Genus Klebsiella Research Articles

Abstract 1973: Patients with operable esophageal cancer and improved responses to combined chemoradiotherapy and immunotherapy display distinct microbiome profiles enriched in multiple Bacteroides species

Abstract Background: Preclinical and clinical data indicate that neoadjuvant chemoradiotherapy (CRT) may prime an anti-tumor immunological response in esophageal cancer driven by intratumoral CD8+ T cells and PD-L1 expression. LAG-3 is also highly expressed in esophagogastric cancers. The microbiome, a novel and potentially modifiable, biomarker of IO response, has not yet been examined in the neoadjuvant setting in esophageal cancer and is the goal of our study. Methods: Fecal samples were collected from patients with stage II/III esophageal or gastroesophageal junction carcinoma eligible for curative resection treated with the standard of care regimen of carboplatin paclitaxel (50mg/m2), radiation 50.4 Gy in 28 fractions and an Ivor-Lewis esophagectomy 6-10 weeks after last CRT and immunotherapy (IO) dose. Patients on arm A (n=11) received 2 cycles of induction with nivolumab plus 3 additional cycles on week 1, 3 and 5 of CRT. Patients on arm B (n=8) received nivolumab plus relatlimab on the same schedule (Clinical trial: NCT03044613). We examined longitudinal fecal samples from n=19 patients across both arms (n=90 samples) using 16S rRNA amplicon sequencing. Patients were classified based on pathological response: complete response (CR) and grades 1, 2, and 3 (G1, G2, G3) with increasing residual tumor visible in the resected specimen. Sequencing data was trimmed and filtered for contaminants, followed by high-resolution taxonomic assignment and normalization of reads across all samples. Analysis was performed using multiple metrics for alpha diversity and beta-diversity, with principal coordinates analysis/PERMANOVA, and pathway analysis using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt). Results: Patients with improved response in the neoadjuvant setting (CR/G1 vs G2/G3) grouped in distinct clusters using Bray-Curtis (p < 0.001). Patients with CR had higher alpha diversity, using both measures of richness and evenness, compared to patients with a G3 responses (p < 0.03). Specifically, family Bacteroidaceae and genus Bacteroides were enriched in patients with CR vs G3 (p < 0.02). At the species level, B. finegoldii, B. ovatus, and B. uniformis were enriched in patients with CR vs G3 (p < 0.02). In contrast, genus Klebsiella and Clostridium termitidis were enriched in patients with a poor response, G3 (p <0.001, both). Pathway analysis found two metabolic pathways enriched in patients with CR: secondary bile acid biosynthesis (p=0.005) and lysine biosynthesis (p=0.02). Conclusions: Patients with operable esophageal cancer and improved responses to combined CRT and IO had distinct microbiome profiles enriched in multiple Bacteroides species. Further analyses and validation efforts are underway to confirm metabolomic pathways. Citation Format: Fyza Y. Shaikh, James R. White, Ronan J. Kelly, Ali H. Zaidi, Jenna V. Canzoniero, Josephine L. Feliciano, Russell K. Hales, K Ranh Voong, Richard J. Battafarano, Blair A. Jobe, Stephen C. Yang, Stephen Broderick, Jinny Ha, Kellie N. Smith, Elizabeth Thompson, Eun J. Shin, Ali I. Amjad, Patrizia Guerrieri, Benny Weksler, Chen Hu, Valsamo Anagnostou, Vincent K. Lam, Cynthia L. Sears. Patients with operable esophageal cancer and improved responses to combined chemoradiotherapy and immunotherapy display distinct microbiome profiles enriched in multiple Bacteroides species [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1973.

Integrated Fecal Microbiome and Metabolomics Reveals a Novel Potential Biomarker for Predicting Tibial Dyschondroplasia in Chickens.

Tibial dyschondroplasia (TD) is a metabolic tibial-tarsal disorder occurring in fast-growing poultry, and its diagnosis is mainly based on an invasive method. Here, we profiled the fecal gut microbiome and metabolome of broilers with and without TD to identify potential non-invasive and non-stress biomarkers of TD. First, TD broilers with the most pronounced clinical signs during the experiment were screened and faecal samples were collected for integrated microbiome and metabolomics analysis. Moreover, the diagnostic potential of identified biomarkers was further validated throughout the experiment. It was noted that the microbial and metabolic signatures of TD broilers differed from those of normal broilers. TD broilers were characterized by enriched bacterial OTUs of the genus Klebsiella, and depleted genera [Ruminococcus], Dorea, Ruminococcus, Oscillospira, Ochrobactrum, and Sediminibacterium. In addition, a total of 189 fecal differential metabolites were identified, mainly enriched in the purine, vitamin and amino acid metabolism, which were closely associated with differential microbiota and tibia-related indicators. Furthermore, three fecal metabolites were screened, including 4-hydroxybenzaldehyde, which distinguished TD from normal broilers with extremely high specificity and was superior to serum bone markers. These results indicated that gut microbiota equilibrium might influence the pathogenesis of TD by modulating host metabolism, and the identified fecal metabolite 4-hydroxybenzaldehyde might be a potential and non-invasive biomarker for predicting TD in chickens.

Interactive analysis of biosurfactants in fruit-waste fermentation samples using BioSurfDB and MEGAN

Agroindustrial waste, such as fruit residues, are a renewable, abundant, low-cost, commonly-used carbon source. Biosurfactants are molecules of increasing interest due to their multifunctional properties, biodegradable nature and low toxicity, in comparison to synthetic surfactants. A better understanding of the associated microbial communities will aid prospecting for biosurfactant-producing microorganisms. In this study, six samples of fruit waste, from oranges, mangoes and mixed fruits, were subjected to autochthonous fermentation, so as to promote the growth of their associated microbiota, followed by short-read metagenomic sequencing. Using the DIAMOND+MEGAN analysis pipeline, taxonomic analysis shows that all six samples are dominated by Proteobacteria, in particular, a common core consisting of the genera Klebsiella, Enterobacter, Stenotrophomonas, Acinetobacter and Escherichia. Functional analysis indicates high similarity among samples and a significant number of reads map to genes that are involved in the biosynthesis of lipopeptide-class biosurfactants. Gene-centric analysis reveals Klebsiella as the main assignment for genes related to putisolvins biosynthesis. To simplify the interactive visualization and exploration of the surfactant-related genes in such samples, we have integrated the BiosurfDB classification into MEGAN and make this available. These results indicate that microbiota obtained from autochthonous fermentation have the genetic potential for biosynthesis of biosurfactants, suggesting that fruit wastes may provide a source of biosurfactant-producing microorganisms, with applications in the agricultural, chemical, food and pharmaceutical industries.

Comparative analysis of the Colistin resistance-regulating gene cluster in Klebsiella species

CrrAB two-component regulatory system is associated with colistin resistance in Klebsiella pneumoniae. Recently, some K. pneumoniae isolates lacking crrAB genes have been identified. In this study, we investigated the distribution and structural variation of the crrBAC-kexD cluster. To evaluate the structural variation of the crrBAC-kexD cluster, we explored 59 clinical K. pneumoniae isolates from Korea, and 508 whole genomes of K. pneumoniae and other strains of Klebsiella sp. Significant structural variations in crrBAC-kexD and its surrounding regions were identified among K. pneumoniae genomes. Within the genus Klebsiella, the cluster was identified only in K. pneumoniae, K. variicola, and K. quasipneumoniae, which form the K. pneumoniae complex. Among the 304 available K. pneumoniae genomes, an intact crrBAC-kexD cluster was identified in 178 isolates (58.6%), while the cluster was absent in 90 isolates (29.6%). Partial deletions within the cluster were identified in 22 genomes (7.2%). The most diverse structural patterns of the crrBAC-kexD cluster were observed in ST11 strains. Some clades lacked the crrBAC-kexD cluster. The crrBAC-kexD cluster was identified in the genomes of other bacterial species, including Citrobacter freundii and Enterobacter ludwigii. The crrBAC-kexD cluster is proposed to have been acquired by the ancestor of the K. pneumoniae complex from other bacterial species and the cluster may have been lost and re-acquired repeatedly in K. pneumoniae strains according to the phylogenetic analysis. The dynamic evolution of the crrBAC-kexD cluster suggests that it may have other roles, in addition to colistin resistance, in bacterial physiology.

Klebsiella species: Taxonomy, hypervirulence and multidrug resistance.

SummaryMembers 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.

Characterization of Halophilic Inulinase Producing Strains of Bacillus and Klebsiella Species Isolated From the Salt Mines of Pakistan

Halophiles are excellent sources of enzymes which are not only salt stable but also perform catalysis under extreme reaction conditions. The habitat of salt mines and its microbial diversity have not been thoroughly explored. In the present study, seven halophilic inulinase-producing bacteria (H1N1–H1N7) were isolated from the Bahadarkhel salt mines of Pakistan, and were identified based on their morphological, biochemical and molecular features. The strains H1N3, H1N4, H1N6, H1N7 were capable of growing at 20% salt concentration and therefore were considered as extremely halotolerant. Inulinase activities of the isolates were detected in inulin-containing broth media, and were further confirmed on inulin-based agar plates. The strains HIN1, H1N3, H1N5, and H1N7 exhibited the maximum zone of clearance (≥15 mm) on inuline agar medium. The strain H1N1 showed the highest zone of clearance of 19 mm. Morphological, biochemical and molecular characterization showed that the inulinase producing isolates were related to Bacillus and Klebsiella genera. The molecular identification was based on 16S rRNA gene sequencing. The strain H1N2 was capable of producing the four tested halozymes including inulinase, amylase, protease, and lipase. The strains HIN3, HIN4, and H1N5 were capable of producing three halozymes, i.e., inulinase, amylase, and protease while the strains H1N6 and H1N7 produced inulinases, lipases and proteases. These salt tolerant bacterial strains exhibiting diverse enzyme characteristics and phylogeny could be highly useful for future practical applications.

Construction of bacterial consortium for efficient degradation of mixed pharmaceutical dyes.

Synthetic dyes are established colorants in the pharmaceutical industries for the coating and coloration of tablets, capsules, etc. to mark the specific formulation and dosage, improve the stability, and esthetic value. The pharmaceuticals manufacturing process releases large volumes of dye-containing wastewaters, contributing to eco-toxic concerns related to different health risks. In this study, biodegradation of amaranth, sunset yellow, tartrazine, indigo carmine, and quinoline yellow dyes was investigated in the mixed environment. Initially, 24 tolerant bacterial strains were isolated from pharmaceutical wastewater samples, among which 19 strains were found to be non-virulent. Five different consortia were constructed by considering the 19 strains among which C1 consisting of strains DY7, 10, 11, and 18 was found to be the most potential consortium with an overall efficiency of 96.65% dye degradation in the presence of glucose after 15days. The strains DY7, 10, 11, and 18 were identified as members of the Klebsiella genus by 16S rRNA partial sequencing. The cell wall structure, carbohydrate utilization profile, and metabolic characterization were conducted on the selected strains of C1. Among the 25 different antibiotics, DY7 and DY11 exhibited the highest zone of growth inhibition in the presence of cefixime, and DY10 was restricted by chloramphenicol whereas DY18 was found sensitive to ceftazidime/tazobactam.

Nitrogen Fixation and Diazotrophic Community in Plastic-Eating Mealworms Tenebrio molitor L.

Mealworms, the larvae of a coleopteran insect Tenebrio molitor L., are capable of eating, living on, and degrading non-hydrolyzable vinyl plastics as sole diet. However, vinyl plastics are carbon-rich but nitrogen-deficient. It remains puzzling how plastic-eating mealworms overcome the nutritional obstacle of nitrogen limitation. Here, we provide the evidence for nitrogen fixation activity within plastic-eating mealworms. Acetylene reduction assays illustrate that the nitrogen-fixing activity ranges from 12.3 ± 0.7 to 32.9 ± 9.3nmol ethylene·h-1·gut-1 and the corresponding fixed nitrogen equivalents of protein are estimated as 8.6 to 23.0µg per day per mealworm. Nature nitrogen isotopic analyses of plastic-eating mealworms provide further evidence for the assimilation of fixed nitrogen as a new nitrogen source. Eliminating the gut microbial microbiota with antibiotics impairs the mealworm's ability to fix nitrogen from the atmosphere, indicating the contribution of gut microbiota to nitrogen fixation. By using the traditional culture-dependent technique, PCR and RT-PCR of nifH gene, nitrogen-fixing bacteria diversity within the gut was detected, and the genus Klebsiella was demonstrated to be an important nitrogen-fixing symbiont. These findings first build the relationship between plastic degradation (carbon metabolism) and nitrogen fixation (nitrogen metabolism) within mealworms. Combined with previously reported plastic-degrading capability and nitrogen-fixing activity, mealworms may be potential candidates for up-recycling of plastic waste to produce protein sources.

Characteristics of intestinal microbiome in children with autism

The aim of the study was to assess the prevalence of dysbiosis in children with autism spectrum disorders (ASD). The study was observational, cohort, comparative. All the patients included in it were divided into 2 groups. The first (main) group (n=46) consisted of children aged 4.92±1.57 years with an established diagnosis of ASD. The second (n=20, comparison group) included conditionally healthy children (1 and 2 health groups) aged 5.10±1.16 years, not suffering from ASD. It was stated that in children with autism a reduced content of lactobacilli (p=0.056), the practical absence of lactic acid streptococci (p=0.019), a rarer detection of individual strains of Enterobacter bacteria (Ent. cloacae; p=0.033), an increase of 2 or more times in the frequency in level of hemolytic Escherichia coli (E. coli; p=0.041), some strains of bacteria of the genus Klebsiella (Kl. Рneumoniae; p=0.080) and Citrobacter (Citr. freundii, p=0.015); Staphylococcus aureus (14% vs. 6%, p>0.050) and Candidae fungi (11% vs. 6%, p>0.050) were found more often than in healthy children. In addition, patients with autism have enough level of Bifidobacterium and Escherichia coli. The study showed the presence of shifts in the gut microbiome in children with autism spectrum disorders. At the same time, structure of microbiome differed significantly from that typical for healthy children

Tetrabromobisphenol A (TBBPA) biodegradation in acidogenic systems: One step further on where and who

The occurrence of brominated flame retardants such as Tetrabromobisphenol A (TBBPA) in water bodies poses a serious threat to aquatic ecosystems. Degradation of TBBPA in wastewater has successfully been demonstrated to occur through anaerobic digestion (AD), although the involved microorganisms and the conditions favouring the conversion remains unclear. In this study, it was observed that bioconversion of TBBPA did not occur during the hydrolytic stage of the AD, but during the strictly fermentative stage. Bioconversion occurred in hydrolytic-acidogenic as well as in strictly acidogenic continuous bioreactors. This indicates that the microorganisms that degrade TBBPA benefit from the electron flux taking place during glycolysis and further transformations into short-chain fatty acids. The degradation kinetics of TBBPA was inversely proportional to the complexity of the wastewater as the apparent kinetics constants were 2.11, 1.86, and 0.52 h−1·gVSS−1 for glucose, starch, and domestic sewage as carbon source, respectively. Additionally, the micropollutant loading rate relative to the overall organic loading rate is of major importance during the investigation of cometabolic transformations. The long-term exposure to TBBPA at environmentally realistic concentrations did not cause any major changes in the microbiome composition. Multivariate statistical analysis of the evolvement of the microbiome throughout the incubation suggested that Enterobacter spp. and Clostridium spp. are the key players in TBBPA degradation. Finally, a batch enrichment was conducted, which showed that concentrations of 0.5 mg·L−1 or higher are detrimental to Clostridium spp., even though these organisms are putative TBBPA degraders. The Clostridium genus was outcompeted by the Enterobacter and Klebsiella genera, hereby highlighting the effect of unrealistic concentrations frequently used in culture-dependent studies on the microbial community composition.

Investigation of blaKPC and blaNDM genes in enterobacteriaceae received in a public health laboratory

Introduction: KPC and NDM carbapenemases production is an important enzymatic mechanism of resistance to carbapenens in bacteria belonging to the Enterobacteriaceae family. These enzymes degrade virtually all beta-lactam antibiotics and are encoded by the blaKPC and blaNDM genes, which can be in mobile genetic elements such as plasmids and transposons. Objectives: This study evaluated the positivity rate of the presence of blaKPC and blaNDM genes in carbapenem-resistant enterobacteria received at the Instituto Adolfo Lutz (IAL) of São José do Rio Preto, Brazil and determined the epidemiological data related to the patients whose isolates were recovered. Methods: From June 2015 to April 2019, bacterial isolates were obtained from different hospitals located in five municipalities in São José do Rio Preto region. In the bacteriology and molecular biology laboratory, DNA extraction and real-time PCR were performed to investigate the blaKPC and blaNDM genes. Afterwards, epidemiological data were surveyed such as the municipality of origin, age, and gender of the patients whose bacterial isolates were recovered. Results: A total of 934 enterobacteria isolates were recovered from the different hospitals. Of these; 93.4% were positive for blaKPC, with 96.3%, 1.85%, and 1.85% of the isolates belonged to the Klebsiella genus, Enterobacter genus, and Escherichia coli species, respectively. Also, 52.5% and 84.4% of the isolates were obtained from women and elderly patients, respectively. The blaNDM gene was detected only in three isolates, two of which originated from surveillance cultures. Conclusion: Therefore, KPC-producing enterobacteria are widespread in all health units of the five municipalities that were studied, suggesting that the blaKPC-carrying Klebsiella sp. isolates may be endemic in these institutions. Additionally, there is a significant role of surveillance cultures in preventing the spread of resistance genes, as observed for blaNDM in this study.

Bioremediation potential of glyphosate-degrading microorganisms in eutrophicated Ecuadorian water bodies

Phosphonate compounds are the basis of many xenobiotic pollutants, such as Glyphosate (N-(phosphonomethyl-glycine). Only procaryotic microorganisms and the lower eukaryotes are capable of phosphonate biodegradation through C–P lyase pathways. Thus, the aim of this study was to determine the presence of C–P lyase genes in Ecuadorian freshwater systems as a first step towards assessing the presence of putative glyphosate degraders. To that end, two Nested PCR assays were designed to target the gene that codifies for the subunit J (phnJ), which breaks the C-P bond that is critical for glyphosate mineralization. The assays designed in this study led to the detection of phnJ genes in 7 out of 8 tested water bodies. The amplified fragments presented 85–100% sequence similarity with phnJ genes that belong to glyphosate-degrading microorganisms. Nine sequences were not reported previously in the GenBank. The presence of phosphonate degraders was confirmed by isolating three strains able to grow using glyphosate as a unique carbon source. According to the 16S sequence, these strains belong to the Pantoea, Pseudomonas, and Klebsiella genera. Performing a Nested PCR amplification of phnJ genes isolated from eutrophicated water bodies, prior to isolation, may be a cost-effective strategy for the bioprospection of new species and/or genes that might have new properties for biotech industries, laying the groundwork for additional research.

Taxonomic composition of bacterioplankton of large freshwater lakes of the Yeravninskaya depression

Bacterioplankton community taxonomic composition of four large freshwater lakes of the Yeravninskaya depression (Bolshoye Yeravnoye, Gunda, Isinga, and Sosnovoye) which have great fishery significance for the region has been studied. The microbial community consists in the main of bacterial phyla Gammaproteobacteria (22-74%), Actinobacteria (4-35%), Firmicutes (1.17-34.6%), Bacteroidetes (0.3-7%), Cyanobacteria (0.9-6.5%) and Planctomycetes (1.5-10.5%). Total dissolved solids and sulphates accounted for 57% of the variation in the microbial community distribution. At the genus level, Acinetobacter dominated (average 21.4-25.2%). Pseudomonas, Exiguobacterium, and Massilia were also widespread. Their predominance may indicate a possible change in the structure of the community caused by natural and anthropogenic factors. In lakes Isinga and Gunda, a large number of bacteria of the genus Klebsiella was observed, Staphylococcus and Streptococcus were detected. These features indicate insufficient water quality as a result of anthropogenic impact.

Metagenomic analysis to identify the causative agents of atypical urogenital tract infections

Background. Research in recent decades has shown that classical microbiological methods can only detect a small, cultivable portion of microorganisms. One of the modern approaches to detect a wide range of bacteria and archaea is presented as a type of metagenomic analysis performed by high-throughput sequencing of ribosomal operon fragment libraries. Objective is to conduct metagenomic analysis of samples from the urogenital tract of patients with chronic inflammation to identify pathogens not detected by other methods. Methods. Taxonomic analysis of the bacterial community was performed by high-throughput sequencing of the V3–V4 hypervariable region of the 16S rRNA gene by using the Illumina HiSeq 3000 platform. Results. The study allowed to identify the taxonomic diversity of microorganisms in samples from the urogenital tract (from 197 to 794 different microorganisms belonging to the Bacteria were identified), as well as to establish differential differences concerning members of the genera Megasphaera, Prevotella, Veillonella, Pedobacter, Mobiluncus, Phobormidium, Sphing Temperatibacter, Oxobacter, Georgenia, Actinobaculum, Varibaculum, Mycobacterium, Rhodococcus, Sediminihabitans, Actinobacter, Actinoplanes, Spirochaeta, Enhydrobacter, Thermacetogenium, Bdellovibrio, Oleibacter, Porphyromonas, Klebsiella, Lachnoclostridium, Caulobacter, Xanthomonas, Novispirillum, Marvinbryantia, Afipia, Shinella, Tepidimonas, Faecalibacterium, Paludibacterium, Aerococcus, Campylobacter, Pasteurella, Rumen, Psychrobacter, Haemophilus, Brevibacillus, Sporosarcina, Yaniella and Lactobacillus between samples from patients with chronic inflammation and apparently healthy individuals. Conclusion. Differential differences were found in the composition of the microbiome from the samples of patients with chronic inflammation and apparently healthy individuals, concerning members of 44 genera, including Megasphaera, Prevotella, Veillonella, Pedobacter, Mobiluncus, Phormidium and Lactobacillus. The inflammatory processes observed in the patient urogenital tract can be associated with imbalanced microflora such as decreased level of typical members of the genera Staphylococcus, Streptococcus and Lactobacillus, but increased number of members from of the genera Klebsiella and Citrobacter.

A Comprehensive Computer Aided Vaccine Design Approach to Propose a Multi-Epitopes Subunit Vaccine against Genus Klebsiella Using Pan-Genomics, Reverse Vaccinology, and Biophysical Techniques.

Klebsiella is a genus of nosocomial bacterial pathogens and is placed in the most critical list of World Health Organization (WHO) for development of novel therapeutics. The pathogens of the genus are associated with high mortality and morbidity. Owing to their strong resistance profile against different classes of antibiotics and nonavailability of a licensed vaccine, urgent efforts are required to develop a novel vaccine candidate that can tackle all pathogenic species of the Klebsiella genus. The present study aims to design a broad-spectrum vaccine against all species of the Klebsiella genus with objectives to identify the core proteome of pathogen species, prioritize potential core vaccine proteins, analyze immunoinformatics of the vaccine proteins, construct a multi-epitopes vaccine, and provide its biophysical analysis. Herein, we investigated all reference species of the genus to reveal their core proteome. The core proteins were then subjected to multiple reverse vaccinology checks that are mandatory for the prioritization of potential vaccine candidates. Two proteins (TonB-dependent siderophore receptor and siderophore enterobactin receptor FepA) were found to fulfill all vaccine parameters. Both these proteins harbor several potent B-cell-derived T-cell epitopes that are antigenic, nonallergic, nontoxic, virulent, water soluble, IFN-γ producer, and efficient binder of DRB*0101 allele. The selected epitopes were modeled into a multi-epitope peptide comprising linkers and Cholera Toxin B adjuvant. For docking with innate immune and MHC receptors and afterward molecular dynamics simulations and binding free energy analysis, the vaccine structure was modeled for tertiary structure and refined for structural errors. To assess the binding affinity and presentation of the designed vaccine construct, binding mode and interactions analysis were performed using molecular docking and molecular dynamics simulation techniques. These biophysical approaches illustrated the vaccine as a good binder to the immune receptors and revealed robust interactions energies. The vaccine sequence was further translated to nucleotide sequence and cloned into an appropriate vector for expressing it at high rate in Escherichia coli K12 strain. In addition, the vaccine was illustrated to generate a good level of primary, secondary, and tertiary immune responses, proving good immunogenicity of the vaccine. Based on the reported results, the vaccine can be a good candidate to be evaluated for effectiveness in wet laboratory validation studies.

Prevalence and antibiotic susceptibility pattern of Klebsiella species isolated from various clinical samples in a tertiary care hospital Coimbatore

Background: The genus Klebsiella of Enterobacteriaceae family is ubiquitous in nature. They cause many nosocomial infections like pneumonia, urinary tract infections, wound infections, bacteremia and septicemia. Multidrug resistance is seen in Klebsiella which serves as the most common cause of increased morbidity and mortality. This study reveals the prevalence and antibiotic sensitivity pattern of Klebsiella species from various clinical samples. The primary objectives are as follows: To isolate and characterize Klebsiella species from various clinical samples. To study the antibiotic susceptibility pattern of Klebsiella isolates.Methods: This prospective study was conducted in our tertiary care hospital during the period from August 2019 to October 2019. A total of 3521 samples were tested during this period. The samples include blood, sputum, urine, and pus.Results: Out of the total samples tested,1106 samples were showing the growth of the organisms. Among this,351 were identified as Klebsiella species and the highest rate of isolation of Klebsiella species is from the sputum sample and also the same was reported high in medical wards. The study also shows that the isolation of Klebsiella species shows male preponderance when compared to females. The antibiotic sensitivity pattern was done by Kirby-Bauer's disc diffusion method and the sensitivity was noted to be higher to amikacin and ciprofloxacin.Conclusions: Thus, this study shows the prevalence rate of Klebsiella species and sensitivity pattern of Klebsiella, which may help select appropriate antibiotics and prevent overuse and misuse of antibiotics.

Novel Drexlerviridae bacteriophage KMI8 with specific lytic activity against Klebsiella michiganensis and its biofilms.

The bacterial genus Klebsiella includes the closely related species K. michiganensis, K. oxytoca and K. pneumoniae, which are capable of causing severe disease in humans. In this report we describe the isolation, genomic and functional characterisation of the lytic bacteriophage KMI8 specific for K. michiganensis. KMI8 belongs to the family Drexlerviridae, and has a novel genome which shares very little homology (71.89% identity over a query cover of only 8%) with that of its closest related bacteriophages (Klebsiella bacteriophage LF20 (MW417503.1); Klebsiella bacteriophage 066039 (MW042802.1). KMI8, which possess a putative endosialidase (depolymerase) enzyme, was shown to be capable of degrading mono-biofilms of a strain of K. michiganensis that carried the polysaccharide capsule KL70 locus. This is the first report of a lytic bacteriophage for K. michiganensis, which is capable of breaking down a biofilm of this species.

Prediction of Minimal Inhibitory Concentration of Meropenem Against Klebsiella pneumoniae Using Metagenomic Data.

Minimal inhibitory concentration (MIC) is defined as the lowest concentration of an antimicrobial agent that can inhibit the visible growth of a particular microorganism after overnight incubation. Clinically, antibiotic doses for specific infections are determined according to the fraction of MIC. Therefore, credible assessment of MICs will provide a physician valuable information on the choice of therapeutic strategy. Early and precise usage of antibiotics is the key to an infection therapy. Compared with the traditional culture-based method, the approach of whole genome sequencing to identify MICs can shorten the experimental time, thereby improving clinical efficacy. Klebsiella pneumoniae is one of the most significant members of the genus Klebsiella in the Enterobacteriaceae family and also a common non-social pathogen. Meropenem is a broad-spectrum antibacterial agent of the carbapenem family, which can produce antibacterial effects of most Gram-positive and -negative bacteria. In this study, we used single-nucleotide polymorphism (SNP) information and nucleotide k-mers count based on metagenomic data to predict MICs of meropenem against K. pneumoniae. Then, features of 110 sequenced K. pneumoniae genome data were combined and modeled with XGBoost algorithm and deep neural network (DNN) algorithm to predict MICs. We first use the XGBoost classification model and the XGBoost regression model. After five runs, the average accuracy of the test set was calculated. The accuracy of using nucleotide k-mers to predict MICs of the XGBoost classification model and XGBoost regression model was 84.5 and 89.1%. The accuracy of SNP in predicting MIC was 80 and 81.8%, respectively. The results show that XGBoost regression is better than XGBoost classification in both nucleotide k-mers and SNPs to predict MICs. We further selected 40 nucleotide k-mers and 40 SNPs with the highest correlation with MIC values as features to retrain the XGBoost regression model and DNN regression model. After 100 and 1,000 runs, the results show that the accuracy of the two models was improved. The accuracy of the XGBoost regression model for k-mers, SNPs, and k-mers & SNPs was 91.1, 85.2, and 91.3%, respectively. The accuracy of the DNN regression model was 91.9, 87.1, and 91.8%, respectively. Through external verification, some of the selected features were found to be related to drug resistance.

Resistance patterns and clinical outcomes of Klebsiella pneumoniae and invasive Klebsiella variicola in trauma patients.

Recent reclassification of the Klebsiella genus to include Klebsiella variicola, and its association with bacteremia and mortality, has raised concerns. We examined Klebsiella spp. infections among battlefield trauma patients, including occurrence of invasive K. variicola disease. Klebsiella isolates collected from 51 wounded military personnel (2009-2014) through the Trauma Infectious Disease Outcomes Study were examined using polymerase chain reaction (PCR) and pulsed-field gel electrophoresis. K. variicola isolates were evaluated for hypermucoviscosity phenotype by the string test. Patients were severely injured, largely from blast injuries, and all received antibiotics prior to Klebsiella isolation. Multidrug-resistant Klebsiella isolates were identified in 23 (45%) patients; however, there were no significant differences when patients with and without multidrug-resistant Klebsiella were compared. A total of 237 isolates initially identified as K. pneumoniae were analyzed, with 141 clinical isolates associated with infections (remaining were colonizing isolates collected through surveillance groin swabs). Using PCR sequencing, 221 (93%) isolates were confirmed as K. pneumoniae, 10 (4%) were K. variicola, and 6 (3%) were K. quasipneumoniae. Five K. variicola isolates were associated with infections. Compared to K. pneumoniae, infecting K. variicola isolates were more likely to be from blood (4/5 versus 24/134, p = 0.04), and less likely to be multidrug-resistant (0/5 versus 99/134, p<0.01). No K. variicola isolates demonstrated the hypermucoviscosity phenotype. Although K. variicola isolates were frequently isolated from bloodstream infections, they were less likely to be multidrug-resistant. Further work is needed to facilitate diagnosis of K. variicola and clarify its clinical significance in larger prospective studies.

Detection of carbapenemase-producing, hypervirulent Klebsiella spp. in wastewater and their potential transmission to river water and WWTP employees

Wastewater treatment plants (WWTPs) release drug-resistant microorganisms to water bodies (with effluents), and WWTP employees are exposed to bioaerosol emissions from the processed wastewater. Bacteria of the genus Klebsiella, in particular carbapenemase-producing (CP), hyper-virulent (Hvr) strains of Klebsiella pneumoniae, play a special role in this process. Klebsiella spp. strains isolated from wastewater, river water and the upper respiratory tract of WWTP employees were analyzed in this study. The isolated strains were identified as K. pneumoniae (K. pn) or K. non-pneumoniae (K. npn). The prevalence of nine types of genes encoding resistance to beta-lactams, nine genes encoding virulence factors and K1/K2 capsular serotypes, three genes encoding multi drug effluent pump systems, and the class 1 integron-integrase gene was determined by PCR. A total of 284 Klebsiella spp. isolates were obtained in the study: 270 environmental strains and 14 strains from the upper respiratory tract. Among environmental isolates 90.7% (245/270) harbored beta-lactam resistance genes, 17.4% (47/270) were classified as CP strains, 11.1% (30/270) were classified as Hvr strains, and 1.9% (5/270) were classified as CP-Hvr strains. CP-Hvr strains were also isolated from WWTP employees. Genes encoding β-lactamases (including carbapenemases), complete efflux pump systems and the K1 serotype were identified more frequently in K. pn strains. In turn, K. npn strains were characterized by a higher prevalence of blaSHV and intI1 genes and K2 serotype gene. The strains isolated from wastewater and river water also differed in the abundance of drug resistance and virulence genes. The results of the study indicate that CP-Hvr K. pn strains are possibly transmitted from wastewater via bioareosol to the upper respiratory tract of WWTP employees. blaGES-type carbapenemases significantly contributed to the spread of drug resistance in the environment.