Effects of oxygen stress on chromate reduction in Enterobacter cloacae strain HO1

Enterobacter cloacae strain HO1 was able to reduce toxic hexavalent chromium (chromate) anaerobically. The reduction of chromate by E. cloacae cells was sensitive to oxygen stress. Cultures under continuous aeration showed no chromate reduction. However, when released from the oxygen stress, the cultures readily resumed chromate reduction.     

Isolation and Characterization of an Enterobacter cloacae Strain That Reduces Hexavalent Chromium under Anaerobic Conditions

An Enterobacter cloacae strain (HO1) capable of reducing hexavalent chromium (chromate) was isolated from activated sludge. This bacterium was resistant to chromate under both aerobic and anaerobic conditions. Only the anaerobic culture of the E. cloacae isolate showed chromate reduction. In the anaerobic culture, yellow turned white with chromate and the turbidity increased as the reduction proceeded, suggesting that insoluble chromium hydroxide was formed. E. cloacae is likely to utilize toxic chromate as an electron acceptor anaerobically because (i) the anaerobic growth of E. cloacae HO1 accompanied the decrease of toxic chromate in culture medium, (ii) the chromate-reducing activity was rapidly inhibited by oxygen, and (iii) the reduction occurred more rapidly in glycerol- or acetate-grown cells than in glucose-grown cells. The chromate reduction in E. cloacae HO1 was observed at pH 6.0 to 8.5 (optimum pH, 7.0) and at 10 to 40°C (optimum, 30°C).     

Class III bacteriocin Helveticin-M causes sublethal damage on target cells through impairment of cell wall and membrane

Helveticin-M, a novel Class III bacteriocin produced by Lactobacillus crispatus exhibited an antimicrobial activity against Staphylococcus aureus, S. saprophyticus, and Enterobacter cloacae. To understand how Helveticin-M injured target cells, Helveticin-M was cloned and heterologously expressed in Escherichia coli. Subsequently, the cell wall organization and cell membrane integrity of target cells were determined. The mechanism of cellular damage differed according to bacterial species. Based on morphology analysis, Helveticin-M disrupted the cell wall of Gram-positive bacteria and disorganized the outer membrane of Gram-negative bacteria, therefore, altering surface structure. Helveticin-M also disrupted the inner membrane, as confirmed by leakage of intracellular ATP from cells and depolarization of membrane potential of target bacteria. Based on cell population analysis, Helveticin-M treatment caused the increase of cell membrane permeability, but the cytosolic enzymes were not influenced, indicating that it was the sublethal injury. Therefore, the mode of Helveticin-M action is bacteriostatic rather than bactericidal.     

Draft Genome Sequence of Enterobacter cloacae subsp. cloacae Strain 08XA1, a Fecal Bacterium of Giant Pandas

Enterobacter cloacae, a common pathogenic bacterium, is a Gram-negative bacillus. We analyzed the draft genome of Enterobacter cloacae subsp. cloacae strain 08XA1 from the feces of a giant panda in China. Genes encoding a β-lactamase and efflux pumps, as well as other factors, have been found in the genome.     

Surface attachment of a sediment isolate ofEnterobacter cloacae

Enterobacter cloacae was recovered from surface sediments of a flood control channel in an area where freshwater runoff mixed with coastal seawater. Cells of this bacterium elaborated an extensive capsule when cultured under laboratory conditions designed to promote extracellular polysaccharide production. Colonization of glass surfaces by cells was similar under aerobic and anaerobic conditions. Temperature exerted little effect on maximum adherent cell density in the range of 15–25°C. The availability of organic nutrients also had little influence on the tendency of cells to adhere to surfaces. Maximum adherent cell densities decreased (76%) as salinity increased from 0 to 12‰ The results suggest that cells ofE. cloacae are suitably adapted to maintain a sessile existence in brackish water sediments of temperate coastal areas.     

Complete Genome Sequence of the Endophytic Enterobacter cloacae subsp. cloacae Strain ENHKU01

Enterobacter cloacae subsp. cloacae strain ENHKU01 is a Gram-negative endophyte isolated from a diseased pepper (Capsicum annuum) plant in Hong Kong. This is the first complete genome sequence report of a plant-endophytic strain of E. cloacae subsp. cloacae.     

Two Novel Bacterial Biosensors for Detection of Nitrate Availability in the Rhizosphere

The nitrate-regulated promoter of narG in Escherichia coli was fused to promoterless ice nucleation (inaZ) and green fluorescent protein (GFP) reporter genes to yield the nitrate-responsive gene fusions in plasmids pNice and pNgfp, respectively. While the promoter of narG is normally nitrate responsive only under anaerobic conditions, the L28H-fnr gene was provided in trans to enable nitrate-dependent expression of these reporter gene fusions even under aerobic conditions in both E. coli DH5α and Enterobacter cloacae EcCT501R. E. cloacae and E. coli cells containing the fusion plasmid pNice exhibited more than 100-fold-higher ice nucleation activity in cultures amended with 10 mM sodium nitrate than in nitrate-free media. The GFP fluorescence of E. cloacae cells harboring pNgfp was uniform at a given concentration of nitrate and increased about 1,000-fold when nitrate increased from 0 to 1 mM. Measurable induction of ice nucleation in E. cloacae EcCT501R harboring pNice occurred at nitrate concentrations of as low as 0.1 μM, while GFP fluorescence was detected in cells harboring pNgfp at about 10 μM. In the rhizosphere of wild oat (Avena fatua), the whole-cell bioreporter E.cloacae(pNgfp) or E. cloacae(pNice) expressed significantly higher GFP fluorescence or ice nucleation activity when the plants were grown in natural soils amended with nitrate than in unamended natural soils. Significantly lower nitrate abundance was detected by the E. cloacae(pNgfp) reporter in the A. fatua rhizosphere compared to in bulk soil, indicating plant competition for nitrate. Ice- and GFP-based bacterial sensors thus are useful for estimating nitrate availability in relevant microbial niches in natural environments.     

Reduction of oxygen by hydrogen in cells of anaerobically grown Proteus mirabilis

Anaerobically grown cells of the enterobacteria Proteus mirabilis, Escherichia coli, Salmonella typhimurium and Enterobacter cloacae catalyse the reduction of oxygen by hydrogen (The Knallgas reaction). We have studied this reaction in detail in P. mirabilis. Oxygen at concentrations above approx. 20 μM inactivates the catalytic pathway for this reaction in a reversible way. The site of inactivation is located in the part of the pathway shared with the reduction of fumarate by hydrogen, possibly hydrogenase. Oxygen exerts its effect via the oxidation state of an unknown component in the bacteria, such that electron transfer from H₂ is blocked when this component is oxidised. We suggest that this component is identical to the regulating factor controlling hydrogen production via the formate-hydrogenlyase reaction (Krab, K., Oltmann, L.F. and Stouthamer, A.H. (1982) Biochim. Biophys. Acta 679, 51–59).     

Enterobacter cloacae A105, isolated from the surface of root nodules of Astragalus sinicus cv. Japan,stimulates nodulation by Rhizobium huakuii bv. renge

Several bacterial strains were isolated from the surface of root nodules of Astragalus sinicus cv. Japan (known as renge-sou in Japanese), a green manure legume that grows in winter and which is used in rice fields to fertilize the soil in both Japan and China. These bacterial strains stimulated the nodulation on renge-sou induced by strains of Rhizobium huakuii bv. renge. From a taxonomic characterization of the isolates, the strains were found to belong to the species Enterobacter cloacae. It was found that strains of E. cloacae increased the number and weight of nodules and the yield of the host plant when these strains were inoculated with a strain of R. huakuii bv. renge both in a test-tube nodulation assay and in soil from a rice field. E. cloacae influenced nodulation at an appropriate ratio of cells of two bacterial strains. The timing of the inoculation of the two strains onto the host plant was also important. The effect of E. cloacae on the nodulation of renge-sou may be due to bacterial products such as exopolysaccharides.     

Genomic diversity within the Enterobacter cloacae complex

Background

Isolates of the Enterobacter cloacae complex have been increasingly isolated as nosocomial pathogens, but phenotypic identification of the E. cloacae complex is unreliable and irreproducible. Identification of species based on currently available genotyping tools is already superior to phenotypic identification, but the taxonomy of isolates belonging to this complex is cumbersome.

Methodology/Principal Findings

This study shows that multilocus sequence analysis and comparative genomic hybridization based on a mixed genome array is a powerful method for studying species assignment within the E. cloacae complex. The E. cloacae complex is shown to be evolutionarily divided into two clades that are genetically distinct from each other. The younger first clade is genetically more homogenous, contains the Enterobacter hormaechei species and is the most frequently cultured Enterobacter species in hospitals. The second and older clade consists of several (sub)species that are genetically more heterogonous. Genetic markers were identified that could discriminate between the two clades and cluster 1.

Conclusions/Significance

Based on genomic differences it is concluded that some previously defined (clonal and heterogenic) (sub)species of the E. cloacae complex have to be redefined because of disagreements with known or proposed nomenclature. However, further improved identification of the redefined species will be possible based on novel markers presented here.     

Cloning and Characterization of Aerobactin Biosynthesis Genes of the Biological Control Agent Enterobacter cloacae

Five strains of Enterobacter cloacae that are biological control agents of Pythium damping-off diseases produced the hydroxamate siderophore aerobactin under iron-limiting conditions. Genes determining aerobactin biosynthesis of the biocontrol strain E. cloacae EcCT-501 were localized to a 12.3-kb region, which conferred aerobactin production to Escherichia coli DH5α. The aerobactin biosynthesis genes of E. cloacae hybridized to those of the pColV-K30 plasmid of E. coli, but restriction patterns of the aerobactin regions of pColV-K30 and E. cloacae differed. A derivative strain with a deletion in the aerobactin biosynthesis locus was as effective as strain EcCT-501 in biological control of Pythium damping-off of cucumber. Thus, aerobactin production did not contribute significantly to the biological control activity of EcCT-501 under the conditions of this study.     

New Delhi Metallo-β-Lactamase 1(NDM-1), the Dominant Carbapenemase Detected in Carbapenem-Resistant Enterobacter cloacae from Henan Province,China

The emergence of New Delhi metallo-β-lactamase 1 (NDM-1) has become established as a major public health threat and represents a new challenge in the treatment of infectious diseases. In this study, we report a high incidence and endemic spread of NDM-1-producing carbapenem-resistant Enterobacter cloacae isolates in Henan province, China. Eight (72.7%) out of eleven non-duplicated carbapenem-resistant E. cloacae isolates collected between June 2011 and May 2013 were identified as NDM-1 positive. The bla _NDM-1 gene surrounded by an entire ISAba125 element and a bleomycin resistance gene ble _MBL in these isolates were carried by diverse conjugatable plasmids (IncA/C, IncN, IncHI2 and untypeable) ranging from ~55 to ~360 kb. Molecular epidemiology analysis revealed that three NDM-1-producing E. cloacae belonged to the same multilocus sequence type (ST), ST120, two of which were classified as extensively drug-resistant (XDR) isolates susceptible only to tigecycline and colistin. The two XDR ST120 E. cloacae isolates co-harbored bla _NDM-1, armA and fosA3 genes and could transfer resistance to carbapenems, fosfomycin and aminoglycosides simultaneously via a conjugation experiment. Our study demonstrated NDM-1 was the most prevalent metallo-β-lactamase (MBL) among carbapenem-resistant E.cloacae isolates and identified a potential endemic clone of ST120 in Henan province. These findings highlight the need for enhanced efforts to monitor the further spread of NDM-1 and XDR ST120 E. cloacae in this region.     

Improved Biosurfactant Production by Enterobacter cloacae B14, Stability Studies,and its Antimicrobial Activity

This work aimed to optimize carbon and nitrogen sources for the growth of Enterobacter cloacae B14 and its biosurfactant (BS) production via One-Variable-At-a-Time (OVAT) method. The BS stability under a range of pH and temperatures was assessed. Antimicrobial activity against Gram-positive and Gram-negative pathogens was determined by the agar well diffusion method. The results showed that the optimum carbon and nitrogen sources for BS production were maltose and yeast extract, respectively, with a maximum BS yield of (39.8 ± 5.2) mg BS/g biomass. The highest emulsification activity (E24) was 79%, which is significantly higher than in the previous studies. We found that B14 BS can withstand a wide range of pH values from 2 to10. It could also function under a range of temperatures from 30–37°C. Thin Layer Chromatography (TLC) and Fourier Transform Infrared Spectrometry (FTIR) analysis confirmed that B14 BS is a glycolipid-like compound, which is rarely found in Enterobacter spp. Cell-free broth showed inhibition against various pathogens, preferable to Gram-positive ones. It had better antimicrobial activity against Bacillus subtilis than a commonly-used antibiotic, tetracycline. Furthermore, B14 broth could inhibit the growth of a tetracycline-resistant Serratia marcescens. Our results showed promising B14 BS applications not only for bioremediation but also for the production of antimicrobial products.Key words: biosurfactant, cultivation media, Enterobacter cloacae, antimicrobial activity, stability     

Bacterial reduction of toxic hexavalent chromium using a fed-batch culture of Enterobacter cloacae strain HOl

Bacterial reduction of hexavalent chromium (chromate: CrO₄²⁻) was investigated using fed-batch cultures of Enterobacter cloacae strain HOl. In the fed-batch cultures, toxic CrO₄²⁻ was continuously added in small doses to minimize the toxic effect. The fed-batch process was proved to be an effective biological method for detoxifying CrO₄²⁻ in aqueous solutions.     

In-Use Contamination of Intravenous Infusion Fluid

During the 1970 to 1971 nationwide epidemic of septicemias caused by Enterobacter cloacae and Enterobacter agglomerans traced to intrinsic contamination of Abbott intravenous infusion products, 94 infusion systems manufactured by Baxter Laboratories were studied microbiologically and epidemiologically during hospital use. Intravenous fluid from 10 systems (11%) contained microorganisms, usually Staphylococcus or Bacillus species; one infusion was heavily contaminated with Klebsiella pneumoniae. No national epidemic organisms, E. cloacae or E. agglomerans (formerly Erwinia), were recovered, suggesting that during this period frequent contamination with these organisms was unique to Abbott's infusion products. Contamination in this study appeared to be extrinsic in origin (introduced during clinical use) and related to the duration of continuous intravenous therapy. Nine of 61 systems (15%) that had been used longer than 48 h were contaminated, whereas only 1 of 33 used less than 48 h (3%) contained microorganisms. This study and the recent national outbreak indicate that contamination of infusion fluid, both from intrinsic and extrinsic sources, must be recognized as an additional risk of intravenous therapy; however, a once-daily replacement of the delivery apparatus can significantly diminish this hazard.     

AiiA Quorum-Sensing Quenching Controls Proteolytic Activity and Biofilm Formation by Enterobacter cloacae

The aim of this work was to evaluate a quorum-quenching approach to identify functions regulated by quorum sensing in Enterobacter cloacae. We employed an aiiA transconjugant strain of E. cloacae that synthesizes a lactonase enzyme that hydrolyzes N-acyl homoserine lactone signaling molecules to compare bacterial phenotypes in the presence and absence of quorum signals. The aiiA-expressing strain displayed increased proteolytic activity and intensity of a milk-clotting reaction when compared to the wild-type strain. Although both strains growing on polystyrene plates in rich media and a minimal medium of salts formed biofilms, the wild-type strain exhibited a higher number of adhered cells. On the surface of stainless steel coupons that were submerged in culture media, the number of adhered cells of the wild type contained up to one log more cells compared with the aiiA transconjugant. However, after 48?h of incubation, there was no significant difference between the strains. The results demonstrated that the quorum-sensing system negatively regulates proteolytic activity and is likely involved in the early steps of biofilm formation by E. cloacae 067.     

Coupled reductive and oxidative degradation of 4-chloro-2-nitrophenol by a co-immobilized mixed culture system

Summary The restriction of oxygen transfer in Ca-alginate beads used for the immobilization of microbial cells was applied to a coupled reductive and oxidative microbial degradation of the xenobiotic 4-chloro-2-nitrophenol (CNP). The conversion of CNP by Enterobacter cloacae under anaerobic conditions led to the formation of 4-chloro-2-aminophenol (CAP, 81%) and 4-chloro-2-acetaminophenol (CAAP, 16%) after 50 h incubation. CAP, the main reduction product, was further degraded under aerobic conditions by Alcaligenes sp. TK-2, a hybrid strain isolated by conjugative in-vivo gene transfer. Whereas both degradation steps excluded one another in homogeneous systems with free cells, a coupled reductive and oxidative degradation of CNP was observed in one aerated reactor system after co-immobilization of both strains in Ca alginate. The diameter of the alginate beads used for immobilization was recognized as one main factor determining the properties of this mixed culture system. Offprint requests to: H.-J. Rehm     

Plantago lanceolata and Plantago rugelii Extracts are Toxic to Meloidogyne incognita but not to Certain Microbes

Extracts from the plants Plantago lanceolata and P. rugelii were evaluated for toxicity to the root-knot nematode Meloidogyne incognita, the beneficial microbes Enterobacter cloacae, Pseudomonas fluorescens and Trichoderma virens, and the plant-pathogenic fungi Fusarium oxysporum f. sp. gladioli, Phytophthora capsici, Pythium ultimum, and Rhizoctonia solani. Wild plants were collected, roots were excised from shoots, and the plant parts were dried and ground to a powder. One set of extracts (10% w/v) was prepared in water and another in methanol. Treatments included extract concentrations of 25%, 50%, 75% and 100%, and water controls. Meloidogyne incognita egg hatch was recorded after 7-day exposure to the treatments, and second-stage juvenile (J2) activity after 48 hours. All extracts were toxic to eggs and J2, with P. lanceolata shoot extract tending to have the most activity against M. incognita. Numbers of active J2 remained the same or decreased in a 24-hour water rinse following the 48-hour extract treatment, indicating that the extracts were lethal. When data from water- and methanol-extracted roots and shoots of both plant species were combined for analysis, J2 tended to be more sensitive than eggs to the toxic compounds at lower concentrations, while the higher concentrations (75% and 100%) were equally toxic to both life stages. The effective concentrations causing 50% reduction (EC₅₀) in egg hatch and in J2 viability were 44.4% and 43.7%, respectively. No extract was toxic to any of the bacteria or fungi in our assays.     

Factors affecting chromate reduction in Enterobacter cloacae strain HO1

Summary Factors affecting chromate reduction by cultures of Enterobacter cloacae HO1 were investigated. The reduction was sensitive to oxygen stress and E. cloacae strain HO1 could reduce chromate only under anaerobic conditions. Rates of reduction of chromate were proportional to cell number. The optimal pH was between 7.0 and 7.8, and the optimal temperature was 30°–37°C. High rates of reduction were observed at levels of 1–2 mM potassium chromate, but concentrations above 5 mM were lethal to growing cells and prevented the reduction. Acetate, ethanol, malate, succinate and glycerol were effective electron donors for chromate reduction. Glucose, citrate, pyruvate and lactate supported anaerobic growth, but only limited amounts of reduction were observed with these organic compounds. Chromate reduction by strain HO1 was inhibited by molybdate, vanadate, tellurate and manganese oxide at concentrations where the cell viability was not significantly affected. Metabolic poisons including carbonylcyanide-m-chlorophenyl hydrazone, sodium cyanide, formaldehyde and zinc sulphate also inhibited chromate reduction.     

Evaluation of the effects of Chlorella vulgaris,Nannochloropsis salina,and Enterobacter cloacae on growth,yield and active compound compositions of Moringa oleifera under salinity stress

Application of Chlorella vulgaris, Nannochloropsis salina and Enterobacter cloacae has been reported to improve the growth of multiple plant species. Moringa oleifera is a medicinal plant found in Saudi Arabia. Its leaves, flowers and fruit have been used as food. Moringa oleifera is rich in rutin and gallic acid and many other bioactive compounds, which collectively contribute to its demonstrated range of pharmacological activities. In Saudi Arabia, the semi-arid and arid weather presents a significant challenge to agriculture. High salinity in cultivated land is a particular threat. We applied Chlorella vulgaris, Nannochloropsis salina, and Enterobacter cloacae at multiple salinities to Moringa oleifera to investigate their effects on the growth, yield, and photosynthetic pigment content. We also examined possible changes in the phytochemical composition. The application of Chlorella vulgaris, Nannochloropsis salina and Enterobacter cloacae enhanced plant growth and yield, while inhibition was observed at high (6000 ppm) salinity. The presence of Chlorella vulgaris and Nannochloropsis salina altered plant growth and yield and rutin and gallic acid content of Moringa oleifera plants grown in saline conditions. Microalgae species were recommended for use as a bio-fertiliser alternative to mainstream synthetic fertilisers.