Isolation and characterization of <i>Salmonella enterica</i> from Antarctic wildlife

In recent years, the human presence in Antarctica has increased and as a consequence, the possibility of microorganisms’ introduction. The aims of this work were to determine the presence of Salmonella enterica in Antarctic seabirds and sea mammals, to characterize the isolates identified, and to determine the genetic relation of Antarctic S. enterica isolates among them and compare with isolates of human, animal, and food sources recovered in Argentina. During the summer 2000 and 2002 in Potter Peninsula, and during the summer 2001 and 2003 in Hope Bay, a total of 1,739 fecal samples from Antarctic animals were collected and analyzed. In summer 2000, S. Newport and S. Enteritidis were isolated from 8.9% of southern giant petrels (Macronectes giganteus). In summer 2003, S. Enteritidis was isolated from 1.5% of Adelie penguins (Pygoscelis adeliae), from 5.5% of skuas (Stercorarius sp.), from 5.4% of kelp gulls (Larus dominicanus), and from 5.6% of Weddell seals (Leptonychotes weddelli). All the isolates belonging to the same serovar showed indistinguishable genomic profiles by Pulse-Field Gel Electrophoresis (PFGE) with XbaI and BlnI restriction enzymes and by Random Amplified Polymorphic DNA (RAPD-PCR). In addition, these Antarctic strains were different from S. enterica isolates from different sources identified in Argentina during the same or close time periods.Facultad de Ciencias Veterinaria

Web-based Surveillance and Global Salmonella Distribution, 2000–2002

Surveillance improves control of Salmonella infections

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.Instituto de Biotecnologia y Biologia Molecula

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.Instituto de Biotecnologia y Biologia Molecula

Salmonella enterica Subclinical Infection: Bacteriological, Serological, Pulsed-Field Gel Electrophoresis, and Antimicrobial Resistance Profiles-Longitudinal Study in a Three-Site Farrow-to-Finish Farm

Fil: Vigo, German B. Universidad Nacional de La Plata. Laboratorio de Diagnóstico e Investigaciones Bacteriológicas; Argentina.Fil: Cappuccio, J. A. Universidad Nacional de La Plata. Cátedra de Patología Especial; Argentina.Fil: Pineyro, Pablo E. Universidad Nacional de La Plata. Cátedra de Patología Especial; Argentina.Fil: Salve, Angela. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología; Argentina.Fil: Machuca, Mariana A. Universidad Nacional de La Plata. Cátedra de Patología Especial; Argentina.Fil: Quiroga, Maria A. Universidad Nacional de La Plata. Cátedra de Patología Especial; Argentina.Fil: Moredo, Fabiana. Universidad Nacional de La Plata. Laboratorio de Diagnóstico e Investigaciones Bacteriológicas; Argentina.Fil: Giacoboni, Gabriel. Universidad Nacional de La Plata. Laboratorio de Diagnóstico e Investigaciones Bacteriológicas; Argentina.Fil: Cancer, Jose L. Private practitioner; Argentina.Fil: Caffer, María Ines. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología; Argentina.Fil: Binsztein, Norma. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología; Argentina.Fil: Pichel, Mariana. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología; Argentina.Fil: Perfumo, Carlos J. Universidad Nacional de La Plata. Cátedra de Patología Especial; Argentina.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 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

Salmonella enterica Subclinical Infection: Bacteriological, Serological, Pulsed-Field Gel Electrophoresis, and Antimicrobial Resistance Profiles-Longitudinal Study in a Three-Site Farrow-to-Finish Farm

Fil: Vigo, German B. Universidad Nacional de La Plata. Laboratorio de Diagnóstico e Investigaciones Bacteriológicas; Argentina.Fil: Cappuccio, J. A. Universidad Nacional de La Plata. Cátedra de Patología Especial; Argentina.Fil: Pineyro, Pablo E. Universidad Nacional de La Plata. Cátedra de Patología Especial; Argentina.Fil: Salve, Angela. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología; Argentina.Fil: Machuca, Mariana A. Universidad Nacional de La Plata. Cátedra de Patología Especial; Argentina.Fil: Quiroga, Maria A. Universidad Nacional de La Plata. Cátedra de Patología Especial; Argentina.Fil: Moredo, Fabiana. Universidad Nacional de La Plata. Laboratorio de Diagnóstico e Investigaciones Bacteriológicas; Argentina.Fil: Giacoboni, Gabriel. Universidad Nacional de La Plata. Laboratorio de Diagnóstico e Investigaciones Bacteriológicas; Argentina.Fil: Cancer, Jose L. Private practitioner; Argentina.Fil: Caffer, María Ines. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología; Argentina.Fil: Binsztein, Norma. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología; Argentina.Fil: Pichel, Mariana. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología; Argentina.Fil: Perfumo, Carlos J. Universidad Nacional de La Plata. Cátedra de Patología Especial; Argentina.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 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

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

Funder: U.S. Department of Health & Human Services | National Institutes of Health (NIH)Abstract: 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

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

Fil: Pichel, Mariana. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas; Argentina.Fil: Brengi, Silvina P. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas; Argentina.Fil: Cooper, Kara L. F. Centers for Disease Control and Prevention; Estados Unidos.Fil: Ribot, Efrain M. Centers for Disease Control and Prevention; Georgia.Fil: Al-Busaidy, Suleiman. Central Public Health Laboratory; Omán.Fil: Araya, Pamela. Instituto de Salud Pública de Chile; Chile.Fil: Fernández, Jorge. Instituto de Salud Pública de Chile; Chile.Fil: Vaz, Tania Ibelli. Instituto Adolfo Lutz; Brazil.Fil: Kam, Kai Man. Public Health Laboratory Centre; Japón.Fil: Morcos, Myriam. Regional Center at the U.S. Naval Medical Research Unit #3 (NAMRU-3). Global Disease Detection (GDD); Egipto.Fil: Nielsen, Eva M. Statens Serum Institut; Dinamarca.Fil: Nadon, Celine. National Microbiology Laboratory; Canadá.Fil: Pimentel, Guillermo. Regional Center at the U.S. Naval Medical Research Unit #3 (NAMRU-3). Global Disease Detection (GDD); Egipto.Fil: Pérez-Gutiérrez, Enrique. PAHO/WHO. Health Surveillance; Panamá.Fil: Gerner-Smidt, Peter. Centers for Disease Control and Prevention; Georgia.Fil: Binsztein, Norma. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas; Argentina.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 pulsedfield 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

CS22, a Novel Human Enterotoxigenic Escherichia coli Adhesin, Is Related to CS15

Enterotoxigenic Escherichia coli (ETEC) expresses a broad spectrum of O:H antigens. Serogroup O20 is one of the most prevalent among the ETEC strains lacking any of the defined colonization factors (CFs), in Argentina. An O20:H− strain, ARG-3, adhered to Caco-2 cells and exhibited a thermoregulated 15.7-kDa protein band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). An antiserum against this protein inhibited ARG-3 adhesion to Caco-2 cells and bound to very thin fibrilla-like structures on the bacterial surface. A 15.7-kDa protein-defective mutant failed to adhere to Caco-2 cells and lacked immunogold-labeled surface structures. The N-terminal amino acid sequence of the structural subunit showed 95% homology to that of CS15 of ETEC (former antigen 8786) and 65% homology with fimbria SEF14 of Salmonella enterica serovar Enteritidis. Nevertheless, the molecular size of ARG-3 adhesin was different from that of CS15, as revealed by SDS-PAGE and mass spectrometry. Both proteins are immunologically related, yet not identical, since an antiserum against the 15.7-kDa protein reacted solely with ARG-3 after absorption with bacteria bearing CS15. Moreover, only under low stringency conditions could DNA from strain ARG-3 be amplified by PCR using primers derived from the nfaA sequence of CS15. Thus, from the DNA sequence obtained from the ARG-3 PCR product, it could be deduced that the subunit protein differed in 30 residues from that of CS15. ARG-3 adhesin was found in 60% of the O20:H- CF-negative ETEC strains from Argentina; however, it appeared restricted to this serotype. We propose the designation CS22 for the herein identified nonfimbrial adhesin of human ETEC