Structural and functional studies of Escherichia coli aggregative adherence fimbriae (AAF/V) reveal a deficiency in extracellular matrix binding.

Enteroaggregative Escherichia coli (EAEC) is an emerging cause of acute and persistent diarrhea worldwide. The pathogenesis of different EAEC stains is complicated, however, the early essential step begins with attachment of EAEC to intestinal mucosa via aggregative adherence fimbriae (AAFs). Currently, five different variants have been identified, which all share a degree of similarity in the gene organization of their operons and sequences. Here, we report the solution structure of Agg5A from the AAF/V variant. While preserving the major structural features shared by all AAF members, only Agg5A possesses an inserted helix at the beginning of the donor strand, which together with altered surface electrostatics, renders the protein unable to interact with fibronectin. Hence, here we characterize the first AAF variant with a binding mode that varies from previously described AAFs.

DFI-seq identification of environment-specific gene expression in uropathogenic Escherichia coli.

BACKGROUND: During infection of the urinary tract, uropathogenic Escherichia coli (UPEC) are exposed to different environments, such as human urine and the intracellular environments of bladder epithelial cells. Each environment elicits a distinct bacterial environment-specific transcriptional response. We combined differential fluorescence induction (DFI) with next-generation sequencing, collectively termed DFI-seq, to identify differentially expressed genes in UPEC strain UTI89 during growth in human urine and bladder cells. RESULTS: DFI-seq eliminates the need for iterative cell sorting of the bacterial library and yields a genome-wide view of gene expression. By analysing the gene expression of UPEC in human urine we found that genes involved in amino acid biosynthesis were upregulated. Deletion mutants lacking genes involved in arginine biosynthesis were outcompeted by the wild type during growth in human urine and inhibited in their ability to invade or proliferate in the J82 bladder epithelial cell line. Furthermore, DFI-seq was used to identify genes involved in invasion of J82 bladder epithelial cells. 56 genes were identified to be differentially expressed of which almost 60% encoded hypothetical proteins. One such gene UTI89_C5139, displayed increased adhesion and invasion of J82 cells when deleted from UPEC strain UTI89. CONCLUSIONS: We demonstrate the usefulness of DFI-seq for identification of genes required for optimal growth of UPEC in human urine, as well as potential virulence genes upregulated during infection of bladder cell culture. DFI-seq holds potential for the study of bacterial gene expression in live-animal infection systems. By linking fitness genes, such as those genes involved in amino acid biosynthesis, to virulence, this study contributes to our understanding of UPEC pathophysiology.

Hypervirulent Klebsiella pneumoniae in California Sea Lions ( Zalophus californianus): Pathologic Findings in Natural Infections.

Tissues of stranded California sea lions ( Zalophus californianus) naturally infected with a hyperviruluent strain of Klebsiella pneumoniae were examined by histopathology and immunohistochemistry against the K. pneumoniae K2 capsular antigen. In 7 of 8 animals, there was severe purulent bronchopneumonia, sometimes complicated by fibrinonecrotizing pleuritis with pyothorax. In affected areas of lung, large numbers of degenerate neutrophils and macrophages were admixed with rare large extracellular and intracellular gram-negative bacilli surrounded by a prominent capsule. Through serotyping, polymerase chain reaction, sequencing, and immunohistochemistry, these bacteria were confirmed to be a K2 serotype of K. pneumoniae. The same bacteria were identified through double immunolabeling within macrophages in blood vessels, lymph nodes, spleen, and liver. Intact K. pneumoniae were identified on epithelial surfaces of the nasopharyngeal, tracheal, and small intestine mucosae and within distal renal tubules. Our findings indicate that hypervirulent K. pneumoniae causes severe respiratory disease and intrahistiocytic bacteremia in California sea lions.

Epidemiology and clinical manifestations of enteroaggregative Escherichia coli.

Enteroaggregative Escherichia coli (EAEC) represents a heterogeneous group of E. coli strains. The pathogenicity and clinical relevance of these bacteria are still controversial. In this review, we describe the clinical significance of EAEC regarding patterns of infection in humans, transmission, reservoirs, and symptoms. Manifestations associated with EAEC infection include watery diarrhea, mucoid diarrhea, low-grade fever, nausea, tenesmus, and borborygmi. In early studies, EAEC was considered to be an opportunistic pathogen associated with diarrhea in HIV patients and in malnourished children in developing countries. In recent studies, associations with traveler's diarrhea, the occurrence of diarrhea cases in industrialized countries, and outbreaks of diarrhea in Europe and Asia have been reported. In the spring of 2011, a large outbreak of hemolytic-uremic syndrome (HUS) and hemorrhagic colitis occurred in Germany due to an EAEC O104:H4 strain, causing 54 deaths and 855 cases of HUS. This strain produces the potent Shiga toxin along with the aggregative fimbriae. An outbreak of urinary tract infection associated with EAEC in Copenhagen, Denmark, occurred in 1991; this involved extensive production of biofilm, an important characteristic of the pathogenicity of EAEC. However, the heterogeneity of EAEC continues to complicate diagnostics and also our understanding of pathogenicity.

Novel aggregative adherence fimbria variant of enteroaggregative Escherichia coli.

Enteroaggregative Escherichia coli (EAEC) organisms belong to a diarrheagenic pathotype known to cause diarrhea and can be characterized by distinct aggregative adherence (AA) in a stacked-brick pattern to cultured epithelial cells. In this study, we investigated 118 EAEC strains isolated from the stools of Danish adults with traveler's diarrhea. We evaluated the presence of the aggregative adherence fimbriae (AAFs) by a multiplex PCR, targeting the four known major subunit variants as well as their usher-encoding genes. Almost one-half (49/118) of the clinical isolates did not possess any known AAF major fimbrial subunit, despite the presence of other AggR-related loci. Further investigation revealed the presence of an AAF-related gene encoding a yet-uncharacterized adhesin, termed agg5A. The sequence of the agg5DCBA gene cluster shared fimbrial accessory genes (usher, chaperone, and minor pilin subunit genes) with AAF/III, as well as the signal peptide present in the beginning of the agg3A gene. The complete agg5DCBA gene cluster from a clinical isolate, EAEC strain C338-14, with the typical stacked-brick binding pattern was cloned, and deletion of the cluster was performed. Transformation to a nonadherent E. coli HB101 and complementation of the nonadherent C338-14 mutant with the complete gene cluster restored the AA adhesion. Overall, we found the agg5A gene in 12% of the 118 strains isolated from Denmark, suggesting that this novel adhesin represents an important variant.

Genomic epidemiology of the Escherichia coli O104:H4 outbreaks in Europe, 2011.

The degree to which molecular epidemiology reveals information about the sources and transmission patterns of an outbreak depends on the resolution of the technology used and the samples studied. Isolates of Escherichia coli O104:H4 from the outbreak centered in Germany in May-July 2011, and the much smaller outbreak in southwest France in June 2011, were indistinguishable by standard tests. We report a molecular epidemiological analysis using multiplatform whole-genome sequencing and analysis of multiple isolates from the German and French outbreaks. Isolates from the German outbreak showed remarkably little diversity, with only two single nucleotide polymorphisms (SNPs) found in isolates from four individuals. Surprisingly, we found much greater diversity (19 SNPs) in isolates from seven individuals infected in the French outbreak. The German isolates form a clade within the more diverse French outbreak strains. Moreover, five isolates derived from a single infected individual from the French outbreak had extremely limited diversity. The striking difference in diversity between the German and French outbreak samples is consistent with several hypotheses, including a bottleneck that purged diversity in the German isolates, variation in mutation rates in the two E. coli outbreak populations, or uneven distribution of diversity in the seed populations that led to each outbreak.

Temporal trends in antimicrobial resistance and virulence-associated traits within the Escherichia coli sequence type 131 clonal group and its H30 and H30-Rx subclones, 1968 to 2012.

To identify possible explanations for the recent global emergence of Escherichia coli sequence type (ST) 131 (ST131), we analyzed temporal trends within ST131 O25 for antimicrobial resistance, virulence genes, biofilm formation, and the H30 and H30-Rx subclones. For this, we surveyed the WHO E. coli and Klebsiella Centre's E. coli collection (1957 to 2011) for ST131 isolates, characterized them extensively, and assessed them for temporal trends. Overall, antimicrobial resistance increased temporally in prevalence and extent, due mainly to the recent appearance of the H30 (1997) and H30-Rx (2005) ST131 subclones. In contrast, neither the total virulence gene content nor the prevalence of biofilm production increased temporally, although non-H30 isolates increasingly qualified as extraintestinal pathogenic E. coli (ExPEC). Whereas virotype D occurred from 1968 forward, virotypes A and C occurred only after 2000 and 2002, respectively, in association with the H30 and H30-Rx subclones, which were characterized by multidrug resistance (including extended-spectrum-beta-lactamase [ESBL] production: H30-Rx) and absence of biofilm production. Capsular antigen K100 occurred exclusively among H30-Rx isolates (55% prevalence). Pulsotypes corresponded broadly with subclones and virotypes. Thus, ST131 should be regarded not as a unitary entity but as a group of distinctive subclones, with its increasing antimicrobial resistance having a strong clonal basis, i.e., the emergence of the H30 and H30-Rx ST131 subclones, rather than representing acquisition of resistance by diverse ST131 strains. Distinctive characteristics of the H30-Rx subclone-including specific virulence genes (iutA, afa and dra, kpsII), the K100 capsule, multidrug resistance, and ESBL production-possibly contributed to epidemiologic success, and some (e.g., K100) might serve as vaccine targets.

Origins of the E. coli strain causing an outbreak of hemolytic-uremic syndrome in Germany.

BACKGROUND: A large outbreak of diarrhea and the hemolytic-uremic syndrome caused by an unusual serotype of Shiga-toxin-producing Escherichia coli (O104:H4) began in Germany in May 2011. As of July 22, a large number of cases of diarrhea caused by Shiga-toxin-producing E. coli have been reported--3167 without the hemolytic-uremic syndrome (16 deaths) and 908 with the hemolytic-uremic syndrome (34 deaths)--indicating that this strain is notably more virulent than most of the Shiga-toxin-producing E. coli strains. Preliminary genetic characterization of the outbreak strain suggested that, unlike most of these strains, it should be classified within the enteroaggregative pathotype of E. coli. METHODS: We used third-generation, single-molecule, real-time DNA sequencing to determine the complete genome sequence of the German outbreak strain, as well as the genome sequences of seven diarrhea-associated enteroaggregative E. coli serotype O104:H4 strains from Africa and four enteroaggregative E. coli reference strains belonging to other serotypes. Genomewide comparisons were performed with the use of these enteroaggregative E. coli genomes, as well as those of 40 previously sequenced E. coli isolates. RESULTS: The enteroaggregative E. coli O104:H4 strains are closely related and form a distinct clade among E. coli and enteroaggregative E. coli strains. However, the genome of the German outbreak strain can be distinguished from those of other O104:H4 strains because it contains a prophage encoding Shiga toxin 2 and a distinct set of additional virulence and antibiotic-resistance factors. CONCLUSIONS: Our findings suggest that horizontal genetic exchange allowed for the emergence of the highly virulent Shiga-toxin-producing enteroaggregative E. coli O104:H4 strain that caused the German outbreak. More broadly, these findings highlight the way in which the plasticity of bacterial genomes facilitates the emergence of new pathogens.

Structural and population characterization of MrkD, the adhesive subunit of type 3 fimbriae.

Type 3 fimbriae are adhesive organelles found in enterobacterial pathogens. The fimbriae promote biofilm formation on biotic and abiotic surfaces; however, the exact identity of the receptor for the type 3 fimbriae adhesin, MrkD, remains elusive. We analyzed naturally occurring structural and functional variabilities of the MrkD adhesin from Klebsiella pneumoniae and Escherichia coli isolates of diverse origins. We identified a total of 33 allelic variants of mrkD among 90 K. pneumoniae isolates and 10 allelic variants among 608 E. coli isolates, encoding 11 and 9 protein variants, respectively. Based on the level of accumulated silent variability between the alleles, mrkD was acquired a relatively long time ago in K. pneumoniae but recently in E. coli. However, unlike K. pneumoniae, mrkD in E. coli is actively evolving under a strong positive selection by accumulation of mutations, often targeting the same positions in the protein. Several naturally occurring MrkD protein variants from E. coli were found to be significantly less adherent when tested in a mannan-binding assay and showed reduced biofilm-forming capacity. Functional examination of the MrkD adhesin in flow chamber experiments determined that it interacts with Saccharomyces cerevisiae cells in a shear-dependent manner, i.e., the binding is catch-bond-like and enhanced under increasing shear conditions. Homology modeling strongly suggested that MrkD has a two-domain structure, comprising a pilin domain anchoring the adhesin to the fimbrial shaft and a lectin domain containing the binding pocket; this is similar to structures found in other catch-bond-forming fimbrial adhesins in enterobacteria.

Role of enteroaggregative Escherichia coli virulence factors in uropathogenesis.

A multiresistant clonal Escherichia coli O78:H10 strain qualifying molecularly as enteroaggregative Escherichia coli (EAEC) was recently shown to be the cause of a community-acquired outbreak of urinary tract infection (UTI) in greater Copenhagen, Denmark, in 1991. This marks the first time EAEC has been associated with an extraintestinal disease outbreak. Importantly, the outbreak isolates were recovered from the urine of patients with symptomatic UTI, strongly implying urovirulence. Here, we sought to determine the uropathogenic properties of the Copenhagen outbreak strain and whether these properties are conferred by the EAEC-specific virulence factors. We demonstrated that through expression of aggregative adherence fimbriae, the principal adhesins of EAEC, the outbreak strain exhibited pronouncedly increased adherence to human bladder epithelial cells compared to prototype uropathogenic strains. Moreover, the strain was able to produce distinct biofilms on abiotic surfaces, including urethral catheters. These findings suggest that EAEC-specific virulence factors increase uropathogenicity and may have played a significant role in the ability of the strain to cause a community-acquired outbreak of UTI. Thus, inclusion of EAEC-specific virulence factors is warranted in future detection and characterization of uropathogenic E. coli.

wzi Gene sequencing, a rapid method for determination of capsular type for Klebsiella strains.

Pathogens of the genus Klebsiella have been classified into distinct capsular (K) types for nearly a century. K typing of Klebsiella species still has important applications in epidemiology and clinical microbiology, but the serological method has strong practical limitations. Our objective was to evaluate the sequencing of wzi, a gene conserved in all capsular types of Klebsiella pneumoniae that codes for an outer membrane protein involved in capsule attachment to the cell surface, as a simple and rapid method for the prediction of K type. The sequencing of a 447-nucleotide region of wzi distinguished the K-type reference strains with only nine exceptions. A reference wzi sequence database was created by the inclusion of multiple strains representing K types associated with high virulence and multidrug resistance. A collection of 119 prospective clinical isolates of K. pneumoniae were then analyzed in parallel by wzi sequencing and classical K typing. Whereas K typing achieved typeability for 81% and discrimination for 94.4% of the isolates, these figures were 98.1% and 98.3%, respectively, for wzi sequencing. The prediction of K type once the wzi allele was known was 94%. wzi sequencing is a rapid and simple method for the determination of the K types of most K. pneumoniae clinical isolates.

Prevalence and characteristics of the epidemic multiresistant Escherichia coli ST131 clonal group among extended-spectrum beta-lactamase-producing E. coli isolates in Copenhagen, Denmark.

We report the characteristics of 115 extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli clinical isolates, from 115 unique Danish patients, over a 1-year study interval (1 October 2008 to 30 September 2009). Forty-four (38%) of the ESBL isolates represented sequence type 131 (ST13)1, from phylogenetic group B2. The remaining 71 isolates were from phylogenetic groups D (27%), A (22%), B1 (10%), and B2 (3%). Serogroup O25 ST131 isolates (n = 42; 95% of ST131) comprised 7 different K antigens, whereas two ST131 isolates were O16:K100:H5. Compared to non-ST131 isolates, ST131 isolates were associated positively with CTX-M-15 and negatively with CTX-M-1 and CTX-M-14. They also were associated positively with 11 virulence genes, including afa and dra (Dr family adhesins), the F10 papA allele (P fimbria variant), fimH (type 1 fimbriae), fyuA (yersiniabactin receptor), iha (adhesin siderophore), iutA (aerobactin receptor), kpsM II (group 2 capsules), malX (pathogenicity island marker), ompT (outer membrane protease), sat (secreted autotransporter toxin), and usp (uropathogenicity-specific protein) and negatively with hra (heat-resistant agglutinin) and iroN (salmochelin receptor). The consensus virulence gene profile (>90% prevalence) of the ST131 isolates included fimH, fyuA, malX, and usp (100% each), ompT and the F10 papA allele (95% each), and kpsM II and iutA (93% each). ST131 isolates were also positively associated with community acquisition, extraintestinal pathogenic E. coli (ExPEC) status, and the O25, K100, and H4 antigens. Thus, among ESBL E. coli isolates in Copenhagen, ST131 was the most prevalent clonal group, was community associated, and exhibited distinctive and comparatively extensive virulence profiles, plus a greater variety of capsular antigens than reported previously.

Enteroaggregative Escherichia coli promotes transepithelial migration of neutrophils through a conserved 12-lipoxygenase pathway.

Enteroaggregative Escherichia coli (EAEC) induces release of pro-inflammatory markers and disruption of intestinal epithelial barriers in vitro, suggesting an inflammatory aspect to EAEC infection. However, the mechanisms underlying EAEC-induced mucosal inflammatory responses and the extent to which these events contribute to pathogenesis is not well characterized. Employing an established in vitro model we demonstrated that EAEC prototype strain 042 induces migration of polymorphonuclear neutrophils (PMNs) across polarized T84 cell monolayers. This event was mediated through a conserved host cell signalling cascade involving the 12/15-LOX pathway and led to apical secretion of an arachidonic acid-derived lipid PMN chemoattractant, guiding PMNs across the epithelia to the site of infection. Moreover, supporting the hypothesis that inflammatory responses may contribute to EAEC pathogenesis, we found that PMN transepithelial migration promoted enhanced attachment of EAEC 042 to T84 cells. These findings suggest that EAEC-induced PMN infiltration may favour colonization and thus pathogenesis of EAEC.

The fimbriae of enteroaggregative Escherichia coli induce epithelial inflammation in vitro and in a human intestinal xenograft model.

BACKGROUND: Enteroaggregative Escherichia coli (EAEC) are increasingly recognized as an important agent of inflammatory and often persistent diarrhea. Although previous studies report on the inflammatory aspects of EAEC pathogenesis, the mechanisms by which EAEC trigger these events are not well understood. METHODS: EAEC strains harboring mutations in known EAEC virulence determinants were tested in an in vitro model of transepithelial migration of polymorphonuclear neutrophils (PMNs) and in human intestinal xenografts in severe-combined immunodeficient (SCID-HU-INT) mice, a novel model for studying EAEC disease in vivo. RESULTS: Expression of aggregative adherence fimbriae (AAFs), the principal adhesins of EAEC, was required for EAEC-induced PMN transepithelial migration in vitro. Moreover, constructed plasmids encoding AAF gene clusters demonstrated that the AAF adhesins are sufficient for triggering this event in a nonpathogenic E. coli background. Furthermore, with use of the SCID-HU-INT mouse model, severe tissue damage and infiltration of inflammatory cells was observed in the human tissue after EAEC infection. These pathological marks were strongly related to AAF expression, thus clearly confirming our in vitro findings. CONCLUSIONS: The present work establishes EAEC as an important inflammatory pathogen and the AAF adhesins as inducers of potentially detrimental immune responses.

Complete Genome Sequences of Extraintestinal Pathogenic Escherichia coli Clinical Isolates from Danish Ulcerative Colitis Patients.

Extraintestinal pathogenic Escherichia coli (ExPEC) is a potential factor in ulcerative colitis etiology. We report here the complete genome and plasmid sequences of three Escherichia coli isolates, C 237-04 (p7), C 236-04A (p10A), and C 691-04A (p19A), obtained from fecal samples from ulcerative colitis patients in Copenhagen, Denmark.

Novel screening assay for in vivo selection of Klebsiella pneumoniae genes promoting gastrointestinal colonisation.

BACKGROUND: Klebsiella pneumoniae is an important opportunistic pathogen causing pneumonia, sepsis and urinary tract infections. Colonisation of the gastrointestinal (GI) tract is a key step in the development of infections; yet the specific factors important for K. pneumoniae to colonize and reside in the GI tract of the host are largely unknown. To identify K. pneumoniae genes promoting GI colonisation, a novel genomic-library-based approach was employed. RESULTS: Screening of a K. pneumoniae C3091 genomic library, expressed in E. coli strain EPI100, in a mouse model of GI colonisation led to the positive selection of five clones containing genes promoting persistent colonisation of the mouse GI tract. These included genes encoding the global response regulator ArcA; GalET of the galactose operon; and a cluster of two putative membrane-associated proteins of unknown function. Both ArcA and GalET are known to be involved in metabolic pathways in Klebsiella but may have additional biological actions beneficial to the pathogen. In support of this, GalET was found to confer decreased bile salt sensitivity to EPI100. CONCLUSIONS: The present work establishes the use of genomic-library-based in vivo screening assays as a valuable tool for identification and characterization of virulence factors in K. pneumoniae and other bacterial pathogens.

Characterization of a novel chaperone/usher fimbrial operon present on KpGI-5, a methionine tRNA gene-associated genomic island in Klebsiella pneumoniae.

BACKGROUND: Several strain-specific Klebsiella pneumoniae virulence determinants have been described, though these have almost exclusively been linked with hypervirulent liver abscess-associated strains. Through PCR interrogation of integration hotspots, chromosome walking, island-tagging and fosmid-based marker rescue we captured and sequenced KpGI-5, a novel genomic island integrated into the met56 tRNA gene of K. pneumoniae KR116, a bloodstream isolate from a patient with pneumonia and neutropenic sepsis. RESULTS: The 14.0 kb KpGI-5 island exhibited a genome-anomalous G + content, possessed near-perfect 46 bp direct repeats, encoded a γ1-chaperone/usher fimbrial cluster (fim2) and harboured seven other predicted genes of unknown function. Transcriptional analysis demonstrated expression of three fim2 genes, and suggested that the fim2A-fim2K cluster comprised an operon. As fimbrial systems are frequently implicated in pathogenesis, we examined the role of fim2 by analysing KR2107, a streptomycin-resistant derivative of KR116, and three isogenic mutants (Δfim, Δfim2 and ΔfimΔfim2) using biofilm assays, human cell adhesion assays and pair-wise competition-based murine models of intestinal colonization, lung infection and ascending urinary tract infection. Although no statistically significant role for fim2 was demonstrable, liver and kidney CFU counts for lung and urinary tract infection models, respectively, hinted at an ordered gradation of virulence: KR2107 (most virulent), KR2107∆fim2, KR2107∆fim and KR2107∆fim∆fim2 (least virulent). Thus, despite lack of statistical evidence there was a suggestion that fim and fim2 contribute additively to virulence in these murine infection models. However, further studies would be necessary to substantiate this hypothesis. CONCLUSION: Although fim2 was present in 13% of Klebsiella spp. strains investigated, no obvious in vitro or in vivo role for the locus was identified, although there were subtle hints of involvement in urovirulence and bacterial dissemination from the respiratory tract. Based on our findings and on parallels with other fimbrial systems, we propose that fim2 has the potential to contribute beneficially to pathogenesis and/or environmental persistence of Klebsiella strains, at least under specific yet-to-be identified conditions.

Intrinsic tet(L) sub-class in Bacillus velezensis and Bacillus amyloliquefaciens is associated with a reduced susceptibility toward tetracycline.

Annotations of non-pathogenic bacterial genomes commonly reveal putative antibiotic resistance genes and the potential risks associated with such genes is challenging to assess. We have examined a putative tetracycline tet(L) gene (conferring low level tetracycline resistance), present in the majority of all publicly available genomes of the industrially important operational group Bacillus amyloliquefaciens including the species B. amyloliquefaciens, Bacillus siamensis and Bacillus velezensis. The aim was to examine the risk of transfer of the putative tet(L) in operational group B. amyloliquefaciens through phylogenetic and genomic position analysis. These analyses furthermore included tet(L) genes encoded by transferable plasmids and other Gram-positive and -negative bacteria, including Bacillus subtilis. Through phylogenetic analysis, we could group chromosomally and plasmid-encoded tet(L) genes into four phylogenetic clades. The chromosomally encoded putative tet(L) from operational group B. amyloliquefaciens formed a separate phylogenetic clade; was positioned in the same genomic region in the three species; was not flanked by mobile genetic elements and was not found in any other bacterial species suggesting that the gene has been present in a common ancestor before species differentiation and is intrinsic. Therefore the gene is not considered a safety concern, and the risk of transfer to and expression of resistance in other non-related species is considered negligible. We suggest a subgrouping of the tet(L) class into four groups (tet(L)1.1, tet(L)1.2 and tet(L)2.1, tet(L)2.2), corresponding with the phylogenetic grouping and tet(L) from operational group B. amyloliquefaciens referred to as tet(L)2.2. Phylogenetic analysis is a useful tool to correctly differentiate between intrinsic and acquired antibiotic resistance genes.

Genetics and pathology associated with Klebsiella pneumoniae and Klebsiella spp. isolates from North American Pacific coastal marine mammals.

Southern sea otters (SSO: Enhydra lutris nereis) are a federally-listed threatened subspecies found almost exclusively in California, USA. Despite their zoonotic potential and lack of host specificity, K. pneumoniae and Klebsiella spp. have largely unknown epizootiology in SSOs. Klebsiella pneumoniae is occasionally isolated at necropsy, but not from live SSOs. Hypermucoviscous (HMV) K. pneumoniae strains are confirmed pathogens of Pacific Basin pinnipeds, but have not been previously isolated from SSOs. We characterized the virulence profiles of K. pneumoniae isolates from necropsied SSOs, evaluated killing of marine mammal K. pneumoniae following in vitro exposure to California sea lion (CSL: Zalophanus californianus) whole blood and serum, and characterized lesion patterns associated with Klebsiella spp. infection in SSOs. Four of 15 SSO K. pneumoniae isolates were HMV and all were recovered from SSOs that stranded during 2005. Many K. pneumoniae infections were associated with moderate to severe pathology as a cause of death or sequela. All HMV infections were assessed as a primary cause of death or as a direct result of the primary cause of death. Klebsiella-infected SSOs exhibited bronchopneumonia, tracheobronchitis and/or pleuritis, enteritis, Profilicollis sp. acanthocephalan peritonitis, septic peritonitis, and septicemia. All SSO HMV isolates were capsular type K2, the serotype most associated with HMV infections in CSLs. Multiplex PCR revealed two distinct virulence gene profiles within HMV isolates and two within non-HMV isolates. In vitro experiments investigating CSL whole blood and serum killing of K. pneumoniae suggest that HMV isolates are more resistant to serum killing than non-HMV isolates.

Complete genome sequence of the N2-fixing broad host range endophyte Klebsiella pneumoniae 342 and virulence predictions verified in mice.

We report here the sequencing and analysis of the genome of the nitrogen-fixing endophyte, Klebsiella pneumoniae 342. Although K. pneumoniae 342 is a member of the enteric bacteria, it serves as a model for studies of endophytic, plant-bacterial associations due to its efficient colonization of plant tissues (including maize and wheat, two of the most important crops in the world), while maintaining a mutualistic relationship that encompasses supplying organic nitrogen to the host plant. Genomic analysis examined K. pneumoniae 342 for the presence of previously identified genes from other bacteria involved in colonization of, or growth in, plants. From this set, approximately one-third were identified in K. pneumoniae 342, suggesting additional factors most likely contribute to its endophytic lifestyle. Comparative genome analyses were used to provide new insights into this question. Results included the identification of metabolic pathways and other features devoted to processing plant-derived cellulosic and aromatic compounds, and a robust complement of transport genes (15.4%), one of the highest percentages in bacterial genomes sequenced. Although virulence and antibiotic resistance genes were predicted, experiments conducted using mouse models showed pathogenicity to be attenuated in this strain. Comparative genomic analyses with the presumed human pathogen K. pneumoniae MGH78578 revealed that MGH78578 apparently cannot fix nitrogen, and the distribution of genes essential to surface attachment, secretion, transport, and regulation and signaling varied between each genome, which may indicate critical divergences between the strains that influence their preferred host ranges and lifestyles (endophytic plant associations for K. pneumoniae 342 and presumably human pathogenesis for MGH78578). Little genome information is available concerning endophytic bacteria. The K. pneumoniae 342 genome will drive new research into this less-understood, but important category of bacterial-plant host relationships, which could ultimately enhance growth and nutrition of important agricultural crops and development of plant-derived products and biofuels.