Characterization of integrative and conjugative element ICEKp1-associated genomic heterogeneity in a Klebsiella pneumoniae strain isolated from a primary liver abscess

Genomic heterogeneity has been shown to be associated with Klebsiella pneumoniae strains causing pyogenic liver abscesses (PLA) and metastatic infections. In order to explore the mechanism responsible for genomic heterogeneity in K. pneumoniae, we compared the complete genomic sequences of strains NTUH-K2044 and MGH78578. An ~76-kbp DNA fragment located adjacent to an asparagine (asn) tRNA gene was present in NTUH-K2044 but not in MGH78578. This fragment could be divided into three regions with different functions, and structurally it resembled a functional integrative and conjugative element (ICE), ICEEc1, in Escherichia coli. The 5′ region of this fragment contained genes similar to a high-pathogenicity island (HPI) of Yersinia pestis and Yersinia pseudotuberculosis. The middle region was similar to part of a large plasmid in K. pneumoniae, and the 3′ region contained genes responsible for DNA conjugative transfer. Therefore, this DNA fragment was designated ICEKp1. Precise excision and extrachromosomal circularization of ICEKp1 were detected in K. pneumoniae wild-type strain NTUH-K2044. ICEKp1 could integrate into the asn tRNA loci of the chromosome of another K. pneumoniae isolate. The prevalence of ICEKp1 was higher in PLA strains (38 of 42 strains) than in non-tissue-invasive strains (5 of 32 strains). Therefore, ICEKp1 may contribute to the transmission of the HPI and result in K. pneumoniae PLA infection-associated genomic heterogeneity.     

Suppression of stop codon UGA in acrB can contribute to antibiotic resistance in Klebsiella pneumoniae ATCC10031

We previously reported that Klebsiella pneumoniae MGH78578 exhibited higher resistance against various antimicrobials than K. pneumoniae ATCC10031. In this study, we showed that the plasmid, pKPN5, in K. pneumoniae MGH78578 played an important role in resistance against aminoglycosides, ampicillin, tetracycline, and chloramphenicol, while genome-derived β-lactamases and drug efflux pumps appeared to be more important in resistance to cloxacillin. acrAB, encoding a potent multidrug efflux pump, was cloned from K. pneumoniae MGH78578 and ATCC10031, to investigate reasons for the high drug resistance of K. pneumoniae MGH78578, and the results revealed that AcrAB from K. pneumoniae ATCC10031 conferred weaker drug resistance than AcrAB from K. pneumoniae MGH78578. DNA sequencing revealed that acrB from K. pneumoniae ATCC10031 carried the nonsense mutation, UGA, which was not found in acrB from K. pneumoniae MGH78578. However, acrB from K. pneumoniae ATCC10031 conferred slightly elevated resistant levels to several antimicrobials. The intact length of AcrB was detected in K. pneumoniae ATCC10031 by Western blot analysis, even though its quantity was small. Therefore, the stop codon UGA in acrB was thought to be overcome to some extent in this strain. We artificially introduced the nonsense mutation, UGA to the cat gene on pACYC184, and the plasmid also elevated the MIC of chloramphenicol in K. pneumoniae ATCC10031. These results suggest that a mechanism to overcome the nonsense mutation in acrB sustained resistance against a few β-lactams, dyes, and cholic acid in K. pneumoniae ATCC10031.     

Enhanced activity of meso-secondary alcohol dehydrogenase from Klebsiella species by codon optimization

Meso-secondary alcohol dehydrogenases (meso-SADH) from Klebsiella oxytoca KCTC1686 and Klebsiella pneumoniae KCTC2242 were codon optimized and expressed in Escherichia coli W3110. The published gene data of K. pneumoniae NTUH-K2044 (NCBI accession number AP006725), K. pneumoniae 342 (NCBI accession number CP000964), and K. pneumoniae MGH 78578 (NCBI accession number CP000647), were compared with the meso-SADH sequences of each strain, respectively. Codon-optimized meso-SADH enzymes of K. oxytoca and K. pneumoniae showed approximately twofold to fivefold increased enzyme activities for acetoin reduction over native enzymes. The highest activities for each strain were obtained at 30–37 °C and pH 6–7 (yielding 203.1 U/mg of protein and 156.5 U/mg of protein, respectively). The increased enzyme activity of the codon-optimized enzymes indicated that these modified enzymes could convert acetoin into 2,3-butanediol with a high yield.     

Genome Sequencing and Comparative Analysis of Klebsiella pneumoniae NTUH-K2044, a Strain Causing Liver Abscess and Meningitis

Nosocomial infections caused by antibiotic-resistant Klebsiella pneumoniae are emerging as a major health problem worldwide, while community-acquired K. pneumoniae infections present with a range of diverse clinical pictures in different geographic areas. In particular, an invasive form of K. pneumoniae that causes liver abscesses was first observed in Asia and then was found worldwide. We are interested in how differences in gene content of the same species result in different diseases. Thus, we sequenced the whole genome of K. pneumoniae NTUH-K2044, which was isolated from a patient with liver abscess and meningitis, and analyzed differences compared to strain MGH 78578, which was isolated from a patient with pneumonia. Six major types of differences were found in gene clusters that included an integrative and conjugative element, clusters involved in citrate fermentation, lipopolysaccharide synthesis, and capsular polysaccharide synthesis, phage-related insertions, and a cluster containing fimbria-related genes. We also conducted comparative genomic hybridization with 15 K. pneumoniae isolates obtained from community-acquired or nosocomial infections using tiling probes for the NTUH-K2044 genome. Hierarchical clustering revealed three major groups of genomic insertion-deletion patterns that correlate with the strains'' clinical features, antimicrobial susceptibilities, and virulence phenotypes with mice. Here we report the whole-genome sequence of K. pneumoniae NTUH-K2044 and describe evidence showing significant genomic diversity and sequence acquisition among K. pneumoniae pathogenic strains. Our findings support the hypothesis that these factors are responsible for the changes that have occurred in the disease profile over time.Klebsiella pneumoniae is a gram-negative bacterium that belongs to the gamma subdivision of the class Proteobacteria and exhibits relatively close genetic relatedness to other genera of the Enterobacteriaceae, including Escherichia, Salmonella, Shigella, and Yersinia (2). The conspicuous difference between K. pneumoniae and the other enterobacteria is the presence of a thick polysaccharide capsule, which is thought to be a significant virulence factor and to help the bacterium avoid phagocytosis (13). Infections caused by K. pneumoniae are seen throughout the world. This organism is a major cause of urinary tract infection and an important source of nosocomial infection (39). Moreover, K. pneumoniae is emerging worldwide as a major cause of bacteremia and drug-resistant infections (25, 38).The clinical pattern of K. pneumoniae infection in humans has changed since this organism was discovered (19, 20) more than 100 years ago. Until the 1960s, K. pneumoniae was an important cause of community-acquired pneumonia in the United States (8) and elsewhere. However, the incidence of this type of infection has dropped to 1 to 3% in the United States and Europe, and hospital-acquired K. pneumoniae infection now predominates (22, 39, 48). The global pattern of community-acquired K. pneumoniae bacteremia varies with geographical area (25). In the United States, Europe, Australia, and Argentina, this condition is associated with urinary tract infection, vascular catheters, and cholangitis. In Asia and South Africa, classic K. pneumoniae pneumonia still exists (25) and has remained important over the past two decades. At the same time, an invasive form of K. pneumoniae infection, which presents as primary bacteremic liver abscesses, endophthalmitis, and meningitis, has been reported almost exclusively in Asia (21), especially in Taiwan (47, 50). Although the reasons for the preponderance of this severe invasive K. pneumoniae infection in Asia are unknown, they are likely to involve both host and microbial factors.Recent studies by several groups have investigated and debated the major virulence factors of K. pneumoniae, including the magA (16) and rmpA (53) genes, capsular serotype K1 or K2 (11, 52), and even hypermucoviscosity (16, 53). In principle, other determinants may also contribute to pyogenic K. pneumoniae infection. To gather sufficient DNA sequence information for a systematic analysis of the genetic features that underlie the diverse clinical manifestations of K. pneumoniae infections, we undertook complete genome sequencing of a pathogenic strain, NTUH-K2044, which had been isolated from a Taiwanese liver abscess case (16). NTUH-K2044 is an appropriate strain because it possesses the magA and rmpA genes, belongs to capsular serotype K1, and has high virulence and hypermucoviscosity; these factors make this isolate very suitable as a model strain for genomic studies. We additionally used a genomic shotgun array (GSA) protocol developed in our laboratory (27) to compare the genome contents of NTUH-K2044 and multiple clinical isolates. The microarray data allowed us to examine the genome evolution of K. pneumoniae and to relate the various genomic signatures to the clinical patterns seen in K. pneumoniae infections.     

Amino Acid Substitutions of MagA in Klebsiella pneumoniae Affect the Biosynthesis of the Capsular Polysaccharide

Mucoviscosity-associated gene A (magA) of Klebsiella pneumoniae contributes to K1 capsular polysaccharide (CPS) biosynthesis. Based on sequence homology and gene alignment, the magA gene has been predicted to encode a Wzy-type CPS polymerase. Sequence alignment with the Wzy_C and RfaL protein families (which catalyze CPS or lipopolysaccharide (LPS) biosynthesis) and topological analysis has suggested that eight highly conserved residues, including G308, G310, G334, G337, R290, P305, H323, and N324, were located in a hypothetical loop region. Therefore, we used site-directed mutagenesis to study the role of these residues in CPS production, and to observe the consequent phenotypes such as mucoviscosity, serum and phagocytosis resistance, and virulence (as assessed in mice) in pyogenic liver abscess strain NTUH-K2044. Alanine substitutions at R290 or H323 abolished all of these properties. The G308A mutant was severely impaired for these functions. The G334A mutant remained mucoid with decreased CPS production, but its virulence was significantly reduced in vivo. No phenotypic change was observed for strains harboring magA G310A, G337A, P305A, or N324A mutations. Therefore, R290, G308, H323, and G334 are functionally important residues of the MagA (Wzy) protein of K. pneumoniae NTUH-K2044, capsular type K1. These amino acids are also likely to be important for the function of Wzy in other capsular types in K. pneumoniae and other species bearing Wzy_C family proteins.     

Role of the two component signal transduction system CpxAR in conferring cefepime and chloramphenicol resistance in Klebsiella pneumoniae NTUH-K2044

Background

Klebsiella pneumoniae is a Gram-negative, non-motile, facultative anaerobe belonging to the Enterobacteriaceae family of the γ-Proteobacteria class in the phylum Proteobacteria. Multidrug resistant K. pneumoniae have caused major therapeutic problems worldwide due to emergence of extended-spectrum β-lactamase producing strains. Two-component systems serve as a basic stimulus-response coupling mechanism to allow organisms to sense and respond to changes in many different environmental conditions including antibiotic stress.

Principal Findings

In the present study, we investigated the role of an uncharacterized cpxAR operon in bacterial physiology and antimicrobial resistance by generating isogenic mutant (ΔcpxAR) deficient in the CpxA/CpxR component derived from the hyper mucoidal K1 strain K. pneumoniae NTUH-K2044. The behaviour of ΔcpxAR was determined under hostile conditions, reproducing stresses encountered in the gastrointestinal environment and deletion resulted in higher sensitivity to bile, osmotic and acid stresses. The ΔcpxAR was more susceptible to β-lactams and chloramphenicol than the wild-type strain, and complementation restored the altered phenotypes. The relative change in expression of acrB, acrD, eefB efflux genes were decreased in cpxAR mutant as evidenced by qRT-PCR. Comparison of outer membrane protein profiles indicated a conspicuous difference in the knock out background. Gel shift assays demonstrated direct binding of CpxR^KP to promoter region of ompC ^KP in a concentration dependent manner.

Conclusions and Significance

The Cpx envelope stress response system is known to be activated by alterations in pH, membrane composition and misfolded proteins, and this systematic investigation reveals its direct involvement in conferring antimicrobial resistance against clinically significant antibiotics for the very first time. Overall results displayed in this report reflect the pleiotropic role of the CpxAR signaling system and diversity of the antibiotic resistome in hyper virulent K1 serotype K. pneumoniae NTUH-K2044.     

Unraveling the dha cluster in Citrobacter werkmanii: comparative genomic analysis of bacterial 1,3-propanediol biosynthesis clusters

In natural 1,3-propanediol (PDO) producing microorganisms such as Klebsiella pneumoniae, Citrobacter freundii and Clostridium sp., the genes coding for PDO producing enzymes are grouped in a dha cluster. This article describes the dha cluster of a novel candidate for PDO production, Citrobacter werkmanii DSM17579 and compares the cluster to the currently known PDO clusters of Enterobacteriaceae and Clostridiaceae. Moreover, we attribute a putative function to two previously unannotated ORFs, OrfW and OrfY, both in C. freundii and in C. werkmanii: both proteins might form a complex and support the glycerol dehydratase by converting cob(I)alamin to the glycerol dehydratase cofactor coenzyme B₁₂. Unraveling this biosynthesis cluster revealed high homology between the deduced amino acid sequence of the open reading frames of C. werkmanii DSM17579 and those of C. freundii DSM30040 and K. pneumoniae MGH78578, i.e., 96 and 87.5 % identity, respectively. On the other hand, major differences between the clusters have also been discovered. For example, only one dihydroxyacetone kinase (DHAK) is present in the dha cluster of C. werkmanii DSM17579, while two DHAK enzymes are present in the cluster of K. pneumoniae MGH78578 and Clostridium butyricum VPI1718.     

Lipopolysaccharide O1 antigen contributes to the virulence in Klebsiella pneumoniae causing pyogenic liver abscess

Klebsiella pneumoniae is the common cause of a global emerging infectious disease, community-acquired pyogenic liver abscess (PLA). Capsular polysaccharide (CPS) and lipopolysaccharide (LPS) are critical for this microorganism's ability to spread through the blood and to cause sepsis. While CPS type K1 is an important virulence factor in K. pneumoniae causing PLA, the role of LPS in PLA is not clear. Here, we characterize the role of LPS O antigen in the pathogenesis of K. pneumoniae causing PLA. NTUH-K2044 is a LPS O1 clinical strain; the presence of the O antigen was shown via the presence of 1,3-galactan in the LPS, and of sequences that align with the wb gene cluster, known to produce O-antigen. Serologic analysis of K. pneumoniae clinical isolates demonstrated that the O1 serotype was more prevalent in PLA strains than that in non-tissue-invasive strains (38/42 vs. 9/32, P<0.0001). O1 serotype isolates had a higher frequency of serum resistance, and mutation of the O1 antigen changed serum resistance in K. pneumoniae. A PLA-causing strain of CPS capsular type K2 and LPS serotype O1 (i.e., O1:K2 PLA strain) deleted for the O1 synthesizing genes was profoundly attenuated in virulence, as demonstrated in separate mouse models of septicemia and liver abscess. Immunization of mice with the K2044 magA-mutant (K(1) (-) O(1)) against LPS O1 provided protection against infection with an O1:K2 PLA strain, but not against infection with an O1:K1 PLA strain. Our findings indicate that the O1 antigen of PLA-associated K. pneumoniae contributes to virulence by conveying resistance to serum killing, promoting bacterial dissemination to and colonization of internal organs after the onset of bacteremia, and could be a useful vaccine candidate against infection by an O1:K2 PLA strain.     

Why Hypothetical Protein KPN00728 of Klebsiella pneumoniae Should Be Classified as Chain C of Succinate Dehydrogenase?

Twenty percent of genes that encode for hypothetical proteins from Klebsiella pneumoniae MGH78578 were identified, leading to KPN00728 and KPN00729 after bioinformatics analysis. Both open reading frames showed high sequence homology to Succinate dehydrogenase Chain C (SdhC) and D (SdhD) from Escherichia coli. Recently, KPN00729 was assigned as SdhD. KPN00728 thus remains of particular interest as no annotated genes from the complete genome sequence encode for SdhC. We discovered KPN00728 has a missing region with conserved residues important for ubiquinone (UQ) and heme group binding. Structure and function prediction of KPN00728 coupled with secondary structure analysis and transmembrane topology showed KPN00728 adopts SDH-(subunit C)-like structure. To further probe its functionality, UQ was docked on the built model (consisting KPN00728 and KPN00729) and formation of hydrogen bonds between UQ and Ser27, Arg31 (KPN00728) and Tyr84 (KPN00729) further reinforces and supports that KPN00728 is indeed SDH. This is the first report on the structural and function prediction of KPN00728 of K. pneumoniae MGH78578 as SdhC.     

Isolation of a Bacteriophage Specific for a New Capsular Type of Klebsiella pneumoniae and Characterization of Its Polysaccharide Depolymerase

Background

Klebsiella pneumoniae is one of the major pathogens causing hospital-acquired multidrug-resistant infections. The capsular polysaccharide (CPS) is an important virulence factor of K. pneumoniae. With 78 capsular types discovered thus far, an association between capsular type and the pathogenicity of K. pneumoniae has been observed.

Methodology/Principal Findings

To investigate an initially non-typeable K. pneumoniae UTI isolate NTUH-K1790N, the cps gene region was sequenced. By NTUH-K1790N cps-PCR genotyping, serotyping and determination using a newly isolated capsular type-specific bacteriophage, we found that NTUH-K1790N and three other isolates Ca0507, Ca0421 and C1975 possessed a new capsular type, which we named KN2. Analysis of a KN2 CPS⁻ mutant confirmed the role of capsule as the target recognized by the antiserum and the phage. A newly described lytic phage specific for KN2 K. pneumoniae, named 0507-KN2-1, was isolated and characterized using transmission electron microscopy. Whole-genome sequencing of 0507-KN2-1 revealed a 159 991 bp double-stranded DNA genome with a G+C content of 46.7% and at least 154 open reading frames. Based on its morphological and genomic characteristics, 0507-KN2-1 was classified as a member of the Myoviridae phage family. Further analysis of this phage revealed a 3738-bp gene encoding a putative polysaccharide depolymerase. A recombinant form of this protein was produced and assayed to confirm its enzymatic activity and specificity to KN2 capsular polysaccharides. KN2 K. pneumoniae strains exhibited greater sensitivity to this depolymerase than these did to the cognate phage, as determined by spot analysis.

Conclusions/Significance

Here we report that a group of clinical strains possess a novel Klebsiella capsular type. We identified a KN2-specific phage and its polysaccharide depolymerase, which could be used for efficient capsular typing. The lytic phage and depolymerase also have potential as alternative therapeutic agents to antibiotics for treating K. pneumoniae infections, especially against antibiotic-resistant strains.     

Virulence of Klebsiella pneumoniae Isolates Harboring bla KPC-2 Carbapenemase Gene in a Caenorhabditis elegans Model

Klebsiella pneumoniae carbapenemase (KPC) is a carbapenemase increasingly reported worldwide in Enterobacteriaceae. The aim of this study was to analyze the virulence of several KPC-2-producing K. pneumoniae isolates. The studied strains were (i) five KPC-2 clinical strains from different geographical origins, belonging to different ST-types and possessing plasmids of different incompatibility groups; (ii) seven transformants obtained after electroporation of either these natural KPC plasmids or a recombinant plasmid harboring only the bla _KPC-2 gene into reference strains K. pneumoniae ATCC10031/{"type":"entrez-protein","attrs":{"text":"CIP53153","term_id":"878514309","term_text":"CIP53153"}}CIP53153; and (iii) five clinical strains cured of plasmids. The virulence of K. pneumoniae isolates was evaluated in the Caenorhabditis elegans model. The clinical KPC producers and transformants were significantly less virulent (LT50: 5.5 days) than K. pneumoniae reference strain (LT50: 4.3 days) (p<0.01). However, the worldwide spread KPC-2 positive K. pneumoniae ST258 strains and reference strains containing plasmids extracted from K. pneumoniae ST258 strains had a higher virulence than KPC-2 strains belonging to other ST types (LT50: 5 days vs. 6 days, p<0.01). The increased virulence observed in cured strains confirmed this trend. The bla _KPC-2 gene itself was not associated to increased virulence.     

Blood Group Antigen Recognition by a Solute-Binding Protein from a Serotype 3 Strain of Streptococcus pneumoniae

Streptococcus pneumoniae is a common bacterial pathogen that is well known for its ability to cause acute respiratory disease (pneumonia), ear infections, and other serious illnesses. This Gram-positive bacterium relies on its carbohydrate-metabolizing capabilities for full virulence in its host; however, the range of glycan targets that it can attack is presently not fully appreciated. S. pneumoniae is known to have a fucose utilization operon that in the TIGR4 strain plays a role in its virulence. Here we identify a second type of fucose utilization operon that is present in a subset of S. pneumoniae strains, including the serotype 3 strain SP3-BS71. This operon contains a transporter with a solute-binding protein, FcsSBP (fucose solute-binding protein), that interacts tightly (K_a ∼ 1 × 10⁶ M^− 1) and specifically with soluble A- and B-antigen trisaccharides but displays no selectivity between these two sugars. The structure of the FcsSBP in complex with the A-trisaccharide antigen, determined to 2.35 Å, reveals its mode of binding to the reducing end of this sugar, thus highlighting this protein's requirement for soluble blood group antigen ligands. Overall, this report exposes a heretofore unknown capability of certain S. pneumoniae strains to transport and potentially metabolize the histo-blood group antigen carbohydrates of its host.     

Characterization of MATE-Type Multidrug Efflux Pumps from Klebsiella pneumoniae MGH78578

We previously described the cloning of genes related to drug resistance from Klebsiella pneumoniae MGH78578. Of these, we identified a putative gene encoding a MATE-type multidrug efflux pump, and named it ketM. Escherichia coli KAM32 possessing ketM on a plasmid showed increased minimum inhibitory concentrations for norfloxacin, ciprofloxacin, cefotaxime, acriflavine, Hoechst 33342, and 4'',6-diamidino-2-phenyl indole (DAPI). The active efflux of DAPI was observed in E. coli KAM32 possessing ketM on a plasmid. The expression of mRNA for ketM was observed in K. pneumoniae cells, and we subsequently disrupted ketM in K. pneumoniae ATCC10031. However, no significant changes were observed in drug resistance levels between the parental strain ATCC10031 and ketM disruptant, SKYM. Therefore, we concluded that KetM was a multidrug efflux pump, that did not significantly contribute to intrinsic resistance to antimicrobial chemicals in K. pneumoniae. MATE-type transporters are considered to be secondary transporters; therefore, we investigated the coupling cations of KetM. DAPI efflux by KetM was observed when lactate was added to produce a proton motive force, indicating that KetM effluxed substrates using a proton motive force. However, the weak efflux of DAPI by KetM was also noted when NaCl was added to the assay mixture without lactate. This result suggests that KetM may utilize proton and sodium motive forces.     

耐碳青霉烯类肺炎克雷伯菌对C57BL/6小鼠肺部菌群的扰动

【背景】肺部菌群与宿主健康和呼吸道疾病密切相关,耐碳青霉烯类肺炎克雷伯菌(carbapenem-resistantKlebsiella pneumonia,CRKP)是临床常见的条件致病菌,感染后对肺部菌群的影响尚不清楚。【目的】探究耐碳青霉烯类肺炎克雷伯杆菌CRKP2对C57BL/6小鼠肺部菌群的扰动。【方法】将C57BL/6小鼠随机分为3组,分别用CRKP2、碳青霉烯类敏感肺炎克雷伯菌KP2044和无菌PBS溶液滴鼻,利用16S rRNA基因的高通量测序技术分析肺部菌群结构。【结果】与健康小鼠相比,菌株KP2044和CRKP2感染后小鼠肺部菌群α多样性和β多样性均显著改变,变形菌门相对丰度显著增加,乳酸杆菌属相对丰度明显下降。与KP2044相比,CRKP2生物膜形成能力较弱,感染后小鼠死亡率较低,对肺部菌群的扰动较小。【结论】虽然肺炎克雷伯菌是条件致病菌,但高剂量耐碳青霉烯类肺炎克雷伯菌CRKP2仍对健康小鼠肺部菌群造成显著影响;尽管菌株CRKP2具有多重耐药性,但与菌株KP2044相比对肺部菌群的扰动较小,因此推测KP菌株感染对肺部菌群的扰动程度可能与菌株毒力有关。     

An improved heuristic for haplotype inference

We cloned a gene, kexD, that provides a multidrug-resistant phenotype from multidrug-resistant Klebsiella pneumoniae MGH78578. The deduced amino acid sequence of KexD is similar to that of the inner membrane protein, RND-type multidrug efflux pump. Introduction of the kexD gene into Escherichia coli KAM32 resulted in a MIC that was higher for erythromycin, novobiocin, rhodamine 6G, tetraphenylphosphonium chloride, and ethidium bromide than that of the control. Intracellular ethidium bromide levels in E. coli cells carrying the kexD gene were lower than that in the control cells under energized conditions, suggesting that KexD is a component of an energy-dependent efflux pump. RND-type pumps typically consist of three components: an inner membrane protein, a periplasmic protein, and an outer membrane protein. We discovered that KexD functions with a periplasmic protein, AcrA, from E. coli and K. pneumoniae, but not with the periplasmic proteins KexA and KexG from K. pneumoniae. KexD was able to utilize either TolC of E. coli or KocC of K. pneumoniae as an outer membrane component. kexD mRNA was not detected in K. pneumoniae MGH78578 or ATCC10031. We isolated erythromycin-resistant mutants from K. pneumoniae ATCC10031, and some showed a multidrug-resistant phenotype similar to the drug resistance pattern of KexD. Two strains of multidrug-resistant mutants were investigated for kexD expression; kexD mRNA levels were increased in these strains. We conclude that changing kexD expression can contribute to the occurrence of multidrug-resistant K. pneumoniae.     

Serotype 1 and 8 Pneumococci Evade Sensing by Inflammasomes in Human Lung Tissue

Streptococcus pneumoniae is a major cause of pneumonia, sepsis and meningitis. The pore-forming toxin pneumolysin is a key virulence factor of S. pneumoniae, which can be sensed by the NLRP3 inflammasome. Among the over 90 serotypes, serotype 1 pneumococci (particularly MLST306) have emerged across the globe as a major cause of invasive disease. The cause for its particularity is, however, incompletely understood. We therefore examined pneumococcal infection in human cells and a human lung organ culture system mimicking infection of the lower respiratory tract. We demonstrate that different pneumococcal serotypes differentially activate inflammasome-dependent IL-1β production in human lung tissue and cells. Whereas serotype 2, 3, 6B, 9N pneumococci expressing fully haemolytic pneumolysins activate NLRP3 inflammasome-dependent responses, serotype 1 and 8 strains expressing non-haemolytic toxins are poor activators of IL-1β production. Accordingly, purified haemolytic pneumolysin but not serotype 1-associated non-haemolytic toxin activates strong IL-1β production in human lungs. Our data suggest that the evasion of inflammasome-dependent innate immune responses by serotype 1 pneumococci might contribute to their ability to cause invasive diseases in humans.     

Virulence and Draft Genome Sequence Overview of Multiple Strains of the Swine Pathogen Haemophilus parasuis

Haemophilus parasuis is the cause of Glässer''s disease in swine, which is characterized by systemic infection resulting in polyserositis, meningitis, and arthritis. Investigation of this animal disease is complicated by the enormous differences in the severity of disease caused by H. parasuis strains, ranging from lethal systemic disease to subclinical carriage. To identify differences in genotype that could account for virulence phenotypes, we established the virulence of, and performed whole genome sequence analysis on, 11 H. parasuis strains. Virulence was assessed by evaluating morbidity and mortality following intranasal challenge of Caesarean-derived, colostrum-deprived (CDCD) pigs. Genomic DNA from strains Nagasaki (serotype 5), 12939 (serotype 1), SW140 (serotype 2), 29755 (serotype 5), MN-H (serotype 13), 84-15995 (serotype 15), SW114 (serotype 3), H465 (serotype 11), D74 (serotype 9), and 174 (serotype 7) was used to generate Illumina paired-end libraries for genomic sequencing and de novo assembly. H. parasuis strains Nagasaki, 12939, SH0165 (serotype 5), SW140, 29755, and MN-H exhibited a high level of virulence. Despite minor differences in expression of disease among these groups, all pigs challenged with these strains developed clinical signs consistent with Glässer''s disease between 1–7 days post-challenge. H. parasuis strains 84-15995 and SW114 were moderately virulent, in that approximately half of the pigs infected with each developed Glässer''s disease. H. parasuis strains H465, D74, and 174 were minimally virulent or avirulent in the CDCD pig model. Comparative genomic analysis among strains identified several noteworthy differences in coding regions. These coding regions include predicted outer membrane, metabolism, and pilin or adhesin related genes, some of which likely contributed to the differences in virulence and systemic disease observed following challenge. These data will be useful for identifying H. parasuis virulence factors and vaccine targets.     

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.     

Functional Characterization of a Novel Outer Membrane Porin KpnO, Regulated by PhoBR Two-Component System in Klebsiella pneumoniae NTUH-K2044

Background

The diffusion of antibiotics through the outer membrane is primarily affected by the porin super family, changes contribute to antibiotic resistance. Recently we demonstrated that the CpxAR two-component signaling system alters the expression of an uncharacterized porin OmpC^KP, to mediate antimicrobial resistance in K. pneumoniae.

Principal Findings

In this study, functional characterization of the putative porin OmpC^KP (denoted kpnO) with respect to antimicrobial susceptibility and virulence was evaluated by generating an isogenic mutant, ΔkpnO in a clinical isolate of K. pneumoniae. Estimation of uronic acid content confirmed that ΔkpnO produced ∼2.0 fold lesser capsular polysaccharide than the wild-type. The ΔkpnO displayed higher sensitivity to hyper osmotic and bile conditions. Disruption of kpnO increased the susceptibility of K. pneumoniae to oxidative and nitrostative stress by ∼1.6 fold and >7 fold respectively. The loss of the Klebsiella porin led to an increase in the minimum inhibitory concentration of tetracycline (3-fold), nalidixic acid (4-fold), tobramycin (4-fold), streptomycin (10-fold), and spectinomycin (10-fold), which could be restored following complementation. The single deletion of kpnO reduced the survival of the pathogen by 50% when exposed to disinfectants. In Caenorhabditis elegans model, the kpnO mutant exhibited significantly (P<0.01) lower virulence. To dissect the role of PhoBR signaling system in regulating the expression of the kpnO, a phoB ^KP isogenic mutant was constructed. The phoB ^KP mutant exhibited impaired gastrointestinal stress response and decreased antimicrobial susceptibility. The mRNA levels of kpnO were found to be 4-fold less in phoB ^KP mutant compared to wild type. A regulatory role of PhoB^KP for the expression of kpnO was further supported by the specific binding of PhoB^KP to the putative promoter of kpnO.

Conclusions and Significance

Loss of PhoBR regulated porin KpnO resulted in increased antimicrobial resistance, increased susceptibility to gastrointestinal stress, and reduced virulence in K. pneumoniae NTUH-K2044.     

外源载体高效转化肺炎克雷伯菌的新途径

研究介绍了提高Klebsiella pneumoniae电转化效率的新途径,即直接从固体平板上收集K.pneumoniae菌落制备电转化感受态细胞,完全不同于传统的试验方法。试验菌株为野生型K.pneumoniae NTUH-K2044和magA—突变型菌株。将大小不同的质粒pIP843T、pIP843TdhaB、pIP843TdhaT电转化K.pneumoniae,计算电转化效率。电转化试验结果表明:K.pneumoniaeNTUH-K2044固体菌电转化效率高达2×105±300转化子/μgDNA,而其液体菌电转化效率仅为150±10转化子/?gDNA;其magA—突变株固体菌的转化效率最高,可以达到3.4×107±500转化子/μgDNA,比液体菌电转化效率提高了104倍。同时发现质粒大小对电转化效率并没有明显影响。此外,激光共聚焦显微镜观察发现固体平板和液体培养基中的菌体存在形态学方面差异,推测固体培养菌电转化效率的显著提高和形态学方面的表现可能具有一定的相关性。