NDM-1对阴沟肠杆菌的致病性及适应性的影响

产生新德里金属β-内酰胺酶-1 (New delhi metallol-β-lactamase 1,NDM-1)的肠杆菌科细菌对大部分β-内酰胺抗生素耐药,其导致的感染难以治疗。细菌中编码NDM-1基因的获得可能会影响它的致病性和适应性,这可能使产NDM-1的细菌的感染治疗和传播更加复杂。产NDM-1阴沟肠杆菌流行率高,现对阴沟肠杆菌的致病过程以及NDM-1对其致病性和适应性的影响作一概述,为产NDM-1阴沟肠杆菌的感染治疗和传播控制提供应对策略。     

Multidrug-resistant phenotype and isolation of a Novel SHV- beta-Lactamase variant in a clinical isolate of Enterobacter cloacae

Background

ESBL-producing bacteria are a clinical problem in the management of diseases caused by these pathogens. Worldwide, systemic infections with BL enzymes are evolving by mutations from classical bla genes in an intensified manner and they continue to be transferred across species.

Results

E.cloacae BF1417 isolate and its transconjugants gave positive results with the DDST, suggesting the presence of ESBL. Sequence analysis revealed a bla_SHV-ESBL-type gene that differs from the gene encoding SHV-1 by five point mutations resulting in three amino acid substitutions in the coding region: C123R, I282T and L286P. This novel SHV-type enzyme was designated SHV-128. The conjugation tests and plasmid characterization showed that the bla_SHV-128 is located on a conjugative plasmid IncFII type. Expression studies demonstrated that the above mutations participated in drug resistance, hydrolysis of extended spectrum β-lactam and the change of the isoelectric point of the protein.

Conclusion

These findings underscore the diversity by which antibiotic resistance can arise and the evolutionary potential of the clinically important ESBL enzymes. In addition, this study highlights the need for systematic surveillance of ESBL-mediated resistance as well as in clinical areas and communities.     

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.     

An outbreak of SHV-5 producing Klebsiella pneumoniae in a neonatal intensive care unit; meropenem failed to avoid fecal colonization

An outbreak of extended-spectrum beta-lactamase (ESBL) producing Klebsiella pneumoniae (ESBL-Kp) in a neonatal intensive care unit prompted a prospective surveillance study between 12th September and 6th October 2003. Surveillance was carried out by obtaining stool samples twice a week. The DNA relatedness of the isolates was shown by random amplified polymorphic DNA comparison (ERIC-PCR). ESBL production was identified by clavulanate synergy, isoelectric focusing, PCR and sequence analysis. During the study period, 49 neonates were hospitalized in the neonatal intensive care unit (NICU). In the first 20-day period, five neonates were infected with ESBL-Kp. The first patient treated with third generation cephalosporin and the second patient treated with meropenem died. While all three infected survivors were clinically improving, the digestive tracts were being colonized by SHV-5 producing Klebsiella. In the next period of the study, five neonates were colonized by ESBL-Kp as well. Univariate comparison of risk factors between colonized and non-colonized neonates was not significant. A total of 24 colonally related ESBL-Kp have been recovered from clinical materials and stool samples. This study demonstrated that parenterally applied meropenem, though successful in treating the systemic illness, might fail to protect the digestive tract from colonization of ESBL-Kp.     

Chromate-resistance in a chromate-reducing strain of Enterobacter cloacae

Resistance to toxic hexavalent chromium (chromate: CrO4(2)) in Enterobacter cloacae strain HO1, isolated from an activated sludge sample, was investigated under aerobic and anaerobic conditions. Decreased uptake of 51CrO4(2-) in E. cloacae strain HO1 was observed under aerobic conditions, when compared with a standard laboratory E. cloacae strain (IAM 1624). Under anaerobic conditions E. cloacae strain HO1 was able to reduce hexavalent chromium to the less toxic trivalent form. When E. clocacae strain HO1 was grown with nitrate anaerobically, the cells were observed to lose simultaneously their chromate-reducing ability and chromate-resistance under anaerobic conditions.     

Emergence of Pseudomonas aeruginosa strains producing high levels of persister cells in patients with cystic fibrosis

The majority of cystic fibrosis (CF) patients succumb to a chronic infection of the airway with Pseudomonas aeruginosa. Paradoxically, pathogenic strains are often susceptible to antibiotics, but the infection cannot be eradicated with antimicrobial therapy. We find that in a majority of patients with airway infections, late isolates of P. aeruginosa produce increased levels of drug-tolerant persister cells. The genomes of a clonal pair of early/late isolates from a single patient have been previously sequenced, and the late isolate (obtained at age 96 months) showed a 100-fold increase in persister levels. The 96-month isolate carries a large number of mutations, including a mutation in mutS that confers a hypermutator phenotype. There is also a mutation in the mexZ repressor controlling the expression of the MexXY-OprM multidrug pump, which results in a moderate increase in the ofloxacin, carbenicillin, and tobramycin MICs. Knocking out the mexXY locus restored the resistance to that of the parent strain but did not affect the high levels of persisters formed by the 96-month isolate. This suggests that the late isolate is a high-persister (hip) mutant. Increased persister formation was observed in exponential phase, stationary phase, and biofilm populations of the 96-month isolate. Analysis of late isolates from 14 additional patients indicated that 10 of them are hip mutants. Most of these hip mutants did not have higher drug resistance. Increased persister formation appears to be their sole mechanism for surviving chemotherapy. Taken together, these findings suggest a link between persisters and recalcitrance of CF infection and identify an overlooked culprit-high-persister mutants producing elevated levels of drug-tolerant cells. Persisters may play a similarly critical role in the recalcitrance of other chronic infections.     

Molecular cloning, characterization, and overexpression of a novel [Fe]-hydrogenase isolated from a high rate of hydrogen producing Enterobacter cloacae IIT-BT 08

Degenerate primers were designed from the conserved zone of hydA structural gene encoding for catalytic subunit of [Fe]-hydrogenase of different hydrogen producing bacteria. A 750 bp of PCR product was amplified by using the above-mentioned degenerate primers and genomic DNA of Enterobacter cloacae IIT-BT 08 as template. The amplified PCR product was cloned and sequenced. The sequence showed the presence of an ORF of 450 bp with significant similarity (40%) with C-terminal end of the conserved zone (H-cluster) of [Fe]- hydrogenase. hydA ORF was then amplified and cloned in-frame with GST in pGEX4T-1 and overexpressed in a non-hydrogen producing Escherichia coli BL-21 to produce a GST-fusion protein of a calculated molecular mass of about 42.1 kDa. Recombinant protein was purified and specifically recognized by anti-GST monoclonal antibody through Western blot. Southern hybridization confirmed the presence of this gene in E. cloacae IIT-BT 08 genome. In vitro hydrogenase assay with the overexpressed hydrogenase enzyme showed that it is catalytically active upon anaerobic adaptation. In vivo hydrogenase assay confirmed the presence of H2 gas in the gas mixture obtained from the batch culture of recombinant E. coli BL-21. A tentative molecular mechanism has been proposed about the transfer of electron from electron donor to H-cluster without the mediation of the F-cluster.     

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.     

Emergence and widespread circulation of a recombinant SARS-CoV-2 lineage in North America

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Temperature sensitivity of a nifA-like gene in Enterobacter cloacae.

Nitrogen fixation (nif) genes of Enterobacter cloacae, a rhizosphere diazotroph of rice plants, were identified by using cloned Klebsiella pneumoniae nif gene fragments as probes for molecular hybridization. The product of a nifA-like gene of E. cloacae appeared less temperature sensitive than the K. pneumoniae nifA gene product. This result correlates with the fact that E. cloacae can fix nitrogen at 39 degrees C, while K. pneumoniae cannot.     

Degradation of pentaerythritol tetranitrate by Enterobacter cloacae PB2.

A mixed microbial culture capable of metabolizing the explosive pentaerythritol tetranitrate (PETN) was obtained from soil enrichments under aerobic and nitrogen-limiting conditions. A strain of Enterobacter cloacae, designated PB2, was isolated from this culture and was found to use PETN as a sole source of nitrogen for growth. Growth yields suggested that 2 to 3 mol of nitrogen was utilized per mol of PETN. The metabolites pentaerythritol dinitrate, 3-hydroxy-2,2-bis-[(nitrooxy)methyl]propanal, and 2,2-bis-[(nitrooxy)methyl]-propanedial were identified by mass spectrometry and 1H-nuclear magnetic resonance. An NADPH-dependent PETN reductase was isolated from cell extracts and shown to liberate nitrite from PETN, producing pentaerythritol tri- and dinitrates which were identified by mass spectrometry. PETN reductase was purified to apparent homogeneity by ion-exchange and affinity chromatography. The purified enzyme was found to be a monomeric flavoprotein with a M(r) of approximately 40,000, binding flavin mononucleotide noncovalently.     

Ultrahigh resolution structure of a class A beta-lactamase: on the mechanism and specificity of the extended-spectrum SHV-2 enzyme

Bacterial beta-lactamases hydrolyze beta-lactam antibiotics such as penicillins and cephalosporins. The TEM-type class A beta-lactamase SHV-2 is a natural variant that exhibits activity against third-generation cephalosporins normally resistant to hydrolysis by class A enzymes. SHV-2 contains a single Gly238Ser change relative to the wild-type enzyme SHV-1. Crystallographic refinement of a model including hydrogen atoms gave R and R(free) of 12.4% and 15.0% for data to 0.91 A resolution. The hydrogen atom on the O(gamma) atom of the reactive Ser70 is clearly seen for the first time, bridging to the water molecule activated by Glu166. Though hydrogen atoms on the nearby Lys73 are not seen, this observation of the Ser70 hydrogen atom and the hydrogen bonding pattern around Lys73 indicate that Lys73 is protonated. These findings support a role for the Glu166-water couple, rather than Lys73, as the general base in the deprotonation of Ser70 in the acylation process of class A beta-lactamases. Overlay of SHV-2 with SHV-1 shows a significant 1-3 A displacement in the 238-242 beta-strand-turn segment, making the beta-lactam binding site more open to newer cephalosporins with large C7 substituents and thereby expanding the substrate spectrum of the variant enzyme. The OH group of the buried Ser238 side-chain hydrogen bonds to the main-chain CO of Asn170 on the Omega loop, that is unaltered in position relative to SHV-1. This structural role for Ser238 in protein-protein binding makes less likely its hydrogen bonding to oximino cephalosporins such as cefotaxime or ceftazidime.     

Transformation of Azospirillum brasilense Cd with an ACC deaminase gene from enterobacter cloacae UW4 fused to the Tet r gene promoter improves its fitness and plant growth promoting ability

It has been reported that PGPB, containing ACC deaminase, can cleave the plant ethylene precursor ACC and thereby lower ethylene concentration in a developing or stressed plant, protecting it against the deleterious effects of stress ethylene and facilitating the formation of longer roots. In a previous work we have demonstrated expression of the ACC deaminase gene (acdS) from Enterobacter cloacae UW4 under the control of the lac promoter in Azospirillum brasilense Cd. With the inference that a construct including the ACC deaminase gene under the control of a constitutive promoter weaker than the lac promoter might impose less metabolic load on Azospirillum and improve its fitness, it was decided to clone acdS under the control of a tetracycline resistance gene promoter. The ACC deaminase structural gene was fused to the Tet ^r gene promoter by overlap extension using PCR, cloned in pRK415, and transferred into A. brasilense Cd. The resulting transformants showed lower ACC deaminase activity than those with the lac promoter controlled acdS gene. However, acdS under the control of the Tet ^r gene promoter imposed lesser metabolic load on Azospirillum brasilense Cd. The result was significantly increased IAA synthesis and greater bacterial growth rate, as well as increased ability to survive on the surface of tomato leaves and to promote the growth of tomato seedlings.