Identification of two new intimin types in atypical enteropathogenic Escherichia coli.

Stool specimens of patients with diarrhea or other gastrointestinal alterations who were admitted to Xeral-Calde Hospital (Lugo, Spain) were analyzed for the prevalence of typical and atypical enteropathogenic Escherichia coli (EPEC). Atypical EPEC strains (eae+ bfp-) were detected in 105 (5.2%) of 2015 patients, whereas typical EPEC strains (eae+ bfp+) were identified in only five (0.2%) patients. Atypical EPEC strains were (after Salmonella) the second most frequently recovered enteropathogenic bacteria. In this study, 110 EPEC strains were characterized. The strains belonged to 43 O serogroups and 69 O:H serotypes, including 44 new serotypes not previously reported among human EPEC. However, 29% were of one of three serogroups (O26, O51, and O145) and 33% belonged to eight serotypes (O10:H-, O26:H11, O26:H-, O51:H49, O123:H19, O128:H2, O145:H28, and O145:H-). Only 14 (13%) could be assigned to classical EPEC serotypes. Fifteen intimin types, namely, alpha1 (6 strains), alpha2 (4 strains), beta1 (34 strains), xiR/b2 (6 strains), gamma1 (13 strains), gamma2/q (16 strains), delta/k (5 strains), epsilon1 (9 strains), nuR/e2 (5 strains), zeta (6 strains), iota1 (1 strain), muR/iota2 (1 strain), nuB (1 strain), xiB (1 strain), and o (2 strains), were detected among the 110 EPEC strains, but none of the strains was positive for intimin types mu1, mu2, lambda, or muB. In addition, in atypical EPEC strains of serotypes O10:H-, O84:H-, and O129:H-, two new intimin genes (eae-nuB and eae-o) were identified. These genes showed less than 95% nucleotide sequence identity with existing intimin types. Phylogenetic analysis revealed six groups of closely related intimin genes: (i) alpha1, alpha2, zeta, nuB, and o; (ii) iota1 and muR/iota2; (iii) beta1, xiR/beta2B, delta/beta2O, and kappa; (iv) epsilon1, xiB, eta1,eta2, and nuR/epsilon2; (v) gamma1, muB, gamma2, and theta; and (vi) lambda. These results indicate that atypical EPEC strains belonging to large number of serotypes and with different intimin types might be frequently isolated from human clinical stool samples in Spain.     

Inhibition and inactivation of pathogenic serogroups of Yersinia enterocolitica by a bacteriocin produced by Yersinia kristensenii

Growth of Yersinia enterocolitica strains representing serogroups O: 3, O: 5, 27, O:6, 30, O:8, O:9 (human isolates) and O:6, 31 (food isolate) were inhibited in the presence of a bacteriocin produced by Yersinia kristensenii at high initial cell count of 10⁶ ml^-1. Complete (100%) inactivation of most Y. enterocolitica cells of different serotypes was observed within 24 h at low initial cell counts of 10⁴ ml^-1. Complete injury of the cells was observed within 4–8 h, with all the serotypes at 10°C and 28°C. The degree of susceptibility to the injury and the recovery of cells from the injury varied from serogroup to serogroup.     

The possession of coli surface antigen CS6 by enterotoxigenicEscherichia coli of serogroups O25, O27, O148, and O159: a possible colonization factor?

A total of 134 enterotoxigenicEscherichia coli (ETEC) of serogroups O25, O27, O148, and O159 were tested in the enzyme-linked immunosorbent assays for the colonization factor antigens I (CFA/I), CFA/II (coli surface antigens CS1, 2 and 3) and putative colonization factor (PCF) 8775 (CS4, 5 and 6). CS6 was detected without CS4 or CS5 in 94% of the strains of serogroup O25, 86% of strains of serogroup O27, 87% of strains of serogroup O148, and 29% of strains of serogroup, O159. The frequency with which CS6 occurs in ETEC of common serotypes without the antigens CS4 or CS5 suggests that it might be a colonization factor.     

Association of pandemic Vibrio parahaemolyticus O3:K6 present in the coastal environment of Northwest Mexico with cases of recurrent diarrhea between 2004 and 2010

In 2004, more than 1,230 cases of gastroenteritis due to pandemic O3:K6 strains of Vibrio parahaemolyticus were reported in southern Sinaloa, a state in Northwestern Mexico. Recurrent sporadic cases arose from 2004 to 2010, spreading from the south to the north. In the present study, Vibrio parahaemolyticus was detected in both environmental samples and clinical cases along the Pacific coast of Sinaloa during 2004 to 2010. An evaluation was made of the serotypes, distribution of virulence genes, and presence of pandemic O3:K6 strains. A total of 144 strains were isolated from environmental samples (from sediment, seawater, and shrimp), and 154 clinical strains were isolated. A total of 10 O serogroups and 30 serovars were identified in the strains. Environmental strains (n = 144) belonged to 10 O serogroups and 28 serovars, while clinical strains (n = 154) belonged to 8 O serogroups and 14 serovars. Ten serovars were shared by both environmental and clinical strains. Among 144 environmental isolates, 4.1% (6/144) belonged to the pandemic clone, with 83.3% containing the orf8 gene and with O3:K6 accounting for 67%. On the other hand, pathogenic strains (tdh and/or trh) accounted for 52% (75/144) of the environmental isolates. Interestingly, among 154 clinical isolates, 80.5% (124/154) were pandemic strains, with O3:K6 (tdh, toxRS(new), and orf8) representing the predominant serovar (99.2%, 123/124). Overall, our results indicate that in spite of a high serodiversity and prevalence of pathogenic Vibrio parahaemolyticus in the environment, the pandemic strain O3:K6 caused >79% of reported cases between 2004 and 2010 in Sinaloa, Mexico.     

Opsonic and protective activity of five human IgM monoclonal antibodies reactive with lipopolysaccharide antigen of Pseudomonas aeruginosa

International Antigen Typing Schema (IATS) serotypes 1, 2, 5, 6, 8 and 11 comprise approximately 80% of Pseudomonas aeruginosa strains isolated from blood, wounds and respiratory specimens. Five human immunoglobulin M (IgM) monoclonal antibodies (MAbs) reactive with lipopolysaccharide O antigens of these IATS serotypes were studied in an opsonophagocytic assay. The assay employed human polymorphonuclear leukocytes, 2% guinea pig serum as the complement source and MAb. Each MAb promoted killing of inoculum of the homologous LPS serotype. The opsonic activity of each MAb was complement-dependent. In a murine model of Pseudomonas burn wound sepsis the LD50 of five strains of P. aeruginosa was increased greater than or equal to 22-fold by MAb-treatment (1.0 mg/kg). The mean effective dose of the five MAbs in mice challenged with approximately 10 LD50 of the homologous LPS serotype ranged from less than 0.01 mg/kg to 1.00 mg/kg.     

Sensitivity to virulent bacteriophages of Pseudomonas aeruginosa strains of different serogroups

The data on the sensitivity of P. aeruginosa clinical strains to pyocyaneum, a therapeutic and prophylactic bacteriophage preparation, and to individual groups of phages contained in this preparation are presented. Out of 549 P. aeruginosa strains, 16% have proved to be nonlysing cultures. The proportion of phage-sensitive strains prevailed in serogroups 01, 03, 06, 09, while phage-resistant strains prevailed in serogroups 04, 07, 011, as well as among O-nontyped cultures. The expediency of introducing P. aeruginosa strains of different serotypes into the collection of cultures used for the production of pyocyaneum has been shown.     

Identification of EPEC and non-EPEC serotypes in the EPEC O serogroups by PCR-RFLP analysis of the fliC gene

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the flagellin gene (fliC) was performed in 233 strains of enteropathogenic Escherichia coli (EPEC) O serogroups for determining their flagellar antigen (H) status. The serological detection of flagellin is the basis for the H-codes typing system in E. coli. Thus, it is impossible to serotype nonmotile bacteria (i.e. to assign H-codes). Twenty-eight fliC restriction patterns were obtained for motile (H2, H4, H6, H7, H8, H9, H10, H11, H12, H18, H21, H27, H32, H34, H35, H40 and H51) and nonmotile serotypes (H(-)). Each motile serotype was characterized by one or two fliC specific restriction patterns. The only exception was serogroup O128ab, where a common restriction pattern was found for serotypes O128ab:H2 and O128ab:H35, even after digestion with RsaI, AluI and Sau3AI endonucleases. These two serotypes were, however, discriminated by single strand conformation polymorphism (SSCP) analysis of RsaI restriction fragments. Nonmotile strains showed fliC restriction patterns identical to some known H serotypes. The PCR-RFLP analysis of fliC gene proved to be a useful method for identifying the H variants in motile and nonmotile EPEC O serogroups.     

Distribution of Pseudomonas syringae pathovars into twenty-three O serogroups.

Serological reactions of Pseudomonas syringae and Pseudomonas viridiflava were studied by Ouchterlony double diffusion. A total of 55 polyclonal antisera, containing anti-lipopolysaccharide (anti-LPS) precipitating antibodies, were cross-tested against antigenic suspensions of 51 strains. Twenty-three O serogroups were defined, primarily on the reaction of the type strains. Two families of O serogroups showed antigenic crossreactivities (PHA, MOP1, MOP2, MOP3, HEL1, HEL2, and SYR1; PERSAVTOM1, PERSAVTOM2, DEL, POR, and SYR2). Ten O serogroups showed a clearcut specificity: APTPIS, TAB, VIR1, VIR2, VIR3, SYR3, SYR4, SYR5, HUS, and LAC. The last serogroup (RIB) contained strains with rough colony morphology and side chain-deficient LPSs, as evidenced by sodium dodecyl sulfate-polycrylamide gel electrophoresis. The LPS basis of the O serogroups was demonstrated by immunoblotting. Serological reference strains were designated for all of the O serogroups and correspondence was established between the O serogroups studied and seven previous serogroups (L. T. Pastushenko and I.D. Simonovich, Mikrobiol, Zh. 41:222-229 and 330-339, 1979). A total of 355 strains of P. syringae (sensu lato) belonging to 15 pathovars, not including pathovar syringae, were typed into the 23 described O serogroups. O serogroups were assigned after double-diffusion reactions, with each strain compared with serological references. The utility of O serogrouping to study P. syringae pathovar structure and diversity is discussed.     

Serogroups of Escherichia coli strains producing cytotoxic necrotizing factors CNF1 and CNF2

The serogroups of 396 necrotizing Escherichia coli of human and bovine origin isolated in Spain between 1979 and 1991 have been determined. The 270 cytotoxic necrotizing factor strains belonged to 22 different O serogroups; however, 84% (226 of 270) were of one of seven serogroups (O2, O4, O6, O14, O22, O75 and O83). Although necrotizing E. coli producing cytotoxic necrotizing factor 2 belonged to 28 different serogroups, only six of them (O1, O3, O15, O55, O88 and O123) accounted for 60% (76 of 126) of cytotoxic necrotizing factor 2 strains. Furthermore, only 3% (4 of 126) of cytotoxic necrotizing factor 2 strains belonged to serogroups most common among strains producing cytotoxic necrotizing factor 1. The majority of necrotizing E. coli producing cytotoxic necrotizing factor 1 were obtained from human extraintestinal infections, whereas cytotoxic necrotizing factor 2 strains were isolated from stools of healthy and diarrhoeic calves.     

Larval susceptibility of the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), to Bacillus thuringiensis H serovars isolated in Japan

A total of 1700 Japanese strains of Bacillus thuringiensis, belonging to at least 47 H serogroups, were examined for insecticidal activity against larvae of the diamondback moth, Plutella xylostella. The high-level toxicity was associated with 612 isolates (36.0%). Of these, 608 isolates (99.3%) fell into 13 H serogroups belonging to the low-numbered H serotypes, H1-H10. Conversely, most isolates belonging to the high-numbered serotypes (>H10) had little or no larvicidal activity; only one isolate of the serovar japonensis H23 was active. P xylostella larvae were susceptible to 89.8% of the serovar morrisoni H8a:8b strains and 85.7% of galleriae H5a:5b strains. High values of 60-80% were also obtained in six serovars (thuringiensis H1, alesti H3a:3c, kurstaki H3a:3b:3c, kenyae H4a:4c, aizawai H7, and tolworhi H9), while relatively low values of <60% in two other common serovars, sotto H4a:4b and darmstadiensis H10a:10b. Five selected isolates, belonging to H serovars other than kurstaki and aizawai, were 10-60 times less toxic than the reference strain HD-1 (serovar kurstaki). Parasporal inclusion proteins of these strains were immunologically unrelated to those of the strain HD-1 and the aizawai type strain.     

Analysis of antigens present in the extracellular products and cell surface of Vibrio anguillarum serotypes O1, O2, and O3.

Antigens present in the extracellular products (ECP) and cell walls of strains of Vibrio anguillarum of serotypes O1, O2, and O3 isolated from different fish species in distinct geographic areas were characterized. The usefulness of slide agglutination, dot blot assay, and quantitative agglutination for subtyping V. anguillarum serovars was also evaluated. The three serological assays used to establish the serogroups within V. anguillarum isolates demonstrated that serotype O1 constitutes a homogeneous group, whereas within serotypes O2 and O3, two different patterns of serological reactions were detected. Among the three serological methods used, only dot blot and quantitative agglutination assays differentiated subgroups within serotypes O2 and O3 with unabsorbed sera. Electrophoretic analysis and immunoblot assays of cell envelope and ECP components showed that strains belonging to serotype O1 possessed immunologically related lipopolysaccharide (LPS) and proteins, while V. anguillarum isolates grouped in serotypes O2 and O3 exhibited internal heterogeneity in their LPS and protein banding patterns. On the other hand, although the LPS present in the ECP and those obtained from cell envelopes of V. anguillarum strains showed apparently different gel patterns, a strong relationship between both types of LPS was seen by immunoblot assay. From these results, it can be concluded that V. anguillarum strains representative of each of the antigenic groups (O1, O2 alpha, O2 beta, O3A, and O3B) and their ECPs should be included in the formulation of vaccines against vibriosis in areas where the three serotypes coexist.     

Establishment of three categories of P-fimbriated Escherichia coli strains that show different toxic phenotypes and belong to particular O serogroups

Eight hundred and nineteen strains of Escherichia coli isolated in Spain between 1986 and 1991 from extraintestinal infections and feces of healthy controls were investigated for expression of P-fimbriae using a particle agglutination test. Among strains causing urinary tract infections, sepsis and other extraintestinal infections, P-fimbriae were found in 31% (130/420) (P < 0.001), 25% (30/118) (P < 0.001) and 12% (11/92) (P < 0.5) respectively. In contrast, only 7% (14/189) of faecal isolates from healthy individuals carried P-fimbriae. According to two more common toxic markers detected in this study (alpha-haemolysin and cytotoxic necrotizing factor type 1), P-fimbriated E. coli strains were grouped into three categories: haemolysin+cytotoxic necrosing factor+ (Hly+CNF1+) (68/185; 37%), haemolysin+cytotoxic necrosing factor- (Hly+CNF1-) (61/185; 33%) and Hly-CNF1- (56/185; 30%). The 185 P-fimbriated strains belonged to 17 different O serogroups. However, 148 (80%) were of one of six serogroups (O1, O2, O4, O6, O7 and O18). The most frequent serogroups determined in the Hly+CNF1+ strains were the O4 and O6 (53/68; 78%), in the Hly+CNF1- strains it was the O18 (27/61; 44%) and in the Hly-CNF1- strains the O1, O2 and O7 (41/56; 73%). The majority (160/185; 86%) of P-fimbriated E. coli expressed the mannose-resistant haemagglutinin type IVa.     

Characterisation of the katA gene encoding a catalase and evidence for at least a second catalase activity in Staphylococcus xylosus,bacteria used in food fermentation

Insertional inactivation of wbpM in Pseudomonas aeruginosa serogroup O11 strain PA103 resulted in mutants exhibiting three distinct lipopolysaccharide (LPS) phenotypes. One mutant, PA103 wbpM-C, had a truncated LPS core and lacked O antigen. These defects were not complemented by the cloned wbpM gene, suggesting a secondary mutation was present. When the wild-type galU gene was introduced into strain PA103 wbpM-C containing the cloned wbpM gene, both LPS defects were corrected. Construction of galU mutants in P. aeruginosa serogroups O11, O5, O6 and O17 strains led to truncation of the LPS core, indicating the involvement of GalU in P. aeruginosa LPS core synthesis.     

Asymptomatic healthy slaughterhouse workers in South Korea carrying Shiga toxin-producing Escherichia coli

A total of 1602 stool samples from healthy employees in a slaughter company were screened by PCR for Shiga toxin (Stx)-producing Escherichia coli (STEC). The PCR product of Stx-encoding genes was detected in 90 (5.6%) of 1602 stool samples. Among the 90 stx -positive workers, the Residual Products Handlers and Slaughterers had rates of 8.0% and 6.0%– higher than Inspectors, Grading Testers and Livestock Hygiene Controllers at 3.3%, 2.0% and 3.5%, respectively. Forty-nine (54.4%) were shown to have stx 2; 25 (27.7%) carried stx 1 and 16 (17.7%) had both stx 1 and 2. Distribution of the stx PCR-positive workers by age revealed an increase in STEC infection with age ( P <0.05). Phenotypic and genotypic traits of nine STEC strains isolated from eight slaughter plant workers were characterized. A variety of serotypes, five O serogroups (O8, O54, O59, O103 and O153) and two H serogroups (H7 and H32) were found, but none of the strains belonged to the serogroup O157. Eight Vero cell cytotoxicity assay-positive strains were isolated from the workers and these workers were asymptomatic and healthy. The results of the study show that slaughter plant workers are at high risk of STEC infection.     

Opsonic and protective activity of five human IgM monoclonal antibodies reactive with lipopolysaccharide antigen of Pseudomonas aeruginosa

Abstract International Antigen Typing Schema (IATS) serotypes 1, 2, 5, 6, 8 and 11 comprise approximately vn80% of Pseudomonas aeruginosa strains isolated from blood, wounds and respiratory specimens. Five human immunolgobulin M (IgM) monoclonal antibodies (MAbs) reactive with lipopolysaccharide O antigens of these IATS serotypes were studies in an opsonophagocytic assay. The assay employed human polymorphonuclear leukocytes, 2% guinea pig serum as the complement source and MAb. Each MAb promoted killing of inoculum of the homologous LPS serotype. The opsonic activity of each MAb was complement-dependent. In a murine model of Pseudomonas burn wound sepsis the LD₅₀ of five strains of P. aeuruginosa was increased ≥ 22-fold by MAb-treatment (1.0 mg/kg). The mean effective dose of the five MA0bs in mice challenged with approximately 10 LD₅₀ of the homologous LPS serotype ranged from < 0.01 mg/kg to 1.00 mg/kg.     

Streptococcus pneumoniae serotypes in children with chronic inflammatory diseases of the respiratory organs

During the 7-year period of observation (1982-1988) the serotypes of 276 pneumococcal strains isolated from children with chronic bronchopulmonary diseases were studied. Among the serotypes of pneumococci under study, serotypes 6, 19 and 15 held the leading place and included a half of all typed pneumococci. Dynamic observation on the serotype composition of pneumococci revealed periodic fluctuations in the occurrence of some types/groups. The regional analysis of different serotypes of pneumococci showed the common occurrence of serogroups 6 and 19, as well as some regional features in the circulation of serotypes 6, 10, 3 and rarely occurring serotypes. The study revealed that any new exacerbation of the chronic bronchopulmonary process is caused by pneumococci of some other serotype. Pneumococcal strains, resistant (3.4%) and sensitive (1.8%) to penicillin, were detected; most of them belonged to serogroup 19.     

Specific gene probe for detection of biotyped and serotyped Listeria strains

A total of 284 strains of Listeria, including all known serovars and biovars together with Listeria grayi and Listeria murrayi, were biotyped and serotyped. Biotyping and serotyping could be done in 2 days. A gene probe encoding a delayed hypersensitivity factor (DTH) was used in the detection of pathogenic biotypes and serotypes of the tested strains. The gene was found in all 117 tested Listeria monocytogenes strains of serogroups 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4c, 4d, 4e, 4ab, and 7. It was also present in Listeria ivanovii. Of 78 L. monocytogenes strains of serogroup 4b, 77 strains contained the gene, whereas it was absent in all 10 tested L. monocytogenes strains of serogroup 4a. Furthermore, the gene was absent in Listeria seeligeri, L. grayi, L. murrayi, and L. innocua of serogroups 3c, 4b, and 6a and in L. welshimeri of serogroups 1/2b, 3b, 6a, and 6b. Since L. monocytogenes and L. ivanovii are the only two biotypes of the genus Listeria considered pathogens, the data obtained indicate that the DNA probe tested may be a useful tool in the detection of virulent Listeria isolates in clinical, environmental, and food samples.     

Immunological analysis of extracellular products and cell surface components of motile Aeromonas isolated from fish

The present work describes the characterization of antigens present in the extracellular products (ECP) and cell wall of strains of motile Aeromonas isolated from rainbow trout culture systems. The relationships among virulence for fish, O-serogroup and profile of LPS were also examined. The slide agglutination test showed that most of the virulent strains of motile Aeromonas (72%) were included in the serotypes O3, O6, O11 and O19 (Guinée and Jansen System). However, there were also non-pathogenic strains within these groups. Electrophoretic analysis of lipopolysaccharides (LPS) and proteins from cell envelope and ECP showed heterogeneity not only among the different serogroups but also within the same serotype. Immunoblot assays of cell envelope components, and of LPS present in the ECP demonstrated a close relationship among Aeromonas strains from the same serotype, while strains from different serotypes were not immunologically related. Moreover, this assay showed that motile Aeromonas belonging to distinct serotypes produced extracellular proteins immunologically related. On the other hand, antigenic cross reactivity was observed between the LPS obtained from cell envelope and those obtained from the ECP. The present results point out the need to include strains representative of each of the serotypes which predominates in a particular area and their ECPs in the formation of vaccines against motile Aeromonas septicaemia.     

Specific gene probe for detection of biotyped and serotyped Listeria strains.

A total of 284 strains of Listeria, including all known serovars and biovars together with Listeria grayi and Listeria murrayi, were biotyped and serotyped. Biotyping and serotyping could be done in 2 days. A gene probe encoding a delayed hypersensitivity factor (DTH) was used in the detection of pathogenic biotypes and serotypes of the tested strains. The gene was found in all 117 tested Listeria monocytogenes strains of serogroups 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4c, 4d, 4e, 4ab, and 7. It was also present in Listeria ivanovii. Of 78 L. monocytogenes strains of serogroup 4b, 77 strains contained the gene, whereas it was absent in all 10 tested L. monocytogenes strains of serogroup 4a. Furthermore, the gene was absent in Listeria seeligeri, L. grayi, L. murrayi, and L. innocua of serogroups 3c, 4b, and 6a and in L. welshimeri of serogroups 1/2b, 3b, 6a, and 6b. Since L. monocytogenes and L. ivanovii are the only two biotypes of the genus Listeria considered pathogens, the data obtained indicate that the DNA probe tested may be a useful tool in the detection of virulent Listeria isolates in clinical, environmental, and food samples.     

Diarrheagenic Escherichia coli categories among the traditional enteropathogenic E. coli O serogroups--a review

The so called enteropathogenic Escherichia coli (EPEC) O serogroups include typical and atypical EPEC, enterohaemorrragic E. coli, enterotoxigenic E. coli, and enteroaggregative E. coli. The aim of this article is to review the composition of each O serogroup and the major serotypes, clones, and additional virulence characteristics of each of these diarrheagenic categories. Their adherence patterns and genetic relationships are also presented. The review is based on the study of 805 strains of serogroups O26, O55, O86, O111, O114, O119, O125, O126, O1127, O128, and O142 most of which isolated in Sao Paulo from children with diarrhea between 1970 and 1990. Since some O serogroups include more than one diarrheagenic category O serogrouping only should be abandoned as a diagnostic method. However serotyping is a reliable method for those serotypes that correspond to clones.