Klebsiella pneumoniae subsp. pneumoniae–bacteriophage combination from the caecal effluent of a healthy woman

A recent study characterizing bacteriophage populations within human caecal effluent demonstrated the presence of numerous Podoviridae, Siphoviridae and Myoviridae within this material (Hoyles et al., 2014, Res Microbiol 165, 803–812). Further to this work, anaerobic bacteria were isolated on fastidious anaerobe agar from the caecal effluent of a healthy 31-year-old woman. Ten colonies were selected at random, streaked to purity and screened against the remaining caecal effluent (filter-sterilized, 0.45 μm pore size) in an attempt to isolate lytic bacteriophages. Bacteriophages within the effluent [2×105 ± 2.65×103 (n=3) pfu/ml] were active against five of the isolates, all identified by 16S rRNA gene sequence analysis as Klebsiella pneumoniae. One of the five isolates, L4-FAA5, was characterized further and found to be K. pneumoniae subsp. pneumoniae capsule type K2 rmpA+, and was used to propagate a bacteriophage (which we named KLPN1) to purity. Bacteriophage KLPN1 was a member of the Siphoviridae with a rosette-like tail tip and exhibited depolymerase activity, demonstrated by the formation of plaque-surrounding haloes that increased in size over the course of incubation. When screened against a panel of 21 clinical strains representing unknown K. pneumoniae subsp. pneumoniae capsule types and types K1, K2, K5, K20, K54 and K57, KLPN1 infected only K2 strains, but did not exhibit depolymerase activity against these. Whole-genome sequence analysis of KLPN1 showed the bacteriophage to have a genome of 49,037 bp (50.53 GC mol%) comprising 73 predicted ORFs, of which 22 encoded genes associated with structure, host recognition, packaging, DNA replication and cell lysis. The host recognition-associated gene was a potential depolymerase. This is the first report of the isolation of a bacterium–bacteriophage combination from the human caecum, and only the third member of the Siphoviridae known to infect K. pneumoniae subsp. pneumoniae.

Laccase bio-grafting a versatile modification tool from green chemistry technologies

Today more than 99% of plastics are petroleum-based because of availability and cost of the raw material. The durability of these disposed plastics contributes to the environmental problems as waste and their persistence in the environment causes deleterious effects on the ecosystem. Environmental pollution awareness and the demand for green technology have drawn considerable attention of both academia and industry into biodegradable polymers. In this regard green chemistry technology has the potential to provide solution to this problematic issue. Laccase bio-grafting has recently been the focus of green chemistry technologies due to the growing environmental concerns, legal restrictions and increasing availability of scientific knowledge. In the last several years, research covering various applications of laccases has been increased rapidly particularly in the field of grafting. In principle, laccase-assisted graft co-polymerization may impart a variety of new functionalities to a polymer. The modified polymers through grafting have a bright future and their development is practically boundless. In present work, novel biodegradable graft copolymers combining the advantages of bacterial cellulose backbone and PHB side chains will be prepared by introducing enzymatic grafting technique. The present research will be a first step in the biopolymer modification. To date no report has been found in literature explaining the enzymatic grafting of PHAs. The technique would also provide an efficient modulation approach to improve the biodegradability and biocompatibility of the graft copolymer. The newly grafted copolymers will exhibit unique functionalities with wider range of potential applications mainly in tissue engineering, biosensors, pharmaceutical industry (drug delivery systems) and bio-plastics.

Klebsiella pneumoniae subsp. pneumoniae–bacteriophage combination from the caecal effluent of a healthy woman

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”.