Characterization of enteroaggregative Escherichia coli isolates

Forty enteraggregative Escherichia coli (EAggEC) previously characterized by their ability to adhere to HEp-2 cells or/and their hybridization with the 1-kb EAggEC DNA probe were investigated for the presence of adherence factors and heat-stable enterotoxin (EAST1)-encoding genes. Only 45% of the isolates harbored the EAST1-encoding genes as detected by polymerase chain reaction. None of them hybridized with an AAF/II-encoding gene specific DNA probe and 35% (14/40) were positive in a PCR assay using primers specific for aggC, an accessory gene of the AAF/I-encoding operon. Cloning and sequence analysis of the aggA variant from one isolate, EAggEC 457, revealed 68.9% identity between its deduced amino acid sequence and those of the aggA product from the AAF/I-producing reference strain, E. coli 17.2. No major protein subunit was detected at the surface of EAggEC 457 compared to the bacterial surface extract of E. coli 17.2.     

Identification of entero-aggregative Escherichia coli based on surface properties

Twenty-nine strains of Escherichia coli that adhere to HEp-2 cells with a'stacked brick' pattern (EAggEC), and four nonadherent control strains, wereexamined for the ability to hybridize with gene probes for aggregative (AA) and diffuse (DA)HEp-2 cell adhesion phenotypes. These strains were also tested for the ability to express an 18 kDa membrane-associated outer- membrane protein (MAP), to agglutinate erythrocytes, and toproduce a pellicle during broth culture. Thirteen of the 29 HEp-2 adherent strains of E. coli hybridized with the gene probes for both AA and DA, and expressed an 18 kDa outermembrane protein (OMP) which was antigenically related to the MAP expressed by strains of E. coli O126:H27. The strains that did not carry the additional DA genes did notexpress an 18 kDa OMP. Although strains of EAggEC share the ability to adhere to HEp-2 cellswith a stacked brick pattern, these strains exhibit a diverse range of physical and biochemicalproperties. From the results of this study, it was concluded that currently, the possession ofEAggEC genes or the ability to adhere to HEp-2 cells in a stacked brick formation, remain theonly reliable means of identifying EAggEC.     

Integration of the DNA of a novel filamentous bacteriophage VSK from Vibrio cholerae 0139 into the host chromosomal DNA

Abstract The surface of three strains of enteroaggregative Escherichia coli (EAggEC) and three strains of enteropathogenic E. coli (EPEC) were examined using the freeze-substitution technique of electron microscopy and as a result an electron dense surface layer was found only on EAggEC strains but not on EPEC strains. The analysis of the outer membrane proteins by polyacrylamide gel electrophoresis revealed the existence of a unique 38 kDa protein in EAggEC strains. The protein could be easily extracted from the bacterial surface with 5 M LiCl treatment at room temperature. The antiserum raised in mice with 38 kDa protein extracted from the electrophoresed gel was immunoreacted with the surface of the bacteria of EAggEC by immunoelectron microscopy. The hydrophobic surface character of the EAggEC strains was lost after the extraction of the protein layer by LiCl. We thus conclude that the surface protein layer therefore plays an important role in the expression of the aggregative phenotype in EAggEC strains.     

Identification of a single-nucleotide insertion in the promoter region affecting the sodC promoter activity in Brucella neotomae

Brucella neotomae is not known to be associated with clinical disease in any host species. Previous research suggested that B. neotomae might not express detectable levels of Cu/Zn superoxide dismutase (SOD), a periplasmic enzyme known to be involved in protecting Brucella from oxidative bactericidal effects of host phagocytes. This study was undertaken to investigate the genetic basis for the disparity in SOD expression in B. neotomae. Our Western blot and SOD enzyme assay analyses indicated that B. neotomae does express SOD, but at a substantially reduced level. Nucleotide sequence analysis of region upstream to the sodC gene identified a single-nucleotide insertion in the potential promoter region. The same single-nucleotide insertion was also detected in the sodC promoter of B. suis strain Thomsen, belonging to biovar 2 in which SOD expression was undetectable previously. Examination of the sodC promoter activities using translational fusion constructs with E. coli β-galactosidase demonstrated that the B. neotomae and B. suis biovar 2 promoters were very weak in driving gene expression. Site-directed mutation studies indicated that the insertion of A in the B. neotomae sodC promoter reduced the promoter activity. Increasing the level of SOD expression in B. neotomae through complementation with B. abortus sodC gene did not alter the bacterial survival in J774A.1 macrophage-like cells and in tissues of BALB/c and C57BL/6 mice. These results for the first time demonstrate the occurrence of a single-nucleotide polymorphism affecting promoter function and gene expression in Brucella.     

Bacterial copper- and zinc-cofactored superoxide dismutase contributes to the pathogenesis of systemic salmonellosis

Copper/zinc-cofactored superoxide dismutase ([Cu,Zn]-SOD) has been found in the periplasm of many bacterial species but its biological function is unknown. Here we report the cloning and characterization of sodC , encoding [Cu,Zn]-SOD, from Salmonella typhimurium . The predicted protein sequence shows only 58% identity to Escherichia coli SodC, and from this its chromosomal location and its immediate proximity to a phage gene, sodC , in Salmonella is speculated to have been acquired by bacteriophage-mediated horizontal transfer from an unknown donor. A sodC mutant of S . typhimurium was unimpaired on aerobic growth in rich medium but showed enhanced sensitivity in vitro to the microbicidal action of superoxide. S . typhimurium , S . choleraesuis and S . dublin sodC mutants showed reduced lethality in a mouse model of oral infection and persisted in significantly lower numbers in livers and spleens after intraperitoneal infection, suggesting that [Cu,Zn]-SOD plays a role in pathogenicity, protecting Salmonella against oxygen radical-mediated host defences. There was, however, no observable difference compared with wild type in the interaction of sodC mutants with porcine pleural, mouse peritoneal or J774 macrophages in vitro , perhaps reflecting the hierarchical capacity of different macrophage lines to kill Salmonella , the most efficient overwhelming the proposed protective effect of periplasmic SOD.     

Virulence gene profiling of enteroaggregative Escherichia coli heat-stable enterotoxin 1-harboring E. coli (EAST1EC) derived from sporadic diarrheal patients

Between 2007 and 2009, a total of 2168 Escherichia coli strains derived from diarrheal patients, defined as putative diarrheagenic E. coli (DEC), were collected from medical institutions in Akita prefecture, Japan. Thirty five of the strains lacked typical pathogenic determinants of DEC other than astA, which encodes enteroaggregative E. coli (EAggEC) heat-stable enterotoxin 1 (EAST1). These E. coli strains are referred to as EAST1EC. Several studies have suggested a role of EAST1 in diarrhea; however, the correlation between diarrhea and the presence of astA remains inconclusive. To investigate whether EAST1EC strains derived from diarrheal patients shared pathogenic factors other than EAST1, virulence gene profiling of 12 virulence genes - iha, lpfA, ldaG, pilS, pic, pet, irp2, daa, aah, aid, cdtB and hlyA - was carried out. PCR analysis revealed that four of the 35 EAST1EC strains harbored only astA, 24 harbored genes associated with adhesins and intestinal colonization, three strains harbored the gene for α-hemolysin, and 24 strains harbored the gene for a siderophore. These results indicated that some EAST1EC strains harbor various virulence genes associated with distinct E. coli pathotypes, primarily enterohemorrhagic E. coli and EAggEC, which may represent additional pathogenic determinants of EAST1EC.     

The role of periplasmic antioxidant enzymes (superoxide dismutase and thiol peroxidase) of the Shiga toxin-producing Escherichia coli O157:H7 in the formation of biofilms

This study examined the role of the periplasmic oxidative defense proteins, copper, zinc superoxide dismutase (SodC), and thiol peroxidase (Tpx), from the Shiga toxin-producing Escherichia coli O157:H7 (STEC) in the formation of biofilms. Proteomic analyses have shown significantly higher expression levels of both periplasmic antioxidant systems (SodC and Tpx) in STEC cells grown under biofilm conditions than under planktonic conditions. An analysis of their growth phase-dependent gene expression indicated that a high level of the sodC expression occurred during the stationary phase and that the expression of the tpx gene was strongly induced only during the exponential growth phase. Exogenous hydrogen peroxide reduced the aerobic growth of the STEC sodC and tpx mutants by more than that of their parental strain. The two mutants also displayed significant reductions in their attachment to both biotic (HT-29 epithelial cell) and abiotic surfaces (polystyrene and polyvinyl chloride microplates) during static aerobic growth. However, the growth rates of both wild-type and mutants were similar under aerobic growth conditions. The formation of an STEC biofilm was only observed with the wild-type STEC cells in glass capillary tubes under continuous flow-culture conditions compared with the STEC sodC and tpx mutants. To the best of our knowledge, this is the first mutational study to show the contribution of sodC and tpx gene products to the formation of an E. coli O157:H7 biofilm. These results also suggest that these biofilms are physiologically heterogeneous and that oxidative stress defenses in both the exponential and stationary growth stages play important roles in the formation of STEC biofilms.     

Identification, cloning and expression of sodC from an alkaline phosphatase gene fusion library of Mycobacterium avium subspecies paratuberculosis

The phoA gene technology was used to investigate secreted proteins of the intracellular pathogen Mycobacteriumn avium subspecies paratuberculosis. This led to the identification of sodC, a gene which codes for a copper and zinc cofactored superoxide dismutase (Cu,ZnSOD) which has been implicated as a virulence factor for some pathogens. The predicted protein possessed a 76% identity with Cu,ZnSOD of Mycobacterium tuberculosis. To characterize Cu,ZnSOD from M. avium subspecies paratuberculosis, the gene was cloned and overexpressed in Escherichia coli. The renatured, affinity-purified recombinant protein possessed enzymatic activity that was inhibited by the presence of cyanide, which is characteristic of a Cu,ZnSOD.     

Expression of antigenically distinct fimbriae with hemagglutination and HeLa cell adherence properties by an enteroaggregative Escherichia coli strain belonging to the enteropathogenic serogroup

Abstract An enteroaggregative Escherichia coli (EAggEC) strain (DS92), isolated from a case of infantile diarrhea, was shown to express mannose-resistant hemagglutination and HeLa cell adhering properties when grown at 37°C but not at 28°C. Cellular adherence properties of DS92, which belonged to enteropathogeci serogroup 0125, were shown to correlate well with the expression of fimbriae that were encoded by a 112 kb plasmid. The fimbriae of the EAggEC strain DS92 were composed of 20 kDa subunit proteins and were serologically distinct from fimbrial or non-fimbrial cell surface antigen(s) of other diarrheagenic E. coli strains including the reference EAggEC strain 17-2. Interestingly, the 20-kDa fimbrial protein was found to be antigenically related to 18- and 14.5-kDa cell surface proteins of two other locally isolated EAggEC strains belonging to the enteropathogenic serogroup 086.     

Periplasmic Cu,Zn superoxide dismutase and cytoplasmic Dps concur in protecting Salmonella enterica serovar Typhimurium from extracellular reactive oxygen species

Several bacteria possess periplasmic Cu,Zn superoxide dismutases which can confer protection from extracellular reactive oxygen species. Thus, deletion of the sodC1 gene reduces Salmonella enterica serovar Typhimurium ability to colonize the spleens of wild type mice, but enhances virulence in p47phox mutant mice. To look into the role of periplamic Cu,Zn superoxide dismutase and into possible additive effects of the ferritin-like Dps protein involved in hydrogen peroxide detoxification, we have analyzed bacterial survival in response to extracellular sources of superoxide and/or hydrogen peroxide. Exposure to extracellular superoxide of Salmonella Typhimurium mutant strains lacking the sodC1 and sodC2 genes and/or the dps gene does not cause direct killing of bacteria, indicating that extracellular superoxide is poorly bactericidal. In contrast, all mutant strains display a sharp hydrogen peroxide-dependent loss of viability, the dps,sodC1,sodC2 mutant being less resistant than the dps or the sodC1,sodC2 mutants. These findings suggest that the role of Cu,Zn superoxide dismutase in bacteria is to remove rapidly superoxide from the periplasm to prevent its reaction with other reactive molecules. Moreover, the nearly additive effect of the sodC and dps mutations suggests that localization of antioxidant enzymes in different cellular compartments is required for bacterial resistance to extracytoplasmic oxidative attack.     

sodC-based real-time PCR for detection of Neisseria meningitidis

Real-time PCR (rt-PCR) is a widely used molecular method for detection of Neisseria meningitidis (Nm). Several rt-PCR assays for Nm target the capsule transport gene, ctrA. However, over 16% of meningococcal carriage isolates lack ctrA, rendering this target gene ineffective at identification of this sub-population of meningococcal isolates. The Cu-Zn superoxide dismutase gene, sodC, is found in Nm but not in other Neisseria species. To better identify Nm, regardless of capsule genotype or expression status, a sodC-based TaqMan rt-PCR assay was developed and validated. Standard curves revealed an average lower limit of detection of 73 genomes per reaction at cycle threshold (C(t)) value of 35, with 100% average reaction efficiency and an average R(2) of 0.9925. 99.7% (624/626) of Nm isolates tested were sodC-positive, with a range of average C(t) values from 13.0 to 29.5. The mean sodC C(t) value of these Nm isolates was 17.6±2.2 (±SD). Of the 626 Nm tested, 178 were nongroupable (NG) ctrA-negative Nm isolates, and 98.9% (176/178) of these were detected by sodC rt-PCR. The assay was 100% specific, with all 244 non-Nm isolates testing negative. Of 157 clinical specimens tested, sodC detected 25/157 Nm or 4 additional specimens compared to ctrA and 24 more than culture. Among 582 carriage specimens, sodC detected Nm in 1 more than ctrA and in 4 more than culture. This sodC rt-PCR assay is a highly sensitive and specific method for detection of Nm, especially in carriage studies where many meningococcal isolates lack capsule genes.     

The regulation and role of the periplasmic copper, zinc superoxide dismutase of Escherichia coli

The discovery of superoxide dismutase (CuZnSOD) within the periplasms of several Gram-negative pathogens suggested that this enzyme evolved to protect cells from exogenous sources of superoxide, such as the oxidative burst of phagocytes. However, its presence in some non-pathogenic bacteria implies that there may be a role for this SOD during normal growth conditions. We found that sodC, the gene that encodes the periplasmic SOD of Escherichia coli, is repressed anaerobically by Fnr and is among the many antioxidant genes that are induced in stationary phase by RpoS. Surprisingly, the entry of wild-type E. coli into stationary phase is accompanied by a several-hour-long period of acute sensitivity to hydrogen peroxide. Induction of the RpoS regulon helps to diminish that sensitivity. While mutants of E. coli and Salmonella typhimurium that lacked CuZnSOD were not detectably sensitive to exogenous superoxide, both were killed more rapidly than their parent strains by exogenous hydrogen peroxide in early stationary phase. This sensitivity required prior growth in air. Evidently, periplasmic superoxide is generated during stationary phase by endogenous metabolism and, if it is not scavenged by CuZnSOD, it causes an unknown lesion that augments or accelerates the damage done by peroxide. The molecular details await elucidation.     

Periplasmic Cu,Zn superoxide dismutase and cytoplasmic Dps concur in protecting Salmonella enterica serovar Typhimurium from extracellular reactive oxygen species

Several bacteria possess periplasmic Cu,Zn superoxide dismutases which can confer protection from extracellular reactive oxygen species. Thus, deletion of the sodC1 gene reduces Salmonella enterica serovar Typhimurium ability to colonize the spleens of wild type mice, but enhances virulence in p47phox mutant mice. To look into the role of periplamic Cu,Zn superoxide dismutase and into possible additive effects of the ferritin-like Dps protein involved in hydrogen peroxide detoxification, we have analyzed bacterial survival in response to extracellular sources of superoxide and/or hydrogen peroxide. Exposure to extracellular superoxide of Salmonella Typhimurium mutant strains lacking the sodC1 and sodC2 genes and/or the dps gene does not cause direct killing of bacteria, indicating that extracellular superoxide is poorly bactericidal. In contrast, all mutant strains display a sharp hydrogen peroxide-dependent loss of viability, the dps,sodC1,sodC2 mutant being less resistant than the dps or the sodC1,sodC2 mutants. These findings suggest that the role of Cu,Zn superoxide dismutase in bacteria is to remove rapidly superoxide from the periplasm to prevent its reaction with other reactive molecules. Moreover, the nearly additive effect of the sodC and dps mutations suggests that localization of antioxidant enzymes in different cellular compartments is required for bacterial resistance to extracytoplasmic oxidative attack.     

Enteroaggregative strains of Escherichia coli belonging to serotypes 0126:H27 and 044:H18 express antigenically similar 18 kDa outer membrane-associated proteins

Abstract Outer membrane-associated proteins of 18 kDa were expressed by enteroaggregative Escherichia coli (EAggEC) belonging to serotypes 0126:H27 and O44:H18, which hybridized with a probe derived from a plasmid necessary for enteroaggregative adhesion. The 18 kDa proteins expressed by strains of E. coli , belonging to these serotypes, were surface exposed and antigenically similar but not structurally identical.     

Salicylate inhibits fimbriae mediated HEp-2 cell adherence of and haemagglutination by enteroaggregative Escherichia coli

Enteroaggregative Escherichia coli (EAggEC) are associated with both acute and persistent diarrhoea in children. Bowel colonisation due to fimbrial adherence factors appears to play a major role in the disease process. In this study, we investigated the effect of sodium salicylate and 5-aminosalicylic acid on adherence of a type strain and 40 clinical isolates of EAggEC to HEp-2 cells and erythrocytes from different species. Growth in the presence of 10 mM salicylate resulted in markedly decreased adherence to tissue culture cells with 33/40 (82.5%) isolates, and was also associated with inhibition of haemagglutination in 20/33 (60.6%) isolates. Complete or partial inhibition of adherence was also seen in two of five isolates showing localised adherence and three of five isolates with diffuse adherence. Decrease in adherence was associated with decreased or absent expression of fimbriae in 28/40 (70%) of the EAggEC isolates, although production of outer membrane proteins was not affected. Salicylates appear to inhibit adherence mediated by fimbrial adhesins.     

Differential contribution of sodC1 and sodC2 to intracellular survival and pathogenicity of Salmonella enterica serovar Choleraesuis

Several of the most virulent Salmonella enterica strains possess two genes encoding periplasmic Cu,Zn superoxide dismutase, sodC1 and sodC2, located on a lambdoid prophage and on the chromosome, respectively. These genes contribute to Salmonella virulence by protecting bacteria from superoxide generated by the host's phagocytes. To investigate the respective contributions of sodC1 and sodC2 to the virulence of a clinical isolate of Salmonella enterica serovar Choleraesuis (S. choleraesuis), we have analyzed both the intracellular survival of wild type and sodC mutant strains within J774 macrophages and Caco-2 cells, and their ability to proliferate in intraperitoneally-infected mice in competition assays. In agreement with previous studies, mutant strains lacking one or both sodC genes were equally impaired in their ability to survive within activated macrophages. However, when macrophage killing experiments were carried out with non-opsonized bacteria, sodC2 contributed to intracellular survival more than sodC1, indicating that changes in the pathways of bacterial uptake can modify the relative role of the two sodC genes. More unexpectedly, we have found that the ability of S. choleraesuis to survive within Caco-2 cells was severely affected by inactivation of sodC genes, sodC2 being more important than sodC1. As Caco-2 cells actively produce superoxide, this suggests that oxygen radical production by colonic cells has a role in controlling proliferation of facultative intracellular bacteria. Mouse infection studies confirmed that, in the S. choleraesuis strain under investigation, both sodC genes are required to confer full virulence, sodC2 contributing slightly more than sodC1 to Salmonella pathogenesis. Our findings contrast with the results of other studies carried out in S. enterica serovar Typhimurium and suggest that the relative contributions of sodC1 and sodC2 to host-pathogen interactive biology may vary depending on the Salmonella serovar or strain.     

Inducible prophages contribute to Salmonella virulence in mice

We show that Salmonella typhimurium harbours two fully functional prophages, Gifsy-1 and Gifsy-2, that can be induced by standard treatments or, more effectively, by exposing bacteria to hydrogen peroxide. Curing bacteria for the Gifsy-2 prophage significantly reduces Salmonella's ability to establish a systemic infection in mice. Cured strains recover their virulence properties upon relysogenization. Phage Gifsy-2 carries the sodC gene for a periplasmic [Cu,Zn]-superoxide dismutase previously implicated in the bacterial defences against killing by macrophages. The contribution of the Gifsy-1 prophage to virulence - undetectable in the presence of Gifsy-2 as prophage - becomes significant in cells that lack Gifsy-2 but carry the sodC gene integrated in the chromosome. This confirms the involvement of Gifsy-2-encoded SodC protein in Salmonella pathogenicity and suggests that the Gifsy-1 prophage carries one or more additional virulence genes that have a functional equivalent on the Gifsy-2 genome.     

Diarrheagenic Escherichia coli in acute gastroenteritis in infants in North-West Italy

Enteropathogenic Escherichia coli may cause diarrhoea in infancy, but it is not routinely detected and regarded as a major causative agent. The aim of the present study was to estimate the incidence of enteropathogenic E. coli infection and to investigate its epidemiology and pathogenesis from faecal specimens in infants hospitalized for acute gastroenteritis. Between March 2008 and June 2009, faecal samples were collected and examined to recognize diarrhoeal aetiology, especially for E. coli, by cultural identification and multiplex-PCR. E. coli were isolated in 75 of 160 collected samples (46,88%); 10 samples of which (6,3%) had been positively recognised for pathogenic genes. Data showed that the presence of diarrheagenic E. coli infection was 6.3%, but it becomes 5% considering E. coli as a unique agent responsible for diarrhoea. The datum is not statistically meaningful because of the small sample (p>0,05). Bacterial pathogens were also isolated in 60 samples (37,5% of the total collected samples): 15 Salmonella spp., 8 Klebsiella pneumoniae, 9 Klebsiella oxytoca, 11 Citrobacter freundii, 5 Pseudomonas aeruginosa, 2 Serratia spp., 7 Enterobacter cloacae, 1 Shigella spp., 2 Campylobacter spp. Rotavirus was the predominant pathogenic single etiologic agent identified. It was found in 35 samples (21.88% of the overall collected samples), while Adenovirus, serotypes 40 or 41, was isolated in 2 samples (1.3%). Rotavirus infection was found predominantly in winter with respect to autumn. Data provide an interesting epidemiologic survey of enteropathogenic E. coli, which is not usually detected, although it may have potential clinical implications. Abbreviations: CDEC, detaching E. coli; DAEC, diffusely adherent E. coli; EAggEC, enteroaggregative E. coli; EHEC, enterohaemorrhagic E. coli; EIEC, enteroinvasive E. coli; EPEC, enteropathogenic E. coli; ETEC, enterotoxigenic E. coli.