Comparative genomics of plant growth promoting phosphobacteria isolated from acidic soils.

Despite being one of the most abundant elements in soil, phosphorus (P) often becomes a limiting macronutrient for plants due to its low bioavailability, primarily locked away in insoluble organic and inorganic forms. Phosphate solubilizing and mineralizing bacteria, also called phosphobacteria, isolated from P-deficient soils have emerged as a promising biofertilizer alternative, capable of converting these recalcitrant P forms into plant-available phosphates. Three such phosphobacteria strains-Serratia sp. RJAL6, Klebsiella sp. RCJ4, and Enterobacter sp. 198-previously demonstrated their particular strength as plant growth promoters for wheat, ryegrass, or avocado under abiotic stresses and P deficiency. Comparative genomic analysis of their draft genomes revealed several genes encoding key functionalities, including alkaline phosphatases, isonitrile secondary metabolites, enterobactin biosynthesis and genes associated to the production of indole-3-acetic acid (IAA) and gluconic acid. Moreover, overall genome relatedness indexes (OGRIs) revealed substantial divergence between Serratia sp. RJAL6 and its closest phylogenetic neighbours, Serratia nematodiphila and Serratia bockelmanii. This compelling evidence suggests that RJAL6 merits classification as a novel species. This in silico genomic analysis provides vital insights into the plant growth-promoting capabilities and provenance of these promising PSRB strains. Notably, it paves the way for further characterization and potential application of the newly identified Serratia species as a powerful bioinoculant in future agricultural settings.

Isolation and Electrochemical Analysis of a Facultative Anaerobic Electrogenic Strain Klebsiella sp. SQ-1.

Electricigens decompose organic matter and convert stored chemical energy into electrical energy through extracellular electron transfer. They are significant biocatalysts for microbial fuel cells with practical applications in green energy generation, effluent treatment, and bioremediation. A facultative anaerobic electrogenic strain SQ-1 is isolated from sludge in a biotechnology factory. The strain SQ-1 is a close relative of Klebsiella variicola. Multilayered biofilms form on the surface of a carbon electrode after the isolated bacteria are inoculated into a microbial fuel cell device. This strain produces high current densities of 625 µA cm-2 by using acetate as the carbon source in a three-electrode configuration. The electricity generation performance is also analyzed in a dual-chamber microbial fuel cell. It reaches a maximum power density of 560 mW m-2 when the corresponding output voltage is 0.59 V. The facultative strain SQ-1 utilizes hydrous ferric oxide as an electron acceptor to perform extracellular electricigenic respiration in anaerobic conditions. Since facultative strains possess better properties than anaerobic strains, Klebsiella sp. SQ-1 may be a promising exoelectrogenic strain for applications in microbial electrochemistry.

Community Structure of Nitrifying and Denitrifying Bacteria from Effluents Discharged into Lake Victoria, Kenya.

An active microbial community of nitrifying and denitrifying bacteria is needed for efficient utilization of nitrogenous compounds from wastewater. In this study, we explored the bacterial community diversity and structure within rivers, treated and untreated wastewater treatment plants (WWTPs) discharging into Lake Victoria. Water samples were collected from rivers and WWTPs that drain into Lake Victoria. Physicochemical analysis was done to determine the level of nutrients or pollutant loading in the samples. Total community DNA was extracted, followed by Illumina high throughput sequencing to determine the total microbial community and abundance. Enrichment and isolation were then done to recover potential nitrifiers and denitrifiers. Physicochemical analysis pointed to high levels total nitrogen and ammonia in both treated and untreated WWTPs as compared to the samples from the lake and rivers. A total of 1,763 operational taxonomic units (OTUs) spread across 26 bacterial phyla were observed with the most dominant phylum being Proteobacteria. We observed a decreasing trend in diversity from the lake, rivers to WWTPs. The genus Planktothrix constituted 19% of the sequence reads in sample J2 collected from the lagoon. All the isolates recovered in this study were affiliated to three genera: Pseudomonas, Klebsiella and Enterobacter in the phylum Proteobacteria. A combination of metagenomic analysis and a culture-dependent approach helped us understand the relative abundance as well as potential nitrifiers and denitrifiers present in different samples. The recovered isolates could be used for in situ removal of nitrogenous compounds from contaminated wastewater.

Klebsiella species: Taxonomy, hypervirulence and multidrug resistance.

Members of the genus Klebsiella have rapidly evolved within the past decade, generating organisms that simultaneously exhibit both multidrug resistance and hypervirulence (MDR-hv) phenotypes; such organisms are associated with severe hospital- and community-acquired infections. Carbapenem-resistant infections with unknown optimal treatment regime were of particular concern among the MDR-hv Klebsiella strains. Recent studies have revealed the molecular features and the mobile resistance elements they harbour, allowing identification of genetic loci responsible for transmission, stable inheritance, and expression of mobile resistance or virulence-encoding elements that confer the new phenotypic characteristics of MDR-hv Klebsiella spp. Here, we provide a comprehensive review on the taxonomic position, species composition and different phylotypes of Klebsiella spp., describing the diversity and worldwide distribution of the MDR-hv clones, the genetic mutation and horizontal gene transfer events that drive the evolution of such clones, and the potential impact of MDR-hv infections on human health.

Evaluation of Rapid Immunochromatographic Tests for the Direct Detection of Extended Spectrum Beta-Lactamases and Carbapenemases in Enterobacterales Isolated from Positive Blood Cultures.

NG-Test CTX-M MULTI and NG-Test Carba 5 (NG Biotech) are two rapid in vitro immunochromatographic assays that are widely used for the detection of the most common extended spectrum beta-lactamases (ESBL) and carbapenemases in Enterobacterales. ESBL and carbapenemases are leading causes of morbidity and mortality worldwide and their rapid detection from positive blood cultures is crucial for early initiation of effective antimicrobial therapy in bloodstream infections (BSI) involving antibiotic-resistant organisms. In this study, we developed a rapid workflow for positive blood cultures for direct identification of Enterobacterales by MALDI-TOF mass-spectrometry, followed by detection of ESBL and carbapenemases using NG-Test CTX-M MULTI and NG-Test Carba 5 (NG Biotech). The workflow was evaluated using Enterobacterales isolates (n = 114), primarily Klebsiella species (n = 50) and Escherichia coli (n = 40). Compared to the standard testing approach in our institution using BD Phoenix, our new testing approach demonstrates 100% sensitivity and specificity for organism identification and detection of ESBL and carbapenemases. Implementation of a rapid workflow in diagnostic microbiology laboratories will enable more effective antimicrobial management of patients with BSI due to ESBL- and carbapenemase-producing Enterobacterales. IMPORTANCE The incidence of bloodstream infections (BSI) with extended spectrum beta-lactamase (ESBL) producing and carbapenemase producing Enterobacterales (CPE) is increasing at an alarming rate, for which only limited therapeutic options remain available. Rapid identification of these bacteria along with their antibiotic resistance mechanisms in positive blood cultures with Gram-negative bacteria will allow for early initiation of effective therapy and limit the overuse of broad-spectrum antibiotics in BSI (1). In this study we evaluated a combined approach of testing positive blood cultures directly, using MALDI-TOF MS followed by rapid immunochromatographic tests, for the detection of ESBLs and CPEs. Our approach demonstrates 100% sensitivity and specificity for the identification of Enterobacterales and detection of ESBLs and CPEs in positive blood culture with a turnaround time (TAT) of ≤60 min compared to a TAT of 48 h required by conventional culture and susceptibility testing methods.

Novel strains of Klebsiella africana and Klebsiella pneumoniae in Australian fruit bats (Pteropus poliocephalus).

Over the past decade human associated multidrug resistant (MDR) and hypervirulent Klebsiella pneumoniae lineages have been increasingly detected in wildlife. This study investigated the occurrence of K. pneumoniae species complex (KpSC) in grey-headed flying foxes (GHFF), an Australian fruit bat. Thirty-nine KpSC isolates were cultured from 275 GHFF faecal samples (14.2%), comprising K. pneumoniae (n = 30), Klebsiella africana (n = 8) and Klebsiella variicola subsp. variicola (n = 1). The majority (79.5%) of isolates belonged to novel sequence types (ST), including two novel K. africana STs. This is the first report of K. africana outside of Africa and in a non-human host. A minority (15.4%) of GHFF KpSC isolates shared STs with human clinical K. pneumoniae strains, of which, none belonged to MDR clonal lineages that cause frequent nosocomial outbreaks, and no isolates were characterised as hypervirulent. The occurrence of KpSC isolates carrying acquired antimicrobial resistance genes in GHFF was low (1.1%), with three K. pneumoniae isolates harbouring both fluoroquinolone and trimethoprim resistance genes. This study indicates that GHFF are not reservoirs for MDR and hypervirulent KpSC strains, but they do carry novel K. africana lineages. Health risks associated with KpSC carriage by GHFF are deemed low for the public and GHFF.

Measurement of Klebsiella Intestinal Colonization Density To Assess Infection Risk.

Klebsiella pneumoniae and the closely related species K. variicola and K. quasipneumoniae are common causes of health care-associated infections, and patients frequently become infected with their intestinal colonizing strain. To assess the association between Klebsiella colonization density and subsequent infections, a case-control study was performed. A multiplex quantitative PCR (qPCR) assay was developed and validated to quantify Klebsiella (K. pneumoniae, K. variicola, and K. quasipneumoniae combined) relative to total bacterial DNA copies in rectal swabs. Cases of Klebsiella infection were identified based on clinical definitions and having a clinical culture isolate and a preceding or coincident colonization isolate with the same wzi capsular sequence type. Controls were colonized patients without subsequent infection and were matched 2:1 to cases based on age, sex, and rectal swab collection date. qPCR from rectal swab samples was used to measure the association between the relative abundance of Klebsiella and subsequent infections. The Klebsiella relative abundance by qPCR was highly correlated with 16S sequencing (ρ = 0.79; P < 0.001). The median Klebsiella relative abundance was higher in cases (15.7% [interquartile range {IQR}, 0.93 to 52.6%]) (n = 83) than in controls (1.01% [IQR, 0.02 to 12.8%]) (n = 155) (P < 0.0001). Adjusting for multiple clinical covariates using inverse probability of treatment weighting, a Klebsiella relative abundance of >22% was associated with infection overall (odds ratio [OR], 2.87 [95% confidence interval {CI}, 1.64 to 5.03]) (P = 0.0003) and with bacteremia in a secondary analysis (OR, 4.137 [95% CI, 1.448 to 11.818]) (P = 0.0084). Measurement of colonization density by qPCR could represent a novel approach to identify hospitalized patients at risk for Klebsiella infection. IMPORTANCE Colonization by bacterial pathogens often precedes infection and offers a window of opportunity to prevent these infections in the first place. Klebsiella colonization is significantly and reproducibly associated with subsequent infection; however, factors that enhance or mitigate this risk in individual patients are unclear. This study developed an assay to measure the density of Klebsiella colonization, relative to total fecal bacteria, in rectal swabs from hospitalized patients. Applying this assay to 238 colonized patients, a high Klebsiella density, defined as >22% of total bacteria, was significantly associated with subsequent infection. Based on widely available PCR technology, this type of assay could be deployed in clinical laboratories to identify patients at an increased risk of Klebsiella infections. As novel therapeutics are developed to eliminate pathogens from the gut microbiome, a rapid Klebsiella colonization density assay could identify patients who would benefit from this type of infection prevention intervention.

Proposal for reunification of the genus Raoultella with the genus Klebsiella and reclassification of Raoultella electrica as Klebsiella electrica comb. nov.

The order Enterobacterales was divided into seven families including the family Enterobacteriaceae in 2016. The genus Klebsiella within the family Enterobacteriaceae was divided into two genera Klebsiella and Raoultella in 2001. Here, our phylogenomic analysis shows that the genus Raoultella is nested within the genus Klebsiella. Klebsiella and Raoultella together are monophyletic and share average amino acid identities (AAIs) of 86.9-89.6% above the AAI threshold (86%) for genus delimitation within the family Enterobacteriaceae. Klebsiella and Raoultella share AAIs of 79.9%-85.0% with the other genera within the subfamily "Klebsiella clade", which are in the range of inter-genus AAIs (74-85%) within the family Enterobacteriaceae. Klebsiella and Raoultella also share six known conserved signature indels. Therefore, we propose to reunify Klebsiella and Raoultella to the single genus Klebsiella and reclassify Raoultella electrica as Klebsiella electrica comb. nov. Our genome-based taxonomic analyses also identified seven potential novel species within the unified genus Klebsiella.

Klebsiella MALDI TypeR: a web-based tool for Klebsiella identification based on MALDI-TOF mass spectrometry.

Klebsiella pathogens affect human and animal health and are widely distributed in the environment. Among these, the Klebsiella pneumoniae species complex, which includes seven phylogroups, is an important cause of community and hospital infections. The Klebsiella oxytoca species complex also causes hospital infections and antibiotic-associated haemorrhagic colitis. The unsuitability of currently used clinical microbiology methods to distinguish species within each of these species complexes leads to high rates of misidentifications that are masking the true clinical significance and potential epidemiological specificities of individual species. We developed a web-based tool, Klebsiella MALDI TypeR, a platform-independent and user-friendly application that enables uploading MALDI-TOF mass spectrometry data in order to identify Klebsiella isolates at the species complex and phylogroup levels. The tool, available at https://maldityper.pasteur.fr/, leverages a database of previously identified biomarkers that are specific for species complexes, individual phylogroups, or related phylogroups. We obtained 84%-100% identification accuracy depending on phylogroup. Identification results are obtained in a few seconds from batches of uploaded spectral data. Klebsiella MALDI TypeR enables fast and reliable identification of Klebsiella strains that are often misidentified with standard microbiological methods. This web-based identification tool may be extended in the future to other human bacterial pathogens.

Screening a Strain of Klebsiella sp. O852 and the Optimization of Fermentation Conditions for Trans-Dihydrocarvone Production.

Flavors and fragrances have high commercial value in the food, cosmetic, chemical and pharmaceutical industries. It is interesting to investigate the isolation and characterization of new microorganisms with the ability to produce flavor compounds. In this study, a new strain of Klebsiella sp. O852 (accession number CCTCC M2020509) was isolated from decayed navel orange (Citrus sinensis (L.) Osbeck), which was proved to be capable of converting limonene to trans-dihydrocarvone. Besides, the optimization of various reaction parameters to enhance the trans-dihydrocarvone production in shake flask was performed for Klebsiella sp. O852. The results showed that the yield of trans-dihydrocarvone reached up to 1 058 mg/L when Klebsiella sp. O852 was incubated using LB-M medium for 4 h at 36 °C and 150 rpm, and the biotransformation process was monitored for 36 h after adding 1680 mg/L limonene/ethanol (final ethanol concentration of 0.8% (v/v)). The content of trans-dihydrocarvone increased 16 times after optimization. This study provided a basis and reference for producing trans-dihydrocarvone by biotransformation.

Decolorization and discovery of metabolic pathway for the degradation of Mordant Black 11 dye by Klebsiella sp. MB398.

Extensive utilization of the synthetic dyes in various industries is leading to water and soil contamination and ultimately impacting the humans. A research study was conducted for investigating the biodecolorization and biotransformation of Mordant Black 11 dye. For this purpose, potential of biofilm forming bacteria Klebsiella pneumoniae MB398 isolated from effluent outlets of Tops Food Industry, Hattar, Pakistan, was assessed to decolorize and transform Mordant Black 11 dye. Bacterial strain MB398 exhibited the capability of growing optimally at acidic pH (pH 6.0). Klebsiella pneumoniae MB398 efficiently decolorized Mordant Black 11 dye (64.55%) in aerobic environment at pH 6.0 and 37 °C over 24 h, which further increased to 75.35% over a period of 72 h of incubation. Strain MB398 also exhibited the capability of decolorizing Mordant Black 11 dye in the presence of cadmium (63.71%), chromium (61.78%), and copper (61.50%), respectively. UV-VIS spectrophotometric analysis, FTIR, and HPLC spectra were also indicative of biotransformation of dye molecules by Klebsiella pneumoniae MB398. GC-MS analysis of Mordant Black 11 dye revealed formation of 9 novel and unique metabolites including phenol,2,4-bis(1,1-dimethylethyl); 9-eicosene, (E); ethanol,2,2-(2-propenyloxy); acetic acid, benzene; 1-naphthol; methyl formate; valeraldehyde,2,4-dimethyl; and 7-hexadecene (Z). A possible metabolic pathway depicting the biotransformation of Mordant Black 11 dye by Klebsiella pneumoniae MB398 was projected. Findings of the current research study strongly suggest application of Klebsiella pneumoniae MB398 for developing large scale bioremediation strategies for the abatement of synthetic dyes to retain environmental sustainability in bioeconomic way.

Comprehensive genomic analysis and characterization of a new ST 174 type Klebsiella variicola strain isolated from chicken embryos.

Klebsiella variicola is a widespread opportunistic pathogen that causes infections in humans and animals. Herein a novel Klebsiella strain, AHKv-S01, was isolated and identified from dead chicken embryos in Anhui, China. Its genome contained a circular chromosome of 5,505,304 bp, with 5244 protein-coding genes, and an integrative conjugative element region containing 79 ORF sequences. AHKv-S01 was given a new sequence type number-174. Phylogenetic analyses showed that rpoB partial nucleotide sequences were highly reliable for identifying Klebsiella spp. Most of the 340 unique genes of AHKv-S01 were involved in cell envelop biogenesis, transcription, transport, and metabolic processes. Moreover, AHKv-S01 was sensitive to several antibiotics, but it showed strong resistance to penicillins, macrolides, and lincosamide. The genome contained three drug efflux pump superfamilies, ß-lactamase genes, and fosfomycin resistance-related genes. Most drug resistance genes showed amino acid mutations. Multiple virulence and pathogenic factors were also identified, and they were mainly related to adhesion, secretion, iron acquisition, and immune evasion. Chicken embryo lethality assay results revealed that the 7-day chicken embryo lethality rate was 80%, 40%, and 50% for AHKv-S01, K. pneumoniae ATCC10031, and K. pneumoniae CICC24714, respectively. The median lethal dose of AHKv-S01 was 39.9 CFU/embryo. Even low infection levels of AHKv-S01 caused a significant reduction in chicken embryo hatchability. Severe pathological changes to the liver, heart, and brain tissues of embryos infected with AHKv-S01 were observed, and these changes appeared earlier in the heart and brain than in the liver. To conclude, our results provide a foundation for further studies aiming to assess the potential risk of K. variicola to poultry populations and production yields.

Caseinolytic Proteins (Clp) in the Genus Klebsiella: Special Focus on ClpK.

Caseinolytic proteins (Clp), which are present in both prokaryotes and eukaryotes, play a major role in cell protein quality control and survival of bacteria in harsh environmental conditions. Recently, a member of this protein family, ClpK was identified in a pathogenic strain of Klebsiella pneumoniae which was responsible for nosocomial infections. ClpK is linked to the thermal stress survival of this pathogen. The genome wide analysis of Clp proteins in Klebsiella spp. indicates that ClpK is present in only 34% of the investigated strains. This suggests that the uptake of the clpk gene is selective and may only be taken up by a pathogen that needs to survive harsh environmental conditions. In silico analyses and molecular dynamic simulations show that ClpK is mainly α-helical and is highly dynamic. ClpK was successfully expressed and purified to homogeneity using affinity and anion exchange chromatography. Biophysical characterization of ClpK showed that it is predominantly alpha-helical, and this is in agreement with in silico analysis of the protein structure. Furthermore, the purified protein is biologically active and hydrolyses ATP in a concentration- dependent manner.

Bacterial contaminants and their antibiotic susceptibility patterns in ready-to-eat foods vended in Ogun state, Nigeria.

Contamination of ready-to-eat (RTE) foods by pathogenic bacteria may predispose consumers to foodborne diseases. This study investigated the presence of bacterial contaminants and their antibiotic susceptibility patterns in three locally processed RTE foods (eko, fufu and zobo) vended in urban markets in Ogun state, Nigeria. Bacteria isolated from a total of 120 RTE food samples were identified by 16S rRNA gene phylogeny while susceptibility patterns to eight classes of antibiotics were determined by the disc diffusion method. Species belonging to the genera Acinetobacter and Enterobacter were recovered from all RTE food types investigated, Klebsiella and Staphylococcus were recovered from eko and fufu samples, while those of Shigella were recovered from eko samples. Enterobacter hormaechei was the most prevalent species in all three RTE food types. Precisely 99% of 149 isolates were multidrug-resistant, suggesting a high risk for RTE food handlers and consumers. Co-resistance to ampicillin and cephalothin was the most frequently observed resistance phenotype. Results demonstrate that improved hygiene practices by food processors and vendors are urgently required during RTE processing and retail. Also, adequate food safety guidelines, regulation and enforcement by relevant government agencies are needed to improve the safety of RTE foods and ensure the protection of consumer health.

Multi-drug resistant, biofilm-producing high-risk clonal lineage of Klebsiella in companion and household animals.

Antimicrobial resistance is a global emergency which needs one health approach to address. The present study was conducted to detect the prevalence of beta-lactamase and biofilm-producing Klebsiella strains in rectal swabs (n = 624) collected from healthy dogs, cats, sheep and goats reared as companion or household animals in India. The dogs and cats were frequently exposed to third- or fourth-generation cephalosporins for therapy. The sheep and goats were occasionally exposed to antibiotics and had environmental exposure. Phenotypical ESBL (n = 93) and ACBL (n = 88)-producing Klebsiella were isolated significantly more (P < 0·05) from companion animals than household animals. Majority of the Klebsiella possessed blaCTX-M-15 . The sequences blaCTX-M-15.2 , blaCTX-M-197 and blaCTX-M-225 are reported first time from the companion animals. All ACBL-producing isolates possessed blaAmpC . The present study detected 65·8% of Klebsiella strains as biofilm producers possessing the studied biofilm associated genes. The isolates showed phenotypical resistance against chloramphenicol, tetracycline, doxycycline, co-trimoxazole, ampicillin, cefotaxime/clavulanic acid. The present study showed that companion and household animals (dogs, cats, sheep, goats) may act as a carrier of ESBL/biofilm-producing, multi-drug resistant, high-risk clonal lineage of Klebsiella.

Assessment of the in vitro activity of ceftazidime/avibactam against a global collection of multidrug-resistant Klebsiella spp. from the INFORM surveillance programme (2015-2017).

Infections caused by Klebsiella spp. are difficult to treat when these pathogens exhibit multidrug resistance (resistance to ≥3 drug classes). This study determined rates of multidrug-resistant (MDR) Klebsiella spp. among clinical isolates collected globally (Africa/Middle East, Asia, Oceania, Europe and Latin America, but not the USA) as part of the INFORM surveillance programme (2015-2017). In vitro antimicrobial activities of ceftazidime/avibactam (CAZ/AVI) and comparator antimicrobials against these MDR isolates were determined using CLSI broth microdilution methodology. MICs were interpreted using EUCAST 2019 breakpoints. By species, 38.4% (4555/11 864) of Klebsiella pneumoniae isolates were MDR, followed by 28.3% (452/1598) of Klebsiella aerogenes. The majority (69.5%) of MDR K. pneumoniae isolates were extended-spectrum ß-lactamase (ESBL)-positive and carbapenemase-negative compared with 1.3% of MDR K. aerogenes. Globally, >92% of MDR K. pneumoniae isolates were resistant to amoxicillin/clavulanic acid, aztreonam, cefepime, ceftazidime or ceftriaxone. CAZ/AVI, colistin and tigecycline MIC90 values were 1-2 mg/L against the global collection of MDR K. pneumoniae and MDR K. aerogenes. Approximately 5% (216/4555) of all MDR K. pneumoniae isolates and 1.1% (5/452) of all MDR K. aerogenes isolates were resistant to CAZ/AVI. Rates of resistance to CAZ/AVI and colistin were low for MDR, ESBL-positive, carbapenemase-negative K. pneumoniae (0.1% and 3.0%, respectively). Resistance to CAZ/AVI was highest among MDR, carbapenemase-positive, metallo-ß-lactamase (MBL)-positive K. pneumoniae (98.0%), whereas colistin resistance was highest among MDR, carbapenemase-positive, MBL-negative isolates (30.2%). The results of this study suggest that CAZ/AVI, colistin and tigecycline are potential treatment options for nosocomial infections caused by MDR Klebsiella spp. isolates.

Genomic surveillance of Escherichia coli and Klebsiella spp. in hospital sink drains and patients.

Escherichia coli and Klebsiella spp. are important human pathogens that cause a wide spectrum of clinical disease. In healthcare settings, sinks and other wastewater sites have been shown to be reservoirs of antimicrobial-resistant E. coli and Klebsiella spp., particularly in the context of outbreaks of resistant strains amongst patients. Without focusing exclusively on resistance markers or a clinical outbreak, we demonstrate that many hospital sink drains are abundantly and persistently colonized with diverse populations of E. coli, Klebsiella pneumoniae and Klebsiella oxytoca, including both antimicrobial-resistant and susceptible strains. Using whole-genome sequencing of 439 isolates, we show that environmental bacterial populations are largely structured by ward and sink, with only a handful of lineages, such as E. coli ST635, being widely distributed, suggesting different prevailing ecologies, which may vary as a result of different inputs and selection pressures. Whole-genome sequencing of 46 contemporaneous patient isolates identified one (2 %; 95 % CI 0.05-11 %) E. coli urine infection-associated isolate with high similarity to a prior sink isolate, suggesting that sinks may contribute to up to 10 % of infections caused by these organisms in patients on the ward over the same timeframe. Using metagenomics from 20 sink-timepoints, we show that sinks also harbour many clinically relevant antimicrobial resistance genes including blaCTX-M, blaSHV and mcr, and may act as niches for the exchange and amplification of these genes. Our study reinforces the potential role of sinks in contributing to Enterobacterales infection and antimicrobial resistance in hospital patients, something that could be amenable to intervention. This article contains data hosted by Microreact.

Klebsiella indica sp. nov., isolated from the surface of a tomato.

A novel bacterial strain, designated TOUT106T, was isolated from the surface of a tomato. The cells were rod-shaped, Gram-negative, encapsulated and non-motile. Strain TOUT106T grows best at 28 °C and pH 7.0 and can tolerate up to 2 % (w/v) NaCl. Based on 16S rRNA gene phylogeny, strain TOUT106T was placed close to the Salmonella clade, with close similarity to Salmonella enterica subsp. arizonae strain NCTC 8297T (98.42 %). Results of genome-based phylogenetic analysis revealed that strain TOUT106T is placed well in the Klebsiella-Raoultella clade, by forming a distinct branch with Klebsiella michiganensis DSM25444T, Klebsiella oxytoca NCTC132727T, Klebsiella grimontii 06D021T and Klebsiella pasteurii SB6412T. The genomic DNA G+C content of strain TOUT106T is 53.53 mol%. The average nucleotide identity values of TOUT106T were less than 86.5 % with closely related members of the family Enterobacteriaceae. The major fatty acids of strain TOUT106T were C16 : 0, C17:0 cyclo, C14:0 3OH/C16:1 iso, C14 : 0, C19:0 cyclo ω8c, C18:1 ω6c/C18:1 ω7c, C12 : 0 and C16:1 ω7c/C16:1 ω6c. Strain TOUT106T showed differences in physiological, phenotypic and protein profiles by MALDI-TOF MS compared to its closest relatives. Based on the results of phenotypic, chemotaxonomic and phylogenetic analyses, strain TOUT106T could be distinguished from the recognized species of the genus Klebsiella. It is suggested to represent a novel species of this genus, for which the name Klebsiella indica sp. nov. is proposed. The type strain is TOUT106T (=MCC 2901T=KACC 21384T=JCM 33718T).

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.

Isolation of an antimicrobial-resistant, biofilm-forming, Klebsiella grimontii isolate from a reusable water bottle.

A reusable water bottle was swabbed as part of the citizen science project "Swab and Send," and a Klebsiella grimontii isolate was recovered on chromogenic agar and designated SS141. Whole-genome sequencing of SS141 showed it has the potential to be a human pathogen as it contains the biosynthetic gene cluster for the potent cytotoxin, kleboxymycin, and genes for other virulence factors. The genome also contains the antibiotic-resistant genes, blaOXY-6-4 , and a variant of fosA, which is likely to explain the observed resistance to ampicillin, amoxicillin, and fosfomycin. We have also shown that SS141 forms biofilms on both polystyrene and polypropylene surfaces, providing a reasonable explanation for its ability to colonize a reusable water bottle. With the increasing use of reusable water bottles as an alternative to disposables and a strong forecast for growth in this industry over the next decade, this study highlights the need for cleanliness comparable to other reusable culinary items.