Automated use of WHONET and SaTScan to detect outbreaks of Shigella spp. using antimicrobial resistance phenotypes.

Antimicrobial resistance is a priority emerging public health threat, and the ability to detect promptly outbreaks caused by resistant pathogens is critical for resistance containment and disease control efforts. We describe and evaluate the use of an electronic laboratory data system (WHONET) and a space-time permutation scan statistic for semi-automated disease outbreak detection. In collaboration with WHONET-Argentina, the national network for surveillance of antimicrobial resistance, we applied the system to the detection of local and regional outbreaks of Shigella spp. We searched for clusters on the basis of genus, species, and resistance phenotype and identified 19 statistical 'events' in a 12-month period. Of the six known outbreaks reported to the Ministry of Health, four had good or suggestive agreement with SaTScan-detected events. The most discriminating analyses were those involving resistance phenotypes. Electronic laboratory-based disease surveillance incorporating statistical cluster detection methods can enhance infectious disease outbreak detection and response.

Characterization and subtyping of Cronobacter spp. from imported powdered infant formulae in Argentina.

Cronobacter spp. (Enterobacter sakazakii), have been associated with severe foodborne infections in neonates and immunocompromised infants. In Argentina, we have isolated Cronobacter spp. from three different brands of imported powdered infant formulae (PIF). The objectives of this work were to characterize the recovered isolates phenotypically and to evaluate the use of a Pulsed-Field Gel Electrophoresis (PFGE) protocol for Cronobacter spp. subtyping. Out of 23 isolates studied from three brands of PIF (20 of brand A, 1 of brand B and 2 of brand C), 22 were identified as C. sakazakii and 1 as C. malonaticus. All isolates were susceptible to twelve antimicrobial agents assayed. The 19 C. sakazakii isolates of brand A showed five XbaI-PFGE patterns and the genetic clusters revealed by XbaI were confirmed with a second restriction enzyme, SpeI. The isolate from brand B showed the same XbaI and SpeI patterns as those of a group of isolates of brand A, suggesting a possible common source of contamination. The C. sakazakii isolates of brand C exhibited two unique XbaI-PFGE patterns, unrelated to the rest. Different genetic subtypes were found among isolates of a single batch of PIF from brand A and the single C. malonaticus strain also showed a distinct XbaI-PFGE pattern.

Pertussis epidemiology in Argentina: trends over 2004-2007.

OBJECTIVES: Pertussis continues causing significant morbidity and mortality worldwide. Although its epidemiology has been studied in many developed countries, the current pertussis situation in South America is scarcely known. This review summarizes the most important recent data concerning pertussis in a country of South America, Argentina. METHODS: CDC criteria were used for pertussis diagnosis. Proportion of pertussis cases by age, immunization status, and immunization coverage rate evaluated at the Argentinean National Pertussis Reference Centers was reported. Bordetella pertussis isolates were characterized and compared with vaccine strains. RESULTS: From 2002 to nowadays, a steady increase of pertussis cases was observed. Most of these cases correspond to patients younger than six months old that received less than three doses of vaccine. However, cases in adolescent and adults have also been detected. For this situation, which is not peculiar to Argentina, several explanations have been proposed. Among them, the inability of current vaccines to induce long-lasting immunity is the most widely accepted as a cause of pertussis resurgence. Furthermore, antigenic divergence between local clinical isolates and vaccine strains may have aggravated the effect of waning immunity. CONCLUSIONS: Pertussis is an important problem for public health in Argentina. Divergence between vaccine strains and local isolates could contribute to the described pertussis epidemiology.

[Characterization of Vibrio cholerae non-O1 and non-O139 isolates associated with diarrhea]. / Caracterización de aislamientos de Vibrio cholerae no-O1, no-O139 asociados a cuadros de diarrea.

Vibrio cholerae, etiologic agent of cholera, is transmitted to humans by ingestion of contaminated food or water. Even though serogroups O1 and O139 are the ones usually associated to epidemic cholera, isolates from other serogroups also cause gastroenteritis and extraintestinal infections. During the period 2003-2005, presence of V. cholerae in stools was investigated in children with diarrhea that seaked assistance at the Niño Jesús Hospital in Tucumán. Thirty four isolates of V. cholerae non-O1, non-O139 were recovered. We characterized the isolates studying its virulence factors by PCR, antimicrobial susceptibility patterns and genetic diversity by pulsed-field gel electrophoresis. Eight virulence patterns were obtained although no isolate was positive for the cholera toxin or the thermostable toxin. Four isolates were positive for the type three secretion system. The 17.6% of the isolates were resistant or intermediate to ampicillin and 5.9% were resistant to trimethoprim-sulfamethoxazole. By Sfil-PFGE, all isolates were genetically very diverse, as 27 different patterns were identified in 29 typeable isolates by pulsed-field gel electrophoresis. Although it has a low incidence, V. cholerae continues to be a causative agent of diarrhea in children, who are affected by a variety of circulating strains of V. cholerae non-O1, non-O139.

Caracterización de aislamientos de Vibrio cholerae no-O1, no-O139 asociados a cuadros de diarrea / Characterization of Vibrio cholerae non-O1 and non-O139 isolates associated with diarrhea

La infección por Vibrio cholerae, el agente causal del cólera, se trasmite al hombre por ingestión de agua y alimentos contaminados. Aunque son los serogrupos O1 y O139 los que habitualmente se asocian al cólera epidémico, los aislamientos de otros serogrupos también son causales de gastroenteritis e infecciones extra-intestinales. Durante el período 2003-2005, se investigó la presencia de V. cholerae en la materia fecal de niños con diarrea atendidos en el Hospital del Niño Jesús, Tucumán. Se recuperaron 34 aislamientos de V. cholerae no-O1, no-O139. Se determinaron sus perfiles de virulencia por PCR, la sensibilidad a los antimicrobianos y la diversidad genética por electroforesis en campo pulsado. Se obtuvieron ocho perfiles de virulencia, aunque ningún aislamiento fue positivo para la toxina colérica ni para la toxina termoestable. Cuatro aislamientos fueron positivos para el sistema de secreción de tipo tres. El 17,6% de los aislamientos fueron resistentes o de sensibilidad intermedia a ampicilina y el 5,9% fueron resistentes a trimetoprima-sulfametoxazol. Los aislamientos resultaron muy diversos: se hallaron 27 patrones distintos en 29 aislamientos tipificables por electroforesis en campo pulsado. A pesar de su baja incidencia, V. cholerae continúa siendo un agente causal de diarrea en niños, los que se ven afectados por una amplia variedad de cepas circulantes.

Vibrio cholerae, etiologic agent of cholera, is transmitted to humans by ingestion of contaminated food or water. Even though serogroups O1 and O139 are the ones usually associated to epidemic cholera, isolates from other serogroups also cause gastroenteritis and extraintestinal infections. During the period 2003-2005, presence of V. cholerae in stools was investigated in children with diarrhea that seaked assistance at the Niño Jesús Hospital in Tucumán. Thirty four isolates of V. cholerae non-O1, non-O139 were recovered. We characterized the isolates studying its virulence factors by PCR, antimicrobial susceptibility patterns and genetic diversity by pulsed-field gel electrophoresis. Eight virulence patterns were obtained although no isolate was positive for the cholera toxin or the thermostable toxin. Four isolates were positive for the type three secretion system. The 17.6% of the isolates were resistant or intermediate to ampicillin and 5.9% were resistant to trimethoprim-sulfamethoxazole. By SfiI-PFGE, all isolates were genetically very diverse, as 27 different patterns were identified in 29 typeable isolates by pulsed-field gel electrophoresis. Although it has a low incidence, V. cholerae continues to be a causative agent of diarrhea in children, who are affected by a variety of circulating strains of V. cholerae non-O1, non-O139.

Environment and virulence factors of Vibrio cholerae strains isolated in Argentina.

AIMS: To determine the presence of Vibrio cholerae in different areas of Argentina in three sample types, to determine the composition of planktonic communities in areas at which this pathogen was detected and to characterize the virulence properties and antimicrobial resistance of the recovered environmental isolates. METHODS AND RESULTS: Water and plankton samples were collected in marine, brackish and freshwater environments. Vibrio cholerae non-O1, non-O139 was isolated in 36.1% of the samples analysed. The micro-organism was detected in freshwater but not in marine or brackish samples. No relationship was found between isolation of V. cholerae and presence of any species of plankton. All the isolates presented very similar virulence profiles by PCR, lacking ctxA and tcpA El Tor and containing hlyA (98.7%), rtxA (99.0%), toxR (98.7%) and stn-sto (1.9%). Resistance to ampicillin was found in both Tucumán (21%) and Buenos Aires isolates (45%). CONCLUSIONS: We identified two geographic areas in Argentina where V. cholerae was present: freshwaters of the rivers from Tucumán and the Río de la Plata. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification of V. cholerae strains in the environment, carrying both virulence factors and resistance to antimicrobial agents, highlight the need for a continuous and active surveillance of this pathogen.

Short report: analysis of clonal relationship among Shigella sonnei isolates circulating in Argentina.

Thirty-five isolates of Shigella sonnei from patients with diarrhoea in three geographic regions of Argentina were examined for genetic diversity by pulsed-field gel electrophoresis (PFGE) and plasmid profile. PFGE of XbaI and BlnI DNA digests confirmed the occurrence of outbreaks in two regions caused by two separate predominant clones of S. sonnei. The third region was characterized by three circulating clones, one of which was possibly associated with an outbreak. Similar plasmids were found in distinct clones and in one outbreak clone five different plasmid profiles were identified. Antimicrobial resistance of the isolates varied from fully susceptible to the agents tested, to resistance to cotrimoxazole, ampicillin and ciprofloxacin. Antibiotic resistance did not correlate with plasmid content. This information will form the basis for active surveillance of shigellosis in Argentina and elsewhere in the region through the PulseNet International Network.

Beyond serotypes and virulence-associated factors: detection of genetic diversity among O153:H45 CFA/I heat-stable enterotoxigenic Escherichia coli strains.

Characterization of enterotoxigenic Escherichia coli (ETEC) has been based almost exclusively on the detection of phenotypic traits such as serotypes and virulence-associated factors: heat-labile (LT) and heat-stable (ST) toxins and colonization factors (CFs). In the present work we show that the analysis of band patterns generated by randomly amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE) of digested chromosomal DNA can be used to detect genetic diversity among ETEC strains expressing identical phenotypic traits. The study included 29 ETEC isolates from Latin America and Spain expressing the phenotype O153:H45 CFA/I ST plus 1 rough derivative, 2 nonmotile derivatives, and 1 O78:H12 CFA/I ST isolate, and a representative of a genetically distinct ETEC group. The results showed that the O153:H45 CFA/I ST ETEC isolates belong to a single clonal cluster whose isolates share on average, 84% of the RAPD bands and 77% of the PFGE restriction fragments, while the O78:H12 isolate shared only 44 and 4% of the RAPD bands and PFGE fragments, respectively, with the isolates of the O153:H45 group. More relevantly, RAPD and PFGE fingerprints disclosed the presence of different clonal lineages among the isolates of the O153:H45 cluster. Some of the genetic variants were isolated from defined geographic areas, while places like São Paulo City in Brazil and the middle-eastern part of Argentina were populated by several genetic variants of related, but not identical, ETEC strains. These results show that molecular biology-based typing methods can disclose strain diversity, which is usually missed in studies restricted to phenotypic typing of ETEC.

Identification of a cluster of strains bearing a new adhesin among genetically diverse enterotoxigenic Escherichia coli isolates of serogroup O20.

About one-third of the enterotoxigenic Escherichia coli isolates lack any of the known colonization factors. Among this group, those of serogroup O20 are the most frequently isolated in Argentina. By combining analysis of adhesion to Caco-2 cells, random amplified polymorphic DNA, and pulsed-field gel electrophoresis techniques, we were able to identify three sets of closely related strains with different binding properties. Further analysis of the most prevalent group revealed that all these isolates expressed the recently described adhesin CS22.

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.

Prospective cohort study of enterotoxigenic Escherichia coli infections in Argentinean children.

In a follow-up study, enterotoxigenic Escherichia coli (ETEC) infections in 145 children from two communities located in northeastern Argentina were monitored for 2 years. The occurrence of diarrhea was monitored by weekly household visits. Of 730 fecal specimens collected, 137 (19%) corresponded to diarrheal episodes. ETEC was isolated from a significantly higher proportion of symptomatic (18.3%) than asymptomatic (13.3%) children (P = 0.04541). Individuals of up to 24 months of age were found to have a higher risk of developing ETEC diarrhea than older children (odds ratio [OR], 3.872; P = 0.00021). When the toxin profiles were considered, only heat stable enterotoxin (ST)-producing ETEC was directly associated with diarrhea (P = 0.00035). Fifty-five percent of the ETEC isolated from symptomatic children and 19% of the ETEC isolated from asymptomatic children expressed one of the colonization factors (CFs) investigated, i.e., CF antigen I (CFA/I), CFA/II, CFA/III, and CFA/IV; coli surface antigens CS7 and CS17; and putative CFs PCFO159, PCFO166, and PCFO20, indicating a clear association between diarrhea and ETEC strains that carry these factors (P = 0.0000034). The most frequently identified CFs were CFA/IV (16%), CFA/I (10%), and CS17 (9%). CFs were mostly associated with ETEC strains that produce ST and both heat-labile enterotoxin and ST. Logistic regression analysis, applied to remove confounding effects, revealed that the expression of CFs was associated with illness independently of the toxin type (OR, 4.81; P = 0.0003). When each CF was considered separately, CS17 was the only factor independently associated with illness (OR, 16.6; P = 0.0151). Most CFs (the exception was CFA/IV) fell within a limited array of serotypes, while the CF-negative isolates belonged to many different O:H types. These results demonstrate that some CFs are risk factors for the development of ETEC diarrhea.

[Antimicrobial resistance among species of Salmonella, Shigella, Escherichia, and aeromonas isolated from children with diarrhea in 7 Argentinian centers]. / Resistencia a los antimicrobianos de especies de Salmonella, Shigella, Escherichia y Aeromonas aisladas de niños diarreicos en siete centros de Argentina.

The increasing levels of resistance of enteropathogenic bacteria against antimicrobial agents present geographic variations. We have analysed the antimicrobial susceptibility of isolates obtained from 4,364 children under 5 years of age with acute diarrhea, in 7 cities of Argentina. Diarrheagenic E. coli exhibited 74.5% of resistance against ampicillin, 64.2% against sulfametoxazole-trimethoprim, and Shigella spp., 62% and 75.6% respectively. Salmonella sp. showed 35%, 14%, 41.8%, 65.4%, 14.5%, and 13.6% of resistance against ampicillin, chloranfenicol, sulfametoxazole-trimetoprim, sulfadiazin, gentamycin, and fosfomycin respectively. These values are higher than the ones observed in developed countries. Aeromonas showed significantly lower resistance percentage. Important differences in our country were observed, consequently, local trials should be carried out in order to apply corrective measures.

Disco fever: epidemic meningococcal disease in northeastern Argentina associated with disco patronage.

Neisseria meningitidis is a leading cause of adult meningitis worldwide. From 5 to 14 August 1996, 8 cases of meningococcal disease occurred in Corrientes city (population 306,000) in northeastern Argentina. Those infected ranged in age from 15 to 45 years (median, 18.5). To determine risk factors for infection, a case-control study was done. Infecting isolates were serogrouped and underwent phenotyping by multilocus enzyme electrophoresis (MLEE) and pulsed-field gel electrophoresis (PFGE). Those infected were significantly more likely than those not infected to have had exposure to passive or active cigarette smoke or to have attended a particular disco. Isolates available from 6 case-patients were all serogroup C; all had identical MLEE and PFGE patterns. These data suggest that dance clubs or discos may be a focus of transmission of N. meningitidis among young people.

Clonal nature of enterotoxigenic Escherichia coli serotype O6:H16 revealed by randomly amplified polymorphic DNA analysis.

The genetic relatedness among 29 enterotoxigenic Escherichia coli strains of serotype O6:H16 was investigated by randomly amplified polymorphic DNA (RAPD) analysis. The strains were isolated in different parts of the world, displayed CS1-CS3 or CS2-CS3 profiles, and expressed heat-labile toxin (LT) and heat-stable toxin; a single strain expressed only LT. Ten RAPD types were distinguished and showed significant similarity, having on average 82% of the amplified bands in common. These results indicated that, irrespective of the different geographical origin or virulence factors, these strains belonged to a widespread clonal group.

Prospective study of enteropathogens in two communities of Misiones, Argentina.

Children under five years of age, from two communities of different socio-economic strata (97 from Zaiman and 55 from Las Dolores) were examined epidemiologically during 2 years, by means of quarterly visits of the working team, who carried out the collection of faecal samples. During the study, one or more enteropathogens were identified in 73.9% of samples in children from Zaiman and in 58.3% of the samples from Las Dolores, being associated to diarrhoea in 70.5% and to asymptomatic infections in 65.7%. The number of diarrheic episodes was higher in Zaiman (15.45%) than in Las Dolores (12.35%), being more frequent in the spring-summer seasons. In Zaiman, the bacterial enteropathogen proportion was relevantly higher (p < 0.005) in children with diarrhoea, whereas the presence of parasites was more frequent in asymptomatic children (p < 0.01). Rotavirus had an even distribution within diarrheic and asymptomatic children. In Las Dolores, no relevant differences were found in the detection of enteroparasites between diarrheic and asymptomatic children. Mixed infections were detected; enterotoxigenic Escherichia coli (ETEC)-rotavirus and ETEC-parasites being the most frequent ones. ETEC was involved in 85% of these infections. These data, together with the high enteropathogen carriage, suggest an elevated level of environmental contamination. The latter plays an important role in diarrheic diseases, and added to the most extreme poverty, it affects children's lives.

Antibody responses against Escherichia coli heat-labile toxin and colonization factor antigens I and II in Argentinian children.

Serum antibody responses against Escherichia coli heat-labile enterotoxin (LT) and colonization factor antigens (CFAs) I and II were studied in 84 children < 5 years old living in two communities. These villages differed in the quality of their drinking water. Children from both communities developed significantly increased antibody titers against LT and CFA/II but not against CFA/I during 3 months of follow-up. The magnitude of the anti-LT response was significantly higher in children from Zaiman than in those from Las Dolores. Antibody titers rose to maximum levels during the second year of age and reached relatively constant levels in children aged 2-5 years, probably due to repeated exposure to enterotoxigenic E. coli strains. Antibody levels of 30 children were followed for 2 years; increases in anti-LT and anti-CFA titers varied in the different age groups.

Epidemiología de los enteropatógenos causantes de diarrea en la ciudad de Rosario, Argentina / Epidemiology of the enteropathogenic cause of the diarrhea at Rosario city, Argentina

La revisión de diferentes estudios efectuados en nuestro medio entre 1970 y 1990 permitieron analizar algunos aspectos epidemiológicos de los M. org. más comunmente hallados en Rosario. La prevalencia de los diversos agentes fue significativamente diferente en pacientes de distintas edades, grados de nutrición, gravedad, fiebre, leucocitos en heces y épocas estacionales. E. Coli enteropatógeno se halló en más del 25 por ciento de los niños de toda edad, pero principalmente en menores de 5 meses y en pacientes internados. E. Coli enterotoxigénico (ECET) predominó en verano y fueron frecuentes las cepas productoras de enterotoxina ST. Entre las especies de Shigella predominó S. flexneri 2. Campylobacter resultó más frecuente que Salmonella, aunque se ha notado un nuevo incremento reciente debido a S. enteritidis. A diferencia de Vibrio, Aeromonas spp aparece con una frecuencia del 0,2-3 por ciento. Otros M. org. importantes fueron Giardia y Rotavirus. Ciertos agentes no habían sido hallados anteriormente, como Salmonella mbandaka, Shigella dysenteriae tipo 2, Vibrio cholerae no 0-1, Campylobacter lari. Algunos serotipos de ECET predominaron en nuestra zona respecto del resto del país

Epidemiología de los enteropatógenos causantes de diarrea en la ciudad de Rosario, Argentina / Epidemiology of the enteropathogenic cause of the diarrhea at Rosario city, Argentina

La revisión de diferentes estudios efectuados en nuestro medio entre 1970 y 1990 permitieron analizar algunos aspectos epidemiológicos de los M. org. más comunmente hallados en Rosario. La prevalencia de los diversos agentes fue significativamente diferente en pacientes de distintas edades, grados de nutrición, gravedad, fiebre, leucocitos en heces y épocas estacionales. E. Coli enteropatógeno se halló en más del 25 por ciento de los niños de toda edad, pero principalmente en menores de 5 meses y en pacientes internados. E. Coli enterotoxigénico (ECET) predominó en verano y fueron frecuentes las cepas productoras de enterotoxina ST. Entre las especies de Shigella predominó S. flexneri 2. Campylobacter resultó más frecuente que Salmonella, aunque se ha notado un nuevo incremento reciente debido a S. enteritidis. A diferencia de Vibrio, Aeromonas spp aparece con una frecuencia del 0,2-3 por ciento. Otros M. org. importantes fueron Giardia y Rotavirus. Ciertos agentes no habían sido hallados anteriormente, como Salmonella mbandaka, Shigella dysenteriae tipo 2, Vibrio cholerae no 0-1, Campylobacter lari. Algunos serotipos de ECET predominaron en nuestra zona respecto del resto del país (AU)

Direct detection of Vibrio cholerae in stool samples.

A direct method to detect Vibrio cholerae in stool samples was developed by using a PCR procedure that did not require a DNA purification step. Dilution (1/100) of stool samples prevented inhibition of the reaction by contaminants, and two consecutive PCRs, the second one with a nested primer, achieved the desired sensitivity. Comparison of the results obtained from stool swab samples processed by the two-step PCR and by an enzyme-linked immunosorbent assay using GM1 as the capture molecule showed that the former is more sensitive and gave positive results even when V. cholerae was not culturable or dead.

A new fimbrial putative colonization factor, PCFO20, in human enterotoxigenic Escherichia coli.

The ability to colonize the small intestine is essential for enterotoxigenic Escherichia coli (ETEC) to cause diarrhea. Several colonization factor antigens (CFAs) and putative colonization factors (PCFs) have been described for ETEC. However, there are still many ETEC strains isolated from patients with diarrhea which do not possess any of these antigens. To identify CFAs in ETEC lacking the above-mentioned antigens, we exploited the ability of ETEC to adhere to tissue-cultured cells from an enterocyte-like cell line, Caco-2. An ETEC strain producing heat-labile toxin and heat-stable toxin of serotype O20:K27:H- (ARG-2) that was isolated from a child with diarrhea in Argentina and bound to Caco-2 cells was studied in further detail. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of this strain revealed a band of 25 kDa when bacteria were grown at 37 degrees C that was missing when the same strain was cultured at 20 degrees C. Furthermore, electron microscopy examination revealed the presence of fimbriae on the surfaces of cells of this strain when cells were grown at 37 degrees C but not at 20 degrees C. Rabbit antiserum raised against purified fimbriae reacted with the 25-kDa protein in immunoblotting and bound specifically to the fimbriae, as shown by immunoelectron microscopy. The presence of fimbriae, adhesion to Caco-2 cells, and the 25-kDa band seen in the SDS-PAGE were all simultaneously lost by single-insertion mutations. The N-terminal amino acid sequence of the protein subunit of the fimbriae showed no relation with those of the known colonization factors of ETEC. Furthermore, the fimbriae of the ARG-2 strain did not cross-react immunologically with any of the previously described adhesive factors in human ETEC when specific antisera against colonization factor antigens and putative colonization factors were used. Moreover, a specific antiserum raised against the fimbriae in ARG-2 did not react with ETEC carrying known colonization factors. We propose to name these new fimbriae PCFO20.