RIBAP: a comprehensive bacterial core genome annotation pipeline for pangenome calculation beyond the species level.

Microbial pangenome analysis identifies present or absent genes in prokaryotic genomes. However, current tools are limited when analyzing species with higher sequence diversity or higher taxonomic orders such as genera or families. The Roary ILP Bacterial core Annotation Pipeline (RIBAP) uses an integer linear programming approach to refine gene clusters predicted by Roary for identifying core genes. RIBAP successfully handles the complexity and diversity of Chlamydia, Klebsiella, Brucella, and Enterococcus genomes, outperforming other established and recent pangenome tools for identifying all-encompassing core genes at the genus level. RIBAP is a freely available Nextflow pipeline at github.com/hoelzer-lab/ribap and zenodo.org/doi/10.5281/zenodo.10890871.

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.

Effects of the quorum sensing related luxS gene and lsr operon on Klebsiella michiganensis resisting copper stress.

Industrial wastewater is a major environmental concern due to its high copper content, which poses significant toxicity to microbial life. Autoinducer-2 (AI-2) can participate in the inter- and intra-species communication and regulate the physiological functions of different bacterial species by producing AI-2 signal molecules. However, there are few research reports on the luxS gene and lsr operon functions for AI-2 in bacteria with a certain tolerance to copper. This study delves into the potential of quorum sensing mechanisms, particularly the AI-2 system, for enhancing microbial resistance to copper toxicity in Klebsiella michiganensis (KM). We detail the critical roles of the luxS gene in AI-2 synthesis and the lsr operon in AI-2 uptake, demonstrating their collective impact on enhancing copper resistance. Our findings show that mutations in the lsr operon, alongside the knockout of the luxS gene in KM strain (KMΔluxSΔlsr), significantly impair the strain's motility (p < 0.0001) and biofilm formation (p < 0.01), underscoring the operon's role in AI-2 transport. These genetic insights are pivotal for developing bioremediation strategies aimed at mitigating copper pollution in wastewater. By elucidating the mechanisms through which KM modulates copper resistance, this study highlights the broader ecological significance of leveraging microbial quorum sensing pathways for sustainable wastewater management.

Production of Exopolysaccharides and Indole Acetic Acid (IAA) by Rhizobacteria and Their Potential against Drought Stress in Upland Rice.

Peatlands are marginal agricultural lands due to highly acidic soil conditions and poor drainage systems. Drought stress is a big problem in peatlands as it can affect plants through poor root development, so technological innovations are needed to increase the productivity and sustainability of upland rice on peatlands. Rhizobacteria can overcome the effects of drought stress by altering root morphology, regulating stress-responsive genes, and producing exopolysaccharides and indole acetic acid (IAA). This study aimed to determine the ability of rhizobacteria in upland rice to produce exopolysaccharides and IAA, identify potential isolates using molecular markers, and prove the effect of rhizobacteria on viability and vigor index in upland rice. Rhizobacterial isolates were grown on yeast extract mannitol broth (YEMB) medium for exopolysaccharides production testing and Nutrient Broth (NB)+L-tryptophan medium for IAA production testing. The selected isolates identify using sequence 16S rRNA. The variables observed in testing the effect of rhizobacteria were germination ability, vigour index, and growth uniformity. EPS-1 isolate is the best production of exopolysaccharides (41.6 mg/ml) and IAA (60.83 ppm). The isolate EPS-1 was identified as Klebsiella variicola using 16S rRNA sequencing and phylogenetic analysis. The isolate EPS-1 can increase the viability and vigor of upland rice seeds. K. variicola is more adaptive and has several functional properties that can be developed as a potential bioagent or biofertilizer to improve soil nutrition, moisture and enhance plant growth. The use of rhizobacteria can reduce dependence on the use of synthetic materials with sustainable agriculture.

The evolution of antibiotic resistance islands occurs within the framework of plasmid lineages.

Bacterial pathogens carrying multidrug resistance (MDR) plasmids are a major threat to human health. The acquisition of antibiotic resistance genes (ARGs) in plasmids is often facilitated by mobile genetic elements that copy or translocate ARGs between DNA molecules. The agglomeration of mobile elements in plasmids generates resistance islands comprising multiple ARGs. However, whether the emergence of resistance islands is restricted to specific MDR plasmid lineages remains understudied. Here we show that the agglomeration of ARGs in resistance islands is biased towards specific large plasmid lineages. Analyzing 6784 plasmids in 2441 Escherichia, Salmonella, and Klebsiella isolates, we quantify that 84% of the ARGs in MDR plasmids are found in resistance islands. We furthermore observe rapid evolution of ARG combinations in resistance islands. Most regions identified as resistance islands are shared among closely related plasmids but rarely among distantly related plasmids. Our results suggest the presence of barriers for the dissemination of ARGs between plasmid lineages, which are related to plasmid genetic properties, host range and the plasmid evolutionary history. The agglomeration of ARGs in plasmids is attributed to the workings of mobile genetic elements that operate within the framework of existing plasmid lineages.

Antimicrobial resistance and AmpC production in ESBL-producing Klebsiella pneumoniae and Klebsiella quasipneumoniae: A retrospective study in Japanese clinical isolates.

INTRODUCTION: The study of Klebsiella quasipneumoniae, Klebsiella variicola, and AmpC production in extended-spectrum ß-lactamase (ESBL)-producing Klebsiella in Japan is limited, and existing data are insufficient. This study aims to characterize Klebsiella species, determine AmpC production rates, and analyze antimicrobial resistance patterns in ESBL-producing Klebsiella isolates in Japan. METHODS: A total of 139 clinical isolates of ESBL-producing Klebsiella were collected in Japan, along with their corresponding antimicrobial susceptibility profiles. The isolates were identified using a web-based tool. ESBL genes within the isolates were identified using multiplex PCR. Screening for AmpC-producing isolates was performed using cefoxitin disks, followed by multiplex PCR to detect the presence of AmpC genes. Antimicrobial resistance patterns were analyzed across the predominant ESBL genotypes. RESULTS: The web-based tool identified 135 isolates (97.1%) as Klebsiella pneumoniae and 4 (2.9%) as K. quasipneumoniae subsp. similipneumoniae, with no instances of K. variicola detected. Among K. pneumoniae, the CTX-M-1 group emerged as the predominant genotype (83/135, 61.5%), followed by K. quasipneumoniae subsp. similipneumoniae (3/4, 75.0%). The CTX-M-9 group was the second most prevalent genotype in K. pneumoniae (45/135, 33.3%). The high resistance rates were observed for quinolones (ranging from 46.7% to 63.0%) and trimethoprim/sulfamethoxazole (78.5%). The CTX-M-1 group exhibited higher resistance to ciprofloxacin (66/83, 79.5%) compared to the CTX-M-9 group (18/45, 40.0%), a trend also observed for levofloxacin and trimethoprim/sulfamethoxazole. Among the 16 isolates that tested positive during AmpC screening, only one K. pneumoniae isolates (0.7%) were confirmed to carry the AmpC gene. CONCLUSION: Klebsiella pneumoniae with the CTX-M-1 group is the most common ESBL-producing Klebsiella in Japan and showed a low proportion of AmpC production. These isolates are resistant to quinolones and trimethoprim/sulfamethoxazole, highlighting the challenge of managing this pathogen. The findings underscore the importance of broader research and continuous monitoring to address the resistance patterns of ESBL-producing Klebsiella.

Antimicrobial Resistance as Risk Factor for Recurrent Bacteremia after Staphylococcus aureus, Escherichia coli, or Klebsiella spp. Community-Onset Bacteremia.

We investigated links between antimicrobial resistance in community-onset bacteremia and 1-year bacteremia recurrence by using the clinical data warehouse of Europe's largest university hospital group in France. We included adult patients hospitalized with an incident community-onset Staphylococcus aureus, Escherichia coli, or Klebsiella spp. bacteremia during 2017-2019. We assessed risk factors of 1-year recurrence using Fine-Gray regression models. Of the 3,617 patients included, 291 (8.0%) had >1 recurrence episode. Third-generation cephalosporin (3GC)-resistance was significantly associated with increased recurrence risk after incident Klebsiella spp. (hazard ratio 3.91 [95% CI 2.32-6.59]) or E. coli (hazard ratio 2.35 [95% CI 1.50-3.68]) bacteremia. Methicillin resistance in S. aureus bacteremia had no effect on recurrence risk. Although several underlying conditions and infection sources increased recurrence risk, 3GC-resistant Klebsiella spp. was associated with the greatest increase. These results demonstrate a new facet to illness induced by 3GC-resistant Klebsiella spp. and E. coli in the community setting.

Effects of Klebsiella michiganensis LDS17 on Codonopsis pilosula growth, rhizosphere soil enzyme activities, and microflora, and genome-wide analysis of plant growth-promoting genes.

Codonopsis pilosula is a perennial herbaceous liana with medicinal value. It is critical to promote Codonopsis pilosula growth through effective and sustainable methods, and the use of plant growth-promoting bacteria (PGPB) is a promising candidate. In this study, we isolated a PGPB, Klebsiella michiganensis LDS17, that produced a highly active 1-aminocyclopropane-1-carboxylate deaminase from the Codonopsis pilosula rhizosphere. The strain exhibited multiple plant growth-promoting properties. The antagonistic activity of strain LDS17 against eight phytopathogenic fungi was investigated, and the results showed that strain LDS17 had obvious antagonistic effects on Rhizoctonia solani, Colletotrichum camelliae, Cytospora chrysosperma, and Phomopsis macrospore with growth inhibition rates of 54.22%, 49.41%, 48.89%, and 41.11%, respectively. Inoculation of strain LDS17 not only significantly increased the growth of Codonopsis pilosula seedlings but also increased the invertase and urease activities, the number of culturable bacteria, actinomycetes, and fungi, as well as the functional diversity of microbial communities in the rhizosphere soil of the seedlings. Heavy metal (HM) resistance tests showed that LDS17 is resistant to copper, zinc, and nickel. Whole-genome analysis of strain LDS17 revealed the genes involved in IAA production, siderophore synthesis, nitrogen fixation, P solubilization, and HM resistance. We further identified a gene (koyR) encoding a plant-responsive LuxR solo in the LDS17 genome. Klebsiella michiganensis LDS17 may therefore be useful in microbial fertilizers for Codonopsis pilosula. The identification of genes related to plant growth and HM resistance provides an important foundation for future analyses of the molecular mechanisms underlying the plant growth promotion and HM resistance of LDS17. IMPORTANCE: We comprehensively evaluated the plant growth-promoting characteristics and heavy metal (HM) resistance ability of the LDS17 strain, as well as the effects of strain LDS17 inoculation on the Codonopsis pilosula seedling growth and the soil qualities in the Codonopsis pilosula rhizosphere. We conducted whole-genome analysis and identified lots of genes and gene clusters contributing to plant-beneficial functions and HM resistance, which is critical for further elucidating the plant growth-promoting mechanism of strain LDS17 and expanding its application in the development of plant growth-promoting agents used in the environment under HM stress.

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.

Whole genome sequencing reveals complex resistome features of Klebsiella pneumoniae isolated from patients at major hospitals in Trinidad, West Indies.

OBJECTIVES: Antibiotic-resistant Klebsiella pneumoniae is a human pathogen of major global concern due to its ability to cause multiple severe diseases that are often difficult to treat therapeutically. This study aimed to investigate the resistome of local clinical K. pneumoniae isolates. METHODS: Herein, we used a whole genome sequencing approach and bioinformatics tools to reconstruct the resistome of 10 clinical K. pneumoniae isolates and one clinical isolate of the closely related Klebsiella quasipneumoniae obtained from patients from three major hospitals in Trinidad, West Indies. RESULTS: The results of the study revealed the presence of a complex antibiotic-resistant armoury among the local isolates with multiple resistance mechanisms involving (i) inactivation of antibiotics, (ii) efflux pumps, (iii) antibiotic target alteration, protection, and replacement against antibiotics, and (iv) altered porin protein that reduced the permeability to antibiotics. Several resistance genes such as blaCTX-M-15, blaTEM-1B, blaSHV-28, blaKPC-2, oqxA, sul1, tetD, aac(6')-Ib-cr5, aph(6)-Id, and fosA6, which are known to confer resistance to antibiotics used to treat K. pneumoniae infections. In most cases, the resistance genes were flanked by mobile elements, including insertion sequences and transposons, which facilitate the spread of these genetic features among related organisms. CONCLUSION: This is the first comprehensive study to thoroughly investigate the resistome of clinical K. pneumoniae isolates and K. quasipneumoniae from Trinidad, West Indies. These findings suggest that monitoring K. pneumoniae and its genome-wide antibiotic resistance features in clinical strains would be of critical importance for guiding antibiotic stewardship programs and improving regional disease management systems for this pathogen.

The outbreak of multispecies carbapenemase-producing Enterobacterales associated with pediatric ward sinks: IncM1 plasmids act as vehicles for cross-species transmission.

BACKGROUND: This study describes an outbreak caused by multispecies carbapenemase-producing Enterobacterales (CPE) occurring in a pediatric ward at an academic medical center in Tokyo. METHODS: The index case involved a 1-year-old boy with Klebsiella variicola (CPE) detected in anal swabs in June 2016. The second case was Klebsiella quasipneumoniae (CPE) occurred in March 2017 followed by further spread, leading to the declaration of an outbreak in April 2017. Extensive environmental and patient microbiological sampling was performed. The relatedness of the isolates was determined using draft-whole-genome sequencing. RESULTS: CPE surveillance cultures of patients and environments were positive in 19 patients and 9 sinks in the ward. The sinks in hospital rooms uninhabited by CPE patients exhibited no positive CPE-positive specimen during the outbreak. All CPE strains analyzed using draft-whole-genome sequencing harbored blaIMP-1, except for one harboring blaIMP-11; these strains harbored identical blaIMP-1-carrying IncM1 plasmids. CPE was detected even after sink replacement; infection-control measures focused on sinks were implemented and the CPE outbreak ended after 7 months. CONCLUSIONS: Multiple bacterial species can become CPE via blaIMP-1-carrying IncM1 plasmids of the same origin and spread through sinks in a hospital ward. Thorough infection-control measures implemented as a bundle might be crucial.

The emergence of highly resistant and hypervirulent Klebsiella pneumoniae CC14 clone in a tertiary hospital over 8 years.

BACKGROUND: Klebsiella pneumoniae is a major bacterial and opportunistic human pathogen, increasingly recognized as a healthcare burden globally. The convergence of resistance and virulence in K. pneumoniae strains has led to the formation of hypervirulent and multidrug-resistant strains with dual risk, limiting treatment options. K. pneumoniae clones are known to emerge locally and spread globally. Therefore, an understanding of the dynamics and evolution of the emerging strains in hospitals is warranted to prevent future outbreaks. METHODS: In this study, we conducted an in-depth genomic analysis on a large-scale collection of 328 multidrug-resistant (MDR) K. pneumoniae strains recovered from 239 patients from a single major hospital in the western coastal city of Jeddah in Saudi Arabia from 2014 through 2022. We employed a broad range of phylogenetic and phylodynamic methods to understand the evolution of the predominant clones on epidemiological time scales, virulence and resistance determinants, and their dynamics. We also integrated the genomic data with detailed electronic health record (EHR) data for the patients to understand the clinical implications of the resistance and virulence of different strains. RESULTS: We discovered a diverse population underlying the infections, with most strains belonging to Clonal Complex 14 (CC14) exhibiting dominance. Specifically, we observed the emergence and continuous expansion of strains belonging to the dominant ST2096 in the CC14 clade across hospital wards in recent years. These strains acquired resistance mutations against colistin and extended spectrum ß-lactamase (ESBL) and carbapenemase genes, namely blaOXA-48 and blaOXA-232, located on three distinct plasmids, on epidemiological time scales. Strains of ST2096 exhibited a high virulence level with the presence of the siderophore aerobactin (iuc) locus situated on the same mosaic plasmid as the ESBL gene. Integration of ST2096 with EHR data confirmed the significant link between colonization by ST2096 and the diagnosis of sepsis and elevated in-hospital mortality (p-value < 0.05). CONCLUSIONS: Overall, these results demonstrate the clinical significance of ST2096 clones and illustrate the rapid evolution of an emerging hypervirulent and MDR K. pneumoniae in a clinical setting.

Characterization of recombinant phytase of Klebsiella sp. and the influence of novel 3-phytase on mineral solubility in broiler diets under an in vitro digestion assay.

Phytate (inositol hexaphosphate) is the major storage form of phosphorus (P) in nature, and phytases catalyze the hydrolysis of P from phytate and the formation of inositol phosphate isomers. In this study, a bacterium that produces phytase was isolated in a phytase screening medium. The bacterium was identified as Klebsiella sp. using phenotypic and molecular techniques. The PhyK phytase gene was successfully amplified from the genome, inserted into the pET-21a (+) vector, and expressed as a recombinant protein in E. Coli BL21. The efficiency of a laboratory phytase (Lab-Ph, PhyK phytase) was determined and compared with a commercial phytase (Com-Ph, Quantum Blue 40P phytase, AB Vista) under an in vitro digestion assay. The native signal peptide effectively facilitated the translocation of the protein to the periplasmic space of E. Coli BL21, resulting in the proper folding of the protein and the manifestation of desirable enzyme activity. The Lab-Ph displayed the temperature and pH optima at 50 °C and 5 respectively. In addition, the Lab-Ph was inactivated at 80 °C. Under an in vitro digestion assay condition, Lab-Ph improved the P solubility coefficient in broiler diets. In comparison, the Com-Ph significantly increased the P solubility coefficient even when compared with the Lab-Ph. In summary, this study has shown that Lab-Ph possesses the necessary biochemical properties to be used in various industrial applications. However, Lab-Ph is extremely sensitive to heat treatment. The Lab-Ph and Com-Ph under an in vitro digestion assay improved the solubility coefficient of P in the broiler diet.

Phenotypic and molecular characterization of ß-lactamase-producing Klebsiella species among children discharged from hospital in Western Kenya.

BACKGROUND: The emergence and spread of ß-lactamase-producing Klebsiella spp. has been associated with a substantial healthcare burden resulting in therapeutic failures. We sought to describe the proportion of phenotypic resistance to commonly used antibiotics, characterize ß-lactamase genes among isolates with antimicrobial resistance (AMR), and assess the correlates of phenotypic AMR in Klebsiella spp. isolated from stool or rectal swab samples collected from children being discharged from hospital. METHODS: We conducted a cross-sectional study involving 245 children aged 1-59 months who were being discharged from hospitals in western Kenya between June 2016 and November 2019. Whole stool or rectal swab samples were collected and Klebsiella spp. isolated by standard microbiological culture. ß-lactamase genes were detected by PCR whilst phenotypic antimicrobial susceptibility was determined using the disc diffusion technique following standard microbiology protocols. Descriptive analyses were used to characterize phenotypic AMR and carriage of ß-lactamase-producing genes. The modified Poisson regression models were used to assess correlates of phenotypic beta-lactam resistance. RESULTS: The prevalence of ß-lactamase carriage among Klebsiella spp. isolates at hospital discharge was 62.9% (154/245). Antibiotic use during hospitalization (adjusted prevalence ratio [aPR] = 4.51; 95%CI: 1.79-11.4, p < 0.001), longer duration of hospitalization (aPR = 1.42; 95%CI: 1.14-1.77, p < 0.002), and access to treated water (aPR = 1.38; 95%CI: 1.12-1.71, p < 0.003), were significant predictors of phenotypically determined ß-lactamase. All the 154 ß-lactamase-producing Klebsiella spp. isolates had at least one genetic marker of ß-lactam/third-generation cephalosporin resistance. The most prevalent genes were blaCTX-M 142/154 (92.2%,) and blaSHV 142/154 (92.2%,) followed by blaTEM 88/154 (57.1%,) and blaOXA 48/154 (31.2%,) respectively. CONCLUSION: Carriage of ß-lactamase producing Klebsiella spp. in stool is common among children discharged from hospital in western Kenya and is associated with longer duration of hospitalization, antibiotic use, and access to treated water. The findings emphasize the need for continued monitoring of antimicrobial susceptibility patterns to inform the development and implementation of appropriate treatment guidelines. In addition, we recommend measures beyond antimicrobial stewardship and infection control within hospitals, improved sanitation, and access to safe drinking water to mitigate the spread of ß-lactamase-producing Klebsiella pathogens in these and similar settings.

Impact of bioelectricity on DNRA process and microbial community composition within cathodic biofilms in dual-chambered bioelectrode microbial fuel cell (MFC).

The synchronous bioelectricity generation and dissimilatory nitrate reduction to ammonium (DNRA) pathway in Klebsiella variicola C1 was investigated. The presence of bioelectricity facilitated cell growth on the anodic biofilms, consequently enhancing the nitrate removal efficiency decreasing total nitrogen levels and causing a negligible accumulation of NO2- in the supernatant. Genomic analysis revealed that K. variicola C1 possessed a complete DNRA pathway and largely annotated electron shuttles. The up-regulated expression of genes narG and nirB, encoding nitrite oxidoreductase and nitrite reductase respectively, was closely associated with increased extracellular electron transfer (EET). High-throughput sequencing analysis was employed to investigate the impact of bioelectricity on microbial community composition within cathodic biofilms. Results indicated that Halomonas, Marinobacter and Prolixibacteraceae were enriched at the cathode electrodes. In conclusion, the integration of a DNRA strain with MFC facilitated the efficient removal of wastewater containing high concentrations of NO3- and enabled the environmentally friendly recovery of NH4+.

Coexistence of blaIMP-4 and blaSFO-1 in an IncHI5B plasmid harbored by tigecycline-non-susceptible Klebsiella variicola strain.

BACKGROUND: Klebsiella variicola is considered a newly emerging human pathogen. Clinical isolates of carbapenemase and broad-spectrum ß-lactamase-producing K. variicola remain relatively uncommon. A strain of K. variicola 4253 was isolated from a clinical sample, and was identified to carry the blaIMP-4 and blaSFO-1 genes. This study aims to discern its antibiotic resistance phenotype and genomic characteristics. METHODS: Species identification was conducted using MALDI-TOF/MS. PCR identification confirmed the presence of the blaIMP-4 and blaSFO-1 genes. Antibiotic resistance phenotype and genomic characteristics were detected by antimicrobial susceptibility testing and whole-genome sequencing. Plasmid characterization was carried out through S1-PFGE, conjugation experiments, Southern blot, and comparative genomic analysis. RESULTS: K. variicola 4253 belonged to ST347, and demonstrated resistance to broad-spectrum ß-lactamase drugs and tigecycline while being insensitive to imipenem and meropenem. The blaIMP-4 and blaSFO-1 genes harbored on the plasmid p4253-imp. The replicon type of p4253-imp was identified as IncHI5B, representing a multidrug-resistant plasmid capable of horizontal transfer and mediating the dissemination of drug resistance. The blaIMP-4 gene was located on the In809-like integrative element (Intl1-blaIMP-4-aacA4-catB3), which circulates in Acinetobacter and Enterobacteriaceae. CONCLUSIONS: This study reports the presence of a strain of K. variicola, which is insensitive to tigecycline, carrying a plasmid harboring blaIMP-4 and blaSFO-1. It is highly likely that the strain acquired this plasmid through horizontal transfer. The blaIMP-4 array (Intl1-blaIMP-4-aacA4-catB3) is also mobile in Acinetobacter and Enterobacteriaceae. So it is essential to enhance clinical awareness and conduct epidemiological surveillance on multidrug-resistant K. variicola, conjugative plasmids carrying blaIMP-4, and the In809 integrative element.

Molecular epidemiology and mechanisms of carbapenem and colistin resistance in Klebsiella and other Enterobacterales from treated wastewater in Croatia.

Among the most problematic bacteria with clinical relevance are the carbapenem-resistant Enterobacterales (CRE), as there are very limited options for their treatment. Treated wastewater can be a route for the release of these bacteria into the environment and the population. The aim of this study was to isolate CRE from treated wastewater from the Zagreb wastewater treatment plant and to determine their phenotypic and genomic characteristics. A total of 200 suspected CRE were isolated, 148 of which were confirmed as Enterobacterales by MALDI-TOF MS. The predominant species was Klebsiella spp. (n = 47), followed by Citrobacter spp. (n = 40) and Enterobacter cloacae complex (cplx.) (n = 35). All 148 isolates were carbapenemase producers with a multidrug-resistant phenotype. Using multi-locus sequence typing and whole-genome sequencing (WGS), 18 different sequence types were identified among these isolates, 14 of which were associated with human-associated clones. The virulence gene analysis of the sequenced Klebsiella isolates (n = 7) revealed their potential pathogenicity. PCR and WGS showed that the most frequent carbapenemase genes in K. pneumoniae were blaOXA-48 and blaNDM-1, which frequently occurred together, while blaKPC-2 together with blaNDM-1 was mainly detected in K. oxytoca, E. cloacae cplx. and Citrobacter spp. Colistin resistance was observed in 40% of Klebsiella and 57% of Enterobacter isolates. Underlying mechanisms identified by WGS include known and potentially novel intrinsic mechanisms (point mutations in the pmrA/B, phoP/Q, mgrB and crrB genes) and acquired mechanisms (mcr-4.3 gene). The mcr-4.3 gene was identified for the first time in K. pneumoniae and is probably located on the conjugative IncHI1B plasmid. In addition, WGS analysis of 13 isolates revealed various virulence genes and resistance genes to other clinically relevant antibiotics as well as different plasmids possibly associated with carbapenemase genes. Our study demonstrates the important role that treated municipal wastewater plays in harboring and spreading enterobacterial pathogens that are resistant to last-resort antibiotics.

Phenotypic and molecular characterization of multi-drug resistant Klebsiella spp. isolates recovered from clinical settings.

Klebsiella pneumoniae is a Gram-negative bacterium that colonizes the gastrointestinal tract and nasopharynx with many being linked to nosocomial infections. Extended-spectrum ß-lactamases (ESBL)-producing and carbapenem-resistant K. pneumoniae is recognized by the World Health Organization (WHO) as a critical public health concern. In this study, whole-genome sequencing (WGS) - based analysis was performed to understand the molecular epidemiology of multi-drug resistant Klebsiella spp. clinical isolates. Genome comparison, multi-locus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and whole-genome-SNP-based phylogenetic analysis (wg-SNP) were used for in-depth molecular characterization. in silico typing was used to determine the resistance genes, virulence factors, Inc. groups, and capsular types. All except one isolate were non-susceptible to meropenem and 89% were non-susceptible to ertapenem and imipenem. blaNDM, blaOXA-48, and blaKPC were the detected carbapenemases with blaNDM-1 found in half of the sequenced genomes. Resistance to colistin was detected in one isolate and was linked to several genetic alterations in crrB, pmrB, and pmrC genes. The most common plasmid type was IncFIB followed by IncR, and the Type 3 fimbriae, encoded by the mrkABCDF operon, was conserved among all isolates. The most common sequence- (ST) and K-type detected were ST147 and K64. The prevelance and the genomic relatedness of ST147 isolates, as shown by the data from SNP-based phylogenetic analysis, PFGE, and genomic clustering, may be an outbreak marker. However, this can only be validated through a more comprehensive study encompassing a wider sampling scheme and over an extended timeframe.

Oral pathobiont Klebsiella chaperon usher pili provide site-specific adaptation for the inflamed gut mucosa.

The ectopic gut colonization by orally derived pathobionts has been implicated in the pathogenesis of various gastrointestinal diseases, including inflammatory bowel disease (IBD). For example, gut colonization by orally derived Klebsiella spp. has been linked to IBD in mice and humans. However, the mechanisms whereby oral pathobionts colonize extra-oral niches, such as the gut mucosa, remain largely unknown. Here, we performed a high-density transposon (Tn) screening to identify genes required for the adaptation of an oral Klebsiella strain to different mucosal sites - the oral and gut mucosae - at the steady state and during inflammation. We find that K. aerogenes, an oral pathobiont associated with both oral and gut inflammation in mice, harbors a newly identified genomic locus named "locus of colonization in the inflamed gut (LIG)" that encodes genes related to iron acquisition (Sit and Chu) and host adhesion (chaperon usher pili [CUP] system). The LIG locus is highly conserved among K. aerogenes strains, and these genes are also present in several other Klebsiella species. The Tn screening revealed that the LIG locus is required for the adaptation of K. aerogenes in its ectopic niche. In particular, we determined K. aerogenes employs a CUP system (CUP1) present in the LIG locus for colonization in the inflamed gut, but not in the oral mucosa. Thus, oral pathobionts likely exploit distinct adaptation mechanisms in their ectopically colonized intestinal niche compared to their native niche.

Bacterial isolation and genome analysis of a novel Klebsiella quasipneumoniae phage in southwest China's karst area.

BACKGROUND: Southwest China is one of the largest karst regions in the world. Karst environment is relatively fragile and vulnerable to human activities. Due to the discharge of sewage and domestic garbage, the karst system may be polluted by pathogenic bacteria. The detection of bacterial distribution and identification of phage capable of infecting them is an important approach for environmental assessment and resource acquisition. METHODS: Bacteria and phages were isolated from karst water in southwest China using the plate scribing and double plate method, respectively. Isolated phage was defined by transmission electron microscopy, one-step growth curve and optimal multiplicity of infection (MOI). Genomic sequencing, phylogenetic analysis, comparative genomic and proteomic analysis were performed. RESULTS: A Klebsiella quasipneumoniae phage was isolated from 32 isolates and named KL01. KL01 is morphologically identified as Caudoviricetes with an optimal MOI of 0.1, an incubation period of 10 min, and a lysis period of 60 min. The genome length of KL01 is about 45 kb, the GC content is 42.5%, and it contains 59 open reading frames. The highest average nucleotide similarity between KL01 and a known Klebsiella phage 6939 was 83.04%. CONCLUSIONS: KL01 is a novel phage, belonging to the Autophagoviridae, which has strong lytic ability. This study indicates that there were not only some potential potentially pathogenic bacteria in the karst environment, but also phage resources for exploration and application.