wherein XbaI and SpeI were the primary and secondary restriction enzymes used, generating an average of 14.7 and 20.3 bands, respectively. The PFGE method developed was both reproducible and discriminatory for subtyping Cronobacter species.

Standardization and international multicenter validation of a PulseNet pulsed-field gel electrophoresis protocol for subtyping Shigella flexneri isolates.

Shigella flexneri is one of the agents most frequently linked to diarrheal illness in developing countries and often causes outbreaks in settings with poor hygiene or sanitary conditions. Travel is one of the means by which S. flexneri can be imported into developed countries, where this pathogen is not commonly seen. A robust and discriminatory subtyping method is needed for the surveillance of S. flexneri locally and regionally, and to aid in the detection and investigation of outbreaks. The PulseNet International network utilizes standardized pulsed-field gel electrophoresis (PFGE) protocols to carry out laboratory-based surveillance of foodborne pathogens in combination with epidemiologic data. A multicenter validation was carried out in nine PulseNet laboratories located in North and South America, Europe, and Asia, and it demonstrated that a new protocol is highly robust and reproducible for subtyping of S. flexneri. This protocol, already approved for PulseNet laboratories, applies NotI and XbaI as primary and secondary restriction enzymes, respectively, under electrophoresis conditions of initial switch time of 5 s to final switch time of 35 s, at 6 volts/cm.

Global Distribution of Shigella sonnei Clones.

To investigate global epidemiology of Shigella sonnei, we performed multilocus variable number tandem repeat analysis of 1,672 isolates obtained since 1943 from 50 countries on 5 continents and the Pacific region. Three major clonal groups were identified; 2 were globally spread. Type 18 and its derivatives have circulated worldwide in recent decades.

Salmonella enterica subclinical infection: bacteriological, serological, pulsed-field gel electrophoresis, and antimicrobial resistance profiles--longitudinal study in a three-site farrow-to-finish farm.

The aim of this surveillance was to study both Salmonella spp. shedding patterns and the time course of serological response in farrow-to-finish reared pigs from a subclinically infected farm. Antimicrobial resistance profile, molecular subtyping, and the relationship among the isolates were determined by pulsed-field gel electrophoresis (PFGE). A farrow-to-finish farm of 6000 sows, with a history of Salmonella Typhimurium septicemia, was selected. A longitudinal bacteriological and serological study was conducted in 25 sows before farrowing (M/S1) and in 50 offspring at 21 (M/S2), 35 (M/S3), 65 (M/S4), 86 (M/S5), 128 (M/S6), and 165 (M/S7) days of age. Serum antibodies were tested using Herdcheck((R)) Swine Salmonella antibody test kit (Idexx Laboratories, ME). Bacteria were isolated from pooled fecal samples. Suspected isolates were confirmed by conventional biochemical assays, and those identified as Salmonella spp. were serotyped. A variation between seropositive percentages and positive fecal samples was observed. Serologically positive pigs decreased from S1 to S4, and subsequently increased from S4 to S7. The percentages of fecal positive culture increased from M1 to M3, and then declined in M4, increased in M5, and were negative in M6 and M7. In the study three serovars, Salmonella 3,10:e,h:-, Salmonella Muenster, and Salmonella Bovismorbificans, were identified with low pathogenicity for swine. Three multidrug resistance strains (one belonged to Salmonella 3,10:e,h:- and two belonged to Salmonella Muenster) were found. PFGE results showed three different but closely related patterns among the 13 isolates of Salmonella Bovismorbificans, and two patterns for the three Salmonella Muenster and Salmonella 3,10:e,h:- isolates. This longitudinal study established critical points of Salmonella spp. infection in the farm and the production stages, where appropriate control measures must be taken. PFGE showed clonal relationships in each serovar. Antibiotic resistance profiles should be periodically included due to public health concerns.

Isolation of Salmonella typhimurium from dead blue and gold macaws (Ara ararauna).

Two blue and gold macaw (Ara ararauna) chicks died of fatal salmonellosis in Buenos Aires Province, Argentina. The birds were histopathologically and microbiologically examined. Salmonella enterica subspecies enterica serovar Typhimurium was isolated from the liver, spleen, heart, lung, kidney, and intestine of both birds. All strains were susceptible to ampicillin, cephalothin, cefotaxime, enrofloxacin, nalidixic acid, gentamicin, streptomycin, chloramphenicol, fosfomycin, tetracycline, nitrofurantoin, and trimethoprim-sulfamethoxazole. The XbaI-PFGE profile of the Salmonella Typhimurium isolated from the two animals, which shared the same cage, was identical and showed a unique pattern compared with 301 isolates included in the PulseNet national database of Salmonella pulsed-field gel electrophoresis patterns. This is the first report that describes fatal cases of salmonellosis from blue and gold macaws.

Lateral gene transfer of O1 serogroup encoding genes of Vibrio cholerae.

In Gram-negative bacteria, the O-antigen-encoding genes may be transferred between lineages, although mechanisms are not fully understood. To assess possible lateral gene transfer (LGT), 21 Argentinean Vibrio cholerae O-group 1 (O1) isolates were examined using multilocus sequence typing (MLST) to determine the genetic relatedness of housekeeping genes and genes from the O1 gene cluster. MSLT analysis revealed that 4.4% of the nucleotides in the seven housekeeping loci were variable, with six distinct genetic lineages identified among O1 isolates. In contrast, MLST analysis of the eight loci from the O1 serogroup region revealed that 0.24% of the 4943 nucleotides were variable. A putative breakpoint was identified in the JUMPstart sequence. Nine conserved nucleotides differed by a single nucleotide from a DNA uptake signal sequence (USS) also found in Pastuerellaceae. Our data indicate that genes in the O1 biogenesis region are closely related even in distinct genetic lineages, indicative of LGT, with a putative DNA USS identified at the defined boundary for the DNA exchange.

Pulsed-field gel electrophoresis, pertactin, pertussis toxin S1 subunit polymorphisms, and surfaceome analysis of vaccine and clinical Bordetella pertussis strains.

To add new insight to our previous work on the molecular epidemiology of Bordetella pertussis in Argentina, the prn and ptxS1 gene sequences and pulsed-field gel electrophoresis (PFGE) profiles of 57 clinical isolates obtained during two periods, 1969 to 1989 and 1997 to 2006, were analyzed. Non-vaccine-type ptxS1A was detected in isolates obtained since 1969. From 1989 on, a shift of predominance from the vaccine prn1 type to the nonvaccine prn2 type was observed. This was also reflected in a transition of PFGE group IV to group VI. These results show that nonvaccine B. pertussis strains are currently circulating. To analyze whether the observed genomic divergences between vaccine strains and clinical isolates have functional implications, protection assays using the intranasal mouse challenge model were performed. For such experiments, the clinical isolate B. pertussis 106 was selected as representative of circulating bacteria, since it came from the major group of the PFGE dendrogram (PFGE group VI). Groups of mice were immunized either with diphtheria-tetanus-whole-cell pertussis vaccine (ptxS1B prn1) or a vaccine prepared by us containing B. pertussis 106. Immunized mice were then challenged with a B. pertussis vaccine strain (Tohama, harboring ptxS1B and prn1) or the clinical isolate B. pertussis 106 (ptxS1A prn2). An adequate bacterial-elimination rate was observed only when mice were immunized and challenged with the same kind of strain. For further characterization, comparative proteomic profiling of enriched membrane proteins was done using three vaccine strains and the selected B. pertussis 106 clinical isolate. By matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis, a total of 54 proteins were identified. This methodology allowed us to detect differing proteins among the four strains studied and, in particular, to distinguish the three vaccine strains from each other, as well as the vaccine strains from the clinical isolate. The differing proteins observed have cellular roles associated with amino acid and carbohydrate transport and metabolism. Some of them have been proposed as novel vaccine candidate proteins for other pathogens. Overall, the global strategy described here is presented as a good tool for the development of next-generation acellular vaccines.

Web-based surveillance and global Salmonella distribution, 2000-2002.

Salmonellae are a common cause of foodborne disease worldwide. The World Health Organization (WHO) supports international foodborne disease surveillance through WHO Global Salm-Surv and other activities. WHO Global Salm-Surv members annually report the 15 most frequently isolated Salmonella serotypes to a Web-based country databank. We describe the global distribution of reported Salmonella serotypes from human and nonhuman sources from 2000 to 2002. Among human isolates, S. Enteritidis was the most common serotype, accounting for 65% of all isolates. Among nonhuman isolates, although no serotype predominated, Salmonella enterica serovar Typhimurium was reported most frequently. Several serotypes were reported from only 1 region of the world. The WHO Global Salm-Surv country databank is a valuable public health resource; it is a publicly accessible, Web-based tool that can be used by health professionals to explore hypotheses related to the sources and distribution of salmonellae worldwide.

Building PulseNet International: an interconnected system of laboratory networks to facilitate timely public health recognition and response to foodborne disease outbreaks and emerging foodborne diseases.

PulseNet USA, the national molecular subtyping network for foodborne disease surveillance, began functioning in the United States in 1996 and soon established itself as a critical early warning system for foodborne disease outbreaks, particularly those in which cases may be geographically dispersed. The PulseNet network is now being replicated in different ways in Canada, Europe, the Asia Pacific region, and Latin America. These independent networks work together in PulseNet International allowing public health officials and laboratorians to share molecular epidemiologic information in real-time and enabling rapid recognition and investigation of multi-national foodborne disease outbreaks. Routine communication between the various international PulseNet networks will provide early warning on foodborne disease outbreaks to participating public health institutions and countries.

Molecular subtyping of Salmonella enterica serovar Typhi isolates from Colombia and Argentina.

Salmonella Typhi is the etiological agent of typhoid fever with 16 million annual cases estimated worldwide. In Colombia and Argentina it is a notifiable disease but many cases have only a clinical diagnosis. Molecular subtyping of S. Typhi is necessary to complement epidemiologic analysis of typhoid fever. The aims of this study were to determine the genetic relationships between the strains circulating in both countries and to evaluate possible variations in the distribution of 12 virulence genes. A total of 136 isolates were analyzed by pulsed-field gel electrophoresis (PFGE) with XbaI following PulseNet protocols and analysis guidelines. Eighty-three different PFGE patterns were identified, showing high diversity among the strains from both countries. Three outbreaks, two in Colombia and one in Argentina, were caused by strains of different PFGE types. In Colombia, two PFGE patterns were found predominantly, which included 36.6% of the isolates from that country. No association was found between the PFGE patterns and the year or place of isolation of the strains, the age of the patients or type of sample. However, several clusters were detected, which included isolates recovered predominantly either from Colombia or Argentina. Most of the strains (97%) exhibited a single virulence profile, suggesting that the pathogenicity markers analyzed are of limited value for strain discrimination and do not correlate with the origin of the isolates (intestinal vs. extra-intestinal). Since the creation of PulseNet Latin America, this was the first international study conducted in South America. The PFGE types identified were incorporated into the Regional S. Typhi PulseNet Database and are now available for comparison with those of strains isolated in other regions. This information will be used for active surveillance, future studies, and outbreak investigations.

Genetic characterization of Vibrio cholerae isolates from Argentina by V. cholerae repeated sequences-polymerase chain reaction.

We have developed a novel typing method based on Vibrio cholerae repeat sequences (VCR) using primers directed out of the VCR sequences. To evaluate the VCR-polymerase chain reaction (PCR) as a typing system, 2 categories, efficacy and efficiency, were analyzed in 69 strains of human and environmental V. cholerae O1 toxigenic and nontoxigenic, and non-O1 strains isolated since 1992-2000 from Argentina. The discriminatory power (0.91), stability (0.95), reproducibility (1), typeability (1), rapidity, accessibility, as well ease of use, indicated that the VCR-PCR method provides an alternative useful tool for molecular epidemiology of V. cholerae. The VCR-PCR of V. cholerae isolates showed 29 patterns, of which pattern 1 represented 68% of the V. cholerae O1 isolates, supporting the hypothesis that a clone with epidemic behavior was responsible for the epidemic in Latin America. These results showed a good correlation and a better epidemiologic analysis when the results were compared in parallel with repetitive extragenic palindromic sequences-PCR. In conclusion, VCR-PCR showed excellent performance as a typing method for cholera surveillance programs.

Viable but nonculturable Vibrio cholerae O1 in the aquatic environment of Argentina.

In Argentina, as in other countries of Latin America, cholera has occurred in an epidemic pattern. Vibrio cholerae O1 is native to the aquatic environment, and it occurs in both culturable and viable but nonculturable (VNC) forms, the latter during interepidemic periods. This is the first report of the presence of VNC V. cholerae O1 in the estuarine and marine waters of the Rio de la Plata and the Argentine shelf of the Atlantic Ocean, respectively. Employing immunofluorescence and PCR methods, we were able to detect reservoirs of V. cholerae O1 carrying the virulence-associated genes ctxA and tcpA. The VNC forms of V. cholerae O1 were identified in samples of water, phytoplankton, and zooplankton; the latter organisms were mainly the copepods Acartia tonsa, Diaptomus sp., Paracalanus crassirostris, and Paracalanus parvus. We found that under favorable conditions, the VNC form of V. cholerae can revert to the pathogenic, transmissible state. We concluded that V. cholerae O1 is a resident of Argentinean waters, as has been shown to be the case in other geographic regions of the world.

Genetic diversity of Vibrio cholerae O1 in Argentina and emergence of a new variant.

The genetic diversity of Vibrio cholerae O1 strains from Argentina was estimated by random amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE). Twenty-nine isolates carrying the virulence genes ctxA, zot, ace, and tcpA appeared to represent a single clone by both typing methods; while 11 strains lacking these virulence genes exhibited several heterogeneous RAPD and PFGE patterns. Among the last group, a set of isolates from the province Tucumán showed a single RAPD pattern and four closely related PFGE profiles. These strains, isolated from patients with diarrhea, did not produce the major V. cholerae O1 virulence determinants, yet cell supernatants of these isolates caused a heat-labile cytotoxic effect on Vero and Y-1 cells and elicited significant variations on the water flux and short-circuit current in human small intestine mounted in an Ussing chamber. All these effects were completely abolished by incubation with a specific antiserum against El Tor hemolysin, suggesting that this virulence factor was responsible for the toxic activity on both the epithelial cells and the small intestine specimens and may hence be involved in the development of diarrhea. We propose "Tucumán variant" as the designation for this new cluster of cholera toxin-negative V. cholerae O1 strains.

Type IV longus pilus of enterotoxigenic Escherichia coli: occurrence and association with toxin types and colonization factors among strains isolated in Argentina.

The longus type IV pilus structural gene (lngA) was sought among 217 clinical enterotoxigenic Escherichia coli (ETEC) strains isolated in Argentina. lngA was present in 20.7% of the isolates and was highly associated with ETEC producing heat-stable toxin and the most common colonization factors. The prevalence of longus among ETEC strains in Argentina was comparable to that of colonization factor antigen I (CFA/I), CFA/II, and CFA/IV in other regions of the world.

Hemolytic uremic syndrome: co-infection with two different serotypes of shiga-like toxin producing Escherichia coli

Se presenta el caso de un niño de 14 meses que desarrolló Sindrome Urémico Hemolítico después de un período prodrómico con vómitos y diarrea mucosanguinolenta del cual se aislaron 2 cepas de E. coli productoras de toxina simil-Shiga (SLT) de diferente serotipo y genotipo. Una de las cepas correspondió al seotipo 0157: H7, biotipo D, productora de SLT II y susceptible a todos los antibióticos probados. Esta cepa hibridizó cona las sondas genéticas para SLT II; la fimbria de adherencia (factor EHEC) y para el factor de fijación y disolución del borde en cepillo ("E. coli attaching and effacing") eae). La otra cepa correspondió al serotipo O25:K2:H2 productora de SLT II en los ensayos de neutralización de efecto citotóxico sobre células VERO utilizando anticuerpos monoclonales, pero negativas para SLT I y SLT II y el factor eae. Sólo fue positiva por hibridización con la sonda para la fimbria de adherencia. Esta cepa presentó además un patron de multiresistencia a los antibióticos probados. Por otra parte, la cepa de E. coli, del serotipo O25: K2: H2 produjo niveles tanto de toxina libre como asociada a células, 20 veces superiores a la cepa del serotipo O157:H7. Esta es la primera publicación de un caso de SUH en el cual se detectó una cepa de E. coli del serotipo O25:H2 produjo niveles tanto de toxina libre como asociada a células, 20 veces superiores a la cepa del serotipo O157:H7. Esta es la primera publicación de un caso de SUH en el cual se detectó una cepa de E. coli del serotipo O25:H2 productora de SLT. Además el hecho de no hibridizar con la sonda genética para SLT II, a pesar de tener una actividad citotóxica neutralizable por el anticuerpo monoclonal (MAb BC5BB12) indicaria que la secuencia que codifica para SLT II no presenta homología con la sonda utilizada. la detección de SLT libre tanto en la materia fecal del niño como en la de su padre (quien padeció diarrea una semana antes), indicaría que el niño adquirió la infección en el entorno familiar (AU)

Hemolytic uremic syndrome: co-infection with two different serotypes of shiga-like toxin producing Escherichia coli

Se presenta el caso de un niño de 14 meses que desarrolló Sindrome Urémico Hemolítico después de un período prodrómico con vómitos y diarrea mucosanguinolenta del cual se aislaron 2 cepas de E. coli productoras de toxina simil-Shiga (SLT) de diferente serotipo y genotipo. Una de las cepas correspondió al seotipo 0157: H7, biotipo D, productora de SLT II y susceptible a todos los antibióticos probados. Esta cepa hibridizó cona las sondas genéticas para SLT II; la fimbria de adherencia (factor EHEC) y para el factor de fijación y disolución del borde en cepillo ("E. coli attaching and effacing") eae). La otra cepa correspondió al serotipo O25:K2:H2 productora de SLT II en los ensayos de neutralización de efecto citotóxico sobre células VERO utilizando anticuerpos monoclonales, pero negativas para SLT I y SLT II y el factor eae. Sólo fue positiva por hibridización con la sonda para la fimbria de adherencia. Esta cepa presentó además un patron de multiresistencia a los antibióticos probados. Por otra parte, la cepa de E. coli, del serotipo O25: K2: H2 produjo niveles tanto de toxina libre como asociada a células, 20 veces superiores a la cepa del serotipo O157:H7. Esta es la primera publicación de un caso de SUH en el cual se detectó una cepa de E. coli del serotipo O25:H2 produjo niveles tanto de toxina libre como asociada a células, 20 veces superiores a la cepa del serotipo O157:H7. Esta es la primera publicación de un caso de SUH en el cual se detectó una cepa de E. coli del serotipo O25:H2 productora de SLT. Además el hecho de no hibridizar con la sonda genética para SLT II, a pesar de tener una actividad citotóxica neutralizable por el anticuerpo monoclonal (MAb BC5BB12) indicaria que la secuencia que codifica para SLT II no presenta homología con la sonda utilizada. la detección de SLT libre tanto en la materia fecal del niño como en la de su padre (quien padeció diarrea una semana antes), indicaría que el niño adquirió la infección en el entorno familiar

Relationship between enterotoxigenic Escherichia coli and diarrhea among children in Buenos Aires

La diarrea aguda infecciosa es el problema de salud más importante en los países en desarrollo. Diversos estudios en Latinoamérica han demonstrado que Escherichia coli enterotoxigénica (ETEC) y rotavirus son los patógenos más frecuentemente asociados con la diarrea infantil. Para causar la diarrea, ETC coloniza, se multiplica en el intestino delgado y produce la enterotoxina lábil (LT) y/o la estable (ST) al calor. La colonización está mediada por estructuras fimbriales de superficie llamadas factores de colonización (CFAs). Tres CFAs han sido descriptos y bien caracterizados: CFA/I, CFA/II y CFA/IV. Las cepas de ETEC aisladas de pacientes con diarrea pertenecen a un número reducido de serotipos y se ha sugerido que existe una relación entre el patrón de fermentación de azúcares y el serotipo. En este trabajo se estudió la incidencia de ETEC en 85 niñoscon diarrea aguda internados en el Hospital de Niños "Pedro de Elizalde" de Buenos Aires y en 38 niños sanos. La edad en ambos grupos, estuvo comprendida entre 0 y 4 años y ninguno de los niños recibió antibioticoterapia en los 7 días previos a la toma de muestra. Se aislaron cepas de ETEC en 9 de los 85(10,6%) niños con diarrea. De estos casos positivos, 6 estuvieron asociados con ETEC productores de ST, 1 con LT y 2 con ambas (LT/ST). Dentro del grupo control sólo en 1 caso (2,6%) se detectó ETEC-LT. En 5 de los 9 casos de diarrea asociados a ETEC (55,5%), se asislaron cepas que expresaron algunos de los CFAs investigados: 4 fueron CFA/I y 1 CFA/II. En 23 cepas de E. coli, aisladas de los 10 niños ETEC positivos, se estudiaron los factores de colonización, el biotipo, serotipo y antibiotipo en relación al perfil toxigénico que presentaban. De 12 cepas productoras sólo de ST, 5 (41,7%) expresaron CFA y 2 (16,7%) CFA/II (CS2 + CS3)...(AU)

Relationship between enterotoxigenic Escherichia coli and diarrhea among children in Buenos Aires

La diarrea aguda infecciosa es el problema de salud más importante en los países en desarrollo. Diversos estudios en Latinoamérica han demonstrado que Escherichia coli enterotoxigénica (ETEC) y rotavirus son los patógenos más frecuentemente asociados con la diarrea infantil. Para causar la diarrea, ETC coloniza, se multiplica en el intestino delgado y produce la enterotoxina lábil (LT) y/o la estable (ST) al calor. La colonización está mediada por estructuras fimbriales de superficie llamadas factores de colonización (CFAs). Tres CFAs han sido descriptos y bien caracterizados: CFA/I, CFA/II y CFA/IV. Las cepas de ETEC aisladas de pacientes con diarrea pertenecen a un número reducido de serotipos y se ha sugerido que existe una relación entre el patrón de fermentación de azúcares y el serotipo. En este trabajo se estudió la incidencia de ETEC en 85 niñoscon diarrea aguda internados en el Hospital de Niños "Pedro de Elizalde" de Buenos Aires y en 38 niños sanos. La edad en ambos grupos, estuvo comprendida entre 0 y 4 años y ninguno de los niños recibió antibioticoterapia en los 7 días previos a la toma de muestra. Se aislaron cepas de ETEC en 9 de los 85(10,6%) niños con diarrea. De estos casos positivos, 6 estuvieron asociados con ETEC productores de ST, 1 con LT y 2 con ambas (LT/ST). Dentro del grupo control sólo en 1 caso (2,6%) se detectó ETEC-LT. En 5 de los 9 casos de diarrea asociados a ETEC (55,5%), se asislaron cepas que expresaron algunos de los CFAs investigados: 4 fueron CFA/I y 1 CFA/II. En 23 cepas de E. coli, aisladas de los 10 niños ETEC positivos, se estudiaron los factores de colonización, el biotipo, serotipo y antibiotipo en relación al perfil toxigénico que presentaban. De 12 cepas productoras sólo de ST, 5 (41,7%) expresaron CFA y 2 (16,7%) CFA/II (CS2 + CS3)...

An evaluation of the pooling culture method for the detection of Escherichia coli enterotoxins

Se estudió la eficiencia de la metodología de inoculación en "pool" de cepas que integran casos de diarrea aguda infantil, para la detección de escherichia coli enteroxigénico (ECET). Se procesaron 6989 cepas de E. coli correspondientes a 1485 casos de diarrea provenientes de 7 centros asistenciales del país. Las cepas de cada caso (3 a 5) se sembraron en "pool". Los cultivos se realizaron en medio de Evans con lincomicina (30 microng/ml) a 37-C durante 18 h con agitación y luego se trataron con sulfato de polimixina B (2200U/ml) por 30 min. Los cultivos se centrifugaron y los sobrenadantes se emplearon para la detección de la enterotoxina lábial al calor (LT) por GMI-ELISA con antisuero a toxina de cólera y la estable al calor (ST) por la prueba del ratón lactante. En aquellos casos que fueron positivos par LT, ST o LT-ST, por la metodología de "pool", se analizaron sus cepas individualmente, como así también en 1 de cada 15 caos negativod para detectar falsos negativos. Se hallaron 57 casos ECET-LT, 61 casos ECET-ST y 15 casos ECET-LT-ST. De los 89 casos negativos seleccionados al azar detectaron 2 casos (pl = 2.25%, intervalo de confianza del 95% entre 0,27% - 7,88) en los cuales una de las cepas no enterotoxigénicas inhibía a la ECET. En otros 5 casos (p2 = 5,62%, intervalo de confianza del 95% entre 1,85% - 12,63%), el falso resultado negativo se debió a la falla en la inoculación del "pool", pues el mismso resultó positivo al repetirse. A partir de los resultados obtenidos que el método de iniculación en "pool" es adecuado cuando se debe analizar un número elevado de casos de diarrea aguda y disponer de un diagnóstico en el menor tiempo posible, con la recomendación de realizar una siembra cuidadosa de las cepas que integran el caso tratando que el inóculo de cada una de ellas sea similar (AU)