Shigatoxigenic Escherichia coli serogroups O157, O111 and O113 in feces, water and milk samples from dairy farms

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

LACK OF VIRULENCE FACTORS IN ESCHERICHIA-COLI STRAINS OF ENTEROPATHOGENIC SEROGROUPS ISOLATED FROM WATER

Thirty-eight Escherichia coli strains belonging to 14 human enteropathogenic serogroups were isolated from 33 of 208 water samples studied. No virulence factor or virulence-related gene sequences were found in any of the 38 strains analyzed. The results point out the importance of detecting specific virulence factors before incriminating water as a source of human diarrhea.

O-serogroups, eae gene and EAF plasmid in Escherichia coli isolates from cases of bovine mastitis in Brazil

Mastitis has been recognized for some time as the most costly disease in dairy herds. From March 1997 to August 1998. 2144 samples of bovine mastitic milk were collected, from which 182 Escherichia coli isolates were made, and from which 14 1 isolates had the somatic anti-en (serogroup) determined. Twelve different serogroups were isolated from mastitic milk, and among them were 026, 055, 0111 and 0 119, all of them classic enteropathogenic E. (oh (EPEC) serogroups. These represented 40.0% of the isolates. The 20 of 57 isolates tested had plasmids and in dot blot hybridization, nine isolates were positive for an EaeA probe and an EPEC adherence factor (EAF) probe while two isolates were negative for EaeA probe but positive for the EAF probe, the nine isolates were characterized as attaching and effacing (A/E) E, coli (AEEC) isolates. (C) 2002 Elsevier B.V. B.V. All rights reserved.

Clonal relationship among invasive and non-invasive strains of enteroinvasive Escherichia coli serogroups

The genetic relatedness among 96 invasive Escherichia coli belonging to several serogroups and 13 non-invasive of several serotypes that share the same O antigen was investigated by multilocus enzyme electrophoresis analysis. The invasive strains were isolated in different parts of the world and most of them recovered from dysentery. Twenty-nine electrophoretic types were distinguished and the most invasive strains were found to belong to two major lineages. These results suggested that the invasive ability in these strains has evolved in divergent chromosomal backgrounds, presumably through the horizontal spread of plasmid-borne invasion genes. The maintenance of invasive phenotypes in separate lineages suggests that this ability confers a selective advantage to invasive strains. Copyright (C) 1999 Federation of European Microbiological Societies.

Overlapped Sequence Types (STs) and Serogroups of Avian Pathogenic (APEC) and Human Extra-Intestinal Pathogenic (ExPEC) Escherichia coli Isolated in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Pathotypes and serogroups of enterotoxigenic Escherichia coli isolated from pre-weaning pigs in north Vietnam

The contribution of enterotoxigenic Escherichia coli (ETEC) to pre-weaning diarrhoea was investigated over a 6 month period at five selected commercial piggeries (CPs) in north Vietnam with at least 100 sows each. Diarrhoea was found to affect 71(.)5% of the litters born during the period of study. Of 406 faecal specimens submitted for bacteriological culture, 200 (49(.)3%) yielded a heavy pure culture of E coli and 126(31 %)were confirmed by PCR to carry at least one of eight porcine ETEC virulence genes. ETEC was responsible for 43% of cases of diarrhoea in neonatal pigs during the first 4 days of life and 23(.)9% of the remaining cases up until the age of weaning. Pathotypes were determined by PCR for the 126 ETEC isolates together with 44 ETEC isolates obtained from village pigs (VPs) raised by smallholder farmers. The CP isolates belonged to five pathotypes, four of which were also identified in VP isolates. Haemolytic serogroup O149: K91 isolates that belonged to F4/STa/STb/LT were most commonly identified in both CPs (33 % of isolates) and VPs (45(.)5%). Other combinations identified in both production systems included O64 (F5/STa), O101 (F4/STa/STb) and O-nontypable (F-/STb). A high proportion of CP isolates (22(.)3 %) possessed all three enterotoxins (STa/STWLT), lacked the genes for all five tested fimbriae (F4, F5, F6, F41 and F18) and belonged to serogroup O8. These unusual 08 F- isolates were haemolytic and were isolated from all ages of diarrhoeic piglets at each CP, suggesting that they have pathogenic potential.

Cross-protection study of the nine serovars of Haemophilus paragallinarum in the Kume haemagglutinin scheme

The cross-protection and haemagglutination-inhibition antibodies present in chickens vaccinated with one of the nine currently recognized Kume haemagglutinin serovars of Haemophilus paragallinarum were investigated. The results confirmed the widely accepted dogma that serogroups A, B, and C represent three distinct immunovars. Within Kume serogroup A, there was generally good cross-protection among all four serovars. However, within Kume serogroup C, there was evidence of a reduced level of cross protection between some of the four serovars. The haemagglutination-inhibition antibody levels generally showed the same trend as with the cross-protection results. This study suggests that some apparent field failures of infectious coryza vaccines may be due to a lack of cross-protection between the vaccine strains and the field strains. Our results will help guide the selection of strains for inclusion in infectious coryza vaccines.

Progressive Decrease in the Potential Usefulness of Meningococcal Serogroup B Vaccine (4CMenB, Bexsero (R)) in Gipuzkoa, Northern Spain

The effectiveness of a vaccine is determined not only by the immunogenicity of its components, but especially by how widely it covers the disease-causing strains circulating in a given region. Because vaccine coverage varies over time, this study aimed to detect possible changes that could affect vaccine protection during a specific period in a southern European region. The 4CMenB vaccine is licensed for use in Europe, Canada, and Australia and is mainly directed against Neisseria meningitidis serogroup B. This vaccine contains four main immunogenic components: three recombinant proteins, FHbp, Nhba and NadA, and an outer membrane vesicle [PorA P1.4]. The allelic distribution of FHbp, Nhba, NadA, and PorA antigens in 82 invasive isolates (B and non-B serogroups) isolated from January 2008 to December 2013 were analyzed. 4CMenB was likely protective against 61.8% and 50% of serogroup B and non-B meningococci, respectively, in the entire period, but between 2012 and 2013, the predicted protection fell below 45% (42.1% for serogroup B isolates). The observed decreasing trend in the predicted protection during the 6 years of the study (X-2 for trend = 4.68, p=0.03) coincided with a progressive decrease of several clonal complexes (e. g., cc11, cc32 and cc41/44), which had one or more antigens against which the vaccine would offer protection.

Leptospirosis among hospitalized febrile patients in northern Tanzania.

We enrolled consecutive febrile admissions to two hospitals in Moshi, Tanzania. Confirmed leptospirosis was defined as a ≥ 4-fold increase in microscopic agglutination test (MAT) titer; probable leptospirosis as reciprocal MAT titer ≥ 800; and exposure to pathogenic leptospires as titer ≥ 100. Among 870 patients enrolled in the study, 453 (52.1%) had paired sera available, and 40 (8.8%) of these met the definition for confirmed leptospirosis. Of 832 patients with ≥ 1 serum sample available, 30 (3.6%) had probable leptospirosis and an additional 277 (33.3%) had evidence of exposure to pathogenic leptospires. Among those with leptospirosis the most common clinical diagnoses were malaria in 31 (44.3%) and pneumonia in 18 (25.7%). Leptospirosis was associated with living in a rural area (odds ratio [OR] 3.4, P < 0.001). Among those with confirmed leptospirosis, the predominant reactive serogroups were Mini and Australis. Leptospirosis is a major yet underdiagnosed cause of febrile illness in northern Tanzania, where it appears to be endemic.

Differential induction, isolation, physicochemical and molecular characterization of temperate phages of environmental Vibrio cholerae as evidence of phage mediated Horizontal Gene Transfer

The resurgence of the enteric pathogen Vibrio cholerae, the causative organism of epidemic cholera, remains a major health problem in many developing countries like India. The southern Indian state of Kerala is endemic to cholera. The outbreaks of cholera follow a seasonal pattern in regions of endemicity. Marine aquaculture settings and mangrove environments of Kerala serve as reservoirs for V. cholerae. The non-O1/non-O139 environmental isolates of V. cholerae with incomplete ‘virulence casette’ are to be dealt with caution as they constitute a major reservoir of diverse virulence genes in the marine environment and play a crucial role in pathogenicity and horizontal gene transfer. The genes coding cholera toxin are borne on, and can be infectiously transmitted by CTXΦ, a filamentous lysogenic vibriophages. Temperate phages can provide crucial virulence and fitness factors affecting cell metabolism, bacterial adhesion, colonization, immunity, antibiotic resistance and serum resistance. The present study was an attempt to screen the marine environments like aquafarms and mangroves of coastal areas of Alappuzha and Cochin, Kerala for the presence of lysogenic V. cholerae, to study their pathogenicity and also gene transfer potential. Phenotypic and molecular methods were used for identification of isolates as V. cholerae. The thirty one isolates which were Gram negative, oxidase positive, fermentative, with or without gas production on MOF media and which showed yellow coloured colonies on TCBS (Thiosulfate Citrate Bile salt Sucrose) agar were segregated as vibrios. Twenty two environmental V. cholerae strains of both O1 and non- O1/non-O139 serogroups on induction with mitomycin C showed the presence of lysogenic phages. They produced characteristic turbid plaques in double agar overlay assay using the indicator strain V. cholerae El Tor MAK 757. PCR based molecular typing with primers targeting specific conserved sequences in the bacterial genome, demonstrated genetic diversity among these lysogen containing non-O1 V. cholerae . Polymerase chain reaction was also employed as a rapid screening method to verify the presence of 9 virulence genes namely, ctxA, ctxB, ace, hlyA, toxR, zot,tcpA, ninT and nanH, using gene specific primers. The presence of tcpA gene in ALPVC3 was alarming, as it indicates the possibility of an epidemic by accepting the cholera. Differential induction studies used ΦALPVC3, ΦALPVC11, ΦALPVC12 and ΦEKM14, underlining the possibility of prophage induction in natural ecosystems, due to abiotic factors like antibiotics, pollutants, temperature and UV. The efficiency of induction of prophages varied considerably in response to the different induction agents. The growth curve of lysogenic V. cholerae used in the study drastically varied in the presence of strong prophage inducers like antibiotics and UV. Bacterial cell lysis was directly proportional to increase in phage number due to induction. Morphological characterization of vibriophages by Transmission Electron Microscopy revealed hexagonal heads for all the four phages. Vibriophage ΦALPVC3 exhibited isometric and contractile tails characteristic of family Myoviridae, while phages ΦALPVC11 and ΦALPVC12 demonstrated the typical hexagonal head and non-contractile tail of family Siphoviridae. ΦEKM14, the podophage was distinguished by short non-contractile tail and icosahedral head. This work demonstrated that environmental parameters can influence the viability and cell adsorption rates of V. cholerae phages. Adsorption studies showed 100% adsorption of ΦALPVC3 ΦALPVC11, ΦALPVC12 and ΦEKM14 after 25, 30, 40 and 35 minutes respectively. Exposure to high temperatures ranging from 50ºC to 100ºC drastically reduced phage viability. The optimum concentration of NaCl required for survival of vibriophages except ΦEKM14 was 0.5 M and that for ΦEKM14 was 1M NaCl. Survival of phage particles was maximum at pH 7-8. V. cholerae is assumed to have existed long before their human host and so the pathogenic clones may have evolved from aquatic forms which later colonized the human intestine by progressive acquisition of genes. This is supported by the fact that the vast majority of V. cholerae strains are still part of the natural aquatic environment. CTXΦ has played a critical role in the evolution of the pathogenicity of V. cholerae as it can transmit the ctxAB gene. The unusual transformation of V. cholerae strains associated with epidemics and the emergence of V. cholera O139 demonstrates the evolutionary success of the organism in attaining greater fitness. Genetic changes in pathogenic V. cholerae constitute a natural process for developing immunity within an endemically infected population. The alternative hosts and lysogenic environmental V. cholerae strains may potentially act as cofactors in promoting cholera phage ‘‘blooms’’ within aquatic environments, thereby influencing transmission of phage sensitive, pathogenic V. cholerae strains by aquatic vehicles. Differential induction of the phages is a clear indication of the impact of environmental pollution and global changes on phage induction. The development of molecular biology techniques offered an accessible gateway for investigating the molecular events leading to genetic diversity in the marine environment. Using nucleic acids as targets, the methods of fingerprinting like ERIC PCR and BOX PCR, revealed that the marine environment harbours potentially pathogenic group of bacteria with genetic diversity. The distribution of virulence associated genes in the environmental isolates of V. cholerae provides tangible material for further investigation. Nucleotide and protein sequence analysis alongwith protein structure prediction aids in better understanding of the variation inalleles of same gene in different ecological niche and its impact on the protein structure for attaining greater fitness of pathogens. The evidences of the co-evolution of virulence genes in toxigenic V. cholerae O1 from different lineages of environmental non-O1 strains is alarming. Transduction studies would indicate that the phenomenon of acquisition of these virulence genes by lateral gene transfer, although rare, is not quite uncommon amongst non-O1/non-O139 V. cholerae and it has a key role in diversification. All these considerations justify the need for an integrated approach towards the development of an effective surveillance system to monitor evolution of V. cholerae strains with epidemic potential. Results presented in this study, if considered together with the mechanism proposed as above, would strongly suggest that the bacteriophage also intervenes as a variable in shaping the cholera bacterium, which cannot be ignored and hinting at imminent future epidemics.

Biofilm formation as a novel phenotypic feature of adherent-invasive Escherichia coli (AIEC)

Crohn's disease (CD) is a high morbidity chronic inflammatory disorder of unknown aetiology. Adherent-invasive Escherichia coli (AIEC) has been recently implicated in the origin and perpetuation of CD. Because bacterial biofilms in the gut mucosa are suspected to play a role in CD and biofilm formation is a feature of certain pathogenic E. coli strains, we compared the biofilmformation capacity of 27 AIEC and 38 non-AIEC strains isolated from the intestinal mucosa. Biofilmformation capacity was then contrasted with the AIEC phenotype, the serotype, the phylotype, andthe presence of virulence genes. Results: Specific biofilm formation (SBF) indices were higher amongst AIEC than non-AIEC strains(P = 0.012). In addition, 65.4% of moderate to strong biofilms producers were AIEC, whereas74.4% of weak biofilm producers were non-AIEC (P = 0.002). These data indicate that AIEC strainswere more efficient biofilm producers than non-AIEC strains. Moreover, adhesion (P = 0.009) andinvasion (P = 0.003) indices correlated positively with higher SBF indices. Additionally, motility(100%, P < 0.001), H1 type flagellin (53.8%, P < 0.001), serogroups O83 (19.2%, P = 0.008) and O22(26.9%, P = 0.001), the presence of virulence genes such as sfa/focDE (38.5%, P = 0.003) and ibeA(26.9%, P = 0.017), and B2 phylotype (80.8%, P < 0.001) were frequent characteristics amongstbiofilm producers.Conclusion: The principal contribution of the present work is the finding that biofilm formationcapacity is a novel, complementary pathogenic feature of the recently described AIEC pathovar. Characterization of AIEC specific genetic determinants, and the regulatory pathways, involved in biofilm formation will likely bring new insights into AIEC pathogenesis

Interaction between attaching-effacing Escherichia coli O26:K60 and O157:H7 in young lambs

Recent surveys have shown that Escherichia coli O26 is prevalent in ruminants compared with E. coli O157. These serogroups share common colonisation factors and we hypothesised that prior colonisation by E. coli O26 may show reduced colonisation by E. coli O157. To test this hypothesis, strains of E. coli O26:K6O and O157:H7 were tested in competitive in vitro and in vivo studies. Using an established 6-week-old lamb model, an experimental group of lambs was dosed orally with E. coli O26:K6O and then E. coli O157:147 four days later. The faecal shedding of O26:K6O and O157:H7 organisms from this experimental group was compared with that from animals dosed with either O26:K6O alone or O157:H7 alone. Shedding data indicated that counts for O157:H7 were unaffected by the competition from O26:K6O, whereas the O26:K6O counts were lower when competing with O157:H7. (C) 2008 Elsevier Ltd. All rights reserved.