virulence genes

streaked on fresh plates. Each colony was independently subjected to Gram staining, microscopic examination, biochemical test and serogrouping. Eventually, 303 E. coli strains from 101 cows (three strains for one cow), were randomly selected for characterizing virulence genes. E. coli strains used as controls were C83922 (O101:K99, F41; st + ), C83902 (O8:K87, K88ac; st + lt + ), E. coli O157 (stx1 + stx2 + ), E. coli O139 (eae + ) and E. coli O127 (eaeA + bfpA + ). All strains were stored at -20 °C in LB broth with 10% glycerol.

Virulence and antibiotic resistance are significant determinants of the types of infections caused by Staphylo- coccus aureus and paediatric groups remain among the most commonly affected populations. The goal of this study was to characterise virulence genes of methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) strains isolated from a paediatric population of a Colombian University Hospital during 2009. Sixty MSSA and MRSA isolates were obtained from paediatric patients between zero-14 years. We identified the genes encoding virulence factors, which included Panton-Valentine leucocidine (PVL), staphylococcal enterotoxins A-E, exfoliative toxins A and B and toxic shock syndrome toxin 1. Typing of the staphylococcal chromosome cassette mec (SCCmec) was performed in MRSA strains. The virulence genes were more diverse and frequent in MSSA than in MRSA iso- lates (83% vs. 73%). MRSA strains harboured SCCmec types IVc (60%), I (30%), IVa (7%) and V (3%). SCCmec type IVc isolates frequently carried the PVL encoding genes and harboured virulence determinants resembling susceptible strains while SCCmec type I isolates were often negative. PVL was not exclusive to skin and soft tissue infections. As previously suggested, these differences in the distribution of virulence factor genes may be due to the fitness cost associated with methicillin resistance.

S. aureus encodes a remarkable number of surface- associated as well as extracellular virulence factors. However, previous studies have shown contradictory results regarding the importance of these different virulence genes for invasiveness. Based on 1γ4 MSSA isolates from three well- characterized patient populations, the aim of the present study was to evaluate the potential association between S. aureus disease and bacterial genotype with a focus on clonality and genes encoding MSCRAMMs. We used DNA microarray based analysis, which offers the great advantage of a simultaneous and fast analysis of a large number of virulence genes as well as assignment to clonal lineages at a very reasonable cost compared to multiple PCRs or whole genome sequencing.

H elicobacter pylori infection significantly increases the risk of development of peptic ulcer disease, distal gastric carcinoma, and gastric lymphoma (1). Infection of the general population with virulent strains, especially those carrying the cagA gene and vacAs1 genotype, is a predictor of increased risk for development of severe H. pylori-associated diseases. However, the majority of the methods used for genotyping H. pylori strains require an in- vasive procedure, endoscopy, for tissue sample collection and are not indicated in epidemiological studies evaluating asymptomatic individuals, especially children. The string test, a minimally inva- sive nonendoscopic procedure, seems to be an accurate method to obtain gastric specimens in order to investigate H. pylori virulence genes. It has been demonstrated that the genotypes of H. pylori strains in DNA from the gastric juice or tissue samples are identi- cal (2).

In poultry, the detection of several virulence factors of E. coli has been associated with clinical signs of APEC. However, no virulence factors have been exclusively linked to pathogenicity (Tivendale et al. 2004). Among the E. coli virulence factors, serum resistance has been shown to have a high correlation with pathogenicity (Mellata et al. 2003). Genes such as the tsh (temperature-sensitive haemagglu- tinin) and iss (serum resistance) have been reported in APEC strains. The iss gene has been associated with cer- tain strains capable to provoke sepsis by providing bacteria with resistance against the host immune bactericidal de- fences (Tivendale et al. 2004). In the present study, the iss gene was detected in E. coli isolated from 9 individuals (11 isolates), all of them in good health conditions. Although the detection of the iss gene alone does not correlate with virulence, turkey faecal samples from asymptomatic and clinical cases of colibacillosis have been tested by PCR. A much higher frequency of iss has been shown to be associ- ated with tsh in symptomatic birds, suggesting that these particular strains do not constitute a major part of the nor- mal enteric microbiota of healthy turkeys (Altekruse et al. 2002). The association of these two genes was not detected in the present study. But tsh gene was detected in associa- tion with iuc and hlyA genes in an E. coli isolated from one currasow. This birds was also healthy, but it should be taken into account that tsh gene has been shown to play a role in mechanisms of adherence to the respiratory tract of poul- try (Dozois et al. 2000). Thus, further studies with in vivo infection should be performed for a better understanding regarding virulence genes and clinical manifestation of E. coli infection.

Escherichia coli isolates from 24 sick psittacine birds were serogrouped and investigated for the presence of genes encoding the following virulence factors: attaching and effacing (eae), enteropathogenic E. coli EAF plasmid (EAF), pili associated with pyelonephritis (pap), S imbriae (sfa), aimbrial adhesin (afa), capsule K1 (neu), curli (crl, csgA), temperature- -sensitive hemagglutinin (tsh), enteroaggregative heat-stable enterotoxin-1 (astA), heat- -stable enterotoxin -1 heat labile (LT) and heat stable (STa and STb) enterotoxins, Shiga-like toxins (stx1 and stx2), cytotoxic necrotizing factor 1 (cnf1), haemolysin (hly), aerobactin production (iuc) and serum resistance (iss). The results showed that the isolates belonged to 12 serogroups: O7; O15; O21; O23; O54; O64; O76; O84; O88; O128; O152 and O166. The virulence genes found were: crl in all isolates, pap in 10 isolates, iss in seven isolates, csgA in ive isolates, iuc and tsh in three isolates and eae in two isolates. The combination of virulence genes revealed 11 different genotypic patterns. All strains were negative for genes encoding for EAF, EAEC, K1, sfa, afa, hly, cnf, LT, STa, STb, stx1 and stx2. Our indings showed that some E. coli isolated from psittacine birds present the same virulence factors as avian pathogenic E. coli (APEC), uropathogenic E. coli (UPEC) and Enteropathogenic E. coli (EPEC) pathotypes. INDEX TERMS: Psittacine birds, Escherichia coli, Virulence factors, Septicemia.

This study provided important information regarding pathogenic E. coli isolated from the feces of dogs. The presence of virulence genes and the phylogenetic classification of E. coli were determined and are important in understanding canine E. coli infections and associated syndromes. There was no association between virulence genes or phylogroups and any of the study’s epidemiological aspects, including age, breed, or gender. Our results indicated that EPEC were the most frequently identified pathovar in the feces of dogs and are mainly classified into the intestinal origin phylogroups, B1 and E. NTEC positive for cnf1 were also identified and were mostly in phylogroups B2 and D, which are associated with ExPEC. Detection of other pathovars was very low, but the ones identified were associated with disease in humans and other animal species. Because of the close contact between dogs and humans and the likely contact with other animal species, these results emphasized the need for further studies to clarify the role of E. coli in dogs.

The prevalence of virulence genes expressing fimbriae, production of hemolysin, colicin and aerobactin, was determined in Escherichia coli isolates from healthy cow’s genital tract not showing clinical signs of infection. The presence of fimbriae expression genes (pap, sfa, afa) was assayed using specific primers in a polymerase chain reaction; none were detected in any of the isolates. Yet, a prevalence of 90.4%, 69.8%, and 28.5% of virulence factors for colicin, hemolysin and aerobactin respectively, was detected in the isolates. Analysis of the bacterial pathogenicity of isolates from the bovine genital tract may contribute towards the understanding of E. coli behavior.

Although no statistically important relation found between carbapenem resistance and virulence factors in this study, the effect of beta-lactam resistance in virulence is known. The relation between the virulence factors and ESBL in E.coli were reported by many researchers. 34,35 Also, Arısoy et al. reported that increase of virulence genes were related with resistance to some antibiotics or sensitivity to others. 13 In recent studies, a few mechanisms were focused for the relation and one of the mechanism is the plasmids carrying antibiotic resistance [36] and others are porin loss 37 , modifications in penicillin binding proteins and efflux pumps mechanism. 38 Efflux pumps are responsible for discharging of molecules containing virulence factors regulated by quorum sensing which has a positive effect on antibiotics resistance and virulence. 39,40 E.coli strain 28 which previously reported with porin loss and OXA-48 resistance 25 , was determined to have gene regions related with siderophore (aer, iutA) in this study. Similarly, virulence factors were highly found in carbapenem susceptible isolates, shows that other mechanisms may have an effect on the relation between carbapenem resistance and virulence. Therefore, determining of beta-lactam group resistance, these mechanisms should be taken into consideration.

Diarrheagenic Escherichia coli (DEC) are considered one of the major causes of human diarrhea in developing countries. Some studies have pointed wild birds as important reservoirs for these pathogens. However, scarce species from the Psittaciformes order have been investigated. This study aimed to evaluate the presence of DEC strains in Psittaciformes from illegal wildlife trade. A total of 78 E. coli strains isolated from cloacal swab samples of 167 Psittaciformes in the Ceará State, Brazil, were evaluated regarding the presence of the following DEC virulence genes by polymerase chain reaction (PCR): eaeA and bfpA genes (Enteropathogenic E. coli – EPEC); stx1 and stx2 (Shiga toxin-producing E. coli - STEC); estA and eltB (Enterotoxigenic E. coli - ETEC); ipaH (Enteroinvasive E. coli - EIEC); aatA and aaiC (Enteroaggregative E. coli - EAEC). Positive strains for eaeA and bfpA genes were considered typical EPEC, while strain positive exclusively for the eae A gene were classified as atypical EPEC. The eae A gene was identified in 20 E. coli strains and bfpA in 22 isolates. In addition, 11 and 9 belonged to tEPEC and aEPEC, respectively. No strain was positive for stx1 or stx2. A total of 47 (60.3%) strains and a total of 136 birds (81.4%) were negative for the remaining DEC pathotypes investigated. In conclusion, psittacine from illegal wildlife trade in Ceará State, Brazil, presented a relevant prevalence of typical and atypical EPEC, potentially playing a role as reservoirs of DEC strains in the environment. Thus, proper control measures must be adopted to block the spread of these pathogens.

Previous studies demonstrated that APEC, NMEC and UPEC strains share common virulence genes, overlapped O serogroups and phylogenetic groups (Rodriguez-Siek et al. 2005, Ewers et al. 2007, Moulin-Schouleur et al. 2007). These characteristics were corroborated by multilocus se- quence typing (MLST) and genome comparison data (John- son et al. 2007, Moulin-Schouleur et al. 2007) and led to a further hypothesis that APEC strains could act as UPEC or NMEC and, therefore, constitute a zoonotic risk. A recent work (Dziva et al. 2013) demonstrated that an APEC strain was more closely related to a human ST23 ETEC (enteroto- xigenic E. coli ) than to strain APEC O1. This suggests that the core genome of ST23 strains has the potential to generate variants able to cause disease on avian or human, depen- ding of the accessory genome. For urinary tract infections and new-born meningitis, it has been hypothesized that APEC could access and colonize the human colon via the in- gestion of contaminated poultry and then reach the urinary tract or the central nervous system (Rodriguez-Siek et al. 2005). Thereby, APEC could act as a cause of human ExPEC infections or as a virulence gene reservoir for human ExPEC strains, thus representing a possible zoonotic risk (Rodri- guez-Siek et al. 2005, Moulin-Schouleur et al. 2007).

Enteroinvasive Escherichia coli (EIEC) and Shigella spp cause bacillary dysentery in humans by invading and multiplying within epithelial cells of the colonic mucosa. Although EIEC and Shigella share many genetic and bio- chemical similarities, the illness caused by Shigella is more severe. Thus, genomic and structure-function molecular studies on the biological interactions of these invasive enterobacteria with eukaryotic cells have focused on Shigella rather than EIEC. Here we comparatively studied the interactions of EIEC and of Shigella flexneri with cultured J774 macrophage-like cells. We evaluated several phenotypes: (i) bacterial escape from macrophages after phago- cytosis, (ii) macrophage death induced by EIEC and S. flexneri, (iii) macrophage cytokine expression in response to infection and (iv) expression of plasmidial (pINV) virulence genes. The results showed that S. flexneri caused macrophage killing earlier and more intensely than EIEC. Both pathogens induced significant macrophage produc- tion of TNF, IL-1 and IL-10 after 7 h of infection. Transcription levels of the gene invasion plasmid antigen-C were lower in EIEC than in S. flexneri throughout the course of the infection; this could explain the diminished virulence of EIEC compared to S. flexneri.

Additionally many of the cephalosporin-resistant strains also display multidrug resistance (MDR) and often these MDR phenotypes can be transferred en masse to susceptible strains [15– 18], which can present an additional challenge for controlling S. Heidelberg in food-animal production settings. Welch et al. (2007) described the sequence of a large transmissible incompatibility group (Inc) A/C plasmid from a S. Newport strain carrying 11 resistance determinants and provided compelling evidence that similar IncA/C plasmids are widely distributed in isolates of various Salmonella serovars, including S. Heidelberg [19]. In 2011, a multi-state outbreak of human S. Heidelberg involving 34 U.S. states was linked to the consumption of ground turkey that resulted in a total of 136 infections (http://www.cdc.gov/salmonella/ heidelberg/111011/index.html). The outbreak strain of S. Heidel- berg was resistant to multiple commonly prescribed antimicrobi- als, which likely increased the risk for treatment failure in the infected individuals. The spread of multidrug resistance among S. Heidelberg isolates can pose a threat to the management of salmonellosis in animal husbandry and human medicine. Since many of antimicrobial resistance and virulence genes are encoded on plasmids, an understanding of plasmid genetics is important for comprehending the factors associated with increased antimicrobial and virulence resistance in S. Heidelberg. The objective of this study was to sequence multidrug resistance plasmids isolated from S. Heidelberg and compare the data to previously sequenced plasmids. These results provide additional data to better understand the genetics of plasmid-associated antimicrobial resistance in S. Heidelberg.

Wassenaar and Gaastra, 2001 [17] proposed some interesting divisions: i) virulence genes from bacteria that are exclusively pathogenic (true virulence genes) such as cholera, anthrax and botulinum toxin; ii) virulence genes from bacteria displaying host- dependent pathogenicity (colonization factors) such adhesins, fimbriae, intimin, invasins and defense system evasion genes, such as immunoglobulin- specific proteases, cytotoxins directed against immune cells, surface layers, slime polysaccharides; and iii) virulence genes from opportunistic pathogens, such as specific proteases, methylases, chaperonins, glycosyltransferases, with virulence life-style genes as a substrate and secretory virulence genes, such as type I and III secretion machinery and virulence housekeeping genes, such as urease, catalase, superoxide dismutase and siderophores, and regulatory genes, such as alternative sigma factors, global regulators, specific transcription activators and regulators of phase variation by gene/promoter inversion.

Diarrheagenic Escherichia coli (DEC) are considered one of the major causes of human diarrhea in developing countries. Some studies have pointed wild birds as important reservoirs for these pathogens. However, scarce species from the Psittaciformes order have been investigated. This study aimed to evaluate the presence of DEC strains in Psittaciformes from illegal wildlife trade. A total of 78 E. coli strains isolated from cloacal swab samples of 167 Psittaciformes in the Ceará State, Brazil, were evaluated regarding the presence of the following DEC virulence genes by polymerase chain reaction (PCR): eaeA and bfpA genes (Enteropathogenic E. coli – EPEC); stx1 and stx2 (Shiga toxin-producing E. coli - STEC); estA and eltB (Enterotoxigenic E. coli - ETEC); ipaH (Enteroinvasive E. coli - EIEC); aatA and aaiC (Enteroaggregative E. coli - EAEC). Positive strains for eaeA and bfpA genes were considered typical EPEC, while strain positive exclusively for the eae A gene were classified as atypical EPEC. The eae A gene was identified in 20 E. coli strains and bfpA in 22 isolates. In addition, 11 and 9 belonged to tEPEC and aEPEC, respectively. No strain was positive for stx1 or stx2. A total of 47 (60.3%) strains and a total of 136 birds (81.4%) were negative for the remaining DEC pathotypes investigated. In conclusion, psittacine from illegal wildlife trade in Ceará State, Brazil, presented a relevant prevalence of typical and atypical EPEC, potentially playing a role as reservoirs of DEC strains in the environment. Thus, proper control measures must be adopted to block the spread of these pathogens.

Here we describe the use of pathogenomics to identify novel potential virulence genes in the pathogenic spirochete Leptospira interrogans. Previous work to identify mechanisms of pathogenesis by gene knockouts and transposon mutagenesis has not yet yielded detailed mechanistic insights into the role of individual genes play in the pathogenesis of leptospirosis. It has long been known in the leptospirosis field that serial in vitro passage of pathogenic Leptospira yielded attenuated organisms; the converse, serial passage of liver homogenates of infected animals selects for virulence. A previous study explored the genomic and proteomic differences between a pathogenic L. interrogans serovar Lai strain 56601, and an avirulent strain IPAV [41]. These data must be carefully considered because the analyzed strains are not isogenic (the IPAV strain is of unknown provenance since details of its original isolation are unavailable) nor do they provide any in vivo relevance for identified genes, focusing instead on proteomic differences between strains during in vitro EMJH culture. Our current study, which employed whole genome sequence comparison of an attenuated strain with its isogenic pathogenic parent, yielded a small set of protein coding genes (CDS) with point mutations. While most of the 11 specific mutations found here cannot be quantitatively attributed to specific aspects of virulence or pathogenicity, our pathogenomic approach yielded the identification of a novel leptospiral AGC

Multiple factors can be involved in the virulence processes of Aeromonas hydrophila. The objective of the present paper was to verify the presence of aerolysin, hidrolipase, elas- tase and lipase virulence genes through the polymerase chain reaction (PCR) in A. hydro- phila isolates obtained from ish of the São Francisco River Valley, and to evaluate virulence according to the presence of these genes in Nile tilapia ingerlings. One hundred and fourte- en isolates from the bacteria were used. DNA was heat extracted and PCR undertaken using speciic primers described in the literature. For in vivo tests Nile tilapia ingerlings were used. From the PCR tests, negative isolates for all genes tested were selected, positive isola- tes for two genes (aerolysin and elastase) and positive for the four genes tested. These were inoculated at a concentration of 10 8 UFC/ml into the tilapias, considered as treatments;

Copyright © 2014 Juliana M. A. Agostinho et al. his is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Pyometra is recognized as one of the main causes of disease and death in the bitch, and Escherichia coli is the major pathogen associated with this disease. In this study, 70 E. coli isolates from the uteri horn, mouth, and rectum of bitches sufering from the disease and 43 E. coli isolates from the rectum of clinically healthy bitches were examined for the presence of uropathogenic virulence genes and susceptibility to antimicrobial drugs. DNA proiles of isolates from uteri horn and mouth in bitches with pyometra were compared by REP, ERIC, and BOX-PCR. Virulence gene frequencies detected in isolates from canine pyometra were as follows: 95.7% im, 27.1% iss, 25.7% hly, 18.5% iuc, and 17.1% usp. Predominant resistance was determined for cephalothin, ampicillin, and nalidixic acid among the isolates from all sites examined. Multidrug resistance was found on ∼50% pyometra isolates. Using the genotypic methods some isolates from uteri, pus, and saliva of the same bitch proved to have identical DNA proiles which is a reason for concern due to the close relationship between household pets and humans.

For decades, the virulence of P. brasiliensis has been a controversial issue because it embodies a great number of variables [30e33] . Being a characteristic agent that is respon- sible for a human disease, its definition necessarily bears a correlation with the disease severity produced by any given isolate. In humans, however, virulence cannot be measured. In animal models, virulence varies from the inoculation site to the animal species used. Furthermore, besides patient’s lungs, isolates come from a variety of different sources such that the proper habitat of the fungus is, as yet, unknown [34]. In order to achieve the goal of identifying a P. brasiliensis virulence biomarker that would provide for some much needed clarity in this field of research, the use of a model in which clearly defined parameters are defined is indispensable. Among these parame- ters are the use of the same animal, the same inoculation route, and the same isolate, whose degree of virulence is clear, even if dependent on artificial procedures such as long times of in vitro cultivation. We closely followed these parameters and proce- dures here. None of these considerations, however, restrained the reporting of several results from different groups that, so far, left us without the identification of any reliable P. brasiliensis virulence marker [30e32,35e37] . The lack of efficient classical genetic techniques for silencing suspected virulence genes contributed to this failure. Characterizing such a molecule or its ligand, for virulent isolate(s), represented the main objective of this work.

epidemiological case-control study using, as a tool of detection, the polymerase chain reaction (PCR). Our other goals were to investigate the nutritional impact of infection (cases) or colonization (controls) for Campylobacter sp., to determine the presence of three virulence genes of C. jejuni cytolethal distending toxin (CDT), and to evaluate the occurrence of inflammation in intestinal infections caused by Campylobacter sp. The study population consisted of 83 cases and 83 controls, where the cases consisted of children with a history of diarrhea in the 14 days prior to selection for the study. We assessed socioeconomic parameters through an epidemiological questionnaire. Anthropometric measurements were collected to determine z-score parameters for assessing the nutritional status of the children. Detection of Campylobacter from frozen samples was performed by enzyme-linked immunosorbent assay (ELISA) and PCR. Also, using PCR technology, we investigated the presence of C. jejuni genes cdtA, cdtB and cdtC. Intestinal inflammation was assessed by semi-quantitative ELISA