Production of α-ketoisovalerate with whey powder by systemic metabolic engineering of Klebsiella oxytoca.

BACKGROUND: Whey, which has high biochemical oxygen demand and chemical oxygen demand, is mass-produced as a major by-product of the dairying industry. Microbial fermentation using whey as the carbon source may convert this potential pollutant into value-added products. This study investigated the potential of using whey powder to produce α-ketoisovalerate, an important platform chemical. RESULTS: Klebsiella oxytoca VKO-9, an efficient L-valine producing strain belonging to Risk Group 1 organism, was selected for the production of α-ketoisovalerate. The leucine dehydrogenase and branched-chain α-keto acid dehydrogenase, which catalyzed the reductive amination and oxidative decarboxylation of α-ketoisovalerate, respectively, were inactivated to enhance the accumulation of α-ketoisovalerate. The production of α-ketoisovalerate was also improved through overexpressing α-acetolactate synthase responsible for pyruvate polymerization and mutant acetohydroxyacid isomeroreductase related to α-acetolactate reduction. The obtained strain K. oxytoca KIV-7 produced 37.3 g/L of α-ketoisovalerate from lactose, the major utilizable carbohydrate in whey. In addition, K. oxytoca KIV-7 also produced α-ketoisovalerate from whey powder with a concentration of 40.7 g/L and a yield of 0.418 g/g. CONCLUSION: The process introduced in this study enabled efficient α-ketoisovalerate production from low-cost substrate whey powder. Since the key genes for α-ketoisovalerate generation were integrated in genome of K. oxytoca KIV-7 and constitutively expressed, this strain is promising in stable α-ketoisovalerate fermentation and can be used as a chassis strain for α-ketoisovalerate derivatives production.

The plasmid-borne hipBA operon of Klebsiella michiganensis encodes a potent plasmid stabilization system.

AIMS: Klebsiella michiganensis is a medically-important bacterium that has been subject to relatively little attention in the literature. Interrogation of sequence data from K. michiganensis strains in our collection has revealed the presence of multiple large plasmids encoding type II toxin-antitoxin (TA) systems. Such TA systems are responsible for mediating a range of phenotypes including plasmid stability ("addiction") and antibiotic persistence. In this work, we characterize the hipBA TA locus found within the Klebsiella oxytoca species complex (KoSC). METHODS AND RESULTS: The HipBA TA system is encoded on a plasmid carried by K. michiganensis PS_Koxy4, isolated from an infection outbreak. Employing viability and plasmid stability assays, we demonstrate that PS_Koxy4 HipA is a potent antibacterial toxin and that HipBA is a functional TA module contributing substantially to plasmid maintenance. Further, we provide in silico data comparing HipBA modules across the entire KoSC. CONCLUSIONS: We provide the first evidence of the role of a plasmid-encoded HipBA system in stability of mobile genetic elements and analyze the presence of HipBA across the KoSC. These results expand our knowledge of both a common enterobacterial TA system and a highly medically-relevant group of bacteria.

Production of exopolysaccharide from Klebsiella oxytoca: Rheological, emulsifying, biotechnological properties, and bioremediation applications.

Bacteria can synthesize a broad spectrum of multifunctional polysaccharides including extracellular polysaccharides (EPS). Bacterial EPS can be utilized in the food, pharmaceutical, and biomedical areas owing to their physical and rheological properties in addition to generally presenting low toxicity. From an ecological viewpoint, EPS are biodegradable and environment compatible, offering several advantages over synthetic compounds. This study investigated the EPS produced by Klebsiella oxytoca (KO-EPS) by chemically characterizing and evaluating its properties. The monosaccharide components of the KO-EPS were determined by HPLC coupled with a refractive index detector and GC-MS. The KO-EPS was then analyzed by methylation analysis, FT-IR and NMR spectroscopy to give a potential primary structure. KO-EPS demonstrated the ability to stabilize hydrophilic emulsions with various hydrophobic compounds, including hydrocarbons and vegetable and mineral oils. In terms of iron chelation capacity, the KO-EPS could sequester 41.9 % and 34.1 % of the most common iron states, Fe2+ and Fe3+, respectively. Moreover, KO-EPS exhibited an improvement in the viscosity of aqueous dispersion, being proportional to the increase in its concentration and presenting a non-Newtonian pseudoplastic flow behavior. KO-EPS also did not present a cytotoxic effect indicating that the KO-EPS could have potential applications as a natural thickener, bioemulsifier, and bioremediation agent.

Epidemiology and antimicrobial resistance of toxin-producing Klebsiella oxytoca clinical isolates from children admitted to the oncology chemotherapy center in Mofid Children's Hospital in Tehran, Iran: A cross-sectional study.

Background and Aims: Klebsiella oxytoca (K. oxytoca) is the second bacterial cause of nosocomial infections in the general population after K. pneumoniae. This study surveyed the frequency of cytotoxin-producing strains of K. oxytoca and their antibiotic susceptibility profile in a cohort of children admitted to a referral hospital with different malignancies. Methods: The Stool samples of children admitted to the Cancer Chemotherapy Unit of the Mofid Children's Hospital, Tehran, Iran were analyzed using conventional biochemical tests and polymerase chain reaction targeting the pehX gene to identify K. oxytoca. The antibiotic susceptibility profile of isolated K. oxytoca against commonly prescribed antibiotics used in treating infection at the facility was determined using the Kirby-Bauer disk diffusion technique. Also, the prevalence of genes encoding toxins among K. oxytoca was identified by PCR assay. Results: The Stool samples of 280 participants were taken for the study of which 38 samples [(55.3% (21/38) 42 males and 44.7% (17/38) females)] tested positive for various Klebsiella spp. Out of this, K. oxytoca was identified in 2.5% (7/280) stools using cultures and conventional biochemical tests. Also, the stools of 2.9% (8/280) of the participants tested positive for K. oxytoca using PCR assay. Using PCR, (2/7) of the K. oxytoca isolates tested positive for the npsA and npsB genes and were identified as toxigenic K. oxytoca strains. Conclusion: The prevalence of toxin-producing K. oxytoca strains in stool samples of children diagnosed with cancer in Iran is relatively low. Most of the K. oxytoca isolates were susceptible to tested antibiotics. Globally, active surveillance of toxigenic K. oxytoca strains in patients with different malignancies or immunocompromised patients is recommended in healthcare settings.

Comprehensive genomics reveals novel sequence types of multidrug resistant Klebsiella oxytoca with uncharacterized capsular polysaccharide K- and lipopolysaccharide O-antigen loci from the National Hospital of Uganda.

The Klebsiella oxytoca complex comprises diverse opportunistic bacterial pathogens associated with hospital and community-acquired infections with growing alarming antimicrobial resistance. We aimed to uncover the genomic features underlying the virulence and antimicrobial resistance of isolates from Mulago National Hospital in Uganda. We coupled whole genome sequencing with Pathogenwatch multilocus sequence typing (MLST) and downstream bioinformatic analysis to delineate sequence types (STs) capsular polysaccharide K- and O-antigen loci, along with antimicrobial resistance (AMR) profiles of eight clinical isolates from the National Referral Hospital of Uganda. Our findings revealed that only two isolates (RSM6774 and RSM7756) possess a known capsular polysaccharide K-locus (KL74). The rest carry various unknown K-loci (KL115, KL128, KLI52, KL161 and KLI63). We also found that two isolates possess unknown loci for the lipopolysaccharide O-antigen (O1/O2v1 type OL104 and unknown O1). The rest possess known O1 and O3 serotypes. From MLST, we found four novel sequence types (STs), carrying novel alleles for the housekeeping genes glyceraldehyde-6-phosphate dehydrogenase A (gapA), glucose-6-phosphate isomerase (pgi), and RNA polymerase subunit beta (rpoB). Our AMR analysis revealed that all the isolates are resistant to ampicillin and ceftriaxone, with varied resistance to other antibiotics, but all carry genes for extended-spectrum beta-lactamases (ESBLs). Notably, one strain (RSM7756) possesses outstanding chromosomal and plasmid-encoded AMR to beta-lactams, cephalosporins, fluoroquinolones and methoprims. Conclusively, clinical samples from Mulago National Referral Hospital harbor novel STs and multidrug resistant K. oxytoca strains, with significant public health importance, which could have been underrated.

Systematic reengineering of Klebsiella oxytoca KC004-TF160 for enhancing metabolic carbon flux towards succinate production pathway.

Klebsiella oxytoca KP001-TF60 (ΔadhEΔpta-ackAΔldhAΔbudABΔpflBΔtdcDΔpmd) was re-engineered to direct more carbon flux towards succinate production with less acetate. Glucose uptake, cell growth, and carbon distribution were restricted by alterations in relative expressions and nucleotide sequences of genes associated with PEP and pyruvate metabolisms. Transcripts of pck, ppc, and frd genes were up-regulated for enhancing NADH reoxidation during succinate production while increased pyk and tdcE transcripts were observed due to maintenance of acetyl-CoA through the oxidative branch of TCA cycle. Based on whole-genome sequencing, several genes in sugars-specific PTS (ptsG, bglF, chbR, fruA, mtlR, and treY), ABC transporters (alsK, and rbsK), Major Facilitator Superfamily (uhpB and setB), and catabolite repression (cyaA and csrB) were found to be mutated. The strain produced succinate yield up to 0.89 g/g (∼80 % theoretical maximum) with acetate < 1 g/L, and may be one of the succinate producers applied in an industrial-production scale with simplified purification processes.

Bacterial septicemia and herpesvirus infection in Antarctic fur seals (Arctocephalus gazella) stranded in the São Paulo coast, Brazil.

In August 2021, two juvenile male Antarctic fur seals (Arctocephalus gazella) stranded in the southeastern Brazilian coast and were referred to rehabilitation centers. The animals presented increased body temperature, prostration, respiratory distress and despite treatment died. A necropsy following a standardized protocol was performed, and formalin-fixed tissues were processed for microscopic examination. Samples were screened for morbillivirus, herpesvirus, and Brucella spp. by molecular analyses (PCR, RT-PCR). Bacteriological culture was performed in samples collected from the lungs, trachea, and lymph nodes of both cases. The main histopathologic findings were of infectious nature, including multifocal necrotizing and fibrinous mixed interstitial pneumonia, bronchiolitis, and bronchitis, with intralesional myriad bacteria associated with vascular fibrinoid necrosis. Pseudomonas aeruginosa was isolated from tracheal and lung swabs of Case 1, and Klebsiella oxytoca was found in nostril swabs, tracheobronchial lymph nodes, and lung of Case 2. Gammaherpesvirus infection was detected in both cases, and the sequences retrieved were classified into the genus Percavirus. All tested samples were PCR-negative for Brucella spp. and morbillivirus. We hypothesize that the deficient immunological status in association with starvation predisposed the reactivation of herpesvirus and secondary bacterial co-infections. To the authors' knowledge, this is the first molecular detection of herpesvirus in an Antarctic pinniped. These findings reinforce that Otariid gammaherpesvirus circulating in the Southern Hemisphere are likely endemic in the Arctocephalus genus. This report contributes to the current knowledge of health aspects affecting wild pinnipeds, especially in the poorly studied Antarctic species.

Clinical differences between Raoultella spp. and Klebsiella oxytoca.

Purpose: Raoultella spp. is a genus of bacteria that is known to be closely related to Klebsiella. It has been debated whether Raoultella should be reclassified as a subgroup of Klebsiella. The aim of this study is to compare clinical aspects of Raoultella and Klebsiella oxytoca, a species of Klebsiella that is known to be bacteriologically similar to Raoultella spp. Methods: Using data collected at a tertiary care hospital in the United States, we identified 43 patients with Raoultella infection and 1173 patients with Klebsiella oxytoca infection. We compared patient demographics (age and sex), hospitalization status, isolation sites and antibiotic resistance profiles between the two species. Results: There was no significant difference in patient demographics between the two bacteria species. The proportions of intensive care unit (ICU) admission were higher among patients with Raoultella infection (p=0.008). The most common site of isolation was urine for both species (39.5% of all patients with Raoultella spp. vs. 59.3% for K. oxytoca). The second most common site of isolation was blood stream for Raoultella spp. (23.3%) and respiratory tract for K. oxytoca (10.8%). Except for the high proportion of resistant isolates of Raoultella spp. for Trimethoprim/sulfamethoxazole, the antibiotic susceptibility profiles were similar between the two bacteria species. Both were susceptible to ciprofloxacin and meropenem. Conclusion: While there are no significant differences in the patient demographics and antibiotic susceptibility profiles between Raoultella spp. and K. oxytoca, Raoultella may cause more serious infection requiring ICU admissions. Also, Raoultella may cause blood stream infection more frequently than K. oxytoca.

Core genome multilocus sequence typing (cgMLST) applicable to the monophyletic Klebsiella oxytoca species complex.

The environmental bacterium Klebsiella oxytoca displays an alarming increase of antibiotic-resistant strains that frequently cause outbreaks in intensive care units. Due to its prevalence in the environment and opportunistic presence in humans, molecular surveillance (including resistance marker screening) and high-resolution cluster analysis are of high relevance. Furthermore, K. oxytoca previously described in studies is rather a species complex (KoSC) than a single species comprising at least six closely related species that are not easily differentiated by standard typing methods. To reach a discriminatory power high enough to identify and resolve clusters within these species, whole genome sequencing is necessary. The resolution is achievable with core genome multilocus sequence typing (cgMLST) extending typing of a few housekeeping genes to thousands of core genome genes. CgMLST is highly standardized and provides a nomenclature enabling cross laboratory reproducibility and data exchange for routine diagnostics. Here, we established a cgMLST scheme not only capable of resolving the KoSC species but also producing reliable and consistent results for published outbreaks. Our cgMLST scheme consists of 2,536 core genome and 2,693 accessory genome targets, with a percentage of good cgMLST targets of 98.31% in 880 KoSC genomes downloaded from the National Center for Biotechnology Information (NCBI). We also validated resistance markers against known resistance gene patterns and successfully linked genetic results to phenotypically confirmed toxic strains carrying the til gene cluster. In conclusion, our novel cgMLST enables highly reproducible typing of four different clinically relevant species of the KoSC and thus facilitates molecular surveillance and cluster investigations.

Kinetic and thermodynamic investigation of Rhodanese synthesized by enhanced Klebsiella oxytoca JCM 1665 strain: a comparative between the free and immobilized enzyme entrapped in alginate beads.

Klebsiella oxytoca JCM 1665 was subjected to extracellular rhodanese production using a submerged fermentation technique. The organism was further engineered for higher cyanide tolerance and rhodanese yield using ethylmethanesulfonate as a mutagen. Mutagenesis resulted in an improved mutant with high cyanide tolerance (100 mM) and rhodanese yield (26.7 ± 0.67 U/mL). This yield was 4.34-fold higher than the wild strain (6.15 ± 0.65 U/mL). At temperatures ranging from 30 to 80 °C, the first-order thermal denaturation constant (Kd) for free enzyme increases from 0.00818 to 0.0333 min-1 while the immobilized enzyme increases from 0.003 to 0.0204 min-1. The equivalent half-life reduces from 99 to 21 minutes and 231 to 35 minutes, respectively. Residual activity tests were used to assess the thermodynamic parameters for both enzyme preparations. For the free enzyme, the parameters obtained were enthalpy (29.40 to 29.06 kJ.mol-1), entropy (-194.24 to -197.50 J.mol-1K-1) and Gibbs free energy (90.20 to 98.80 kJ.mol-1). In addition, for immobilized rhodanese, we obtained enthalpy (40.40 to 40.07 kJ.mol-1), entropy (-164.21 to - 165.20 J.mol-1K-1) and Gibbs free energy (91.80 to 98.40 kJ.mol-1. Regarding its operational stability, the enzyme was able to maintain 63% of its activity after being used for five cycles. Immobilized K. oxytoca rhodanese showed a marked resistance to heat inactivation compared to free enzyme forms; making it of utmost significance in many biotechnological applications.

Adhesion of Klebsiella oxytoca to bladder or lung epithelial cells is promoted by the presence of other opportunistic pathogens.

The intestinal and respiratory tracts of healthy individuals serve as habitats for a diverse array of microorganisms, among which Klebsiella oxytoca holds significance as a causative agent in numerous community- and hospital-acquired infections, often manifesting in polymicrobial contexts. In specific circumstances, K. oxytoca, alongside other constituents of the gut microbiota, undergoes translocation to distinct physiological niches. In these new environments, it engages in close interactions with other microbial community members. As this interaction may progress to co-infection where the virulence of involved pathogens may be promoted and enhance disease severity, we investigated how K. oxytoca affects the adhesion of commonly co-isolated bacteria and vice versa during co-incubation of different biotic and abiotic surfaces. Co-incubation was beneficial for the adhesion of at least one of the two co-cultured strains. K. oxytoca enhanced the adhesion of other enterobacteria strains to polystyrene and adhered more efficiently to bladder or lung epithelial cell lines in the presence of most enterobacteria strains and S. aureus. This effect was accompanied by bacterial coaggregation mediated by carbohydrate-protein interactions occurring between bacteria. These interactions occur only in sessile, but not planktonic populations, and depend on the features of the surface. The data are of particular importance for the risk assessment of the urinary and respiratory tract infections caused by K. oxytoca, including those device-associated. In this paper, we present the first report on K. oxytoca ability to acquire increased adhesive capacities on epithelial cells through interactions with common causal agents of urinary and respiratory tract infections.

Scolopax rusticola Carrying Enterobacterales Harboring Antibiotic Resistance Genes.

The Eurasian woodcock (Scolopax rusticola) belongs to those bird species that make systematic migratory flights in spring and autumn in search of favorable breeding and wintering areas. These specimens arrive in the Mediterranean Area from northeastern European countries during the autumn season. The purpose of this study was to assess whether woodcocks can carry antibiotic resistance genes (ARGs) along their migratory routes. Although the role of migratory birds in the spread of some zoonotic diseases (of viral and bacterial etiology) has been elucidated, the role of these animals in the spread of antibiotic resistance has not yet been clarified. In this study, we analyzed the presence of beta-lactam antibiotic resistance genes. The study was conducted on 69 strains from 60 cloacal swabs belonging to an equal number of animals shot during the 2022-2023 hunting season in Sicily, Italy. An antibiogram was performed on all strains using the microdilution method (MIC) and beta-lactam resistance genes were investigated. The strains tested showed no phenotypic resistance to any of the 13 antibiotics tested; however, four isolates of Enterobacter cloacae and three of Klebsiella oxytoca were found to carry the blaIMP-70, blaVIM-35, blaNDM-5 and blaOXA-1 genes. Our results confirm the importance of monitoring antimicrobial resistance among migratory animals capable of long-distance bacteria spread.

An adapted method for Cas9-mediated editing reveals the species-specific role of ß-glucoside utilization driving competition between Klebsiella species.

Cas9-based gene editing tools have revolutionized genetics, enabling the fast and precise manipulation of diverse bacterial species. However, widely applicable genetic tools for non-model gut bacteria are unavailable. Here, we present a two-plasmid Cas9-based system designed for gene deletion and knock-in complementation in three members of the Klebsiella oxytoca species complex (KoSC), which we applied to study the genetic factors underlying the role of these bacteria in competition against Klebsiella pneumoniae. Firstly, the system allowed efficient and precise full-length gene deletion via enhanced lambda Red expression. Furthermore, we tested the efficiency of two independent, functionally validated complementation strategies. Ultimately, the insertion of universal "bookmark" targets during gene deletion subsequently allows the most optimal genetic complementation in K. oxytoca, Klebsiella michiganensis, and Klebsiella grimontii. This approach offers a significant advantage by enabling the use of a single high-efficiency "bookmark" for complementing other loci or strains, eliminating the need for site-specific design. We revealed that the carbohydrate permease CasA is critical in ex vivo assays for K. pneumoniae inhibition by K. oxytoca but is neither sufficient nor required for K. michiganensis and K. grimontii. Thus, the adaptation of state-of-the-art genetic tools to KoSC allows the identification of species-specific functions in microbial competition. IMPORTANCE: Cas9-based gene editing tools have revolutionized bacterial genetics, yet, their application to non-model gut bacteria is frequently hampered by various limitations. We utilized a two-plasmid Cas9-based system designed for gene deletion in Klebsiella pneumoniae and demonstrate after optimization its utility for gene editing in three members of the Klebsiella oxytoca species complex (KoSC) namely K. oxytoca, Klebsiella michiganensis, and Klebsiella grimontii. We then adapted a recently developed protocol for functional complementation based on universal "bookmark" targets applicable to all tested species. In summary, species-specific adaptation of state-of-the-art genetic tools allows efficient gene deletion and complementation in type strains as well as natural isolates of KoSC members to study microbial interactions.

Prevalence, regional distribution, and trends of antimicrobial resistance among female outpatients with urine Klebsiella spp. isolates: a multicenter evaluation in the United States between 2011 and 2019.

BACKGROUND: Antimicrobial resistance research in uncomplicated urinary tract infection typically focuses on the main causative pathogen, Escherichia coli; however, little is known about the antimicrobial resistance burden of Klebsiella species, which can also cause uncomplicated urinary tract infections. This retrospective cohort study assessed the prevalence and geographic distribution of antimicrobial resistance among Klebsiella species and antimicrobial resistance trends for K. pneumoniae in the United States (2011-2019). METHODS: K. pneumoniae and K. oxytoca urine isolates (30-day, non-duplicate) among female outpatients (aged ≥ 12 years) with presumed uUTI at 304 centers in the United States were classified by resistance phenotype(s): not susceptible to nitrofurantoin, trimethoprim/sulfamethoxazole, or fluoroquinolone, extended-spectrum ß-lactamase-positive/not susceptible; and multidrug-resistant based on ≥ 2 and ≥ 3 resistance phenotypes. Antimicrobial resistance prevalence by census division and age, as well as antimicrobial resistance trends over time for Klebsiella species, were assessed using generalized estimating equations. RESULTS: 270,552 Klebsiella species isolates were evaluated (250,719 K. pneumoniae; 19,833 K. oxytoca). The most frequent resistance phenotypes in 2019 were nitrofurantoin not susceptible (Klebsiella species: 54.0%; K. pneumoniae: 57.3%; K. oxytoca: 15.1%) and trimethoprim/sulfamethoxazole not susceptible (Klebsiella species: 10.4%; K. pneumoniae: 10.6%; K. oxytoca: 8.6%). Extended-spectrum ß-lactamase-positive/not susceptible prevalence was 5.4%, 5.3%, and 6.8%, respectively. K. pneumoniae resistance phenotype prevalence varied (p < 0.0001) geographically and by age, and increased over time (except for the nitrofurantoin not susceptible phenotype, which was stable and > 50% throughout). CONCLUSIONS: There is a high antimicrobial resistance prevalence and increasing antimicrobial resistance trends among K. pneumoniae isolates from female outpatients in the United States with presumed uncomplicated urinary tract infection. Awareness of K. pneumoniae antimicrobial resistance helps to optimize empiric uncomplicated urinary tract infection treatment.

Efficient L-valine production using systematically metabolic engineered Klebsiella oxytoca.

L-Valine, a branched-chain amino acid with diversified applications, is biosynthesized with α-acetolactate as the key precursor. In this study, the metabolic flux in Klebsiella oxytoca PDL-K5, a Risk Group 1 organism producing 2,3-butanediol as the major fermentation product, was rearranged to L-valine production by introducing exogenous L-valine biosynthesis pathway and blocking endogenous 2,3-butanediol generation at the metabolic branch point α-acetolactate. After further enhancing L-valine efflux, strengthening pyruvate polymerization and selecting of key enzymes for L-valine synthesis, a plasmid-free K. oxytoca strain VKO-9 was obtained. Fed-batch fermentation with K. oxytoca VKO-9 in a 7.5 L fermenter generated 122 g/L L-valine with a yield of 0.587 g/g in 56 h. In addition, repeated fed-batch fermentation was conducted to prevent precipitation of L-valine due to oversaturation. The average concentration, yield, and productivity of produced L-valine in three cycles of repeated fed-batch fermentation were 81.3 g/L, 0.599 g/g, and 3.39 g/L/h, respectively.

Multicenter comparative genomic study of Klebsiella oxytoca complex reveals a highly antibiotic-resistant subspecies of Klebsiellamichiganensis.

BACKGROUND: The Klebsiella oxytoca complex is an opportunistic pathogen that has been recently identified as an actual complex. However, the characteristics of each species remain largely unknown. We aimed to study the clinical prevalence, antimicrobial profiles, genetic differences, and interaction with the host of each species of this complex. METHODS: One hundred and three clinical isolates of the K. oxytoca complex were collected from 33 hospitals belonging to 19 areas in China from 2020 to 2021. Species were identified using whole genome sequencing based on average nucleotide identity. Clinical infection characteristics of the species were analyzed. Comparative genomics and pan-genome analyses were performed on these isolates and an augmented dataset, including 622 assemblies from the National Center for Biotechnology Information. In vitro assays evaluating the adhesion ability of human respiratory epithelial cells and survivability against macrophages were performed on randomly selected isolates. RESULTS: Klebsiella michiganensis (46.6%, 48/103) and K. oxytoca (35.92%, 37/103) were the major species of the complex causing human infections. K. michiganensis had a higher genomic diversity and larger pan-genome size than did K. oxytoca. K. michiganensis isolates with blaoxy-5 had a higher resistance rate to various antibiotics, antimicrobial gene carriage rate, adhesion ability to human respiratory epithelial cells, and survival rate against macrophages than isolates of other species. CONCLUSION: Our study revealed the genetic diversity of K. michiganensis and firstly identified the highly antimicrobial-resistant profile of K. michiganensis carrying blaoxy-5.

An efficient production of bio-succinate in a novel metabolically engineered Klebsiella oxytoca by rational metabolic engineering and evolutionary adaptation.

Klebsiella oxytoca KC004 (ΔadhEΔpta-ackAΔldhAΔbudABΔpflB) was engineered to enhance succinate production. The strain exhibited poor growth without succinate production due to its deficiencies in ATP production and NADH reoxidation. To overcome obstacles, evolutionary adaptation with over 6,000 generations of growth-based selection was conducted. Under anaerobic conditions, enhanced productions of ATP for growth and succinate for NADH reoxidation by the evolved KC004-TF160 strain were coupled to an increased transcript of PEP carboxykinase (pck) while those of genes in the oxidative branch of TCA cycle (gltA, acnAB, and icd), and pyruvate and acetate metabolisms (pykA, acs, poxB and tdcD) were alleviated. The expression of pyruvate dehydrogenase repressor (pdhR) decreased whereas threonine decarboxylase (tdcE) increased. KC004-TF160 produced succinate at 84 g/L (0.84 g/g, 79 % theoretical maximum). KC004-TF160 produced succinate at 0.87 g/g non-pretreated sugarcane molasses without addition of nutrients and buffers. KC004-TF160 may be a microbial platform for commercial production of bio-succinate.

Sepsis probada a Klebsiella oxytoca en un recién nacido

La sepsis neonatal constituye una de las principales causas de muertes neonatales en los países en desarrollo, con datos que estiman más de un millón de muertes en todo el mundo cada año. Se persigue presentar un caso, dada la infrecuencia de la sepsis neonatal tardía por Klebsiella oxytoca. Se trata de una paciente femenina, pretérmino y de bajo peso al nacer, que a los 17 días de vida comenzó con deterioro de su estado clínico dado por hipoactividad, palidez cutánea, succión morosa e incremento de la circunferencia abdominal, acompañados de disfunción hematológica severa dada por anemia, trombocitopenia y neutropenia, que requirió varias transfusiones con hemoderivados y terapéutica antimicrobiana combinada (primero con meronem y amikacina, luego con ciprofloxacina y vancomicina). Se trató también con antifúngicos, diuréticos, drogas vasoactivas, ventilación mecánica y eritropoyetina. Se interconsultó con Cardiología e Infectología pediátricas. Tuvo finalmente una evolución satisfactoria, con lactancia materna efectiva. El incremento de la sepsis en neonatos hospitalizados y la resistencia bacteriana son problemas de salud pública. Es importante reconocer los factores de riesgo para la sepsis en este grupo de pacientes, para su tratamiento oportuno.

Neonatal sepsis is one of the main causes of neonatal deaths in developing countries, with data estimating more than one million deaths around the world every year. The aim is to present case a case, given the infrequency of late neonatal sepsis by Klebsiella oxytoca. This is the case of a pre-term female patient, with low weight at birth, who at 17 days of birth began with deterioration of her clinical status due to hypo-activity, skin paleness, morose suction and increase in abdominal circumference, accompanied by severe hematological dysfunction given by anemia, thrombocytopenia and neutropenia, which required several transfusions with blood products and combined antimicrobial therapeutic (first with meronem and amikacin, then with ciprofloxacin and vancomycin). She was also treated antifungals, diuretics, vasoactive drugs, mechanical ventilation and erythropoietin. She was consulted with Pediatric Cardiology and Infectious diseases. Finally she had a satisfactory evolution, with effective maternal breastfeeding. Sepsis increase in hospitalized neonates and bacterial resistance are public health problems. It is important to recognize the risk factors for sepsis in this group of patients, for their timely treatment.

Pyogenic liver abscess associated with Klebsiella oxytoca: Mimicking invasive liver abscess syndrome.

A pyogenic liver abscess (PLA) is a space-occupying lesion in the liver that is associated with significant morbidity and mortality. We herein present the case of a Japanese 76-year-old man who visited our hospital with fever and back pain lasting 3 weeks after endoscopic treatment for common bile duct stones. He was accompanied by poorly controlled diabetes mellitus (DM) with an HbA1c of 9.7 %. Laboratory tests disclosed elevated C-reactive protein level (22.1 mg/dL) and white cell count (11,910/µL). Abdominal computed tomography (CT) revealed hypodense lesions in the right liver lobe, with abdominal ultrasonography showing an echogenicity-mixed hypoechoic lesion. Percutaneous needle aspiration of a liver lesion was performed under suspicion of a PLA. Subsequent enhanced CT and magnetic resonance imaging confirmed the hepatic lesions in the right lobe as well as a septic pulmonary embolism, right hepatic vein thrombosis, spondylodiscitis, and a retroperitoneal abscess. Gram staining of the abscess drainage revealed gram-negative bacteria. The above findings indicated invasive liver abscess syndrome (ILAS) caused by Klebsiella pneumoniae. However, further examination of blood, urine, and abscess drainage cultures revealed positivity for Klebsiella oxytoca. This case illustrates that K. oxytoca may cause ILAS-like symptoms. Screening for systemic metastatic infection should be considered in patients with PLA due to K. oxytoca in whom therapeutic intervention has been delayed, especially in patients with poorly controlled DM.

Extended spectrum beta-lactamase carriage among elderly residents of a long-term care facility in Beirut.

BACKGROUND: Antimicrobial resistance is an emerging problem worldwide, endangering antimicrobials efficacy and resulting in high rates of morbidity and mortality. It is one of the major concerns that health care facilities are facing nowadays. Mainly, extended-spectrum beta-lactamases (ESBL)-producing Enterobacterales play a role in hydrolyzing ß-lactams, specifically the third-generation cephalosporins. This study aimed to investigate the prevalence of fecal carriage and molecular characterization of ESBL-producing Enterobacterales among Lebanese elderly residents in a long-term care facility (Dar Al-Ajaza Al Islamia Hospital). METHODS: Rectal culture swab specimens were collected from 132 patients at Dar Al Ajaza Al Islamia hospital between January 2019 till June 2020. The phenotype of ESBL producers was confirmed by a modified double disc synergy test and antibiotic susceptibility was determined using the Kirby-Bauer disk diffusion method. Genotypically, multiplex polymerase chain reaction was used to detect the ESBL genes. RESULTS: The main Enterobacterales strain observed was E coli (90.15%) followed by Klebsiella pneumoniae (4.54%) and Klebsiella oxytoca (3.80%). It has been found that the ESBL percentage rate has decreased when compared to a study conducted previously at the same hospital. Moreover, the predominant ESBL gene was CTX-M (cefotaximase). CONCLUSIONS: This study is the first to demonstrate the improved current status of ESBL in one long-term care facility. In addition, the CTX-M is still the major type in ESBL-producing organisms.