Construction of Otherwise Isogenic Serotype 6B, 7F, 14, and 19F Capsular Variants of Streptococcus pneumoniae Strain TIGR4

The polysaccharide capsule is the primary virulence factor in Streptococcus pneumoniae. There are at least 90 serotypes of S. pneumoniae, identified based on the immunogenicity of different capsular sugars. The aim of this study was to construct pneumococcal strains that are isogenic except for capsular type. Serotype 4 strain TIGR4 was rendered unencapsulated by recombinational replacement of the capsular polysaccharide synthesis (cps) locus with the bicistronic Janus cassette (C. K. Sung, J. P. Claverys, and D. A. Morrison, Appl. Environ. Microbiol. 67:5190-5196, 2001). In subsequent transformation with chromosomal DNA, the cassette was replaced by the cps locus derived from a strain of a different serotype, either 6B, 7F, 14, or 19F. To minimize the risk of uncontrolled recombinational replacements in loci other than cps, the TIGRcps::Janus strain was “backcross” transformed three times with chromosomal DNA of subsequently constructed capsular type transformants. Capsular serotypes were confirmed in all new capsule variants by the Quellung reaction. Restriction fragment length polymorphism (RFLP) analysis of the cps locus confirmed the integrity of the cps region transformed into the TIGR strain, and RFLP of the flanking regions confirmed their identities with the corresponding regions of the recipient. Transformants had in vitro growth rates greater than or equal to that of TIGR4. All four strains were able to colonize C57BL/6 mice (female, 6 weeks old) for at least 7 days when mice were intranasally inoculated with 6 × 10⁶ to 8 × 10⁶ CFU. The constructed capsular variants of TIGR4 are suitable for use in studies on the role of S. pneumoniae capsular polysaccharide in immunity, colonization, and pathogenesis.     

Identification of a phage-derived depolymerase specific for KL47 capsule of Klebsiella pneumoniae and its therapeutic potential in mice

Klebsiella pneumoniae is one of the major pathogens causing global multidrug-resistant infections. Therefore, strategies for preventing and controlling the infections are urgently needed. Phage depolymerase, often found in the tail fiber protein or the tail spike protein, is reported to have antibiofilm activity. In this study, phage P560 isolated from sewage showed specific for capsule locus type KL47 K. pneumoniae, and the enlarged haloes around plaques indicated that P560 encoded a depolymerase. The capsule depolymerase, ORF43, named P560dep, derived from phage P560 was expressed, purified, characterized and evaluated for enzymatic activity as well as specificity. We reported that the capsule depolymerase P560dep, can digest the capsule polysaccharides on the surface of KL47 type K. pneumoniae, and the depolymerization spectrum of P560dep matched to the host range of phage P560, KL47 K. pneumoniae. Crystal violet staining assay showed that P560dep was able to significantly inhibit biofilm formation. Further, a single dose (50 μg/mouse) of depolymerase intraperitoneal injection protected 90%–100% of mice from lethal challenge before or after infection by KL47 carbapenem-resistant K. pneumoniae. And pathological changes were alleviated in lung and liver of mice infected by KL47 type K. pneumoniae. It is demonstrated that depolymerase P560dep as an attractive antivirulence agent represents a promising tool for antimicrobial therapy.     

Identification of three podoviruses infecting Klebsiella encoding capsule depolymerases that digest specific capsular types

Klebsiella pneumoniae is an important human pathogen causing opportunistic nosocomial and community-acquired infections. A major public health concern regarding K. pneumoniae is the increasing incidence of multidrug-resistant strains. Here, we isolated three novel Klebsiella bacteriophages, KN1-1, KN3-1 and KN4-1, which infect KN1, KN3 and K56, and KN4 types respectively. We determined their genome sequences and conducted a comparative analysis that revealed a variable region containing capsule depolymerase-encoding genes. Recombinant depolymerase proteins were produced, and their enzymatic activity and specificity were evaluated. We identified four capsule depolymerases in these phages that could only digest the capsule types of their respective hosts. Our results demonstrate that the activities of these capsule depolymerases were correlated with the host range of each phage; thus, the capsule depolymerases are host specificity determinants. By generating a capsule mutant, we demonstrate that capsule was essential for phage adsorption and infection. Further, capsule depolymerases can enhance bacterial susceptibility to serum killing. The discovery of these phages and depolymerases lays the foundation for the typing of KN1, KN3, KN4 and K56 Klebsiella and could be useful alternative therapeutics for the treatment of K. pneumoniae infections.     

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.     

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.     

Capsular Types of Klebsiella pneumoniae Revisited by wzc Sequencing

Capsule is an important virulence factor in bacteria. A total of 78 capsular types have been identified in Klebsiella pneumoniae. However, there are limitations in current typing methods. We report here the development of a new genotyping method based on amplification of the variable regions of the wzc gene. Fragments corresponding to the variable region of wzc were amplified and sequenced from 76 documented capsular types of reference or clinical strains. The remaining two capsular types (reference strains K15 and K50) lacked amplifiable wzc genes and were proven to be acapsular. Strains with the same capsular type exhibited ≧94% DNA sequence identity across the variable region (CD1-VR2-CD2) of wzc. Strains with distinct K types exhibited <80% DNA sequence identity across this region, with the exception of three pairs of strains: K22/K37, K9/K45, and K52/K79. Strains K22 and K37 shared identical capsular polysaccharide synthesis (cps) genes except for one gene with a difference at a single base which resulted in frameshift mutation. The wzc sequences of K9 and K45 exhibited high DNA sequence similarity but possessed different genes in their cps clusters. K52 and K79 exhibited 89% wzc DNA sequence identity but were readily distinguished from each other at the DNA level; in contrast, strains with the same capsular type as K52 exhibited 100% wzc sequence identity. A total of 29 strains from patients with bacteremia were typed by the wzc system. wzc DNA sequences confirmed the documented capsular type for twenty-eight of these clinical isolates; the remaining strain likely represents a new capsular type. Thus, the wzc genotyping system is a simple and useful method for capsular typing of K. pneumoniae.     

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.     

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.     

A method for generation phage cocktail with great therapeutic potential

Background

Bacteriophage could be an alternative to conventional antibiotic therapy against multidrug-resistant bacteria. However, the emergence of resistant variants after phage treatment limited its therapeutic application.

Methodology/Principal Findings

In this study, an approach, named “Step-by-Step” (SBS), has been established. This method takes advantage of the occurrence of phage-resistant bacteria variants and ensures that phages lytic for wild-type strain and its phage-resistant variants are selected. A phage cocktail lytic for Klebsiella pneumoniae was established by the SBS method. This phage cocktail consisted of three phages (GH-K1, GH-K2 and GH-K3) which have different but overlapping host strains. Several phage-resistant variants of Klebsiella pneumoniae were isolated after different phages treatments. The virulence of these variants was much weaker [minimal lethal doses (MLD)>1.3×10⁹ cfu/mouse] than that of wild-type K7 countpart (MLD = 2.5×10³ cfu/mouse). Compared with any single phage, the phage cocktail significantly reduced the mutation frequency of Klebsiella pneumoniae and effectively rescued Klebsiella pneumoniae bacteremia in a murine K7 strain challenge model. The minimal protective dose (MPD) of the phage cocktail which was sufficient to protect bacteremic mice from lethal K7 infection was only 3.0×10⁴ pfu, significantly smaller (p<0.01) than that of single monophage. Moreover, a delayed administration of this phage cocktail was still effective in protection against K7 challenge.

Conclusions/Significance

Our data showed that the phage cocktail was more effective in reducing bacterial mutation frequency and in the rescue of murine bacteremia than monophage suggesting that phage cocktail established by SBS method has great therapeutic potential for multidrug-resistant bacteria infection.     

Comparison of virulence between matt and mucoid colonies of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> coproducing NDM-1 and OXA-232 isolated from a single patient

Nine Klebsiella pneumoniae isolates coproducing NDM-1 and OXA-232 carbapenemases were successively isolated from a single patient. Although they were isolated simultaneously and were isogenic, they presented different colony phenotypes (matt and mucoid). All nine isolates were resistant to most antibiotics except colistin and fosfomycin. In addition, matt-type isolates were resistant to tigecycline. No differences were detected in the cps cluster sequences, except for the insertion of IS5 in the wzb gene of two matt-type isolates. In vitro virulence assays based on production of capsular polysaccharide, biofilm formation, and resistance to human serum indicated that the mucoid-type isolates were significantly more virulent than the matt-type. In addition, mucoid-type isolates showed higher survival rates than the matt-type ones in infection experiments in the fruit fly, suggesting a higher virulence of K. pneumoniae isolates with a mucoid phenotype. To our knowledge, this is the first report of K. pneumoniae colonies with different phenotypes being isolated from the same sample. In addition, we show that virulence varies with colony phenotype. Dissemination of K. pneumoniae isolates expressing both antibiotic resistance and high virulence would constitute a great threat.     

Effects of Streptococcus pneumoniae strain background on complement resistance

Background

Immunity to infections caused by Streptococcus pneumoniae is dependent on complement. There are wide variations in sensitivity to complement between S. pneumoniae strains that could affect their ability to cause invasive infections. Although capsular serotype is one important factor causing differences in complement resistance between strains, there is also considerable other genetic variation between S. pneumoniae strains that may affect complement-mediated immunity. We have therefore investigated whether genetically distinct S. pneumoniae strains with the same capsular serotype vary in their sensitivity to complement mediated immunity.

Methodology and Principal Findings

C3b/iC3b deposition and neutrophil association were measured using flow cytometry assays for S. pneumoniae strains with different genetic backgrounds for each of eight capsular serotypes. For some capsular serotypes there was marked variation in C3b/iC3b deposition between different strains that was independent of capsule thickness and correlated closely to susceptibility to neutrophil association. C3b/iC3b deposition results also correlated weakly with the degree of IgG binding to each strain. However, the binding of C1q (the first component of the classical pathway) correlated more closely with C3b/iC3b deposition, and large differences remained in complement sensitivity between strains with the same capsular serotype in sera in which IgG had been cleaved with IdeS.

Conclusions

These data demonstrate that bacterial factors independent of the capsule and recognition by IgG have strong effects on the susceptibility of S. pneumoniae to complement, and could therefore potentially account for some of the differences in virulence between strains.     

Non-typeable pneumococci circulating in Portugal are of cps type NCC2 and have genomic features typical of encapsulated isolates

Background

Pneumococcus is a major human pathogen and the polysaccharide capsule is considered its main virulence factor. Nevertheless, strains lacking a capsule, named non-typeable pneumococcus (NT), are maintained in nature and frequently colonise the human nasopharynx. Interest in these strains, not targeted by any of the currently available pneumococcal vaccines, has been rising as they seem to play an important role in the evolution of the species. Currently, there is a paucity of data regarding this group of pneumococci. Also, questions have been raised on whether they are true pneumococci. We aimed to obtain insights in the genetic content of NT and the mechanisms leading to non-typeability and to genetic diversity.

Results

A collection of 52 NT isolates representative of the lineages circulating in Portugal between 1997 and 2007, as determined by pulsed-field gel electrophoresis and multilocus sequence typing, was analysed. The capsular region was sequenced and comparative genomic hybridisation (CGH) using a microarray covering the genome of 10 pneumococcal strains was carried out. The presence of mobile elements was investigated as source of intraclonal variation. NT circulating in Portugal were found to have similar capsular regions, of cps type NCC2, i.e., having aliB-like ORF1 and aliB-like ORF2 genes. The core genome of NT was essentially similar to that of encapsulated strains. Also, competence genes and most virulence genes were present. The few virulence genes absent in all NT were the capsular genes, type-I and type-II pili, choline-binding protein A (cbpA/pspC), and pneumococcal surface protein A (pspA). Intraclonal variation could not be entirely explained by the presence of prophages and other mobile elements.

Conclusions

NT circulating in Portugal are a homogeneous group belonging to cps type NCC2. Our observations support the theory that they are bona-fide pneumococcal isolates that do not express the capsule but are otherwise essentially similar to encapsulated pneumococci. Thus we propose that NT should be routinely identified and reported in surveillance studies.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-863) contains supplementary material, which is available to authorized users.     

Population-Based Analysis of Invasive Nontypeable Pneumococci Reveals That Most Have Defective Capsule Synthesis Genes

Since nasopharyngeal carriage of pneumococcus precedes invasive pneumococcal disease, characteristics of carriage isolates could be incorrectly assumed to reflect those of invasive isolates. While most pneumococci express a capsular polysaccharide, nontypeable pneumococci are sometimes isolated. Carriage nontypeables tend to encode novel surface proteins in place of a capsular polysaccharide synthetic locus, the cps locus. In contrast, capsular polysaccharide is believed to be indispensable for invasive pneumococcal disease, and nontypeables from population-based invasive pneumococcal disease surveillance have not been extensively characterized. We received 14,328 invasive pneumococcal isolates through the Active Bacterial Core surveillance program during 2006–2009. Isolates that were nontypeable by Quellung serotyping were characterized by PCR serotyping, sequence analyses of the cps locus, and multilocus sequence typing. Eighty-eight isolates were Quellung-nontypeable (0.61%). Of these, 79 (89.8%) contained cps loci. Twenty-two nontypeables exhibited serotype 8 cps loci with defects, primarily within wchA. Six of the remaining nine isolates contained previously-described aliB homologs in place of cps loci. Multilocus sequence typing revealed that most nontypeables that lacked capsular biosynthetic genes were related to established non-encapsulated lineages. Thus, invasive pneumococcal disease caused by nontypeable pneumococcus remains rare in the United States, and while carriage nontypeables lacking cps loci are frequently isolated, such nontypeable are extremely rare in invasive pneumococcal disease. Most invasive nontypeable pneumococci possess defective cps locus genes, with an over-representation of defective serotype 8 cps variants.     

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.     

PspA Family Distribution,Antimicrobial Resistance and Serotype of Streptococcus pneumoniae Isolated from Upper Respiratory Tract Infections in Japan

Background

The protection against pneumococcal infections provided by currently available pneumococcal polysaccharide conjugate vaccines are restricted to the limited number of the serotypes included in the vaccine. In the present study, we evaluated the distribution of the pneumococcal capsular type and surface protein A (PspA) family of pneumococcal isolates from upper respiratory tract infections in Japan.

Methods

A total of 251 S. pneumoniae isolates from patients seeking treatment for upper respiratory tract infections were characterized for PspA family, antibiotic resistance and capsular type.

Results

Among the 251 pneumococci studied, the majority (49.4%) was identified as belonging to PspA family 2, while most of the remaining isolates (44.6%) belonged to family 1. There were no significant differences between the distributions of PspA1 versus PspA2 isolates based on the age or gender of the patient, source of the isolates or the isolates’ susceptibilities to penicillin G. In contrast, the frequency of the mefA gene presence and of serotypes 15B and 19F were statistically more common among PspA2 strains.

Conclusion

The vast majority of pneumococci isolated from the middle ear fluids, nasal discharges/sinus aspirates or pharyngeal secretions represented PspA families 1 and 2. Capsular serotypes were generally not exclusively associated with certain PspA families, although some capsular types showed a much higher proportion of either PspA1 or PspA2. A PspA-containing vaccine would potentially provide high coverage against pneumococcal infectious diseases because it would be cross-protective versus invasive disease with the majority of pneumococci infecting children and adults.     

Streptococcus pneumoniae Serotype 11D Has a Bispecific Glycosyltransferase and Expresses Two Different Capsular Polysaccharide Repeating Units

Streptococcus pneumoniae (pneumococcus) expresses a capsular polysaccharide (CPS) that protects against host immunity and is synthesized by enzymes in the capsular polysaccharide synthesis (cps) locus. Serogroup 11 has six members (11A to -E) and the CPS structure of all members has been solved, except for serotype 11D. The cps loci of 11A and 11D differ by one codon (N112S) in wcrL, which putatively encodes a glycosyltransferase that adds the fourth sugar of the CPS repeating unit (RU). Gas chromatography and nuclear magnetic resonance analysis revealed that 11A and 11D PSs contain identical CPS RUs that contain αGlc as the fourth sugar. However, ∼25% of 11D CPS RUs contain instead αGlcNAc as the fourth sugar, suggesting that 11D wcrL encodes a bispecific glycosyltransferase. To test the hypothesis that codon 112 of WcrL determines enzyme specificity, and therefore the fourth sugar in the RU, we generated three isogenic pneumococcal strains with 11A cps loci containing wcrL encoding Ser-112 (MBO128) or Ala-112 (MBO130). MBO128 was serologically and biochemically identical to serotype 11D. MBO130 has a unique serologic profile; has as much αGlcNAc as 11F, 11B, and 11C CPS do; and may represent a new serotype. These findings demonstrate how pneumococci alter their CPS structure and their immunologic properties with a minimal genetic change.     

Unusual features in organisation of capsular polysaccharide-related genes of C. jejuni strain X

PCR probing of the genome of Campylobacter jejuni strain X using conserved capsular polysaccharide (CPS)-related genes allowed elucidation of a complete sequence of the respective gene cluster (cps). This is the largest known Campylobacter cps cluster (38 kb excluding flanking kps regions), which includes a number of genes not detected in other Campylobacter strains. Sequence analysis suggests genetic rearrangements both within and outside the cps gene cluster, a mechanism which may be responsible for mosaic organisation of sugar transferase-related genes leading to structural variability of the capsular polysaccharide (CPS).     

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.