Klebsiella pneumoniae subsp. pneumoniae-bacteriophage combination from the caecal effluent of a healthy woman.
ABSTRACT: A sample of caecal effluent was obtained from a female patient who had undergone a routine colonoscopic examination. Bacteria were isolated anaerobically from the sample, and screened against the remaining filtered caecal effluent in an attempt to isolate bacteriophages (phages). A lytic phage, named KLPN1, was isolated on a strain identified as Klebsiella pneumoniae subsp. pneumoniae (capsular type K2, rmpA (+)). This Siphoviridae phage presents a rosette-like tail tip and exhibits depolymerase activity, as demonstrated by the formation of plaque-surrounding haloes that increased in size over the course of incubation. When screened against a panel of clinical isolates of K. pneumoniae subsp. pneumoniae, phage KLPN1 was shown to infect and lyse capsular type K2 strains, though it did not exhibit depolymerase activity on such hosts. The genome of KLPN1 was determined to be 49,037 bp (50.53 %GC) in length, encompassing 73 predicted ORFs, of which 23 represented genes associated with structure, host recognition, packaging, DNA replication and cell lysis. On the basis of sequence analyses, phages KLPN1 (GenBank: KR262148) and 1513 (a member of the family Siphoviridae, GenBank: KP658157) were found to be two new members of the genus "Kp36likevirus."
Project description:The emergence of multidrug-resistant bacteria is a major global health concern. The search for new therapies has brought bacteriophages into the spotlight, and new phages are being described as possible therapeutic agents. Among the bacteria that are most extensively resistant to current antibiotics is Klebsiella pneumoniae, whose hypervariable extracellular capsule makes treatment particularly difficult. Here, we describe two new K. pneumoniae phages, ?VLC5 and ?VLC6, isolated from environmental samples. These phages belong to the genus Drulisvirus within the family Podoviridae. Both phages encode a similar tail spike protein with putative depolymerase activity, which is shared among other related phages and probably determines their ability to specifically infect K. pneumoniae capsular types K22 and K37. In addition, we found that phage ?VLC6 also infects capsular type K13 and is capable of striping the capsules of K. pneumoniae KL2 and KL3, although the phage was not infectious in these two strains. Genome sequence analysis suggested that the extended tropism of phage ?VLC6 is conferred by a second, divergent depolymerase. Phage ?VLC5 encodes yet another putative depolymerase, but we found no activity of this phage against capsular types other than K22 and K37, after testing a panel of 77 reference strains. Overall, our results confirm that most phages productively infected one or few Klebsiella capsular types. This constitutes an important challenge for clinical applications.
Project description:Two Klebsiella bacteriophages K5-2 and K5-4, which are able to infect and grow on either capsular types K30/K69 and K5 or K8 and K5 of Klebsiella strains, were isolated and characterized. Each phage contained two open reading frames (ORFs), which encoded two putative capsule depolymerases, respectively. The first ORF encoded tail fiber proteins, which have K30/K69 depolymerase and K8 depolymerase activities. The second ORF encoded hypothetical proteins, which are almost identical in amino acid sequences, and have K5 depolymerase activity. Alcian blue staining of enzyme-treated capsular polysaccharides (CPS) showed that purified depolymerases can cleave purified Klebsiella CPS in vitro and liberate monosaccharaides. Capsule K5 deletion mutants were not lysed by either phage, suggesting that the capsule was essential for phage infection. Bacterial killing was observed when incubated Klebsiella strains with phages but not with purified depolymerases. Treatment with the K5-4 phage significantly increased the survival of mice infected with a K. pneumoniae K5 strain. In conclusion, two dual host-specific Klebsiella phages and their tailspikes exhibit capsule depolymerase activity were characterized. Each phage and phage-encoded depolymerase has specificity for capsular type K30/K69, K8 or K5, and could be used for the typing and treatment of K. pneumoniae infection.
Project description: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.
Project description:Klebsiella pneumoniae carries a thick polysaccharide capsule. This highly variable chemical structure plays an important role in its virulence. Many Klebsiella bacteriophages recognize this capsule with a receptor binding protein (RBP) that contains a depolymerase domain. This domain degrades the capsule to initiate phage infection. RBPs are highly specific and thus largely determine the host spectrum of the phage. A majority of known Klebsiella phages have only one or two RBPs, but phages with up to 11 RBPs with depolymerase activity and a broad host spectrum have been identified. A detailed bioinformatic analysis shows that similar RBP domains repeatedly occur in K. pneumoniae phages with structural RBP domains for attachment of an RBP to the phage tail (anchor domain) or for branching of RBPs (T4gp10-like domain). Structural domains determining the RBP architecture are located at the N-terminus, while the depolymerase is located in the center of protein. Occasionally, the RBP is complemented with an autocleavable chaperone domain at the distal end serving for folding and multimerization. The enzymatic domain is subjected to an intense horizontal transfer to rapidly shift the phage host spectrum without affecting the RBP architecture. These analyses allowed to model a set of conserved RBP architectures, indicating evolutionary linkages.
Project description:To overcome antibiotic resistance in biofilms, enzymes aimed at biofilm dispersal are under investigation. In the present study, applicability of an Aeromonas punctata derived depolymerase capable of degrading the capsular polysaccharide (CPS) of Klebsiella pneumoniae, in disrupting its biofilm and increasing gentamicin efficacy against biofilm was investigated.Intact biofilm of K. pneumoniae was recalcitrant to gentamicin due to lack of antibiotic penetration. On the other hand, gentamicin could not act on disrupted biofilm cells due to their presence in clusters. However, when depolymerase (20 units/ml) was used in combination with gentamicin (10 ?g/ml), dispersal of CPS matrix by enzyme facilitated gentamicin penetration across biofilm. This resulted in significant reduction (p?<?0.05) in bacterial count in intact and disrupted biofilms. Reduction in CPS after treatment with depolymerase was confirmed by confocal microscopy and enzyme linked lectinosorbent assay. Furthermore, to substantiate our study, the efficacy of bacterial depolymerase was compared with a phage borne depolymerase possessing similar application against K. pneumoniae. Although both were used at same concentration i.e. 20 units/ml, but a higher efficacy of bacterial depolymerase particularly against older biofilms was visibly clear over its phage counterpart. This could be explained due to high substrate affinity (indicated by Km value) and high turnover number (indicated by Kcat value) of the bacterial depolymerase (Km?=?89.88 ?M, Kcat?=?285 s(-1)) over the phage derived one (Km?=?150 ?M, Kcat?=?107 s(-1)).Overall the study indicated that, the A. punctata derived depolymerase possesses antibiofilm potential and improves gentamicin efficacy against K. pneumoniae. Moreover, it can serve as a potential substitute to phage borne depolymerases for treating biofilms formed by K. pneumoniae.
Project description:Acinetobacter baumannii is emerging as a major nosocomial pathogen in intensive care units. The bacterial capsules are considered major virulence factors, and the particular A. baumannii capsular type K2 has been associated with high antibiotic resistance. In this study, we identified a K2 capsule-specific depolymerase in a bacteriophage tail spike C terminus, a fragment that was heterologously expressed, and its antivirulence properties were assessed by in vivo experiments. The K2 depolymerase is active under a broad range of environmental conditions and is highly thermostable, with a melting point (Tm ) at 67°C. In the caterpillar larva model, the K2 depolymerase protects larvae from bacterial infections, using either pretreatments or with single-enzyme injection after bacterial challenge, in a dose-dependent manner. In a mouse sepsis model, a single K2 depolymerase intraperitoneal injection of 50??g is able to protect 60% of mice from an otherwise deadly infection, with a significant reduction in the proinflammatory cytokine profile. We showed that the enzyme makes bacterial cells fully susceptible to the host complement system killing effect. Moreover, the K2 depolymerase is highly refractory to resistance development, which makes these bacteriophage-derived capsular depolymerases useful antivirulence agents against multidrug-resistant A. baumannii infections.IMPORTANCE Acinetobacter baumannii is an important nosocomial pathogen resistant to many, and sometimes all, antibiotics. The A. baumannii K2 capsular type has been associated with elevated antibiotic resistance. The capsular depolymerase characterized here fits the new trend of alternative antibacterial agents needed against multidrug-resistant pathogens. They are highly specific, stable, and refractory to resistance, as they do not kill bacteria per se; instead, they remove bacterial surface polysaccharides, which diminish the bacterial virulence and expose them to the host immune system.
Project description:Forty strains of Salmonella enterica (S. enterica) subspecies salamae (II), arizonae (IIIa), diarizonae (IIIb), and houtenae (IV) were isolated from human or environmental samples and tested for bacteriophage production. Production of bacteriophages was observed in 15 S. enterica strains (37.5%) belonging to either the subspecies salamae (8 strains) or diarizonae (7 strains). Activity of phages was tested against 52 pathogenic S. enterica subsp. enterica isolates and showed that phages produced by subsp. salamae had broader activity against pathogenic salmonellae compared to phages from the subsp. diarizonae. All 15 phages were analyzed using PCR amplification of phage-specific regions and 9 different amplification profiles were identified. Five phages (SEN1, SEN4, SEN5, SEN22, and SEN34) were completely sequenced and classified as temperate phages. Phages SEN4 and SEN5 were genetically identical, thus representing a single phage type (i.e. SEN4/5). SEN1 and SEN4/5 fit into the group of P2-like phages, while the SEN22 phage showed sequence relatedness to P22-like phages. Interestingly, while phage SEN34 was genetically distantly related to Lambda-like phages (Siphoviridae), it had the morphology of the Myoviridae family. Based on sequence analysis and electron microscopy, phages SEN1 and SEN4/5 were members of the Myoviridae family and phage SEN22 belonged to the Podoviridae family.
Project description:The emergence of multi-drug-resistant bacteria represents a major public-health threat. Phages constitute a promising alternative to chemical antibiotics due to their high host specificity, abundance in nature, and evolvability. However, phage host specificity means that highly diverse bacterial species are particularly difficult to target for phage therapy. This is the case of Klebsiella pneumoniae, which presents a hypervariable extracellular matrix capsule exhibiting dozens of variants. Here, we report four novel phages infecting K. pneumoniae capsular type K22 which were isolated from environmental samples in Valencia, Spain. Full genome sequencing showed that these phages belong to the Podoviridae family and encode putative depolymerases that allow digestion of specific K22 K. pneumoniae capsules. Our results confirm the capsular type-specificity of K. pneumoniae phages, as indicated by their narrow infectivity in a panel of K. pneumoniae clinical isolates. Nonetheless, this work represents a step forward in the characterization of phage diversity, which may culminate in the future use of large panels of phages for typing and/or for combating multi-drug-resistant K. pneumoniae.
Project description:Carbapenem-resistant <i>Klebsiella pneumoniae</i> (CRKP), one of the major nosocomial pathogens, is increasingly becoming a serious threat to global public health. There is an urgent need to develop effective therapeutic and preventive approaches to combat the pathogen. Here, we identified and characterized a novel capsule depolymerase (K64-ORF41) derived from <i>Klebsiella</i> phage SH-KP152410, which showed specific activities for <i>K. pneumoniae</i> K64-serotype. We showed that this depolymerase could be used in the identification of K64 serotypes based on the capsular typing, and the results agreed well with those from the conventional serotyping method using antisera. From this study, we also identified K64 mutant strains, which showed typing discrepancy between <i>wzi</i>-sequencing based genotyping and depolymerase-based or antiserum-based typing methods. Further investigation indicated that the mutant strain has an insertion sequence (IS) in <i>wcaJ</i>, which led to the alteration of the capsular serotype structure. We further demonstrated that K64-ORF41 depolymerase could sensitize the bacteria to serum or neutrophil killing by degrading the capsular polysaccharide. In summary, the identified K64 depolymerase proves to be an accurate and reliable tool for capsular typing, which will facilitate the preventive intervention such as vaccine development. In addition, the polymerase may represent a potential and promising therapeutic biologics against CRKP-K64 infections.
Project description:The increased prevalence of drug-resistant, nosocomial Acinetobacter infections, particularly from pathogenic members of the Acinetobacter calcoaceticus-baumannii complex, necessitates the exploration of novel treatments such as phage therapy. In the present study, we characterized phage Petty, a novel podophage that infects multidrug-resistant Acinetobacter nosocomialis and Acinetobacter baumannii Genome analysis reveals that phage Petty is a 40,431-bp ?KMV-like phage, with a coding density of 92.2% and a G+C content of 42.3%. Interestingly, the lysis cassette encodes a class I holin and a single-subunit endolysin, but it lacks canonical spanins to disrupt the outer membrane. Analysis of other ?KMV-like genomes revealed that spaninless lysis cassettes are a feature of phages infecting Acinetobacter within this subfamily of bacteriophages. The observed halo surrounding Petty's large clear plaques indicated the presence of a phage-encoded depolymerase capable of degrading capsular exopolysaccharides (EPS). The product of gene 39, a putative tail fiber, was hypothesized to possess depolymerase activity based on weak homology to previously reported phage tail fibers. The 101.4-kDa protein gene product 39 (gp39) was cloned and expressed, and its activity against Acinetobacter EPS in solution was determined. The enzyme degraded purified EPS from its host strain A. nosocomialis AU0783, reducing its viscosity, and generated reducing ends in solution, indicative of hydrolase activity. Given that the accessibility to cells within a biofilm is enhanced by degradation of EPS, phages with depolymerases may have enhanced diagnostic and therapeutic potential against drug-resistant Acinetobacter strains.IMPORTANCE Bacteriophage therapy is being revisited as a treatment for difficult-to-treat infections. This is especially true for Acinetobacter infections, which are notorious for being resistant to antimicrobials. Thus, sufficient data need to be generated with regard to phages with therapeutic potential, if they are to be successfully employed clinically. In this report, we describe the isolation and characterization of phage Petty, a novel lytic podophage, and its depolymerase. To our knowledge, it is the first phage reported to be able to infect both A. baumannii and A. nosocomialis The lytic phage has potential as an alternative therapeutic agent, and the depolymerase could be used for modulating EPS both during infections and in biofilms on medical equipment, as well as for capsular typing. We also highlight the lack of predicted canonical spanins in the phage genome and confirm that, unlike the rounding of lambda lysogens lacking functional spanin genes, A. nosocomialis cells infected with phage Petty lyse by bursting. This suggests that phages like Petty employ a different mechanism to disrupt the outer membrane of Acinetobacter hosts during lysis.