Cloacae Strain Research Articles

Epidemiology of Enterobacter cloacae strains producing a carbapenemase or metallo-beta-lactamase in Vietnamese clinical settings in 2014-2017.

Introduction. Little is known about the epidemiology of Enterobacter cloacae strains producing a carbapenemase or metallo-beta-lactamase in Vietnamese hospitals.Aim. This study analysed E. cloacae strains resistant to imipenem or meropenem that had been isolated from patients admitted to one of the largest hospitals in Vietnam in 2014-2017.Methodology. Eighteen Vietnamese (VN) strains were subjected to whole-genome sequencing and their sequences compared with those of 17 E. cloacae strains carrying a carbapenemase or metallo-beta-lactamase in the database (db strains).Results. Although the distribution of virulence factors did not differ significantly between VN and db strains, all 18 VN isolates harboured blaNDM-1, phylogenetic analysis revealed a high clonality of the VN strains. Bayesian phylogenetic analysis suggested that the VN strains speciated relatively recently.Conclusions. Several prevalent clones of carbapenem-resistant E. cloacae have circulated within Vietnamese hospitals. Adequate measures are needed to prevent their further spread.

A Multispecies Cluster of VIM-1 Carbapenemase-Producing Enterobacterales Linked by a Novel, Highly Conjugative, and Broad-Host-Range IncA Plasmid Forebodes the Reemergence of VIM-1.

In this study, we investigated VIM-1-producing Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Citrobacter freundii, and Enterobacter cloacae strains, isolated in 2019 during a period of active surveillance of carbapenem-resistant Enterobacterales in a large university hospital in Italy. VIM-1-producing strains colonized the gut of patients, with up to three different VIM-1-positive bacterial species isolated from a single rectal swab, but also caused bloodstream infection in one colonized patient. In the multispecies cluster, blaVIM-1 was identified in a 5-gene cassette class 1 integron, associated with several genetic determinants, including the blaSHV-12, qnrS1, and mph(A) genes, located on a highly conjugative and broad-host-range IncA plasmid. The characteristics and origin of this IncA plasmid were studied.

The whole-genome sequence analysis of Enterobacter cloacae strain Ghats1: insights into endophytic lifestyle-associated genomic adaptations.

Enterobacter cloacae is normally considered to be an opportunistic human pathogen. Here, we report on the whole-genome sequence of an endophytic E. cloacae, strain "Ghats1", isolated from leaves of the medicinal plant Coscinium fenestratum Gaertn. Functional analysis of the Ghats1 genome revealed an enrichment for genes involved in the uptake and exchange of nutrients, for chemotaxis and for plant colonization. Unexpectedly though, there were no ORFs belonging to the "virulence factors and antibiotic resistance". Moreover, the presence of hydrolytic enzymes and motility functions reveals the characteristics of an endophyte lifestyle of a bacterium that can colonize and adapt to plant environment. These results provide a better understanding of an endophytic lifestyle through plant-microbe interaction, which can be further exploited as a biocontrol agent.

Tetracycline Resistance Associated with tet Genes or Integrons among Enterobacter cloacae Strains Isolated from Patients with Urinary Tract Infections

Tetracycline Resistance Associated with tet Genes or Integrons among Enterobacter cloacae Strains Isolated from Patients with Urinary Tract Infections

ISOLATION, IDENTIFICATION AND SENSITIVITY OF AMILOLITIC BACTERIA FROM MANGROVE ECOSYSTEM SEDIMENT IN PURNAMA MARINE STATION DUMAI ON THE PATHOGENIC BACTERIA

Litter from the weathering of dead mangrove stems and leaves contains a lot of starch which has potential to be degraded by amylolytic bacteria into simple compounds with the help of the amylase enzyme. Amylolytic bacteria are bacteria that hydrolyze starch into simpler compounds namely glucose with the help of the amylase enzyme. This study aims to 1) isolate, identify and test sensitivity of amylolytic bacterial isolates found at the Purnama Dumai Marine Station, 2) the ability of amylolytic bacterial isolates to inhibit the growth of pathogenic bacteria (Escherichia coli, Pseudomonas aeruginosa and Vibrio alginolyticus) and 3) to determine the of amylolytic bacterial species by 16S rRNA sequence analysis. The results showed 10 bacterial isolates (TR 2, TR 6, TR 7, TR 9, TR 11, TR 13, TR 15, TR 16, TR 18 and TR 20) were able to inhibit the growth of pathogenic bacteria (E.coli, P.aeruginosa and V.alginolyticus). The sensitivity test of isolate TR 20 against E.coli was categorized into weak with inhibition zone diameter of 4.65 mm. Sensitivity of isolate TR 6 against P.aeruginosa was categorized into medium with inhibition zone diameter of 5.22 mm. Then sensitivity of isolate TR 11 against V.algynolyticus was categorized into medium with inhibition zone diameter of 5.55 mm. DNA analysis using 16S rRNA method and BLAST analysis showed similarity of each isolate. Isolate TR 6 was similar to Bacillus paramycoides strain MCCC 1A04098, isolate TR 11 was in a group of Enterobacter cloacae strain ATCC 13047 and TR 20 was in a group of Vibrio harveyi strains of NBRC 15634.

Cleaner strategies on the effective elimination of toxic chromium from wastewater using coupled electrochemical/biological systems

AbstractHexavalent chromium is a toxic pollutant and carcinogen, which affects the organs internally as well as externally in our body. In this research, a combination of three‐dimensional (3D)‐electrochemical treatment followed by biological treatment was done for chromium removal in simulated wastewater. Surface‐modified Strychnos potatorum (SMSp) seeds and surface‐modified tamarind pod (SMTp) shell were used as particle electrodes, and the experiments were conducted for different pH, voltage, and particle electrode followed by biological treatment. In biological treatment, Enterobacter cloacae strain was isolated from a common effluent treatment plant (CETP) and it was utilized. The molecular characterization of E. cloacae (live biomass) was done using 16s rRNA sequencing. In 3D‐electrochemical treatment with SMSp, efficiency was found to be the best when compared with SMTp under optimum condition (6 V, 3 pH, 15 g of SMSp) at a working period of 60 min, and the removal of hexavalent chromium from aqueous solution using SMSp was successfully applied to semi‐empirical kinetic model, which is based on Fermi's equation. This semi‐empirical kinetic model effectively depicts the hexavalent chromium concentration evolution in 3D‐electrochemical treatment. The combined treatment reduced the amount of chemical oxygen demand as well as the hexavalent chromium‐ion concentration in aqueous solution.

Multi-trait PGP rhizobacterial endophytes alleviate drought stress in a senescent genotype of sorghum [Sorghum bicolor (L.) Moench

Root-tissue colonizing bacteria demonstrated with multiple PGP traits from sorghum plants were identified as Ochrobactrum sp. EB-165, Microbacterium sp. EB-65, Enterobacter sp. EB-14 and Enterobacter cloacae strain EB-48 on the basis of 16S rRNA gene sequencing. Here, the in vivo experiments using ½-MS media and ½-MS media + 15% PEG 8000 (for inducing drought stress) indicated stress tolerance imparting ability of these rhizobacterial endophytes in a non-stay green and senescent genotype (R-16) of sorghum. In the experiment with sterile soilrite mix base, seed bacterization with these isolates showed improved plant growth specifically the roots, in terms of root length (~ 44.2 to 50.8% over controls), root dry weight (~ 91.3 to 99.8% over controls) and root surface area (~ 1 to 1.5 fold over controls) under drought stress. Rhizobacterial endophytes were successful, not only in providing better cellular osmotic adjustment in leaves (≥ 1-fold increase in proline accumulation over controls), but favorable physiological responses like Relative Water Content (RWC) and cell Membrane Stability Index (MSI) in the inoculated plants during the drought stress induction. Up-regulation of drought responsive genes like sbP5CS2 and sbP5CS1 was observed in these endophytes-treated plants as compared to untreated control and Escherichia coli DH5α (negative control)-treated plants. Interestingly, the stress imparting traits of rhizobacterial endophytes, including up-regulation of specific genes, were observed during sorghum seedling growth only under drought stresses. The results of this study lead to the conclusion that the potential endophytic rhizobacterial interactions can contribute to plant growth promotion as well as induced stress tolerance in sorghum.

Biofilm-Forming by Carbapenem Resistant Enterobacteriaceae May Contribute to the Blood Stream Infection.

Bloodstream infection (BSI) due to carbapenem-resistant Enterobacteriaceae (CRE) has a high mortality rate and is a serious threat worldwide. Ten CRE strains (eight Enterobacter cloacae, one Klebsiella pneumoniae and one Citrobacter freundii) were isolated from the blood of nine patients, a percentage of whom had been treated with indwelling devices. The steps taken to establish cause included minimum inhibitory concentration (MIC) tests, a pulsed-field gel electrophoresis (PFGE), biofilm study, a multiplex PCR for resistant genes of carbapenemases and extended-spectrum beta-lactamases (ESBLs), and plasmid incompatibility typing. All strains showed a tendency toward resistance to multiple antibiotics, including carbapenems. Frequently isolated genes of ESBLs and carbapenemases include blaTEM-1 (four strains), blaSHV-12 (four strains) and blaIMP-1 (six strains). A molecular analysis by PFGE was used to divide the XbaI-digested genomic DNAs of 10 CRE strains into eight patterns, and the analysis showed that three E. cloacae strains detected from two patients were either identical or closely related. The biofilm production of all CRE strains was examined using a microtiter biofilm assay, and biofilm growth in continuous flow chambers was observed via the use of a confocal laser scanning microscope. Our study indicates that biofilm formation on indwelling devices may pose a risk of BSI due to CRE.

Engineering a bioluminescent bioreporter from an environmentally sourced mercury-resistant Enterobacter cloacae strain for the detection of bioavailable mercury.

Escherichia coli is the conventional choice as the host strain for whole-cell bioreporter construction due to its well-understood genetics and well-established cloning protocols. However, for real-world environmental biosensing applications, it is often beneficial to use a bacterial strain derived directly from the environment under study to better ensure chemical target specificity and optimal response time. The aim of this study was to develop a whole-cell bioreporter for detection of bioavailable mercury by replacing E. coli with a wild-type bacterial host derived from a soil environment. In this study, an Enterobacter cloacae strain isolated from soil derived from a municipal and electronic waste dumping site was engineered to serve as a bioluminescent bioreporter for mercury toxicity by linking its merR-like gene and promoter sequence to a reorganized luxABCDE gene cassette from Photorhabdus luminescens. This bioreporter, designated as E. cloacae DWH4lux , detected mercury (HgCl2 ) at a minimum concentration of 0·2µgl-1 with a linear response profile being maintained between a range of 0·4-1600µgl-1 (R2 =0·9604) with a peak bioluminescent response occurring within 1h after exposure. No significant synergistic or antagonistic influences were observed on the bioluminescent response by other contaminating metal elements. Enterobacter cloacae DWH4lux was also demonstrated to detect mercury effectively in artificially contaminated water sample with linear correlation (R2 =0·9623). The results indicated that E. cloacae DWH4lux could detect mercury in quantities below the US Environmental Protection Agency's permitted limit values (2µgl-1 ). Hence, it is concluded that E. cloacae DWH4lux has the potential to serve as an effective whole-cell bioreporter for the environmental monitoring of mercury contamination. This study provides new insight into the recruitment of mercury-tolerant bacterial hosts derived from environmental samples over the conventional lab-based E. coli host for the construction of mercury bioreporters. With improved response time and selectivity, the environmentally sourced bacteria can serve as an alternative host choice to improve biosensing technology in the near future.

Comparative Susceptibility Study Against Pathogens Using Fermented Cranberry Juice and Antibiotics.

In the present study, unfermented and fermented cranberry juice in combination with the Antibiotics vancomycin and tigecycline were tested for their antimicrobial activity. Cranberry juice was fermented with a recently isolated potentially probiotic Lactobacillus paracasei K5. The tested strains selected for this purpose were Enterococcus faecalis, E. faecium, Enterobacter cloacae and Staphylococcus aureus. The methods followed were the determination of zones inhibition, Minimum Inhibitory Concentration (MIC) and Fractional Inhibitory Concentration Index (FICI). Tigecycline together with fermented juice exhibited larger Zones of Inhibition (ZOI) in strains of E. faecium (65 ± 4.8 mm) compared to the respective ZOI with tigecycline and unfermented juice (no zone). The same outcome was also obtained with E. cloacae. Vancomycin together with fermented juice exhibited larger ZOI in strains of E. faecium (28 ± 2.2 mm) compared to the respective ZOI with vancomycin and unfermented juice (24 ± 2.3 mm). The lowest MIC values were recorded when tigecycline was combined with fermented cranberry juice against S. aureus strains, followed by the same combination of juice and antibiotic against E. cloacae strains. FICI revealed synergistic effects between fermented juice and tigecycline against a strain of E. faecium (A2020) and a strain of E. faecalis (A1940). Such effects were also observed in the case of fermented juice in combination with vancomycin against a strain of S. aureus (S18), as well as between fermented juice and tigecycline against E. cloacae (E1005 and E1007) strains. The results indicate that the antibacterial activity of juice fermented with the potentially probiotic L. paracasei K5 may be due to synergistic effects between some end fermentation products and the antibiotic agents examined.

Enterobacter timonensis sp. nov., a new bacterial species isolated from a sample of human stool

Strain Marseille-P2201T is a new species from the order Enterobacteriales that was isolated from a stool sample of a 38-month-old healthy girl from Senegal. It is a facultative anaerobic Gram-negative rod-shaped bacterium. Strain Marseille-P2201T exhibits 98.4% similarity levels with the Enterobacter cloacae strain ATCC 13047T, a phylogenetically closely related species with standing in nomenclature. The draft genome size of strain Marseille-P2201T is 4.19 Mb with 56.8% G + C content. We propose that strain Marseille-P2201T (= CSURP2201) is the type strain of the new species Enterobacter timonensis sp. nov.

Antiobesity Effects of Lactobacillus plantarum LMT1-48 Accompanied by Inhibition of Enterobacter cloacae in the Intestine of Diet-Induced Obese Mice.

The gut microbiota is the most important environmental factor that plays a role in inducing obesity. The gram-negative bacteria, Enterobacter cloacae strains, recently identified in obese mice are considered to be pathogenic bacteria in the gut. Probiotics are important members of the gut microbiota and exert beneficial effects, including inhibiting the growth of potential pathogenic bacteria. Therefore, we isolated a total of 230 lactic acid bacteria from traditional, Korean fermented foods and fecal samples from newborn infants, including Lactobacillus plantarum LMT1-48, which exhibited maximal antimicrobial activity against E. cloacae. We next investigated the functional antiobesity effects of L. plantarum LMT1-48 in an E. cloacae-induced high-fat diet (HFD)-fed animal obesity model. To this end, the L. plantarum LMT1-48 showed antiobesity effects, including body weight loss and reduction of abdominal fat volume, which was accompanied by a decrease in leptin and total cholesterol levels in E. cloacae-induced HFD-fed mice. Notably, gut microbiota diversity also increased after long-term ingestion of L. plantarum LMT1-48, resulting in amelioration of obesity in E. cloacae-induced HFD-fed mice. Accordingly, results suggest that dietary intake of L. plantarum LMT1-48 protects against the onset of E. cloacae-induced obesity.

Genomic analysis of two bacterial strains co-isolated from a urinary tract infection: NDM-1-producing Enterobacter cloacae accompanied by extended-spectrum β-lactamase-producing Escherichia coli.

Escherichia coli and Enterobacter cloacae are two major pathogens causing urinary tract infection (UTI). Here we characterised the genome of an NDM-1-producing E. cloacae strain and an extended-spectrum β-lactamase (ESBL)-producing E. coli strain that were co-isolated from a UTI. The genomes of E. cloacae strain EC32 and E. coli strain EC33 were sequenced using an Illumina HiSeqTM platform. The whole genome sequences were assembled using CLC Genomics Workbench and were annotated by the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) server. Genomic analysis was further performed. The draft genome sequences of E. cloacae EC32 (ST66) and E. coli EC33 (ST1139) consisted of 89 contigs comprising 5 178 393 bp and 62 contigs comprising 5 057 666 bp, respectively. Including the blaNDM-1 gene, in total 13 resistance genes were identified in E. cloacae EC32 conferring resistance to β-lactams, rifampicin, phenicols, fosfomycin, macrolides, quinolones, sulfonamides and tetracycline. Nine resistance genes were identified in E. coli EC33 including the ESBL-encoding gene blaCTX-M-27. To our knowledge, this is the first report of genomic characterisation of NDM-1-producing E. cloacae and ESBL-producing E. coli co-isolated from a UTI in China. The two strains inhabiting in the same environment may allow the possibility of horizontal transfer of blaNDM-1 and blaCTX-M-27, making clinical treatment even difficult. This report also sheds light on geographically distinct Enterobacteriaceae strains from China enabling a comparative analysis of NDM-1- or ESBL-producing strains.

A rhizosphere isolate from Oryza sativa, Enterobacter cloacae VITTPN2, as a potential plant growth promoting rhizobacteria; an in vitro study

The increasing need for Plant Growth Promoting Rhizobacteria (PGPR) for biofertilizer development is warranted owing to the environmental hazards caused by chemical fertilizers. Our investigation was to isolate, screen and characterize PGPR from rhizospheric soil with potential PGPR properties. Oryza sativa and Saccharum officinarum rhizosphere were collected from the agricultural research station, Virinjipuram, Vellore (12.9202N, 79.1333E), Tamil Nadu, India for PGPR isolation. Eleven distinct isolates of bacteria were grown on Jensen’s (seven) and Pikovskaya’s media (four). Among these, four isolates (TPN1 to TPN4) showed phosphate solubilisation activity. And one isolate TPN2 particularly showed both nitrogen fixation and phosphate solubilization with other PGPR properties. Furthermore, the isolate TPN2 demonstrated promising results in Indole 3-Acetic Acid production (99.29±0.945µg ml-1). Since the isolate TPN2 displayed all PGPR characteristics under study, it was selected for pot culture studies. The seeds treated with TPN2 revealed an increase of 63.6% in shoot length and 14.63% in root length of the okra plant. There was a 74.6% increase in shoot length and a 16% increase in the root length of the tomato plant. Additionally, there was extensive development of lateral roots in okra plant. Henceforth TPN2 was identified as Enterobacter cloacae VITTPN2 (ku951582). This report produced remarkable results which promise the bacterial strain Enterobacter cloacae strain VITTPN2 can be further studied as a prospective biofertilizer.

Investigation of a hospital Enterobacter cloacae NDM-1 outbreak using whole genome sequencing

Caprbapenemase-producing Gram-negative micro-organisms are emerging as a major clinical problem. The infections caused by these highly resistant and hospital-adapted pathogens may become untreatable using existing antibiotics. Over a three year period, six patients at a large UK tertiary-referral hospital were colonised or infected with carbapenem-resistant Enterobacter cloacae carrying the blaNDM-1 metallo-β-lactamase gene. Environmental isolates were also obtained from a clinical wash-hand basin and taps. The isolates had very similar pulsed-field gel electrophoresis profiles, suggesting they were related, although only four of the cases had epidemiological links. Whole genome sequencing showed the isolates had the same genomic background (sequence type ST114). Genes encoding seven different extended-spectrum and inhibitor-resistant β-lactamase and carbapenemase enzymes (blaNDM-1; blaCTX-M-15; blaACT-16; blaVEB-1; blaTEM-1; blaOXA-1 and blaOXA-10) were present, in addition to multiple genes and mutations conferring resistance to aminoglycosides, quinolones, trimethoprim, tetracycline, sulphonamide, chloramphenicol, rifampicin and fosfomycin. Phenotypic testing indicated sensitivity only to colistin and tigecycline. Genome-wide single nucleotide polymorphism analysis showed the four linked isolates were closely related, and differed from the unlinked isolates by 16–24 SNPs. Moreover, resistance encoding plasmids had been lost in the two unlinked isolates. This suggested these isolates, although sharing a recent common ancestor, had evolved in different environments. Whole genome sequencing allowed resolution of very closely related E. cloacae strains, and confirmed the outbreak did not extend beyond the linked patients. Sequencing also confirmed the same highly resistant E. cloacae strain had persisted within a clinical unit for over two years, despite rigorous efforts to eradicate it.

High distribution of 16S rRNA methylase genes rmtB and armA among Enterobacter cloacae strains isolated from an Ahvaz teaching hospital, Iran.

The emergence of 16S rRNA methylase genes encoded on plasmids confers high-level aminoglycoside resistance (HLAR). This study aimed to investigate the prevalence of 16S rRNA methylases among Enterobacter cloacae strains isolated from an Ahvaz teaching hospital, Iran. A total of 68 E. cloacae clinical strains were collected between November 2017 and September 2018. The MICs of aminoglycosides were assessed using the agar dilution method. The presence of 16S rRNA methylase genes, including armA, rmtA to rmtH, and nmpA was evaluated by PCR. The transferability of 16S rRNA methylase-harboring plasmids was evaluated by conjugation assay. The genetic diversity of all isolates was evaluated by ERIC-PCR. The armA and rmtB genes were the only 16S rRNA methylase genes detected in this study (29 out of 68 isolates; 42.64%). The transferability by conjugation was observed in 23 rmtB or/and armA positive donors. HLAR phenotype was in 33 of 68 strains. Ten clonal types were obtained by ERIC-PCR and significant associations (p < 0.05) were between the clone types and aminoglycoside susceptibility, as well as with profile of the 16S rRNA methylase genes. In conclusion, both horizontal transfer and clonal spread are responsible for dissemination of the rmtB and armA genes among E. cloacae strains.

Study on microbial communities in domestic kitchen sponges: Evidence of Cronobacter sakazakii and Extended Spectrum Beta Lactamase (ESBL) producing bacteria.

Domestic environment, in particular, kitchen setting is a well-established source of microbial contamination. Kitchen sponges represent an important vehicle of microbial transmission and maintenance of spoilage bacteria and pathogenic strains responsible for food borne diseases. The aim of this study was to evaluate the microbial communities of 100 ‘in-use’ kitchen sponges, improving the knowledge on their role in cross-contamination in domestic environment and transmission of ESBLproducing strains. Sponges were processed for: aerobic mesophilic bacteria (AMB), Enterobacteriaceae (EB), yeasts and molds (YM), coagulase-positive staphylococci (CPS), micrococci (MCC), anaerobic sulfite reducing bacteria (ASR), and for the detection of Listeria monocytogenes, Salmonella spp. and Yersinia enterocolitica. A total of 309 enterobacteria strains were identified and then processed for ESBL (Extended Spectrum Beta Lactamase) phenotypical expression. A high contamination level of kitchen sponges was observed (mean value AMB 8.25±1.1; EB 5.89±1.2; YM 5.57±1.1; MCC 4.82±0.1 log CFU/g). Identified enterobacteria strains revealed several opportunistic and pathogenic agents such as Enterobacter cloacae (28%), Citrobacter freundii (23.3%), Cronobacter sakazakii (14.6%) and other strains in lower percentage. Listeria monocytogenes was found in only one sponge (1%). A total of 69 (22.3%) enterobacteria resulted ESBL+, with the following prevalence: P. rettgeri (50%), L. adenocarboxilata (30%), K. pneumoniae (25%), K. oxytoca (25%), C. sakazakii (20%), E. cloacae (20.7%), C. freundii (20.1%). Results confirm the potential role of kitchen sponges as vehicle for food-borne pathogens such as, C. sakazakii for the first time, infectious agents and spoilage microorganisms. The observed high contamination level and the presence of several ESBLs opportunistic pathogens, stresses the necessity to improve a proper education of the consumers on the effective treatment to reduce their microbial loads.

Genetic Diversity of Carbapenem-Resistant Enterobacteriaceae (CRE) Clinical Isolates From a Tertiary Hospital in Eastern China.

The prevalence of carbapenem-resistant Enterobacteriaceae (CRE) is increasing globally, with different molecular mechanisms described. Here we studied the molecular mechanisms of carbapenem resistance, including clonal and plasmid dissemination, of 67 CRE isolates collected between 2012 and 2016 from a tertiary hospital in Eastern China, an CRE endemic region. Species identification and susceptibility testing were performed using the BD Phoenix Automated Microbiology System. Isolates were characterized by PCR (for carbapenemases, ESBLs, AmpC and porin genes), multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and conjugation transfer experiments. Selected blaKPC-2 -harboring plasmids were subjected to next-generation sequencing using the Illumina Miseq platform. Among the 67 CRE isolates, 42 Klebsiella pneumoniae, 10 Serratia marcescens, 6 Enterobacter cloacae, 2 Raoultella ornithinolytica, 2 K. oxytoca, 1 K. aerogenes, and 4 Escherichia coli isolates were identified. Six different carbapenemases were detected, including blaKPC-2 (n = 45), blaKPC-3 (n = 1), blaNDM-1 (n = 6), blaNDM-5 (n = 1), blaIMP-4 (n = 2), and blaVIM-1 (n = 2); blaOXA-48-like genes were not detected. One E. cloacae strain possessed both blaNDM-1 and blaKPC-3, while two E. cloacae isolates harbored blaNDM-1 and blaVIM-1. ESBLs (CTX-M, SHV, and TEM) and/or AmpC (CMY, DHA, and ACT/MIR) genes were also identified in 59 isolates, including 13 strains that lacked carbapenemases. Several insertions or stop codon mutations were found within porin genes of K. pneumoniae, E. coli and S. marcescens isolates, both with and without carbapenemases. The 42 K. pneumoniae isolates belonged to 12 different sequence types (ST), with ST11 being the most common, while the 6 E. cloacae isolates comprised 4 different STs. The 10 S. marcescens all shared the same PFGE pulsotype, suggestive of clonal spread. Complete plasmid sequencing and PCR screening revealed both intra-strain and inter-species spread of a common blaKPC-2-harboring plasmid in our hospital. Taken together, our study revealed extensive genetic diversity among CRE isolates form a single Chinese hospital. CRE isolates circulating in the hospital differ significantly in their species, STs, porin genes, carbapenemase genes, and their plasmid content, highlighting the complex dissemination of CRE in this endemic region.

Inhibition of Wild Enterobacter cloacae Biofilm Formation by Nanostructured Graphene- and Hexagonal Boron Nitride-Coated Surfaces.

Although biofilm formation is a very effective mechanism to sustain bacterial life, it is detrimental in medical and industrial sectors. Current strategies to control biofilm proliferation are typically based on biocides, which exhibit a negative environmental impact. In the search for environmentally friendly solutions, nanotechnology opens the possibility to control the interaction between biological systems and colonized surfaces by introducing nanostructured coatings that have the potential to affect bacterial adhesion by modifying surface properties at the same scale. In this work, we present a study on the performance of graphene and hexagonal boron nitride coatings (h-BN) to reduce biofilm formation. In contraposition to planktonic state, we focused on evaluating the efficiency of graphene and h-BN at the irreversible stage of biofilm formation, where most of the biocide solutions have a poor performance. A wild Enterobacter cloacae strain was isolated, from fouling found in a natural environment, and used in these experiments. According to our results, graphene and h-BN coatings modify surface energy and electrostatic interactions with biological systems. This nanoscale modification determines a significant reduction in biofilm formation at its irreversible stage. No bactericidal effects were found, suggesting both coatings offer a biocompatible solution for biofilm and fouling control in a wide range of applications.

Significant role of Asn-247 and Arg-64 residues in close proximity of the active site in maintaining the catalytic function of CTX-M-15 type β-lactamase.

Members of Enterobacteriaceae cause antibiotic-resistant infections worldwide. One such marker, CTX-M-15, expressed by Enterobacteriaceae produces β-lactamases, which hydrolyze the cephalosporin group of antibiotics, such as cefotaxime, used in the treatment of both Gram-positive and negative bacterial infections. Amino acid residues present in close proximity of the active site might also play a major role in the structure and function of CTX-M-15, hence the objective of this study was to investigate the significance of two amino acid residues, Asn-247 and Arg-64, present near to the active site in the hydrolysis of cefotaxime. blaCTX-M-15, cloned from the E. cloacae strain, and using Polymerase Chain Reaction (PCR)-based site-directed mutagenesis, Asn247Val and Arg64Leu mutations were introduced. The minimum inhibitory concentrations of cefotaxime for the CTX-M-15 (N247V) and CTX-M-15 (R64L) mutants were reduced by 512 and 128 fold, respectively. Proteins/enzymes of wild-type CTX-M-15, CTX-M-15 (N247V) and CTX-M-15 (R64L) mutants were expressed and purified. Kinetic studies showed that the catalytic efficiencies of the N247V mutant and R64L mutant enzymes in the hydrolysis of cefotaxime were reduced by 89.66% and 71.11%, respectively. Circular dichroism spectroscopic studies showed considerable changes in the α-helical content of the mutant enzymes. A fluorescence study showed that N247V mutant-cefotaxime and R64L mutant-cefotaxime underwent complex formation with strong interactions. The study provides an understanding of the crucial role of the amino acid residues asparagine 247 and arginine 64 present in close proximity of the active site in the hydrolytic mechanism of CTX-M-15 type β-lactamases. Hence, Asn-247 and Arg-64 can be used as potential target sites for the design of inhibitory molecules against CTX-M-15-producing bacterial strains.