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.

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.

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.

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.

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.

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.

Origin of the plasmid-mediated fosfomycin resistance gene fosA3.

Background: fosA3 is the most commonly reported plasmid-mediated fosfomycin resistance gene among Enterobacteriaceae. Objectives: To identify the origin of fosA3. Methods: The chromosome of Kluyvera georgiana clinical strain YDC799 was fully sequenced with single-molecule real-time sequencing. Comparative genetic analysis was performed for K. georgiana YDC799, K. georgiana type strain ATCC 51603 and representative fosA3-carrying plasmids. fosA genes were cloned in Escherichia coli to confirm function. Results: K. georgiana YDC799 harboured fosA (designated fosAKG) and blaCTX-M-8 on the chromosome. The genetic environments surrounding fosA3 and bounded by IS26 were nearly identical with the corresponding regions of K. georgiana YDC799 and ATCC 51603. The amino acid sequence of FosAKG from YDC799 and K. georgiana ATCC 51603 shared 99% and 94% identity with FosA3, respectively. Cloned FosAKG conferred fosfomycin resistance with an MIC of >1024 mg/L for E. coli. Conclusions: The plasmid-mediated fosA3 gene was likely mobilized from the chromosome of K. georgiana by an IS26-mediated event.

Concentration and Variety of Carbapenemase Producers in Recreational Coastal Waters Showing Distinct Levels of Pollution.

Carbapenemase-producing bacteria cause difficult-to-treat infections related to increased mortality in health care settings. Their occurrence has been reported in raw sewage, sewage-impacted rivers, and polluted coastal waters, which may indicate their spread to the community. We assessed the variety and concentration of carbapenemase producers in coastal waters with distinct pollution levels for 1 year. We describe various bacterial species producing distinct carbapenemases not only in unsuitable waters but also in waters considered suitable for primary contact.

Kluyvera ascorbata Strain from Hospital Sewage Carrying the mcr-1 Colistin Resistance Gene.

The newly identified plasmid-borne colistin resistance gene mcr-1 was found in a Kluyvera ascorbata isolate from hospital sewage in China. mcr-1 was carried by a 57-kb self-transmissible IncI2 plasmid. Unlike in a previous report, mcr-1 was not associated with ISApl1 and was inserted into a gene encoding a putative membrane protein by an unknown mechanism. This study highlights that mcr-1 has spread to multiple bacterial species.

Bacteriophage ϕMAM1, a viunalikevirus, is a broad-host-range, high-efficiency generalized transducer that infects environmental and clinical isolates of the enterobacterial genera Serratia and Kluyvera.

Members of the enterobacterial genus Serratia are ecologically widespread, and some strains are opportunistic human pathogens. Bacteriophage ϕMAM1 was isolated on Serratia plymuthica A153, a biocontrol rhizosphere strain that produces the potently bioactive antifungal and anticancer haterumalide oocydin A. The ϕMAM1 phage is a generalized transducing phage that infects multiple environmental and clinical isolates of Serratia spp. and a rhizosphere strain of Kluyvera cryocrescens. Electron microscopy allowed classification of ϕMAM1 in the family Myoviridae. Bacteriophage ϕMAM1 is virulent, uses capsular polysaccharides as a receptor, and can transduce chromosomal markers at frequencies of up to 7 × 10(-6) transductants per PFU. We also demonstrated transduction of the complete 77-kb oocydin A gene cluster and heterogeneric transduction of a plasmid carrying a type III toxin-antitoxin system. These results support the notion of the potential ecological importance of transducing phages in the acquisition of genes by horizontal gene transfer. Phylogenetic analyses grouped ϕMAM1 within the ViI-like bacteriophages, and genomic analyses revealed that the major differences between ϕMAM1 and other ViI-like phages arise in a region encoding the host recognition determinants. Our results predict that the wider genus of ViI-like phages could be efficient transducing phages, and this possibility has obvious implications for the ecology of horizontal gene transfer, bacterial functional genomics, and synthetic biology.

Quorum sensing activity of a Kluyvera sp. isolated from a Malaysian waterfall.

In many species of bacteria, the quorum sensing mechanism is used as a unique communication system which allows them to regulate gene expression and behavior in accordance with their population density. N-Acylhomoserine lactones (AHLs) are known as diffusible autoinducer molecules involved in this communication network. This finding aimed to characterize the production of AHL of a bacterial strain ND04 isolated from a Malaysian waterfall. Strain ND04 was identified as Kluyvera sp. as confirmed by molecular analysis of its 16S ribosomal RNA gene sequence. Kluyvera sp. is closely related to the Enterobacteriaceae family. Chromobacterium violaceum CV026 was used as a biosensor to detect the production of AHL by strain ND04. High resolution triple quadrupole liquid chromatography-mass spectrometry analysis of strain ND04 showed our isolate produced two AHLs which are N-(3-oxohexanoyl)homoserine lactone (3-oxo-C6 HSL) and N-3-oxo-octanoyl-L-homoserine lactone (3-oxo-C8 HSL).

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.

Misidentification and misreporting of antibiotic resistance in Kluyvera bacteremia by blood culture molecular identification panels.

The use of molecular identification panels has advanced the diagnosis for blood stream infections with fast turnaround time and high accuracy. Yet, this technology cannot completely replace conventional blood culture and standardized antibiotic susceptibility testing (AST) given its limitations and occasional false results. Here we present two cases of bacteremia caused by Kluyvera. Its identification and antibiotic resistance were at least partially mispresented by blood culture molecular identification panels on ePlex, Verigene, and Biofire. The detection of CTX-M resistance marker did not align with the susceptibility to the third generation cephalosporins among a wide range of antibiotics for this organism. Conventional extended-spectrum beta-lactamase (ESBL) testing was used to confirm the absence of ESBL. Caution should be taken when managing cases with CTX-M or ESBL detection in blood culture caused by uncommon pathogens. Conventional culture with microbial identification and standardized AST should continue to be the gold standard for routine patient care. IMPORTANCE: This is the first report that highlights the limitations of blood culture molecular identification panels on identifying Kluyvera and its associated antibiotic resistance patterns. Both the false identification and overreporting of antibiotic resistance could mislead the treatment for bacteremia caused by this pathogen. Patient isolation could have been avoided due to the lack of extended-spectrum beta-lactamase (ESBL) activity of the organism. This report emphasizes the importance of confirming rapid identification and antibiotic resistance by molecular technologies with standardized methods. It also provides insight into the development of new diagnostic panels.

Cloning, overexpression, crystallization and preliminary X-ray crystallographic analysis of a slow-processing mutant of penicillin G acylase from Kluyvera citrophila.

Kluyvera citrophila penicillin G acylase (KcPGA) has recently attracted increased attention relative to the well studied and commonly used Escherichia coli PGA (EcPGA) because KcPGA is more resilient to harsh conditions and is easier to immobilize for the industrial hydrolysis of natural penicillins to generate the 6-aminopenicillin (6-APA) nucleus, which is the starting material for semi-synthetic antibiotic production. Like other penicillin acylases, KcPGA is synthesized as a single-chain inactive pro-PGA, which upon autocatalytic processing becomes an active heterodimer of α and ß chains. Here, the cloning of the pac gene encoding KcPGA and the preparation of a slow-processing mutant precursor are reported. The purification, crystallization and preliminary X-ray analysis of crystals of this precursor protein are described. The protein crystallized in two different space groups, P1, with unit-cell parameters a = 54.0, b = 124.6, c = 135.1 Å, α = 104.1, ß = 101.4, γ = 96.5°, and C2, with unit-cell parameters a = 265.1, b = 54.0, c = 249.2 Å, ß = 104.4°, using the sitting-drop vapour-diffusion method. Diffraction data were collected at 100 K and the phases were determined using the molecular-replacement method. The initial maps revealed electron density for the spacer peptide.

Isolation and characterization of Kluyvera georgiana strain with the potential for acrylamide biodegradation.

Worldwide contamination by acrylamide, a neurotoxicant and carcinogen in animals, is becoming a significant problem. We isolated three novel acrylamide-degrading bacteria from domestic wastewater in Chonburi, Thailand. Using biochemical characteristics and 16S rRNA gene sequencing, the strains were identified as Klebsiella pneumoniae, Kluyvera georgiana and Enterococcus faecalis. K. georgiana strain No. 2 was selected for further characterization due to its degradation potential of high concentrations of acrylamide at the mesophilic temperatures. The strain grew well in the presence of acrylamide at concentrations to 0.5 % (w/v), pH 5.0 to 7.0 and 37°C. Degradation of acrylamide to acrylic acid began after 30 min of cultivation as a biomass-dependent manner. Mass balance analysis revealed 92.3 % conversion of acrylamide to acrylic acid and two lower polarity compounds. Strain No. 2 degraded many aliphatic amides but not iodoacetamide and thioacetamide. High degradation level (>80 %) was found with propionamide, cyanoacetamide and acetamide. Moderate degradation was obtained in the order of formamide > butyramide > lactamide > urea while sodium azide provided 34 % degradation. These findings render this novel bacterium attractive for biodegradation of acrylamide and other aliphatic amides in the environment.