Activity of the Antimicrobial Peptide and Thanatin Analog S-thanatin on Clinical Isolates of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> Resistant to Conventional Antibiotics with Different Structures

The treatment of infections caused by bacteria resistant to the vast majority of antibiotics is a challenge worldwide. To evaluate the effect of S-thanatin (an analog of thanatin, a cationic antimicrobial peptide isolated from the hemipteran insect Podisus maculiventris) against microbial resistant to antibiotics, we studied its bactericidal kinetics, synergistic effect, resistance, and activity on clinical isolates of Klebsiella pneumoniae resistant to conventional antibiotics with different structures. The bactericidal rate of S-thanatin was more than 99% against K. pneumoniae ATCC 700603 when bacterial cultures were monitored for 60 min. The peptide was synergistic with β-lactam cefepime in most of the clinical MDR isolates tested (7/8). An average value of FIC was 0.3708. No synergy was found between the peptide and amoxicillin, gentamycin, tetracycline, or ciprofloxacin in all bacteria tested. A total of 48 isolates of K. pneumoniae with different resistance spectrum tested was susceptible to S-thanatin. The MICs were 6.25–25 μg/ml. No significant difference in the MICs of S-thanatin between the sensitive isolates and the resistant isolates to single antibiotic was observed (P > 0.05). The resistance of K. pneumoniae ATCC 700603 to S-thanatin was slightly higher, when cultured at sub-inhibitory concentration for 5 days. S-thanatin may be an attractive candidate for developing into an antimicrobial agent.     

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.     

Streptococcus pneumoniae Phosphoglycerate Kinase Is a Novel Complement Inhibitor Affecting the Membrane Attack Complex Formation

The Gram-positive bacterium Streptococcus pneumoniae is a major human pathogen that causes infections ranging from acute otitis media to life-threatening invasive disease. Pneumococci have evolved several strategies to circumvent the host immune response, in particular the complement attack. The pneumococcal glycolytic enzyme phosphoglycerate kinase (PGK) is both secreted and bound to the bacterial surface and simultaneously binds plasminogen and its tissue plasminogen activator tPA. In the present study we demonstrate that PGK has an additional role in modulating the complement attack. PGK interacted with the membrane attack complex (MAC) components C5, C7, and C9, thereby blocking the assembly and membrane insertion of MAC resulting in significant inhibition of the hemolytic activity of human serum. Recombinant PGK interacted in a dose-dependent manner with these terminal pathway proteins, and the interactions were ionic in nature. In addition, PGK inhibited C9 polymerization both in the fluid phase and on the surface of sheep erythrocytes. Interestingly, PGK bound several MAC proteins simultaneously. Although C5 and C7 had partially overlapping binding sites on PGK, C9 did not compete with either one for PGK binding. Moreover, PGK significantly inhibited MAC deposition via both the classical and alternative pathway at the pneumococcal surface. Additionally, upon activation plasmin(ogen) bound to PGK cleaved the central complement protein C3b thereby further modifying the complement attack. In conclusion, our data demonstrate for the first time to our knowledge a novel pneumococcal inhibitor of the terminal complement cascade aiding complement evasion by this important pathogen.     

Application of S-thanatin,an antimicrobial peptide derived from thanatin,in mouse model of Klebsiella pneumoniae infection

Thanatin was first discovered from the hemipteran insect Podisus maculiventris and showed a promising antimicrobial activity. Multidrug-resistant (MDR) clinical isolates of Klebsiella pneumoniae have developed resistance to current therapies. As an attempt to resolve this problem, the efficacy of thanatin and its analogues against clinical isolates of K. pneumoniae was studied in vitro and in vivo. S-thanatin showed an improved antimicrobial activity with the tested MIC values was 2–8-fold lower than those of other thanatin analogs. Antimicrobial assay indicated a high activity of S-thanatin against K. pneumoniae in vitro with MIC between 4 and 8 μg/ml. Its in vivo activity was evaluated using a K. pneumoniae-infected mice model. Adult male ICR mice were randomly grouped and given an intraperitoneal (i.p.) administration of 2 × 10¹⁰ colony-forming units of K. pneumoniae (CI 120204205). Afterwards, mouse groups were subjected to i.p. administration of saline or S-thanatin (5, 10, or 15 mg/kg). After an inspection of 72 h, the mice were finally sacrificed for analysis of in vivo bacterial growth and plasma endotoxin level. The results showed that S-thanatin administration apparently improved the survival rate and reduced the bacterial CFU from intra-abdominal fluid in mice. The plasma endotoxin level was improved as well. All above implied that S-thanatin, as an alternative, may provide a novel strategy for treating K. pneumoniae infection and other infections due to multidrug-resistant bacteria.     

Human L-ficolin,a Recognition Molecule of the Lectin Activation Pathway of Complement,Activates Complement by Binding to Pneumolysin,the Major Toxin of Streptococcus pneumoniae

The complement system is an essential component of the immune response, providing a critical line of defense against different pathogens including S. pneumoniae. Complement is activated via three distinct pathways: the classical (CP), the alternative (AP) and the lectin pathway (LP). The role of Pneumolysin (PLY), a bacterial toxin released by S. pneumoniae, in triggering complement activation has been studied in vitro. Our results demonstrate that in both human and mouse sera complement was activated via the CP, initiated by direct binding of even non-specific IgM and IgG3 to PLY. Absence of CP activity in C1q^−/− mouse serum completely abolished any C3 deposition. However, C1q depleted human serum strongly opsonized PLY through abundant deposition of C3 activation products, indicating that the LP may have a vital role in activating the human complement system on PLY. We identified that human L-ficolin is the critical LP recognition molecule that drives LP activation on PLY, while all of the murine LP recognition components fail to bind and activate complement on PLY. This work elucidates the detailed interactions between PLY and complement and shows for the first time a specific role of the LP in PLY-mediated complement activation in human serum.     

Opc Expression,LPS Immunotype Switch and Pilin Conversion Contribute to Serum Resistance of Unencapsulated Meningococci

Neisseria meningitidis employs polysaccharides and outer membrane proteins to cope with human serum complement attack. To screen for factors influencing serum resistance, an assay was developed based on a colorimetric serum bactericidal assay. The screening used a genetically modified sequence type (ST)-41/44 clonal complex (cc) strain lacking LPS sialylation, polysaccharide capsule, the factor H binding protein (fHbp) and MutS, a protein of the DNA repair mechanism. After killing of >99.9% of the bacterial cells by serum treatment, the colorimetric assay was used to screen 1000 colonies, of which 35 showed enhanced serum resistance. Three mutant classes were identified. In the first class of mutants, enhanced expression of Opc was identified. Opc expression was associated with vitronectin binding and reduced membrane attack complex deposition confirming recent observations. Lipopolysaccharide (LPS) immunotype switch from immunotype L3 to L8/L1 by lgtA and lgtC phase variation represented the second class. Isogenic mutant analysis demonstrated that in ST-41/44 cc strains the L8/L1 immunotype was more serum resistant than the L3 immunotype. Consecutive analysis revealed that the immunotypes L8 and L1 were frequently observed in ST-41/44 cc isolates from both carriage and disease. Immunotype switch to L8/L1 is therefore suggested to contribute to the adaptive capacity of this meningococcal lineage. The third mutant class displayed a pilE allelic exchange associated with enhanced autoaggregation. The mutation of the C terminal hypervariable region D of PilE included a residue previously associated with increased pilus bundle formation. We suggest that autoaggregation reduced the surface area accessible to serum complement and protected from killing. The study highlights the ability of meningococci to adapt to environmental stress by phase variation and intrachromosomal recombination affecting subcapsular antigens.     

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.     

First Emergence of acrAB and oqxAB Mediated Tigecycline Resistance in Clinical Isolates of Klebsiella pneumoniae Pre-Dating the Use of Tigecycline in a Chinese Hospital

Tigecycline is one of the few therapeutic options for treating infections caused by some multi-drug resistant pathogens, such as Klebsiella pneumoniae. However, tigecycline-resistant K. pneumoniae has been discovered recently in China. From 2009 to 2013, nine tigecycline-resistant K. pneumoniae isolates were identified in our hospital. Six of nine strains were identified before using tigecycline. To investigate the efflux-mediated resistance mechanisms of K. pneumoniae, the expression of efflux pump genes (acrA, acrB, tolC, oqxA and oqxB) and pump regulators (acrR, marA, soxS, rarA, rob and ramA) were examined by real-time RT-PCR. Molecular typing of the tigecycline resistant strains was performed. ST11 was the predominant clone of K. pneumoniae strains, while ST1414 and ST1415 were novel STs. Efflux pump inhibitor (EPI)-carbonyl cyanide chlorophenylhydrazone (CCCP) was able to reverse the resistance patterns of 5 resistant K. pneumoniae strains. In comparison with strain A111, a tigecycline-susceptible strain (negative control), we found that the expression levels of efflux pump genes and pump regulators were higher in a majority of resistant strains. Higher expression levels of regulators rarA (2.41-fold, 9.55-fold, 28.44-fold and 18.31-fold, respectively) and pump gene oqxB (3.87-fold, 31.96-fold, 50.61-fold and 29.45-fold, respectively) were observed in four tigecycline resistant strains (A363, A361, A368, A373, respectively). Increased expression of acrB was associated with ramA and marA expression. To our knowledge, studies on tigecycline resistance mechanism in K. pneumoniae are limited especially in China. In our study, we found that both efflux pump AcrAB-TolC and OqxAB contributed to tigecycline resistance in K. pneumoniae isolates.     

Role of type 1 and type 3 fimbriae in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> biofilm formation

Background  

Klebsiella pneumoniae is an important gram-negative opportunistic pathogen causing primarily urinary tract infections, respiratory infections, and bacteraemia. The ability of bacteria to form biofilms on medical devices, e.g. catheters, has a major role in development of many nosocomial infections. Most clinical K. pneumoniae isolates express two types of fimbrial adhesins, type 1 fimbriae and type 3 fimbriae. In this study, we characterized the role of type 1 and type 3 fimbriae in K. pneumoniae biofilm formation.     

Incidence of Klebsiella Species in Surface Waters and Their Expression of Virulence Factors

To investigate the occurrence of different Klebsiella spp. in aquatic environments, a total of 208 samples of natural surface waters was examined. From half (53%) of these samples, 123 Klebsiella strains were isolated, the most common species being Klebsiella pneumoniae. A comparison of these isolates to a group of 207 clinical K. pneumoniae isolates demonstrated that water isolates of K. pneumoniae, unlike those of K. oxytoca and K. planticola, are as capable as clinical isolates of expressing putative virulence factors such as serum resistance and capsular polysaccharides, pili, and siderophores.     

Streptococcus iniae: serological differences,presence of capsule and resistance to immune serum killing

The biochemical profiles, presence of capsule, outer membrane protein profiles and serological interactions of isolates of Streptococcus iniae obtained from different geographical and fish host origins were examined. The isolates had very similar biochemical profiles using API 20 Strep but varied as to whether they were arginine dihydrolase-negative, -positive or -intermediate (AD-ve, AD+ve, AD+/-ve, respectively). Representatives of each AD type were compared in subsequent experiments. All types possessed a polysaccharide capsule. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of outer membrane proteins or whole cells revealed no difference in banding patterns between isolates. All isolates were resistant to trout normal and specific immune serum and grew well in the presence of added fresh normal serum. Serological analyses of the isolates revealed antigenic differences. Trout antiserum against the AD+ve isolate did not agglutinate the AD-ve or AD+/-ve isolates, while antisera against the latter 2 types showed low agglutinating activity with all 3 isolates. When whole live cells of AD-ve and AD+ve isolates were dot-blotted, antiserum to the AD+ve isolate did not stain the AD-ve isolate, but antiserum to the AD-ve isolate stained both AD types. However, if the cells were pre-treated with Proteinase K (to remove surface-exposed protein antigens), the AD+ve isolate was stained only by its homologous antiserum. These results suggest that while certain protein antigens of the different AD type strains are immunologically cross-reactive, the capsular antigens appear to be AD type-specific. Furthermore, the results suggest that the cross-reactive antigens on the AD-ve isolate are effectively hidden by the strain-specific capsule, while they are partially exposed on the AD+ve isolate.     

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.     

Development of a Multiplex-PCR probe system for the proper identification of <Emphasis Type="Italic">Klebsiella variicola</Emphasis>

Background

Klebsiella variicola was very recently described as a new bacterial species and is very closely related to Klebsiella pneumoniae; in fact, K. variicola isolates were first identified as K. pneumoniae. Therefore, it might be the case that some isolates, which were initially classified as K. pneumoniae, are actually K. variicola. The aim of this study was to devise a multiplex-PCR probe that can differentiate isolates from these sister species.

Result

This work describes the development of a multiplex-PCR method to identify K. variicola. This development was based on sequencing a K. variicola clinical isolate (801) and comparing it to other K. variicola and K. pneumoniae genomes. The phylogenetic analysis showed that K. variicola isolates form a monophyletic group that is well differentiated from K. pneumoniae. Notably, the isolate K. pneumoniae 342 and K. pneumoniae KP5-1 might have been misclassified because in our analysis, both clustered with K. variicola isolates rather than with K. pneumoniae. The multiplex-PCR (M-PCR-1 to 3) probe system could identify K. variicola with high accuracy using the shared unique genes of K. variicola and K. pneumoniae genomes, respectively. M-PCR-1 was used to assay a collection of multidrug-resistant (503) and antimicrobial-sensitive (557) K. pneumoniae clinical isolates. We found K. variicola with a prevalence of 2.1% (23/1,060), of them a 56.5% (13/23) of the isolates were multidrug resistant, and 43.5% (10/23) of the isolates were antimicrobial sensitive. The phylogenetic analysis of rpoB of K. variicola-positive isolates identified by multiplex-PCR support the correct identification and differentiation of K. variicola from K. pneumoniae clinical isolates.

Conclusions

This multiplex-PCR provides the means to reliably identify and genotype K. variicola. This tool could be very helpful for clinical, epidemiological, and population genetics studies of this species. A low but significant prevalence of K. variicola isolates was found, implying that misclassification had occurred previously. We believe that our multiplex-PCR assay could be of paramount importance to understand the population dynamics of K. variicola in both clinical and environmental settings.

     

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.     

Screening of mcr-1 Among Gram-Negative Bacteria from Different Clinical Samples from ICU Patients in Alexandria,Egypt: One-Year Study

Antimicrobial resistance represents a global dilemma. Our present study aimed to investigate the presence of mcr-1 among different Gram-negative bacteria including Enterobacteriaceae (except intrinsically resistant to colistin) and Pseudomonas aeruginosa. Gram-negative bacterial isolates were collected from different ICUs in several Alexandria hospitals from June 2019 to June 2020. The identification of these Gram-negative isolates was made using the VITEK-2^® system (BioMérieux, France). SYBR Green-based PCR was used to screen for the presence of mcr-1 using a positive control that we amplified and sequenced earlier in our pilot study. All isolates were screened for the presence of mcr-1 regardless of their colistin susceptibility. Isolates that harbored mcr-1 were tested for colistin susceptibility and for the presence of some beta-lactamase genes. Klebsiella pneumoniae isolates harboring mcr-1 were capsule typed using the wzi sequence analysis. Four hundred eighty isolates were included in this study. Only six isolates harbored mcr-1.1. Of these, four were resistant to colistin, while two (K. pneumoniae and P. aeruginosa) were susceptible to colistin. Five of the six isolates were resistant to carbapenems. They harbored bla_OXA-48, and three of them co-harbored bla_NDM-1. K-58 was the most often found among our K. pneumoniae harboring mcr-1.1. To our knowledge, this is the first time to report colistin susceptible P. aeruginosa and K. pneumoniae harboring the mcr-1.1 gene in Egypt. Further studies are needed to investigate the presence of the mcr genes among colistin susceptible isolates to shed more light on its significance as a potential threat. Open in a separate window     

Occurrence of Plasmid-Mediated AmpC β-Lactamases Among Escherichia coli and Klebsiella pneumoniae Clinical Isolates in a Tertiary Care Hospital in Bangalore

Therapeutic options for infections caused by gram-negative organisms expressing plasmid-mediated AmpC β-lactamases are limited because these organisms are usually resistant to all the β-lactam antibiotics, except for cefepime, cefpirome and the carbapenems. These organisms are a major concern in nosocomial infections and should therefore be monitored in surveillance studies. Hence, this study was aimed out to determine the prevalence of plasmid-mediated AmpC β-lactamases in E. coli and K. pneumoniae from a tertiary care in Bangalore. A total of 63 E. coli and 27 K. pneumoniae were collected from a tertiary care hospital in Bangalore from February 2008 to July 2008. The isolates with decreased susceptibility to cefoxitin were subjected to confirmation test with three dimensional extract tests. Minimum inhibitory concentrations (MICs) were determined by agar dilution method. Conjugation experiments, plasmid profiling and susceptibility testing were carried out to investigate the underlying mechanism of resistance. In our study, 52 (57.7%) isolates showed resistance to cefoxitin, the occurrence of AmpC was found to be 7.7% of the total isolates. Plasmid analysis of the selected isolates showed the presence of a single plasmid of 26 kb in E. coli and 2 Kb in K. pneumoniae. Plasmid-mediated AmpC β-lactamases were found in 11.1% of K. pneumoniae and in 6.3% of E. coli. Curing and conjugation experiments showed that resistance to cephamycins and cephalosporins was plasmid-mediated. Our study has demonstrated the occurrence of plasmid-mediated AmpC in E. coli and K. pneumoniae which illustrates the importance of molecular surveillance in tracking AmpC-producing strains at general hospitals and emphasizes the need for epidemiological monitoring.     

Seroepidemiology of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> colonizing the intestinal tract of healthy chinese and overseas chinese adults in Asian countries

Background  

Capsular serotypes K1 and K2 of Klebsiella pneumoniae are thought to the major virulence determinants responsible for liver abscess. The intestine is one of the major reservoirs of K. pneumoniae, and epidemiological studies have suggested that the majority of K. pneumoniae infections are preceded by colonization of the gastrointestinal tract. The possibility of fecal-oral transmission in liver abscess has been raised on the basis of molecular typing of isolates. Data on the serotype distribution of K. pneumoniae in stool samples from healthy individuals has not been previously reported. This study investigated the seroepidemiology of K. pneumoniae isolates from the intestinal tract of healthy Chinese in Asian countries. Stool specimens from healthy adult Chinese residents of Taiwan, Japan, Hong Kong, China, Thailand, Malaysia, Singapore, and Vietnam were collected from August 2004 to August 2010 for analysis.     

Klebsiella pneumoniae triggers a cytotoxic effect on airway epithelial cells

Background  

Klebsiella pneumoniae is a capsulated Gram negative bacterial pathogen and a frequent cause of nosocomial infections. Despite its clinical relevance, little is known about the features of the interaction between K. pneumoniae and lung epithelial cells on a cellular level, neither about the role of capsule polysaccharide, one of its best characterised virulence factors, in this interaction.     

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.     

Comparative genomics of closely related strains of Klebsiella pneumoniae reveals genes possibly involved in colistin resistance

Strains of colistin-resistant Klebsiella pneumoniae are emerging worldwide, due to the increased use of this molecule in antibiotic-resistant nosocomial infections. Comparative genomics was performed on three closely related K. pneumoniae strains isolated from three patients in a single hospital in Bologna, Italy. Two of these isolates are colistin-resistant, while the third is sensitive to this antibiotic. The designed bioinformatic approach detected, among the three analyzed genomes, single nucleotide polymorphisms, insertions and deletions, specific patterns of gene presence and absence, in a total of 270 genes. These genes were analyzed by automatic and manual methods, to identify those potentially involved in colistin resistance, based on the data available in the literature and on the mechanism of action of colistin, the alteration of the outer membrane. Three of the identified genes (waaL, rfbA, vacJ), all presenting non-synonymous substitutions in the colistin resistant strains, resulted to be of special interest, due to the specific function of their protein products, involved in the biosynthesis of the outer bacterial membrane.