Clinical Isolates Of Enterobacter Cloacae Research Articles

Quantitative Analysis of Swertiamarin Content from Fagraea fragrans Leaf Extract using HPLC Technique and its Correlation to Antibacterial Activity

The rise in drug resistance poses escalating challenges for antibacterial medications, leading to an urgent demand for the exploration and innovation of new antibacterial drugs. Fagraea fragrans Roxb., belonging to the Gentianaceae family, is one of the common herbal medicines which can be found abundantly in Southeast Asia. The secoiridoid glucoside swertiamarin, one of the major compounds in F. fragrans leaf, exhibits antimicrobial effects. To guarantee the medicinal effectiveness of F. fragrans leaves, it is essential to identify a standardized analytical method for quantifying the active compound. In this study, the optimized HPLC method following ICH guideline was validated for the quantitative analysis of swertiamarin content in F. fragrans leaf in terms of linearity (y = 5733.5x - 369.1; R2 = 0.9999), accuracy (93.57-96.39% recovery), precision (0.91% RSD for repeatability precision; 1.19% RSD for intermediate precision), limit of detection (0.73 µg/mL), limit of quantitation (2.23 µg/mL), specificity (peak purity index = 0.999995) and robustness (% RSD <1).The maximum wavelength of swertiamarin was found to be at 238 nm. The amount of swertiamarin content in F. fragrans leaf extract conducted from the validated HPLC method was found to be 0.0259 ± 0.0005 g/100 g crude drug. The leaf extract exhibited antimicrobial activity against clinical isolates of Enterobacter cloacae, Klebsiella pneumonia, and Escherichia coli at 0.125 mg/mL, and Pseudomonas aeruginosa at 0.5, showing minimum inhibitory concentration (MIC) values. Whereas swertiamarin exhibited even lower MIC values. The developed HPLC analysis effectively determines swertiamarin content as a chemical marker to ensure the antimicrobial potential of F. fragrans leaves.

The first report of the mobile colistin resistance gene, mcr-10.1, in Kenya and a novel mutation in the phoQ gene (S244T) in a colistin-resistant Enterobacter cloacae clinical isolate.

This study describes the identification of the mcr-10.1 gene in a clinical isolate of an ST1 Enterobacter cloacae isolate cultured in 2015 in Kenya. The isolate was multidrug resistant, phenotypically non-susceptible to various antibiotics, including colistin. Whole genome sequence analyses indicated carriage of chromosomally encoded antimicrobial resistance genes and the colistin-resistant gene mcr-10.1 located on a 72-kb plasmid designated pECC011b with an IncFIA(HI1) replicon directly adjacent to tyrosine recombinase gene, xerC, and downstream of an ISKPn26 insertion sequence. Studies have shown that expression of mcr-10.1 may not be sufficient to confer colistin resistance, but a novel non-synonymous mutation (S244T) was identified in the phoQ gene known to influence colistin resistance within lipid modification pathways, which could have complemented the mcr-10.1 resistance mechanism. In silico analysis of the mutant phoQ protein shows the location of the mutation to be at the Histidine kinases, Adenyl cyclases, Methyl-accepting proteins and Phosphatases (HAMP) region, which plays a crucial role in the protein's activity. This study and our previous report of mcr-8 in Klebsiella pneumoniae indicate the presence of mobile mcr genes in the Enterobacterales order of bacteria in Kenya. The study points to the importance of regulation of colistin in the animal industry and enhancing surveillance in both human and animal health to curb the spread of mcr genes and accurately assess the risks posed by these mobile genetic elements in both sectors.IMPORTANCEThis paper reports the detection of new colistin resistance mechanisms in Kenya in a clinical isolate of Enterobacter cloacae in a patient with a healthcare-associated infection. The plasmid-mediated resistance gene, mcr-10.1, and a novel amino acid mutation S244T in the phoQ gene, located in a region of the protein involved in membrane cationic stability contributing to colistin resistance, were detected. Colistin is a critical last-line drug for multidrug-resistant (MDR) gram-negative human infections and is used for treatment and growth promotion in the animal industry. The emergence of the resistance mechanisms points to the potential overuse of colistin in the animal sector in Kenya, which enhances resistance, threatens the utility of colistin, and limits treatment options for MDR infections. This study highlights the need to enhance surveillance of colistin resistance across sectors and strengthen One Health policies that ensure antimicrobial stewardship and implementation of strategies to mitigate the spread of antibiotic resistance.

Antibacterial Activities and Phytochemical Composition of Sunflower (Tithonia diversifolia) on Clinical Isolates of Enterobacter cloacae

Antibacterial Activities and Phytochemical Composition of Sunflower (Tithonia diversifolia) on Clinical Isolates of Enterobacter cloacae

Impact of CLSI Break Point Changes Over the Past Decade on Antimicrobial Susceptibility in Gram-Negative Bacteria

Background: Over the past decade, the CLSI has updated susceptibility break points for several antimicrobial agents. The purpose of this study was to evaluate the impact of these changes against gram-negative bacteria at our academic medical center. Methods: In this retrospective, IRB-approved study, we collected consecutive, nonduplicate clinical isolates of Enterobacter cloacae, Escherichia coli, Klebsiella aerogenes, K. oxytoca, K. pneumoniae, and Pseudomonas aeruginosa for the past decade (2010–2019) at our academic medical center and 3 adult ICUs. Susceptibility testing was performed using the BD Phoenix automated system. For these isolates, susceptibilities for 7 β-lactams (aztreonam, ceftriaxone, ceftazidime, cefepime, piperacillin/tazobactam, ertapenem, and meropenem) and 2 fluoroquinolones (levofloxacin, ciprofloxacin) were calculated based upon CLSI break points in 2010 and current CLSI break points in 2020. Any change >5% in susceptibility was deemed significant for this analysis. Results: In 17.5% of Enterobacteriales isolates tested, at least 1 antimicrobial demonstrated significant decline. Ertapenem was the most commonly affected antimicrobial (45% of the isolates) followed by ceftriaxone (35%) and cefepime (25%). Susceptibilities of aztreonam, ceftazidime, and meropenem were not affected for any of the Enterobacteriales. The most common organism demonstrating a significant impact on change in susceptibility among the Enterobacteriales was E. cloacae (41.7% of the time) followed by E. aerogenes (20.8%), K. oxytoca (12.5%), K. pneumoniae (8.3%) and E. coli (4.2%). Most of the impact was observed hospital-wide (33.3%), followed closely by the MICU (28.6%), the NSICU (23.8%) and the CVICU (14.3%). For P. aeruginosa, the impact of the antimicrobial break-point changes on susceptibility was more pronounced than the Enterobacteriales. Overall, 93.8% of the time there was a significant decline in antimicrobial susceptibility. Each antimicrobial (ciprofloxacin, levofloxacin, meropenem, and piperacillin/tazobactam) demonstrated a significant decline in susceptibility hospital-wide and in each ICU except for the susceptibility of meropenem in the NSICU. Conclusions: Changes in break points had a significant impact on the susceptibility of all antimicrobials for P. aeruginosa at our institution, both hospital-wide and in the adult ICUs. Although the impact was less for the Enterobacteriales, ertapenem, ceftriaxone, and cefepime demonstrated significant susceptibility changes, especially with E. cloacae. Understanding and evaluating the impact of the break-point changes may lead to changes in empiric therapy in other institutions.Funding: NoDisclosures: None

Green tea extract-mediated augmentation of imipenem antibacterial activity against Enterobacter cloacae clinical isolates

The emergence of pathogenic bacteria with β-lactam antibiotics-resistant profile has threatened the continued use of such antibiotics in the future. This research was conducted to investigate the antimicrobial activity of green tea ethanol extract (GTE) and its ability to improve the antibacterial action of several β-lactam antibiotics against Enterobacter cloacae clinical isolates. The simplicia of green tea was extracted by sonication for 30 minutes using 50% ethanol solvent, and the total phenolic content of the GTE was subsequently determined. Next, the GTE used in testing against two clinical isolates of E. cloacae was obtained from the Pathology Laboratory of Wahidin Sudiro Husodo Hospital in Makassar. The sensitivity of bacteria to GTE was confirmed using the agar diffusion method, the Vitek® rapid method, and the double-disk synergistic test. Antibacterial activity of antibiotics, GTE, and combination of antibiotics with GTE were then tested against clinical isolates of E. cloacae using the checkerboard microdilution assay. The results showed that GTE contained 51.64 ± 0.21 % measured as gallic acid equivalent and 37.95 + 5.17 % Epigallocatechin gallate (EGCG). The confirmatory test results indicated that one clinical isolate of E. cloacae (code 13/04) was resistant to amoxicillin-clavulanate but did not produce an extended-spectrum β-lactamase (ESBL). Another clinical E. cloacae isolate (code 275B/06) was indicated to produce ESBL and demonstrated to yield resistance to amoxicillin-clavulanate and cefotaxime. The minimum inhibitory concentration of GTE against the two clinical isolates of E. cloacae was >8000 ppm (8 mg/ml). In conclusion, GTE could not increase the antibacterial activity of amoxicillin and cefotaxime, but it was sufficient to improve the activity of imipenem against the tested isolates of E. cloacae .

Identification of AGI1-A, a variant of Acinetobacter genomic island 1 (AGI1), in a French clinical isolate belonging to the Enterobacter cloacae complex.

To characterize the structure of a variant of Acinetobacter genomic island 1 (AGI1) in the Enterobacter cloacae clinical isolate EclC2185 responsible for an 8 year outbreak in Dijon University Hospital. The genome was sequenced (Miseq) and de novo assembled (Velvet). PCR and Sanger sequencing were performed to determine the sequence of the genomic island. The new variant of AGI1 named AGI1-A (41.7 kb), located at the 3' end of the chromosomal trmE, was detected in an E. cloacae complex isolate identified as Enterobacter hormaechei subsp. oharae. The backbone of AGI1-A lacked A008, part of A025, A026 and the resolvase gene. Its MDR region (19.7 kb) contained two integrons followed by a hybrid transposon Tn502/Tn5053. The former integron was a typical In4-type class 1 integron carrying aadA1 and the latter integron carrying dfrA1 did not belong to a class described to date. The nucleotide sequence intI-dfrA1 was surrounded by a 78 bp imperfect repeat sequence in inverse orientation. An external circular form of AGI1-A was detected, suggesting potential mobility. AGI1-A was also detected in 15 isolates from the outbreak selected at random. They belonged to the ST114 high-risk clone. AGI1-A, a variant of AGI1 described in Acinetobacter baumannii, is the first genomic resistance island belonging to the Salmonella genomic island/Proteus genomic island/Acinetobacter genomic island family detected in E. cloacae. One might, therefore, fear for interspecies dissemination of genomic islands from this family.

Effect of increasing meropenem MIC on the killing activity of meropenem in combination with amikacin or polymyxin B against MBL- and KPC-producing Enterobacter cloacae

Carbapenem resistant Enterobacteriaceae (CRE) are a growing threat worldwide. Infections caused by these organisms have exhibited high rates of mortality (50%) for which there is no standard of care and a dearth of clinical trials. Most in vitro data on CRE focus on Klebsiella pneumoniae, but it is known that effective therapy may depend on species or even strain. To address this, meropenem, amikacin, and polymyxin B alone and in combination were evaluated by time kill against four carbapenem-producing Enterobacter cloacae clinical isolates representing a range of meropenem nonsusceptibility (2–32 mg/L) and resistance mechanisms (KPC 2 and/or VIM 1). As meropenem minimum inhibitory concentration (MIC) increased, bactericidal activity and synergy were maintained for 48 hours in isolates exposed to meropenem and amikacin, but synergy and bactericidal activity were not maintained in all isolates exposed to meropenem and polymyxin B.

Characterization of a Novel Lytic Bacteriophage EC14 that Infects Enterobacter cloacae Clinical Isolates

Characterization of a Novel Lytic Bacteriophage EC14 that Infects Enterobacter cloacae Clinical Isolates

A hemin auxotrophic Enterobacter cloacae clinical isolate with increased resistance to carbapenems and aminoglycosides.

Small-colony variants (SCVs) were obtained from an Enterobacter cloacae clinical isolate in Okinawa, Japan. One variant showed auxotrophy for hemin with a deletion of 20 365 nucleotides, dosC-ydiK-mmuP-mmuM-tauA-tauB-tauC-tauD-hemB-yaiT-yaiV-ampH-yddQ-sbmA-yaiW-yaiY-yaiZ, including hemB, and was more resistant to aminoglycosides and carbapenems, but more susceptible to aztreonam, than the parent strain.

What does the susceptible-dose dependent interpretive category for cefepime in ESBL-producing bacteria?

In this study, we investigated the frequency of isolates included in the susceptible-dose dependent (SDD) category, according to the Clinical and Laboratory Standards Institute guidelines, carrying blaTEM, blaSHV and blaCTX genes among 92 Klebsiella pneumoniae and 80 Enterobacter cloacae clinical isolates. The presence of one or more extended-spectrum β-lactamases (ESBLs) genes was observed in 64% K. pneumoniae and 69% E. cloacae isolates. Nineteen isolates were included in SDD interpretive category criteria, of which 15 carried ESBL genes (seven K. pneumoniae and eight E. cloacae). Considering the high proportion of ESBL gene-containing isolates included in the SDD category (79%), we recommend that physicians exercise caution in the use of cefepime for treatment of infections caused by these isolates, reducing possible therapeutic failure, particularly in cases of ESBL-producing bacterial strains.

Medicinal Plants Used by a Mbyá-Guarani Tribe against Infections: Activity on KPC-Producing Isolates and Biofilm-Forming Bacteria

The traditional use of medicinal plants for treatment of infectious diseases by an indigenous Mbyá-Guarani tribe from South Brazil was assessed by evaluating the antibiotic and antibiofilm activities against relevant bacterial pathogens. Aqueous extracts from 10 medicinal plants were prepared according to indigenous Mbyá-Guarani traditional uses. To evaluate antibiotic (OD600) and antibiofilm (crystal violet method) activities, Pseudomonas aeruginosa ATCC 27853, Staphylococcus epidermidis ATCC 35984 and seven multi-drug resistant Klebsiella pneumoniae carbapenemase (KPC)-producing bacterial clinical isolates were challenged with the extracts. Furthermore, the susceptibility profile of KPC-producing bacteria and the ability of these isolates to form biofilm were evaluated. The plants Campomanesia xanthocarpa, Maytenus ilicifolia, Bidens pilosa and Verbena sp. showed the best activity against bacterial growth and biofilm formation. The majority of KPC-producing isolates, which showed strong ability to form biofilm and a multidrug resistance profile, was inhibited by more than 50% by some extracts. The Enterobacter cloacae (KPC 05) clinical isolate was the only one resistant to all extracts. This study confirms the importance of indigenous traditional medicinal knowledge and describes for the first time the ability of these plants to inhibit biofilm formation and/or bacterial growth of multi-drug resistant KPC-producing isolates.

Emergence of TEM, SHV, and CTX-M-Extended Spectrum β-Lactamases and Class 1 Integron AmongEnterobacter cloacaeIsolates Collected from Hospitals of Tehran and Qazvin, Iran

Extended-spectrum β-lactamase (ESBL) production is an important resistance mechanism among clinical isolates of Enterobacter cloacae. TEM, SHV, and CTX-M are the most common ESBL genotypes among Enterobacter spp. The main aims of this study were to determine the antimicrobial susceptibility pattern and to detect ESBL-encoding genes as well as intI1 genes. One hundred twenty isolates of E. cloacae were collected from hospitals of Tehran and Qazvin, Iran. The isolates were identified by standard laboratory methods and API 20E strips. ESBL screening was performed by the combined disk method. PCR and sequencing were conducted for detection of ESBL-encoding genes as well as intI1 genes. Clonal relatedness of ESBL-producing isolates was assessed by the enterobacterial repetitive intergenic consensus (ERIC)-PCR method. Of 120 isolates, 57 (47.5%) were characterized as multidrug resistant among those 48 (84.2%) isolates carried class 1 integron. Fifty-three (44.2%) isolates were found to be ESBL producers, in which bla(CTX-M-15) (60.4%) was the most common gene followed by bla(TEM-1) (32.1%), bla(TEM-169) (13.2%), and bla(SHV-12) (7.5%) either alone or in combination. Forty-four of the 53 (83.01%) ESBL-producing isolates were genetically unrelated. For the first time, this study describes the emergence of TEM-169, SHV-12, and CTX-M-15 ESBL genotypes in E. cloacae isolates in Iran.

First Report of a Nonmetallocarbapenemase Class A Carbapenemase in an Enterobacter cloacae Isolate from Colombia

Carbapenemases are a growing concern worldwide since they threaten the efficacy of carbapenems, the most potent β-lactam antibiotics. Nonmetallocarbapenemase class A (NMC-A) was identified in 1990 from a clinical isolate of Enterobacter cloacae, and since its first report in France ([1][1]), this

Susceptibility patterns to extended-spectrum cephalosporins among Enterobacteriaceae harbouring extended-spectrum β-lactamases using the updated Clinical and Laboratory Standards Institute interpretive criteria

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.

Genetic characterization of tigecycline resistance in clinical isolates of Enterobacter cloacae and Enterobacter aerogenes

The intrinsically encoded ramA gene has been linked to tigecycline resistance through the up-regulation of efflux pump AcrAB in Enterobacter cloacae. The molecular basis for increased ramA expression in E. cloacae and Enterobacter aerogenes, as well as the role of AraC regulator rarA, has not yet been shown. To ascertain the intrinsic molecular mechanism(s) involved in tigecycline resistance in Enterobacter spp., we analysed the expression levels of ramA and rarA and corresponding efflux pump genes acrAB and oqxAB in Enterobacter spp. clinical isolates. The expression levels of ramA, rarA, oqxA and acrA were tested by quantitative real-time RT-PCR. The ramR open reading frames of the ramA-overexpressing strains were sequenced; strains harbouring mutations were transformed with wild-type ramR to study altered ramA expression and tigecycline susceptibility. Tigecycline resistance was mediated primarily by increased ramA expression in E. cloacae and E. aerogenes. Only the ramA-overexpressing E. cloacae isolates showed increased rarA and oqxA expression. Upon complementation with wild-type ramR, all Enterobacter spp. containing ramR mutations exhibited decreased ramA and acrA expression and increased tigecycline susceptibility. Exceptions were one E. cloacae strain and one E. aerogenes strain, where a decrease in ramA levels was not accompanied by lower acrA expression. Increased ramA expression due to ramR deregulation is the primary mediator of tigecycline resistance in clinical isolates of E. cloacae and E. aerogenes. However, some ramA-overexpressing isolates do not show changes in ramR, suggesting alternate pathways of ramA regulation; the rarA regulator and the oqxAB efflux pump may also play a role in tigecycline resistance in E. cloacae.

Emergence of SHV-12 extended spectrum beta-lactamase among clinical isolates of Enterobacter cloacae in Tunisia

A collection of seven multidrug-resistant clinical isolates of Enterobacter cloacae with reduced susceptibility to ceftazidime and cefepime recovered from 2009 to 2010 at the University Hospital of Mahdia, Tunisia, was analysed. PCR analysis and sequencing demonstrated that all study isolates harbored SHV-12 β-lactamase that was transferred by conjugation. Characterization of the regions surrounding the blaSHV-12 showed that this gene was flanked by two IS26 elements. pulsed-field gel electrophoresis (PFGE) revealed differents profiles indicating that the study isolates were not clonally related. Diffusion of E. cloacae producing SHV-12 ESBL in our hospital is the consequence of disseminations of plasmids harboring the SHV-12 gene.

Emergence of carbapenem-resistant Enterobacter cloacae carrying IMP-26 metallo-β-lactamase in class 1 integron and TEM-104 extended-spectrum β- Lactamase in conjugative plasmid

A clinical isolate of Enterobacter cloacae exhibiting reduced susceptibility to carbapenem and a positive EDTA-disc synergy test was studied for carbapenemase production. MICs were determined with standard procedures. Isoelectric focusing of cell extracts was used for detection of β-lactamases. PCR assays with primers specific for the blaIMP-26 gene and the conserved segments of class 1 integrons and sequence analyses were carried out to identify the gene and to map the metallo-β-lactamase encoding integron. Transferability of the gene was assessed with conjugation experiments. To identify the location of the blaTEM-104 gene, conjugation experiments and plasmid electrophoresis were carried out and amplified by PCR. A carbapenem-resistant E. cloacae was found to carry IMP-26 metallo-β-lactamase in class 1 integron and TEM-104 extended-spectrum β- lactamase in conjugative plasmid. Existence of multiple resistant genes on plasmids and integrons may pose a serious epidemiological, clinical, and public health threat. Key words: Metallo-β-lactamases, carbapenemases, Enterobacter cloacae

Detection and Characterization of VIM-31, a New Variant of VIM-2 with Tyr224His and His252Arg Mutations, in a Clinical Isolate of Enterobacter cloacae

We report the first description of the metallo-β-lactamase VIM-31, a new variant of VIM-2 with Tyr224His and His252Arg mutations, in Enterobacter cloacae 11236, which was isolated from blood specimens of a patient with colonic adenocarcinoma in Belgium. bla(VIM-31) was found on a class 1 integron located on a self-transferable but not typeable 42-kb plasmid. Compared to values published elsewhere for VIM-2, the purified VIM-31 enzyme showed weaker catalytic efficiency against all the tested beta-lactam agents (except for ertapenem), resulting from lower k(cat) (except for ertapenem) and higher K(m) values for VIM-31.

Involvement of curli fimbriae in the biofilm formation of Enterobacter cloacae

In this study, we examined the biofilm forming ability, the mRNA expression of curli genes and the morphologies of curli fimbriae and biofilms in clinical isolates of Enterobacter cloacae. The csgBA operon was found in 11 (78.6%) of the 14 isolates. The ability of E. cloacae isolates to form biofilms was significantly correlated with the mRNA expression level of the csgA and csgD genes. The curli protein fimbriae appeared as tangled fibers and the curli-proficient strain formed mature biofilms. Our data suggest that the expression of the curli fimbriae play an important role in biofilm formation in E. cloacae.

The cyclohexane tolerance and Phe-Arg-b-naphtylamide susceptibility of multidrug-resistant Enterobacter cloacae clinical isolates, and the predominance of one PFGE clone in Hungary

The report concerns the molecular epidemiology, cyclohexane tolerance and Phe-Arg-β-naphtylamide (PAβN) susceptibility of multidrug-resistant Enterobacter cloacae isolates, with high-level fluoroquinolone resistance collected from healthcare facilities in a nationwide survey. A total of 113 multidrug-resistant E. cloacae isolates (recovered in 1997–2005) were subjected to disc diffusion tests, ERIC-PCR and XbaI PFGE. Representatives of the ERIC-types (n = 67) were tested further with cyclohexane and PAβN, using ciprofloxacin as the substrate. Forty-four per cent of the isolates were derived from the urinary tract, 19% from the bloodstream, 17% from the respiratory tract, and 15% from wound infections. Four ERIC-types (A, B, C and D) were distinguished, but 109 isolates were found to belong to a single, epidemic ERIC type: A. PFGE results suggested that the epidemic-type isolates were of monoclonal origin. Forty-two patients were involved in four outbreaks caused by the epidemic-type strains. Eighty-one cases were found to be nosocomial. At least fourfold reduction in ciprofloxacin MICs was found in the presence of PAβN in 79% of representative isolates (representing types A, C and D); an eightfold or greater reduction in ciprofloxacin MICs in the presence of PAβN (PAβN+) was found in 37% of representative isolates, representing types A and C. Eighty-five per cent of the representative isolates were found to be cyclohexane-tolerant, representing types A, C and D. This is the first report of a wide distribution of cyclohexane-tolerant or PAβN+ strains of E. cloacae. These featureindicators of adaptive mechanisms that help bacteria to survive in hospital wards may have contributed to the nationwide spread of type A strains.