Distinct but Spatially Overlapping Intestinal Niches for Vancomycin-Resistant Enterococcus faecium and Carbapenem-Resistant Klebsiella pneumoniae

Antibiotic resistance among enterococci and γ-proteobacteria is an increasing problem in healthcare settings. Dense colonization of the gut by antibiotic-resistant bacteria facilitates their spread between patients and also leads to bloodstream and other systemic infections. Antibiotic-mediated destruction of the intestinal microbiota and consequent loss of colonization resistance are critical factors leading to persistence and spread of antibiotic-resistant bacteria. The mechanisms underlying microbiota-mediated colonization resistance remain incompletely defined and are likely distinct for different antibiotic-resistant bacterial species. It is unclear whether enterococci or γ-proteobacteria, upon expanding to high density in the gut, confer colonization resistance against competing bacterial species. Herein, we demonstrate that dense intestinal colonization with vancomycin-resistant Enterococcus faecium (VRE) does not reduce in vivo growth of carbapenem-resistant Klebsiella pneumoniae. Reciprocally, K. pneumoniae does not impair intestinal colonization by VRE. In contrast, transplantation of a diverse fecal microbiota eliminates both VRE and K. pneumoniae from the gut. Fluorescence in situ hybridization demonstrates that VRE and K. pneumoniae localize to the same regions in the colon but differ with respect to stimulation and invasion of the colonic mucus layer. While VRE and K. pneumoniae occupy the same three-dimensional space within the gut lumen, their independent growth and persistence in the gut suggests that they reside in distinct niches that satisfy their specific in vivo metabolic needs.     

Identification of Antigenic Proteins of the Nosocomial Pathogen Klebsiella pneumoniae

The continuous expansion of nosocomial infections around the globe has become a precarious situation. Key challenges include mounting dissemination of multiple resistances to antibiotics, the easy transmission and the growing mortality rates of hospital-acquired bacterial diseases. Thus, new ways to rapidly detect these infections are vital. Consequently, researchers around the globe pursue innovative approaches for point-of-care devices. In many cases the specific interaction of an antigen and a corresponding antibody is pivotal. However, the knowledge about suitable antigens is lacking. The aim of this study was to identify novel antigens as specific diagnostic markers. Additionally, these proteins might be aptly used for the generation of vaccines to improve current treatment options. Hence, a cDNA-based expression library was constructed and screened via microarrays to detect novel antigens of Klebsiella pneumoniae, a prominent agent of nosocomial infections well-known for its extensive antibiotics resistance, especially by extended-spectrum beta-lactamases (ESBL). After screening 1536 clones, 14 previously unknown immunogenic proteins were identified. Subsequently, each protein was expressed in full-length and its immunodominant character examined by ELISA and microarray analyses. Consequently, six proteins were selected for epitope mapping and three thereof possessed linear epitopes. After specificity analysis, homology survey and 3d structural modelling, one epitope sequence GAVVALSTTFA of KPN_00363, an ion channel protein, was identified harboring specificity for K. pneumoniae. The remaining epitopes showed ambiguous results regarding the specificity for K. pneumoniae. The approach adopted herein has been successfully utilized to discover novel antigens of Campylobacter jejuni and Salmonella enterica antigens before. Now, we have transferred this knowledge to the key nosocomial agent, K. pneumoniae. By identifying several novel antigens and their linear epitope sites, we have paved the way for crucial future research and applications including the design of point-of-care devices, vaccine development and serological screenings for a highly relevant nosocomial pathogen.     

Sequencing and Genetic Variation of Multidrug Resistance Plasmids in Klebsiella pneumoniae

Background

The development of multidrug resistance is a major problem in the treatment of pathogenic microorganisms by distinct antimicrobial agents. Characterizing the genetic variation among plasmids from different bacterial species or strains is a key step towards understanding the mechanism of virulence and their evolution.

Results

We applied a deep sequencing approach to 206 clinical strains of Klebsiella pneumoniae collected from 2002 to 2008 to understand the genetic variation of multidrug resistance plasmids, and to reveal the dynamic change of drug resistance over time. First, we sequenced three plasmids (70 Kb, 94 Kb, and 147 Kb) from a clonal strain of K. pneumoniae using Sanger sequencing. Using the Illumina sequencing technology, we obtained more than 17 million of short reads from two pooled plasmid samples. We mapped these short reads to the three reference plasmid sequences, and identified a large number of single nucleotide polymorphisms (SNPs) in these pooled plasmids. Many of these SNPs are present in drug-resistance genes. We also found that a significant fraction of short reads could not be mapped to the reference sequences, indicating a high degree of genetic variation among the collection of K. pneumoniae isolates. Moreover, we identified that plasmid conjugative transfer genes and antibiotic resistance genes are more likely to suffer from positive selection, as indicated by the elevated rates of nonsynonymous substitution.

Conclusion

These data represent the first large-scale study of genetic variation in multidrug resistance plasmids and provide insight into the mechanisms of plasmid diversification and the genetic basis of antibiotic resistance.     

Role of Novel Multidrug Efflux Pump Involved in Drug Resistance in Klebsiella pneumoniae

Background

Multidrug resistant Klebsiella pneumoniae have caused major therapeutic problems worldwide due to the emergence of the extended-spectrum β-lactamase producing strains. Although there are >10 major facilitator super family (MFS) efflux pumps annotated in the genome sequence of the K. pneumoniae bacillus, apparently less is known about their physiological relevance.

Principal Findings

Insertional inactivation of kpnGH resulting in increased susceptibility to antibiotics such as azithromycin, ceftazidime, ciprofloxacin, ertapenem, erythromycin, gentamicin, imipenem, ticarcillin, norfloxacin, polymyxin-B, piperacillin, spectinomycin, tobramycin and streptomycin, including dyes and detergents such as ethidium bromide, acriflavine, deoxycholate, sodium dodecyl sulphate, and disinfectants benzalkonium chloride, chlorhexidine and triclosan signifies the wide substrate specificity of the transporter in K. pneumoniae. Growth inactivation and direct fluorimetric efflux assays provide evidence that kpnGH mediates antimicrobial resistance by active extrusion in K. pneumoniae. The kpnGH isogenic mutant displayed decreased tolerance to cell envelope stressors emphasizing its added role in K. pneumoniae physiology.

Conclusions and Significance

The MFS efflux pump KpnGH involves in crucial physiological functions besides being an intrinsic resistance determinant in K. pneumoniae.     

Tigecycline Susceptibility and the Role of Efflux Pumps in Tigecycline Resistance in KPC-Producing Klebsiella pneumoniae

KPC-producing Klebsiella pneumoniae isolates have emerged as important pathogens of nosocomial infections, and tigecycline is one of the antibiotics recommended for severe infections caused by KPC-producing K. pneumoniae. To identify the susceptibility profile of KPC-producing K. pneumoniae to tigecycline and investigate the role of efflux pumps in tigecycline resistance, a total of 215 KPC-producing K. pneumoniae isolates were collected. The minimum inhibitory concentration (MIC) of tigecycline was determined by standard broth microdilution tests. Isolates showing resistance to tigecycline underwent susceptibility test with efflux pump inhibitors. Expression levels of efflux pump genes (acrB and oqxB) and their regulators (ramA, marA, soxS and rarA) were examined by real-time PCR, and the correlation between tigecycline MICs and gene expression levels were analysed. Our results show that the tigecycline resistance rate in these isolates was 11.2%. Exposure of the tigecycline-resistant isolates to the efflux pump inhibitor NMP resulted in an obvious decrease in MICs and restored susceptibility to tigecycline in 91.7% of the isolates. A statistically significant association between acrB expression and tigecycline MICs was observed, and overexpression of ramA was found in three tigecycline-resistant isolates, further analysis confirmed ramR mutations existed in these isolates. Transformation of one mutant with wild-type ramR restored susceptibility to tigecycline and repressed overexpression of ramA and acrB. These data indicate that efflux pump AcrAB, which can be up-regulated by ramR mutations and subsequent ramA activation, contributed to tigecycline resistance in K. pneumoniae clinical isolates.     

Glycerol Dehydrogenase Plays a Dual Role in Glycerol Metabolism and 2,3-Butanediol Formation in Klebsiella pneumoniae

Glycerol dehydrogenase (GDH) is an important polyol dehydrogenase for glycerol metabolism in diverse microorganisms and for value-added utilization of glycerol in the industry. Two GDHs from Klebsiella pneumoniae, DhaD and GldA, were expressed in Escherichia coli, purified and characterized for substrate specificity and kinetic parameters. Both DhaD and GldA could catalyze the interconversion of (3R)-acetoin/(2R,3R)-2,3-butanediol or (3S)-acetoin/meso-2,3-butanediol, in addition to glycerol oxidation. Although purified GldA appeared more active than DhaD, in vivo inactivation and quantitation of their respective mRNAs indicate that dhaD is highly induced by glycerol and plays a dual role in glycerol metabolism and 2,3-butanediol formation. Complementation in K. pneumoniae further confirmed the dual role of DhaD. Promiscuity of DhaD may have vital physiological consequences for K. pneumoniae growing on glycerol, which include balancing the intracellular NADH/NAD⁺ ratio, preventing acidification, and storing carbon and energy. According to the kinetic response of DhaD to modified NADH concentrations, DhaD appears to show positive homotropic interaction with NADH, suggesting that the physiological role could be regulated by intracellular NADH levels. The co-existence of two functional GDH enzymes might be due to a gene duplication event. We propose that whereas DhaD is specialized for glycerol utilization, GldA plays a role in backup compensation and can turn into a more proficient catalyst to promote a survival advantage to the organism. Revelation of the dual role of DhaD could further the understanding of mechanisms responsible for enzyme evolution through promiscuity, and guide metabolic engineering methods of glycerol metabolism.     

Multilocus Sequence Typing (MLST) for Characterization of Enterobacter cloacae

Enterobacter cloacae is an important emerging pathogen, which sometime causes respiratory infection, surgical site infection, urinary infection, sepsis, and outbreaks at neonatal units. We have developed a multilocus sequence typing (MLST) scheme utilizing seven housekeeping genes and evaluated the performance in 101 clinical isolates. The MLST scheme yielded 83 sequence types (ST) including 78 novel STs found in the clinical isolates. These findings supported the robustness of the MLST scheme developed in this study.     

Toward Repurposing Ciclopirox as an Antibiotic against Drug-Resistant Acinetobacter baumannii,Escherichia coli,and Klebsiella pneumoniae

Antibiotic-resistant infections caused by gram-negative bacteria are a major healthcare concern. Repurposing drugs circumvents the time and money limitations associated with developing new antimicrobial agents needed to combat these antibiotic-resistant infections. Here we identified the off-patent antifungal agent, ciclopirox, as a candidate to repurpose for antibiotic use. To test the efficacy of ciclopirox against antibiotic-resistant pathogens, we used a curated collection of Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae clinical isolates that are representative of known antibiotic resistance phenotypes. We found that ciclopirox, at 5–15 µg/ml concentrations, inhibited bacterial growth regardless of the antibiotic resistance status. At these same concentrations, ciclopirox reduced growth of Pseudomonas aeruginosa clinical isolates, but some of these pathogens required higher ciclopirox concentrations to completely block growth. To determine how ciclopirox inhibits bacterial growth, we performed an overexpression screen in E. coli. This screen revealed that galE, which encodes UDP-glucose 4-epimerase, rescued bacterial growth at otherwise restrictive ciclopirox concentrations. We found that ciclopirox does not inhibit epimerization of UDP-galactose by purified E. coli GalE; however, ΔgalU, ΔgalE, ΔrfaI, or ΔrfaB mutant strains all have lower ciclopirox minimum inhibitory concentrations than the parent strain. The galU, galE, rfaI, and rfaB genes all encode enzymes that use UDP-galactose or UDP-glucose for galactose metabolism and lipopolysaccharide (LPS) biosynthesis. Indeed, we found that ciclopirox altered LPS composition of an E. coli clinical isolate. Taken together, our data demonstrate that ciclopirox affects galactose metabolism and LPS biosynthesis, two pathways important for bacterial growth and virulence. The lack of any reported fungal resistance to ciclopirox in over twenty years of use in the clinic, its excellent safety profiles, novel target(s), and efficacy, make ciclopirox a promising potential antimicrobial agent to use against multidrug-resistant problematic gram-negative pathogens.     

Genomic Epidemiology of an Endoscope-Associated Outbreak of Klebsiella pneumoniae Carbapenemase (KPC)-Producing K. pneumoniae

Increased incidence of infections due to Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae (KPC-Kp) was noted among patients undergoing endoscopic retrograde cholangiopancreatography (ERCP) at a single hospital. An epidemiologic investigation identified KPC-Kp and non-KPC-producing, extended-spectrum β-lactamase (ESBL)-producing Kp in cultures from 2 endoscopes. Genotyping was performed on patient and endoscope isolates to characterize the microbial genomics of the outbreak. Genetic similarity of 51 Kp isolates from 37 patients and 3 endoscopes was assessed by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Five patient and 2 endoscope isolates underwent whole genome sequencing (WGS). Two KPC-encoding plasmids were characterized by single molecule, real-time sequencing. Plasmid diversity was assessed by endonuclease digestion. Genomic and epidemiologic data were used in conjunction to investigate the outbreak source. Two clusters of Kp patient isolates were genetically related to endoscope isolates by PFGE. A subset of patient isolates were collected post-ERCP, suggesting ERCP endoscopes as a possible source. A phylogeny of 7 Kp genomes from patient and endoscope isolates supported ERCP as a potential source of transmission. Differences in gene content defined 5 ST258 subclades and identified 2 of the subclades as outbreak-associated. A novel KPC-encoding plasmid, pKp28 helped define and track one endoscope-associated ST258 subclade. WGS demonstrated high genetic relatedness of patient and ERCP endoscope isolates suggesting ERCP-associated transmission of ST258 KPC-Kp. Gene and plasmid content discriminated the outbreak from endemic ST258 populations and assisted with the molecular epidemiologic investigation of an extended KPC-Kp outbreak.     

Multilocus Sequence Typing as a Replacement for Serotyping in Salmonella enterica

Salmonella enterica subspecies enterica is traditionally subdivided into serovars by serological and nutritional characteristics. We used Multilocus Sequence Typing (MLST) to assign 4,257 isolates from 554 serovars to 1092 sequence types (STs). The majority of the isolates and many STs were grouped into 138 genetically closely related clusters called eBurstGroups (eBGs). Many eBGs correspond to a serovar, for example most Typhimurium are in eBG1 and most Enteritidis are in eBG4, but many eBGs contained more than one serovar. Furthermore, most serovars were polyphyletic and are distributed across multiple unrelated eBGs. Thus, serovar designations confounded genetically unrelated isolates and failed to recognize natural evolutionary groupings. An inability of serotyping to correctly group isolates was most apparent for Paratyphi B and its variant Java. Most Paratyphi B were included within a sub-cluster of STs belonging to eBG5, which also encompasses a separate sub-cluster of Java STs. However, diphasic Java variants were also found in two other eBGs and monophasic Java variants were in four other eBGs or STs, one of which is in subspecies salamae and a second of which includes isolates assigned to Enteritidis, Dublin and monophasic Paratyphi B. Similarly, Choleraesuis was found in eBG6 and is closely related to Paratyphi C, which is in eBG20. However, Choleraesuis var. Decatur consists of isolates from seven other, unrelated eBGs or STs. The serological assignment of these Decatur isolates to Choleraesuis likely reflects lateral gene transfer of flagellar genes between unrelated bacteria plus purifying selection. By confounding multiple evolutionary groups, serotyping can be misleading about the disease potential of S. enterica. Unlike serotyping, MLST recognizes evolutionary groupings and we recommend that Salmonella classification by serotyping should be replaced by MLST or its equivalents.     

Optimized Multilocus Sequence Typing (MLST) Scheme for Trypanosoma cruzi

Trypanosoma cruzi, the aetiological agent of Chagas disease possess extensive genetic diversity. This has led to the development of a plethora of molecular typing methods for the identification of both the known major genetic lineages and for more fine scale characterization of different multilocus genotypes within these major lineages. Whole genome sequencing applied to large sample sizes is not currently viable and multilocus enzyme electrophoresis, the previous gold standard for T. cruzi typing, is laborious and time consuming. In the present work, we present an optimized Multilocus Sequence Typing (MLST) scheme, based on the combined analysis of two recently proposed MLST approaches. Here, thirteen concatenated gene fragments were applied to a panel of T. cruzi reference strains encompassing all known genetic lineages. Concatenation of 13 fragments allowed assignment of all strains to the predicted Discrete Typing Units (DTUs), or near-clades, with the exception of one strain that was an outlier for TcV, due to apparent loss of heterozygosity in one fragment. Monophyly for all DTUs, along with robust bootstrap support, was restored when this fragment was subsequently excluded from the analysis. All possible combinations of loci were assessed against predefined criteria with the objective of selecting the most appropriate combination of between two and twelve fragments, for an optimized MLST scheme. The optimum combination consisted of 7 loci and discriminated between all reference strains in the panel, with the majority supported by robust bootstrap values. Additionally, a reduced panel of just 4 gene fragments displayed high bootstrap values for DTU assignment and discriminated 21 out of 25 genotypes. We propose that the seven-fragment MLST scheme could be used as a gold standard for T. cruzi typing, against which other typing approaches, particularly single locus approaches or systematic PCR assays based on amplicon size, could be compared.     

Genome-Wide Assessment of Outer Membrane Vesicle Production in Escherichia coli

The production of outer membrane vesicles by Gram-negative bacteria has been well documented; however, the mechanism behind the biogenesis of these vesicles remains unclear. Here a high-throughput experimental method and systems-scale analysis was conducted to determine vesiculation values for the whole genome knockout library of Escherichia coli mutant strains (Keio collection). The resultant dataset quantitatively recapitulates previously observed phenotypes and implicates nearly 150 new genes in the process of vesiculation. Gene functional and biochemical pathway analyses suggest that mutations that truncate outer membrane structures such as lipopolysaccharide and enterobacterial common antigen lead to hypervesiculation, whereas mutants in oxidative stress response pathways result in lower levels. This study expands and refines the current knowledge regarding the cellular pathways required for outer membrane vesiculation in E. coli.     

Draft Genome Sequence of an Extremely Drug-Resistant KPC-Producing Klebsiella pneumoniae ST258 Epidemic Strain

Extremely drug-resistant (XDR) Klebsiella pneumoniae carbapenemase-producing clone ST258 has rapidly disseminated worldwide. We report here the draft genome sequence of the K. pneumoniae ST258 XDR clinical strain from Israel.     

Multilocus Sequence Typing and Further Genetic Characterization of the Enigmatic Pathogen,Staphylococcus hominis

Staphylococcus hominis is a commensal resident of human skin and an opportunistic pathogen. The species is subdivided into two subspecies, S. hominis subsp. hominis and S. hominis subsp. novobiosepticus, which are difficult to distinguish. To investigate the evolution and epidemiology of S. hominis, a total of 108 isolates collected from 10 countries over 40 years were characterized by classical phenotypic methods and genetic methods. One nonsynonymous mutation in gyrB, scored with a novel SNP typing assay, had a perfect association with the novobiocin-resistant phenotype. A multilocus sequence typing (MLST) scheme was developed from six housekeeping gene fragments, and revealed relatively high levels of genetic diversity and a significant impact of recombination on S. hominis population structure. Among the 40 sequence types (STs) identified by MLST, three STs (ST2, ST16 and ST23) were S. hominis subsp. novobiosepticus, and they distinguished between isolates from different outbreaks, whereas 37 other STs were S. hominis subsp. hominis, one of which was widely disseminated (ST1). A modified PCR assay was developed to detect the presence of ccrAB4 from the SCCmec genetic element. S. hominis subsp. novobiosepticus isolates were oxacillin-resistant and carriers of specific components of SCCmec (mecA class A, ccrAB3, ccrAB4, ccrC), whereas S. hominis subsp. hominis included both oxacillin-sensitive and -resistant isolates and a more diverse array of SCCmec components. Surprisingly, phylogenetic analyses indicated that S. hominis subsp. novobiosepticus may be a polyphyletic and, hence, artificial taxon. In summary, these results revealed the genetic diversity of S. hominis, the identities of outbreak-causing clones, and the evolutionary relationships between subspecies and clones. The pathogenic lifestyle attributed to S. hominis subsp. novobiosepticus may have originated on more than one occasion.     

Multilocus Sequence Type Analysis Reveals both Clonality and Recombination in Populations of Candida glabrata Bloodstream Isolates from U.S. Surveillance Studies

The human commensal yeast Candida glabrata is becoming increasingly important as an agent of nosocomial bloodstream infection. However, relatively little is known concerning the genetics and population structure of this species. We have analyzed 230 incident bloodstream isolates from previous and current population-based surveillance studies by using multilocus sequence typing (MLST). Our results show that in the U.S. cities of Atlanta, GA; Baltimore, MD; and San Francisco, CA during three time periods spanning 1992 to 2009, five populations of C. glabrata bloodstream isolates are defined by a relatively small number of sequence types. There is little genetic differentiation in the different C. glabrata populations. We also show that there has been a significant temporal shift in the prevalence of one major subtype in Atlanta. Our results support the concept that both recombination and clonality play a role in the population structure of this species.In the most recently available survey of nosocomial bloodstream infections, Candida species were the fourth most common organism, surpassed only by Staphylococcus and Enterococcus species (24). Although Candida albicans remains the most commonly isolated Candida species worldwide, the incidence of Candida glabrata infection has been increasing steadily so that it is now the second most common cause of Candida infection in the United States (14). C. glabrata is considered a normal component of the human epithelial flora but is capable of causing serious systemic infections in susceptible hosts. This increase in the relative proportion of infections due to C. glabrata has come during the period of the introduction and prophylactic use of azole antifungal drugs (21) and may be a reflection of the decreased susceptibility of C. glabrata to these azole antifungal drugs (7, 15). Many questions regarding the epidemiology of C. glabrata infections have a direct impact on public health and still remain unanswered. Is the decreased susceptibility due to a small number of clones expanding in a population, or are all isolates capable of developing resistance to azole drugs? Are some isolates more virulent than others and therefore more prevalent in a population? Can we monitor the expansion of clonal isolates that may be more virulent or have increased drug resistance? A better understanding of the population genetics of C. glabrata may allow us to answer some of these questions.Many DNA fingerprinting methods have been developed for the investigation of the population genetics of Candida species (19). Two of the most important aspects of a typing system are reproducibility between laboratories and the ability to archive strain types. Multilocus sequence typing (MLST) has been developed as a typing system which allows highly reproducible strain discrimination as well as the development of genotypic strain archives that can be stored digitally for both prospective and retrospective analysis of isolates (13, 22). An MLST system which utilizes six housekeeping genes on six separate chromosomes was developed for C. glabrata (4), and an online archive of sequence types (STs) was established (http://cglabrata.mlst.net). Several studies utilizing this typing system have described the molecular population structure of both regional and worldwide collections of C. glabrata isolates (4, 5, 11, 12).During the past 2 decades, the Centers for Disease Control and Prevention (CDC) and our partners have undertaken three active, population-based surveillance studies in order to determine the incidence of candidemia, the distribution of species causing bloodstream infection, and the prevalence of antifungal drug resistance (8, 10). In each case, two major metropolitan areas were included: San Francisco, CA, and Atlanta, GA (1992 to 1993); Baltimore, MD, and the state of Connecticut (1998 to 2000); and Atlanta, GA, and Baltimore, MD (2008 to 2010). Population-based surveillance is unique in that it includes the total population of a particular geographic area and avoids the biases associated with single or select institutional studies. During each of the surveillance studies, incident bloodstream isolates from all hospitals within each defined geographic area were collected and identified to the species level. While C. glabrata isolates comprised a smaller percentage of the isolates in the 1992-to-1993 and 1998-to-2000 surveillance studies (8, 10), they represent almost a third of the isolates collected during the current surveillance (N. Iqbal and S. Lockhart, unpublished observations).In the present work, we have characterized by MLST analysis 230 isolates of C. glabrata from five populations (excluding Connecticut) separated both geographically and temporally. This unique collection of isolates allowed an analysis of the changing population genetics of this organism. We identified 31 unique STs and showed the maintenance of a major ST both geographically and temporally that is unique to the United States. An analysis of the relatedness of specific C. glabrata populations and a strong indication for recombination within and between populations are provided.     

A Type IV Pilus Mediates DNA Binding during Natural Transformation in Streptococcus pneumoniae

Natural genetic transformation is widely distributed in bacteria and generally occurs during a genetically programmed differentiated state called competence. This process promotes genome plasticity and adaptability in Gram-negative and Gram-positive bacteria. Transformation requires the binding and internalization of exogenous DNA, the mechanisms of which are unclear. Here, we report the discovery of a transformation pilus at the surface of competent Streptococcus pneumoniae cells. This Type IV-like pilus, which is primarily composed of the ComGC pilin, is required for transformation. We provide evidence that it directly binds DNA and propose that the transformation pilus is the primary DNA receptor on the bacterial cell during transformation in S. pneumoniae. Being a central component of the transformation apparatus, the transformation pilus enables S. pneumoniae, a major Gram-positive human pathogen, to acquire resistance to antibiotics and to escape vaccines through the binding and incorporation of new genetic material.     

Diversity of Capsular Polysaccharide Gene Clusters in Kpc-Producing Klebsiella pneumoniae Clinical Isolates of Sequence Type 258 Involved in the Italian Epidemic

Strains of Klebsiella pneumoniae producing KPC-type beta-lactamases (KPC-Kp) are broadly disseminating worldwide and constitute a major healthcare threat given their extensively drug resistant phenotypes and ability to rapidly disseminate in healthcare settings. In this work we report on the characterization of two different capsular polysaccharide (CPS) gene clusters, named cps _BO-4 and cps _207-2, from two KPC-Kp clinical strains from Italy belonging in sequence type (ST) 258, which is one of the most successful ST of KPC-Kp spreading worldwide. While cps _BO-4 was different from known 78 K-types according to the recently proposed typing schemes based on the wzi or wzc gene sequences, cps _207-2 was classified as K41 by one of these methods. Bioinformatic analysis revealed that they were represented in the genomic sequences of KPC-Kp from strains of ST258 from different countries, and cps _BO-4 was also detected in a KPC-Kp strain of ST442 from Brazil. Investigation of a collection of 46 ST258 and ST512 (a single locus variant of ST258) clinical strains representative of the recent Italian epidemic of KPC-Kp by means of a multiplex PCR typing approach revealed that cps _BO-4 was the most prevalent type, being detected both in ST258 and ST512 strains with a countrywide distribution, while cps _207-2 was only detected in ST258 strains with a more restricted distribution.     

多位点测序分型技术在病原微生物分型鉴定中的应用

多位点测序分型(Multilocus sequence typing,MLST)技术是一种以核苷酸序列为基础的病原菌分型方法,它是高通量测序技术与成熟的群体遗传学相结合的产物。该方法简单易行,重复性强,可以通过国际互联网对某一致病菌株在全球范围内的传播分布情况进行追踪监控。目前,MLST技术已被广泛应用于原核病原菌及一些真核病原菌(如真菌)的分型鉴定中。主要对MLST技术的原理及其在一些常见病原菌分型鉴定中的应用进行了简要的阐述。