The accessory genomes of many pathogenic bacteria include ABC transporters that scavenge metal by siderophore uptake and ABC transporters that contribute to antimicrobial resistance by multidrug efflux. There are mechanistic and recently recognized structural similarities between siderophore importer proteins and efflux pumps. Here we investigated the influence of siderophore importer YbtPQ on antimicrobial resistance of Klebsiella pneumoniae. YbtPQ is encoded in the yersiniabactin cluster in a prevalent mobile genetic element ICEKp, and is also common in pathogenicity islands of Escherichia coli and Yersinia species, where yersiniabactin enhances virulence. Deletion of ICEKp increased the susceptibility of K. pneumoniae to all antimicrobials tested. The mechanism was dependent on the yersiniabactin importer YbtPQ and may involve antimicrobial efflux, since it was affected by the inhibitor reserpine. The element ICEKp is naturally highly mobile, indeed the accessory genome of K. pneumoniae is recognized as a reservoir of genes for the emergence of hospital outbreak strains and for transfer to other Gram-negative pathogens. Introduction of ICEKp, or a plasmid encoding YbtPQ, to E. coli decreased its susceptibility to a broad range of antimicrobials. Thus a confirmed siderophore importer, on a rapidly evolving and highly mobile element capable of interspecies transfer, may have a secondary function exporting antimicrobials.

The accessory genome of the human pathogen Klebsiella pneumoniae is large, variable and highly mobile. This reservoir of genes leads to the emergences of hospital outbreak strains with enhanced virulence and multidrug resistance, such as ST258, and acts as a source for transfer of these traits to other Gram negative pathogens. One such mobile genetic element, ICEKp, is prevalent in isolates of invasive disease where it enhances iron acquisition by the siderophore yersiniabactin. Yersiniabactin is also a virulence factor in pathogenic Escherichia coli and Yersinia species. Similarities between siderophore transporters and drug efflux pumps led us to postulate that the yersiniabactin transport proteins could contribute to antimicrobial resistance. We determined the effect of loss and gain of ICEKp, or the transporters alone, on iron acquisition and drug sensitivity of K. pneumoniae and Escherichia coli. Deletion of ICEKp impaired iron acquisition of clinical isolate K. pneumoniae HS11286 due to reduced siderophore secretion and reduced ability to acquire iron from siderophores. A simultaneous increase in sensitivity to a broad range of antimicrobials could be complemented by reintroduction of the ybtPQ ABC transporter. Furthermore, transfer of ICEKp to E. coli occurred efficiently by conjugation and conferred a similar decrease in sensitivity to antimicrobials.

The accessory genome of many pathogenic bacteria includes ABC transporters that scavenge metal by siderophore uptake and ABC transporters that contribute to antimicrobial resistance by multidrug efflux. There are mechanistic and recently recognised structural similarities between siderophore importer proteins and efflux pumps. Here we investigated the influence of siderophore importer YbtPQ on antimicrobial resistance of Klebsiella pneumoniae . YbtPQ is encoded in the yersiniabactin cluster in a prevalent mobile genetic element ICEKp, and is also common in pathogenicity islands of Escherichia coli and Yersinia species, where yersiniabactin enhances virulence. Deletion of ICEKp increased the sensitivity of K. pneumoniae to all antimicrobials tested. The mechanism was dependent on the yersiniabactin importer YbtPQ and involved antimicrobial efflux, since it was affected by the inhibitor reserpine. The element ICEKp is naturally highly mobile, indeed the accessory genome of K. pneumoniae is recognised as a reservoir of genes for the emergence of hospital outbreak strains and for transfer to other Gram-negative pathogens. Introduction of ICEKp, or a plasmid encoding YbtPQ, to E. coli decreased its sensitivity to a broad range of antimicrobials. Thus, a confirmed siderophore importer, on a rapidly evolving and highly mobile element capable of interspecies transfer, may have a secondary function exporting antimicrobials. ### Competing Interest Statement The authors have declared no competing interest.

Fimbriae mediate adhesion of Salmonella enterica organisms to the intestinal epithelium, which is an essential step in the pathogenesis process preceding invasion and/or systemic spread. In addition, Salmonella fimbrial genes transcripts were detected in the blood samples from Salmonella infected human patients, which supports the proposal that fimbriae play a role in invasive Salmonella infections. In this study, BlastN-based interrogation of the NCBI bacterial genome database and PCR investigation of Salmonella serovars have shown that the S. Paratyphi A stkF gene and/or the whole stk fimbrial gene cluster is present in about ~30% of S. enterica serovars investigated up to date. Furthermore, bioinformatics and phenotypic characterization have revealed that the stk fimbrial operon belongs to the chaperone/usher-γ4- fimbrial clade and that it encodes a mannose-sensitive hemagglutinating fimbrial structure. The latter trait is typical of type 1 fimbriae, in which fimbrial phase variation is common. The observed intragenic, 26 bp tandem repeat triplication event in stkF would suggest that slipped-strand mispairing and/or recombination within a signature stkF-borne tandem repeat motif as a likely mechanism for a form of low-frequency phase switching at the translational level leading to allelic OFF forms, hence the inability of production and/or absence of fimbriae by EM-examination on E. coli HB101/pUCstk-stkFOFFv2. The in vitro profile of marked anti-StkF-mediated opsonophagocytosis and complement-mediated killing activity observed coupled with the mice immunogenicity profile strongly supports further investigation of StkF as a potential Salmonella vaccine candidate.

Klebsiella pneumoniae is a human pathogen, prominent in antimicrobial-resistant and nosocomial infection. The integrative and conjugative element ICEKp1 is present in a third of clinical isolates and more prevalent in invasive disease; it provides genetic diversity and enables the spread of virulence-associated genes. We report a second integrative conjugative element that can co-occur with ICEKp1 in K. pneumoniae. This element, ICEKp2, is similar to the Pseudomonas aeruginosa pathogenicity island PAPI. We identified ICEKp2 in K. pneumoniae sequence types ST11, ST258 and ST512, which are associated with carbapenem-resistant outbreaks in China and the US, including isolates with and without ICEKp1. ICEKp2 was competent for excision, but self-mobilisation to recipient Escherichia coli was not detected. In an isolate with both elements, ICEKp2 positively influenced the efficiency of plasmid mobilisation driven by ICEKp1. We propose a putative mechanism, in which a Mob2 ATPase of ICEKp2 may contribute to the ICEKp1 conjugation machinery. Supporting this mechanism, mob2, but not a variant with mutations in the ATPase motif, restored transfer efficiency to an ICEKp2 knockout. This is the first demonstration of the interaction between integrative and conjugative genetic elements in a single Gram-negative bacterium with implications for understanding evolution by horizontal gene transfer.

Acinetobacter baumannii among other pathogens is capable of acquiring new virulence determinants via mobile genetic elements (MGE). Analysis of the distribution of genomic islands (GIs) has shown in many species that most MGEs cluster tightly with specific lineages on a phylogenetic core genome tree. This study aims to address the questions on the association between GIs distribution among certain clonal lineagesand to test the contribution of G08 and G62 in A. baumannii to metal susceptibility phenotypes. The whole genome phylogenetic single nucleotide polymorphism (SNP) tree was build using the feature frequency profile. As input, the 101 complete A. baumannii genomes deposited in GenBank were used. The matrix was generated using command line blast and the R platform to generate the output. Susceptibility testing of heavy metals was performed on wild-type and GI-deletion mutants using eight different heavy metals. Whole genome phylogenetic SNP tree constructed on all complete deposited A. baumannii genomes showed that the integrase of the metal resistance G08 and G62 are present only in single or very few sequence types (STs). To test the hypothesis that these GI seven if present only in defined lineage are still mobile, G08 and G62 were selected respectively in strains AB0057 and ATCC17978. Phylogenetic analysis of the respective GI integrases confirmed that the G08int and G62int belong to a separate clade within the Acinetobacter tyrosine recombinases. Susceptibility testing in A. baumannii strain ATCC 17978, AB0057 and their respective ΔG62 and ΔG08 mutants confirmed the contribution of the GI-encoded efflux transporters to heavy metal decreased susceptibility.

Genomic islands (GIs) are discrete gene clusters encoding for a variety of functions including antibiotic and heavy metal resistance, some of which are tightly associated to lineages of the core genome phylogenetic tree. We have investigated the functions of two distinct integrase genes in the mobilization of two metal resistant GIs, G08 and G62, of Acinetobacter baumannii. Real-time PCR demonstrated integrase-dependent GI excision, utilizing isopropyl β-d-1-thiogalactopyranoside IPTG-inducible integrase genes in plasmid-based mini-GIs in Escherichia coli. In A. baumannii, integrase-dependent excision of the original chromosomal GIs could be observed after mitomycin C induction. In both E. coli plasmids and A. baumannii chromosome, the rate of excision and circularization was found to be dependent on the expression level of the integrases. Susceptibility testing in A. baumannii strain ATCC 17978, A424, and their respective ΔG62 and ΔG08 mutants confirmed the contribution of the GI-encoded efflux transporters to heavy metal decreased susceptibility. In summary, the data evidenced the functionality of two integrases in the excision and circularization of the two Acinetobacter heavy-metal resistance GIs, G08 and G62, in E. coli, as well as when chromosomally located in their natural host. These recombination events occur at different frequencies resulting in genome plasticity and may participate in the spread of resistance determinants in A. baumannii.

Plasmids play critical roles in bacterial fitness and evolution of Pseudomonas aeruginosa. Here two plasmids found in a drug-resistant P. aeruginosa clinical isolate HS87 were completely sequenced. The pHS87b plasmid (11.2 kb) carries phage-related genes and function-unknown genes. Notably, pHS87b encodes an integrase and has an adjacent tRNAThr-associated attachment site. A corresponding integrated form of pHS87b at the tRNAThr locus was identified on the chromosome of P. aeruginosa, showing that pHS87b is able to site-specifically integrate into the 3’-end of the tRNAThr gene. The pHS87a plasmid (26.8 kb) displays a plastic structure containing a putative replication module, stability factors and a variable region. The RepA of pHS87a shows significant similarity to the replication proteins of pPT23A-family plasmids. pHS87a carries a transposon Tn6049, a truncated insertion sequence ΔIS1071 and a Tn402-like class 1 integron which contains an aacA4 cassette that may confer aminoglycoside resistance. Thus, pHS87b is a site-specific integrative plasmid whereas pHS87a is a plastic antibiotic resistance plasmid. The two native plasmids may promote the fitness and evolution of P. aeruginosa.

OBJECTIVES:This study aims to investigate the landscape of the mobile genome, with a focus on antibiotic resistance-associated factors in carbapenem-resistant Klebsiella pneumoniae.METHODS:The mobile genome of the completely sequenced K. pneumoniae HS11286 strain (an ST11, carbapenem-resistant, near-pan-resistant, clinical isolate) was annotated in fine detail. The identified mobile genetic elements were mapped to the genetic contexts of resistance genes. The blaKPC-2 gene and a 26 kb region containing 12 clustered antibiotic resistance genes and one biocide resistance gene were deleted, and the MICs were determined again to ensure that antibiotic resistance had been lost.RESULTS:HS11286 contains six plasmids, 49 ISs, nine transposons, two separate In2-related integron remnants, two integrative and conjugative elements (ICEs) and seven prophages. Sixteen plasmid-borne resistance genes were identified, 14 of which were found to be directly associated with Tn1721-, Tn3-, Tn5393-, In2-, ISCR2- and ISCR3-derived elements. IS26 appears to have actively moulded several of these genetic regions. The deletion of blaKPC-2, followed by the deletion of a 26 kb region containing 12 clustered antibiotic resistance genes, progressively decreased the spectrum and level of resistance exhibited by the resultant mutant strains.CONCLUSIONS:This study has reiterated the role of plasmids as bearers of the vast majority of resistance genes in this species and has provided valuable insights into the vital role played by ISs, transposons and integrons in shaping the resistance-coding regions in this important strain. The 'resistance-disarmed' K. pneumoniae ST11 strain generated in this study will offer a more benign and readily genetically modifiable model organism for future extensive functional studies.

SecReT6 (http://db-mml.sjtu.edu.cn/SecReT6/) is an integrated database providing comprehensive information on type VI secretion systems (T6SSs) in bacteria. T6SSs are a class of sophisticated cell contact-dependent apparatuses involved in mediating antagonistic or synergistic communications between bacteria and/or bacteria and eukaryotes. These apparatuses have recently been found to be widely distributed among Gram-negative bacterial species. SecReT6 offers a unique, readily explorable archive of known and putative T6SSs, and cognate effectors found in bacteria. It currently contains data on 11 167 core T6SS components mapping to 906 T6SSs found in 498 bacterial strains representing 240 species, as well as a collection of over 600 directly relevant references. Also collated and archived were 1340 diverse candidate secreted effectors which were experimentally shown and/or predicted to be delivered by T6SSs into target eukaryotic and/or prokaryotic cells as well as 196 immunity proteins. A broad range of T6SS gene cluster detection and comparative analysis tools are readily accessible via SecReT6, which may aid identification of effectors and immunity proteins around the T6SS core components. This database will be regularly updated to ensure its ongoing maximal utility and relevance to the scientific research community.

Staphylococcus aureus bacteremia cases are complicated by bacterial persistence and treatment failure despite the confirmed in vitro susceptibility of the infecting strain to administered antibiotics. A high incidence of methicillin-resistant S. aureus (MRSA) bacteremia cases are classified as persistent and are associated with poorer patient outcomes. It is still unclear how S. aureus evades the host immune system and resists antibiotic treatment for the prolonged duration of a persistent infection. In this study, the genetic changes and associated phenotypic traits specific to S. aureus persistent bacteremia were identified by comparing temporally dispersed isolates from persistent infections (persistent isolates) originating from two independent persistent S. aureus bacteremia cases with the initial infection isolates and with three resolved S. aureus bacteremia isolates from the same genetic background. Several novel traits were associated specifically with both independent sets of persistent S. aureus isolates compared to both the initial isolates and the isolates from resolved infections (resolved isolates). These traits included (i) increased growth under nutrient-poor conditions; (ii) increased tolerance of iron toxicity; (iii) higher expression of cell surface proteins involved in immune evasion and stress responses; and (iv) attenuated virulence in a Galleria mellonella larva infection model that was not associated with small-colony variation or metabolic dormancy such as had been seen previously. Whole-genome sequence analysis identified different single nucleotide mutations within the mprF genes of all the isolates with the adaptive persistence traits from both independent cases. Overall, our data indicate a novel role for MprF function during development of S. aureus persistence by increasing bacterial fitness and immune evasion.

Introduction: Helicobacter pylori is a gram-negative urease-producing bacterium causally linked with gastritis, peptic ulcer disease and gastric adenocarcinoma. Infection is more frequent and acquired at an earlier age in developing countries compared to European populations. The incidence of Helicobacter pylori infection in dyspeptic Ghanaian patients was 75.4 %. However, epidemiological factors associated with infection vary across populations. Methods: This study used a cross-sectional design to consecutively sample dyspeptic patients at the Endoscopy Unit of the Korle-Bu Teaching Hospital, Accra between 2010 and 2012. The study questionnaire elicited their epidemiological clinical characteristics. Helicobacter pylori infection was confirmed by rapid-urease examination of antral biopsies at upper Gastro-intestinal endoscopy. Results: The sample population of dyspeptic patients attending the Endoscopy Unit for upper GI endoscopy yielded 242 patients of which 47.5 % were females. The age distribution of H. pylori-infection was even across most age – groups, ranging from 69.2% (61 – 70) years to 80% (21 – 30) years. Helicobacter pylori prevalence decreased across areas mapping to the three residential classes in accordance with increasing affluence with rural areas having the highest prevalence. The unemployed and patients in farming had relatively high Helicobacter pylori infection rates of 92.3 % and 91.7 % respectively. Conclusion: Helicobacter pylori is endemic in Ghana but the persistently high prevalence across age groups despite significant community anti-microbial use suggests likely re-crudescence or re-infection from multiple sources in a developing country. Socio-cultural factors such as residential class and farming may be facilitating factors for its continued prevalence. Pan African Medical Journal. 2015; 20:178 doi:10.11604/pamj.2015.20.178.5024 This article is available online at: http://www.panafrican-med-journal.com/content/article/20/178/full/ © Timothy Nii Akushe Archampong et al. The Pan African Medical Journal ISSN 1937-8688. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Pan African Medical Journal – ISSN: 19378688 (www.panafrican-med-journal.com) Published in partnership with the African Field Epidemiology Network (AFENET). (www.afenet.net) Research Open Access

INTRODUCTION:Helicobacter pylori is a gram-negative urease-producing bacterium causally linked with gastritis, peptic ulcer disease and gastric adenocarcinoma. Infection is more frequent and acquired at an earlier age in developing countries compared to European populations. The incidence of Helicobacter pylori infection in dyspeptic Ghanaian patients was 75.4%. However, epidemiological factors associated with infection vary across populations.METHODS:This study used a cross-sectional design to consecutively sample dyspeptic patients at the Endoscopy Unit of the Korle-Bu Teaching Hospital, Accra between 2010 and 2012. The study questionnaire elicited their epidemiological clinical characteristics. Helicobacter pylori infection was confirmed by rapid-urease examination of antral biopsies at upper Gastro-intestinal endoscopy.RESULTS:The sample population of dyspeptic patients attending the Endoscopy Unit for upper GI endoscopy yielded 242 patients of which 47.5% were females. The age distribution of H. pylori-infection was even across most age - groups, ranging from 69.2% (61 - 70) years to 80% (21 - 30) years. Helicobacter pylori prevalence decreased across areas mapping to the three residential classes in accordance with increasing affluence with rural areas having the highest prevalence. The unemployed and patients in farming had relatively high Helicobacter pylori infection rates of 92.3% and 91.7% respectively.CONCLUSION:Helicobacter pylori is endemic in Ghana but the persistently high prevalence across age groups despite significant community anti-microbial use suggests likely recrudescence or re-infection from multiple sources in a developing country. Socio-cultural factors such as residential class and farming may be facilitating factors for its continued prevalence.

Dietary intake of L-carnitine can promote cardiovascular diseases in humans through microbial production of trimethylamine (TMA) and its subsequent oxidation to trimethylamine N-oxide by hepatic flavin-containing monooxygenases. Although our microbiota are responsible for TMA formation from carnitine, the underpinning molecular and biochemical mechanisms remain unclear. In this study, using bioinformatics approaches, we first identified a two-component Rieske-type oxygenase/reductase (CntAB) and associated gene cluster proposed to be involved in carnitine metabolism in representative genomes of the human microbiota. CntA belongs to a group of previously uncharacterized Rieske-type proteins and has an unusual "bridging" glutamate but not the aspartate residue, which is believed to facilitate intersubunit electron transfer between the Rieske center and the catalytic mononuclear iron center. Using Acinetobacter baumannii as the model, we then demonstrate that cntAB is essential in carnitine degradation to TMA. Heterologous overexpression of cntAB enables Escherichia coli to produce TMA, confirming that these genes are sufficient in TMA formation. Site-directed mutagenesis experiments have confirmed that this unusual "bridging glutamate" residue in CntA is essential in catalysis and neither mutant (E205D, E205A) is able to produce TMA. Taken together, the data in our study reveal the molecular and biochemical mechanisms underpinning carnitine metabolism to TMA in human microbiota and assign the role of this novel group of Rieske-type proteins in microbial carnitine metabolism.

Chlorite dismutases (Clds) convert chlorite to O2 and Cl(-), stabilizing heme in the presence of strong oxidants and forming the O═O bond with high efficiency. The enzyme from the pathogen Klebsiella pneumoniae (KpCld) represents a subfamily of Clds that share most of their active site structure with efficient O2-producing Clds, even though they have a truncated monomeric structure, exist as a dimer rather than a pentamer, and come from Gram-negative bacteria without a known need to degrade chlorite. We hypothesized that KpCld, like others in its subfamily, should be able to make O2 and may serve an in vivo antioxidant function. Here, it is demonstrated that it degrades chlorite with limited turnovers relative to the respiratory Clds, in part because of the loss of hypochlorous acid from the active site and destruction of the heme. The observation of hypochlorous acid, the expected leaving group accompanying transfer of an oxygen atom to the ferric heme, is consistent with the more open, solvent-exposed heme environment predicted by spectroscopic measurements and inferred from the crystal structures of related proteins. KpCld is more susceptible to oxidative degradation under turnover conditions than the well-characterized Clds associated with perchlorate respiration. However, wild-type K. pneumoniae has a significant growth advantage in the presence of chlorate relative to a Δcld knockout strain, specifically under nitrate-respiring conditions. This suggests that a physiological function of KpCld may be detoxification of endogenously produced chlorite.

ABSTRACT In order to understand the genetic background and dissemination mechanism of carbapenem resistance and fosfomycin resistance in Enterobacteriaceae isolates, we studied a clinical Escherichia coli strain HS102707 isolate and an Enterobacter aerogenes strain HS112625 isolate, both of which were resistant to carbapenem and fosfomycin and positive for the bla KPC-2 and fosA3 genes. In addition, a clinical Klebsiella pneumoniae strain HS092839 isolate which was resistant to carbapenem was also studied. A 70-kb plasmid was successfully transferred to recipient E. coli J53 by a conjugation test. PCR and Southern blot analysis showed that bla KPC-2 was located on this plasmid. The complete sequence of pHS102707 showed that this plasmid belongs to the P11 subfamily (IncP1) and has a replication gene, several plasmid-stable genes, an intact type IV secretion system gene cluster, and a composite transposon Tn 1721 -Tn 3 that harbored bla KPC-2 . Interestingly, a composite IS 26 transposon carrying fosA3 was inserted in the Tn 1721-tnpA gene in pHS102707 and pHS112625, leading to the disruption of Tn 1721-tnpA and the deletion of Tn 1721-tnpR . However, only IS 26 with a truncated Tn 21-tnpR was inserted in pHS092839 at the same position. To our knowledge, this is the first report of fosA3 and bla KPC-2 colocated in the same Tn 1721 -Tn 3 –like composite transposon on a novel IncP group plasmid.