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Uropatogene Escherichia coli (UPEC) povzročajo okužbe sečil. Ključno vlogo pri tem imajo virulentni dejavniki, med katerimi najpogosteje zasledimo fimbrije, toksin hemolizin ter sisteme za privzem železa. Izražanje genov za virulentne dejavnike bi lahko bilo podvrženo heterogenosti, pojavu ki ga zasledimo v populaciji genetsko enakih celic in je posledica okoljskih dejavnikov, ki vplivajo na razlike v translaciji, transkripciji in topologiji DNA. Da bi natančneje preučili izražanje genov za virulentne dejavnike na ravni posameznih celic, smo na plazmidnem vektorju pBAC, spojili promotorje genov za fimbrije tipa 1 (fimA), ? hemolizina (hlyA) in za kelator železa, aerobaktin (iucD) z genom za fluorescentni protein Gfp brez lastnega promotorja. Na izražanje preiskovanih promotorjev smo s fluorescentno mikroskopijo spremljali vpliv hranil, pomanjkanja železa, pH in rast na trdni podlagi. Ugotovili smo, da sta se promotorja genov iucD in hlyA izražala v višji meri na minimalnem gojišču s kelatorjem železa dipiridilom, medtem ko smo za promotor gena za fimbrije fimA pokazali višjo raven izražanja v hranilnem gojišču LB. Izražanje vseh treh preiskovanih promotorjev je bilo načeloma homogeno, vendar smo zasledili primer heterogenega izražanja promotorja iucD v pogojih, ko izražanje sicer ni bilo inducirano. Uropathogenic Escherichia coli (UPEC) are a leading cause of urinary tract infections and virulence factors play a key role in pahogenesis - fimbriea, toxins such as hemolysin and iron uptake systems such as aerobactin, being the most common. The expression of virulence factor genes might be heterogenic, meaning that all genetically identical cells in a population, do not express the same genes. It is belived that heterogeneity is affected by differences in rates of translation, transcription and DNA topology. To further understand the expression of virulence factor genes we constructed gene fusions using the pBAC plasmid, the gfp gene for the Gfp fluorescent protein without its promoter and fimA, hlyA and iucD promotors for fimbriae, hemolysin and aerobactin, respectively. Using fluorescent microscopy and a variety of different growth conditions we were able to study gene expression at a single cell level. The employed growth media were: rich LB medium, minimal media with an added iron chelating ligand, as well as a low pH medium, all in liquid and firm form. Results showed higher promotor iucD expression when cells were grown on minimal media with iron deficiency, whereas the fimA fimbriae promotor showed higher expression upon growth on rich medium. In principle, expression was found to be homogenous however, an example of heterogenous expression of iucD was observed under noninducing conditions.

Hypervirulent K. pneumoniae (hvKp) is an evolving pathotype that is more virulent than classical K. pneumoniae (cKp). hvKp usually infects individuals from the community, who are often healthy. Infections are more common in the Asian Pacific Rim but are occurring globally. hvKp infection frequently presents at multiple sites or subsequently metastatically spreads, often requiring source control. hvKp has an increased ability to cause central nervous system infection and endophthalmitis, which require rapid recognition and site-specific treatment. The genetic factors that confer hvKp’s hypervirulent phenotype are present on a large virulence plasmid and perhaps integrative conjugal elements. Increased capsule production and aerobactin production are established hvKp-specific virulence factors. Similar to cKp, hvKp strains are becoming increasingly resistant to antimicrobials via acquisition of mobile elements carrying resistance determinants, and new hvKp strains emerge when extensively drug-resistant cKp strains acquire hvKp-specific virulence determinants, resulting in nosocomial infection. Presently, clinical laboratories are unable to differentiate cKp from hvKp, but recently, several biomarkers and quantitative siderophore production have been shown to accurately predict hvKp strains, which could lead to the development of a diagnostic test for use by clinical laboratories for optimal patient care and for use in epidemiologic surveillance and research studies.

Background Salmonella enterica possess several iron acquisition systems, encoded on the chromosome and plasmids. Recently, we demonstrated that incompatibility group (Inc) FIB plasmid-encoded iron acquisition systems (Sit and aerobactin) likely play an important role in persistence of Salmonella in human intestinal epithelial cells (Caco-2). In this study, we sought to determine global transcriptome analyses of S. enterica in iron-rich (IR) and iron-depleted (ID) growth conditions. Results The number of differentially-expressed genes were substantially higher for recipient (SE819) (n = 966) and transconjugant (TC) (n = 945) compared to the wild type (WT) (SE163A) (n = 110) strain in ID as compared to IR growth conditions. Several virulence-associated factors including T3SS, flagellin, cold-shock protein (cspE), and regulatory genes were upregulated in TC in ID compared to IR conditions. Whereas, IS1 and acrR/tetR transposases located on the IncFIB plasmid, ferritin and several regulatory genes were downregulated in TC in ID conditions. Enterobactin transporter (entS), iron ABC transporter (fepCD), colicin transporter, IncFIB-encoded enolase, cyclic di-GMP regulator (cdgR) and other regulatory genes of the WT strain were upregulated in ID compared to IR conditions. Conversely, ferritin, ferrous iron transport protein A (feoA), IncFIB-encoded IS1 and acrR/tetR transposases and ArtA toxin of WT were downregulated in ID conditions. SDS-PAGE coupled with LC-MS/MS analyses revealed that siderophore receptor proteins such as chromosomally-encoded IroN and, IncFIB-encoded IutA were upregulated in WT and TC in ID growth conditions. Both chromosome and IncFIB plasmid-encoded SitA was overexpressed in WT, but not in TC or recipient in ID conditions. Increased expression of flagellin was detected in recipient and TC, but not in WT in ID conditions. Conclusion Iron concentrations in growth media influenced differential gene expressions both at transcriptional and translational levels, including genes encoded on the IncFIB plasmid. Limited iron availability within the host may promote pathogenic Salmonella to differentially express subsets of genes encoded by chromosome and/or plasmids, facilitating establishment of successful infection. Electronic supplementary material The online version of this article (10.1186/s12864-019-5768-0) contains supplementary material, which is available to authorized users.

Objectives To describe the population structure, molecular epidemiology and genetic context of blaKPC-2-bearing Klebsiella pneumoniae. Methods Isolates (n = 157) were retrospective, phenotypically carbapenem-resistant blaKPC-positive K. pneumoniae, collected from public hospitals. WGS was performed on the Illumina platform. Phylogenomic analysis, screening of resistance and virulence genes, and comparison of the genetic environment of blaKPC were carried out. Results Based on core-tree phylogeny, 67.5% of the isolates were K. pneumoniae and the remainder comprised Klebsiella quasipneumoniae. No Klebsiella variicola strains were observed. Only a single K. pneumoniae carbapenemase (KPC) variant type, blaKPC-2, was seen. MLSTs were diverse and did not comprise the ‘traditional’ KPC clonal group (CG) 258. blaKPC-2 was associated with a non-Tn4401 element (NTE) in >99% of genomes. Screening for four key virulence loci: yersiniabactin (ybt), aerobactin (iuc), salmochelin (iro) and colibactin (clb) as well as ICEKp (virulence-associated integrative conjugative element of K. pneumoniae), revealed the lack of virulence factors and ICEKp within K. quasipneumoniae. Amongst the K. pneumoniae, there were 32 ybt+ isolates (32/106, 30.2%) and, of these, 8 isolates were also clb+ (7.5%). K. pneumoniae serotypes K1 and K2, the majority of capsular serotype seen in patients with invasive liver abscess syndrome, were detected at 4.5% (7/157). Conclusions Results suggest that dissemination of blaKPC-2 is driven by NTEKPC in non-ST258 isolates. The detection of blaKPC-2K. pneumoniae serotypes K1/K2 carrying virulence factors, albeit in low numbers, reflects the worrisome convergence of carbapenem resistance and hypervirulence in K. pneumoniae.

BACKGROUND: Carbapenem-resistant hypervirulent (hypermucoviscous) Klebsiella pneumoniae (CR-HMKP) poses a significant public health challenge. We investigated its epidemiology and molecular characteristics in a tertiary care hospital in eastern China. METHODS: CR-HMKP were identified among 106 non-duplicated carbapenem-resistant K. pneumoniae isolates (from June 2013 to September 2017) using the string test. The pulsotype (PT) and sequence type (ST) of CR-HMKP isolates were determined using pulsed-field gel electrophoresis and multilocus sequence typing. Resistance determinants, capsular serotypes, and virulence genes were detected by PCR and sequencing. Representative isolates from each PT were selected, and their virulence phenotypes were established using the serum killing and Galleria mellonella lethality assays. RESULTS: Of the 106 isolates, 13 (12.3%) were CR-HMKP. Seven were positive for blaNDM-1 and shared the same genotype (PT5/ST1764); the others were positive for blaKPC-2, belonged to ST11, and were divided into four different PTs. The serotype of all blaNDM-1-positive isolates was K64, while that of blaKPC-2-positive isolates were K47 (N=4) and K64 (N=2). The NDM-1-producing HMKP isolates were positive for aerobactin, exhibited high serum resistance, and elicited significantly increased larval mortality compared with the other isolates. All patients had received invasive treatment prior to infection by NDM-1-producing HMKP. The infections occurred between July and August 2016 and were hospital-acquired. CONCLUSIONS: NDM-1-producing HMKP ST1764 isolates were identified; this is the first report worldwide on an outbreak of nosocomial infection caused by these isolates. Effective surveillance and strict infection control strategies should be implemented to prevent CR-HMKP dissemination. ispartof: ANNALS OF LABORATORY MEDICINE vol:39 issue:2 pages:167-+ ispartof: location:Korea (South) status: published

AIMS In this study, the association between multidrug resistance (MDR) and the expression of some virulence factors were evaluated in Escherichia coli strains isolated from infant faeces and fresh green vegetables. The effect of isolate origin on associated virulence factors was evaluated. In addition, genetic fingerprinting of a sample of these isolates (10 isolates from each group) was studied in order to detect any genetic relatedness among these isolates. METHODS AND RESULTS Escherichia coli isolates were divided into four groups based on their origin (human faeces or plant) and their antibiotic resistance (multiresistance or susceptible). PCR was used to investigate heat-labile and heat-stable enterotoxin genes, and four siderophore genes (aerobactin, enterobactin, salmochelin and yersiniabactin). Genetic fingerprinting of the isolates was performed using enterobacterial repetitive intergenic consensus PCR. Siderophore production was measured by a colorimetric method. Biofilm formation was evaluated by a crystal violet assay. The results of the study showed that the expression of MDR is not significantly associated with an increase in these virulence factors or with biofilm formation. However, the origin of isolates had a significant association with siderophore gene availability and consequently on the concentrations of siderophores released. Genetic fingerprinting indicated that human and plant isolates have the same clonal origin, suggesting their circulation among humans and plants. CONCLUSION Antibiotic-susceptible strains of E. coli may be as virulent as MDR strains. Results also suggest that the environment can play a potential role in selection of strains with specific virulence factors. SIGNIFICANCE AND IMPACT OF THE STUDY Antibiotic-susceptible isolates of Escherichia coli from plant or human origin can be as virulent as the multidrug resistance (MDR) ones. Genetic relatedness was detected among the isolates of plant and human origin, indicating the circulation of these bacteria among human and plants. This could imply a potential role for environmental antimicrobial resistant bacteria in human infection.

Background Endogenous endophthalmitis caused by hypervirulent Klebsiella pneumoniae (HVKP) is an emerging infectious disease commonly with a devastating visual outcome. Most HVKP strains display a wild-type susceptibility profile to antibiotics. However, reports of antimicrobial-resistant HVKP have increased over time, which poses a serious therapeutic dilemma. Case presentation A 25-year-old man with a liver abscess and poorly controlled diabetes mellitus was admitted for endophthalmitis due to K. pneumoniae. The isolate displayed hypermucoviscosity as determined by a positive string test and exhibited resistance to ceftazidime, piperacillin-tazobactam and amikacin. Whole genome sequencing (WGS) analysis demonstrated the isolate to be a K1-serotype strain and belong to a novel single locus variant of ST23, ST2922. In addition to the virulence genes linked to HVKP, rmpA, magA, iucABCDiutA (aerobactin), ybtAPSTUX (yersiniabactin) and iroBDN (salmochelin), it was found to harbor extended-spectrum β-lactamase (ESBL) gene (bla CTX-M-14), AmpC β-lactamase gene (bla DHA), and 16S rRNA methylase gene (armA). Conclusions This is the first known case of endogenous endophthalmitis caused by a multidrug-resistant HVKP strain ever reported in China. Early diagnosis and treatment with intravenous and intravitreal injection of carbapenem were essential for a favorable visual outcome.

Iron is a vital mineral nutrient required by virtually all life forms to prosper; pathogenic bacteria are no exception. Despite the abundance of iron within the human host, highly regulated iron physiology can result in exceedingly low levels of iron bioavailable to prospective invading bacteria. To combat this scarcity of iron, many pathogenic bacteria have acquired specific and efficient iron acquisition systems, which allow them to thrive in iron-deficient host environments. One of the more prominent bacterial iron acquisition systems involves the synthesis, secretion, and reuptake of small-molecule iron chelators known as siderophores. Aerobactin, a citrate-hydroxamate siderophore originally isolated nearly 50 years ago, is produced by a number of pathogenic Gram-negative bacteria. Aerobactin has recently been demonstrated to play a pivotal role in mediating the enhanced virulence of a particularly invasive pathotype of Klebsiella pneumoniae (hvKP). Toward further understanding of this key virulence factor, we report the structural and functional characterization of aerobactin synthetase IucA from a strain of hvKP. The X-ray crystal structures of unliganded and ATP-bound forms of IucA were solved, forming the foundation of our structural analysis. Small angle X-ray scattering (SAXS) data suggest that, unlike its closest structurally characterized homologues, IucA adopts a tetrameric assembly in solution. Finally, we employed activity assays to investigate the substrate specificity and determine the apparent steady-state kinetic parameters of IucA.

Purpose: Escherichia coli isolated from asymptomatic bacteriuria (ABU) correlated genotypically and phenotypically with cystitis isolates may help in distinguishing urovirulence determinants from 'fitness factors', latter necessary only for survival of E. coli in urinary tract; for gaining insight into the pathogenesis of urinary tract infection. Materials and Methods: In this cross-sectional study, we compared genotypic (phylogroups and 15 putative virulence genes), and phenotypic profiles of ABU E. coli strains with our previously genotyped collection of cystitis isolates. Virulence score was calculated for each isolate as a number of virulence genes detected. Results: Significant differences were observed in the proportion of four phylogenetic groups (P = 0.009) amongst cystitis and ABU isolates. Average virulence score was higher for ABU isolates (6.6) than cystitis strains (4.2); and hlyA (P = 0.001), cytotoxic necrotising factor 1 (P = 0.00), fyuA (P = 0.00), ibeA (P = 0.00), kpsMII (P = 0.01), and malX/pathogenicity-associated island (P = 0.01) were more frequently present in ABU strains. Conclusions: The expression of adhesins, haemolysin, aerobactin, and capsule synthesis gene were similar in two groups suggesting their role as fitness factors. ABU isolates were better biofilm producers, reflecting its importance in silent persistence. Serum resistance gene which was more expressed in cystitis isolates may represent virulence determinant. Genetic makeup of E. coli does not change much rather genes helping in survival and colonisation are expressed equally in ABU and cystitis isolates as opposed to phenotypic attenuation of those that helps in invasion or inflammation in ABU isolates.

Uropathogenic Escherichia coli is the common pathogen to cause urinary tract infections (UTIs) and have become multidrug-resistant (MDR) extended-spectrum β-lactamase (ESBL) producers. The differences in the antimicrobial susceptibility, 5 bla genes, 12 virulence genes of 87 clinical ESBL-producing E. coli isolates and genomic variations and sequence types of 18 recurrent and repeated isolates from 9 patients were investigated. The 87 MDR-ESBL isolates collected mainly from indwelling urinary catheters (IUCs) and UTIs were highly resistant to fluoroquinolones, with over 50% of the isolates being resistant to cefepime and piperacillin/tazobactam and a few being resistant to carbapenem. These isolates carried at least two of the five bla genes examined, with the highest prevalence (87.4%) found for blaCTX-M (blaCTX-M3-like and blaCTX-M14-like), followed by blaCMY-2 (80.5%) and blaSHV (56.3%). The predominant virulence genes were the fimbriae gene fimH and the toxin genes cnf1 and hlyA in blood isolates and the capsule gene kpsMTII in UTI and blood isolates. Over 80% of the isolates carried yersiniabactin and aerobactin of siderophores. In 18 isolates, the fluoroquinolone-resistant ST131 isolate of pulsotypes I and II with blaCTX-M-15 was clonally disseminated in the hospital. The genomic plasticity of these ST131 occurred mainly through the conjugative plasmids with differences in replicon types A/C, I1, FIA, FIB and Y, size and number. In conclusion, MDR ESBL-producing E. coli isolates differed in virulence genes of UPEC and antibiotic resistance associated with the sources. Plasmid acquisition and chromosomal variations increase the spread of fluoroquinolone-resistant UPEC ST131 worldwide.

Fifteen hypermucoviscous isolates (13 blaNDM-1-positive) obtained from 11 oncology patients were analyzed by whole genome sequencing, and selected isolates were assessed in a murine model of sepsis. ST395/K2 isolates harboring rmpA, rmpA2, peg-344, aerobactin, enterobactin, yersiniabactin, type I fimbriae, etc. displayed maximal virulence in the mouse lethality assay (LD50 = 102 CFU). ST147/K20 isolates lacking yersiniabactins were relatively less virulent (LD50 = 104 CFU), ST395/K2 isolates lacking rmpA, rmpA2, peg-344, and aerobactin, but harboring yersiniabactin demonstrated minimal virulence (LD50 = 105 CFU). Isolates represent various paths and stages of evolution directed towards convergence of multidrugresistant classical Klebsiella pneumoniae and hypervirulent K. pneumoniae.

BackgroundKlebsiella pneumoniaeis a recognised agent of multidrug-resistant (MDR) healthcare-associated infections, however individual strains vary in their virulence potential due to the presence of mobile accessory genes. In particular, gene clusters encoding the biosynthesis of siderophores aerobactin (iuc) and salmochelin (iro) are associated with invasive disease and are common amongst hypervirulentK. pneumoniaeclones that cause severe community-associated infections such as liver abscess and pneumonia. Concerninglyiuchas also been reported in MDR strains in the hospital setting, where it was associated with increased mortality, highlighting the need to understand, detect and track the mobility of these virulence loci in theK. pneumoniaepopulation.MethodsHere we examined the genetic diversity, distribution and mobilisation ofiucandiroloci among 2503K. pneumoniaegenomes using comparative genomics approaches, and developed tools for tracking them via genomic surveillance.ResultsIroandiucwere detected at low prevalence (iroand sixiuclineages that show distinct patterns of mobilisation and dissemination in theK. pneumoniaepopulation. The major burden ofiucandiroamongst the genomes analysed was due to two linked lineages (iuc1/iro1, 74% andiuc2/iro2, 14%), each carried by a distinct non-self-transmissible IncFIBKvirulence plasmid type that we designate KpVP-1 and KpVP-2. These dominant types also carry hypermucoidy (rmpA) determinants and include all previously described virulence plasmids ofK. pneumoniae. The otheriucandirolineages were associated with diverse plasmids, including some carrying FII conjugative transfer regions and some imported fromE. coli; the exceptions wereiro3(mobilised byICEKp1), andiuc4(fixed in the chromosome ofK. pneumoniaesubspeciesrhinoscleromatis).Iro/iucMGEs appear to be stably maintained at high frequency within known hypervirulent strains (ST23, ST86, etc), but were also detected at low prevalence in others such as MDR strain ST258.ConclusionsIucandiroare mobilised inK. pneumoniaevia a limited number of MGEs. This study provides a framework for identifying and tracking these important virulence loci, which will be important for genomic surveillance efforts including monitoring for the emergence of hypervirulent MDRK. pneumoniaestrains.

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.

WOS: 000443307900005, PubMed ID: 30155366, Background. Extraintestinal pathogenic Escherichia coli (ExPEC) is an important bacterium and responsible for many bloodstream infections, including urinary tract infections and even fatal bacteremia. The aim of this research was to investigate whether ExPEC strains isolated from Turkish blood cultures have a relationship between 16S rRNA based phylogenetic clusters and antibiotic resistance profiles, virulence factors or clonal lineages. Methods. Phenotypically identified ExPEC blood culture isolates (n = 104) were included in this study. The 16S rRNA partial sequence analysis was performed for genotypic identification of ExPEC isolates. Antibiotic susceptibility and Extended Spectrum beta-Lactamase testing of isolates were performed. Phylogenetic classification (A, B1, B2 and D), Multi Locus Sequence Typing analysis and virulence-associated genes were investigated. Results. Based on 16S rRNA partial sequence analysis, 97 out of 104 (93.26%) ExPEC isolates were confirmed as E. coli. Ampicillin (74.22%) and cefuroxime axetil (65.97%) resistances had the highest frequencies among the ExPEC isolates. In terms of phylogenetic classification of ExPEC, D (38.14%, 37/97) was the most prevalent group after A (29.89%, 29/97), B2 (20.61%, 20/97), and B1 (11.34%, 11/97). The sequence types of the 20 ExPEC isolates belonging to the B2 phylogenetic group were analyzed by Multi Locus Sequence Typing. Ten isolates out of 20 (50.0%) were identified as ST131. The other STs were ST95 (n=1), ST14 (n=1), ST10 (n=1), ST69 (n=1), ST1722 (n=2), ST141 (n=1), ST88 (n=1), ST80 (n=1), and ST998 (n=1). Of the ST131 strains, six (60%, 6/10) represented serogroup O25. The most common virulence factor genes were serum resistance factor gene, traT (55.7%) aerobactin siderophore receptor and yersiniabactin encoding genes iutA (45.3%) and fyuA (50.5%), respectively. In addition, PAI (41.2%), iroN (23.7%), hlyA (15.4%), kpsII (13.4%), ompT (13.4%), papG (12.4%), iss (9.3%), cnf1 (7.2%), ibeA (2.06%), and sfaS (2.06%) genes were present in the ExPEC isolates. Conclusion. The 16S rRNA-based phylogenetic relationship tree analysis showed that a large cluster was present among 97 ExPEC isolates along with related reference strains. There were 21 main clusters with 32 closely related subclusters. Based on our findings, different clonal lineages of ExPEC can display different antibiotic susceptibilities and virulence properties. We also concluded that virulence factors were not distributed depending on phylogenetic groups (A, B1, B2, and D). The ExPEC isolates belonging to the same phylogenetic group and sequence type could display different resistance and virulence characteristics., Scientific Research Unit of the Istanbul University [FUA-2016-20386, BYP-2016-2305], This study was supported by The Scientific Research Unit of the Istanbul University with project ID: FUA-2016-20386 and ID: BYP-2016-2305. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The relevance ofEscherichia coliassociated bacteriuria infection in pregnant women is poorly understood, despite these strains sharing a similar virulence profile with other extra intestinal pathogenicE. coliproducing severe obstetric and neonatal infections. We characterized and determined the antimicrobial susceptibility, resistant genes and virulence profiles of 82E. coliisolates associated with asymptomatic bacteriuria in some pregnant in five very distinct hospitals in the Volta region from January, 2016 to April, 2016 using Kirby-Bauer disc diffusion and polymerase chain reaction.High levels of antimicrobial resistance was observed to Ampicillin (79.3%), Tetracycline (70.7%) and Cotrimoxazole (59.8%), except for Cefuroxime (32.9%). Resistant genes analyses revealed 58.5% were positive forBlaTEMand 14.6% foraph(3)-Ia(aphA2). Virulence factors (VFs) was more widespread in pregnant women in the 2ndand 3rdtrimesters than 1sttrimester. VFs relating to adhesion (papC andiha), Protectins (traT), aerobactin acquisition (iutA) and iron acquisition systems (fyuA andirp2) were more prevalent in the resistantE. coliisolates. This study provides additional evidence for a link in bacteriuria and transmission of extra-intestinalE. coliin pregnant women to cause multi-resistant severe obstetric or neonatal infections. Considering the involvement of extra-intestinalE. coliin infections, our results may be helpful to develop strategies to prevent maternal and/ neonatal infections. In addition continuous surveillance is required to guide appropriate antibiotic usage in pregnant women.

For many pathogenic members of the Enterobacterales, siderophores play an important role in virulence, yet the siderophores of the host-associating members of the genus Pantoea remain unexplored. We conducted a genome-wide survey of environmental and host-associating strains of Pantoea to identify known and candidate siderophore biosynthetic clusters. Our analysis identified three clusters homologous to those of enterobactin, desferrioxamine, and aerobactin that were prevalent among Pantoea species. Using both phylogenetic and comparative genomic approaches, we demonstrate that the enterobactin-like cluster was present in the common ancestor of all Pantoea, with evidence for three independent losses of the cluster in P. eucalypti, P. eucrina, and the P. ananatis—P. stewartii lineage. The desferrioxamine biosynthetic cluster, previously described and characterized in Pantoea, was horizontally acquired from its close relative Erwinia, with phylogenetic evidence that these transfer events were ancient and occurred between ancestral lineages. The aerobactin cluster was identified in three host-associating species groups, P. septica, P. ananatis, and P. stewartii, with strong evidence for horizontal acquisition from human-pathogenic members of the Enterobacterales. Our work identifies and describes the key siderophore clusters in Pantoea, shows three distinct evolutionary processes driving their diversification, and provides a foundation for exploring the roles that these siderophores may play in human opportunistic infections.

Background A colistin-resistant mucoid Klebsiella strain was recovered from the blood of a patient in China. Hypervirulence has been reported in Klebsiella pneumoniae, but not in other Klebsiella spp. The strain was suspected to be hypervirulent and was therefore characterized. Methods The strain was subjected to genome sequencing using both the short-read Illumina HiSeq X10 Sequencer and the long-read MinION sequencer. Precise species identification was established using average nucleotide identity based on genome sequences. Virulence and antimicrobial resistance genes were identified using ResFinder and the bigsdb database. Conjugation experiments were performed. Virulence was assessed using wax moth (Galleria mellonella) larvae with control Klebsiella strains of low virulence and hypervirulence. Results The strain had a 5 553 341 bp circular chromosome and a 236 355 bp large plasmid. It was identified as Klebsiella variicola. The strain had multiple virulence genes encoding mucoid phenotype regulator (rmpA and rmpA2), aerobactin (iucABCD-iutA), salmochelin (iroBCDN) and yersiniabactin (irp1-2 and ybtAEPQSTUX) on the plasmid, which was not self-transmissible. It exhibited enhanced virulence in the larvae model, suggesting that the strain was hypervirulent. It was resistant to colistin (MIC = 8 mg/L) but was susceptible to amikacin, aztreonam, cefotaxime, ceftazidime, gentamicin, imipenem, meropenem, moxifloxacin, piperacillin/tazobactam, trimethoprim/sulfamethoxazole and tigecycline. The D150G substitution in PhoP, part of the PhoP-Q two-component system, which is known to mediate colistin resistance, was present in the strain. Conclusions Hypervirulence is not restricted to K. pneumoniae; it is also seen in other Klebsiella spp. The convergence of colistin resistance and hypervirulence in K. variicola represents a new challenge for health.

Shiga toxin-producing E. coli (STEC) O80:H2 is an uncommon hybrid pathotype that has recently emerged in France. We analysed 18 STEC O80:H2 isolated from humans in Switzerland during 2010-2017. All isolates carried stx2a or stx2d, the rare eae variant eae-ξ and at least seven virulence genes associated with pS88, a plasmid that is found in extraintestinal pathogenic E. coli (ExPEC). Whole genome sequencing (WGS) identified additional chromosomal extraintestinal virulence genes encoding for type 1 fimbria (fimA, fimC and fimH), aerobactin (iuc/iutA) and afimbrial adhesins (afaA/C/D/E-VIII). Core genome multi-locus sequence typing (cgMLST) detected two closely related but distinct subclusters with different stx2 and iuc/iutA genotypes. All isolates were multidrug resistant (MDR), but susceptible to third generation cephalosporins and azithromycin. STEC/ExPEC hybrid pathotypes such as STEC O80:H2 represent a therapeutical challenge in the event of extraintestinal infection.

We found that Vibrio furnissii can utilize aerobactin (AERO) as a xenosiderophore. A homology search of its genome revealed that this bacterium possesses genes encoding an AERO-mediated iron acquisition system similar to that of V. vulnificus. The system consists of the ABC transporter gene vatCDB, the GntR-type transcriptional repressor gene iutR, and the outer membrane receptor gene iutA. The functions of the vatCDB operon and iutA in V. furnissii were confirmed by the inability of the corresponding deletion mutants to utilize AERO. Reverse transcription-quantitative PCR revealed that iutA transcription under iron-limiting conditions was extensively activated by the addition of AERO to the growth medium; therefore, we focused on elucidating this phenomenon. Electrophoretic mobility shift and DNase I footprinting assays revealed that glutathione S-transferase-fused IutR (GST-IutR) bound directly to a specific palindromic sequence in the iutA promoter region. However, GST-IutR did not bind to this sequence when either AERO or ferric AERO was present in the assay mixture. These in vitro findings suggest that, under iron-limiting conditions, iutA transcription in V. furnissii is artfully regulated both by IutR, acting as a direct repressor of iutA, and by AERO, acting as an effector for IutR, leading to the derepression of iutA transcription.

The increasing prevalence of KPC-producing Klebsiella pneumoniae strains in clinical settings has been largely attributed to dissemination of organisms of specific multilocus sequence types, such as ST258 and ST11. Compared with the ST258 clone, which is prevalent in North America and Europe, ST11 is common in China but information regarding its genetic features remains scarce. In this study, we performed detailed genetic characterization of ST11 K. pneumoniae strains by analyzing whole-genome sequences of 58 clinical strains collected from diverse geographic locations in China. The ST11 genomes were found to be highly heterogeneous and clustered into at least three major lineages based on the patterns of single-nucleotide polymorphisms. Exhibiting five different capsular types, these ST11 strains were found to harbor multiple resistance and virulence determinants such as the blaKPC-2 gene, which encodes carbapenemase, and the yersiniabactin-associated virulence genes irp, ybt and fyu. Moreover, genes encoding the virulence factor aerobactin and the regulator of the mucoid phenotype (rmpA) were detectable in six genomes, whereas genes encoding salmochelin were found in three genomes. In conclusion, our data indicated that carriage of a wide range of resistance and virulence genes constitutes the underlying basis of the high level of prevalence of ST11 in clinical settings. Such findings provide insight into the development of novel strategies for prevention, diagnosis and treatment of K. pneumoniae infections.

Background Hypervirulent K. pneumoniae (hvKp) causes severe community acquired infections, predominantly in Asia. Though initially isolated from liver abscesses, they are now prevalent among invasive infections such as bacteraemia. There have been no studies reported till date on the prevalence and characterisation of hvKp in India. The objective of this study is to characterise the hypervirulent strains isolated from bacteraemic patients for determination of various virulence genes and resistance genes and also to investigate the difference between healthcare associated and community acquired hvKp with respect to clinical profile, antibiogram, clinical outcome and molecular epidemiology. Results Seven isolates that were susceptible to all of the first and second line antimicrobials and phenotypically identified by positive string test were included in the study. They were then confirmed genotypically by presence of rmpA and rmpA2 by PCR. Among the study isolates, four were from patients with healthcare associated infections; none were fatal. All patients with community acquired infection possessed chronic liver disease with fatal outcome. Genes encoding for siderophores such as aerobactin, enterobactin, yersiniabactin, allantoin metabolism and iron uptake were identified by whole genome sequencing. Five isolates belonged to K1 capsular type including one K. quasipneumoniae. None belonged to K2 capsular type. Four isolates belonged to the international clone ST23 among which three were health-care associated and possessed increased virulence genes. Two novel sequence types were identified in the study; K. pneumoniae belonging to ST2319 and K. quasipneumoniae belonging to ST2320. Seventh isolate belonged to ST420. Conclusion This is the first report on whole genome analysis of hypervirulent K. pneumoniae from India. The novel sequence types described in this study indicate that these strains are evolving and hvKp is now spread across various clonal types. Studies to monitor the prevalence of hvKp is needed since there is a potential for the community acquired isolates to develop multidrug resistance in hospital environment and may pose a major challenge for clinical management.

Source at http://doi.org/10.1371/journal.pone.0191860 Siderophores are small molecules synthesized and secreted by bacteria and fungi to scavenge iron. Extracellular ferri-siderohores are recognized by cognate receptors on the cell surface for transport over membranes. Several siderophore systems from Vibrionaceae representatives are known and well understood, e.g., the molecular structure of the siderophore, the biosynthesis gene cluster and pathway, and the gene expression pattern. Less is known about how these systems are distributed among the ~140 Vibrionaceae species, and which evolutionary processes contributed to the present-day distribution. In this work, we compiled existing knowledge on siderophore biosynthesis systems and siderophore receptors from Vibrionaceae and used phylogenetic analyses to investigate their organization, distribution, origin and evolution. Through literature searches, we identified nine different siderophore biosynthesis systems and thirteen siderophore receptors in Vibrionaceae. Homologs were identified by BLAST searches, and the results were mapped onto a Vibrionaceae phylogeny. We identified 81 biosynthetic systems distributed in 45 Vibrionaceae species and 16 unclassified Vibrionaceae strains, and 409 receptors in 89 Vibrionaceae species and 49 unclassified Vibrionaceae strains. The majority of taxa are associated with at least one type of siderophore biosynthesis system, some (e.g., aerobactin and vibrioferrin) of which are widely distributed in the family, whereas others (i.e., bisucaberin and vibriobactin) are found in one lineage. Cognate receptors are found more widespread. Phylogenetic analysis of three siderophore systems (piscibactin, vibrioferrin and aerobactin) show that their present-day distribution can be explained by an old insertion into Vibrionaceae, followed mainly by stable vertical evolution and extensive loss, and some cases of horizontal gene transfers. The present work provides an up to date overview of the distribution of siderophore-based iron acquisition systems in Vibrionaceae, and presents phylogenetic analysis of these systems. Our results suggest that the present-day distribution is a result of several evolutionary processes, such as old and new gene acquisitions, gene loss, and both vertical and horizontal gene transfers.

Klebsiella pneumoniae strains that are commonly recognized by clinicians and microbiologists are termed as classical K. pneumoniae (cKP). A strain with capsule-associated mucopolysaccharide web is known as hypervirulent K. pneumoniae (hvKP) as it enhances the serum resistant and biofilm production. Aim is to determine and correlate various virulence genes, ESBL, serum bactericidal effect and biofilm formation of clinical isolated cKP and hvKP from respiratory tract infected patients. A total of 96 K. pneumoniae strains were isolated from sputum of respiratory tract infected patients. The isolates were performed string test, AST, ESBL virulence gene, serum bactericidal and biofilm assays. Out of 96 isolates, 39 isolates (40.6%) were identified with hypervirulent phenotypes. The number of cKP exhibiting resistance to the tested antimicrobials and ESBLs were significantly higher than that of the hvKP strains. The virulence genes of K. pneumoniae such as K1, K2, rmpA, uge, kfu and aerobactin were strongly associated with hvKP than cKP. However, no significant difference was found in FIM-1 and MrKD3 genes. ESBL producing cKP and hvKP were significantly associated with strong biofilm formation (both P < 0.05) and highly associated with bactericidal effect of serum (both P < 0.05) than cKP strains. However, neither biofilm formation nor bactericidal effect of serum was found with significant difference in between ESBL producing cKP and ESBL producing hvKP strains (both P > 0.05). Although the hvKP possess more virulence gene, but they didn’t show any significant difference between biofilm formation and bactericidal effect of serum compared with ESBL producing cKP strains.

The siderophore aerobactin is the dominant siderophore produced by hypervirulent Klebsiella pneumoniae (hvKP) and was previously shown to be a major virulence factor in systemic infection. However, strains of hvKP commonly produce the additional siderophores yersiniabactin, salmochelin, and enterobactin. The roles of these siderophores in hvKP infection have not been optimally defined. To that end, site-specific gene disruptions were created in hvKP1 (wild type), resulting in the generation of hvKP1Δ iucA (aerobactin deficient), hvKP1Δ iroB (salmochelin deficient), hvKP1Δ entB (enterobactin and salmochelin deficient), hvKP1Δ irp2 (yersiniabactin deficient), and hvKP1Δ entB Δ irp2 (enterobactin, salmochelin, and yersiniabactin deficient). The growth/survival of these constructs was compared to that of their wild-type parent hvKP1 ex vivo in human ascites fluid, human serum, and human urine and in vivo in mouse systemic infection and pulmonary challenge models. Interestingly, in contrast to aerobactin, the inability to produce enterobactin, salmochelin, or yersiniabactin individually or in combination did not decrease the ex vivo growth/survival in human ascites or serum or decrease virulence in the in vivo infection models. Surprisingly, none of the siderophores increased growth in human urine. In human ascites fluid supplemented with exogenous siderophores, siderophores increased the growth of hvKP1Δ iucA , with the relative activity being enterobactin > aerobactin > yersiniabactin > salmochelin, suggesting that the contribution of aerobactin to virulence is dependent on both innate biologic activity and quantity produced. Taken together, these data confirm and extend a role for aerobactin as a critical virulence factor for hvKP. Since it appears that aerobactin production is a defining trait of hvKP strains, this factor is a potential antivirulence target.

The expression of aerobactin accounts for much of the pathogenesis of avian pathogenic Escherichia coli (APEC). iucA, iucB, iucC and iucD are involved in aerobactin synthesis and iutA is responsible for the expression of a specific outer membrane receptor protein for ferric aerobactin. Knockout mutants of iucD and iucDiutA in the APEC O2 strain E058 were constructed and named E058ΔiucD and E058ΔiucDΔiutA, respectively. To evaluate the pathogenicity of these mutants, we used multiple approaches to assess the effects of mutations on the virulence of APEC E058. Aerobactin-defective mutants E058ΔiucD and E058ΔiucDΔiutA showed significantly decreased pathogenicity compared with the wild-type strain E058, evidenced by the low extent of colonization in selected organs or being outcompeted by E058 in vivo. Chickens challenged with APEC E058 exhibited typical signs and lesions of avian colibacillosis, while those inoculated with the mutants E058ΔiucD or E058ΔiucDΔiutA showed moderate airsacculitis, mild pericarditis and perihepatitis. However, no significant differences in resistance to specific-pathogen-free chicken serum, ingestion by HD-11 cells, and growth rates in LB were observed between the mutants and the wild-type strain. These results indicated that the IucD- and IutA-related virulence factors play a significant role in the pathogenesis of the APEC strain E058.

Iron is a key micronutrient for microbial growth but is often present in low concentrations or in biologically unavailable forms. Many microorganisms overcome this challenge by producing siderophores, which are ferric-iron chelating compounds that enable the solubilization and acquisition of iron in a bioactive form. Pantoea stewartii subsp. stewartii , the causal agent of Stewart's wilt of sweet corn, produces a siderophore under iron-limiting conditions. The proteins involved in the biosynthesis and export of this siderophore are encoded by the iucABCD-iutA operon, which is homologous to the aerobactin biosynthetic gene cluster found in a number of enteric pathogens. Mutations in iucA and iutA resulted in a decrease in surface-based motility that P. stewartii utilizes during the early stages of biofilm formation, indicating that active iron acquisition impacts surface motility for P. stewartii . Furthermore, bacterial movement in planta is also dependent on a functional siderophore biosynthesis and uptake pathway. Most notably, siderophore-mediated iron acquisition is required for full virulence in the sweet corn host, indicating that active iron acquisition is essential for pathogenic fitness for this important xylem-dwelling bacterial pathogen.

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.

A direct and selective method for the functionalization of aerobactin has been described. Selectivity was achieved by masking the functioning carboxylate via iron-chelation, leaving the two remaining carboxylates for direct modification. Both mono- and bis-functionalized aerobactin effectively targeted pathogenic bacteria, showing a facile method with prospective applications.

Hypervirulent and multidrug resistant Klebsiella pneumoniae strains pose a significant threat to the public health. In the present study, 21 carbapenem-resistant K. pneumoniae isolates (CRKP) were determined by the string test as hypermucoviscous K. pneumoniae (HMKP), with the prevalence of 15.0% (21/140) among CRKP, and 1.1% (21/1838) among all K. pneumoniae isolates. Among them, 7 (33.3%), and 1 (4.76%) isolate belonged to capsular serotype K20 and K2 respectively, while 13 (61.9%, 13/21) weren't successfully typed by capsular serotyping. All the 21 isolates were carbapenemase-producers and were positive for blaKPC-2. In addition to blaKPC-2, all the 21 isolates except one harbor blaSHV-11, and 15 carry extended-spectrum β-lactamase gene blaCTX-M-65. The virulence-associated genes with more than 90% of positive rates among 21 isolates included ureA (100%, 21/21), wabG (100%, 21/21), fimH (95.2%, 20/21), entB (95.2%, 20/21), ycf (95.2%, 20/21), ybtS (95.2%, 20/21), and iutA (90.5%, 19/21). rmpA and aerobactin were found in 57.1% (12/21) isolates. Five sequence types (STs) were identified by multilocus sequence typing (MLST), including ST11 (11 K-non capsule typable and 5 K20 isolates), ST268 (1 K20 isolate and 1 K-non capsule typable isolate), ST65 (1 K2 isolate), ST692 (1 K-non capsule typable isolate), and ST595, a novel sequence type (1 K-non capsule typable isolate). Pulsed-field gel electrophoresis (PFGE) results showed two major PFGE clusters, of which cluster A accounts for 6 ST11 isolates (28.6%) and cluster B includes 8 ST11 isolates (38.1%, 8/21). Ten and six ST11 isolates were isolated from 2014 and 2015, respectively, while 8 were isolated from the same month of December in 2014. Ten isolates were collected from the intensive care unit (ICU), and all except one belonged to ST11. Additional 4 ST11 isolates were collected from patients in non-ICU wards, who had more than 10 days of ICU stay history in 2014 prior to transfer to their current wards where the isolates were recovered. Taken together, the present study showed a hospital outbreak and dissemination of ST11 HMKP with carbapenem resistance caused by KPC-2. Effective surveillance and strict infection control strategies should be implemented to prevent outbreak by HMKP with carbapenem resistance in hospitals.

Background We describe the first two multifocal invasive infections due to Klebsiella pneumoniae recently observed in Switzerland. Methods Phenotypic (MIC assays and string test) and molecular analyses (PCR/Sequencing for bla, virulence factor genes and whole genome sequencing for one strain) were performed to characterize the causative K. pneumoniae isolates. Results Both K. pneumoniae isolates (Kp1 and Kp2) were pan-susceptible to antibiotics and produced narrow-spectrum SHV β-lactamases. However, only Kp1 was string test positive. Kp1 was of ST380 and caused liver abscess as well as pneumonia and orbital phlegmon in an Eritrean patient. It belonged to the hypervirulent capsular serotype K2 and harboured the classic virulence-associated rmpA and aerobactin genes, fulfilling both the clinical and microbiological definitions for an invasive K. pneumoniae syndrome. Kp2 was of ST1043 and caused both liver abscess and endocarditis in a Swiss patient. Moreover, it did not possess the classic virulence-associated genes. Whole genome sequencing identified less well-known virulence factors in Kp2 that might have contributed to its virulence. Among these there were genes important for intestinal colonization and/or invasion, such as genes involved in adhesion (e.g., fimABCD and mrkABCD), regulation of capsule polysaccharide biosynthesis (e.g., evgS-evgA), as well as iron uptake (iroN), energy conversion, and metabolism. Discussion This report confirms the continuous dissemination of hypervirulent K. pneumoniae strains among patients of non-Asian descent in Europe. Moreover, it highlights the genetic background of an atypical hypervirulent K. pneumoniae causing a severe invasive infection despite not possessing the classical virulence characteristics of hypermucoviscous strains.

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.

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.

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.

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.