Complete Genome Sequence of Cronobacter sakazakii Bacteriophage vB_CsaM_GAP161

Cronobacter sakazakii is an opportunistic pathogen that causes infant meningitis and is often associated with milk-based infant formula. We have fully sequenced the genome of a newly isolated lytic C. sakazakii myovirus, vB_CsaM_GAP161, briefly named GAP161. It consists of 178,193 bp and has a G+C content of 44.5%. A total of 277 genes, including 275 open reading frames and two tRNA-encoding genes, were identified. This phage is closely related to coliphages RB16 and RB43 and Klebsiella pneumoniae phage KP15.     

Isolation and characterisation of KP34--a novel φKMV-like bacteriophage for Klebsiella pneumoniae

Bacteriophage KP34 is a novel virus belonging to the subfamily Autographivirinae lytic for extended-spectrum ??-lactamase-producing Klebsiella pneumoniae strains. Its biological features, morphology, susceptibility to chemical and physical agents, burst size, host specificity and activity spectrum were determined. As a potential antibacterial agent used in therapy, KP34 molecular features including genome sequence and protein composition were examined. Phylogenetic analyses and clustering of KP34 phage genome sequences revealed its clear relationships with ??phiKMV-like viruses??. Simultaneously, whole-genome analyses permitted clustering and classification of all phages, with completely sequenced genomes, belonging to the Podoviridae.     

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.     

Characterization of five novel endolysins from Gram-negative infecting bacteriophages

We here characterize five globular endolysins, encoded by a set of Gram-negative infecting bacteriophages: BcepC6gp22 (Burkholderia cepacia phage BcepC6B), P2gp09 (Escherichia coli phage P2), PsP3gp10 (Salmonella enterica phage PsP3), K11gp3.5 and KP32gp15 (Klebsiella pneumoniae phages K11 and KP32, respectively). In silico, BcepC6gp22, P2gp10 and PsP3gp10 are predicted to possess lytic transglycosylase activity, whereas K11gp3.5 and KP32gp15 have putative amidase activity. All five endolysins show muralytic activity on the peptidoglycan of several Gram-negative bacterial species. In vitro, Pseudomonas aeruginosa PAO1 is clearly sensitive for the antibacterial action of the five endolysins in the presence of the outer membrane permeabilizer EDTA: reductions are ranging from 1.89 to 3.08 log units dependent on the endolysin. The predicted transglycosylases BcepC6gp22, P2gp10 and PsP3gp10 have a substantially higher muralytic and in vitro antibacterial activity compared to the predicted amidases K11gp3.5 and KP32gp15, highlighting the impact of the catalytic specificity on endolysin activity. Furthermore, initial data exclude the synergistic lethal effect of a combination of the predicted transglycosylase PsP3gp10 and the predicted amidase K11gp3.5 on PAO1. As these globular endolysins show a lower enzymatic and antibacterial activity, in comparison to modular endolysins, we suggest that the latter should be favored for antibacterial applications.     

Complete Genome Sequence of Wide-Host-Range Staphylococcus aureus Phage JD007

Methicillin-resistant Staphylococcus aureus-related infections have become a serious problem worldwide. Bacteriophage therapy is an alternative approach against this threat. S. aureus phage JD007, which belongs to the Myoviridae family according to transmission electron microscopic imaging, could lyse nearly 30% of the S. aureus strains from Ruijin Hospital, Shanghai, China, and was isolated from chicken feces in Shanghai, China. The complete genome showed that JD007 is a linear, double-stranded DNA phage 141,836 bp in length with a GC content of 30.4% encoding 217 open reading frames. A BLAST search of the JD007 genome revealed that it was very similar to that of phage GH15.     

PhREEPred: Phage Resistance Emergence Prediction Web Tool to Foresee Encapsulated Bacterial Escape from Phage Cocktail Treatment

Phages, as well as phage-derived proteins, especially lysins and depolymerases, are intensively studied to become prospective alternatives or supportive antibacterials used alone or in combination. In the common phage therapy approach, the unwanted emergence of phage-resistant variants from the treated bacterial population can be postponed or reduced by the utilization of an effective phage cocktail. In this work, we present a publicly available web tool PhREEPred (Phage Resistance Emergence Prediction) (https://phartner.shinyapps.io/PhREEPred/), which will allow an informed choice of the composition of phage cocktails by predicting the outcome of phage cocktail or phage/depolymerase combination treatments against encapsulated bacterial pathogens given a mutating population that escapes single phage treatment. PhREEPred simulates solutions of our mathematical model calibrated and tested on the experimental Klebsiella pneumoniae setup and Klebsiella-specific lytic phages: K63 type-specific phage KP34 equipped with a capsule-degrading enzyme (KP34p57), capsule-independent myoviruses KP15 and KP27, and recombinant capsule depolymerase KP34p57. The model can calculate the phage-resistance emergence depending on the bacterial growth rate and initial density, the multiplicity of infection, phage latent period, its infectiveness and the cocktail composition, as well as initial depolymerase concentration and activity rate. This model reproduced the experimental results and showed that (i) the phage cocktail of parallelly infecting phages is less effective than the one composed of sequentially infecting phages; (ii) depolymerase can delay or prevent bacterial resistance by unveiling an alternative receptor for initially inactive phages. In our opinion, this customer-friendly web tool will allow for the primary design of the phage cocktail and phage-depolymerase combination effectiveness against encapsulated pathogens.     

Targeting Enterococcus faecalis Biofilms with Phage Therapy

Phage therapy has been proven to be more effective, in some cases, than conventional antibiotics, especially regarding multidrug-resistant biofilm infections. The objective here was to isolate an anti-Enterococcus faecalis bacteriophage and to evaluate its efficacy against planktonic and biofilm cultures. E. faecalis is an important pathogen found in many infections, including endocarditis and persistent infections associated with root canal treatment failure. The difficulty in E. faecalis treatment has been attributed to the lack of anti-infective strategies to eradicate its biofilm and to the frequent emergence of multidrug-resistant strains. To this end, an anti-E. faecalis and E. faecium phage, termed EFDG1, was isolated from sewage effluents. The phage was visualized by electron microscopy. EFDG1 coding sequences and phylogeny were determined by whole genome sequencing (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"KP339049","term_id":"761545084","term_text":"KP339049"}}KP339049), revealing it belongs to the Spounavirinae subfamily of the Myoviridae phages, which includes promising candidates for therapy against Gram-positive pathogens. This analysis also showed that the EFDG1 genome does not contain apparent harmful genes. EFDG1 antibacterial efficacy was evaluated in vitro against planktonic and biofilm cultures, showing effective lytic activity against various E. faecalis and E. faecium isolates, regardless of their antibiotic resistance profile. In addition, EFDG1 efficiently prevented ex vivo E. faecalis root canal infection. These findings suggest that phage therapy using EFDG1 might be efficacious to prevent E. faecalis infection after root canal treatment.     

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.     

耐碳青霉烯类肺炎克雷伯菌对C57BL/6小鼠肺部菌群的扰动

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

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.     

Genomic characterization of four novel bacteriophages infecting the clinical pathogen Klebsiella pneumoniae

Bacteriophages are an invaluable source of novel genetic diversity. Sequencing of phage genomes can reveal new proteins with potential uses as biotechnological and medical tools, and help unravel the diversity of biological mechanisms employed by phages to take over the host during viral infection. Aiming to expand the available collection of phage genomes, we have isolated, sequenced, and assembled the genome sequences of four phages that infect the clinical pathogen Klebsiella pneumoniae: vB_KpnP_FBKp16, vB_KpnP_FBKp27, vB_KpnM_FBKp34, and Jumbo phage vB_KpnM_FBKp24. The four phages show very low (0–13%) identity to genomic phage sequences deposited in the GenBank database. Three of the four phages encode tRNAs and have a GC content very dissimilar to that of the host. Importantly, the genome sequences of the phages reveal potentially novel DNA packaging mechanisms as well as distinct clades of tubulin spindle and nucleus shell proteins that some phages use to compartmentalize viral replication. Overall, this study contributes to uncovering previously unknown virus diversity, and provides novel candidates for phage therapy applications against antibiotic-resistant K. pneumoniae infections.     

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.     

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.     

Whole genome sequence of Klebsiella pneumoniae U25, a hypermucoviscous,multidrug resistant,biofilm producing isolate from India

Klebsiella pneumoniae U25 is a multidrug resistant strain isolated from a tertiary care hospital in Chennai, India. Here, we report the complete annotated genome sequence of strain U25 obtained using PacBio RSII. This is the first report of the whole genome of K. pneumoniaespecies from Chennai. It consists of a single circular chromosome of size 5,491,870-bp and two plasmids of size 211,813 and 172,619-bp. The genes associated with multidrug resistance were identified. The chromosome of U25 was found to have eight antibiotic resistant genes [blaOXA-1,blaSHV-28, aac(6’)1b-cr,catB3, oqxAB, dfrA1]. The plasmid pMGRU25-001 was found to have only one resistant gene (catA1) while plasmid pMGRU25-002 had 20 resistant genes [strAB, aadA1,aac(6’)-Ib, aac(3)-IId,sul1,2, blaTEM-1A,1B,blaOXA-9, blaCTX-M-15,blaSHV-11, cmlA1, erm(B),mph(A)]. A mutation in the porin OmpK36 was identified which is likely to be associated with the intermediate resistance to carbapenems in the absence of carbapenemase genes. U25 is one of the few K. pneumoniaestrains to harbour clustered regularly interspaced short palindromic repeats (CRISPR) systems. Two CRISPR arrays corresponding to Cas3 family helicase were identified in the genome. When compared to K. pneumoniaeNTUHK2044, a transposase gene InsH of IS5-13 was found inserted.     

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.     

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.

     

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

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

The novel Shewanella putrefaciens-infecting bacteriophage Spp001: genome sequence and lytic enzymes

Shewanella putrefaciens has been identified as a specific spoilage organism commonly found in chilled fresh fish, which contributes to the spoilage of fish products. Limiting S. putrefaciens growth can extend the shelf-life of chilled fish. Endolysins, which are lytic enzymes produced by bacteriophages, have been considered an alternative to control bacterial growth, and have been useful in various applications, including food preservation. We report here, for the first time, the complete genome sequence of a novel phage Spp001, which lyses S. putrefaciens Sp225. The Spp001 genome comprises a 54,789-bp DNA molecule with 67 open reading frames and an average total G + C content of 49.42 %. In silico analysis revealed that the Spp001 open reading frames encode various putative functional proteins, including an endolysin (ORF 62); however, no sequence for genes encoding the holin polypeptides, which work in concert with endolysins, was identified. To examine further the lytic activity of Spp001, we analyzed the lytic enzyme-containing fraction from phages released at the end of the phage lytic cycle in S. putrefaciens, using diffusion and turbidimetric assays. The results show that the partially purified extract contained endolysin, as indicated by a high hydrolytic activity towards bacterial peptidoglycan decrease in the OD₅₉₀ value by 0.160 in 15 min. The results will allow further investigation of the purification of natural Spp001 endolysin, the extension of Spp001 host range, and the applications of the phage-encoded enzymes.     

Pneumococcal Bacteriophage Cp-1 Encodes Its Own Protease Essential for Phage Maturation

The major capsid protein of the pneumococcal phage Cp-1 that accounts for 90% of the total protein found in the purified virions is synthesized by posttranslational processing of the product of the open reading frame (ORF) orf9. Cloning of different ORFs of the Cp-1 genome in Escherichia coli and Streptococcus pneumoniae combined with Western blot analysis of the expressed products led to the conclusion that the product of orf13 is an endoprotease that cleaves off the first 48 amino acid residues of the major head protein. This protease appears to be a key enzyme in the morphopoietic pathway of the Cp-1 phage head. To our knowledge, this is the first case of a bacteriophage infecting gram-positive bacteria that encodes a protease involved in phage maturation.