Clinical Isolates Of Enterobacter Cloacae Research Articles

Genetic organization of plasmid-mediated Qnr determinants in cefotaxime-resistant Enterobacter cloacae isolates in Korea

Because of the strong association between qnr genes and plasmids carrying β-lactamase genes, we screened 176 clinical isolates of Enterobacter cloacae with cefotaxime MICs of ≥16 μg/mL for qnr genes. The qnrA, qnrB, and qnrS genes were detected in 18 (10.2%), 11 (6.2%), and 1 (0.56%) of the isolates, respectively. The genetic environments of the plasmids encoding these qnr genes were analyzed.

Sensitization of Microcin J25-Resistant Strains by a Membrane-Permeabilizing Peptide

Microcin J25 (MccJ25) is a plasmid-encoded, 21-amino-acid, antibacterial peptide produced by Escherichia coli. MccJ25 inhibits RNA polymerase and the membrane respiratory chain. MccJ25 uptake into E. coli-sensitive strains is mediated by the outer membrane receptor FhuA and the inner membrane proteins TonB, ExbB, ExbD, and SbmA. This peptide is active on some E. coli, Salmonella, and Shigella species strains, while other Gram-negative bacteria, such as clinical isolates of Enterobacter cloacae, Citrobacter freundii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Moraxella catarrhalis, and Salmonella enterica serovar Typhimurium, are completely resistant. In the present work, we demonstrated that the membrane-permeabilizing peptide (KFF)₃K made some resistant strains sensitive to MccJ25, among them S. Typhimurium, where the antibiotic inhibits in vitro cell growth and bacterial replication within macrophages. The results demonstrate that the membrane permeabilization induced by (KFF)₃K allows MccJ25 penetration in an FhuA and SbmA-independent manner and suggest that the combination of both peptides could be considered as a therapeutic agent against pathogenic Salmonella strains.

Decreased quinolone susceptibility in high percentage of Enterobacter cloacae clinical isolates caused only by Qnr determinants

The prevalence of qnr genes was similar in the ciprofloxacin-resistant and ciprofloxacin-susceptible Enterobacter cloacae isolates, and no amino acid change in quinolone resistance-determining regions was found in any of the 21 qnr-positive but ciprofloxacin-susceptible strains. Qnr may thus be present before the development of higher-level resistance from target mutations.

Emergence of AcrAB-mediated tigecycline resistance in a clinical isolate of Enterobacter cloacae during ciprofloxacin treatment

Tigecycline resistance remains rare amongst Enterobacteriaceae in the UK, as elsewhere, but has been associated with upregulation of the AcrAB efflux system. Using isolates of an Enterobacter cloacae strain that developed tigecycline resistance in vivo during ciprofloxacin therapy as well as laboratory-selected mutants, we investigated the role of this pump and the global regulator RamA in tigecycline resistance. Laboratory mutants were selected from a susceptible clinical isolate in vitro by exposure to increasing concentrations of tigecycline. Expression of the acrAB operon and the ramA gene was monitored by real-time reverse-transcription polymerase chain reaction (RT-PCR). Overexpression of ramA was achieved using the pBAD expression vector, whilst insertional inactivation of acrB with a gentamicin resistance cassette was achieved with the bacteriophage λ Red recombination system. Increased tigecycline minimum inhibitory concentrations in the clinical isolate and a laboratory mutant were associated with increases in acrAB and ramA transcripts. Induction of increased ramA expression resulted in increased acrAB expression, whilst insertional inactivation of acrB restored full susceptibility to tigecycline. Treatment with ciprofloxacin, a substrate of AcrAB in E. cloacae, possibly selected for cross-resistance to tigecycline as a result of RamA-mediated AcrAB upregulation.

First report of CTX-M-9 in a clinical isolate of Enterobacter cloacae in a Tunisian hospital

β-lactamases are one of several mechanisms of bacterial resistance to β-lactam antibiotics. The aim of this study was to analyze the resistance to extended-spectrum cephalosporins of a multidrug-resistant isolate of Enterobacter cloacae, BF5011. This strain was isolated from a stool culture, during the nosocomials infections occurring in the intensive care unit of the Military Hospital of Tunis in 2005. Analysis of E. cloacae BF5011 by double-disk synergy test yielded a positive result suggesting the production of extended-spectrum-β-lactamases. Cell sonicate of this isolate is very active against cefotaxime and showed a specific activity (AS) of 7,54 U/mg for the same antibiotic. This activity was inhibited by the sulbactam and the clavulanic acid. By polymerase chain reaction and sequencing, the isolate was found to produce extended-spectrum-β-lactamase, CTX-M-9. The bla CTX-M-9 gene was transferred from E. cloacae by conjugation. Isoelectric focusing method revealed one band with a pI of about 8. This is the first report of CTX-M-9 in Tunisia.

Detection of Plasmid-Mediated Quinolone Resistance Genes in Clinical Isolates of Enterobacter spp. in Spain

We have studied by PCR and DNA sequencing the presence of the qnrA, qnrB, qnrS, aac(6')-Ib-cr, qepA, intI1, and ISCR1 genes in 200 clinical isolates of Enterobacter cloacae (n = 153) and E. aerogenes (n = 47) consecutively collected between January 2004 and October 2005 in two hospitals located in Santander (northern Spain) and Seville (southern Spain). Mutations in the quinolone resistance-determining region of gyrA and parC also were investigated in organisms containing plasmid-mediated quinolone resistance genes. The isolates had different resistant phenotypes, including AmpC hyperproduction, extended-spectrum beta-lactamase production, resistance or decreased susceptibility to quinolones, and/or resistance to aminoglycosides. Among the 116 E. cloacae isolates from Santander, qnrS1, qnrB5, qnrB2, and aac(6')-Ib-cr were detected in 22 (19%), 1 (0.9%), 1 (0.9%), and 3 (2.6%) isolates, respectively. Twenty-one, 17, and 2 qnrS1-positive isolates also contained bla(LAP-1), intI1, and ISCR1, respectively. A qnrB7-like gene was detected in one E. aerogenes isolate from Santander. No plasmid-mediated quinolone resistance gene was detected in the isolates from Seville. The qnrS1-containing isolates corresponded to four pulsed-field gel electrophoresis patterns and showed various levels of resistance to quinolones. Six isolates were susceptible to nalidixic acid and presented reduced susceptibility to ciprofloxacin. The qnrS1 gene was contained in a conjugative plasmid of ca. 110 kb, and when the plasmid was transferred to recipient strains that did not have a specific mechanism of quinolone resistance, the ciprofloxacin MICs ranged from 0.047 to 0.125 microg/ml.

Prevalence and characteristics of aac(6′)-Ib-cr in AmpC-producing Enterobacter cloacae, Citrobacter freundii, and Serratia marcescens: a multicenter study from Korea

We investigated the prevalence of aac(6′)-Ib-cr and its association with other resistance genes in AmpC-producing Enterobacteriaceae without any selection criteria. A total of 479 clinical isolates of Enterobacter cloacae (179), Citrobacter freundii (134), and Serratia marcescens (166) from 12 laboratories between March and July 2005 were examined. We performed polymerase chain reaction for aac(6′)-Ib, bla OXA-1, IS Ecp1, and class 1 integron. The aac(6′)-Ib-cr was further identified by digestion with BstF5I and sequencing. The aac(6′)-Ib was detected in 110 (23%) of 479 isolates, and 15 isolates (3.1%) were cr variants (8 E. cloacae, 5 C. freundii, and 2 S. marcescens). The aac(6′)-Ib-cr was significantly associated with various resistance genes ( bla OXA-1, qnrS, qnrA, bla CTX-M-3, and bla CTX-M-14), mobile elements (IS Ecp1, IS CR1, and class 1 integron), and quinolone resistance. Eleven of 15 aac(6′)-Ib-cr producers coharbored qnr genes. Although aac(6′)-Ib-cr was uncommon in Korean AmpC producers, its association with various resistance genes and mobile elements would facilitate the dissemination of this variant.

Molecular Characterization of Extended-Spectrum β-Lactamases Produced by Clinical Isolates of <i>Enterobacter cloacae</i> from a Teaching Hospital in China

Of 59 clinical isolates of Enterobacter cloacae from a teaching hospital in Sichuan, China, 18 isolates were shown to be resistant to oxyimino cephalosporins and aztreonam. Enterobacterial repetitive consensus PCR revealed that these isolates comprised 7 distinct genotypes. The presence of plasmids in the 18 clinical isolates was revealed by conjugational transfer of plasmids from E. cloacae to Escherichia coli with the further isolation of the plasmids in the transconjugants. Subsequent nucleotide sequencing and β-lactamase isoelectric focusing indicated that the plasmids encoded blaSHV, blaCTX-M and/or blaTEM genes, including genes for CTX-M-22 (13 strains), TEM-1 (12 strains), TEM-29 (1 strain), TEM-141 (1 strain), TEM-157 (1 strain), SHV-5 (1 strain), SHV-12 (1 strain), and SHV-70 (1 strain). The widespread presence of extended-spectrum β-lactamases in E. cloacae isolated from the southwest of China was likely due to the dissemination of resistance plasmids with the predominant genotype of blaCTX-M-22.

A novel β-lactamase gene, LAP-2, produced by an Enterobacter cloacae clinical isolate in China

Many β-lactamases classified by their amino acid sequences and functional substrate specificity profiles in various Gram-negative bacilli such as Pseudomonas spp. and members of the family Enterobacteriaceae have been documented.1 Since the late 1980s, extended-spectrum β-lactamases (ESBLs) derived from TEM- and SHV-type penicillinases capable of hydrolysing the oxymino-cephalosporins, have been spreading globally, mainly in Enterobacteriaceae, including Klebsiella pneumoniae and Escherichia coli.

Dissemination of ESBL and Qnr determinants in Enterobacter cloacae in Algeria

The aim of this study is to evaluate the prevalence and diversity of extended-spectrum beta-lactamases (ESBLs) in Enterobacter cloacae clinical isolates collected from Algerian hospitals and to verify the association with qnr genes. MICs were determined by Etest for isolates giving positive double-disc synergy tests, and all isolates were screened by PCR and sequenced, respectively, for bla(TEM), bla(CTX-M), bla(SHV) and bla(VEB) genes and for qnr genes (qnrA, qnrB, qnrS), using specific primers. The prevalence of ESBLs was 25/141 (17.7%) with 11, 9, 4 and 1 isolates testing positive for genes encoding CTX-M-15, CTX-M-3, SHV-12 and VEB-1, respectively. Two SHV-12 producers and one CTX-M-15 producer expressed QnrS1, one isolate produced CTX-M-15 and QnrB1 and one SHV-12 producer co-expressed QnrS1 and QnrB4. qnrA was not detected in our collection, and qnr alleles were not detected in non-ESBL-producing isolates. SHV-12, QnrS1, QnrB1 and QnrB4 were reported for the first time in Algeria. This study also described a co-expression of qnrS1 and qnrB4 by an SHV-12 producer isolate.

O207 Plasmid-mediated quinolone resistance determinants, QnrA, QnrB, and QnrS, among clinical isolates of Enterobacter cloacae in a Taiwanese hospital

O207 Plasmid-mediated quinolone resistance determinants, QnrA, QnrB, and QnrS, among clinical isolates of Enterobacter cloacae in a Taiwanese hospital

First Isolation of bla IMI-2 in an Enterobacter cloacae Clinical Isolate from China

Carbapenem resistance mediated by acquired carbapenemases is a growing concern worldwide. Acquired class A carbapenemases of the NMC/IMI, SME, and KPC types remain infrequent in Enterobacteriaceae (2, 6-8), but some of them have increasingly been reported recently (10). IMI-1 was first described in 1996 in clinical isolates of Enterobacter cloacae (9). Recently, IMI-2, a variant that differs from IMI-1 by 2 amino acid residues, was found in a strain of Enterobacter asburiae isolates in U.S. rivers (1). In this study, we report on the detection of IMI-2 in a clinical isolate of Enterobacter cloacae from China. An imipenem-resistant E. cloacae isolate (isolate 8) was obtained from the blood of a patient in our hospital in August 2001. The patient had previously been treated with ampicillin-sulbactam and cefotaxime. E. cloacae isolate 8 was resistant to imipenem, meropenem, and ertapenem; had intermediate resistance to cefotaxime and ceftazidime; and was susceptible to cefepime (Table ​(Table1).1). Conjugation was carried out by a broth method as previously described (11). Transconjugant clones were selected on Mueller-Hinton agar containing rifampin (256 mg/liter) and imipenem (4 mg/liter). Imipenem resistance was transferred to a transconjugant along with a plasmid with a size of about 80 kb. The MIC for the transconjugant (Escherichia coli C600E8) showed that it has resistance to carbapenems but not to expanded-spectrum cephalosporins (Table ​(Table1).1). The β-lactamase activity was determined by UV spectrophotometry with imipenem as a substrate (1, 5). In E. coli C600E8, expression of carbapenemase activity increased from 643 to 1,352 U/mg of protein upon exposure to imipenem (4 mg/liter) as an inducer. These results demonstrated that the β-lactamase was produced at a high basal level and that expression was very poorly inducible. Isoelectric focusing was performed according to a published protocol (3). The result demonstrated that E. cloacae 8 had 5 pI bands at pI 5.1, 5.4, 6.9, 8.1, and 8.6, while E. coli C600E8 had only one band at pI 8.1, which was inhibited by clavulanic acid. TABLE 1. MICs of selected antimicrobial agents for various strains determined by Etest The imipenem resistance gene was cloned into pGEM-T Easy (Promega, Madison, Wis.) as a 10.6-kb EcoRI fragment. A 10,629-bp stretch of DNA sequence was obtained by nucleotide sequencing on both strands (GenBank accession no. {type:entrez-nucleotide,attrs:{text:AY780889,term_id:55845893,term_text:AY780889}}AY780889). In the cloned fragment, blaIMI-2 is preceded by a gene encoding a protein 97% identical to ImiR (9). The regions flanking the blaIMI-R2-blaIMI-2 gene contain sequences related to Tn903 (4) and IS2 (Fig. ​(Fig.1).1). Their structure apparently differs from that previously reported to be flanking the blaIMI-R2-blaIMI-2 gene complex from E. asburiae (1). The transposable elements flanking the blaIMI-R2-blaIMI-2 gene complex could be involved in mobilization of the carbapenemase gene to enterobacterial plasmids. FIG. 1. Schematic representation of the structure of the region containing the blaIMI-R2-blaIMI-2 gene complex from the E. cloacae 8 plasmid. The two sides of the blaIMI-R2-blaIMI-2 gene are inverted repeat sequences. “Tn903-like” downstream is ...

Gene Mutations Responsible for Overexpression of AmpC β-Lactamase in Some Clinical Isolates ofEnterobacter cloacae

AmpC regulatory genes in 21 ceftazidime-resistant clinical isolates of Enterobacter cloacae (MICs of > or = 16 microg/ml) were characterized. All isolates exhibited AmpC overproduction due to AmpD mutation. Additionally, we found two AmpR mutants among the isolates. This is the first report of chromosomal ampR mutation in clinical isolates of E. cloacae.

Characterization of a new integron containing blaVIM-1 and aac(6′)-IIc in an Enterobacter cloacae clinical isolate from Greece

A clinical isolate of Enterobacter cloacae exhibiting reduced susceptibility to imipenem and a positive EDTA-disc synergy test was studied for carbapenemase production. MICs were determined with standard procedures as well as using a higher inoculum. Isoelectric focusing of cell extracts was used for detection of beta-lactamases. PCR assays with primers specific for the bla(VIM) gene and the conserved segments of class 1 integrons and sequence analyses were carried out to identify the gene and to map the metallo-beta-lactamase encoding integron. Transferability of the gene was assessed with conjugation experiments using the filter mating technique. To identify the location of the bla(VIM-1) gene, Southern hybridization was carried out in genomic DNA using an internal fragment of the bla(VIM-1) gene as a probe, amplified by PCR. The isolate was resistant to extended-spectrum beta-lactams. The MICs of carbapenems were below the resistance breakpoints but rose above resistance breakpoints when an inoculum of 10(8) cfu/mL was used. Isoelectric focusing detected a beta-lactamase with a pI of 6.1, which exhibited imipenem-hydrolysing activity in a microbiological assay. Ceftazidime and imipenem resistance were not transferable by conjugation. PCR assays identified the bla(VIM-1) gene in the variable region of a class 1 integron which also carried the aac(6')-IIc gene. The bla(VIM-1) probe hybridized with an approximately 130 kb fragment of genomic DNA, suggesting a chromosomal location of the gene. We describe a novel class 1 integron containing bla(VIM-1) and aac(6')-IIc genes in an E. cloacae clinical isolate.

Relationship between ceftriaxone use and resistance to third-generation cephalosporins among clinical strains of Enterobacter cloacae.

To investigate the potential correlation between the use of extended-spectrum cephalosporins (ESCs) and resistance to this antibiotic class among clinical isolates of Enterobacter cloacae in a university-affiliated hospital. Data on antimicrobial resistance and antimicrobial use concerning E. cloacae and ESCs were collected over a 4 year period. Various statistical tools were used to explore the potential relationship. From 1999 to 2002, the proportion of E. cloacae isolates resistant to ESCs increased from 24.3% to 29.6%. (P=0.04), and the quantity of ESCs prescribed and given did not change. Within the subclass constituted by first-line ESCs, the proportion of ceftriaxone increased from 64.3% to 77.6% and the proportion of cefotaxime decreased accordingly, from 35.7% to 22.4%. Statistical analyses showed that E. cloacae resistance to ESCs correlated with ceftriaxone use regardless of the other ESCs. For every defined daily dose of ceftriaxone per 1000 patient days used in our hospital, resistance of E. cloacae isolates to ESCs increased by 1.36%. This study demonstrates a specific correlation between ceftriaxone use and the development of resistance in E. cloacae clinical isolates. The high biliary elimination of ceftriaxone compared with other ESCs may be responsible for a greater impact of this antibiotic on the digestive flora.

Diversity of chromosomal AmpC β-lactamases from Enterobacter cloacae isolates in a Portuguese hospital

Six clinical isolates of Enterobacter cloacae isolated in a Portuguese hospital, between April 1999 and November 2000, demonstrated resistance to almost all broad-spectrum cephalosporins, except to cefepime. These isolates were susceptible to quinolones and to aminoglycosides. Isoelectric focusing demonstrated production of β-lactamases with p Is>8.0 and by all six isolates, exhibiting a cephalosporinase phenotype. The results of pulsed field gel electrophoresis revealed that these isolates were genetically unrelated. The amino acid sequence of six AmpC β-lactamases (Eclo1FF, Eclo6FF, Eclo9FF, Eclo10FF, Eclo11FF and Eclo15FF) shared 97–99% homology with the chromosomal AmpC β-lactamase from E. cloacae P99 and 86–87% homology with those of two plasmid-mediated AmpC β-lactamases, MIR-1 and ACT-1. This is the first report of chromosomal AmpC β-lactamase production by E. cloacae isolates in a Portuguese hospital.

KMP048 Five percent of Enterobacter cloacae strains produce Extended spectrum β-lactamases (ESBLs) in Germany

We examined the effect of applying the updated 2010 Clinical and Laboratory Standards Institute (CLSI) susceptibility breakpoints for extended-spectrum cephalosporins (ESCs) to detect extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae. In total, 202 ESBL-producing, plasmidic AmpC- and carbapenemase-negative isolates derived from separate patients were collected from three Greek hospitals during 2007–2011, including 150 Escherichia coli, 43 Klebsiella pneumoniae and 9 Enterobacter cloacae clinical isolates. ESBLs were detected using the ESBL CLSI confirmatory test and PCR assays. Sequencing analysis showed that 91 (45.0%) of the ESBL-producers carried the blaCTX-M-3 gene, 66 (32.7%) carried the blaCTX-M-15 gene and the remaining 45 (22.3%) carried the blaSHV-5 gene. Minimum inhibitory concentrations for cefotaxime, ceftazidime and cefepime were determined by the agar dilution method. Based on the new CLSI breakpoints, 13 (6.4%) of the ESBL-producers were susceptible to cefotaxime, 90 (44.6%) to ceftazidime and 112 (55.4%) to cefepime; as many as 145 (71.8%) were susceptible to at least one ESC. Among the 150 E. coli, 12 (8.0%), 87 (58.0%) and 79 (52.7%) were susceptible to cefotaxime, ceftazidime and cefepime, respectively, whilst among the 43 K. pneumoniae, 1 (2.3%), 3 (7.0%) and 25 (58.1%) were susceptible to the above ESCs, respectively. None of the nine E. cloacae were susceptible to cefotaxime and ceftazidime, but all except one were susceptible to cefepime. By implementation of the new 2010 CLSI breakpoints, a considerable proportion of ESBL-possessing Enterobacteriaceae would be reported as susceptible, mostly to ceftazidime and cefepime, leading to possible infection control and therapeutic implications.

Hospital epidemiology and antibiotic resistance in clinical isolates of Enterobacter cloacae

In thIe latter years Enterobacter cloacae has been counted as a very important occasional pathogen, above all in causing hospital infections.The micro-organism’s interesting increases because of its high resistance in vitro to many antimicrobial agents. For this reason it would be possible to find out others resistance mutant phenotypes. The results (2000-2001 and the first six months of 2002), carried out 341 isolated samples from hospital patients, show that the percentage of these E. cloacae is little more than 1%, increasing prevalence of patients belonging to General Surgery and Intensive Care Unit. The susceptible tests suggest (without important differences during the whole year) that most of the clinical isolates have moderate or high level resistance to various antimicrobial agents (penicillins, cephalosporins, aminoglycosides, quinolones, monobactam) and almost all were susceptible to amikacin, carbapenems (imipenem) and trimethoprim/sulfmamethoxazolo. Always regarding the resistance of these pathogens, it was possible to find out some phenotypes, and one of these, among 172 isolates belonging to Intensive Care Unit, was a particular multiresistance phenotype. The VITEK ESßL detection test and the conventional double disk synergy test for detection of ESßL find out some phenotypes showing expression of stably derepressed beta-lactamase.

Increase in MICs of ciprofloxacin in vivo in two closely related clinical isolates of Enterobacter cloacae.

The mechanisms of fluoroquinolone resistance in two isolates of Enterobacter cloacae, Ecl#1 and Ecl#2, from the same patient and with identical pulsed-field gel electrophoresis patterns, have been analysed. MICs of ciprofloxacin were 0.25 and 1 mg/L for Ecl#1 and Ecl#2, respectively. Ecl#2 was also more resistant to chloramphenicol and organic solvents. The quinolone resistance determining regions of gyrA/B and parC/E, and the marORA and acrB genes, were sequenced. Expression of marR, acrB, soxS, robA, ramA and fis was analysed by northern blotting. The activity of a 90 bp E. cloacae mar promoter fragment was examined with the reporter plasmid pIGJ-1mar. Sequencing the gyrAB and parCE genes revealed a single amino acid substitution in GyrA (corresponding to position 83 in GyrA of Escherichia coli) in Ecl#1 and Ecl#2 (Phe83) compared with reference strain E. cloacae DSMZ 3264 (Thr83). Ecl#2 accumulated significantly less norfloxacin and displayed higher levels of expression of marR and acrB than Exl#1, indicative of greater fluoroquinolone efflux activity. Sequencing gyrB, parC/E and marORA, and northern blotting of robA, ramA and fis, did not reveal any further differences between the two strains. No homologue of soxRS was detected in E. cloacae. Expression of GFP from pIGJ1-mar in Ecl#2 was higher than in Ecl#1. In these two closely related clinical isolates of E. cloacae, a target mutation in GyrA (Ecl#1 and Ecl#2) and increased fluoroquinolone efflux by AcrAB (Ecl#2) contribute to the resistance phenotypes, corroborating findings in vitro and in vivo about the sequential development of fluoroquinolone resistance.

Retro-nitroreductase, a putative evolutionary precursor to Enterobacter cloacae strain 96-3 nitroreductase.

Enterobacter cloacae strain 96-3 nitroreductase (NR) is a homodimeric flavoenzyme that catalyzes the pyridine nucleotide-dependent four-electron reduction of a variety of nitroaromatic compounds, including the explosives TNT (2,4,6-trinitrotoluene), RDX (1,3,5-trinitro-1,3,5-triazine), tetryl (2,4,6-trinitrophenyl-N-methylnitramine), and pentryl (2,4,6-trinitrophenyl-N-nitroaminoethylnitrate). The enzyme was initially characterized by Bryant et al. from a strain of Enterobacter that had been isolated from a weapons dump in La Jolla, CA. The enzyme displays a catalytic efficiency for nitroreduction at least 10-fold higher than that of several highly homologous bacterial nitroreductases and has long been thought to have evolved to be a more efficient nitroreductase due to the high nitroaromatic compound concentrations in its environment. We report the cloning and biochemical characterization of a nitroreductase gene from a clinical isolate of Enterobacter cloacae, a strain that presumably had not encountered high concentrations of nitroaromatics. The new enzyme, which we term retro-nitroreductase, had an amino acid sequence 96.7% identical to NR, and most differences are relatively conservative. The catalytic efficiency of the new enzyme is twofold less than that of NR for the oxidation of NADH and is not significantly different from the value observed for NR for the reduction of dinitrobenzyl alcohol. We conclude that NR has not significantly evolved to be a more efficient nitroreductase as a result of its environment, and the relatively high catalytic activity of the enzyme is a general property of Enterobacter cloacae nitroreductases.