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Objective The toxin-producing Klebsiella oxytoca causes antibiotic-associated haemorrhagic colitis (AAHC). The disease-relevant cytotoxins tilivalline and tilimycine produced by certain K. oxytoca isolates are encoded by the non-ribosomal peptide synthetase genes A (npsA) and B (npsB). In this study, the new LightMix® Modular kit for detection of relevant K. oxytoca sensu lato (s.l.) toxin genes was evaluated. Methods DNA was extracted on the automated EMAG® platform. Amplification was done on the Light Cycler® 480 II instrument. A total of 130 residual faecal specimens that had been collected from patients with antibiotic-associated diarrhoea were studied to determine the clinical sensitivity and specificity. Toxigenic culture served as reference method. Results With the new kit, the limit of detection was 15 CFU/mL for all targets. For the K. oxytoca s.l. specific pehX target, 65 of 130 clinical specimens were positive while toxin specific targets (npsA/npsB) were positive in 47 of 130. The npsA/npsB PCR targets showed a clinical sensitivity of 100% (95% CI of 80.5% to 100%) and a specificity of 73.5% (95% CI of 64.3% to 81.3%) with a positive predictive value of 16.5% (95% CI of 12.7% to 21.2%) and a negative predictive value of 100%. Conclusion Compared with culture, additional K. oxytoca s.l.-positive clinical specimens were detected with real-time PCR. The specificity of the toxin targets appears moderate due to the inferior sensitivity of the culture-based reference method. Since the developed assay is highly sensitive, it may be used as first-line method to improve AAHC diagnosis.

Summary New Delhi metallo-β-lactamase (NDM) gene confers high-level resistance to an array of β-lactams including carbapenems. Short- and long-read sequencing was used to investigate outbreaks of NDM-positive Enterobacterales including a potential horizontal gene transfer (HGT) event of an NDM-positive plasmid between Salmonella enterica and Klebsiella pneumoniae. Genomic analysis demonstrated a high degree of similarity between NDM-carrying plasmids from patient 1 in K. pneumoniae and patient 2 with S. enterica, K. pneumoniae and Klebsiella oxytoca, confirming an inter-species HGT event. The utility of whole-genome sequencing was demonstrated for in-hospital outbreaks, previously undetected using traditional infection-control surveillance.

The rapid detection and characterization of carbapenemases in isolates of Enterobacterales are crucial for precise antibiotic administration and infection control. This article reports the findings from a parallel evaluation of the NG-Test Carba 5 (NG Biotech, Guipry, France) and Xpert Carba-R (Cepheid, Sunnyvale, CA) assays in the detection and differentiation of five carbapenemases [imipenem-resistant phenotype (IMP), Klebsiella pneumoniae carbapenemase, New Delhi metallo-β-lactamase (NDM), oxacillin-hydrolyzing β-lactamase (OXA)-48-like, and Verona integron-encoded metallo-β-lactamase] or the genes that encode them. A total of 122 isolates recovered from blood cultures and 106 positive blood culture broth (BCB) specimens, including 134 Klebsiella pneumoniae, 54 Escherichia coli, 27 Enterobacter cloacae, 8 Klebsiella oxytoca, 2 Klebsiella aerogenes, and 3 Citrobacter freundii, were collected from two tertiary hospitals (Xi'an, China). Using PCR sequencing techniques, 89 isolates and 29 BCB specimens were determined to be Enterobacterales harboring carbapenem-resistance genes. In comparison to the PCR sequencing results, the specificities with both the NG-Test Carba 5 and Xpert Carba-R assays were 100%; the sensitivities were 92.1% and 100%, respectively, for recovered isolates and 79.3% and 100% for BCB specimens. The NG-Test Carba 5 missed eight NDM, four OXA-48-like, and one IMP β-lactamases in specimens containing two or three carbapenemase types. In summary, the NG-Test Carba 5 assay may yield false-negative results if isolates or BCB specimens contain two or three carbapenemases.

Objective The production of β-lactamase enzymes such as AmpC β-lactamases and extended-spectrum β-lactamases (ESBLs) is among the main mechanisms for resistance to expanded-spectrum cephalosporins. The present study was conducted to investigate the prevalence and molecular epidemiology of plasmid-mediated AmpC beta (β)-lactamase in ESBL co-producing Escherichia coli (E. coli) and Klebsiella spp. (Klebsiella pneumoniae and Klebsiella oxytoca) clinical isolates in the northeast of Iran. Methods A total of 602 E. coli and Klebsiella spp. clinical isolates were collected from three hospitals in Mashhad (northeast of Iran). A combination disk test (CDT) was performed for the phenotypic detection of ESBLs. Screening for the detection of AmpC β-lactamases was performed by a susceptibility test to a cefoxitin disc among ESBL producing isolates. A confirmatory test for AmpC β-lactamases was performed using the Mast® D68C test. Identification of plasmid-mediated AmpC cluster genes was done by multiplex polymerase chain reaction (PCR). Results Among 336 ESBL-producing strains, 230 (68.4%) isolates were resistant to cefoxitin. Results of the Mast® D68C test showed that 30% (69/230) of cefoxitin-resistant isolates simultaneously exhibited ESBL and AmpC activity and 22% (51/230) of isolates probably showed multi-drug resistant (MDR) phenotype. Results of multiplex PCR among ESBL-positive isolates showed that, 16.7% (56/336) of isolates were positive for plasmid-borneampC cluster genes, and CMY (38%) was the most frequent genotype of plasmid mediated AmpC. Conclusion Findings of the study revealed that an increase in the prevalence of ESBL and AmpC co-producer in E. coli and Klebsiella spp. strains may become an important public health issue. Therefore, there is a vital need for surveillance of spread of these clinical isolates.

Objectives The aim of this study was to investigate the molecular mechanisms of imipenem resistance in Enterobacteriaceae and to assess the antimicrobial activities of ceftazidime/avibactam (CAZ/AVI) and aztreonam/avibactam (ATM/AVI) against imipenem-resistant clinical isolates in a tertiary hospital in China. Methods A total of 91 imipenem-resistant Enterobacteriaceae were collected and genes encoding carbapenemases, ESBLs, AmpC β-lactamases and porins were detected using PCR. MICs and susceptibility were determined using in-house-prepared broth microdilution panels and were interpreted according to CLSI breakpoints. Results Imipenem-resistant isolates comprising 54 Klebsiella pneumoniae, 18 Escherichia coli, 8 Enterobacter cloacae, 6 Serratia marcescens, 3 Klebsiella oxytoca and 2 Klebsiella aerogenes were collected independently. Five different carbapenemase genes were identified, namely blaKPC-2 (n = 60), blaNDM-5 (n = 14), blaNDM-1 (n = 11), blaKPC-3 (n = 4) and blaIMP-4 (n = 1). Among the 91 carbapenem-resistant Enterobacteriaceae (CRE), 85 isolates harboured at least one ESBL and/or AmpC gene, including 5 strains without carbapenemase-encoding genes. Moreover, 31 K. pneumoniae carried ompK35 and/or ompK36 mutations. MLST results showed that the K. pneumoniae belonged to 12 different STs, with ST11 being predominant (29/54; 53.7%). Overall, 17.6%, 25.3%, 41.8%, 65.9% and 100% of the CRE strains were susceptible to amikacin, trimethoprim/sulfamethoxazole, tetracycline, CAZ/AVI and ATM/AVI, respectively. Conclusion This study revealed that CRE isolates differ significantly in their species, STs, porins and carbapenemase genes in a single Chinese hospital. ATM/AVI exhibited potent activity against CRE isolates, even for the most notorious double-carbapenemase-producers with porin defects, whereas CAZ/AVI was active against all the non-metallo-β-lactamase-producing isolates.

Background Epidemiological data on secondary bacteremia associated with nosocomial urinary tract infections generally include adult patients with urinary catheters. Aim To evaluate the frequency and outcome of secondary bacteremia complicating healthcare-associated urinary tract infections. Material and methods This study was conducted between May 2013 and December 2017 at the Dr. Behcet Uz Children's Hospital and included symptomatic nosocomial urinary tract infections. A total of 117 patients with positive blood cultures were enrolled in the study. Results Six patients had bacteremia associated with nosocomial urinary tract infections yielding an incidence of 5.1%. The pathogens responsible for secondary bacteremia were: Klebsiella pneumonia in two patients, Enterococcus faecium in two patients, Klebsiella oxytoca in one patient, and Pseudomonas aeruginosa in one patient. Conclusion The incidence of bacteremia associated with nosocomial urinary tract infections was not different from bacteremia associated with community-acquired urinary tract infections, and was approximately 5%.

Objectives Recently, the Gram-negative bacterium Klebsiella oxytoca has been identified as an emerging pathogen. Here we report the draft genome of a 2,3-butanediol-producing strain, K. oxytoca CCTCC M207023, isolated from soil in Nanjing, China. The tetracycline-resistant phenotype and the high yield of 2,3-butanediol was demonstrated. Methods The draft genome of K. oxytoca CCTCC M207023 was determined using an Illumina NovaSeq™ 6000 next-generation DNA sequencing platform. Clean sequencing data were subsequently assembled using SOAPdenovo. Results The draft genome of K. oxytoca CCTCC M207023, comprising 5 658 144 bp and with a GC content of 56.50%, was assembled into 5262 open-reading frames (ORFs). Antimicrobial resistance genes were also annotated. Conclusions The draft genome sequence of K. oxytoca CCTCC M207023 reported here will be a reference for comparative analysis with the antimicrobial resistance mechanisms for the safety of 2,3-butanediol industrial production.

Introduction: Carbapenemase-producing Enterobacteriaceae (CPE) are of global concern due to the growing number of patients who acquire them and their association with high mortality rates. Although there are some reports of endemicity in developing countries, little is known about this microorganism, and Ecuador is not an exception. Subsequently, our objective was to clinically and molecularly characterize carbapenemase producing-Enterobacteriaceae in intensive care units (ICUs) in Guayaquil, Ecuador. Methods: To determine CPE colonization, we obtained perineal and inguinal swabs from patients admitted to seven intensive-care adult units in Guayaquil-Ecuador between February and April 2016. The Centers for Disease Control and Prevention (CDC) laboratory protocol and chromogenic agar were used to process the cultures. Polymerase chain reaction was used to confirm carbapenemase production. Genotypic analysis was performed by Multilocus Sequence Typing (MLST) and pulsed-field electrophoresis (PFEG). Demographic and clinical data were obtained from the electronic charts and patient’s relatives. Results: Six hundred seventy-seven patients were included in the study, of whom 255 were colonized/infected by CPE. The CPE prevalence was 37.67%. Previous use of antimicrobials, use of invasive procedures and being burned at admission were associated with CPE. The most frequent infection was found after a surgical procedure. Klebsiella pneumoniae (n = 249) was the predominant microorganism harbouring blaKPC, followed by Enterobactercloacae (n = 8), Klebsiella aerogenes (n = 4), Escherichia coli (n = 4) and Klebsiella oxytoca (n = 1). NDM was present in Proteus mirabilis. The strains were distributed in 19 sequence types (ST), and 10 were not reported previously in Ecuador. ST 258 was the sequence type isolated most frequently. Conclusion: This study shows a high prevalence of CPE in ICUs, particularly K. pneumoniae blaKPC ST 258. The identification of KPC alleles may help to understand the routes of dissemination and control spread within ICUs in Guayaquil, Ecuador. Keywords: Carbapenemase, Intensive care units, Prevalence

We report the asymmetric catalysis mediated by a newly isolated strain from soil, Klebsiella oxytoca, for the preparation of the chiral intermediate of antiviral agent lamivudine which is proven to be one of most potent medicines for the treatment of human chronic hepatitis B and HIV infection. To improve the efficiency of the transformation, firstly the carbon and nitrogen sources of the medium were screened and the best performance was achieved with glucose (5 g/L) as carbon source and tryptone (5 g/L) as nitrogen source. Secondly, optimization of the reaction condition was carried out. A series of parameters were investigated including temperature, pH, metal ions, surfactant, and substrate concentration. The best outcome [enantiomeric excess (ee) of 99.9% and yield of 24%] of the enantioselective resolution of the racemic substrate catalyzed by whole cell of Klebsiella oxytoca was reached at 30 ℃, pH 7.0, substrate concentration of 1.5 g/L, and no additives. As a comparation to most of the lipase-catalyzed systems for the production of chiral oxathiolane moiety of lamivudine, the reaction occurred in a single-phase aqueous system which meets the golden and stringent criteria of green chemistry. To our knowledge, this is the first study using Klebsiella oxytoca as catalyst for the preparation of the chiral moiety of lamivudine.

The structure of the di-cationic complexes [M(II)(C8H10N4O2)4](PF6)2, M = Fe(II), Co(II), Mn(II), Cd(II), Zn(II), Cu(II), Ni(II) were prepared and characterized by molar conductance, FTIR, UV–visible, EPR and 31P, 19F, 1H, 13C NMR. The molar conductance data show that the complexes are electrolyte of the type 2:1. As well as, the results of infrared spectroscopic analysis shows the characteristic bands of caffeine and hexafluorophosphate (PF6−). Moreover, the multiplicity of signals in the spectrum of 1H NMR, exhibit that the cationic metals surrounded by four caffeine. Furthermore, the analysis electronic spectra of complexes shows that the complexes have tetrahedral geometry, except the copper complex, it has a square planar geometry. The antibacterial activity of the complexes was evaluated against some bacteria Escherichia coli, Staphylococcus aureus, Klebsiella pneumonia, Klebsiella oxytoca and Pseudomonas putida species. The results of the molecular docking show that the complexes have good affinity with the protein PI3Kγ.

A novel bioflocculant, A-GS408, produced by Klebsiella oxytoca GS-4-08 cultured in acetonitrile (ACN) as sole nitrogen source was investigated in this study. A complete degradation of 1 g l−1 of ACN was achieved in 350 h, and 4.6 g of crude A-GS408 can be obtained in one litter of synthetic medium. The as-prepared bioflocculant exhibits good flocculation efficiency (over 90%) toward Kaolin solution with the aid of Fe3+. Chemical analysis showed that the bioflocculant was mainly composed of polysaccharides (46.3%) and proteins (20.6%). FTIR and XPS results indicated the abundant carboxyl, amine and hydroxyl groups in A-GS408 s, which play an important role on Pd2+ and Cu2+ adsorption. The adsorption of heavy metals can be well stimulated by Freundlich isotherm equation, and the Kf was up to 439.2 mg1-1/n l1/n g−1 and 112.2 mg1-1/n l1/n g−1 for Pb2+ and Cu2+, respectively. The kinetic fitting results proved that the adsorption of heavy metals by A-GS408 was chemisorption. This study may provide a new method for preparation of bioflocculant, which can not only degrade toxic compound i.e., acetonitrile, but also can reuse considerable nitrogen source from nitrile-containing wastewater.

Klebsiella is an opportunistic human pathogen and responsible for many of nosocomial infections. Gram-negative bacteria can acquire antibiotic resistance genes through several mechanisms including integrons. This study aimed to determine the prevalence of antibiotic resistance pattern, and class 1–3 integrons in clinical isolates of Klebsiella among Iranian patients. From March to September 2016, plasmid DNA of 169 Klebsiella isolates was extracted after determining their resistance pattern and ESBL production by disk diffusion method. The presence of class 1–3 integrons was detected by targeting integrase genes with polymerase chain reaction (PCR) method. Out of 169 Klebsiella, 41.1% of Klebsiella pneumoniae and 18.2% of Klebsiella oxytoca isolates were ESBL-positive. The most resistance rate in both species was against ampicillin. Class 1 and 2 integrons were detected in 15.8% and 5.9% of ESBL-producing K. pneumoniae, respectively. In case of K. oxytoca, class 1 and 2 integrons were positive equally in 18.2% of isolates. The results of our study showed a significant association between the presence of integrons and antibiotic resistance in clinical isolates of Klebsiella. Drug-resistant Klebsiella strains complicate the management and treatment of nosocomial infections, hence prudent use of antibiotics therapy and effective control measures of infection are mandatory.

Summary To study intestinal colonization by OXA-48-producing Klebsiella pneumoniae (KpO48) after hospital discharge, stool samples from 22 previously colonized subjects were collected. Time from discharge was 33–611 days, without readmissions. Eight subjects (36%) were identified as blaOXA-48 gene carriers. In all of them the hospital-acquired strain of KpO48 had been lost, and the gene was harboured by other strains of K. pneumoniae, Klebsiella oxytoca and/or Escherichia coli. Our findings show intestinal persistence for several months of a plasmid harbouring the OXA-48 carbapenemase gene in a significant proportion of individuals in the absence of antibiotic treatment.

The study aimed to evaluate the influence of rotating magnetic field on the growth rate and cell metabolic activity of Gram-positive (Staphylococcus aureus, ATCC 43300, Enterococcus faecalis, ATCC 29212, Streptococcus mutans ATCC 35668) and Gram-negative bacteria (Escherichia coli ATCC 8739, Serratia marcescens ATCC 274, Klebsiella oxytoca PCM 22). Liquid cultures of these bacteria were exposed to the rotating magnetic field with frequency f = 50 Hz and magnetic induction Bmax = 18 m T for 8 h at 37 °C. The obtained results showed the stimulated growth and metabolic activity of Gram-positive bacteria (up to 25%) and inhibited proliferation of Gram-negative bacteria (up to 17%). Obtained data suggest that the rotating magnetic field influenced on the cell wall transport mechanisms and this effect was different for these two groups of bacteria.

Selected marine fishes with different feeding habits were screened for the presence of symbiotic cellulolytic bacteria in their gut. Four cellulolytic species of symbiotic bacteria were isolated from GI tract of marine fishes namely Carangoides praeustus, Filimanus similis, Sardinella longiceps and Sillago sihama. The strains were identified after polyphasic phenotypic and genotypic (16S rRNA gene) characterization as Bacillus subtilis strain TCPC1, Vibrio alginolyticus strain CFSS2C2, Pseudomonas stutzeri strain KSLS4C3 and Klebsiella oxytoca strain MSSC4. (Genbank Accession nos.: JN710380, JN710378, JN710377, JN712301).The results indicated the presence of cellulolytic bacteria in GI tract of marine fishes of carnivorous, phytoplanktivorous and omnivorous feeding habits. Cellulolytic activity was the maximum for B. subtilis strain TCPC1 (0.45 mg glucose ml−1) and V. alginolyticus strain CFSS2C2 (0.24 mg glucose ml−1) at 234 h. While P. stutzeri strain KSLS4C3 showed the maximum utilization (0.22 mg glucose ml−1) from 240 to 258 h. K. oxytoca strain MSSC4 (0.47 mg glucose ml−1) showed three peaks during the study. The maximum rate of cellulose utilization was shown by P. stutzeri strain KSLS4C3 (0.05 mg glucose ml−1 medium h−1) followed by K. oxytoca strain MSSC4 (0.03 mg glucose ml−1 medium h−1).

The aim of the present study was to determine whether there is the mechanistic connection between antibacterial-dependent gut microbiota disturbance and anxiety. First, exposure of mice to ampicillin caused anxiety and colitis and increased the population of Proteobacteria, particularly Klebsiella oxytoca, in gut microbiota and fecal and blood lipopolysaccharide levels, while decreasing lactobacilli population including Lactobacillus reuteri. Next, treatments with fecal microbiota of ampicillin-treated mouse (FAP), K. oxytoca, or lipopolysaccharide isolated from K. oxytoca (KL) induced anxiety and colitis in mice and increased blood corticosterone, IL-6, and lipopolysaccharide levels. Moreover, these treatments also increased the recruitment of microglia (Iba1+), monocytes (CD11b+/CD45+), and dendritic cells (CD11b+/CD11c+) to the hippocampus, as well as the population of apoptotic neuron cells (caspase-3+/NeuN+) in the brain. Furthermore, ampicillin, K. oxytoca, and KL induced NF-κB activation and IL-1β and TNF-α expression in the colon and brain as well as increased gut membrane permeability. Finally, oral administration of L. reuteri alleviated ampicillin-induced anxiety and colitis. These results suggest that ampicillin exposure can cause anxiety through neuro-inflammation which can be induced by monocyte/macrophage-activated gastrointestinal inflammation and elevated Proteobacteria population including K. oxytoca, while treatment with lactobacilli suppresses it.

The potential human health risk of Japanese ready-to-eat (RTE) foods was investigated by determining the contamination by foodborne bacterial pathogens, the prevalence of opportunistic and nosocomial pathogens, and the antibiotic susceptibility of the isolates recovered from 96 samples of lightly pickled vegetables, 88 samples of Western-style desserts, and 98 samples of RTE fish and seafood products sold at retail in Osaka, Japan. Staphylococcus aureus, including isolates producing staphylococcal enterotoxin (SE), were isolated from six lightly pickled vegetable products, seven Western-style dessert products, and three RTE fish and seafood products. Of these isolates, one SEC-producing isolate from a cake was identified as community-acquired methicillin-resistant S. aureus, which was multilocus sequence type 8 and staphylococcal cassette chromosome mec type IV. Enterobacteriaceae species, including Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Proteus mirabilis, Serratia marcescens, Citrobacter freundii-Citrobacter braakii, and/or the Enterobacter cloacae complex, were isolated from 92 (95.8%) of the lightly pickled vegetable products, 39 (44.3%) of the Western-style dessert products, and 74 (75.5%) of the RTE fish and seafood products. On the basis of the antimicrobial susceptibility profiles of the opportunistic and nosocomial Enterobacteriaceae pathogens, the third-generation cephalosporin, fosfomycin, and quinolone resistance determinants were investigated. We detected AmpC products of the CIT group and a qnrB9 product in 5 and 1 C. freundii-C. braakii isolates, respectively, and fosA gene products in 15 E. cloacae complex isolates. Because RTE foods are consumed without a heating process, the spread of bacterial pathogens from contaminated food to human consumers is possible. RTE foods must be handled using hygienic procedures from the processing steps to the table to reduce the prevalence of potentially pathogenic or pathogenic bacteria and to prevent bacterial growth.

Four coordination complexes, [2-mimH]2[Co(pydc)2][ClO4]. H2O (1), [2-mimH]2[Zr(pydc)3] (2), [imiH]2[Zr(pydc)3].4H2O (3) and [Ca(pydcH2)2(H2O)]2[Co(pydc)2]2·6H2O (4) have been prepared from the reaction between proton-transfer compounds containing pyridine-2,6-dicarboxylic acid (pydcH2) with 2-methylimidazole (2-mim) and imidazole (imi) in 1:2 M ratio and metal salts. These compounds have been characterized by spectroscopic methods and single crystal X-ray diffraction technique was used to determine their structures. Also, thermal analyses (TGA/DTA) were carried out on all the synthetic compounds. Then the agar dilution and diffusion methods were used to determine the antibacterial activity of the ligands and their complexes on the growth profile of two pathogenic Gram-positive bacteria namely Staphylococcus aureus and Bacillus cereus and also two Gram- negative bacteria namely Escherichia coli and Klebsiella oxytoca. The highest antibacterial activity was exhibited by (pydcH2) and complex (2) at MIC values between 75 and 175 μg/mL. These two compounds exhibited stronger effects than gentamicin as a standard drug toward Klebsiella oxytoca. The anti-proliferative activity of compounds was evaluated in vitro using oxaliplatin as a positive control against MCF7 (a human breast cancer), HL60 (a human lymphocyte) and HT29 (a human colon adenocarcinoma) cell lines. Selective and significant cytotoxic effect of complex (4) was observed on MCF7 cells (IC50 = 10 μM, Cell viability 62.12%).

The emergence of extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E) infections requires re-assessment of therapeutic choices. Here we report the efficacy of cefoxitin-based antibiotic therapy for ESBL-E prostatitis. A prospective study including patients with ESBL-E prostatitis resistant to trimethoprim/sulfamethoxazole and fluoroquinolones from January 2014 to March 2016 was conducted. Cefoxitin was administered by continuous infusion for 3 weeks in the case of acute bacterial prostatitis or 6 weeks in the case of chronic bacterial prostatitis (CBP), with intravenous fosfomycin for the first 5 days. Urological investigations were performed to diagnose underlying urinary tract pathology. Clinical and microbiological efficacy were evaluated 3 months (M3) and 6 months (M6) after the end of therapy. A total of 23 patients were included in the study. The median patient age was 74 years (range 48-88 years). Of the 23 infections, 14 (61%) were CBP and 12 (52%) were healthcare-associated infections. The bacteria involved were Escherichia coli in 11 cases, Klebsiella pneumoniae in 10 cases and Klebsiella oxytoca in 2 cases. Clinical cure was observed in 19/23 patients (83%) at M3 and in 17/22 patients (77%) at M6. Urocultures were sterile in 13/23 patients (57%) at M3 and in 9/19 patients (47%) and M6. Urinary colonisation was observed in 6/19 patients (32%) with clinical cure at M3 and 5/14 patients (36%) with clinical cure at M6. No resistance to cefoxitin was detected. Surgical treatment was required for 7/23 patients (30%). In conclusion, cefoxitin-based antibiotic therapy is suitable for difficult-to-treat ESBL-E infections such as prostatitis.

Background and objectives A study of the susceptibility to antimicrobials of the extended spectrum beta-lactamase phenotypes (ESBL) in Escherichia coli and Klebsiella spp. was performed to discover the evolution of this type of resistance from urinary tract infections. Material and method A retrospective study was carried out between 2012 and 2016. Susceptibility to ciprofloxacin, tobramycin, cefoxitin, fosfomycin, nitrofurantoin, co-trimoxazole, and carbapenems was analysed using MicroScan® system. Results A total of 95,399 samples were processed and 9772 E. coli, 1784 Klebsiella pneumoniae and 248 Klebsiella oxytoca were isolated. ESBL strains were more frequent in women, although they decreased during 2015 and 2016 (65.7–67.2%). The prevalence of K. pneumoniae ESBL increased annually (28.1% in 2016). The average prevalence of E. coli ESBL was 10.5% with few oscillations. Higher resistance occurred to ciprofloxacin and cotrimoxazole, 89.5 and 94.7% in 2015, respectively, and there was lesser resistance to imipenem. Fosfomycin and nitrofurantoin were very active on E. coli ESBL. Conclusions ESBL producing E. coli and K. pneumoniae were prevalent, especially the latter, with a significant resistance to ciprofloxacin and cotrimoxazole. Susceptibility to imipenem was high.

Bacteria of the genera Klebsiella and Raoultella, which are present in foods and the natural environment, are associated with health hazards in humans. In the present study, two types of strips-based methods were developed to detect these bacteria simply and quickly. One method used lateral-flow test strips (LFTS) in combination with anti-Klebsiella antibodies labeled with palladium nanoparticles that bind to target bacteria, allowing their visualization. In the other, the antibodies were immobilized on nitrocellulose membranes, and urease activity was measured using an ion sensitive field effect transistor pH sensor. Testing of a combination of these two methods on 72 cultured strains successfully identified all 25 strains of Klebsiella pneumoniae, Klebsiella oxytoca and Raoultella ornithinolytica. This approach also accurately identified 76 of 77 (99%) strains isolated from meats and pastries. When combined with preculture, this method was accurate in identifying 19 of 26 (73%) target bacteria in food. These results suggest that the novel combination of strip-based assays may be effective for the on-site monitoring of food production plants, and thereby enhance food safety.

A facultative anaerobic iron reducing bacterium IMCUGIR-ZA was isolated from high arsenic (As) groundwater in Hetao Basin of Inner Mongolia and was identified as Klebsiella oxytoca. Strain IMCUGIR-ZA could reduce more than 96.8% of 2.78 mmol L−1 soluble Fe(III)-citrate in 7 days and 0.42 mmol solid iron from ferrihydrite into aqueous phase in 10 days using glucose as an electron donor under strictly anaerobic conditions. Appropriate concentrations of As could improve microbially-mediated iron reduction and release from ferrihydrite. Nearly threefold iron was reduced and released by strain IMCUGIR-ZA from As(V)-doped ferrihydrite (As 100 μmol L−1). When the strain was incubated with high arsenic sediments from Hetao Basin of Inner Mongolia under anaerobic conditions, 0.894 mmol L−1 Fe(III) and 12.44 μmol L−1 As were released to aqueous phase in 7 days. The results provided evidence of microbially-mediated mobilization of As in high arsenic groundwater aquifer in Hetao Basin of Inner Mongolia, China.

Klebsiella spp. are ubiquitous bacteria capable of colonizing humans and animals, and sometimes leading to severe infections in both. Due to their high adaptability against environmental/synthetic conditions as well as their potential in aquiring antimicrobial/metal/biocide resistance determinants, Klebsiella spp. are recognized as an emerging threat to public health, worldwide. Currently, scarce information on the impact of livestock for the spread of pathogenic Klebsiella spp. is available. Thus, the phenotypic and genotypic properties of extended-spectrum β-lactamase-producing, and colistin-resistant Klebsiella strains (n = 185) from process- and wastewater of two poultry and pig slaughterhouses as well as their receiving municipal wastewater treatment plants (mWWTPs) were studied to determine the diversity of isolates that might be introduced into the food-production chain or that are released into the environment by surviving the wastewater treatment. Selectively-isolated Klebsiella spp. from slaughterhouses including effluents and receiving waterbodies of mWWTPs were assigned to various lineages, including high-risk clones involved in human outbreaks, and exhibited highly heterogeneous antibiotic-resistance patterns. While isolates originating from poultry slaughterhouses showed the highest rate of colistin resistance (32.4%, 23/71), carbapenem-resistant Klebsiella spp. were only detected in mWWTP samples (n = 76). The highest diversity of resistance genes (n = 77) was detected in Klebsiella spp. from mWWTPs, followed by isolates from pig (n = 56) and poultry slaughterhouses (n = 52). Interestingly, no carbapenemase-encoding genes were detected and mobile colistin resistance genes were only obeserved in isolates from poultry and pig slaughterhouses. Our study provides in-depth information into the clonality of livestock-associated Klebsiella spp. and their determinants involved in antimicrobial resistance and virulence development. On the basis of their pathogenic potential and clinical importance there is a potential risk of colonization and/or infection of wildlife, livestock and humans exposed to contaminated food and/or surface waters.

Summary Gut colonization with multidrug-resistant (MDR) bacteria enhances the risk of bloodstream infections in susceptible individuals. We demonstrate highly variable degrees of ex vivo colonization resistance against a carbapenem-resistant Klebsiella pneumoniae strain in human feces samples and subsequently isolate diverse K. oxytoca strains from protected donors. Several of these K. oxytoca strains reduce gut colonization of MDR K. pneumoniae strains in antibiotic-treated and gnotobiotic mouse models. Comparative analysis of K. oxytoca strains coupled with CRISPR-Cas9-mediated deletion of casA, a protein essential for utilization of selected beta-glucosides, identified competition for specific carbohydrates as key in promoting colonization resistance. In addition to direct competition between K. oxytoca and K. pneumoniae, cooperation with additional commensals is required to reestablish full colonization resistance and gut decolonization. Finally, humanized microbiota mice generated from K. pneumoniae-susceptible donors are protected by K. oxytoca administration, demonstrating the potential of commensal K. oxytoca strains as next-generation probiotics.

Parkia biglobosa wastewater (PBWW), a waste in the local production of a condiment ‘iru’ was used to synthesize AgNPs, acting as bio-reductant and stabilizer to fabricate AgNPs. The particles were characterized by UV–visible spectroscopy, Fourier transform infrared spectroscopy (FTIR) along with electron microscopic investigations. The effectiveness of PBWW-AgNPs as antimicrobial agents was determined using multi-drug resistant (MDR) clinical bacterial isolates and some fungi. Also, the mechanisms of actions of the AgNPs on bacterial cells were studied via release of protein and nucleic acids. In an applied investigation, cotton and silk were functionalized with the AgNPs, and antimicrobial properties determined up to 5th wash cycle. The brown colloidal AgNPs depicted maximum absorption at 390 nm, while the FTIR spectra revealed O-H, N-H, and O = C = O bonds as the functional groups present. Optimal production of PBWW-AgNPs occurred at the pH 10 and concentration of 1 mM AgNO 3 . The particles were mainly spherical having size range of 11.00–83.30 nm with face-centred crystallinity. PBWW-AgNPs had inhibitions of 8–26 nm against MDR strains of Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Klebsiella oxytoca, K. pneumoniae and Proteus mirabilis with MIC of 80–100 μ g/ml. Evidences exist for the leakage of proteins and nucleic acids upon exposure of bacteria to AgNPs. The functionalized-fabrics inhibited bacterial growth and achieved 100% antifungal activity. This report is the first on valorization of P. biglobosa wastewater for nanobiotechnological exploration with potentials for biomedical applications in producing specialized fabrics with immense antimicrobial properties.

The Rapid Polymyxin NP test has been recently developed to rapidly detect polymyxin resistance in Enterobacteriaceae. Here we evaluated this test for detecting MCR-1/MCR-2-producing Enterobacteriaceae using a collection of 70 non-redundant strains either recovered from the environment, animals, or humans. Sensitivity and specificity were found to be 100%.

Accumulation of p-hydroxybenzoic acid (PHBA) in soil causes autotoxicity stress in cucumber. When the stress is mitigated by PHBA-degrading bacteria, plant metabolites have not been detected. To explore mechanisms underlining the mitigation, plant metabolites have not been combined with rhizospheric microbes, antioxidant and soil enzymes. In this study, a strain P620 of Klebsiella decomposed PHBA to acetyl CoA. Cucumber was sown into soil supplemented with P620 and/or PHBA. After addition with P620, P620 colonization and the enriched bacterial genera were observed in rhizosphere. Compared to PHBA stress alone, the combination of P620 application and PHBA stress improved plant growth, decreased PHBA concentration in soil, and increased the activities of five soil enzymes and eight antioxidant enzymes in leaves. Metabolomic and transcriptomic analysis highlighted that P620 application decreased the intensities of MAG(18:3) isomer 4, MAG(18:3) isomer 2, lysoPC 18:3 (2n isomer), 2'-deoxyadenosine-5'-monophosphate, pyridoxine, and glucarate O-phosphoric acid in PHBA-stressed leaves and down-regulated the expression of genes related to these metabolites. We propose a mechanism that P620 application alters microbial communities in PHBA-contaminated soil. Thus, the application reduces PHBA concentration in soil, activates antioxidant and soil enzymes, and also influences metabolites in leaves by affecting plant transcriptome, mitigating PHBA stress in cucumber.

Under the guidance of strain tolerance, a new combination method for crude oil-degrading bacterial consortium was studied. Firstly, more than 50 efficient crude oil-degrading and biosurfactant producing bacteria were isolated from petroleum-contaminated soil and water in Tianjin Binhai New Area Oilfield, China. Twenty-four of them were selected for further study. These strains were identified as belonging Pseudomonas aeruginosa, Bacillus subtilis, Brevibacillus brevis, Achromobacter sp., Acinetobacter venetianus, Lysinibacillus macroides, Klebsiella oxytoca, Stenotrophomonas rhizophila, Rhodococcus sp. and Bacillus thuringiensis. A shake-flask degradation test revealed that 12 of these strains could degrade over 50% of 1% crude oil concentration in 7 d. Of these, 8 strains were able to produce biosurfactants. Furthermore, environmental tolerance experiments indicated that the majority of the strains had the ability to adapt to extreme environments including high temperatures, alkaline environments and high salinity environments. A mixed bacterial agent comprising the strains WB2, W2, W3 and HA was developed based on the environmental tolerance tests and subjected to the pilot-scale degradation test indicating that this bacterial agent could degrade 85.2% of 0.8% crude oil concentration in 60 d. Our results suggest that the application of this mixed agent could remediate crude oil polluted soils in saline and alkaline environments.

Background The cytotoxic effects on epithelial cells of the human are not observed in other strains of Klebsiella spp and are only observed in K. oxytoca strains. MTT assay was used to evaluate cytotoxic activity. In this study, colorimetric method was used to evaluate the cytotoxic effect of cytotoxin-producing isolates on Hep-2 cell line and determines the percentage of surviving cells. Materials and methods In this study, we collected a total of 75 K. oxytoca strains isolate and we detected the production of toxins and their cytotoxic effects on HEp-2 cells. Colorimetric method such as MTT assay was used to evaluate the cytotoxic effect of cytotoxin-producing isolates on Hep-2 cell line and determines the percentage of surviving cells. Results Nine isolates had cytotoxic effects on HEp-2 cells. The results of MTT assay showed that the isolated strains were different from the control stain in terms of toxinogenicity and cytotoxic effects on HEp-2 cells at the studied dilutions (1:3, 1:6, 1:12, 1:24, 1:48, and 1:96). Conclusions In the current study, Percentage of Hep-2 surviving cells exposed to 1:3, 1:6, 1:12, 1:24, 1:48, and 1:96 supernatant dilutions of cytotoxin-producing Klebsiella oxytoca isolates was different.

Background Klebsiella oxytoca is an opportunistic pathogen which damages intestinal epithelium through producing cytotoxin tilivalline. This toxin plays a role in the pathogenesis of bacteria and is the main virulence factor which leads to antibiotic-associated hemorrhagic colitis progress. Materials and methods In this study, we collected a total of 75 K. oxytoca strains isolated from the stool, urine, blood, wounds, and sputum and evaluated them in terms of the production of toxins; we detected their cytotoxic effects on HEp-2 cells. Results Of all the isolates, five K. oxytoca strains isolated from the stool cultures, two strains isolated from the blood cultures, one strains isolated from the wound cultures, and one strains isolated from the urine cultures had cytotoxic effects on HEp-2 cells. The strains isolated from sputum cultures had no cytotoxic effects on HEp-2 cells. Conclusions In the current study, the majority of strains isolated from the stool of the patients included cytopathic effects on HEp-2 cells.

Meat wastes and losses were associated with spoilage microorganisms, usually sourcing from the biofilm cross-contamination. In this study, the attachment and biofilm formation of five meat-borne Enterobacteriaceae strains ( Enterobacter cloacae, Hafnia alvei, Proteus mirabilis, Citrobacter freundii and Klebsiella oxytoca ) were characterized by two simulated contamination scenarios. There were great variations in surface hydrophobicity, motility and adhesion ability on a polystyrene surface of 96-well microplates among these tested strains. P. mirabilis and C. freundii showed higher adhesion than other strains. More than 4.5 log CFU/cm 2 cells were transferred to stainless steel surface under a short-term (5 h) attachment scenario. More than 8 log CFU/cm 2 of biofilm formation on stainless steel surface under a long-term (7 d) growth scenario were found for all tested strains, and nearly 10.5 log CFU/cm 2 cells were observed for E . cloacae after 5-day incubation. A mature biofilm architecture was observed by confocal laser scanning microscopy (CLSM), consisting of multilayer aggregates of more than 30 μm thicknesses. This was further confirmed by attenuated total reflectance fourier transform infrared spectroscopy (ATR-FTIR), and the dominant peaks in biofilm spectra were mainly associated with amide, polysaccharides and glycosidic linkage. This finding could provide the basic data for developing novel disinfectant strategies.

A total of 82 extended spectrum beta-lactamase (ESBL) producing Klebsiella pneumoniae and 4 Klebsiella oxytoca isolates were collected in 2014 from four geographical areas in Bulgaria and their multilocus sequence type (MLST) and transferability of the ESBL encoding genes were investigated. The predominant type was CTX-M-15 (87%), followed by CTX-M-3 (9%), SHV-12 or SHV-2 (2%) and CTX-M-14 (1%). The CTX-M-15 producers belonged to ST15 (34.1%) and to a lesser extent to CC17 (ST16, ST17, ST336). The CTX-M-15 transconjugants showed a presence of R, A/C 2 and F replicons. The CTX-M-3 producers were assigned to ST29, ST70, ST432, ST542 and ST15 types and the transconjugants carried M 2 replicons. To the best of our knowledge, this is the first report that fully describes the MLST types among Bulgarian ESBL producing K. pneumoniae and the first report of the detection of IncR plasmid replicon type in our country.

Cytotoxin-producing Klebsiella oxytoca is the causative agent of antibiotic-associated hemorrhagic colitis (AAHC). Recently, the cytotoxin associated with AAHC was identified as tilivalline, a known pentacyclic pyrrolobenzodiazepine (PBD) metabolite produced by K. oxytoca. Although this assertion of tilivalline's role in AAHC is supported by evidence from animal experiments, some key aspects of this finding appear to be incompatible with toxicity mechanisms of known PBD toxins. We therefore hypothesized that K. oxytoca may produce some other uncharacterized cytotoxins. To address this question, we investigated whether tilivalline alone is indeed necessary and sufficient to induce cytotoxicity or whether K. oxytoca also produces other cytotoxins. LC-MS- and NMR-based metabolomic analyses revealed the presence of an abundant tricyclic PBD, provisionally designated kleboxymycin, in the supernatant of toxigenic K. oxytoca strains. Moreover, by generating multiple mutants with gene deletions affecting tilivalline biosynthesis, we show that a tryptophanase-deficient, tilivalline-negative K. oxytoca mutant induced cytotoxicity in vitro similar to tilivalline-positive K. oxytoca strains. Furthermore, synthetic kleboxymycin exhibited greater than 9-fold higher cytotoxicity than tilivalline in TC50 cell culture assays. We also found that the biosynthetic pathways for kleboxymycin and tilivalline appear to overlap, as tilivalline is an indole derivative of kleboxymycin. In summary, our results indicate that tilivalline is not essential for inducing cytotoxicity observed in K. oxytoca-associated AAHC and that kleboxymycin is a tilivalline-related bacterial metabolite with even higher cytotoxicity.

Primordial enzymes are proposed to possess broad specificities. Through divergence and evolution, enzymes have been refined to exhibit specificity towards one reaction or substrate, and are thus commonly assumed as "specialists". However, some enzymes are "generalists" that catalyze a range of substrates and reactions. This property has been defined as enzyme promiscuity and is of great importance for the evolution of new functions. The promiscuities of two enzymes, namely glycerol dehydratase and diol dehydratase, were herein exploited for catalyzing long-chain polyols, including 1,2-butanediol, 1,2,4-butanetriol, erythritol, 1,2-pentanediol, 1,2,5-pentanetriol, and 1,2,6-hexanetriol. The specific activities required for catalyzing these six long-chain polyols were studied via in vitro enzyme assays, and the catalytic efficiencies were increased through protein engineering. The promiscuous functions were subsequently applied in vivo to establish 1,4-butanediol pathways from lignocellulose derived compounds, including xylose and erythritol. In addition, a pathway for 1-pentanol production from 1,2-pentanediol was also constructed. The results suggest that exploiting enzyme promiscuity is promising for exploring new catalysts, which would expand the repertoire of genetic elements available to synthetic biology and may provide a starting point for designing and engineering novel pathways for valuable chemicals.

Klebsiella oxytoca causes several diseases in immunocompromised as well as healthy individuals. Increasing resistance to a number of antibiotics makes treatment options limited. Prevention using vaccine could be an important solution to get rid of infections caused by Klebsiella oxytoca. In recent time, genome based approaches have contributed significantly in vaccine development. Our aim was to identify the most conserved and immunogenic antigens that can be considered as potential vaccine candidates. KEGG database was used to find out pathways unique to the bacteria. Subcellular localization of the protein sequences taken from the selected 36 pathways were predicted using PSORTb v3.0.2 and CELLO v2.5. Prediction of B cell epitope and the probability of the antigenicity were evaluated by using IEDB and Vaxijen respectively. BLASTp was done to find out the similarity of the selected proteins with the human proteome. Proteins failing to comply with the set parameters were filtered at each step. Finally, we identified 6 surface exposed proteins as potential vaccine candidates against Klebsiella oxytoca.

For this study, 2,3-butanediol (BD) fermentation from pure and biomass-derived sugar were optimized in shake-flask and 5-L bioreactor levels using Klebsiella oxytoca ATCC 8724. The results showed that 70 g/L of single sugar (glucose or xylose) and 90 g/L of mixed-sugar (glucose:xylose = 2:1) were optimum concentrations for efficient 2,3-BD fermentation. At optimum sugar concentrations, 2,3-BD productivities were 1.03, 0.64 and 0.50 gL −1 h −1 , and yields were 0.43, 0.36 and 0.35 g/g in glucose, xylose and mixed-sugar medium, respectively. The lack of simultaneous utilization of glucose and xylose led to the lowest productivity in the mixed-sugar medium. Detoxification of biomass hydrolyzates was necessary for efficient 2,3-BD fermentation when sugar concentrations in the medium was 90 g/L or higher, but not with sugar concentrations of 30 g/L or less. A fed-batch fermentation using glucose medium led to an increase 2,3-BD titer to 79.4 g/L and yields 0.47 g/g, while productivity decreased to 0.79 gL −1 h −1 . However, the fed-batch process was inefficient using mixed-sugar and biomass hydrolyzates because of poor xylose utilization. These results indicated that appropriate biomass processing technologies must be developed to generate separate glucose and xylose streams to produce high 2,3-BD titer from biomass-derived sugar using a fed-batch process.

Alcohol dehydrogenases can catalyze the inter-conversion of aldehydes and alcohols. The t -butyl 6-chloro-(3 R ,5 S )-dihydroxyhexanoate is a key chiral intermediate in the synthesis of statin-type drugs such as Crestor (rosuvastatin calcium) and Lipitor (atorvastatin). Herein, a novel alcohol dehydrogenase (named as KleADH) discovered from Klebsiella oxytoca by a genome mining method was cloned and characterized. The KleADH was functionally overexpressed in Escherichia coli Rosetta (DE3) and the whole cell biocatalyst was able to convert t -butyl 6-chloro-(5 S )-hydroxy-3-oxohexanoate to t -butyl 6-chloro-(3 R ,5 S )-dihydroxyhexanoate with more than 99% diastereomeric excess (de) and 99% conversion in 24 h without adding any expensive cofactors. Several factors influencing the whole cell catalyst activity such as temperature, pH, the effects of metal ions and organic solvent were determined. The optimum enzyme activity was achieved at 30 °C and pH 7.0 and it was shown that 1 mM Fe 3+ can increase the enzyme activity by 1.2 times. N-hexane/water and n-heptane/water biphasic systems can also increase the activity of KleADH. Substrate specificity studies showed that KleADH also exhibited notable activity towards several aryl ketones with high stereoselectivity. Our investigation on this novel alcohol dehydrogenase KleADH reveals a promising biocatalyst for producing chiral alcohols for preparation of valuable pharmaceuticals.

A strain of Klebsiella oxytoca DSM 29614 is grown on sodium citrate in the presence of 50 mg l−1 of Hg as Hg(NO3)2. During growth, the strain produces an extracellular polymeric substance (EPS), constituted by a mixture of proteins and a specific exopolysaccharide. The protein components, derived from the outer membrane of cells, are co-extracted with the extracellular exopolysaccharide using ethanol. The extracted EPS contains 7.5% of Hg (total amount). This indicates that EPS is an excellent material for the biosorption of Hg2+, through chemical complexation with the EPS components. The binding capacity of these species towards Hg2+ is studied by cyclic voltammetry, and Hg L3-edge XANES and EXAFS spectroscopy. The results found indicate that Hg2+ is mainly bound to the nitrogen of the imidazole ring or other N-heterocycle compounds. The hydroxyl moities of sugars and/or the carboxyl groups of two glucuronic acids in the polysaccharide can also play an important role in sequestring Hg2+ ions. However, N-heterocyclic groups of proteins bind Hg2+ faster than hydroxyl and carboxyl groups of the polysaccharide.

Objectives This study aimed to describe the characteristics of clinical isolates of extended-spectrum β-lactamase (ESBL)-producing enterobacteria (EPE) in Uruguay’s paediatric hospital. Methods ESBLs, qnr alleles and aac(6′)-Ib-cr were sought and characterised in EPE isolated between March 2010 and March 2012. Transfer of resistance determinants was assessed by conjugation. Incompatibility (Inc) groups, plasmid toxin–antitoxin systems (TAS) and plasmid size were determined in transconjugants. Clonality was analysed by pulsed-field gel electrophoresis. Multilocus sequence typing was done for ESBL-producing Klebsiella pneumoniae. Results A total of 77 EPE isolates were characterised, comprising 43% K. pneumoniae, 19.5% Serratia marcescens, 19.5% Escherichia coli, 17% Enterobacter cloacae and 1% Klebsiella oxytoca. ESBLs belonged mainly to the blaCTX-M family (69.6%) [blaCTX-M-15 (45%) and blaCTX-M-2 (31%)]. The aac(6′)-Ib-cr/qnrB duplex was the most frequently detected plasmid-mediated quinolone resistance mechanism; this association was detected in K. pneumoniae harbouring blaCTX-M-15. Transconjugants were obtained for 71% of the EPE. Amongst transconjugants, certain combinations were found between ESBLs and Inc group, e.g. IncA/C–blaCTX-M-2, IncHI1/HI2–blaCTX-M-9 and IncHI1/HI2–blaSHV-12. In addition, the combination ccdAB–blaCTX-M-15 was also found. K. pneumoniae isolates harbouring blaCTX-M-15/aac(6′)-Ib-cr/qnrB showed allodemic behaviour, with a predominance of ST14, ST45 and ST48. Conclusions In this study, epidemiological changes in ESBL distribution could be explained by the spread of K. pneumoniae harbouring blaCTX-M-15/aac(6′)-Ib-cr/qnrB, encoded mainly on conjugative plasmids featuring ccdAB TAS. Since reports of TAS in K. pneumoniae plasmids are scarce, new strategies are needed to combat intrinsic selection pressure exerted by the association, in conjugative plasmids, of resistance mechanisms with TAS.

Metabolic engineering has emerged as a powerful tool for bioproduction of both fine and bulk chemicals. The natural coordination among different metabolic pathways contributes to the complexity of metabolic modification, which hampers the development of biorefineries. Herein, the coordination between the oxidative and reductive branches of glycerol metabolism was rearranged in Klebsiella oxytoca to improve the 1,3-propanediol production. After deliberating on the product value, carbon conservation, redox balance, biological compatibility and downstream processing, the lactate-producing pathway was chosen for coupling with the 1,3-propanediol-producing pathway. Then, the other pathways of 2,3-butanediol, ethanol, acetate, and succinate were blocked in sequence, leading to improved d-lactate biosynthesis, which as return drove the 1,3-propanediol production. Meanwhile, efficient co-production of 1,3-propanediol and l-lactate was also achieved by replacing ldhD with ldhL from Bacillus coagulans. The engineered strains PDL-5 and PLL co-produced over 70g/L 1,3-propanediol and over 100g/L optically pure d-lactate and l-lactate, respectively, with high conversion yields of over 0.95mol/mol from glycerol.

Recently, carbapenemase-producing Enterobacteriaceae (CPE) have been spreading worldwide and have become a threat in healthcare systems. We investigated the isolation frequency and molecular epidemiological characteristics of CPE isolated from clinical samples collected at a primary care hospital over the four years of 2010–2013 in Japan. CPE were detected in 17 (0.34%) of 4875 isolates by the broth microdilution method, sodium mercaptoacetate inhibition test, and modified Hodge test using meropenem disks. The frequency of CPE isolates was 0.09% in 2010, 0.17% in 2011, 0.16% in 2012 and 0.82% in 2013. Isolates positive for carbapenemase included Klebsiella pneumoniae (0.92%), Escherichia coli (0.12%), Enterobacter cloacae (0.80%), Klebsiella oxytoca (0.55%), Enterobacter aerogenes (0.81%) and Proteus mirabilis (0.08%). Antimicrobial susceptibility testing showed low MICs for piperacillin-tazobactam, amikacin, ciprofloxacin and levofloxacin, and only one multidrug-resistant strain. The carbapenemase genotype of all strains was IMP-6, and 94% of the strains were simultaneous CTX-M-2 producers. Two K. pneumoniae and 3 E. coli isolates showed the same pulsed-field gel electrophoresis group. Multilocus sequence typing detected no international high-risk clone types. Plasmid replicon typing detected IncN from all CPE strains, and IncF and IncFIB were simultaneously detected in 24% and 18%, respectively. All patients with detected CPE were inpatients, and many were elderly long-term hospitalized patients or had a history of prior vancomycin or levofloxacin antibiotic administration. The rapid spread of CPE is a concern in Japan. Preventive measures must be implemented against the spread of CPE after considering the epidemiological trend of CPE detection, antibiograms, and risk factors.

Wastewater from coconut processing industry contains carbohydrates which are potential substrates for butanol and ethanol fermentation process. In this research, five isolates of anaerobic bacteria were screened from coconut wastewater. To identify the species and solvent production, the morphological characterization, 16S rRNA gene identification and biochemical test were performed. It was discovered that the isolate COCON2 belongs to the species Pectinatus frisingensis with 16S rRNA gene sequence identity of 98%, whereas COCON4, COCON9 and COCON11 are closely related to Pectinatus portalensis (with 16S rRNA sequence identity percentage of 98, 99 and 99% respectively). As for COCON6, it is similar to Klebsiella oxytoca with 98% 16S rRNA sequence similarity. The solvent production of Pectinatus spp. under anaerobic condition from 20 g/L mannose in 30 mL synthetic medium showed acetate as the main product (1.877–2.146 g/L). In conclusion, naturally selected bacteria have the ability to convert sugar into acetate and ethanol which are metabolites from cellular respiration in optimum condition.

The spread of Extended-Spectrum β-Lactamases (ESBLs) or AmpC β-Lactamases (AmpC) encoding genes in healthy human populations is of major concern. The role of the food chain has been questioned since numerous studies reported surface contamination of retail meat or crude vegetables with ESBL/AmpC-producing Enterobacteriaceae (ESBL/AmpC-E). Nonetheless, these food products are intended to be cooked or washed before consumption so that the risk of human transfer might be low. Here, the presence of ESBL/AmpC-E was investigated in ready-to-eat (RTE) sandwiches purchased in the street in the city of Bejaia, Algeria. Thirty ESBL/AmpC-producing E. coli (n=18), K. pneumoniae (n=11) and K. oxytoca (n=1) were recovered from 21 sandwiches purchased in 14 of the 100 shops that were visited (14%). Twenty-four isolates (13 E. coli, 10 K. pneumoniae, 1 K. oxytoca) produced one or two ESBLs, while 5 E. coli and 1 K. pneumoniae isolates produced an AmpC. Among those, 12 E. coli harbored blaCTX-M-1 (n=7), blaCTX-M-15 (n=3), blaCTX-M-14 (n=1) or blaCTX-M-2 (n=1) and one E. coli co-harbored the blaCTX-M-15 and blaSHV-2 genes. The 10 K. pneumoniae displayed blaCTX-M-15 (n=7), blaSHV-2 (n=3), blaSHV-12 (n=1) or blaCTX-M-1 (n=1), including two isolates presenting a blaCTX-M-15/blaSHV-2 or blaCTX-M-1/blaSHV-2 combination. The K. oxytoca harbored the blaSHV-2 gene, and one K. pneumoniae and four E. coli displayed blaDHA and blaCMY-2, respectively. Most isolates (26/30, n=87%) also possessed the aac(6')-Ib-cr gene. Identical ESBL/AmpC-producing E. coli or K. pneumoniae clones were detected at different places across the city. This may reflect cross-contamination through poor handling practices, contaminated equipment, common ingredients or environmental factors. Of note, the emergent ST405 K. pneumoniae human clone was identified as a CTX-M-15 producer. This study highlights the presence of ESBL/AmpC-E in RTE sandwiches, which are a source of direct transfer to the human gut. These data indicate that fast food shops should be regarded as ESBL/AmpC reservoirs, and a risk for humans. Major efforts should be made in Algeria through guidelines on good practices in the food chain, and more globally in all countries sharing similar poor levels of food hygiene worldwide.

Nitrile compounds are common toxic and refractory organic wastes in industrial wastewater, and can be efficiently treated through biodegradation. The strain Klebsiella oxytoca GS-4-08 could utilize nitriles as the sole nitrogen source for growth, and the nitrile biodegradation kinetics showed good linear correlation with cell growth and glucose consumption. Degradation kinetics showed that the reaction order was dependent on initial cell concentration, and the degradation rates were increased when more easily degradable substrate was available. Higher acetonitrile (ACN) concentration inhibited not only the cell growth but also the ACN degradation. Haldane model constants indicated a high tolerance ability of K. oxytoca to acetonitrile. The formation of acetate during High Performance Liquid Chromatography (HPLC) wastewater treatment showed that the strain has a great potential for treating real nitriles-containing wastewater, and for organic acid production.

Multidrug-resistant (MDR) bacteria are widespread in hospitals and have been increasingly isolated from aquatic environments. The aim of the present study was to characterize extended-spectrum β-lactamase (ESBL) and quinolone-resistant Enterobacteriaceae from a hospital effluent, sanitary effluent, inflow sewage, aeration tank, and outflow sewage within a wastewater treatment plant (WWTP), as well as river water upstream and downstream (URW and DRW, respectively), of the point where the WWTP treated effluent was discharged. β-lactamase (bla) genes, plasmid-mediated quinolone resistance (PMQR), and quinolone resistance-determining regions (QRDRs) were assessed by amplification and sequencing in 55 ESBL-positive and/or quinolone-resistant isolates. Ciprofloxacin residue was evaluated by high performance liquid chromatography. ESBL-producing isolates were identified in both raw (n=29) and treated (n=26) water; they included Escherichia coli (32), Klebsiella pneumoniae (22) and Klebsiella oxytoca (1). Resistance to both cephalosporins and quinolone was observed in 34.4% of E. coli and 27.3% of K. pneumoniae. Resistance to carbapenems was found in 5.4% of K. pneumoniae and in K. oxytoca. Results indicate the presence of blaCTX-M (51/55, 92.7%) and blaSHV (8/55, 14.5%) ESBLs, and blaGES (2/55, 3.6%) carbapenemase-encoding resistance determinants. Genes conferring quinolone resistance were detected at all sites, except in the inflow sewage and aeration tanks. Quinolone resistance was primarily attributed to amino acid substitutions in the QRDR of GyrA (47%) or to the presence of PMQR (aac-(6′)-Ib-cr, oqxAB, qnrS, and/or qnrB; 52.9%) determinants. Ciprofloxacin residue was absent only from URW. Our results have shown strains carrying ESBL genes, PMQR determinants, and mutations in the gyrA QRDR genes mainly in hospital effluent, URW, and DRW samples. Antimicrobial use, and the inefficient removal of MDR bacteria and antibiotic residue during sewage treatment, may contribute to the emergence and spreading of resistance in the environment, making this a natural reservoir.

The stability of Clostridium butyricum TISTR 1032 during biohydrogen fermentation of non-sterile food waste in 5-L semi-batch operation was investigated under controlled and uncontrolled pH conditions at the initial pH 6, and 37 °C. Profiles of carbon mass balance, microbial community and metabolite dynamics were used to evaluate the process efficiency. The results showed that under the uncontrolled pH condition, the maximum hydrogen yield, production rate and specific production rate were 362 mL H2 g−1 VS, 695 mL h−1and 174 mL h−1 L−1, respectively while under the controlled pH condition, those were 350 mL H2 g−1 VS, 1092 mL h−1 and 273 mL h−1 L−1, respectively. Regarding the carbon distribution, food waste was still remained in the solid fraction more than 30% at which the maximum hydrogen production was achieved for all cases. The main factor, which controlled the route of fermentative process under uncontrolled pH condition was acidic condition while acetogenesis was the major effect for the production stability in the controlled pH condition. DGGE profiles showed that C. butyricum TISTR 1032 was still the dominant group in the reactor in both conditions. However, Klebsiella oxytoca, Straphylococcus spp., Enterobacter spp., Lactococcus spp. and Acinetobacter sp. were observed for all cases. The metabolite analysis revealed the correlation of K. oxytoca and solventoganesis process under the uncontrolled pH condition while the presence of Lactococcus spp. was related to lower yield of the hydrogen production under the controlled pH condition.

3-Phenyllactic acid (PLA) and slightly acid electrolyzed water (SAEW) are effective antimicrobial agents with broad-spectrum activity. The combined effects of PLA and SAEW on the inactivation of Klebsiella oxytoca planktonic and biofilm cells were evaluated in this study. The K. oxytoca K6 planktonic and biofilm cells were treated with PLA (0.5%, 1% and 2%), SAEW (30 and 60 mg/L), 0.5% PLA plus 30 mg/L SAEW, and 1% PLA plus 30 mg/L SAEW for 5, 10, 20, 30, and 60 min. The planktonic and biofilm cell assays showed that 2% PLA was more effective for the inactivation of K. oxytoca planktonic and biofilm cells than the other agents, and the combination of PLA and SAEW was more effective than the single agents at the same concentrations. Confocal laser scanning microscopy and ATP leakage analysis showed that the combined treatment of PLA and SAEW at different concentrations destroyed the cell membrane integrity of K. oxytoca planktonic and biofilm cells and caused ATP leakage at different levels. Scanning electron microscopy (SEM) assays showed that the morphologies of K. oxytoca planktonic and biofilm cells changed after treatment with the antibacterial agents at different levels. The cells were seriously damaged after treatment with 1% PLA and 30 mg/L SAEW for 60 min. The biofilm polysaccharide detection assays showed that SAEW-containing antibacterial agents were effective for disrupting the biofilm extracellular polymeric matrix, as shown in SEM images. The combination of PLA and SAEW effectively inactivated K. oxytoca planktonic and biofilm cells by damaging the cell membrane and disrupting the biofilm matrix.

Some species of Klebsiella, such as Klebsiella pneumoniae and Klebsiella oxytoca, are important nosocomial pathogens frequently involved in outbreaks in Neonatal Intensive Care Units (NICU) and have the ability to form a biofilm. This study aims to evaluate the biofilm production of K. pneumoniae and K. oxytoca isolates collected from the hands of health professionals, neonates' blood and the environment of a Brazilian NICU, using three colorimetric methods and a classical method of counting the colony-forming units and compare the analysis among these techniques. The biofilm formation was carried out by the microplate technique, using three colorimetric assays: crystal violet, safranin and 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl) -5 [(phenylamino) arbonyl] - 2H-tetrazolium hydroxide (XTT). Also, colony-forming units were determined. Twenty-eight isolates of K. pneumoniae were collected from the blood, hands and environment and five of K. oxytoca from the hands and environment. All of them were strong biofilm producers, but K. pneumoniae isolates produced more biofilm than K. oxytoca when compared to the American Type Culture Collection (ATCC) strains used as positive controls. The number of viable cells in the biofilm produced by K. pneumoniae isolated from blood was significantly higher than in the control sample. Regarding the three colorimetric tests used in the study, the violet crystal obtained a higher absorbance average. The use of crystal-violet and XTT in the evaluation of biofilm in vitro make possible a complete analysis, since that it can quantify the total biomass (including the extracellular matrix) and evaluate the metabolic activity. In conclusion, this study identified isolates of K. pneumoniae and K. oxytoca that produce biofilms in the NICU and the bloodstream of neonates. This fact deserves attention since these patients are immunocompromised. The best methods will be chosen to answer research questions by always adopting more than one method so that more than one parameter or component of the biofilm is analyzed.