Genomic insights into the evolution, pathogenicity, and extensively drug-resistance of emerging pathogens Kluyvera and Phytobacter.

Introduction: Kluyvera is a Gram-negative, flagellated, motile bacillus within the Enterobacteriaceae. The case reports of clinical infections shed light on the importance of this organism as an emerging opportunistic pathogen. The genus Phytobacter, which often be misidentified with Kluyvera, is also an important clinically relevant member of the Enterobacteriaceae. However, the identification of Kluyvera and Phytobacter is problematic, and their phylogenetic relationship remains unclear. Methods: Here, 81 strains of Kluyvera and 16 strains of Phytobacter were collected. A series of comparative genomics approaches were applied to the phylogenetic relationship reconstruction, virulence related genes profiles description, and antibiotic resistance genes prediction. Results: Using average nucleotide identity (ANI) and in silico DNA-DNA hybridization (isDDH), we offered reliable species designations of 97 strains, in which 40 (41.24%) strains were incorrectly labeled. A new Phytobacter genomospecies-1 were defined. Phytobacter and Kluyvera show great genome plasticity and inclusiveness, which may be related to their diverse ecological niches. An intergenomic distances threshold of 0.15875 was used for taxonomy reassignments at the phylogenomic-group level. Further principal coordinates analysis (PCoA) revealed 11 core genes of Kluyvera (pelX, mdtL, bglC, pcak-1, uhpB, ddpA-2, pdxY, oppD-1, cptA, yidZ, csbX) that could be served as potential identification targets. Meanwhile, the Phytobacter specific virulence genes clbS, csgA-C, fliS, hsiB1_vipA and hsiC1_vipB, were found to differentiate from Kluyvera. We concluded that the evolution rate of Kluyvera was 5.25E-6, approximately three times higher than that of Phytobacter. Additionally, the co-existence of ESBLs and carbapenem resistance genes were present in approximately 40% strains, suggesting the potential development of extensively drug-resistant or even fully drug-resistant strains. Discussion: This work provided a better understanding of the differences between closely related species Kluyvera and Phytobacter. Their genomes exhibited great genome plasticity and inclusiveness. They not only possess a potential pathogenicity threat, but also a risk of multi-drug resistance. The emerging pathogens Kluyvera and Phytobacter warrant close attention.

Detection of Drug-Resistant Kluyvera intermedia from Zoacys dhumnades.

Zoacys dhumnades is native to china and has important economic and medicinal value, but the pathogenic microorganisms have been reported rarely. Kluyvera intermedia is usually considered a commensal. In this study, Kluyvera intermedia was first isolated from Zoacys dhumnades identical by the 16SrDNA sequence, phylogenetic tree analysis, and biochemical tests. Cell infection experimental did not find cell morphology change significantly compared to control with pathological organs homogenates from Zoacys dhumnades. Antibiotic susceptibility shown Kluyvera intermedia isolates were sensitive to 12 kinds of antibiotics and resistant to 8 kinds of antibiotics. Resistant antibiotic genes screening display gyrA, qnrB, and sul2 were found in Kluyvera intermedia. This is the first report of Kluyvera intermedia associated fatality with Zoacys dhumnades suggesting the need for continuous monitoring of nonpathogenic bacteria antimicrobial susceptibility from human, domestic animals and wildlife.

Redefining the Origin and Evolution of Chromosomally Encoded blaCTX-M/KLU in the Context of a Revised Taxonomy of Genus Kluyvera.

Changes in Kluyvera taxonomy may clarify each species contribution for recruitment and dissemination of their relevant ß-lactamases. The CTX-M-2 subgroup is linked to Kluyvera ascorbata, KLUC to Kluyvera cryocrescens, and CTX-M-25 to Kluyvera georgiana. The CTX-M-8 subgroup can be linked to Kluyvera genomospecies 3 and CTX-M-9 to Kluyvera genomospecies 2. Kluyvera sichuanensis and Kluyvera genomospecies 1 harbor new subgroups. The CTX-M-1 subgroup has a direct counterpart in an isolate proposed as a new genomospecies 5.

Screening of natural compounds for identification of novel inhibitors against ß-lactamase CTX-M-152 reported among Kluyvera georgiana isolates: An in vitro and in silico study.

Multidrug resistance due to the expression of extended spectrum ß-lactamases (ESBLs) by bacterial pathogens is an alarming health concern with huge socio-economic burden. Here, 102 bacterial isolates from Wastewater treatment plants (WTPs) were screened for resistance to different antibiotics. Kirby-Bauer method and phenotypic disc confirmatory test confirmed the prevalence of 20 ESBLs. Polymerase chain reaction-based detection confirmed 11 blaCTX-M positive bacterial isolates. Genotyping of bacterial isolates by 16S rRNA gene sequencing showed the dissemination of blaCTX-M in Escherichia fergusonii, Escherichia coli, Shigella sp., Kluyvera georgiana and Enterobacter sp. Amongst Kluyvera georgiana isolates, two were harboring blaCTX-M-152. The 3D model of CTX-M-152 protein was generated using SwissProt and characterized by Ramachandran plot and SAVES. A library of natural compounds was screened to identify novel CTX-M-152 inhibitor(s). High-throughput virtual screening (HTVS), standard precision (SP) and extra precision (XP) docking led to the identification of five natural compounds (Naringin dihydrochalcone, Salvianolic acid B, Inositol, Guanosine and Ellagic acid) capable of binding to active site of CTX-M-152. Futher, characterization by MM-GBSA (Molecular Mechanism General Born Surface Area), and ADMET (Adsorption, Distribution, Metabolism, Excretion and Toxicity) showed that Ellagic acid was the most potent inhibitor of CTX-M-152. Molecular dynamics simulation also confirmed that Ellagic acid form a stable complex with CTX-M-152. The ability of Ellagic acid to inhibit growth of bacteria harboring CTX-M-152 was confirmed by MIC (Minimum Inhibitory Concentration; broth dilution method) and Zone of Inhibition (ZOI) studies with respect to Cefotaxime. The identification of a novel inhibitor of CTX-M-152 from a natural source holds promise for employment in the control of bacterial infections.

First isolation of an IMP-1 metallo-ß-lactamase-producing Kluyvera ascorbata in Japan.

OBJECTIVES: The emergence of carbapenemase-producing Enterobacterales has been increasing globally, causing growing concerns. Although Kluyvera ascorbata is not known as a metallo-ß-lactamase (MBL)-producer, in the present study we isolated a K. ascorbata strain producing IMP-1 MBL from catheter-associated urine of a paediatric patient and performed whole-genome analysis to elucidate the features of this strain and the origin of IMP-1. METHODS: Carbapenemase production was confirmed by a modified carbapenemase inactivation method. Whole-genome sequencing was performed using NovaSeq 6000 and GridION. Conjugation ability was evaluated using Escherichia coli ML1410 by a broth mating assay. RESULTS: TheblaIMP-1 gene was located on a 149 316-bp transferable plasmid (pKATP2) and formed a class 1 integron structure. In addition, this plasmid had two types of repA genes as well as astA encoding a putative heat-stable enterotoxin. Comparison with other plasmids from Enterobacterales and Pseudomonas aeruginosa suggested that this plasmid might have originated by the integration of multiple plasmids. In addition, pKATP2 harboured conjugation-associated genes and was transferable. CONCLUSION: This is the first report of MBL-producing K. ascorbata. Therefore, our findings suggest that species which do not typically produce MBL could acquire the corresponding genes, attracting attention as potential MBL-producing pathogens.

Classificação dos graus de lesões de aerossaculite em perus associadas com enterobactérias / [Classification of the levels of the airsacculitis injuries in turkeys associated with Enterobacteriaceae]

Foram padronizados os graus de lesões dos sacos aéreos em perus com aerossaculite, associadas com a presença de isolados de enterobactérias nesses órgãos. Um total de 110 amostras de sacos aéreos de perus machos com aerossaculite foi coletado para o estudo. Durante o processo de abate, as amostras foram coletadas por meio de swabs e submetidas a três métodos de armazenamento (imediato, congelado ou pré-incubado após congelamento) para posterior comparação das suas eficiências de isolamento. Os gêneros da família Enterobacteriaceae foram identificados pelas séries bioquímicas EPM, MILi e citrato de Simmons. O crescimento bacteriano ocorreu em 43,64% das amostras. Neste estudo, quatro padrões de lesões de aerossaculite foram identificados de acordo com as características patológicas dos sacos aéreos. Os principais gêneros de enterobactérias identificadas foram: Escherichia coli, Citrobacter, Proteus, Edwardsiella, Morganella, Kluyvera, Salmonella e Klebsiella. Foi observado que os graus padronizados como 3 e 4 apresentaram maior variedade de gêneros bacterianos. O armazenamento imediato apresentou maior porcentagem de positividade, 41,82%, no entanto o pré-incubado após congelamento se apresentou mais eficaz em relação à quantidade de colônias.(AU)

The degrees of air sac lesions in turkeys with airsacculitis were standardized, associated with the presence of Enterobacteriaceae isolated from these organs. A total of 110 samples of air sacs from male turkeys with airsacculitis were collected and analyzed. During the slaughtering process, the sample collection was done using swabs and submitted to three storage methods (immediate, frozen, or pre incubated after freezing) for further comparison of their isolated efficiency. The bacterial genera of the family Enterobacteriaceae were identified biochemical series EPM, MILi and Simmons citrate. Bacterial growth occurred in 43.64% of samples. In this study, four patterns of aerossaculitis lesions were identified according to the pathological characteristics of air sacs. The frequencies of the Enterobacteriaceae isolated identified in the samples were: Escherichia coli, Citrobacter, Proteus, Edwardsiella, Morganell, Kluyvera, Salmonella and Klebsiella. Otherwise, it was observed that the levels already standardized as level three and four showed higher variety of genus. The immediate storage showed higher percentage of positivity at 41.82%, however, the pre incubated after freezing showed more efficiency in relation to the quantity of colonies.(AU)

Secretory expression and characterization of a novel amidase from Kluyvera cryocrescens in Bacillus subtilis.

OBJECTIVE: Cloning and secretory expression of an amidase from Kluyvera cryocrescens and characterization of its potential in preparation of chiral amino acids. RESULTS: An amidase belonging to the Ntn-hydrolase superfamily was identified from Kluyvera cryocrescens ZJB-17005 (Kc-Ami). The maximum activity of Kc-Ami was observed at pH 8.5 and 55 °C. Remarkably, Kc-Ami showed an excellent enantioselectivity (99% ee) using rac-4-(hydroxy(methyl)phosphoryl)-2-(2-phenylacetamido) butanoic acid as substrate. Kc-Ami remained stable at pH 7.0-9.0 and exhibited prominent thermostability with a half-life time of 59.1, 47.4 and 20.4 h at 50, 55 and 60 °C, respectively. Kc-Ami could be appllied to synthesize chiral amino acids and its derivatives with excellent enantioselectivity (> 99% ee). The synthesized chiral amino acids could contain short or long side chain, and further the side chain could be replaced with -OH, -COOH or benzene ring. CONCLUSIONS: Kc-Ami exhibited remarkable thermostability and excellent enantioselectivity for synthesizing chiral amino acids and its derivatives. This specific characteristic provides great potential for industrial application in preparation of chiral amino acids and its derivatives.

Double-Sided Microwells with a Stepped Through-Hole Membrane for High-Throughput Microbial Assays.

To improve the throughput of microwell arrays for identifying immense cellular diversities even at a single-bacteria level, further miniaturization or densification of the microwells has been an obvious breakthrough. However, controlling millions of nanoliter samples or more at the microscale remains technologically difficult and has been spatially restricted to a single open side of the microwells. Here we employed a stepped through-hole membrane to utilize the bottom as well as top side of a high-density nanoliter microwell array, thus improving spatial efficiency. The stepped structure shows additional effectiveness for handling several millions of nanoliter bacterial samples in the overall perspectives of controllability, throughput, simplicity, versatility, and automation by using novel methods for three representative procedures in bacterial assays: partitioning cells, manipulating the chemical environment, and extracting selected cells. As a potential application, we show proof-of-concept isolation of rare cells in a mixed ratio of 1 to around 106 using a single chip. Our device can be further applied to various biological studies pertaining to synthetic biology, drug screening, mutagenesis, and single-cell heterogeneity.

Urosepsis caused by Kluyvera Ascorbata in a Pregnant Woman.

Among the Kluyvera species, K. ascorbata has been isolated from only a few adult cases. Furthermore, there is little or no information in the literature as to whether the species of Kluyvera can cause a clinically significant infection in pregnant women. We report a case of urosepsis caused by K. ascorbata in a 23-year pregnant woman at 26 weeks of gestation who presented with left flank pain. Ultrasonography showed left grade 3 hydronephrosis, ureteral dilatation, and a 10-mm distal ureteral stone. The patient underwent laser lithotripsy and JJ placement. Ten days later, she was readmitted with urosepsis and empirical antibiotherapy and aggressive hydration were initiated. On the third day, K. ascorbata growth was detected in the urine culture. Based on the clinical status of the patient and the antimicrobial susceptibility testing, the treatment was switched to ertapenem 1×1 g/day and was continued for 14 days. Among the Kluyvera species, K. ascorbata is the most frequent pathogen which may be isolated from pregnant women and can cause urosepsis. To the best of authors' knowledge, this is the first report showing the isolation of K. ascorbata in a pregnant woman which caused urosepsis.

Efficient biocatalyst of L-DOPA with Escherichia coli expressing a tyrosine phenol-lyase mutant from Kluyvera intermedia.

L-DOPA (L-dihydroxyphenylalanine) is a promising drug for Parkinson's disease and thereby has a growing annual demand. Tyrosine phenol-lyase (TPL)-based catalysis is considered to be a low-cost yet efficient route for biosynthesis of L-DOPA. TPL is a tetrameric enzyme that catalyzes the synthesis of L-DOPA from pyrocatechol, sodium pyruvate, and ammonium acetate. The implementation of TPL for L-DOPA production has been hampered and the need for the most efficient TPL source with higher L-DOPA production and substrate conversion rate is prevailing. This study involves identifying a novel TPL from Kluyvera intermedia (Ki-TPL) and displayed a robust expression in Escherichia coli. The recombinant strain YW000 carrying Ki-TPL proved strong catalytic activity with a highest L-DOPA yield compared with 16 other TPLs from different organisms. With a further aim to improve this efficiency, random mutagenesis of Ki-TPL was performed and a mutant namely YW021 was obtained. The whole cells of YW021 as biocatalyst yielded 150.4 g L-1 of L-DOPA with a 99.99 % of pyrocatechol conversion at the optimum condition of pH 8.0 at 25 °C, which is the highest level reported to date. Further, the homology modeling and structural analysis revealed the mutant residues responsible for the extensive L-DOPA biosynthesis.

Biotreatment of restaurant wastewater with an oily high concentration by newly isolated bacteria from oily sludge.

High concentration restaurant oily wastewater from restaurants and food processing industries discharged into water environment usually results in environment pollution and inhibits the activity of microorganisms in biological wastewater treatment systems. In this study, 75 strains from oily sludge were isolated with oil degradation activity for edible oil-contained wastewater. Eight isolates were able to grow well in liquid cultures with edible oil as the sole carbon source and discovered with high efficient oil-degrading ability. Seven out of eight isolates were identified as Acinetobacter and one isolate as Kluyvera cryocrescens, based on their 16S rRNA gene sequences. Three highly efficient oil degrading bacteria (Acinetobacter dijkshoorniae LYC46-2, Kluyvera cryocrescens LYC50-1a and Acinetobacter pittii LYC73-4b) were selected and their degradation characteristic were examined, the results showed that the three isolates were effective under pH range from 7.0 to 10.0, and temperature from 25 to 35 °C. For degradation of 2-4% (v/v) of vegetable oil, > 85% degradation percentage were obtained within 30 h. Degradation of the higher concentration oil (6-8%, v/v) result in 50-70% degradation percentage within 72 h, and the degradation percentage for the isolated strains were decreased about 50% for the degradation of 10% oil (< 45%) compared to 2% oil. Different type of oils were also tested, > 90% of degradation percentage were obtained by the three isolates, implied that these strains are capable of removing various oils efficiently. These results suggested that Acinetobacter dijkshoorniae LYC46-2, Kluyvera cryocrescens LYC50-1a and Acinetobacter pittii LYC73-4b are potential species could be efficiently used for high concentration restaurant oily wastewater treatment and might be applicable to a wastewater treatment system for the removal of oil.

Kluyvera georgiana MCC 3673: A Novel Electrogen Enriched in Microbial Fuel Cell Fed with Oilseed Cake.

A novel electrogenic bacterial species, Kluyvera georgiana MCC 3673, was isolated by enrichment in microbial fuel cells (MFC) using oilseed cake as a growth substrate. CHNS analyses showed that oilseed cakes are rich in carbon and nitrogen content. Utilization of these compounds by bacteria was evident from 50% reduction in chemical oxygen demand. The maximum power density of 379 ± 8 mW/m2 was produced from sesame seed cake media with mixed consortia inoculum from lake sediment. Enrichment was carried out for over 15 cycles by renewing the media periodically on drop of the voltage. A pure culture of enriched electrogen was isolated by dilution plate technique. Physiological and biochemical studies were performed on the isolate as per standard methods. Genetic analysis by 16S rDNA sequencing revealed that this organism is closely related to Kluyvera georgiana. When inoculated in MFC as pure culture, the maximum power density of 158 ± 11 mW/m2 and 172 ± 13 mW/m2 was produced with sesame and groundnut oilseed cake media, respectively. The performance increased in LB media producing maximum power density of 394 ± 6 mW/m2. This bacterium has also scope for application in wide range of MFC as it can produce electricity even in suspended culture. To our knowledge, this is the first report on bio-electricity generation using oilseed cake as substrate in MFC.

Kluyvera ascorbata as a Pathogen in Adults and Children: Clinical Features and Antibiotic Susceptibilities in a Single Center Study.

To assess the clinical characteristics of the rare Kluyvera ascorbata infection, we reviewed the medical records of patients from whom K. ascorbata was isolated from 2010 to 2016, and conducted a systematic review of the English and Spanish literature in PubMed for reports of K. ascorbata infection in humans from 1971 to 2018. A total of 43 cases (24 adults and 19 children) were enrolled: 3 at our hospital and 40 from the literature review. The urinary tract was the most common site of infection (44.2%, 19/43), followed by the bloodstream (27.9%, 12/43). There was no significant difference in the frequency of urinary tract infections (50% vs 36.8%; P = 0.388) and bloodstream infections (25% vs 31.6%; P = 0.633) in adults and children. Seventeen (60.7%, present in 28 of 43 cases) had nosocomial or healthcare-associated infections: 72.7% among children and 60% among adults. Superinfection developed in 20% (6 in 30 cases). The overall mortality was 12.1%. The antimicrobial agents mainly used in these 43 cases were third-generation cephalosporin, cefepime, piperacillin-tazobactam, ciprofloxacin, amikacin, and carbapenem. Most strains were resistant to ampicillin and first- and second-generation cephalosporins. K. ascorbata is a rare but significant clinical pathogen in adults and children.

First Report of an OXA-48- and CTX-M-213-Producing Kluyvera Species Clone Recovered from Patients Admitted in a University Hospital in Madrid, Spain.

Enterobacterales species other than Klebsiella pneumoniae also contribute to OXA-48 carbapenemase endemicity. We studied the emergence of an OXA-48-producing Kluyvera species clone, which expresses the novel CTX-M-213 enzyme, colonizing patients in our hospital. Rectal swabs from patients admitted in four wards (March 2014 to March 2016; R-GNOSIS project) were seeded onto Chromo ID-ESBL) and Chrom-CARB/OXA-48 chromogenic agar plates. Carbapenemases and extended-spectrum ß-lactamases (ESBLs) were characterized (PCR, sequencing, cloning, and site-directed mutagenesis), and antibiotic susceptibility was determined. Clonal relatedness was established (XbaI pulsed-field gel electrophoresis [XbaI-PFGE]), and plasmid content was studied (transformation, S1 nuclease digestion-PFGE, SB-hybridization, restriction fragment length polymorphism [RFLP] analysis [DraI and HpaI], and PCR [incompatibility group and repA, traU, and parA genes]). Whole-genome sequencing (WGS) (Illumina HiSeq-2500) and further bioinformatics analysis of plasmids (PLACNET and plasmidSPAdes) were performed. Patients' charts were reviewed. Six unrelated patients (median age, 75 years [range, 59 to 81 years]; 4/6 male patients) colonized with OXA-48-producing Kluyvera species isolates (>95% similarity of the PFGE pattern) were identified. Nosocomial acquisition was demonstrated. In two patients, OXA-48-producing Kluyvera species isolates coexisted with OXA-48-producing Raoultella ornithinolytica, K. pneumoniae, and Escherichia coli The blaOXA-48 gene was located on an ∼60-kb IncL plasmid related to IncL/M-pOXA-48a and the novel blaCTX-M-213 gene in a conserved chromosomal region of Kluyvera species isolates. CTX-M-213, different from CTX-M-13 (K56E) but conferring a similar ß-lactam resistance profile, was identified. Genomic analysis also revealed a 177-kb IncF plasmid (class I integron harboring sul1 and aadA2) and an 8-kb IncQ plasmid (IS4-blaFOX-8). We describe the first blaOXA-48 plasmid in Kluyvera spp. and the novel chromosomal CTX-M-213 enzyme and highlight further nosocomial dissemination of blaOXA-48 through clonal lineages or plasmids related to IncL/M-pOXA-48a.

Proposing Kluyvera georgiana as the Origin of the Plasmid-Mediated Resistance Gene fosA4.

A putative fosA gene in Kluyvera georgiana 14751 showed 99% nucleotide identity with plasmid-encoded fosA4 Due to a single-nucleotide insertion translating to a truncated protein, K. georgiana 14751 fosA does not confer fosfomycin resistance. However, analysis of another genome deposit (Kluyvera ascorbata WCH1410) that could be recategorized as K. georgiana after phylogenetic analysis revealed a fosA gene 100% identical to the plasmid-borne fosA4 gene. We suggest that Kluyvera georgiana represents the most probable origin of fosA4.