Bacteriophages of Klebsiella spp., their diversity and potential therapeutic uses.

Klebsiella spp. are commensals of the human microbiota, and a leading cause of opportunistic nosocomial infections. The incidence of multidrug resistant (MDR) strains of Klebsiella pneumoniae causing serious infections is increasing, and Klebsiella oxytoca is an emerging pathogen. Alternative strategies to tackle infections caused by these bacteria are required as strains become resistant to last-resort antibiotics such as colistin. Bacteriophages (phages) are viruses that can infect and kill bacteria. They and their gene products are now being considered as alternatives or adjuncts to antimicrobial therapies. Several in vitro and in vivo studies have shown the potential for lytic phages to combat MDR K. pneumoniae infections. Ready access to cheap sequencing technologies has led to a large increase in the number of genomes available for Klebsiella-infecting phages, with these phages being heterogeneous at the whole-genome level. This review summarizes our current knowledge on phages of Klebsiella spp. and highlights technological and biological issues relevant to the development of phage-based therapies targeting these bacteria.

Preterm infants harbour diverse Klebsiella populations, including atypical species that encode and produce an array of antimicrobial resistance- and virulence-associated factors.

Klebsiella spp. are frequently enriched in the gut microbiota of preterm neonates, and overgrowth is associated with necrotizing enterocolitis (NEC), nosocomial infections and late-onset sepsis. Little is known about the genomic and phenotypic characteristics of preterm-associated Klebsiella, as previous studies have focused on the recovery of antimicrobial-resistant isolates or culture-independent molecular analyses. The aim of this study was to better characterize preterm-associated Klebsiella populations using phenotypic and genotypic approaches. Faecal samples from a UK cohort of healthy and sick preterm neonates (n=109) were screened on MacConkey agar to isolate lactose-positive Enterobacteriaceae. Whole-genome sequences were generated for Klebsiella spp., and virulence and antimicrobial resistance genes identified. Antibiotic susceptibility profiling and in vitro macrophage and iron assays were undertaken for the Klebsiella strains. Metapangenome analyses with a manually curated genome dataset were undertaken to examine the diversity of Klebsiella oxytoca and related bacteria in a publicly available shotgun metagenome dataset. Approximately one-tenth of faecal samples harboured Klebsiella spp. (Klebsiella pneumoniae, 7.3 %; Klebsiella quasipneumoniae, 0.9 %; Klebsiella grimontii, 2.8 %; Klebsiella michiganensis, 1.8 %). Isolates recovered from NEC- and sepsis-affected infants and those showing no signs of clinical infection (i.e. 'healthy') encoded multiple ß-lactamases. No difference was observed between isolates recovered from healthy and sick infants with respect to in vitro siderophore production (all encoded enterobactin in their genomes). All K. pneumoniae, K. quasipneumoniae, K. grimontii and K. michiganensis faecal isolates tested were able to reside and persist in macrophages, indicating their immune evasion abilities. Metapangenome analyses of published metagenomic data confirmed our findings regarding the presence of K. michiganensis in the preterm gut. There is little difference in the phenotypic and genomic characteristics of Klebsiella isolates recovered from healthy and sick infants. Identification of ß-lactamases in all isolates may prove problematic when defining treatment regimens for NEC or sepsis, and suggests that healthy preterm infants contribute to the resistome. Refined analyses with curated sequence databases are required when studying closely related species present in metagenomic data.

Rapid MinION profiling of preterm microbiota and antimicrobial-resistant pathogens.

The MinION sequencing platform offers near real-time analysis of DNA sequence; this makes the tool attractive for deployment in fieldwork or clinical settings. We used the MinION platform coupled to the NanoOK RT software package to perform shotgun metagenomic sequencing and profile mock communities and faecal samples from healthy and ill preterm infants. Using Nanopore data, we reliably classified a 20-species mock community and captured the diversity of the immature gut microbiota over time and in response to interventions such as probiotic supplementation, antibiotic treatment or episodes of suspected sepsis. We also performed rapid real-time runs to assess gut-associated microbial communities in critically ill and healthy infants, facilitated by NanoOK RT software package, which analysed sequences as they were generated. Our pipeline reliably identified pathogenic bacteria (that is, Klebsiella pneumoniae and Enterobacter cloacae) and their corresponding antimicrobial resistance gene profiles within as little as 1 h of sequencing. Results were confirmed using pathogen isolation, whole-genome sequencing and antibiotic susceptibility testing, as well as mock communities and clinical samples with known antimicrobial resistance genes. Our results demonstrate that MinION (including cost-effective Flongle flow cells) with NanoOK RT can process metagenomic samples to a rich dataset in < 5 h, which creates a platform for future studies aimed at developing these tools and approaches in clinical settings with a focus on providing tailored patient antimicrobial treatment options.

Draft Genome Sequence of Raoultella ornithinolytica P079F W, Isolated from the Feces of a Preterm Infant.

Here, we describe the draft genome sequence of Raoultella ornithinolytica P079F W, isolated from the feces of an infant residing in a neonatal intensive care unit during an ongoing study to characterize the neonate gut microbiota. P079F W will be used in studies investigating the role of the microbiome in neonatal infections.

Draft Genome Sequences of Citrobacter freundii and Citrobacter murliniae Strains Isolated from the Feces of Preterm Infants.

Here, we describe the draft genome sequences of three strains of Citrobacter isolated from feces of preterm neonates with suspected sepsis. Strains P106E PI and P079F I were Citrobacter freundii Strain P080C CL represents the first draft genome sequence of Citrobacter murliniae.