Glutamate production from aerial nitrogen using the nitrogen-fixing bacterium Klebsiella oxytoca.

Glutamate is an essential biological compound produced for various therapeutic and nutritional applications. The current glutamate production process requires a large amount of ammonium, which is generated through the energy-consuming and CO2-emitting Haber-Bosch process; therefore, the development of bio-economical glutamate production processes is required. We herein developed a strategy for glutamate production from aerial nitrogen using the nitrogen-fixing bacterium Klebsiella oxytoca. We showed that a simultaneous supply of glucose and citrate as carbon sources enhanced the nitrogenase activity of K. oxytoca. In the presence of glucose and citrate, K. oxytoca strain that was genetically engineered to increase the supply of 2-oxoglutarate, a precursor of glutamate synthesis, produced glutamate extracellularly more than 1 g L-1 from aerial nitrogen. This strategy offers a sustainable and eco-friendly manufacturing process to produce various nitrogen-containing compounds using aerial nitrogen.

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.

Molecular characterization and virulence profile of Klebsiella pneumoniae and Klebsiella oxytoca isolated from ill cats and dogs in Portugal.

Klebsiella spp. are important pathogens of humans and companion animals such as cats and dogs, capable of causing severe life-threatening diseases. The aim of this study was to characterize the molecular and phenotypic properties of Klebsiella pneumoniae and Klebsiella oxytoca isolated from ill companion animals by whole genome sequencing, followed by in vitro assessment of biofilm formation and in vivo pathogenicity using the Galleria mellonella model. Two LPS O-types were identified for all the K. pneumoniae isolates tested (O3B and O1/O2v2) and only one for K. oxytoca isolates (OL104), and the most common STs found were ST11 and ST266. Furthermore, a high diversity of K-locus types was found for K. pneumoniae (KL102; KL105; KL31, and KL13). Within K. pneumoniae, one specific O/K/ST-types combination (i.e., KL105-ST11-O1/O2v2) showed results that were of concern, as it exhibited a high inflammatory response at 12 h post-infection in G. mellonella with 80% of the larvae dead at 72 h post-infection. This virulence potential, on the other hand, did not appear to be directly related to the biofilm-forming capacity. Also, virulence and resistance scores obtained for this set of strains did surpass score 1. The present study demonstrated that Klebsiella spp. isolated from companion animals belonging to STs that can cause human infections and present virulence on an invertebrate model. Thus, this study underscores the role of dogs and cats as reservoirs of resistant Klebsiella spp. that could potentially be transmitted to humans.

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.

Country-wide expansion of a VIM-1 carbapenemase-producing Klebsiella oxytoca ST145 lineage in Poland, 2009-2019.

PURPOSE: To elucidate the role of the Klebsiella oxytoca species complex (KoSC) in epidemiology of VIM-type MBL-producing Enterobacterales in Poland. METHODS: The study comprised all 106 VIM-positive KoSC isolates collected by the Polish National Reference Centre for Susceptibility Testing during 2009-2019 from 60 institutions in 35 towns. All isolates were sequenced by Illumina MiSeq, followed by MinION sequencing of selected organisms. Genomes were subjected to bioinformatic analysis, addressing taxonomy, clonality, phylogeny and structural characterisation of key resistance determinants within their chromosomal and plasmidic loci. RESULTS: Among five species identified, K. oxytoca was predominant (n = 92), followed by Klebsiella michiganensis (n = 11). MLST distinguished 18 STs, with the most prevalent Klebsiella oxytoca ST145 (n = 83). The clone segregated a lineage with the In237-like integron [blaVIM-1-aacA4 genes; n = 78], recorded in 28 cities almost all over the country. The integron was located in a ~ 49-50 kb chromosomal mosaic region with multiple other resistance genes, linked to a ~ 51 kb phage-like element. The organism might have originated from Greece, and its evolution in Poland included several events of chromosomal ~ 54-258 kb deletions, comprising the natural ß-lactamase blaOXY gene. A group of other isolates of various species and clones (n = 12) carried the integron In916 on self-transmissible IncA-type plasmids, effectively spreading in Italy, France and Poland. CONCLUSION: KoSC has been one of the major VIM producers in Poland, owing largely to clonal expansion of the specific K. oxytoca-In237-like lineage. Its apparently enhanced epidemic potential may create a danger on international scale.

Characterization and diversity of CRISPR/Cas systems in Klebsiella oxytoca.

CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated protein) system is a crucial adaptive immune system for bacteria to resist foreign DNA infection. In this study, we investigated the prevalence and diversity of CRISPR/Cas systems in 175 Klebsiella oxytoca (K. oxytoca) strains. Specifically, 58.86% (103/175) of these strains possessed at least one confirmed CRISPR locus. Two CRISPR/Cas system types, I-F and IV-A3, were identified in 69 strains. Type I-F system was the most prevalent in this species, which correlated well with MLST. Differently, type IV-A3 system was randomly distributed. Moreover, the type IV-A3 system was separated into two subgroups, with subgroup-specific cas genes and repeat sequences. In addition, spacer origin analysis revealed that approximately one-fifth of type I-F spacers and one-third of type IV-A3 spacers had a significant match to MGEs. The phage tail tape measure protein and conjunctive transfer system protein were important targets of type I-F and IV-A3 systems in K. oxytoca, respectively. PAM sequences were inferred to be 5'-NCC-3' for type I-F, 5'-AAG-3' for subgroup IV-A3-a, and 5'-AAN-3' for subgroup IV-A3-b. Collectively, our findings will shed light on the prevalence, diversity, and functional effects of the CRISPR/Cas system in K. oxytoca.

Prevalence, molecular characterization of integrons and its associated gene cassettes in Klebsiella pneumoniae and K. oxytoca recovered from diverse environmental matrices.

The high prevalence of infections arising from Klebsiella species is related to their ability to acquire and disseminate exogenous genes associated with mobile genetic elements such as integrons. We assessed the prevalence, diversity, and associated gene cassettes (GCs) of integrons in Klebsiella species. The isolates recovered from wastewater and hospital effluents, rivers, and animal droppings were identified using the conventional Polymerase Chain Reaction (PCR) with primers targeting the gryA, pehX, and 16S-23S genes. The antimicrobial resistance profile and the Extended-Spectrum and Metallo ß-lactamases production were carried out using standard microbiological techniques. PCR, DNA sequencing analyses, and Restriction Fragment Length Polymorphism were used to characterize the integrons and their associated GCs. Furthermore, the genotypic relationships between the different isolated K. pneumoniae were determined using Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR. About 98% (51/52) of the confirmed isolates harboured an integrase gene, with 80% intI1, while the remaining 20% concurrently harboured intI1 and intI2, with no intI3 observed. About 78% (40/51) of the bacterial strains were positive for a promoter, the P2R2, investigated, while 80% (41/51) harboured at least one of the qacEΔ1 and sul1. Three different GCs arrangements identified were aac(6')-Ib, aadA1-dfrA1, and dfrA1-sat2. At a similarity index of 60%, the ERIC-PCR fingerprints generated were categorized into nine clusters. Our study is the first to reveal the features of integrons in Klebsiella spp. recovered from environmental sources in the Eastern Cape Province, South Africa. We conclude that the organisms' sources are repositories of integrons harbouring various gene cassettes, which can be readily mobilized to other microorganisms in similar or varied niches.

Chronic Diarrhea Caused by a Klebsiella oxytoca Toxin Producer Strain Following Antibiotic-Associated Hemorrhagic Colitis: Successful Treatment by Fecal Microbiota Transplant.

Klebsiella oxytoca is a gram-negative bacterium found in fecal microbiota and known to cause several infections in humans, including antibiotic-associated hemorrhagic colitis. We present here a case of colitis caused by K. oxytoca toxin-producing strains that evolved in chronic diarrhea successfully treated by fecal microbiota transplant.

Genomic features of Klebsiella isolates from artisanal ready-to-eat food production facilities.

Increasing reports on K. pneumoniae strains with antimicrobial resistance and virulence traits from food and farm animals are raising concerns about the potential role of Klebsiella spp. as a foodborne pathogen. This study aimed to report and characterize Klebsiella spp. isolates from two artisanal ready-to-eat food (soft cheese and salami) producing facilities, and to track similar genotypes in different ecological niches. Over 1170 samples were collected during the whole production chain of different food batches. The overall Klebsiella prevalence was 6%. Strains were classified into the three Klebsiella species complexes: K. pneumoniae (KpSC, n = 17), K. oxytoca (KoSC, n = 38) and K. planticola (KplaSC, n = 18). Despite high genetic diversity we found in terms of known and new sequence types (STs), core genome phylogeny revealed clonal strains persisting in the same processing setting for over 14 months, isolated from the environment, raw materials and end-products. Strains showed a natural antimicrobial resistance phenotype-genotype. K. pneumoniae strains showed the highest virulence potential, with sequence types ST4242 and ST107 strains carrying yersiniabactin ybt16 and aerobactin iuc3. The latter was detected in all K. pneumoniae from salami and was located on a large conjugative plasmid highly similar (97% identity) to iuc3+ plasmids from human and pig strains circulating in nearby regions of Italy. While identical genotypes may persist along the whole food production process, different genotypes from distinct sources in the same facility shared an iuc3-plasmid. Surveillance in the food chain will be crucial to obtain a more comprehensive picture of the circulation of Klebsiella strains with pathogenic potential.

Binary CuO\CoO nanoparticles inhibit biofilm formation and reduce the expression of papC and fimH genes in multidrug-resistant Klebsiella oxytoca.

BACKGROUND AND AIM: Binary copper-cobalt oxide nanoparticles (CuO\CoO NPs) are modern kinds of antimicrobials, which may get a lot of interest in clinical application. This study aimed to detect the effect of the binary CuO\CoO NPs on the expression of papC and fimH genes in multidrug-resistant (MDR) isolates of Klebsiella oxytoca to reduce medication time and improve outcomes. METHODS: Ten isolates of K. oxytoca were collected and identified by different conventional tests besides PCR. Antibiotic sensitivity and biofilm-forming ability were carried out. The harboring of papC and fimH genes was also detected. The effect of binary CuO\CoO nanoparticles on the expression of papC and fimH genes was investigated. RESULTS: Bacterial resistance against cefotaxime and gentamicin was the highest (100%), while the lowest percentage of resistance was to amikacin (30%). Nine of the ten bacterial isolates had the ability to form a biofilm with different capacities. MIC for binary CuO\CoO NPs was 2.5 µg/mL. Gene expression of papC and fimH was 8.5- and 9-fold lower using the NPs. CONCLUSION: Binary CuO\CoO NPs have a potential therapeutic effect against infections triggered by MDR K. oxytoca strains due to the NPs-related downregulation ability on the virulence genes of K. oxytoca.

French national epidemiology of bacterial superinfections in ventilator-associated pneumonia in patients infected with COVID-19: the COVAP study.

BACKGROUND: Description and comparison of bacterial characteristics of ventilator-associated pneumonia (VAP) between critically ill intensive care unit (ICU) patients with COVID-19-positive, COVID + ; and non-COVID-19, COVID-. METHODS: Retrospective, observational, multicenter study that focused on French patients during the first wave of the pandemic (March-April 2020). RESULTS: 935 patients with identification of at least one bacteriologically proven VAP were included (including 802 COVID +). Among Gram-positive bacteria, S. aureus accounted for more than two-thirds of the bacteria involved, followed by Streptococcaceae and enterococci without difference between clinical groups regarding antibiotic resistance. Among Gram-negative bacteria, Klebsiella spp. was the most frequently observed bacterial genus in both groups, with K. oxytoca overrepresented in the COVID- group (14.3% vs. 5.3%; p < 0.05). Cotrimoxazole-resistant bacteria were over-observed in the COVID + group (18.5% vs. 6.1%; p <0.05), and after stratification for K. pneumoniae (39.6% vs. 0%; p <0.05). In contrast, overrepresentation of aminoglycoside-resistant strains was observed in the COVID- group (20% vs. 13.9%; p < 0.01). Pseudomonas sp. was more frequently isolated from COVID + VAPs (23.9% vs. 16.7%; p <0.01) but in COVID- showed more carbapenem resistance (11.1% vs. 0.8%; p <0.05) and greater resistance to at least two aminoglycosides (11.8% vs. 1.4%; p < 0.05) and to quinolones (53.6% vs. 7.0%; p <0.05). These patients were more frequently infected with multidrug-resistant bacteria than COVID + (40.1% vs. 13.8%; p < 0.01). CONCLUSIONS: The present study demonstrated that the bacterial epidemiology and antibiotic resistance of VAP in COVID + is different from that of COVID- patients. These features call for further study to tailor antibiotic therapies in VAP patients.

Exploring the prevalence and antibiotic resistance profile of Klebsiella pneumoniae and Klebsiella oxytoca isolated from clinically ill companion animals from North of Portugal.

Klebsiella spp. is an important pathogen in humans and animals and due to the indiscriminate use of antibiotics, its prevalence and antibiotic resistance has increased in companion animals. The main goal of this study was to investigate the prevalence and antibiotic resistance of Klebsiella spp. isolated from clinically ill cats and dogs admitted in veterinary clinics in the North of Portugal. A total of 255 clinical specimens were collected and, after isolation, the identification of Klebsiella strains was performed using the BBL Crystal™ identification system and confirmed by PCR-based sequencing with specific primers. Antibiotic resistance profile was determined through the disc diffusion method. Beta-lactam resistance genes were screened through a multiplex PCR assay. Fifty Klebsiella strains were isolated and, 39 were identified as Klebsiella pneumoniae and 11 as Klebsiella oxytoca. Thirty-one were recovered from dogs and 19 from cats. The Klebsiella isolates were recovered mainly from skin wounds, respiratory tract, and from urine. Fifty percent of K. oxytoca and K. pneumoniae isolates revealed to be Multidrug Resistant (MDR) strains, with most of them positive for the presence of blaTEM-like and blaSHV genes. This data shows that MDR Klebsiella are highly disseminated in companion animals and that extended-spectrum beta-lactamases can be easily found among these isolates. This highlights the potential role of dogs and cats as a reservoir of resistant Klebsiella spp. that have the potential to be transmitted to humans.

Whole-genome sequence analysis reveals phenanthrene and pyrene degradation pathways in newly isolated bacteria Klebsiella michiganensis EF4 and Klebsiella oxytoca ETN19.

Polycyclic aromatic hydrocarbons (PAHs) are diverse pollutants of significant environmental concerns, requiring effective biodegradation. This study used different bioinformatics tools to conduct whole-genome sequencing of two novel bacterial strains, Klebsiella michiganensis EF4 and K. oxytoca ETN19, to improve our understanding of their many genomic functions and degradation pathways of phenanthrene and pyrene. After 28 days of cultivation, strain EF4 degraded approximately 80% and 60% of phenanthrene and pyrene, respectively. However, their combinations (EF4 +ETN19) showed tremendous phenanthrene degradation efficiency, supposed to be at the first-level kinetic model with a t1/2 value of approximately 6 days. In addition, the two bacterial genomes contained carbohydrate-active enzymes and secondary metabolites biosynthetic gene clusters associated with PAHs degradation. The two genomes contained the bZIP superfamily of transcription factors, primarily the cAMP-response element-binding protein (CREB), which could regulate the expression of several PAHs degradation genes and enzymes. Interestingly, the two genomes were found to uniquely degrade phenanthrene through a putative pathway that catabolizes 2-carboxybenzalpyruvate into the TCA cycle. An operon containing multicomponent proteins, including a novel gene (JYK05_14550) that could initiate the beginning step of phenanthrene and pyrene degradation, was found in the EF4 genome. However, the degradation pathway of ETN19 showed that the yhfP gene encoding putative quinone oxidoreductase was associated with phenanthrene and pyrene catabolic processes. Furthermore, the significant expression of catechol 1,2-dioxygenase and quinone oxidoreductase genes in EF4 +ETN19 and ETN19 following the quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis confirmed the ability of the bacteria combination to degrade pyrene and phenanthrene effectively. These findings present new insight into the possible co-metabolism of the two bacterial species in the rapid biodegradation of phenanthrene and pyrene in soil environments.

Lytic bacteriophage vB_KmiS-Kmi2C disrupts biofilms formed by members of the Klebsiella oxytoca complex, and represents a novel virus family and genus.

AIMS: This study aimed to characterize the lytic phage vB_KmiS-Kmi2C, isolated from sewage water on a GES-positive strain of Klebsiella michiganensis. METHODS AND RESULTS: Comparative phylogenetic and network-based analyses were used to characterize the genome of phage vB_KmiS-Kmi2C (circular genome of 42 234 bp predicted to encode 55 genes), demonstrating it shared little similarity with other known phages. The phage was lytic on clinical strains of K. oxytoca (n = 2) and K. michiganensis (n = 4), and was found to both prevent biofilm formation and disrupt established biofilms produced by these strains. CONCLUSIONS: We have identified a phage capable of killing clinically relevant members of the K. oxytoca complex (KoC). The phage represents a novel virus family (proposed name Dilsviridae) and genus (proposed name Dilsvirus).

Evaluation of Phenotypic Tests to Detect Extended-Spectrum ß-Lactamase (ESBL)-Producing Klebsiella oxytoca Complex Strains.

Klebsiella oxytoca complex (KoC) species may overproduce their chromosomal class A OXY ß-lactamases, conferring reduced susceptibility to piperacillin-tazobactam, expanded-spectrum cephalosporins and aztreonam. Moreover, since clavulanate maintains its ability to inhibit these enzymes, the resulting resistance phenotype may falsely resemble the production of acquired extended-spectrum ß-lactamases (ESBLs). In this work, a collection of 44 KoC strains of human and animal origin was characterized with whole-genome sequencing (WGS) and broth microdilution (BMD) susceptibility testing. Comparison of ESBL producers (n = 11; including CTX-M-15 [n = 6] and CTX-M-1 [n = 5] producers) and hyperproducers of OXYs (n = 21) showed certain phenotypic differences: piperacillin-tazobactam (MIC90s: 16 versus >64 µg/mL), cefotaxime (MIC90s: 64 versus 4 µg/mL), ceftazidime (MIC90s: 32 versus 4 µg/mL), cefepime (MIC90s: 8 versus 4 µg/mL) and associated resistance to non-ß-lactams (e.g., trimethoprim-sulfamethoxazole: 90.9% versus 14.3%, respectively). However, a clear phenotype-based distinction between the two groups was difficult. Therefore, we evaluated 10 different inhibitor-based confirmatory tests to allow such categorization. All tests showed a sensitivity of 100%. However, only combination disk tests (CDTs) with cefepime/cefepime-clavulanate and ceftazidime/ceftazidime-clavulanate or the double-disk synergy test (DDST) showed high specificity (100%, 95.5%, and 100%, respectively). All confirmatory tests in BMD or using the MIC gradient strip did not perform well (specificity, ≤87.5%). Of note, ceftazidime/ceftazidime-avibactam tests also exhibited low specificity (CDT, 87.5%; MIC gradient strip, 77.8%). Our results indicate that standard antimicrobial susceptibility profiles can raise some suspicion, but only the use of cefepime/cefepime-clavulanate CDT or DDST can guarantee distinction between ESBL-producing KoC strains and those hyperproducing OXY enzymes.

Characteristics and comparisons of the aerobic and anaerobic denitrification of a Klebsiella oxytoca strain: Performance, electron transfer pathway, and mechanism.

The performance and electron (e-) transfer mechanisms of anaerobic and aerobic denitrification by strain Klebsiella were investigated in this study. The RT-PCR results demonstrated that the membrane bound nitrate reductase gene (narG) and Cu-nitrite reductase gene (nirK) were responsible for both aerobic and anerobic denitrification. The extreme low gene relative abundance of nirK might be responsible for the severe accumulation of NO2--N (nitrogen in the form of NO2- ion) under anaerobic condition. Moreover, the nitrite reductase (Nir) activity was 0.31 µg NO2--N min-1 mg-1 protein under anaerobic conditions, which was lower than that under aerobic conditions (0.38 µg NO2--N min-1 mg-1 protein). By using respiration chain inhibitors, the e- transfer pathways of anaerobic and aerobic denitrification of Klebsiella strain were constructed. Fe-S protein and Complex III were the core components under anaerobic conditions, while Coenzyme Q (CoQ), Complexes I and III played a key role in aerobic denitrification. Nitrogen assimilation was found to be the main way to generate NH4+-N (nitrogen in the form of NH4+ ion) during anaerobic denitrification, and also served as the primary nitrogen removal way under aerobic condition. The results of this study may help to improve the understanding of the core components of strain Klebsiella during aerobic and anaerobic denitrifications, and may suggest potential applications of the strain for nitrogen-containing wastewater.

Clinical characteristics of Raoultella ornithinolytica bacteremia and antimicrobial susceptibility of Raoultellaornithinolytica.

Raoultella ornithinolytica (R. ornithinolytica) is a gram-negative rod that was considered related to Klebsiella oxytoca and was classified as R. ornithinolytica in 2001. R. ornithinolytica is known as a histamine-producing bacterium that causes mackerel poisoning. Although only few clinical cases of R. ornithinolytica infection in humans have been reported, the number of diagnosed cases is expected to increase owing to the advancements in identification methods. In the present study, we performed a retrospective analysis of cases of R. ornithinolytica infection detected at our hospital. From September 2019 to July 2021, 62 specimens positive for R. ornithinolytica were obtained after removing duplicates. The clinical courses of these cases were investigated retrospectively based on electronic medical records. Of the 62 specimens, 24 were sputum, 19 were urine, three were stool, six were blood, four were bile, and six were other specimens. All the six blood culture specimens in which R. ornithinolytica was detected were from male patients, and the causative diseases were cholangitis in four cases and complicated pyelonephritis in two cases. Of these, two patients with cholangitis succumbed to death due to the worsening of underlying cancer. Identification of R. ornithinolytica is reportedly difficult, and some instruments may misidentify it as Klebsiella oxytoca. The prognosis of R. ornithinolytica infection has been reported to be good when susceptible drugs are used. However, high mortality rates were also reported despite the use of these drugs, suggesting the need for further investigation of clinical features of R. ornithinolytica infection.

I shall be released: Assembly platform dynamics during type II secretion.

In this issue of Structure, Dazzoni et al. solve the high-resolution homo- and hetero-dimeric structures of the Klebsiella oxytoca PulL and PulM C-terminal domains and unravel an uncharacterized dynamic interaction interface that is required for correct function of the type II secretion system.