Bacteriophage cocktail shows no toxicity and improves the survival of Galleria mellonella infected with Klebsiella spp.

Klebsiella spp. are causative agents of healthcare-associated infections in patients who are immunocompromised and use medical devices. The antibiotic resistance crisis has led to an increase in infections caused by these bacteria, which can develop into potentially life-threatening illnesses if not treated swiftly and effectively. Thus, new treatment options for Klebsiella are urgently required. Phage therapy can offer an alternative to ineffective antibiotic treatments for antibiotic-resistant bacteria infections. The aim of the present study was to produce a safe and effective phage cocktail treatment against Klebsiella pneumoniae and Klebsiella oxytoca, both in liquid in vitro culture and an in vivo Galleria mellonella infection model. The phage cocktail was significantly more effective at killing K. pneumoniae and K. oxytoca strains compared with monophage treatments. Preliminary phage cocktail safety was demonstrated through application in the in vivo G. mellonella model: where the phage cocktail induced no toxic side effects in G. mellonella. In addition, the phage cocktail significantly improved the survival of G. mellonella when administered as a prophylactic treatment, compared with controls. In conclusion, our phage cocktail was demonstrated to be safe and effective against Klebsiella spp. in the G. mellonella infection model. This provides a strong case for future treatment for Klebsiella infections, either as an alternative or adjunct to antibiotics.IMPORTANCEKlebsiella infections are a concern in individuals who are immunocompromised and are becoming increasingly difficult to treat with antibiotics due to their drug-resistant properties. Bacteriophage is one potential alternative therapy that could be used to tackle these infections. The present study describes the design of a non-toxic phage cocktail that improved the survival of Galleria mellonella infected with Klebsiella. This phage cocktail demonstrates potential for the safe and effective treatment of Klebsiella infections, as an adjunct or alternative to antibiotics.

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.

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 Detection and Phylogenetic Analysis of the alkB Gene in Klebsiella oxytoca Strains Isolated from the Gut of Tenebrio molitor.

The challenge in polystyrene disposal has caused researchers to look for urgent innovative and ecofriendly solutions for plastic degradation. Some insects have been reported to use polystyrene as their sole carbon source, and this has been linked to the presence of microbes in their guts that aid in plastic digestion. Thus, this study focuses on the molecular detection and phylogenetic analysis of the alkane-1-monooxygenase (alkB) gene in Klebsiella oxytoca strains isolated from the gut of Tenebrio molitor. The alkB gene encodes for alkane-1-monooxygenase, an enzyme involved in the oxidation of inactivated alkanes. This gene can be used as a marker to assess bacteria's ability to biodegrade polystyrene. Three bacterial strains were isolated from the guts of T. molitor mealworms and were confirmed using polymerase chain reaction (PCR) of the 16S ribosomal RNA gene. The primers used in the amplification of the 16S ribosomal RNA region were designed using NCBI, a bioinformatics tool. To detect the presence of the alkB gene in the isolated bacterial strains, a set of primers used in the amplification of this gene was manually designed from the conserved regions of the alkB nucleotide sequences of eleven bacterial species from GenBank. TCOFFE online tool was used to align the alkB sequences of the bacteria, while Jalview and ConSurf were used to view the alignment. The amplified alkB gene was then sequenced using the Sanger sequencing technique, blasted on NCBI to look for similar sequences, and a phylogenetic tree was constructed. Based on the 16S ribosomal RNA gene sequences, the isolated bacterial strains were confirmed to be Klebsiella oxytoca NBRC 102593, Klebsiella oxytoca JCM 1665, and Klebsiella oxytoca ATCC 13182. The alkB gene sequence identical to fourteen alkB gene sequences derived from Actinobacteria whole genome was detected in Klebsiella oxytoca for the first time to the best of our knowledge. The novel nucleotide sequence was published in the NCBI database under accession number OP959069. This gene sequence was found to be for the enzyme alkane-1-monooxygenase and may be one of the enzymes responsible for polystyrene degradation by the putative Klebsiella oxytoca ATCC 13182 in T. molitor.

Klebsiella oxytoca Complex: Update on Taxonomy, Antimicrobial Resistance, and Virulence.

Klebsiella oxytoca is actually a complex of nine species-Klebsiella grimontii, Klebsiella huaxiensis, Klebsiella michiganensis, K. oxytoca, Klebsiella pasteurii, Klebsiella spallanzanii, and three unnamed novel species. Phenotypic tests can assign isolates to the complex, but precise species identification requires genome-based analysis. The K. oxytoca complex is a human commensal but also an opportunistic pathogen causing various infections, such as antibiotic-associated hemorrhagic colitis (AAHC), urinary tract infection, and bacteremia, and has caused outbreaks. Production of the cytotoxins tilivalline and tilimycin lead to AAHC, while many virulence factors seen in Klebsiella pneumoniae, such as capsular polysaccharides and fimbriae, have been found in the complex; however, their association with pathogenicity remains unclear. Among the 5,724 K. oxytoca clinical isolates in the SENTRY surveillance system, the rates of nonsusceptibility to carbapenems, ceftriaxone, ciprofloxacin, colistin, and tigecycline were 1.8%, 12.5%, 7.1%, 0.8%, and 0.1%, respectively. Resistance to carbapenems is increasing alarmingly. In addition to the intrinsic blaOXY, many genes encoding ß-lactamases with varying spectra of hydrolysis, including extended-spectrum ß-lactamases, such as a few CTX-M variants and several TEM and SHV variants, have been found. blaKPC-2 is the most common carbapenemase gene found in the complex and is mainly seen on IncN or IncF plasmids. Due to the ability to acquire antimicrobial resistance and the carriage of multiple virulence genes, the K. oxytoca complex has the potential to become a major threat to human health.

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.

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.

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.

A newly isolated bacteriophage vB8388 and its synergistic effect with aminoglycosides against multi-drug resistant Klebsiella oxytoca strain FK-8388.

The bacteriophage vB8388 can lyse multi-drug resistant Klebsiella oxytoca strain FK-8388 and maintain stability in a wide range of temperatures (from 4 °C to 80 °C) and pHs (3-11). Bioinformatics analysis showed that vB8388 is a linear double-stranded DNA virus that is 39,750 long with 50.65% G + C content and 44 putative open reading frames (ORFs). Phage vB8388 belongs to the family Autographviridae and possesses a non-contractile tail. The latency period of vB8388 was approximately 20 min. The combination of phage vB8388 and gentamicin, amikacin, or tobramycin could effectively inhibit the growth of K. oxytoca strain FK-8388, with a decrease of more than 4 log units within 12 h in vitro. Phage vB8388 showed a strong synergistic effect with gentamicin that could enhance the anti-biofilm effect of vB8388. The phage + gentamicin combination also showed synergy in vivo in the larval infection model of Galleria mellonella. In conclusion, the findings of this study suggest the potential of phage + antibiotic combination therapy to be used as an alternative therapeutic approach for treating infectious diseases caused by multidrug-resistant bacteria.

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.

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.

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).

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.

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.

In Vitro Activity of Fosfomycin on Multidrug-Resistant Strains of Klebsiella pneumoniae and Klebsiella oxytoca Causing Urinary Tract Infection.

With the emergence of multi-drug resistant strains among Klebsiella isolates, the use of old drugs such as fosfomycin has been considered. In this context, we investigated the effect of fosfomycin on biofilm-producing Klebsiella pneumoniae and Klebsiella oxytoca strains isolated from ICU patients. A total of 90 isolates of Klebsiella pneumoniae and 30 isolates of Klebsiella oxytoca were collected from the ICU ward. All isolates were confirmed by biochemical and genotypic methods. Antibiotic susceptibility testing was performed by disc diffusion method and for fosfomycin and colistin, minimum inhibitory concentration (MIC) was done using micro broth dilution. The presence of the beta-lactamase encoding genes, biofilm-related genes, and fosfomycin resistance-related genes was detected by PCR. Finally, for fosfomycin-resistant isolates, we determined the sequence type by the MLST method. Sensitivity rate to fosfomycin in Klebsiella pneumoniae and Klebsiella oxytoca isolates was 92.2% and 100%, respectively. Fosfomycin was the most active antimicrobial agent with 96% sensitivity among all tested antibiotics. All tested isolates could produce biofilm. The frequency of biofilm-related genes for Klebsiella pneumoniae was as follows: 95.5% fimH, 86.6% mrkD, 77.7% mrkA, and 50% wcaG. The frequency of these genes for Klebsiella oxytoca was as follows: 56.6% fimA, 46.6% mrkA, 93.3% matB, and 90% pilQ. Only 4.4% of Klebsiella pneumoniae isolates showed resistance to fosfomycin, and the fosA gene was detected in all of them. Our results showed that fosfomycin effectively inhibits multidrug-resistant (MDR) strains of Klebsiella pneumoniae and Klebsiella oxytoca.

Using synthetic biology to overcome barriers to stable expression of nitrogenase in eukaryotic organelles.

Engineering biological nitrogen fixation in eukaryotic cells by direct introduction of nif genes requires elegant synthetic biology approaches to ensure that components required for the biosynthesis of active nitrogenase are stable and expressed in the appropriate stoichiometry. Previously, the NifD subunits of nitrogenase MoFe protein from Azotobacter vinelandii and Klebsiella oxytoca were found to be unstable in yeast and plant mitochondria, respectively, presenting a bottleneck to the assembly of active MoFe protein in eukaryotic cells. In this study, we have delineated the region and subsequently a key residue, NifD-R98, from K. oxytoca that confers susceptibility to protease-mediated degradation in mitochondria. The effect observed is pervasive, as R98 is conserved among all NifD proteins analyzed. NifD proteins from four representative diazotrophs, but not their R98 variants, were observed to be unstable in yeast mitochondria. Furthermore, by reconstituting mitochondrial-processing peptidases (MPPs) from yeast, Oryza sativa, Nicotiana tabacum, and Arabidopsis thaliana in Escherichia coli, we demonstrated that MPPs are responsible for cleavage of NifD. These results indicate a pervasive effect on the stability of NifD proteins in mitochondria resulting from cleavage by MPPs. NifD-R98 variants that retained high levels of nitrogenase activity were obtained, with the potential to stably target active MoFe protein to mitochondria. This reconstitution approach could help preevaluate the stability of Nif proteins for plant expression and paves the way for engineering active nitrogenase in plant organelles.

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.

Genome Analysis of Klebsiella oxytoca Complex for Antimicrobial Resistance and Virulence Genes.

Klebsiella oxytoca complex comprises nine closely related species causing human infections. We curated genomes labeled Klebsiella (n = 14,256) in GenBank and identified 588 belonging to the complex, which were examined for precise species, sequence types, K- and O-antigen types, and virulence and antimicrobial resistance genes. The complex and Klebsiella pneumoniae share many K- and O-antigen types. Of the complex, K. oxytoca and Klebsiella michiganensis appear to carry more virulence genes and be more commonly associated with human infections.

Identification of multiple transfer units and novel subtypes of tmexCD-toprJ gene clusters in clinical carbapenem-resistant Enterobacter cloacae and Klebsiella oxytoca.

OBJECTIVES: Tigecycline is a last-resort antibiotic used to treat lethal infections caused by carbapenem-resistant Enterobacterales; however, plasmid-borne tigecycline resistance tmexCD-toprJ gene clusters can confer tigecycline resistance. The aim of the study was to identify novel subtypes and the spread of tmexCD-toprJ. METHODS: Five non-duplicate isolates of different species, carrying tmexCD-toprJ gene clusters or novel subtypes, were isolated from patients across China between November 2018 and June 2019. WGS was performed using Illumina and Nanopore platforms. A phylogenetic tree was constructed using a dataset of 77 sequences carrying the tmexCD-toprJ gene clusters, 72 of which were downloaded from NCBI with a blastn identity cut-off of 95%. RESULTS: We detected six different transfer units and two novel subtypes (tmexC1D1.2-toprJ1 and tmexC2D2.2-toprJ2) of the tmexCD-toprJ gene clusters. Among the six transfer units, three were mediated by IS26, while the rest were presumably mediated by Tn5393, hypothetical integrases (xerD-hp clusters-umuC-integrases-tnfxB2-tmexC2D2-toprJ2-umuC) and hypothetical units (hp-hp-hp-tnfxB2-tmexC2D2.2-toprJ2-ΔTn5393-Tn6292). Moreover, two tmexCD-toprJ-like gene clusters co-located on the same plasmid with blaNDM in five isolates. Phylogenetic analysis revealed that tmexCD-toprJ gene clusters may have originated in Pseudomonas spp., being mainly distributed in Pseudomonas spp. and Klebsiella spp. (64/77). Most tmexCD-toprJ gene clusters in Enterobacterales were located on plasmids, indicating that the gene clusters have a high inter-species transfer risk after transfer to Enterobacterales. CONCLUSIONS: In summary, to the best of our knowledge, this is the first report of tmexCD-toprJ gene clusters being isolated from Enterobacter cloacae and Klebsiella oxytoca, revealing that these multiple transfer units should be further studied because of their clinical significance.

Klebsiella oxytoca: an efficient pyrene-degrading bacterial strain isolated from petroleum-contaminated soil.

Polycyclic aromatic hydrocarbons (PAHs) are the hazardous xenobiotic agents of oil production. One of the methods to eliminate hazardous compounds is bioremediation, which is the most efficient and cost-effective method to eliminate the harmful byproducts of crude petroleum processing. In this study, five pure bacterial isolates were isolated from petroleum-contaminated soil, four of which showed a robust growth on the PAH pyrene, as a sole carbon source. Various methods viz mass spectroscopy, biochemical assays, and 16S RNA sequencing employed to identify the isolates ascertained the consistent identification of Klebsiella oxytoca by all three methods. Scanning electron microscopy and Gram staining further demonstrated the characterization of the K. oxytoca. High-performance liquid chromatography of the culture supernatant of K. oxytoca grown in pyrene containing media showed that the cells started utilizing pyrene from the 6th day onwards and by the 12th day of growth, 70% of the pyrene was completely degraded. A genome search for the genes predicted to be involved in pyrene degradation using Kyoto Encyclopedia of Genes and Genomes (KEGG) confirmed their presence in the genome of K. oxytoca. These results suggest that K. oxytoca would be a suitable candidate for removing soil aromatic hydrocarbons.