Characterising the biology of novel lytic bacteriophages infecting multidrug resistant Klebsiella pneumoniae

Virology Journal Characterising the biology of novel lytic bacteriophages infecting multidrug resistant Klebsiella pneumoniae Agata Ksik-Szeloch 0 Zuzanna Drulis-Kawa 0 Beata Weber-Dbrowska Jerzy Kassner 0 Grayna Majkowska-Skrobek 0 Daria Augustyniak 0 Marzanna usiak-Szelachowska Maciej aczek Andrzej Grski Andrew M Kropinski 0 Institute of Genetics and Microbiology, University of Wroclaw , Przybyszewskiego 63/77, Wroclaw 51-148 Poland Background: Members of the genus Klebsiella are among the leading microbial pathogens associated with nosocomial infection. The increased incidence of antimicrobial resistance in these species has propelled the need for alternate/combination therapeutic regimens to aid clinical treatment. Bacteriophage therapy forms one of these alternate strategies. Methods: Electron microscopy, burst size, host range, sensitivity of phage particles to temperature, chloroform, pH, and restriction digestion of phage DNA were used to characterize Klebsiella phages. Results and conclusions: Of the 32 isolated phages eight belonged to the family Myoviridae, eight to the Siphoviridae whilst the remaining 16 belonged to the Podoviridae. The host range of these phages was characterised against 254 clinical Enterobacteriaceae strains including multidrug resistant Klebsiella isolates producing extended-spectrum beta-lactamases (ESBLs). Based on their lytic potential, six of the phages were further characterised for burst size, physicochemical properties and sensitivity to restriction endonuclease digestion. In addition, five were fully sequenced. Multiple phage-encoded host resistance mechanisms were identified. The Siphoviridae phage genomes (KP16 and KP36) contained low numbers of host restriction sites similar to the strategy found in T7-like phages (KP32). In addition, phage KP36 encoded its own DNA adenine methyltransferase. The KMV-like KP34 phage was sensitive to all endonucleases used in this study. Dam methylation of KP34 DNA was detected although this was in the absence of an identifiable phage encoded methyltransferase. The Myoviridae phages KP15 and KP27 both carried Dam and Dcm methyltransferase genes and other anti-restriction mechanisms elucidated in previous studies. No other anti-restriction mechanisms were found, e.g. atypical nucleotides (hmC or glucosyl hmC), although Myoviridae phage KP27 encodes an unknown anti-restriction mechanism that needs further investigation. Bacteriophage; Klebsiella spp; Multidrug resistance; Restriction endonuclease patterns; Myoviridae; Siphoviridae; Podoviridae - Background Bacteriophages, or phages, are viruses that infect bacteria. They are the most abundant and the most genetically diverse biological entities on Earth, with global numbers estimated at 1030 to 1032 [1,2]. These viruses are ubiquitous throughout the environment and are found in all environments that support bacterial proliferation [3,4]. It is now realized that phages play an important role in the cycling of organic matter in the biosphere and play a significant role in bacterial diversity [5,6]. Successful infection by lytic bacteriophages requires attachment to a susceptible host cell through specific binding to a surface epitope, injection of the phage nucleic acid, replication, virion assembly and, finally, release of infectious progeny [7]. In the case of Gram-negative bacteria phage adsorption to specific receptors such as pili, flagella, capsules, outer membrane proteins and lipopolysaccharides has been demonstrated [8]. The specificity of interaction between phage tail structures and host receptor defines the host range of these viruses [9]. Some phages have specificity at the strain level whereas some have broader host ranges and can infect multiple bacterial strains within a single species or even multiple related species [10]. To achieve successful infection and propagation, bacteriophages need to overcome the host resistance mechanisms that target foreign DNA on entry into the bacterium. Four types of resistance mechanisms have been categorised through their mode of action and how they target the phage life cycle. These include: adsorption inhibition, blocking of DNA injection, restriction-modification (RM) and abortive infection [11]. Arguably, the most well-studied anti-phage defence mechanism is the restriction-modification system, which is present in over 90% of sequenced bacterial genomes [12]. There are several antirestriction mechanisms developed by bacteriophages [13,14]: (i) counter selection against relevant restriction sites in phage genome exemplified by coliphage T7; (ii) inhibitors of host restriction enzyme Ocr proteins of phages T7 and T3 block type I and type III of RM systems; (iii) hydrolysis of RM system cofactors e.g. hydrolysis of S-adenosylmethionine by T3 resulting in blockage of type I and type III RM systems; (iv) co-injection of DNA and RM inhibitors the DarA and DarB proteins of P1 block the type I RM system; (v) stimulation of host methyltransferase activity (type I system) Ral and Lar proteins of -like phages; (vi) DNA modifying enzymes acquisition of Dam and Dcm methyltransferase genes by T4-like phages; (vii) incorporation of hypermodified nucleotides into DNA hydroxymethylcytosine (hmC) or glucosylated hydroxymethylcytosine (glucosyl hmC) in T4-like phages [15]. Bacteriophages have been of interest to scientists as tools to understand fundamental molecular biology processes, as vectors for horizontal gene transfer and drivers of bacterial evolution. Moreover, bacterial viruses are convenient sources of diagnostic and genetic tools and have the potential to be used as novel therapeutic agents [4,16]. With the increased incidence of multidrug resistance in bacteria, therapeutic and preventive options have become limited. One of the possible alternatives to antibiotics is the application of bacteriophages or phage proteins. The idea/implementation of using phages as a therapeutic intervention is well known. As antimicrobial drugs entering the pharmaceutical market is limited, there is a need for novel therapeutics where phages with lytic potential and generally regarded as safe (GRAS) status by the FDA fall into this category. Multidrug resistant Klebsiella pneumoniae isolates carrying extendedspectrum beta-lactamases (ESBLs) encoding plasmids are becoming increasingly associated with nosocomial infection. At present the prevalence of ESBL-producing Klebsiella strains in Europe has reached 10-30% of invasive isolates [17]. Antibiotic usage in clinical settings, and also in animal husbandry, has led to the maintenance of ESBLencoding bacteria in the environment [18]. It is now well documented that ESBL-producing bacteria may also have a zoonotic origin with strains isolated from poultry, a pig farm and retail meat [19-21]. The high incidence of multidrug resistant bacteria has resulted in limited efficacy of treatment with current antibiotics, and a high probability of patient colonizatio (...truncated)