Kegg Pathway: Pathogenic Escherichia coli infection - EPEC

J Bacteriol. 2009 Nov 6;
Petty NK, Bulgin R, Crepin VF, Cerdeño-Tárraga AM, Schroeder GN, Quail MA, Lennard N, Corton C, Barron A, Clark L, Toribio AL, Parkhill J, Dougan G, Frankel G, Thomson NR

Citrobacter rodentium (formally C. freundii Biotype 4280) is a highly infectious pathogen that causes colitis and transmissible colonic hyperplasia in mice. In common with enteroPathogenic and enterhemorrhagic Escherichia coli (EPEC and EHEC respectively), C. rodentium exploits a type III secretion system (T3SS) to induce attaching and effacing (A/E) lesions that are essential for virulence. Here we report the fully annotated genome sequence of the 5.3 Mb chromosome and four plasmids harboured by C. rodentium strain ICC168. The genome sequence revealed key information about the phylogeny of C. rodentium and identified 1585 C. rodentium-specific (without orthologues in EPEC or EHEC) coding sequences, 10 prophage-like regions and 17 genomic islands, including the LEE region, which encodes a T3SS and effector proteins. Among the 29 T3SS effectors found in C. rodentium are all the core 22 effectors of EPEC strain E2348/69. In addition, we identified a novel C. rodentium effector, named EspS. C. rodentium harbours two type VI secretion systems (T6SS) (CTS1 and CTS2), while EHEC contains only one T6SS (EHS). Our analysis suggests that C. rodentium and EPEC/EHEC have converged on a common host infection strategy through access to a common pool of mobile DNA, and that C. rodentium has lost gene functions associated with a previous Pathogenic niche.

Can J Microbiol. 2009 Jun; 55(6): 672-9
Pitondo-Silva A, Minarini LA, Camargo IL, Darini AL

EnteroPathogenic Escherichia coli (EPEC) infections are a leading cause of infantile diarrhea in developing nations. Multilocus sequence typing (MLST) characterizes bacterial strains based on the sequences of internal fragments in housekeeping genes. Little is known about strains of EPEC analyzed by MLST from Brazil. In this study, a diverse collection of 29 EPEC strains isolated from patients with diarrhea, admitted to the University Hospital of Ribeirao Preto, was characterized by MLST. Strain analysis demonstrated 22 different sequence types (STs), of which almost half (48%) were new, indicating a high genotype diversity. The 22 STs were divided by eBURST into 12 clonal complexes. It was not possible to correlate typical and atypical EPEC with other strains in the MLST database. This is the first study that analyzed EPEC strains from South America that are included in the E. coli MLST database. Nine (31%) out of 29 strains are part of the CC10 clonal complex, the major clonal complex in the database, which comprises 174 strains and 86 different STs, suggesting that these strains might be the most important intestinal Pathogenic E. coli worldwide. Genetic relationships between typical and atypical EPEC, enterohemorrhagic E. coli, and enteroaggregative E. coli strains were not established by MLST.

Int J Food Microbiol. 2009 Sep 15; 134(3): 196-200
Lee GY, Jang HI, Hwang IG, Rhee MS

Foodborne diseases occur worldwide, including through the consumption of contaminated meat. This study was conducted to investigate the prevalence of Escherichia coli contamination in fresh beef, poultry, and pork, and to determine whether any isolated E. coli possessed genes associated with Pathogenicity. Three thousand meat samples were collected from 2004 to 2006 and were tested for the presence of E. coli. Two hundred and seventy-three E. coli isolates were obtained from beef, poultry, and pork, resulting in an overall isolation rate of 9.1%. Of these isolates, 201 were obtained from 1350 pork samples (14.9%), followed by 41 of 900 poultry samples (4.6%) and 31 of 750 beef samples (4.1%). A total of 39 Pathogenic E. coli isolates from the three meat types were categorized into three virulence groups, namely enterotoxigenic E. coli (43.6%), enterohemorrhagic E. coli (EHEC) (35.9%; 22.6% of beef, 7.3% of poultry, and 2.0% of pork), and enteroPathogenic E. coli (20.5%). Fourteen strains were identified as belonging to the EHEC, which included O18, O136, O119, O86, O8, O111, O15, O128, and O6. This study demonstrated that Pathogenic E. coli are found in meat in Korea, and could act as a transmission vehicle for human infection as suggested by the occurrence and classification of Pathogenic E. coli in retail meats. Furthermore, the data from this study could be used in the risk assessment of foodborne illnesses linked to meat consumption.

Jpn J Infect Dis. 2009 Jul; 62(4): 289-93
Usein CR, Tatu-Chitoiu D, Ciontea S, Condei M, Damian M

To document the association of Pathogenic Escherichia coli with diarrhea in Romanian children, 250 E. coli fecal isolates from children under 5 years of age were PCR-screened for well-recognized virulence determinants, as well as for their phylogenetic background. The putative diarrheagenic E. coli (DEC) were investigated for susceptibility to various antibiotics. Overall, 61 E. coli isolates were classified as enteroaggregative E. coli (29 isolates), atypical enteroPathogenic E. coli (22 isolates), enterotoxigenic E. coli (8 isolates), and verotoxin-producing E. coli (1 isolate), and one isolate was categorized as unconventional DEC. Only 8 of the PCR-positive isolates would have been assumed to be Pathogenic based on their O antigenicity, which highlights the limited effectiveness of serotyping. More than a half (51%) of the Pathogenic isolates expressed a multidrug-resistant phenotype, which raises concerns about the therapeutic pediatric approach. The DEC isolates were heterogeneous phylogenetically, deriving from all four major groups: A (31 isolates), B2 (14 isolates), B1 (10 isolates), and D (6 isolates), respectively. Thus, the phylogenetic descent was less significant than the virulence gene content. Our findings document the importance of DEC as a cause of childhood diarrhea in Romania, providing evidence that efforts should be made to estimate the burden of infections by etiology for a better medical approach.

Am J Physiol Gastrointest Liver Physiol. 2009 Jul 23;
Ashokkumar B, Kumar JS, Hecht GA, Said HM

infection with the gram-negative enteroPathogenic Escherichia coli (EPEC), a food-borne pathogen, represents a significant risk to human health. While diarrhea is a major consequence of this infection, mal-nutrition also occurs especially in severe and prolonged cases, which may aggravate the health status of the infected hosts. Here we examined the effect of EPEC infection on the intestinal uptake of the water-soluble vitamin B1 (thiamin) using an established human intestinal epithelial Caco-2 cell model. The results showed that infecting Caco-2 cells with wild-type EPEC (but not with non-Pathogenic E. coli, killed EPEC or filtered supernatant) leads to a significant (P < 0.01) inhibition in thiamin uptake. Kinetic parameters of both the nanomolar (mediated by THTR-2) and the micromolar (mediated by THTR-1) saturable thiamin uptake processes were affected by EPEC infection. Cell surface expression of hTHTR-1 & 2 proteins, (determined by biotinylation method) showed a significantly (P < 0.01) lower expression in EPEC- treated cells compared to controls. EPEC infection also affected the steady state mRNA levels as well as promoter activity of the SLC19A2 and SLC19A3 genes. Infecting Caco-2 cells with EPEC mutants that harbor mutations in the escN gene (which encodes a putative ATPase for the EPEC type III secretion system, TTSS), or the espA, espB, or espD genes (which encode structural components of the TTSS) did not affect thiamin uptake. On the other hand, mutations in espF and espH genes (which encode effector proteins) exhibited partial inhibition in thiamin uptake. These results demonstrate for the first time that EPEC infection of human intestinal epithelial cells leads to inhibition in thiamin uptake via effects on physiological and molecular parameters of hTHTR-1 & 2. Further, the inhibition appears to be dependent on a functional TTSS of EPEC. Key words: Thiamin transport, hTHTR-1, hTHTR-2, EPEC.

Zh Mikrobiol Epidemiol Immunobiol. 2009 May-Jun; 98-100
Utemuradova GE

AIM: To study etiologic structure of acute enteric infections (AEI) with isolation of hemolytic and enteroPathogenic Escherichia and to assess the efficacy of probiotic therapy during infections caused by E. coli. MATERIALS AND METHODS: Bacteriologic tests were performed in 245 children < 3 years old with acute enteric infection. Influence of colibacterin and Bifidumbacterin preparations on the dynamics of infectious process was assessed. RESULTS: Hemolytic Escherichia, enteroPathogenic E. coli, Shigella spp., and Salmonella spp. were isolated in 47.3%, 12.2%, 18.3%, and 1.8% of patients with AEI, respectively. The cause of AEI was identified in 77.1% of cases. Bifidumbacterin had good therapeutic effect on infections caused by Pathogenic E. coli including its hemolytic forms. CONCLUSION: Treatment with Bifidumbacterin improved state of the patients and resulted in suppression of growth of hemolytic Escherichia in the gut. colibacterin had a good therapeutic effect in the group of patients with domination of staphylococci, enterococci, Proteus in the gut as well as in patients with isolated Shigella and Salmonella. colibacterin is contraindicated in cases of colonization of the intestine by hemolytic E. coli.

Infect Immun. 2009 Sep; 77(9): 3639-50
Borenshtein D, Schlieper KA, Rickman BH, Chapman JM, Schweinfest CW, Fox JG, Schauer DB

Citrobacter rodentium causes epithelial hyperplasia and colitis and is used as a model for enteroPathogenic and enterohemorrhagic Escherichia coli infections. Little or no mortality develops in most inbred strains of mice, but C3H and FVB/N mice exhibit fatal outcomes of infection. Here we test the hypothesis that decreased intestinal transport activity during C. rodentium infection results in fatality in C3H/HeOu and FVB/N mice. Susceptible strains were compared to resistant C57BL/6 mice and to inbred strains SWR and SJL of Swiss origin, which have not been previously characterized for outcomes of C. rodentium infection. Mortality in susceptible strains C3H/HeOu and FVB/N was associated with significant fluid loss in feces, a remarkable downregulation of Slc26a3 and carbonic anhydrase IV (CAIV) message and protein expression, retention of chloride in stool, and hypochloremia, suggesting defects in intestinal chloride absorption. SWR, SJL, and C57BL/6 mice were resistant and survived the infection. Fluid therapy fully prevented mortality in C3H/HeOu and FVB/N mice without affecting clinical disease. Common Pathogenic mechanisms, such as decreased levels of expression of Slc26a3 and CAIV, affect intestinal ion transport in C. rodentium-infected FVB and C3H mice, resulting in profound electrolyte loss, dehydration, and mortality. Intestinal chloride absorption pathways are likely a potential target for the treatment of infectious diarrhea.

FEMS Microbiol Lett. 2009 Aug; 297(2): 137-49
Hernandes RT, Elias WP, Vieira MA, Gomes TA

The enteroPathogenic Escherichia coli (EPEC) pathotype is currently divided into two groups, typical EPEC (tEPEC) and atypical EPEC (aEPEC). The property that distinguishes these two groups is the presence of the EPEC adherence factor plasmid, which is only found in tEPEC. aEPEC strains are emerging enteropathogens that have been detected worldwide. Herein, we review the serotypes, virulence properties, genetic relationships, epidemiology, reservoir and diagnosis of aEPEC, including those strains not belonging to the classical EPEC serogroups (nonclassical EPEC serogroups). The large variety of serotypes and genetic virulence properties of aEPEC strains from nonclassical EPEC serogroups makes it difficult to determine which strains are truly Pathogenic.

Curr Microbiol. 2009 Sep; 59(3): 302-8
Silva VL, Nicoli JR, Nascimento TC, Diniz CG

Urban pigeons (Columba livia) come into close contact with humans and animals, and may contribute to the spread of infectious agents. These may include human pathogens such as diarrheagenic Escherichia coli strains, which are able to survive in pigeon feces, thus creating potential for human exposure and infection. Our objectives were to determine the occurrence of diarrheagenic E. coli strains in fresh feces from urban pigeons and their drug susceptibility patterns. E. coli strains were isolated from 100 fresh feces samples and presumptive phenotypic species identification was carried out, confirmed by amplification of specific 16S ribosomal RNA encoding DNA. Multiplex PCR was performed to characterize Pathogenic strains. Drug susceptibility patterns were determined by the agar dilution method. Enteroinvasive E. coli, Shiga toxin-producing E. coli, enteroPathogenic E. coli, and enterotoxigenic E. coli were detected at an overall rate of 12.1%. Among the isolated E. coli strains, 62.1% were susceptible to all tested drugs, whereas 37.9% were resistant to at least one of the antimicrobials tested. Amikacin was the less effective drug (36.8% resistance), followed by ampicillin (7.8%). No resistance was detected to gentamicin, ceftriaxone, and ceftazidime and almost all the isolates were susceptible to ampicillin-sulbactam (98.4%), levofloxacin (97.8%), and trimethoprim-sulfamethoxazole (96.1%). Since these pigeons may harbor multidrug-resistant pathogens, their presence in an urban environment could be an important component of infection spread, with impact on public health.

Nature. 2009 May 28; 459(7246): 578-82
Kim M, Ogawa M, Fujita Y, Yoshikawa Y, Nagai T, Koyama T, Nagai S, Lange A, Fässler R, Sasakawa C

The rapid turnover and exfoliation of mucosal epithelial cells provides an innate defence system against bacterial infection. Nevertheless, many Pathogenic bacteria, including Shigella, are able to surmount exfoliation and colonize the epithelium efficiently. Here we show that the Shigella flexneri effector OspE (consisting of OspE1 and OspE2 proteins), which is highly conserved among enteroPathogenic Escherichia coli, enterohaemorrhagic E. coli, Citrobacter rodentium and Salmonella strains, reinforces host cell adherence to the basement membrane by interacting with integrin-linked kinase (ILK). The number of focal adhesions was augmented along with membrane fraction ILK by ILK-OspE binding. The interaction between ILK and OspE increased cell surface levels of 1 integrin and suppressed phosphorylation of focal adhesion kinase and paxillin, which are required for rapid turnover of focal adhesion in cell motility. Nocodazole-washout-induced focal adhesion disassembly was blocked by expression of OspE. Polarized epithelial cells infected with a Shigella mutant lacking the ospE gene underwent more rapid cell detachment than cells infected with wild-type Shigella. infection of guinea pig colons with Shigella corroborated the pivotal role of the OspE-ILK interaction in suppressing epithelial detachment, increasing bacterial cell-to-cell spreading, and promoting bacterial colonization. These results indicate that Shigella sustain their infectious foothold by using special tactics to prevent detachment of infected cells.

Cell Microbiol. 2009 Aug; 11(8): 1254-71
Brandt S, Kenny B, Rohde M, Martinez-Quiles N, Backert S

Many Gram-negative Pathogenic bacteria possess type-III or type-IV secretion systems to inject 'effector' proteins directly into host cells to modulate cellular processes in their favour. A common target is the actin-cytoskeleton linked to the delivery of a single (CagA) effector by Helicobacter pylori and multiple effectors by enteroPathogenic Escherichia coli (EPEC) respectively. Here we report co-infection as a powerful strategy for defining effector protein function and mechanisms by which they modulate cellular responses. This is exemplified by our finding that EPEC inhibits H. pylori-induced AGS cell elongation, a disease-related event linked to Rac1 activation. While this inhibitory process is dependent on the translocated Intimin receptor, Tir, and the outer-membrane protein, Intimin, it unexpectedly revealed evidence for Tir signalling independent of Intimin interaction and tyrosine phosphorylation of Tir. Furthermore, the work defined a long awaited role for protein kinase A (PKA)-mediated phosphorylation of Tir at serine-434 and serine-463. Our data are consistent with a model whereby EPEC activates PKA for Tir phosphorylation. Activated PKA then phosphorylates Rac1 at serine-71 associated with reduced GTP-load and inhibited cell elongation. Thus, the co-infection approach is a powerful strategy for defining novel effector function with important implications for characterizing mechanisms and regulatory signalling pathways in bacterial pathogenesis.

Nature. 2009 May 6;
Kim M, Ogawa M, Fujita Y, Yoshikawa Y, Nagai T, Koyama T, Nagai S, Lange A, Fässler R, Sasakawa C

The rapid turnover and exfoliation of mucosal epithelial cells provides an innate defence system against bacterial infection. Nevertheless, many Pathogenic bacteria, including Shigella, are able to surmount exfoliation and colonize the epithelium efficiently. Here we show that the Shigella flexneri effector OspE (consisting of OspE1 and OspE2 proteins), which is highly conserved among enteroPathogenic Escherichia coli, enterohaemorrhagic E. coli, Citrobacter rodentium and Salmonella strains, reinforces host cell adherence to the basement membrane by interacting with integrin-linked kinase (ILK). The number of focal adhesions was augmented along with membrane fraction ILK by ILK-OspE binding. The interaction between ILK and OspE increased cell surface levels of beta1 integrin and suppressed phosphorylation of focal adhesion kinase and paxillin, which are required for rapid turnover of focal adhesion in cell motility. Nocodazole-washout-induced focal adhesion disassembly was blocked by expression of OspE. Polarized epithelial cells infected with a Shigella mutant lacking the ospE gene underwent more rapid cell detachment than cells infected with wild-type Shigella. infection of guinea pig colons with Shigella corroborated the pivotal role of the OspE-ILK interaction in suppressing epithelial detachment, increasing bacterial cell-to-cell spreading, and promoting bacterial colonization. These results indicate that Shigella sustain their infectious foothold by using special tactics to prevent detachment of infected cells.

Lett Appl Microbiol. 2009 Jul; 49(1): 53-9
Vettorato MP, de Castro AF, Cergole-Novella MC, Camargo FL, Irino K, Guth BE

AIMS: Sheep are important carriers of Shiga toxin-producing Escherichia coli (STEC) in several countries. However, there are a few reports about ovine STEC in American continent. METHODS AND RESULTS: About 86 E. coli strains previously isolated from 172 healthy sheep from different farms were studied. PCR was used for detection of stx(1), stx(2), eae, ehxA and saa genes and for the identification of intimin subtypes. Restriction fragment length polymorphism (RFLP)-PCR was performed to investigate the variants of stx(1) and stx(2), and the flagellar antigen (fliC) genes in nonmotile isolates. Five isolates were eae(+) and stx(-), and belonged to serotypes O128:H2/beta-intimin (2), O145:H2/gamma, O153:H7/beta and O178:H7/epsilon. Eighty-one STEC isolates were recovered, and the stx genotypes identified were stx(1c)stx(2d-O118) (46.9%), stx(1c) (27.2%), stx(2d-O118) (23.4%), and stx(1c)stx(2dOX3a) (2.5%). Pulsed-field gel electrophoresis (PFGE) revealed 27 profiles among 53 STEC and atypical enteroPathogenic Escherichia coli (EPEC) isolates. CONCLUSIONS: This study demonstrated that healthy sheep in São Paulo, Brazil, can be carriers of potential human Pathogenic STEC and atypical EPEC. SIGNIFICANCE AND IMPACT OF THE STUDY: As some of the STEC serotypes presently found have been involved with haemolytic uraemic syndrome (HUS) in other countries, the important role of sheep as sources of STEC infection in our settings should not be disregarded.

Cell Host Microbe. 2009 Mar 19; 5(3): 244-58
Weiss SM, Ladwein M, Schmidt D, Ehinger J, Lommel S, Städing K, Beutling U, Disanza A, Frank R, Jänsch L, Scita G, Gunzer F, Rottner K, Stradal TE

Actin pedestal formation by Pathogenic E. coli requires signaling by the bacterial intimin receptor Tir, which induces host cell actin polymerization mediated by N-WASP and the Arp2/3 complex. Whereas canonical enteroPathogenic E. coli (EPEC) recruit these actin regulators through tyrosine kinase signaling cascades, enterohemorrhagic E. coli (EHEC) O157:H7 employ the bacterial effector EspF(U) (TccP), a potent N-WASP activator. Here, we show that IRSp53 family members, key regulators of membrane and actin dynamics, directly interact with both Tir and EspF(U). IRSp53 colocalizes with EspF(U) and N-WASP in actin pedestals. In addition, targeting of IRSp53 is independent of EspF(U) and N-WASP but requires Tir residues 454-463, previously shown to be essential for EspF(U)-dependent actin assembly. Genetic and functional loss of IRSp53 abrogates actin assembly mediated by EHEC. Collectively, these data indentify IRSp53 family proteins as the missing host cell factors linking bacterial Tir and EspF(U) in EHEC pedestal formation.

Dtsch Med Wochenschr. 2009 Feb; 134(9): 417-20
Klapproth JM, Meyer F

INTRODUCTION: Gastrointestinal infections are a significant cause of diarrhea and a worldwide problem with annually one billion illnesses and 3 to 4 million deaths. Gram negative bacteria like EnteroPathogenic E. coli (EPEC) in developing countries and Enterohemorrhagic E. coli (EHEC) in the developed world are responsible for the majority of acute diarrheal episodes, especially among children less than three years of age. Pathogenic E. coli are supplied with an arsenal of effector proteins to modify and neutralize specific cellular functions of the host organism. METHODS: Based on personal and extensively published results we provide a selected overview of Gram negative virulence functions with a focus on lymphostatin. RESULTS: Lymphostatin is an effector protein encoded by lifA/efa-1 (lymphocyte inhibitory factor A/ EHEC factor for adherence). lifA/efa-1 and homologous genes have been identified in EPEC, EHEC, and mouse pathogen Citrobacter rodentium, as well as various Chlamydia strains. Multiple groups have shown that in various EHEC strains lifA/efa-1 is part of a larger Pathogenicity island responsible for increased virulence. Statistically, DNA Microarray analysis associated lifA/efa-1 as the single most imortant gene with diarrhea caused by EPEC. Interestingly, lifA/efa-1 encodes for two critical enzymatic activities that have been identified in other Pathogenic bacteria: glucosyltransferase- in Clostridium- and protease activity in Yersinia strains. In vitro studies identified lymphostatin as an effector protein with an immunosuppressive effect on peripheral blood and gastrointestinal mucosa T lymphocytes. Further, lymphostatin regulates the barrier function of epithelial monolayer cultures: activation of small GTPase RhoA and inhibition of Cdc42 lead to disassembly of adherens junctions and tight junctions, respectively. Besides an effect on immune and epithelial barrier function, lymphostatin also functions as an adhesion factor for EPEC and EHEC, is essential for colonization of mouse and calf intestine, and regulates bacterial effector proteins. SUMMARY: Lymphostatin is a common toxin in Gram negative bacteria with multiple functions: cell adhesion, immunosuppression, disruption of epithelial barrier function, and intestinal colonization.

J Infect. 2009 Jan; 58(1): 21-7
Jafari F, Garcia-Gil LJ, Salmanzadeh-Ahrabi S, Shokrzadeh L, Aslani MM, Pourhoseingholi MA, Derakhshan F, Zali MR

INTRODUCTION: Acute diarrhea disease is the second cause of death among all infectious diseases in children younger than 5 years of age worldwide. The aim of this study was to employ a combination of biochemical, microbiological and molecular diagnostic techniques to investigate the stools of Iranian children with acute diarrhea for bacterial enteropathogens. METHOD: Diarrheagenic Escherichia coli, Shigella spp., Salmonella spp., Campylobacter spp. and Yersinia spp., were investigated from June 2003 to June 2005, in 1087 children less than 5 years old with acute diarrhea. Stool specimens from children were studied for enteropathogens both by standard culturing and molecular methods. This study was designed on hospital based. RESULT: The highest incidence values were found in the summer and in children less than 1-year-old (42.7%). The Pathogenic bacteria recovered out from fecal samples of 555 (55.1%) patients had the following profile: Shigella spp. (26.7%) was the most prevalent bacterial pathogen and Shiga-like toxin producing E. coli (STEC) and Enteroaggregative E. coli (EAEC) 105 (18.9%) and 92 (16.6%) had the second and third highest prevalence, respectively. EnteroPathogenic E. coli (EPEC), Campylobacter, Salmonella and Enterotoxigenic E. coli (ETEC) were found in 70 (12.6%), 60 (10.8%), 42 (7.6%), and 38 (6.8%) positive samples, respectively. In this study neither Yersinia nor E. coli O157:H7 were found. Of the 30 co-infections detected, Shigella flexneri and Campylobacter jejuni accounted for more than 50%. CONCLUSION: Information about the prevalence of wide-range Shigella and STEC may facilitate the control and management of infant diarrhea diseases in Iran. The results of this study suggest that comprehensive surveys are needed in different parts of the country in order to identify the incidence of different enteroPathogenic diarrhea, especially diarrheagenic E. coli in children in Iran.

Microb Pathog. 2008 Nov-Dec; 45(5-6): 361-9
Antão EM, Glodde S, Li G, Sharifi R, Homeier T, Laturnus C, Diehl I, Bethe A, Philipp HC, Preisinger R, Wieler LH, Ewers C

E. coli infections in avian species have become an economic threat to the poultry industry worldwide. Several factors have been associated with the virulence of E. coli in avian hosts, but no specific virulence gene has been identified as being entirely responsible for the Pathogenicity of avian Pathogenic E. coli (APEC). Needless to say, the chicken would serve as the best model organism for unravelling the Pathogenic mechanisms of APEC, an extraintestinal pathogen. Five-week-old white leghorn SPF chickens were infected intra-tracheally with a well characterized APEC field strain IMT5155 (O2:K1:H5) using different doses corresponding to the respective models of infection established, that is, the lung colonization model allowing re-isolation of bacteria only from the lung but not from other internal organs, and the systemic infection model. These two models represent the crucial steps in the pathogenesis of APEC infections, including the colonization of the lung epithelium and the spread of bacteria throughout the bloodstream. The read-out system includes a clinical score, pathomorphological changes and bacterial load determination. The lung colonization model has been established and described for the first time in this study, in addition to a comprehensive account of a systemic infection model which enables the study of severe extraintestinal Pathogenic E. coli (ExPEC) infections. These in vivo models enable the application of various molecular approaches to study host-pathogen interactions more closely. The most important application of such genetic manipulation techniques is the identification of genes required for extraintestinal virulence, as well as host genes involved in immunity in vivo. The knowledge obtained from these studies serves the dual purpose of shedding light on the nature of virulence itself, as well as providing a route for rational attenuation of the pathogen for vaccine construction, a measure by which extraintestinal infections, including those caused by APEC, could eventually be controlled and prevented in the field.

J Microbiol Methods. 2008 Dec; 75(3): 566-71
You Y, Fu C, Zeng X, Fang D, Yan X, Sun B, Xiao D, Zhang J

A microarray technique for the detection and identification of enteroPathogenic bacteria at the species and subspecies levels was developed in this study, and the target bacteria included Pathogenic Escherichia coli, Vibrio cholerae, Vibrio parahaemolyticus, Salmonella enterica, Campylobacter jejuni, Shigellae, Yersinia enterocolitica, and Listeria monocytogenes. The virulence gene of each pathogen was chosen as the amplification target, labeled with a fluorescence dye by multiplex polymerase chain reaction (PCR), and hybridized to the specific virulence gene probes that had been immobilized on a microchip. Stool specimens from 34 patients with diarrhea were tested in this study. Five were positive for multiple genera. Nested PCRs and sequencing were used to amplify and identify the related genes, which were found to share 95.8% to 100% of the nucleotide identity with the corresponding regions in the Genbank database. Real-time PCR was used to determine the number of gene copies to determine the sensitivity of this technique, which was shown to be 58 copies/microl. The results indicated that the microarray technique which targets multiple virulence genes of enteroPathogenic bacteria at the species and subspecies levels is an attractive diagnostic tool for rapidly and simultaneously identifying multiple enteroPathogenic pathogens in clinical practice, especially in patients with infectious diarrhea.

PLoS Negl Trop Dis. 2008; 2(7): e266
Galván-Moroyoqui JM, Del Carmen Domínguez-Robles M, Franco E, Meza I

BACKGROUND: Mixed intestinal infections with Entamoeba histolytica, Entamoeba dispar and bacteria with exacerbated manifestations of disease are common in regions where amoebiasis is endemic. However, amoeba-bacteria interactions remain largely unexamined. METHODOLOGY: Trophozoites of E. histolytica and E. dispar were co-cultured with enteroPathogenic bacteria strains Escherichia coli (EPEC), Shigella dysenteriae and a commensal Escherichia coli. Amoebae that phagocytosed bacteria were tested for a cytopathic effect on epithelial cell monolayers. Cysteine proteinase activity, adhesion and cell surface concentration of Gal/GalNAc lectin were analyzed in amoebae showing increased virulence. Structural and functional changes and induction of IL-8 expression were determined in epithelial cells before and after exposure to bacteria. Chemotaxis of amoebae and neutrophils to human IL-8 and conditioned culture media from epithelial cells exposed to bacteria was quantified. PRINCIPAL FINDINGS: E. histolytica digested phagocytosed bacteria, although S. dysenteriae retained 70% viability after ingestion. Phagocytosis of Pathogenic bacteria augmented the cytopathic effect of E. histolytica and increased expression of Gal/GalNAc lectin on the amoebic surface and increased cysteine proteinase activity. E. dispar remained avirulent. Adhesion of amoebae and damage to cells exposed to bacteria were increased. Additional increases were observed if amoebae had phagocytosed bacteria. Co-culture of epithelial cells with enteroPathogenic bacteria disrupted monolayer permeability and induced expression of IL-8. Media from these co-cultures and human recombinant IL-8 were similarly chemotactic for neutrophils and E. histolytica. CONCLUSIONS: Epithelial monolayers exposed to enteroPathogenic bacteria become more susceptible to E. histolytica damage. At the same time, phagocytosis of Pathogenic bacteria by amoebae further increased epithelial cell damage. SIGNIFICANCE: The in vitro system presented here provides evidence that the Entamoeba/enteroPathogenic bacteria interplay modulates epithelial cell responses to the pathogens. In mixed intestinal infections, where such interactions are possible, they could influence the outcome of disease. The results offer insights to continue research on this phenomenon.

Appl Environ Microbiol. 2008 Sep; 74(18): 5635-44
Bag PK, Bhowmik P, Hajra TK, Ramamurthy T, Sarkar P, Majumder M, Chowdhury G, Das SC

Vibrio cholerae non-O1, non-O139 was isolated from natural surface waters from different sites sampled in diarrhea endemic zones in Kolkata, India. Twenty-one of these isolates were randomly selected and included in the characterization. The multiserogroup isolates were compared by their virulence traits with a group of clinical non-O1, non-O139 isolates from the same geographic area. Of the 21 environmental isolates, 6 and 14 strains belonged to Heiberg groups I and II, respectively. Three of the environmental isolates showed resistance to 2,2-diamine-6,7-diisopropylpteridine phosphate. All of the non-O1, non-O139 strains were positive for toxR, and except for one environmental isolate, none of them were positive for tcpA in the PCR assay. None of the isolates were positive for genes encoding cholera toxin (ctxA), heat-stable toxin (est), heat-labile toxin (elt), and Shiga toxin variants (stx) of Escherichia coli. Additionally, except for one environmental isolate (PC32), all were positive for the gene encoding El Tor hemolysin (hly). The culture supernatants of 86% (18 of 21) of the environmental isolates showed a distinct cytotoxic effect on HeLa cells, and some of these strains also produced cell-rounding factor. The lipase, protease, and cell-associated hemagglutination activities and serum resistance properties of the environmental and clinical isolates did not differ much. However, seven environmental isolates exhibited very high hemolytic activities (80 to 100%), while none of the clinical strains belonged to this group. The environmental isolates manifested three adherence patterns, namely, carpet-like, diffuse, and aggregative adherence, and the clinical isolates showed diffuse adherence on HeLa cells. Of the 11 environmental isolates tested for enteroPathogenic potential, 8 (73%) induced positive fluid accumulation (>/=100) in a mouse model, and the reactivities of these isolates were comparable to those of clinical strains of non-O1, non-O139 and toxigenic O139 V. cholerae. Comparison of the counts of the colonized environmental and clinical strains in the mouse intestine showed that the organisms of both groups had similar colonizing efficiencies. These findings indicate the presence of potentially Pathogenic V. cholerae non-O1, non-O139 strains in surface waters of the studied sites in Kolkata.