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Klebsiella pneumoniae genellikle immünsupresif hastaları etkileyen ve hastane kaynaklı enfeksiyonlara neden olan fırsatçı bir patojendir. K.pneumoniae özellikle kapsül polisakkaridi, hipermukoviskozite (HV), fimbria, toksinler ve demir alım determinantları gibi pek çok virülans faktörüne sahiptir. Bu çalışmanın amacı, iki yıl içerisinde hastane kaynaklı enfeksiyonlardan izole edilen K.pneumoniae izolatlarında çeşitli virülans faktörlerini araştırmaktır. Selçuk Üniversitesi Tıp Fakültesi Hastanesi Tıbbi Mikrobiyoloji Laboratuvarında 2011-2013 yılları arasında çeşitli kliniklerde hastane enfeksiyonu tanısı almış hastalara ait örneklerden izole edilen 53 adet K.pneumoniae izolatı çalışmaya alınmıştır. İzolatların tanımlama ve antimikrobiyal duyarlılık testleri VITEK 2 otomatik sistemi (bioMerieux, Almanya) ile yapılmıştır. Biyofilm oluşturma yetenekleri, alfa hemolizin üretimi, kapsül ve hipermukoviskozite özellikleri fenotipik yöntemlerle araştırılmıştır. Adezin kodlayan virülans genleri (fimH-1, mrkD, kpn, ycfM), siderofor genleri (entB: enterobactin, iutA: aerobactin, irp-1, irp-2, ybtS, fyuA: yersiniabactin, iroN: catechols receptor) protektin veya invazin (rmpA, magA, traT) ve toksin (hlyA, cnf-1) virülans genlerinin varlığı polimeraz zincir reaksiyonu (PCR) ile araştırılmıştır. Toplam 53 K.pneumoniae izolatının 12 (%22.6)'si reanimasyon yoğun bakım ünitesi, 8 (%15.1)'i tıbbi onkoloji servisi, 7 (%13.2)'si yenidoğan yoğun bakım ünitesi ve 26 (%49)'sı diğer servislerde yatan hastalardan izole edilmiştir. Örneklere göre izolatların dağılımı şu şekildedir: 14 (%26.4)'ü idrar, 13 (%24.5)'ü kan, 10 (%18.9)'u drenaj sıvısı, 8 (%15.1)'i yara, 7 (%13.2)'si bronkoalveoler lavaj ve 1 (%19)'i beyin omurilik sıvısından üretilmiştir. İzolatların %5.7'si meropeneme dirençli, %71.7'si genişlemiş spektrumlu beta-laktamaz üretimi pozitif olarak bulunmuştur. Kapsül, biyofilm formasyonu ve HV görülme sıklığı sırasıyla %100, %79.2 ve %1.9 olarak saptanmıştır. Alfa hemolizin varlığı tespit edilmemiştir. entB (%96.2), ycfM (%86.8) ve mrkD (%83.0) genleri en yüksek düzeyde saptanan genler olmuştur. Araştırılan diğer genler; fimH-1 (%64.2), fyuA (%54.7), kpn (%49.1), ybtS (%41.5), irp-1(%41.5), irp-2 (%37.7), traT (%11.3) ve iutA (%5.7%) farklı sıklıklarda saptanmıştır. İzolatlarda iroN, rmpA, magA, hlyA ve cnf-1 genleri tespit edilmemiştir. Enterobaktin geni sideroforlar arasında, ycfM ve mrkD genleri adezinler arasında en sık saptanan virülans genleri olmuştur. Kapsül ve biyofilm oluşumu izolatlarda sıklıkla belirlenmiştir. Hipermukoviskozite yalnızca bir izolatta belirlenmiş ancak ilişkili genler tespit edilmemiştir. Sonuç olarak; alfa hemolizin üretimi, hlyA ve cnf-1 genleri hiçbir izolatta gözlenmemiştir. Hastane enfeksiyonu etkeni K.pneumoniae izolatlarında kapsül, adezinler, enterobaktin ve biyofilm oluşturma bu izolatların patojenitelerinin temelini oluşturmuştur. K.pneumoniae'ya bağlı hastane enfeksiyonlarının kontrolünde antibiyotik direncinin yanı sıra toksin ve invazyon yeteneğinin sürekli takip edileceği yeni çalışmalara ihtiyaç vardır., Klebsiella pneumoniae is an opportunistic pathogen that commonly affects immunosuppressed patients and causes nosocomial infections. K.pneumoniae has a variety of virulence factors, especially capsule polysaccharide, hypermucoviscosity (HV), fimbriae, toxins and determinants for iron acquisition. The aim of this study was to detect the virulence factors in K.pneumoniae strains isolated from nosocomial infections in two years. Fifty three K.pneumoniae strains isolated from the samples of patients with nosocomial infections in the Medical Microbiology Laboratory of Selcuk University Faculty of Medicine Hospital between 2011 and 2013 were included in the study. Identification and antimicrobial susceptibilities of the isolates were performed by VITEK 2 automatic system. Biofilm formation,?-hemolysin, capsule and HV were investigated by phenotypic methods. Polymerase chain reaction (PCR) was used to detect virulence genes encoding adhesins (fimH-1, mrkD, kpn, ycfM), siderophores (entB: enterobactin, iutA: aerobactin, irp-1, irp-2, ybtS, fyuA: yersiniabactin, iroN: catechols receptor), protectines or invasins (rmpA, magA, traT) and toxins (hlyA, cnf-1). Of the 53 K.pneumoniae isolates,12 (22.6%) were isolated from in patients of reanimation intensive care unit, 8 (15.1%) medical oncology, 7 (13.2%) newborn intensive care unit and 26 (49%) other clinics. The distribution of the isolates according to the samples was as follows: urine (n 14), blood (n 13), wound (n 8), drainage fluid (n 10), broncho-alveolar lavage (n 7), and cerebrospinal fluid (n 1). Isolates which were resistant to meropenem were 5.7% and production of extended spectrum beta-lactamase (ESBL) was 71.7%. The capsule, biofilm formation, and HV were observed in 100%, 79.2%, and 1.9% of the isolates, respectively. Production of ?-hemolysin was not detected in any of the isolates. The genes; entB (96.2%), ycfM (86.8%), and mrkD (83.0%) showed high prevalence. The other genes were detected in different ratios: fimH-1 (64.2%), fyuA (54.7%), kpn (49.1%), ybtS (41.5%), irp-1(41.5%), irp-2 (37.7%), traT (11.3%) and iutA (5.7%). Virulence genes; iroN, rmpA, magA, hlyA and cnf-1 were not detected in any of the isolates. Enterobactin had the highest rate among siderophores, and ycfM and mrkD in adhesins. The capsule and biofilm formation were commonly found in the isolates. Hypermucoviscosity was only found in one isolate but associated genes were not detected. Alfa hemolysin production and hlyA gene were not determined. As a result, it seems that the basis of the pathogenicity of K.pneumoniae strains isolated from nosocomial infections are capsule, adhesins, enterobactin and ability of biofilm formation. There is a need for new studies for the continuous monitoring of toxin and invasion ability as well as antibiotic resistance in the control of hospital infection caused by K.pneumoniae.

Foodstuffs of animal origin may present hazards, due to bacterial contamination. This study was conducted to determine Escherichia coli in some types of animal source foods (raw milk, raw beef meat and raw poultry meat). Bacteriological examination of total 246 raw food samples of animal origin, raw milk, (105); beef meat (31) and poultry meat (110) were collected from different localities in Ismailia City showed that 64/246 (26.01%) of samples were infected with E. coli. Serological identification of (10) E. colistrains revealed that they were belonged to O111 polyvalent 1; O44 polyvalent 2 (2 strains for each); O78 polyvalent 4; O146 polyvalent 2; O26 polyvalent 1; O119 polyvalent 1; O86 polyvalent 1 and O18 polyvalent 3 (1 strain for each). E. coli strains showed high susceptible rate to Ciprofloxacin (CIP), (100%) and high resistance (100%) to Ampicillin (AMP), Metronidazole (MTZ), Amoxicillin (AML), and Vancomycin (VAS). Four strains of the isolated E. coli were submitted to molecular studies for detection of the eaeA (protein intimin), iutA (encoding aerobactin) and iss (increased serum survival protein) genes, by using PCR technique.

Vibrio mimicus is a bacterium that causes gastroenteritis; it is closely related to Vibrio cholerae, and can cause acute diarrhea like cholera- or dysentery-type diarrhea. It is distributed worldwide. Factors associated with virulence (such as hemolysins, enterotoxins, proteases, phospholipases, aerobactin, and hemagglutinin) have been identified; however, its pathogenicity mechanism is still unknown. In pathogenic Vibrio species such as V. cholerae, Vibrio. parahaemolyticus and Vibrio vulnificus, capsule, biofilms, lateral flagellum, and type IV pili are structures described as essential for pathogenicity. These structures had not been described in V. mimicus until this work. We used 20 V. mimicus strains isolated from water (6), oyster (9), and fish (5) samples and we were able to identify the capsule, biofilm, lateral flagellum, and type IV pili through phenotypic tests, electron microscopy, PCR, and sequencing. In all tested strains, we observed and identified the presence of capsular exopolysaccharide, biofilm formation in an in vitro model, as well as swarming, multiple flagellation, and pili. In addition, we identified homologous genes to those described in other bacteria of the genus in which these structures have been found. Identification of these structures in V. mimicus is a contribution to the biology of this organism and can help to reveal its pathogenic behavior.

Avian pathogenic Escherichia coli (APEC) is one of the most economically devastating pathogens affecting the poultry industry. This group of extra-intestinal E. coli causes a variety of clinical conditions including airsacculitis and cellulitis. The economic impact of APEC is mainly due to mortality, slower growth rates, and carcass downgrading. In commercial broiler operations, APEC infections are controlled indirectly by vaccination against other respiratory diseases and minimizing stress conditions, and directly by administration of antimicrobial agents to suppress the infection in already infected flocks. The fact that most APEC strains possess some common virulence factors suggests that an effective vaccine against APEC is a viable option. The most important virulence factors that have been investigated over the years include type I and P fimbriae, aerobactin iron-acquisition system, and serum resistance traits. Despite the potential for developing an efficacious vaccine to combat this economically important poultry disease, several obstacles hinder such efforts. Those obstacles include the cost, vaccine delivery method and timing of vaccination as the birds should be immune to APEC by 21 days of age. Herein, we review the various attempts to develop an effective vaccine against the respiratory form of APEC diseases in poultry. We also discuss in-depth the potentials and limitations of such vaccines.

There is conflicting data regarding whether commercial chilling has any effect on persistence of Salmonella serovars, including antibiotic resistant variants, on chicken carcasses. A total of 309 Salmonella Typhimurium and Salmonella Kentucky isolates recovered from pre- and post-chill whole broiler carcasses were characterized for genetic relatedness using Pulsed Field Gel Electrophoresis (PFGE) and for the presence of virulence factors (invA, pagC, spvC) by PCR and for aerobactin and colicin production by bioassays. A subset of these isolates (n ¼ 218) displaying resistance to either sulfisoxazole and/or ceftiofur [S. Typhimurium (n ¼ 66) and S. Kentucky (n ¼ 152)] were further tested for the presence of associated antibiotic resistance elements (class-I integrons and blaCMY genes) by PCR. All 145 ceftiofur resistant S. Kentucky and S. Typhimurium isolates possessed blaCMY genes. Class-I integrons were only detected in 6.1% (n ¼ 4/66) of sulfisoxazole resistant S. Typhimurium isolates. The PFGE analysis revealed the presence of genetically diverse populations within the recovered isolates but clusters were generally concordant with serotypes and antimicrobial resistance profiles. At a 100% pattern similarity index, thirty-six percent of the undistinguishable S. Typhimurium and 22% of the undistinguishable S. Kentucky isolates were recovered from the same chilling step. All isolates possessed the invA and pagC genes, but only 1.4%possessed spvC. Irrespective of the chilling step, there was a significant difference (P < 0.05) in the production of aerobactin and colicin between S. Typhimurium and S. Kentucky isolates. Taken together, these results indicate that chilling impacted the recovery of particular Salmonella clonal groups but had no effect on the presence of class-I integrons, blaCMY genes, and tested virulence factors.

Extraintestinal pathogenicEscherichia coli(ExPEC) is one of the main etiological agents of bloodstream infections caused by Gram-negative bacilli. In the present study, 20E. coliisolates from human hemocultures were characterized to identify genetic features associated with virulence (pathogenicity islands markers, phylogenetic group, virulence genes, plasmid profiles, and conjugative plasmids) and these results were compared with commensal isolates. The most prevalent pathogenicity island, in strains from hemoculture, were PAI IV536, described by many researchers as a stable island in enterobacteria. Among virulence genes,iutA gene was found more frequently and this gene enconding the aerobactin siderophore receptor. According to the phylogenetic classification, group B2 was the most commonly found. Additionally, through plasmid analysis, 14 isolates showed plasmids and 3 of these were shown to be conjugative. Although in stool samples of healthy people the presence of commensal strains is common, human intestinal tract may serve as a reservoir for ExPEC.

We examined 50 Escherichia coli (E. coli) strains isolated from broiler chickens between January 2013 to March 2014 in order to evaluate the epidemiological prevalence of avian pathogenic E. coli (APEC) in Jordan by multiplex PCR and random amplification of polymorphic DNA (RAPD) tests. The multiplex polymerase chain reaction (PCR) which was used as tentative criteria of APEC targets 8 virulence associated genes; enteroaggregative toxin (astA), Type 1 fimbria adhesion (fimH), iron-repressible protein (irp2), P fimbriae (papC), aerobactin (iucD), temperature-sensitive hemagglutinin (tsh), vacuolating autotransporter toxin (vat), and colicin V plasmid operon (cva/cvi) genes. The number of detected genes could be used as a reliable index of their virulence. E. coli strains already typed as an APEC always harbor 5 to 8 genes, but non-APEC strains harbor less than 4 genes. Assuming the criteria of an APEC is possession of 5 or more virulence associated genes; we found that all 50 E. coli strains were classified as APEC strains. The RAPD analysis showed that the E. coli strains could be grouped into 35 of RAPD types by using these two different RAPD primer sets, RAPD analysis primer 4 5'AAGAGCCCGT5', and RAPD analysis primer 6 5'CCCGTCAGCA3'. The current study confirmed the endemic nature of APEC in broiler flocks in Jordan. It is essential that the biosecurity on poultry farms should be improved to prevent the introduction and dissemination of APEC and other agents. Furthermore, farmers need to be educated about the signs, lesions, and the importance of this agent.

Multidrug-resistant and hypervirulent Klebsiella pneumoniae (hvKP) poses a significant risk to public health. To better understand the molecular characteristics of multidrug-resistant and hypervirulent K. pneumoniae of animal origin, fifteen K. pneumoniae strains from the liver, blood of sick pigs and chicken feces were collected. All K. pneumoniae isolates were subjected to antimicrobial susceptibility testing, string test, multi-locus sequence typing and whole genome sequencing. Seven K. pneumoniae isolates were found carrying the mcr-1.1 gene. Among them, a multidrug-resistant and hypervirulent K. pneumoniae strain SCsl1 isolated from the liver of a diseased pig was found to harbor 16 resistance genes (e.g., mcr-1.1) and 16 virulence genes including aerobactin. Moreover, a novel integrative and conjugative element, named ICEKpSL1, was identified in SCsl1, which contains a full Yersinia high-pathogenicity island (HPI). This element could be excised from the chromosome to form a circular intermediate, indicating potential transmission of the Yersinia pathogenicity island. The emergence of multidrug-resistance and hypervirulence in K. pneumoniae from animals warrants further surveillance.

Woodybactins A-D are a suite of new fatty acyl siderophores produced by the luminous marine bacterium Shewanella woodyi MS32. While this bacterium has a set of genes homologous to the biosynthetic gene cluster for aerobactin, aerobactin is not produced. The arrangement of these genes within the genome differs in S. woodyi MS32 when compared to E. coli and other species producing siderophores similar to aerobactin, and one synthetase gene which would append a second acyl-hydroxylysine to the terminal carboxylate of citrate is not functional. Within the suite of woodybactins A-D, which differ by the fatty acid appendage, one contains an unusual C9 (9:0) fatty acid and one contains a unique branched C9 iso (9:0 iso) fatty acid, as well as a C8 (8:0) and C10 (10:0) fatty acid.

Graphical abstract, Highlights • Ligand recognition by antibacterial Siderocalin controls the competition for iron during infection. • We determined nine crystal structures of Siderocalin mutants with ligands. • We determined three candidate ligands did not bind. • We determined the crystal structure of SBP YfiY. • Multiplexed specificity of Siderocalin was determined., Siderocalin/Lipocalin 2/Neutrophil Gelatinase Associated Lipocalin/24p3 is an innate immune system protein with bacteriostatic activity, acting by tightly binding and sequestering diverse catecholate and mixed-type ferric siderophores from enteric bacteria and mycobacteria. Bacterial virulence achieved through siderophore modifications, or utilization of alternate siderophores, can be explained by evasion of Siderocalin binding. Siderocalin has also been implicated in a wide variety of disease processes, though often in seemingly contradictory ways, and has been proposed to bind to a broader array of ligands beyond siderophores. Using structural, directed mutational, and binding studies, we have sought to rigorously test, and fully elucidate, the Siderocalin recognition mechanism. Several proposed ligands fail to meet rigorous binding criteria, including the bacterial siderophore pyochelin, the iron-chelating catecholamine hormone norepinephrine, and the bacterial second messenger cyclic diguanylate monophosphate. While possessing a remarkably rigid structure, in principle simplifying analyses of ligand recognition, understanding Scn recognition is complicated by the observed conformational and stoichiometric plasticity, and instability, of its bona fide siderophore ligands. Since the role of Siderocalin at the early host/pathogen interface is to compete for bacterial ferric siderophores, we also analyzed how bacterial siderophore binding proteins and enzymes alternately recognize siderophores that efficiently bind to, or evade, Siderocalin sequestration – including determining the crystal structure of Bacillus cereus YfiY bound to schizokinen. These studies combine to refine the potential physiological functions of Siderocalin by defining its multiplexed recognition mechanism.

The composition of Escherichia coli in the neonatal gut has rarely been studied in developing countries. To gain insight into the composition of E. coli in the neonatal gut and to assess factors that could influence colonization by E. coli, analysis of the phylogenetic groups and virulence determinants of E. coli isolated from the guts of neonates in a tertiary care hospital was carried out. The distribution of the phylogroups of 124 E. coli isolates recovered showed that phylogroups A (23 %) and B1 (49 %) accounted for 72 % of the isolates. Isolates of the phylogenetic group B2 were rare (8 %). Virulence factors were also rare with the exception of aerobactin (iucC), which was detected in 45 % of the isolates and was significantly associated with phylogroup B1. Multinomial logistic regression established that colonization with phylogroup B1 was associated with a stay in the neonatal intensive care unit; phylogroup A was associated with a stay on the ward; and phylogroups B2 and D were associated with neonates delivered vaginally. Evaluation of the effect of different E. coli phylogroups, with and without identified virulence determinants, on the gut of neonatal mice showed histopathological changes in the mucosa. The severity of the changes could be correlated with the presence of virulence determinants, irrespective of the phylogroup.

Bdellovibrio and like organisms (BALO) are obligate predators of Gram-negative bacteria, belonging to the α- and δ-proteobacteria. BALO prey using either a periplasmic or an epibiotic predatory strategy, but the genetic background underlying these phenotypes is not known. Here we compare the epibiotic Bdellovibrio exovorus and Micavibrio aeruginosavorus to the periplasmic B. bacteriovorus and Bacteriovorax marinus. Electron microscopy showed that M. aeruginosavorus, but not B. exovorus, can attach to prey cells in a non-polar manner through its longitudinal side. Both these predators were resistant to a surprisingly high number of antibiotic compounds, possibly via 26 and 19 antibiotic-resistance genes, respectively, most of them encoding efflux pumps. Comparative genomic analysis of all the BALOs revealed that epibiotic predators have a much smaller genome (ca. 2.5 Mbp) than the periplasmic predators (ca. 3.5 Mbp). Additionally, periplasmic predators have, on average, 888 more proteins, at least 60% more peptidases, and one more rRNA operon. Fifteen and 219 protein families were specific to the epibiotic and the periplasmic predators, respectively, the latter clearly forming the core of the periplasmic ‘predatome', which is upregulated during the growth phase. Metabolic deficiencies of epibiotic genomes include the synthesis of inosine, riboflavin, vitamin B6 and the siderophore aerobactin. The phylogeny of the epibiotic predators suggests that they evolved by convergent evolution, with M. aeruginosavorus originating from a non-predatory ancestor while B. exovorus evolved from periplasmic predators by gene loss.

Avian pathogenic Escherichia coli (APEC) are known to cause extraintestinal disease in poultry, leadingto substantial losses in the industry. IutA, iron-regulated aerobactin receptor is firmly associated with APEC. To assessthe potential of IutA to induce protective immune responses, attenuated Salmonella Typhimurium strain expressingIutA was constructed and administered orally to BALB/c mice. The IutA-specific immune responses were measuredwith sera, vaginal and fecal samples by an enzyme-linked immunosorbent assay. We found that the Salmonella-IutAvaccine induced significantly higher immune responses as compared to the control inoculated with the attenuated S.Typhimurium containing the plasmid only. The IutA-specific immune responses were increased by second immunizationat third week after initial immunization, whereas triple immunization induced lower immune responses than thoseinduced by the double immunization. The Salmonella-IutA vaccine induced a nature of immunity biased to the Th1-type, as judged by the ratio of IutA-specific IgG isotypes (IgG2a/IgG1). Overall, these results suggest that theSalmonella-IutA vaccine appear to be suitable candidate for a vaccine against APEC.Keywords : avian pathogenic Escherichia coli, boost immunization, IutA, oral vaccination

Summary Background Escherichia coli is the predominant pathogen causing urinary tract infection (UTI), the most common bacterial infectious disease encountered in clinical practice, accounting for significant morbidity and high medical costs. The severity of UTI produced by E. coli is due to the expression of a wide spectrum of virulence factors. In this study we evaluated the role of E. coli virulence determinants in the pathogenesis of UTI. Methods A total of 90 uropathogenic E. coli strains were screened by PCR for the prevalence of seven virulence genes encoding type 1 fimbriae ( fimH ), pili associated with pyelonephritis ( pap ), S and F1C fimbriae ( sfa and foc ), afimbrial adhesins ( afa ), cytotoxic necrotizing factor ( cnf ), hemolysin ( hly ), and aerobactin ( aer ). Results The prevalence of genes coding for fimbrial adhesive systems was 68% for fimH , 41% for pap , and 34% for sfa/foc . The operons coding for afa afimbrial adhesins were identified in 20% of strains. The hly and cnf genes coding for toxins were amplified in 19% and 3% of strains, respectively. A prevalence of 52% was found for the aer gene. The various combinations of detected genes were designated as virulence patterns. The strains isolated from hospitalized patients displayed a great diversity of gene associations compared to those isolated from ambulatory patients. Conclusions Our study showed that investigation of the bacterial pathogenicity associated with UTI may contribute to a better medical intervention.

Birds of the Cracidae family (curassows, guans, and chachalacas) are endemic of the Neotropics and 50 species are currently classified. Brazil has 22 species, seven of which are considered threatened. The Alagoas Curassow (Pauxi mitu) species is considered extinct in the wild; but about 120 birds are alive in captivity. Conservation of this species depends entirely on correct management. Health reports of both wildlife and captive curassows are rare. In this study the presence of Escherichia coli was evaluated in 23 healthy Alagoas Curassows from two private breeding centres. E. coli was isolated from cloacal swabs, and the presence of genes encoding cytotoxic necrotising factor 1 (cnf1), alpha-haemolysin (hly), aerobactin (iuc), serum resistance (iss) and the following adhesions: S fimbriae (sfa), pili associated with pyelonephritis (pap) and temperature-sensitive haemagglutinin (tsh) were investigated. E. coli was isolated from 78.3% (18/23) of the birds, and the percentage of curassows colonized by E. coli was similar between the two facilities. From the 22 E. coli isolates, 15 (68.2%) were positive for at least one virulence factor by PCR, and the most frequently found gene was iss (50%). No curassows had clinical signs of disease. Nevertheless, the presence of some E. coli strains may be a concern to the wildlife in captivity. Additional health surveillance studies are essential to guarantee successful conservation programmes for threatened cracids in Brazil. Aves da família Cracidae (mutuns, jacutingas e aracuãs) são endêmicas da região Neotropical com 50 espécies atualmente classificadas. O Brasil possui 22 espécies nesta família e sete delas são consideradas ameaçadas de extinção. O mutum-do-nordeste (Pauxi mitu) é considerado extinto na natureza, no entanto, aproximadamente 120 indivíduos são mantidos em cativeiro. A conservação desta espécie depende inteiramente de um manejo correto. Informações sobre o status sanitário de mutuns são raras, tanto em vida livre quanto em cativeiro. Neste estudo a presença de Escherichia coli foi avaliada em 23 mutuns-do-nordeste sadios de dois criatórios particulares. E. coli foi isolada a partir de suabes cloacais, em seguida, foi avaliada a presença de genes que codificam fator citotóxico necrotizante 1 (cnf1), alfa-hemolisina (hly), produção de aerobactina (iuc) e resistência sérica (iss) e genes que codificam os seguintes fatores de virulência: fímbria S (sfa), pili associado à pielonefrite (pap) e hemaglutinina termosensível (tsh). E. coli foi isolada de 78,3% (18/23) das aves e o percentual de mutuns positivos para E. coli foi semelhante entre as duas criações. De 22 isolados de E. coli, 15 (68,2%) foram positivos para pelo menos um fator de virulência pela PCR e o gene mais frequente foi o iss (50%). Nenhuma ave apresentava sinal clínico de doença, no entanto, a presença de determinadas cepas de E. coli pode representar uma preocupação em relação às aves silvestres mantidas em cativeiro. Estudos adicionais de monitoria do status sanitário do plantel são essenciais para garantir o sucesso de futuros programas de conservação de cracídeos ameaçados no Brasil.

An attenuated Salmonella (deltalon, deltacpxR, and deltaasdA16) delivery system containing the genes encoding P-fimbriae (papa and papG), aerobactin receptor (iutA), and CS31A surface antigen (clpG) of avian pathogenic Escherichia coli (APEC) was constructed, and its potential as a vaccine candidate against APEC infection in chickens was evaluated. The birds were divided into three groups designated group A (nonvaccinated control), group B (given a single immunization), and group C (administered prime and boost immunizations). Prime and booster vaccinations with the constructions were administered to 1-day-old and 14-day-old birds, respectively. Immune responses were measured postimmunization, and the birds were challenged via an intra-air sac route with a virulent APEC strain at the second, third, and fourth weeks of age. Group B birds were partially protected against the challenge and showed increased levels of plasma immunoglobulin (Ig)G, mucosal IgA antibodies, and lymphocyte proliferation. Group C birds showed greater protection against the challenge, with significantly stronger immune responses compared with the birds in the other groups. Overall, our data suggest that the Salmonella delivery system with recombinant constructs is capable of inducing robust immune responses and induces effective protection against colibacillosis caused by APEC.

Avian pathogenic Escherichia coli (APEC) causes respiratory disease and sepsis in poultry. To persist in its host, E. coli requires essential nutrients including iron. Since iron is limited in extra-intestinal tissues, E. coli produces siderophores, small molecules with high affinity for ferric iron, to sequester this essential nutrient. To counter bacterial siderophore systems, mammalian hosts secrete siderocalin (also called lipocalin 2 or NGAL), which binds ferric-siderophore complexes rendering them unavailable to bacteria. In humans and mice, siderocalin is known to play a role in primary defense against bacterial infections. In poultry, 4 proteins display homology to the human NGAL (CALβ, CALγ, Ggal-C8GC and Ex-FABP). The function and expression of the genes coding for these 4 proteins during infection by APEC is still unknown. Expression levels of these genes were determined by quantitative RT-PCR using RNA extracted from lungs, livers and spleens of healthy 3-week-old chickens and chickens infected with APEC. The gene coding for Ex-FABP was overexpressed in all organs tested. It was significantly more overexpressed in the lungs and liver than in the spleen (37.3 and 27.3 times versus 11.5 times, respectively). The genes coding for Calβ and Calγ were also found significantly overexpressed in the liver (27 and 8.2 times, respectively). To confirm the function of Ex-FABP as a siderocalin, the gene coding for this protein was cloned in an expression vector and the protein was purified. In vitro growth inhibition of E. coli strains by Ex-FABP was assayed in parallel with growth inhibition caused by human siderocalin. Purified Ex-FABP inhibited growth of E. coli K-12, which only produces the siderophore enterobactin. However, E. coli strains producing pathogen-associated siderophores including salmochelins (glucosylated enterobactin), aerobactin and yersiniabactin grew normally in the presence of Ex-FABP. These results indicate that Ex-FABP is an avian siderocalin with a siderophore-binding activity similar to that of human siderocalin and that pathogen-specific siderophores are required by APEC to overcome this innate defense protein in poultry.

BackgroundEscherichia coli bovine mastitis is a disease of significant economic importance in the dairy industry. Molecular characterization of mastitis-associated E. coli (MAEC) did not result in the identification of common traits. Nevertheless, a mammary pathogenic E. coli (MPEC) pathotype has been proposed suggesting virulence traits that differentiate MAEC from commensal E. coli. The present study was designed to investigate the MPEC pathotype hypothesis by comparing the genomes of MAEC and commensal bovine E. coli.ResultsWe sequenced the genomes of eight E. coli isolated from bovine mastitis cases and six fecal commensal isolates from udder-healthy cows. We analyzed the phylogenetic history of bovine E. coli genomes by supplementing this strain panel with eleven bovine-associated E. coli from public databases. The majority of the isolates originate from phylogroups A and B1, but neither MAEC nor commensal strains could be unambiguously distinguished by phylogenetic lineage. The gene content of both MAEC and commensal strains is highly diverse and dominated by their phylogenetic background. Although individual strains carry some typical E. coli virulence-associated genes, no traits important for pathogenicity could be specifically attributed to MAEC. Instead, both commensal strains and MAEC have very few gene families enriched in either pathotype. Only the aerobactin siderophore gene cluster was enriched in commensal E. coli within our strain panel.ConclusionsThis is the first characterization of a phylogenetically diverse strain panel including several MAEC and commensal isolates. With our comparative genomics approach we could not confirm previous studies that argue for a positive selection of specific traits enabling MAEC to elicit bovine mastitis. Instead, MAEC are facultative and opportunistic pathogens recruited from the highly diverse bovine gastrointestinal microbiota. Virulence-associated genes implicated in mastitis are a by-product of commensalism with the primary function to enhance fitness in the bovine gastrointestinal tract. Therefore, we put the definition of the MPEC pathotype into question and suggest to designate corresponding isolates as MAEC.

Vibrio vulnificus, an opportunistic pathogen that causes a serious, often fatal, infection in humans, requires iron for its growth. This bacterium utilizes iron from the environment via the vulnibactin-mediated iron uptake system. The mechanisms of vulnibactin biosynthesis, vulnibactin export, and ferric-vulnibactin uptake systems have been reported, whereas the ferric-vulnibactin reduction mechanism in the cell remains unclear. The results of our previous study showed that VuuB, a member of the flavin adenine dinucleotide-containing siderophore-interacting protein family, is a ferric-vulnibactin reductase, but there are other reductases that can complement for the defective vuuB. The aim of this study was to identify these proteins that can complement the loss of function of VuuB. We constructed mutants of genes encoding putative reductases in V. vulnificus M2799, and analyzed their growth under low-iron conditions. Complementation analyses confirmed that IutB, which functions as a ferric-aerobactin reductase, participates in ferric-vulnibactin reduction in the absence of VuuB. This is the first genetic evidence that ferric-vulnibactin is reduced by a member of the ferric-siderophore reductase protein family. In the aerobactin-utilization system, IutB plays a major role in ferric-aerobactin reduction in V. vulnificus M2799, and VuuB and DesB can compensate for the defect of IutB. Furthermore, the expression of iutB and desB was found to be regulated by iron and a ferric uptake regulator.

Klebsiella pneumoniae with hypermucoviscosity (HM) phenotype is generally more virulent than HM-negative strains. The aim of this study was to investigate the prevalence of HM phenotype among urinary isolates and to compare the virulence factors, antimicrobial susceptibility patterns and clinical characteristics of HM-positive and -negative K. pneumoniae isolated from urine of hospitalized patients.From June to October 2013, a total of 81 non-repetitive K. pneumoniae strains were isolated from urine. HM phenotype was determined by a string test. The K1 and K2 genotypes, the allS, kfu, rmpA, rmpA2 and wabG, aerobactin gene were detected by polymerase chain reaction.Of the 81 K. pneumoniae isolates, 12.3% produced a positive string test. The aerobactin (80.0%[8/10] vs. 15.5%[11/71], p = .0001), allS (40.0%[4/10] vs. 9.9%[7/71], p = .009), rmpA (70.0%[7/10] vs. 14.1%[10/71], p = .0001) and rmpA2 (60.0%[6/10] vs. 16.9%[12/71], p = .002) genes were more prevalent in HM positive than in HM negative strains. The K1 (20.0%[2/10) vs. 8.5%[6/71]] and K2 (30.0%[3/10] vs. 4.2%[3/71]) capsular serotypes were more common in HM strains than in non-HM strains (p = .0001). HM-positive K. pneumoniae isolates were more susceptible to amoxicillin/clavulanic acid (p = .02), cefazolin (p = .03), cefotaxime (p = .02) and ciprofloxacin (p = .03) than HM-negative isolates. Multivariate analysis showed that HM phenotype (OR, 23.87; 95% CI, 3.91-145.4, p .01) and age60 years (OR, 8.33; 95% CI, 1.25-55.31, p = .03) were significant risk factors for concurrent bacteraemia.Klebsiella pneumoniae with expression of HM phenotype isolated from urine were more likely to be associated with concurrent bacteraemia than isolates without the HM phenotype, and were more susceptible to antibiotics. Physicians have to be aware of the possibility of bacteraemia in patients with K. pneumoniae bacteriuria, especially if strains are HM positive and the patient is60 years old.

P fimbriae, enabling adherence to colonic and urinary epithelium, and aerobactin, an iron sequestering system, are both colonization factors in the human colon and virulence factors for urinary tract infection. The colonic microbiota is suggested to be a site suitable for the transfer of antibiotic resistance genes. We investigated whether phenotypic resistance to antibiotics in commensal and uropathogenic Escherichia coli from infants and young children is associated with carriage of virulence genes and to phylogenetic group origin and, in the case of fecal strains, to persistence in the gut and fecal population levels. The commensal strains (n = 272) were derived from a birth cohort study, while the urinary isolates (n = 205) were derived from outpatient clinics. Each strain was assessed for phenotypic antibiotic resistance and for carriage of virulence genes (fimA, papC, sfaD/E, hlyA, iutA, kfiC, and neuB), phylogenetic group (A, B1, B2, or D), and markers of particular virulent clones (CGA-D-ST69, O15:H1-D-ST393, and O25b:H4-B2-ST131). Resistance to ampicillin, tetracycline, and trimethoprim was most prevalent. Multivariate analysis showed that resistance to any antibiotic was significantly associated with carriage of genes encoding P fimbriae (papC) and aerobactin (iutA), and a phylogenetic group D origin. Neither fecal population numbers nor the capacity for long-term persistence in the gut were related to antibiotic resistance among fecal strains. Our study confirms the importance of phylogenetic group D origin for antibiotic resistance in E. coli and identifies the virulence genes papC and iutA as determinants of antibiotic resistance. The reason for the latter association is currently unclear.

Introduction: This retrospective study investigated the clinical etiology of community-acquired bacteremic Klebsiella pneumoniae infections, and characterized laboratory and genetic markers which may be associated with primary liver abscess (PLA). Methods: Community-onset K. pneumoniae bacteremic episodes from 2010 to 2011 were identified from the laboratory information system. Isolates were retrieved for susceptibility testing, hypermucoviscosity testing, PCR-based serotyping (K1, K2 and K5) and PCR detection of virulence genes (rmpA, alls, kfu and aerobactin). Clinical data collected from electronic medical records included primary and secondary diagnoses, co-existing morbidities, antibiotic therapy, and in-patient mortality. Results: 129 bacteremic episodes were identified. The most common primary infections were pneumonia (n = 24, 18.6%), primary liver abscess (n = 21, 16.3%) and urinary tract infections (n = 21, 16.3%). Hypermucoviscosity was present in 55 isolates (42.6%). The most commonly detected virulence genes were aerobactin (n = 63, 48.8%) and rmpA (n = 59, 45.7%). Isolates causing liver abscess were significantly associated with a positive string test, rmpA, aerobactin gene, and capsular serotype K1 (all p

Background: Acinetobacter baumannii which is a Gram-negative bacterium can cause several different infections. The appearance of carbapenemase-producing A. baumannii in recent years has made the treatment process more difficult. The identification of virulence factors (VFs), such as nonadhesives in A. baumannii, helps to fight against related infections. Materials and Methods: A total of 104 samples from teaching hospitals in Kermanshah, Iran, were collected during a 24 months period (2011-2013). Sample identification was first carried out by biochemical tests, and then their susceptibility to carbapenems was determined using the Kirby-Bauer method. For confirmation of carbapenemase-producing A. baumannii, polymerase chain reaction (PCR) was done for carbapenemase-encoding genes. In addition, the frequency of nonadhesive VFs in carbapenemase-producing isolates was determined by PCR. Results: There were 50 isolates that were identified as carbapenemase-producing A. baumannii. The PCR results showed; 40 isolates (80%) for traT, 17 isolates (34%) for cvaC, and 8 isolates (16%) for iutA, and these encode serum resistance, colicin V and aerobactin, respectively. No significant correlation was observed between these three genes. Conclusions: The mechanism of A. baumannii virulence has always been in question. The role of VFs has also been recognized in other Gram-negative bacteria. According to the prevalence of traT, cvaC and iutA, as nonadhesive VFs, we can suggest that they could be the main mechanism of carbapenemase-producing A. baumannii pathogenesis.

Urinary tract infections impose substantial health burdens on women worldwide. Urinary tract infections often incur a high risk of recurrence and antibiotic resistance, and uropathogenic E. coli accounts for approximately 80% of clinically acquired cases. The diagnosis of, treatment of, and drug development for urinary tract infections remain substantial challenges due to the complex pathogenesis of this condition. The clinically isolated UPEC 83972 strain was found to produce four siderophores: yersiniabactin, aerobactin, salmochelin, and enterobactin. The biosyntheses of some of these siderophores implies that the virulence of UPEC is mediated via the targeting of primary metabolism. However, the differential modulatory roles of siderophore biosyntheses on the differential metabolomes of UPEC and non-UPEC strains remain incompletely understood. In the present study, we sought to investigate how the differential metabolomes can be used to distinguish UPEC from non-UPEC strains and to determine the associated regulatory roles of siderophore biosynthesis. Our results are the first to demonstrate that the identified differential metabolomes strongly differentiated UPEC from non-UPEC strains. Furthermore, we performed metabolome assays of mutants with different patterns of siderophore deletions; the data revealed that the mutations of all four siderophores exerted a stronger modulatory role on the differential metabolomes of the UPEC and non-UPEC strains relative to the mutation of any single siderophore and that this modulatory role primarily involved amino acid metabolism, oxidative phosphorylation in the carbon fixation pathway, and purine and pyrimidine metabolism. Surprisingly, the modulatory roles were strongly dependent on the type and number of mutated siderophores. Taken together, these results demonstrated that siderophore biosynthesis coordinately modulated the differential metabolomes and thus may indicate novel targets for virulence-based diagnosis, therapeutics, and drug development related to urinary tract infections.

Background: Uropathogenic Escherichia coli (UPEC) are a common causative agent of urinary tract infections. Strains of UPEC encode a number of virulence factors that facilitate their dissemination and persistence within the host. To diminish the burden of UPEC, using effective preventive measures, data on virulence factor prevalence in different geographic regions must be assessed. Objectives: As no such data was available for this geographic region of Iran, the purpose of this study was to analyze the prevalence of ten UPEC virulence genes among 100 E. coli isolates collected from patients with urinary tract infections (UTI) in Zabol, Iran. Patients and Methods: One hundred UPEC obtained from patients with urinary tract infection were screened by the polymerase chain reaction (PCR) with primers specific for the following UPEC virulence genes: astA (enterotoxins), cdtB (enterotoxins), cvi/cva (colicin V operon), ibeA (an invasive protein), iss (increased serum survival protein), iutA (aerobactin), kpsII (group 2 capsule), neuS (K1 polysialyltransferase), tsh (an adhesive and proteolytic protein), and vat (vacuolating autotransporter toxin). Results: Amongst the total of 100 UPEC isolates, 99 (99%) isolates were found to carry the studied virulence genes. Twenty-six different virulence patterns were identified. The prevalence of astA, cdtB, cvi/cva, ibeA, iss, iutA, kpsII, neuS, tsh and vat were 29%, 0%, 19%, 67%, 47%, 99%, 98% 96%, 1% and 18%, respectively. Conclusions: We concluded that major differences exist in the prevalence of virulence factors between different UPEC isolated from different countries. Detecting these genes as primary controllers of UPEC virulence may aid in better management of related infections.

Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae infections are associated with high mortality; however, little is known about the virulence determinants of KPC-producing K. pneumoniae. At the Veterans Affairs New Jersey Healthcare System (VA NJHCS), we investigated the virulence and plasmid transferability of 60 clinically unique KPC-containing K. pneumoniae isolates. All 60 isolates were negative for known virulence factors K1, K2, and K5 capsular antigens; rmpA; and the aerobactin gene by polymerase chain reaction. Isolates varied in their susceptibility to neutrophil phagocytosis, but were less resistant than the virulent serotype K1 isolate. Additionally, no deaths were seen on murine lethality studies. Conjugation results of this study showed that the bla(KPC) gene can be transferred into an Escherichia coli J-53 strain but not to E. coli JP-995. However, the stability is very limited as E. coli J-53 does not retain bla(KPC)-containing plasmids for any period of time. The lack of virulence factors in the set of KPC-producing K. pneumoniae studied suggests that morbidity and mortality may be due to detection issues or lack of effective antibiotics.

A total of 145 Escherichia coli strains causing pyelonephritis in children were investigated for the prevalence of genes encoding the following virulence factors (VFs): P fimbria (67.6 %), S fimbria (53.8 %), AFA adhesins (2.8 %), cytotoxic necrotizing factor 1 (37.9 %), α-hemolysin (41.4 %), and aerobactin (71.7 %). One hundred and thirty-six (93.8 %) isolates harbored at least one of the virulence genes detected in the present study. Statistically significant co-occurrent presence of two VF genes was found for α-hly–cnf1, α-hly–sfa, cnf1–sfa (p < 0.001), and α-hly–pap (p = 0.001). Twenty-six profiles of VF genes were detected in this study. The combinations of aer–pap and aer–pap–sfa–α-hly–cnf1 were presented with the highest frequency—both of them in 28 isolates (19.3 %). All E. coli strains included in the study were susceptible to meropenem, amikacin, and tobramycin; the highest frequency resistance was found toward ampicillin (43.4 %), piperacillin (31.7 %), tetracycline (15.9 %), and trimethoprim/sulfamethoxazole (11.7 %). The resistance to the other tested antimicrobial drugs did not exceed 3 % incidence. Overall, 55.9 % strains were susceptible to all tested anti-infective agents. Antimicrobial resistance of E. coli strains toward trimethoprim/sulfamethoxazole statistically significantly correlated with the presence of α-hly (p < 0.001), sfa (p < 0.01), and cnf1 (p < 0.05).

The TonB system of proteins is required for the energy-dependent active transport of iron-bound substrates across the outer membrane of Gram-negative bacteria. We have identified three TonB systems within the human pathogen Vibrio vulnificus . The TonB1 system contains the TonB1, ExbD1, and ExbB1 proteins, whereas both the TtpC2-TonB2 and TtpC3-TonB3 systems contain an additional fourth protein, TtpC. Here we report that TtpC3, although highly related to TtpC2, is inactive in iron transport, whereas TtpC2 is essential for the function of the TtpC2-TonB2 system in V. vulnificus . This protein, together with TonB2, is absolutely required for both the uptake of endogenously produced iron-bound siderophores as well as siderophores produced from other organisms. Through complementation we show that V. vulnificus is capable of using different TtpC2 proteins from other Vibrio species to drive the uptake of multiple siderophores. We have also determined that aerobactin, a common bacterial siderophore involved in virulence of enteric bacteria, can only be brought into the cell using the TtpC2-TonB2 system, indicating an important evolutionary adaptation of TtpC2 and TonB2. Furthermore, in the absence of TonB1, TtpC2 is essential for a fully virulent phenotype as demonstrated using 50% lethal dose (LD 50 ) experiments in mice.

SUMMARYHypermucoviscous (HV) isolates ofKlebsiella pneumoniaehave been linked to virulence potential in experimental infections. We examined 33 isolates ofK. pneumoniaefrom patients with bacteraemia for the HV phenotype on agar culture, and determined their virulence potential by screening for capsular (K) serotype by polymerase chain reaction and the presence of seven virulence factor genes. Fourteen (42·4%) isolates expressed the HV phenotype and 11 of these were serotype K1 or K2; these serotypes were not identified in HV-negative isolates. The genesrmpA,rmpA2,aerobactin,wabGandallSwere significantly more frequent in HV than non-HV isolates. Multilocus sequence typing identified 21 sequence types (ST), eight of which were found in HV-positive isolates and the clonal relatedness of isolates of the most frequent types (ST23 and ST11) from different hospitals was confirmed by pulsed-field gel electrophoresis. The HV phenotype was more associated with community-acquired infection with a lower frequency of fatal underlying illness, but with significantly more focal infections, notably liver abscesses. Clinicians should be aware of such clinical impacts of the HV phenotype.

Successful international clones have recently emerged among Escherichia coli that produce CTX-M beta-lactamases as important causes of community-onset urinary tract and bloodstream infections. One hundred and seven isolates that belong to sequence types (STs) ST38, ST131, ST405, ST648, and 38 nonrelated CTX-M producing E. coli from Canada and the Netherlands were assigned to phylogenetic groups and tested for the presence of genes encoding for virulence factors (VFs) using established multiplex polymerase chain reaction. The STs E. coli were significantly more resistant to antibiotics-ST38, ST405, and ST648 belonged to phylogenetic group D while ST131 belonged to B2. Secreted autotransporter toxin (sat), aerobactin receptor, and pathogenicity island marker were significantly more common among the STs; the heat-resistant agglutinin (hra) was present in ST38, sat, and uropathogenic-specific protein, and putative adhesin-siderophore receptor was more common in ST131, while outer membrane protease T was present in ST648. ST131 had a significantly higher VF score. In conclusion, the precise role of these VFs remains to be elucidated; however, we have identified certain putative VFs that possibly contribute to the fitness and success of certain sequence types. (C) 2012 Elsevier Inc. All rights reserved.

Avian pathogenic Escherichia coli (APEC) strains have multiple iron-uptake systems that facilitate adaptation to iron-restricted environments and are believed to assist in colonisation of the host. These systems include several TonB-dependent transporters of ferri-siderophores encoded by the chromosome and the large virulence plasmid common to APECs. The tonB gene of the virulent APEC strain E956 was replaced with a selectable antibiotic resistance marker using Lambda Red recombinase mutagenesis. The phenotype of the ΔtonB E956 mutant was compared to the parent strain under various culture conditions and in chickens experimentally infected via the respiratory route. The mutant was resistant to streptonigrin, impaired in its ability to adapt to growth in iron-depleted medium and had greater tolerance of oxidative stress than the parental strain. The mutant was avirulent in chickens, did not affect the growth of chicks and colonisation was mostly limited to the trachea. This study has demonstrated that TonB is essential for virulence in APEC.

Nearly all microbes require iron for growth. The low concentration of iron found in the ocean makes iron acquisition a particularly difficult task. In response to these low iron conditions, many bacteria produce low-molecular-weight iron-binding molecules called siderophores to aid in iron uptake. We report herein the isolation and structural characterization of a suite of amphiphilic siderophores called the ochrobactins-OH, which are produced by a Vibrio species isolated from the Gulf of Mexico after the 2010 Deepwater Horizon oil spill. The citrate-based ochrobactins-OH are derivatives of aerobactin, replacing the acetyl groups with fatty acid appendages ranging in size from C8 to C12, and are distinctly different from the ochrobactins in that the fatty acid appendages are hydroxylated rather than unsaturated. The discovery of the marine amphiphilic ochrobactin-OH suite of siderophores increases the geographic and phylogenetic diversity of siderophore-producing bacteria.

The molecular mechanisms that define asymptomatic bacteriuria (ABU) Escherichia coli colonization of the human urinary tract remain to be properly elucidated. Here, we utilize ABU E. coli strain 83972 as a model to dissect the contribution of siderophores to iron acquisition, growth, fitness, and colonization of the urinary tract. We show that E. coli 83972 produces enterobactin, salmochelin, aerobactin, and yersiniabactin and examine the role of these systems using mutants defective in siderophore biosynthesis and uptake. Enterobactin and aerobactin contributed most to total siderophore activity and growth in defined iron-deficient medium. No siderophores were detected in an 83972 quadruple mutant deficient in all four siderophore biosynthesis pathways; this mutant did not grow in defined iron-deficient medium but grew in iron-limited pooled human urine due to iron uptake via the FecA ferric citrate receptor. In a mixed 1:1 growth assay with strain 83972, there was no fitness disadvantage of the 83972 quadruple biosynthetic mutant, demonstrating its capacity to act as a “cheater” and utilize siderophores produced by the wild-type strain for iron uptake. An 83972 enterobactin/salmochelin double receptor mutant was outcompeted by 83972 in human urine and the mouse urinary tract, indicating a role for catecholate receptors in urinary tract colonization.

Fifty-five clinical isolates of avian pathogenic Escherichia coli (APEC) from seven outbreaks of acute haemorrhagic septicaemia in turkeys were characterized by serotyping, plasmid profiling including restriction analysis with HindIII, ribotyping with EcoRI and HindIII, multilocus sequence typing (MLST) and virulence profiling. A clonal relationship was demonstrated for each outbreak according to serotype, plasmid profiling, ribotyping, and MLST. In addition, isolates demonstrated highly similar virulence profiles, as all isolates were positive for F11 pili and possessed genes encoding aerobactin (iucD), increased serum survival (iss), temperature-sensitive haemagglutinin (tsh) and colicin V plasmid operon genes (cva/cvi). However, only 20% of the isolates produced colicin V and 42% exhibited serum resistance. All strains with O group O111 and a single O18ac strain (demonstrating non-clonal DNA profiles) were positive for enteroaggregative heat-stabile toxin (EAST1), while isolates of a single outbreak all possessed the enteroaggregative toxin gene (astA). All isolates were negative for genes encoding verocytotoxins (vtx/stx), iron-repressible protein (irp2), P-fimbria (papC), invasion plasmid antigen (ipaH), attaching and effacing gene (eae), enterohaemolysin (ehxA), and enterotoxins LT, STIa (ST(p)) and STIb (ST(h)). In conclusion, highly similar virulence profiles were demonstrated for isolates of E. coli associated with a single well-defined lesion type of colibacillosis in turkeys; acute haemorrhagic septicaemia. The isolates obtained, however, demonstrated a different phylogenetic background, underlining the importance of using well-defined strain collections for characterization of APEC pathotypes.

Trichodesmium colonies contain an abundant microbial consortium that is likely to play a role in nutrient cycling within the colony. This study used laboratory cultures of Trichodesmium and two genome‐sequenced strains of bacteria typical of Trichodesmium‐associated microbes to develop an understanding of the cycling of iron, a potentially limiting micronutrient, within Trichodesmium colonies. We found that the ferric siderophores desferrioxamine B and aerobactin were not readily bioavailable to Trichodesmium, relative to ferric chloride or citrate‐associated iron. In contrast, the representative bacterial strains we studied were able to acquire iron from all of the iron sources, implying that naturally occurring Trichodesmium‐associated bacteria may be capable of utilizing a more diverse array of iron sources than Trichodesmium. From the organism‐specific uptake data collected in this study, a theoretical Trichodesmium colony was designed to model whole colony iron uptake. The bacteria accounted for most (> 70%) of the iron acquired by the colony, highlighting the importance of determining organism‐specific uptake in a complex environment. Our findings suggest that, although they may share the same micro‐environment, Trichodesmium and its colony‐associated microbial cohort may differ substantially in terms of iron acquisition strategy.

Extraintestinal pathogenic E. coli (ExPEC) are responsible for many infectious diseases in livestock, such as airsacculitis in poultry, acute mastitis in dairy animals and neonatal septicaemia and urinary tract infections (UTI) in pigs and cattle. In their animal hosts, ExPEC have to cope with low iron availability. By using different strategies, ExPEC strains are able to retrieve iron sequestered by host proteins. One of these strategies is the use of siderophores, which are small secreted molecules with high affinity for iron. ExPEC are known to synthesize up to four different types of siderophores: enterobactin, salmochelins, yersiniabactin and aerobactin. Steps required for iron acquisition by siderophores include (1) siderophore synthesis in the cytoplasm, (2) siderophore secretion, (3) ferri-siderophore reception, (4) ferri-siderophore internalization and (5) iron release in the cytoplasm. Each siderophore has specific properties and may be differentially regulated to provide different advantages, potentially allowing ExPEC to adapt to different environmental conditions or to overcome host innate immunity. Iron acquisition by siderophores plays a significant role in ExPEC virulence and, as it requires outer membrane receptors, it constitutes an interesting target for the development of vaccines that could be used to limit the number of infectious diseases due to ExPEC in livestock.

Serotype K1 Klebsiella pneumoniae with multilocus sequence type 23 (ST23) has been strongly associated with liver abscess in Taiwan. Few data regarding the strain types and virulence of this serotype from other Asian countries are available. Serotype K1 K. pneumoniae strains isolated from liver abscess and stool samples from subjects hospitalized in Hong Kong, Singapore, and Taiwan hospitals were examined. Forty-seven serotype K1 isolates were identified: 26 from liver abscess samples and 21 from stool samples. MLST revealed 7 sequence types: 85.1% (40 of 47 isolates) belonged to ST23, 1 isolate belonged to ST163 (a single-locus variant of ST23), and 2 isolates were ST249 (a 3-locus variant of ST23). New STs, namely, ST367, ST425, and ST426, were allocated to 3 of 4 isolates from stool samples. The virulence of these strains was determined by neutrophil phagocytosis and mouse infection models. Except for two ST23 isolates, all Klebsiella pneumoniae isolates were resistant to phagocytosis. Resistance to serum killing varied in isolates of ST23, while all non-ST23 strains were susceptible to serum killing except one with ST249 from a liver abscess. All hypervirulent isolates with a 50% lethal dose of 2 CFU were from ST23, were resistant to phagocytosis and serum killing, and also carried both virulence-associated genes, rmpA and aerobactin. Multilocus sequence typing genotype 23 was the most prevalent sequence type among serotype K1 K. pneumoniae isolates from both liver abscess and stool samples in the Asia Pacific region. Serotype K1 K. pneumoniae isolates with capsule expression leading to phagocytic resistance and with the aerobactin gene were associated with hypervirulence.

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 fimbriae (sfa), afimbrial 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 five 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 findings 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.

Klebsiella pneumoniae liver abscess syndrome (KLAS) is an emerging invasive infection caused by highly virulent community-acquired strains of K. pneumoniae displaying hypermucoviscosity. The salient features of this syndrome include the presence of bacteremia, primary monomicrobial liver abscess, and metastatic complications. A previously healthy Argentinean man presented with fever and found to have liver abscess caused by K. pneumoniae with metastatic seeding of gastric wall. Cultures from blood and liver abscess grew hypermucoviscous K1 K. pneumoniae with sequence type (ST) 23 by multilocus sequence typing (MLST), positive for rmpA (regulator of mucoid phenotype A), wzy(KpK1) (capsular polymerase) and aerobactin genes. The hypermucoviscous phenotype of this K. pneumoniae isolate was readily identified by the "string test" (colonies formed a long string when touched with a loop). The patient responded favourably to percutaneous drainage of the abscess and antibiotics. This is the first documented report of KLAS described in Argentina, and may signal the emergence of this syndrome in South America.

Capsular serotypes K1 and K2, the rmpA gene (a regulator of the mucoid phenotype) and aerobactin from Klebsiella pneumoniae have been identified as the major virulence factors for pyogenic liver abscesses with high morbidity, mortality and severe complications. The pathological mechanisms remain unclear. In this study, we compared liver immune responses and pathological changes in response to different serotypes of K. pneumoniae infections. A mouse model was used to investigate cytokine and chemokine production, histopathology findings, phagocytic uptake and mortality induced by serotypes K1 (magA(+), rmpA(+), aerobactin(+)), K2 (magA(-), rmpA(+), aerobactin(+)), K62 (magA(-), rmpA(-), aerobactin(-)) and an acapsulated isogenic K1 mutant (ΔK1, magA(+), rmpA(+), aerobactin(+)). K. pneumoniae serotypes K1 and K2 showed lower 50% lethal dose values and more phagocytic resistance to neutrophils than K62 and the ΔK1 mutant. In sequential liver samples, viable bacteria counts increased 3 h to 3 days after low-dose inoculation (10(1) colony-forming unit (cfu)) with K1 and K2, while K62 and ΔK1 cleared rapidly and became undetectable even with high-dose inoculation (∼2.9 × 10(5) cfu). Time-dependent increases in cytokines and chemokines, including tumor necrosis factor-α, interleukin (IL)-1β, IL-6, IL-10, keratinocyte-derived chemokines and macrophage inflammatory protein-2, were observed in the serum and liver tissue of K1- and K2-infected mice, and severe disease progression manifesting as microabscesses was also identified. K62 and ΔK1 inoculation did not result in similar immune responses and histological changes. These findings illustrate the critical role of phagocytic resistance against innate immunological defense mechanisms as well as its contribution to the development of liver abscesses.

Escherichia coli is a normal inhabitant of the large intestine, but certain strains also cause extraintestinal infections when they spread from their primary niche. Such pathogenic E. coli strains express several virulence-associated traits that contribute to the disease process, including adhesins, certain O serotypes and capsular antigens, iron-trapping compounds (such as aerobactin), and cytolytic toxins (such as hemolysin) (17). Pathogenicity-associated islands (PAIs) are particular regions on the bacterial chromosome where virulence genes have accumulated. PAIs, and their associated virulence genes, have spread among bacterial populations by horizontal transfer (15). Several PAIs were previously identified in uropathogenic E. coli strains such as E. coli 536, E. coli J96, and E. coli CFT073. PAIs I to IV from strain 536 (I536 to IV536) encode a range of virulence factors, including P fimbriae, P-related fimbriae, α-hemolysin, S fimbriae, and the yersiniabactin siderophore system. PAI IJ96 and IIJ96 encode P fimbriae, P-related fimbriae, and α-hemolysin; PAI ICFT073 and IICFT073 encode P fimbriae, α-hemolysin, and aerobactin (42) (Table ​(Table1).1). usp is prevalent among strains causing urinary tract infections (24). It enhances infectivity in a mouse ascending urinary tract infection (UTI) model (48), displays homology with S-type pyocin, and may function as a bacteriocin (34). malX codes for a phosphotransferase system enzyme II that recognizes maltose and glucose (37) and is enriched in strains causing extraintestinal infections (18, 19, 41). TABLE 1. The pathogenicity islands and the functions encoded E. coli segregate into four main phylogenetic groups, termed A, B1, B2, and D (16). Strains of group A and B1 seldom cause extraintestinal infections and carry few virulence factor genes (4, 7). Extraintestinal pathogenic E. coli strains belong mostly to group B2 (4, 18) and to a lesser extent to group D and usually carry several PAIs (5, 33, 40). Intestinal E. coli strains differ widely in colonization capacity; resident strains have the ability to persist in the gut microbiota, while transient strains disappear from the microbiota within a short time (44-46). Previously, in several epidemiological studies, we demonstrated that genes for certain virulence-associated factors, such as P fimbriae, type 1 fimbriae, hemolysin, capsular polysaccharides (K1 and K5), and aerobactin, are significantly enriched in resident strains (27-29). Furthermore, strains of B2 origin have an enhanced ability to persist in the intestinal microbiotas of infants (32). The present study was designed to investigate whether several markers for pathogenicity islands and the virulence-associated genes malX and usp are associated with an increased capacity of E. coli to persist in the infantile intestinal microbiota. In regard to this, 273 E. coli strains obtained from 130 healthy Swedish infants, monitored longitudinally over the first year of life, were assessed for carriage of markers for PAIs I536, II536, III536, IV536, IICFT073, and IIJ96 and for malX and usp. Carriage of PAI-associated markers was compared with a strain's persistence.

Uropathogenic Escherichia coli (UPEC), the predominant cause of uncomplicated urinary tract infection (UTI), utilizes an array of outer membrane iron receptors to facilitate siderophore and heme import from within the iron-limited urinary tract. While these systems are required for UPEC in vivo fitness and are assumed to be functionally redundant, the relative contributions of specific receptors to pathogenesis are unknown. To delineate the relative roles of distinct UPEC iron acquisition systems in UTI, isogenic mutants in UPEC strain CFT073 or 536 lacking individual receptors were competed against one another in vivo in a series of mixed infections. When combinations of up to four mutants were coinoculated using a CBA/J mouse model of ascending UTI, catecholate receptor mutants (Δ fepA , Δ iha , and Δ iroN mutants) were equally fit, suggesting redundant function. However, noncatecholate siderophore receptor mutants, including the Δ iutA aerobactin receptor mutant and the Δ fyuA yersiniabactin receptor mutant, were frequently outcompeted by coinoculated mutants, indicating that these systems contribute more significantly to UPEC iron acquisition in vivo . A tissue-specific preference for heme acquisition was also observed, as a heme uptake-deficient Δ hma Δ chuA double mutant was outcompeted by siderophore receptor mutants specifically during kidney colonization. The relative contribution of each receptor to UTI only partially correlated with in vivo levels of receptor gene expression, indicating that other factors likely contributed to the observed fitness differences. Overall, our results suggest that UPEC iron receptors provide both functional redundancy and niche specificity for this pathogen as it colonizes distinct sites within the urinary tract.

F. Bert, B. Huynh, F. Dondero, J.R. Johnson, C. Paugam-Burtz, F. Durand, J. Belghiti, D. Valla, R. Moreau, M.-H. Nicolas-Chanoine. Molecular epidemiology of Escherichia coli bacteremia in liver transplant recipients. Transpl Infect Dis 2011: 13: 359–365. All rights reserved Abstract: The characteristics of Escherichia coli strains causing bacteremia in profoundly immunosuppressed patients such as transplant recipients are undefined. The phylogenetic group and the virulence genotype of 57 distinct E. coli strains that caused bacteremia in 53 liver transplant recipients were investigated, and the association of these characteristics with host factors and in-hospital mortality was examined. Phylogenetic groups A, B1, B2, and D accounted for 39%, 10%, 25%, and 26% of the isolates, respectively. The most prevalent virulence genes were fyuA (yersiniabactin system: 70%) and iutA (aerobactin system: 63%), whereas hlyA (alpha-hemolysin) and cnf1 (cytotoxic necrotizing factor 1) occurred in only 14% and 12% of isolates, respectively. Most virulence genes were significantly more prevalent among group B2 and D isolates, vs. group A and B1 isolates. The overall rate of in-hospital mortality after E. coli bacteremia was 20%. Predictors of mortality included onset of bacteremia within 30 days of transplantation or during the intensive care unit stay, and non-urinary source and cutaneous source, but not E. coli phylogenetic group or virulence profile. Compared with historical E. coli bloodstream isolates from non-transplant patients, those from liver transplant recipients are characterized by a higher prevalence of groups A and B1 isolates and reduced virulence gene content. This finding can be explained by the severely immunocompromised status of the patients and the predominance of abdominal-source bacteremic episodes. Time of onset and source of bacteremia, not bacterial characteristics, predict mortality.

Extraintestinal pathogenic Escherichia coli (ExPEC) use siderophores to sequester iron during infection. Enterobactin and salmochelins are catecholate siderophores produced by some ExPEC strains and other pathogenic enterobacteria. Siderophore export and synthesis mutants of avian ExPEC strain χ7122 were tested in a chicken infection model. In single-strain infections, siderophore-negative (ΔentDΔiuc), ΔentS and ΔentSΔiroC export mutants were attenuated in tissues and blood, whereas the ΔiroC export mutant was only attenuated in blood. Interestingly, the ΔentD mutant, producing only aerobactin, retained full virulence, and loss of entD in the ΔentSΔiroC mutant restored virulence. LC-MS/MS quantification of siderophores in export mutants demonstrated that loss of entS impaired enterobactin and mono-glucosylated enterobactin secretion, whereas loss of iroC impaired di- and tri-glucosylated enterobactin secretion. Loss of entS and/or iroC resulted in intracellular accumulation and increased secretion of siderophore monomers. Catecholate siderophore export mutants also demonstrated decreased fitness in a co-challenge infection model. By contrast, catecholate siderophore synthesis mutants (ΔentD and ΔiroB) competed as well as the wild-type strain. Results establish that EntS and IroC mediate specific export of catecholate siderophores and the role of these exporters for ExPEC virulence is contingent on enterobactin synthesis, which is not required when other siderophores like aerobactin are functional.

Eight virulence factors associated with uropathogenic Escherichia coli (UPEC) were investigated in 204 clinical isolates of E. coli recovered from urine cultures at counts ≥10(5). The bacteria were classified into two groups according to the number of leukocytes in urine samples from which they were isolated: group I ≤8 leukocytes/hpf, 104 strains; group II8 leukocytes/hpf, 100 strains. Two multiplex PCR systems were used to detect genes encoding adhesin P (pap), adhesin S (sfa), afimbrial adhesin I (afa), siderophore aerobactin (aer), alpha-hemolysin (hly), cytotoxic necrotizing factor type 1 (cnf1), and traT associated with serum resistance. The PAI marker for the virulence island identified in strains CFT072 and CVD432, a marker of enteroaggregative E. coli, was also investigated using PCR. The susceptibility profile of E. coli strains was determined by disk diffusion method. Ninety percent UPEC showed at least one of the virulence genes, the prevalence being traT (76%), aer (41%), PAI (32%), sfa (26%), pap (25%), cnf1 (18%), afa (6%), and hly (5%). There was no significant difference in the distribution of virulence genes between groups I and II. A significantly higher degree of virulence was detected in UPEC group II. The CVD432 gene was not detected in any of the UPECs. Fifty-nine percent of the strains were resistant to at least one of the antimicrobials that we tested; the most common being resistance to ampicillin (51%) and trimethoprim-sulfamethoxazole (44%).

Vibrio vulnificus possesses multiple iron-uptake systems which are mediated by VuuA (vulnibactin receptor), IutA (aerobactin receptor) and HupA (heme receptor). In this study, we determined the effect of a mutation of luxS encoding autoinducer-2 (AI-2) synthase on the expressions of the three receptors. A mutation and an in trans complementation of luxS did not affect the growing ability of V. vulnificus in iron-deficient conditions. Nevertheless, the luxS mutation slightly decreased vuuA expression, but slightly increased iutA and hupA expressions in the transcriptional reporter assay or Western blot analysis. These changes were all recovered by the luxS complementation. These results suggest that AI-2-mediated quorum sensing system may be involved in the fine modulation of V. vulnificus iron-uptake systems, positively affecting vuuA expression but negatively affecting iutA and hupA expressions.

The aims of this study were molecular identification some of virulence genes in Escherichia coli isolated from milk of bovines with clinical mastitis. (n = 60) E. coli isolates from acute clinical mastitis were examined for detect the presence of the genes encodingshigatoxin1 (stx1), intimin (eaeA), cytotoxicnecrotizingfactor 2 (cnf2), aerobactin (iucD) and P fimberiae (pap). The majors finding in the PCR assays were: 8 isolates (13.33%) had at least one virulence gene, None of isolates contained the genes for stx1, eaeA , the most common gene in the examined isolate was iucD which was positive in 6 isolates (10%). One isolate (1.66%) was positive for both iucD and pap genes and one isolate (1.66%) for cnf2 gene. In this study similar to previous investigations indicated that prevalence virulence genes in E. coli isolated of bovine mastitis is deferent. The results of this investigate similar to previous studies indicated none of the potential virulence genes or specific pathotype was observed in E. coli isolates from bovine clinical mastitis. DOI: http://dx.doi.org/10.3126/ijls.v8i2.10219 International Journal of Life Sciences Vol.8(2): 2014; 1-3