Type I nitroreductases in soil enterobacteria reduce TNT (2,4,6,-trinitrotoluene) and RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine)

Many enteric bacteria express a type I oxygen-insensitive nitroreductase, which reduces nitro groups on many different nitroaromatic compounds under aerobic conditions. Enzymatic reduction of nitramines was also documented in enteric bacteria under anaerobic conditions. This study indicates that nitramine reduction in enteric bacteria is carried out by the type I, or oxygen-insensitive nitroreductase, rather than a type II enzyme. The enteric bacterium Morganella morganii strain B2 with documented hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) nitroreductase activity, and Enterobacter cloacae strain 96-3 with documented 2,4,6-trinitrotoluene (TNT) nitroreductase activity, were used here to show that the explosives TNT and RDX were both reduced by a type I nitroreductase. Morganella morganii and E. cloacae exhibited RDX and TNT nitroreductase activities in whole cell assays. Type I nitroreductase, purified from E. cloacae, oxidized NADPH with TNT or RDX as substrate. When expression of the E. cloacae type I nitroreductase gene was induced in an Escherichia coli strain carrying a plasmid, a simultaneous increase in TNT and RDX nitroreductase activities was observed. In addition, neither TNT nor RDX nitroreductase activity was detected in nitrofurazone-resistant mutants of M. morganii. We conclude that a type I nitroreductase present in these two enteric bacteria was responsible for the nitroreduction of both types of explosive.     

Characteristics of selected virulence factors of Enterobacter cloacae strains isolated from clinical specimens

We demonstrated that Enterobacter cloacae possesses a selective haemolytic activity on sheep erythrocytes. All the screened strains showed a haemolytic activity on sheep erythrocytes when cultures were preincubated with beta-mercaptoethanol. The investigation circulation of the genes encoding extended spectrum beta-lactamases (ESBL) shows that beta-lactamase producers can be ascribed to specific patterns of plasmids. We also demonstrated that genetic material from E. coli can be transferred and established in selected Enterobacter cloacae strains. In a survival tests we demonstrated that similarly to Salmonella or Vibrio clinical isolates Enterobacter cloacae doesn't demonstrate acid tolerance.     

Release of the type I secreted alpha-haemolysin via outer membrane vesicles from Escherichia coli

The alpha-haemolysin is an important virulence factor commonly expressed by extraintestinal pathogenic Escherichia coli. The secretion of the alpha-haemolysin is mediated by the type I secretion system and the toxin reaches the extracellular space without the formation of periplasmic intermediates presumably in a soluble form. Surprisingly, we found that a fraction of this type I secreted protein is located within outer membrane vesicles (OMVs) that are released by the bacteria. The alpha-haemolysin appeared very tightly associated with the OMVs as judged by dissociation assays and proteinase susceptibility tests. The alpha-haemolysin in OMVs was cytotoxically active and caused lysis of red blood cells. The OMVs containing the alpha-haemolysin were distinct from the OMVs not containing alpha-haemolysin, showing a lower density. Furthermore, they differed in protein composition and one component of the type I secretion system, the TolC protein, was found in the lower density vesicles. Studies of natural isolates of E. coli demonstrated that the localization of alpha-haemolysin in OMVs is a common feature among haemolytic strains. We propose an alternative pathway for the transport of the type I secreted alpha-haemolysin from the bacteria to the host cells during bacterial infections.     

Frequency of Escherichia coli strains producing the cytotoxic necrotizing factor (CNF1) in nosocomial urinary tract infections

The presence of cytotoxic necrotizing factor 1 (CNF1), together with various associated virulence factors (alpha-haemolysin, P-, S- and A-fimbriae), was screened in 175 uropathogenic Escherichia coli strains isolated from hospitalized adult patients. The cnf1 gene was detected in 30% of the selected strains independently of the severity of the clinical urinary infection. A significant association between CNF1, haemolytic activity and the products of the pap/sfa genes was found. However, CNF1 appeared not to play a major role in nosocomial E. coli urinary tract infections.     

Haemolytic activity of Morganella morganii strains

Haemolytic activity on solid and liquid media of 103 Morganella morganii strains isolated from clinical sources was investigated. The ability to produce haemolysin was found in 42.7% of strains. All strains capable to produce haemolysin on blood agar media also revealed haemolytic activity in some liquid media. Haemolysins were found in the supernatants and filtrates of the cultures in peptone water but not in Brain Heart Infusion and Trypticase Soy Broth. The maximal titer of haemolysin was observed in the logarithmic phase of growth. Heating and incubation with trypsin led to complete loss of haemolytic activity.     

Recognition of Morganella subspecies, with proposal of Morganella morganii subsp. morganii subsp. nov. and Morganella morganii subsp. sibonii subsp. nov.

The genus name Morganella was established within the family Enterobacteriaceae in 1978. Morganella morganii is the only species described thus far within this genus, and the name M. morganii has been accepted by usage in the scientific community for strains previously known as Proteus morganii. M. morganii isolates differ in their abilities to ferment trehalose and exhibit variable lysine and ornithine decarboxylase patterns, emphasizing the phenotypic heterogeneity within this species. Previous genetic studies failed to reveal separate entities within the genus Morganella. We observed some trehalose-fermenting strains with different lysine and ornithine decarboxylase patterns. Two strains were lysine and ornithine positive, 3 were lysine positive and ornithine negative, and 29 were lysine negative and ornithine positive. These strains and 25 non-trehalose-fermenting strains with different lysine and ornithine decarboxylase patterns were investigated. DNA-DNA hybridization studies and phenotypic characterizations revealed that M. morganii can be separated into three DNA relatedness groups and seven biogroups. Strains from DNA relatedness group 1 were trehalose negative, and strains from DNA relatedness groups 2 and 3 were trehalose positive. One biogroup from DNA relatedness group 2 was phenotypically indistinguishable from DNA relatedness group 3. On the basis of these studies, we propose that M. morganii be subdivided into M. morganii subsp. morganii (type strain ATCC 25830) containing biogroups A, B, C, and D (DNA relatedness group 1) and M. morganii subsp. sibonii (type strain 8103-85; = ATCC 49948) containing biogroups E, F, and G (DNA relatedness groups 2 and 3).     

Population genetics of the nomenspecies Enterobacter cloacae

The genetic heterogeneity of the nomenspecies Enterobacter cloacae is well known. Enterobacter asburiae, Enterobacter cancerogenus, Enterobacter dissolvens, Enterobacter hormaechei, Enterobacter kobei, and Enterobacter nimipressuralis are closely related to it and are subsumed in the so-called E. cloacae complex. DNA-DNA hybridization studies performed previously identified at least five DNA-relatedness groups of this complex. In order to analyze the genetic structure and the phylogenetic relationships between the clusters of the nomenspecies E. cloacae, 206 strains collected from 22 hospitals, a veterinarian, and an agricultural center in 11 countries plus all 13 type strains of the genus and reference strain CDC 1347-71(R) were examined with a combination of sequence and PCR-restriction fragment length polymorphism (PCR-RFLP) analyses of the three housekeeping genes hsp60, rpoB, and hemB as well as ampC, the gene of a class C beta-lactamase. Based on the neighbor-joining tree of the hsp60 sequences, 12 genetic clusters (I to XII) and an unstable sequence crowd (xiii) were identified. The robustness of the genetic clusters was confirmed by analyses of rpoB and hemB sequences and ampC PCR-RFLPs. Sequence crowd xiii split into two groups after rpoB analysis. Only three strains formed a cluster with the type strain of E. cloacae, indicating that the minority of isolates identified as E. cloacae truly belong to the species; 13% of strains grouped with other type strains of the genus, suggesting that the phenotypes of these species seem to be more heterogeneous than so far believed. Three clusters represented 70% of strains, but none of them included a type or reference strain. The genetic clustering presented in this study might serve as a framework for future studies dealing with taxonomic, evolutionary, epidemiological, or pathogenetic characteristics of bacteria belonging to the E. cloacae complex.     

Chromosomal mutations that increase the production of a plasmid-encoded haemolysin in Escherichia coli

Transposon mutagenesis was used to isolate two Escherichia coli mutants which express very large amounts of haemolysin when carrying the multicopy plasmid pANN202-312. E. coli strain Hha-2 was isolated by Mud1 mutagenesis, and strain Hha-3 by Tn5 mutagenesis. The transposon insertion was chromosomal in both mutants and could be demonstrated to be unrelated to the haemolytic region of the plasmid. The substantial increase in both extracellular and intracellular haemolysin production was dependent upon plasmid copy number and was drastically reduced when either mutant carried the low-copy-number haemolytic plasmid pHly152. In both mutants, the marked increase in extracellular production was dependent upon the specific haemolysin transport genes, hlyB and hlyD. The lack of either gene function resulted in no external haemolysin production. SDS-PAGE analysis showed no change in the pattern of outer-membrane proteins of the mutants, although changes (differing between the two mutants) were seen in their periplasmic proteins. The mutations of both strains (termed hha-2 and hha-3) were mapped at minute 10.5 of the E. coli chromosome. No relation to any known gene affecting gene regulation in E. coli could be found.     

A toxaphene-degrading bacterium related to Enterobacter cloacae, strain D1 isolated from aged contaminated soil in Nicaragua

Enterobacter sp. strain D1 is a facultative anaerobic, Gram-negative heterotrophic bacterium isolated from toxaphene-contaminated soil. This organism was identified and characterized through phylogenetic and taxonomic studies. Based on 16S rDNA analysis, the strain D1 was clustered closely with the species Enterobacter cloacae subsp. dissolvens (LMG 2683) and E. cloacae (ATCC 13047T). Strain D1 resembled these E. cloacae strains with respect to various biochemical and nutritional characteristics, but also exhibited differences. Moreover, strain D1 is able to grow and survive with toxaphene supplied in the medium in the range 3-96 mg/L. Amongst the chemical components of toxaphene, octachlorocamphenes, nonachlorobornanes and decachlorobornanes were seen to be rapidly metabolized, although levels of hexachlorocamphenes and heptachlorobornanes were found to be slowly degraded, and subsequently accumulated during the last stage of the cultivation.     

62株阴沟肠杆菌的生化特性和药敏结果

目的探讨临床分离阴沟肠杆菌的实用鉴定方法.方法对VITEK-32微生物分析仪鉴定所得的62株阴沟肠杆菌的生化反应和药敏结果作回顾性分析.结果 62株阴沟肠杆菌用鉴定卡鉴定的可信度在94%～99%,其中51株为99%、占82.3%,4株为95%、占6.5%,其他7株、占11.2%.阴沟肠杆菌对除外亚胺培南的20种抗生素均有不同程度的耐药.结论脲酶、侧金盏花醇、精氨酸水解和赖氨酸脱羧等试验对肠杆菌属中5个常见菌种(阴沟、坂崎、产气、聚团和格高菲肠杆菌)的鉴别有重要意义.     

Escherichia coli alpha-haemolysin induces focal leaks in colonic epithelium: a novel mechanism of bacterial translocation

Extraintestinal pathogenic Escherichia coli (ExPEC) are usually harmless colonizer of the intestinal microflora. However, they are capable to translocate and cause life-threatening disease. Translocation of ExPEC isolates was quantified in colonic monolayers. Transepithelial resistance (R(t)) was monitored and local changes in conductivity analysed with conductance scanning. Confocal microscopy visualized the translocation route. Corroboratory experiments were performed on native rat colon. One translocating strain E. coli O4 was identified. This translocation process was associated with an R(t) decrease (36 +/- 1% of initial resistance) beginning only 2 h after inoculation. The sites of translocation were small defects in epithelial integrity (focal leaks) exhibiting highly increased local ion permeability. Translocation was enhanced by preincubation of monolayers with tumour necrosis factor-alpha or interleukin-13. Mutant strains lacking alpha-haemolysin lost the ability to induce focal leaks, while this effect could be restored by re-introducing the haemolysin determinant. Filtrate of a laboratory strain carrying the alpha-haemolysin operon was sufficient for focal leak induction. In native rat colon, E. coli O4 decreased R(t) and immunohistology demonstrated focal leaks resembling those in cell monolayers. E. coli alpha-haemolysin is able to induce focal leaks in colonic cell cultures as well as in native colon. This process represents a novel route of bacterial translocation facilitated by pro-inflammatory cytokines.     

Rapid detection of phylloplane bacterium Enterobacter cloacae based on chitinase gene transformation and lytic infection by specific bacteriophages

AIMS: To establish a rapid and efficient method for detecting Enterobacter cloacae based on chitinase gene transformation and lytic infection by virulent bacteriophages. METHODS AND RESULTS: A phylloplane strain of E. cloacae was isolated from tomato leaves and transformed with a chitinase gene. Transformed bacteria were collected from single colonies and infected with newly isolated, virulent bacteriophages in the presence of the chitinase substrate 4-methylumbelliferon (4MU)-(GlcNac)3. To assay chitinase activity in the lysates, the product 4MU was measured spectrofluorophotometrically or visibly detected under u.v. irradiation. Chitinase gene-transformed bacteria obtained from single colonies could be specifically identified in 30 min by the emission of 4MU fluorescence following lysis caused by phage infection. CONCLUSIONS: The chitinase gene was used as a reporter gene to construct a new system for easy and rapid monitoring of transgenic strains of E. cloacae released in the environment, in combination with specific recognition by virulent bacteriophages. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay is simple, rapid, inexpensive, easy to perform and applicable to other strains. The system can be used for the routine monitoring of bacteria, which is important because of the increased use of transgenic strains of E. cloacae as an antagonistic biological control agent for plant diseases.     

Differential interference with Pythium ultimum sporangial activation and germination by Enterobacter cloacae in the corn and cucumber spermospheres

Differential protection of plants by Enterobacter cloacae was studied by investigating early sensing and response behavior of Pythium ultimum sporangia toward seeds in the presence or absence of E. cloacae. Ten percent of P. ultimum sporangia were activated within the first 30 min of exposure to cucumber seeds. In contrast, 44% of the sporangia were activated as early as 15 min after exposure to corn seeds with over 80% sporangial activation by 30 min. Germ tubes emerged from sporangia after 2.5 and 1.0 h in the cucumber and corn spermospheres, respectively. Seed application of the wild-type strain of E. cloacae (EcCT-501R3) reduced sporangial activation by 45% in the cucumber spermosphere, whereas no reduction was observed in the corn spermosphere. Fatty acid transport and degradation mutants of E. cloacae (strains EcL1 and Ec31, respectively) did not reduce sporangial activation in either of the spermospheres. Although wild-type or mutant strains of E. cloacae failed to reduce seed colonization incidence, pathogen biomass on cucumber seeds was reduced in the presence of E. cloacae strains EcCT-501R3 and Ec31 by 4 and 8 h after sowing, respectively. By 12 h, levels of P. ultimum on cucumber seeds treated with E. cloacae EcCT-501R3 did not differ from levels on noninoculated seeds. On corn seeds, P. ultimum biomass was not affected by the presence of any E. cloacae strain. When introduced after sporangial activation had occurred, E. cloacae failed to reduce P. ultimum biomass on cucumber seeds compared with that on nontreated seeds. Also, increasing numbers of sporangia used to inoculate seeds yielded increased pathogen biomass at each sampling time. This indicates a direct link between the level of seed-colonizing biomass of P. ultimum and the number of activated and germinated sporangia in the spermosphere, suggesting that E. cloacae suppresses P. ultimum seed infections by reducing sporangial activation and germination within the first 30 to 90 min after sowing.     

Overexpression of serine hydroxymethyltransferase from halotolerant cyanobacterium in Escherichia coli results in increased accumulation of choline precursors and enhanced salinity tolerance

A real-time PCR procedure targeting the gene of the molecular cochaperon DnaJ (dnaJ) was developed for specific detection of strains belonging to the Enterobacter cloacae group. The inclusivity and exclusivity of the real-time PCR assay were assessed with seven reference strains of E.?cloacae, 12 other Enterobacter species and 41 non-Enterobacter strains. Inclusivity as well as exclusivity of the duplex real-time PCR was 100%. In contrast, resolution of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was inadequate for delineation of Enterobacter asburiae, Enterobacter hormaechei, Enterobacter kobei and Enterobacter ludwigii from E.?cloacae. Eleven of 56 (20%) clinical isolates of the E.?cloacae group could not be clearly identified as a certain species using MALDI-TOF MS. In summary, the combination of MALDI-TOF MS with the E.?cloacae-specific duplex real-time PCR is an appropriate method for identification of the six species of the E.?cloacae complex.     

GASP phenotype: presence in enterobacteria and independence of sigmaS in its acquisition

The appearance of growth advantage in stationary phase or GASP was originally detected in Escherichia coli. The presence of this phenotype in other enterobacteria such as Enterobacter cloacae, Salmonella typhimurium, Providencia stuartii and Shigella dysenteriae is described in this work. E. cloacae GASP strains presented lower levels of RpoS than the parental strain, although no mutation in the gene or its promoter was detected. This work offers evidence of GASP rpoS-independent pathways as GASP was also acquired in knock-out rpoS E. cloacae and E. coli strains.     

Secretion of cyclolysin, the calmodulin-sensitive adenylate cyclase-haemolysin bifunctional protein of Bordetella pertussis.

The calmodulin-sensitive adenylate cyclase of Bordetella pertussis, a 45 kd secreted protein, is synthesized as a 1706 amino acid precursor. We have shown that this precursor is a bifunctional protein, carrying both adenylate cyclase and haemolytic activities. The 1250 carboxy-terminal amino acids of the precursor showed 25% similarity with Escherichia coli alpha-haemolysin (HlyA) and 22% similarity with Pasteurella haemolytica leucotoxin. Three open reading frames were identified downstream from the cyaA gene: cyaB, cyaD and cyaE, coding for polypeptides of 712, 440 and 474 amino acid residues, respectively. As for E. coli alpha-haemolysin, secretion of B.pertussis adenylate cyclase and haemolysin requires the expression of additional genes. The gene products of cyaB and cyaD are highly similar to HlyB and HlyD, known to be necessary for the transport of HlyA across the cell envelope and for its release into the external medium. Complementation and functional studies indicate that the B.pertussis adenylate cyclase-haemolysin bifunctional protein is secreted by a mechanism similar to that described for E.coli alpha-haemolysin, requiring, in addition to the cyaB and cyaD gene products, the presence of a third gene product specified by the cyaE gene.     

prbA,a gene coding for an esterase hydrolyzing parabens in enterobacter cloacae and Enterobacter gergoviae strains

The new gene prbA encodes an esterase responsible for the hydrolysis of the ester bond of parabens in Enterobacter cloacae strain EM. This gene is located on the chromosome of strain EM and was cloned by several PCR approaches. The prbA gene codes for an immature protein of 533 amino acids, the first 31 of which represent a proposed signal peptide yielding a mature protein of a putative molecular mass of 54.6 kDa. This enzyme presents analogies with other type B carboxylesterases, mainly of eukaryotic origin. The cloning and expression of the prbA gene in a strain of Escherichia coli previously unable to hydrolyze parabens resulted in the acquisition of a hydrolytic capacity comparable to the original activity of strain EM, along with an increased resistance of the transformed strain to methyl paraben. The presence of homologues of prbA was tested in additional ubiquitous bacteria, which may be causative factors in opportunistic infections, including Enterobacter gergoviae, Enterobacter aerogenes, Pseudomonas agglomerans, E. coli, Pseudomonas aeruginosa, and Burkholderia cepacia. Among the 41 total strains tested, 2 strains of E. gergoviae and 1 strain of Burkholderia cepacia were able to degrade almost completely 800 mg of methyl paraben liter(-1). Two strains of E. gergoviae, named G1 and G12, contained a gene that showed high homology to the prbA gene of E. cloacae and demonstrated comparable paraben esterase activities. The significant geographical distance between the locations of the isolated E. cloacae and E. gergoviae strains suggests the possibility of an efficient transfer mechanism of the prbA gene, conferring additional resistance to parabens in ubiquitous bacteria that represent a common source of opportunistic infections.     

Expression of AmpC beta-lactamase in Enterobacter cloacae isolated from retail ground beef, cattle farm and processing facilities

AIMS: To better understand antibiotic resistance of Enterobacter cloacae isolates originated from food animals, the phenotypic and genotypic resistance of Ent. cloacae isolates from retail ground beef, cattle farm, processing facilities and clinical settings were investigated. METHODS AND RESULTS: The ampC, ampD and ampR genes in the isolates were sequenced and analysed. beta-Lactamase activities and beta-lactamase profiles of the isolates were analysed by the enzymatic hydrolysis of nitrocefin and isoelectric focussing, respectively. The ampC gene of the Ent. cloacae isolate was cloned and transformed into Escherichia coli strains. The genomic DNA profiles of Ent. cloacae isolates were analysed by using pulse field gel electrophoresis (PFGE). Mutation at one residue (Val-54-->Ile) in the AmpR amino acid sequence was consistently found in Ent. cloacae isolates that were resistant to a broadspectrum of beta-lactam agents. The enzyme activity in the isolates was induced by cefoxitin. The pI (isoelectric point) of the enzymes produced by the test strains ranged from 8.4 to 8.9. Cloning of ampC gene of the Ent. cloacae isolate conferred the resistance to ampicillin, cephalothin and amoxicillin in recipient E. coli strains. One recipient of E. coli O157:H7 strain additionally acquired resistance to ceftiofur. The genomic analysis of Ent. cloacae isolates by PFGE showed that the isolates from various sources were genetically unrelated. CONCLUSIONS: The spread of diverse clones of AmpC-producing Ent. cloacae occurred in the ecosystem and retail products. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings suggested that AmpC-producing Ent. cloacae could be a contributor in spreading beta-lactamase genes in farm environments and food processing environments.     

Evaluation of a commercial beta-glucuronidase test for the rapid and economical identification of Escherichia coli

A commercial beta-glucuronidase (beta-GUR) test for the rapid and economical identification of Escherichia coli was evaluated. A total of 762 clinical strains and 228 environmental isolates were studied. More than 95% of the E. coli strains were found to be beta-GUR positive. Thirty-one clinical isolates of Shigella sonnei, 10 of Enterobacter cloacae, eight of Enterobacter aerogenes, nine of Citrobacter freundii and one of Salmonella enteritidis also gave positive results. The enzyme beta-GUR was also detected in two environmental strains of E. cloacae and one C. freundii. A comparative study between the beta-GUR test and the conventional identification system was carried out in 233 consecutive isolates of lactose positive enterobacteria. Agreement was observed in 223 cases and 190 E. coli strains were correctly identified using this test. Discrepancies were found in 10 cases: nine E. coli were beta-GUR negative and one C. freundii was beta-GUR positive. Escherichia coli was the only species positive for both beta-GUR and indole tests. This procedure permits a rapid, easy, precise and inexpensive identification of E. coli. beta-GUR positive Enterobacter strains have not previously been described.