Cloacae Isolates Research Articles

Bacillus subtilis and Enterobacter cloacae endophytes from healthy Theobroma cacao L. trees can systemically colonize seedlings and promote growth

Clonal genotypes resistant to fungal diseases are an important component of the cocoa production system in southeastern Bahia state (Brazil), so that technologies for faster production of stronger and healthier plantlets are highly desirable. In this study, the effects of inoculated bacterial endophytes isolated from healthy adult cacao plants on seedlings, and aspects related to inoculation methods, colonization patterns, and photosynthesis were investigated. Sequencing of 16S rRNA, hsp-60, and rpo-B genes placed the wild-type isolates within the species Enterobacter cloacae (isolates 341 and 344) and Bacillus subtilis (isolate 629). Spontaneous rifampicin-resistant (rif(R)) variants for 344 were also produced and tested. Endophytic application was either by immersion of surface sterilized seeds in bacterial suspensions or direct inoculation into soil, 20days after planting non-inoculated seeds into pots. Results from in vitro recovery of inoculated isolates showed that the wild-type endophytes and rif(R) variants systemically colonized the entire cacao seedlings in 15-20days, regardless of the inoculation method. Some endophytic treatments showed significant increases in seedlings' height, number of leaves, and dry matter. Inoculation methods affected the combined application of endophytes, which maintained the growth-promotion effects, but not in the same manner as in single applications. Interestingly, the 344-3.2 rif(R) variant showed improved performance in relation to both the wild type and another related variant. Photosynthetic rates and stomatal conductance increased significantly for some endophytic treatments, being partially associated with effects on growth and affected by the inoculation method. The results suggest that E. cloacae and B. subtilis endophytes from healthy adult plants (not transmitted by seeds) were able to promote vegetative growth on cacao seedlings. The development of products for large-scale use in seedlings/plantlets production systems was discussed.

Prevalence of Plasmid-Mediated Quinolone Resistance and Aminoglycoside Resistance Determinants among Carbapeneme Non-Susceptible Enterobacter cloacae

BackgroundSimultaneous resistance to aminoglycosides and fluoroquinolones in carbapeneme non-susceptible (CNS) isolates will inevitably create problems. The present study was performed to characterize the prevalence of the plasmid-mediated quinolone resistance determinants (QRDs) and aminoglycoside resistance determinants (ARDs) among the CNS Enterobacter cloacae (E. cloacae) isolates in a Chinese teaching hospital, and to acquire their molecular epidemiological characteristics.MethodsThe β-lactamases genes (including class A carbapenemase genes blaKPC and blaSME, metallo-β-lactamase genes (MBLs) blaIMP, blaVIM and blaNDM, and extended spectrum β-lactamases (ESBLs),blaCTX-M, blaTEM and blaSHV), QRDs (including qnrA, qnrB, qnrS and aac(6′)-Ib-cr) and ARDs (including aac(6′)-Ib, armA and rmtB) of these 35 isolates were determined by PCR and sequenced bidirectionally. The clonal relatedness was investigated by pulsed-field gel electrophoresis (PFGE).ResultsOf the 35 isolates, 9 (25.7%) harbored a carbapenemase gene; 23 (65.7%) carried ESBLs; 24 (68.6%) were QRD positive; and 27 (77.1%) were ARD positive. Among the 5 blaIMP-8 positive strains, 4 (80%) contained both ESBL and QRD genes, and all the 5 (100%) harbored ARD genes. Of the 23 ESBLs positive isolates, 6 (26.1%) were carbapenemase positive, 14 (60.9%) were QRD positive, and 18 (78.3%) were ARD positive. PFGE revealed genetic diversity among the 35 isolates, indicating that the high prevalence of CNS E. cloacae isolates was not caused by clonal dissemination.ConclusionQRD and ARD genes were highly prevalent among the CNS E. cloacae isolates. Multiple resistant genes were co-expressed in the same isolates. The CNS E. cloacae isolate co-expressing blaNDM-1, blaIMP-26, qnrA1 and qnrS1 was first reported.

Characterization of fluoroquinolone and cephalosporin resistance mechanisms in Enterobacteriaceae isolated in a Dutch teaching hospital reveals the presence of an Escherichia coli ST131 clone with a specific mutation in parE

To characterize the mechanisms of fluoroquinolone and cephalosporin resistance in Enterobacteriaceae from a Dutch teaching hospital in 2008. We sequenced gyrA, gyrB, parC and parE. The presence of plasmid-encoded genes qnrA, qnrB, qnrS, aac(6')-Ib, qepA, bla(TEM), bla(SHV,) bla(OXA), bla(CTX-M) and bla(AmpC) was studied by PCR. Escherichia coli isolates were further characterized by AFLP and multilocus sequence typing (MLST). In total, 49 E. coli, 16 Klebsiella pneumoniae and 3 Enterobacter cloacae isolates were investigated. Mutations in gyrA were found in all E. coli isolates. Forty-five (92%) E. coli isolates carried at least one point mutation in parC. Most E. coli isolates (59%) also carried mutations in parE, of which I529L was the most prevalent. I529L was unequivocally associated with E. coli sequence type (ST) 131. This single-nucleotide polymorphism (SNP) was later also found in eight out of nine ST131 strains from another collection. Twenty-nine E. coli isolates carried extended-spectrum β-lactamase (ESBL) genes, predominantly bla(CTX-M-15). In E. coli, aac(6')-Ib-cr was the predominant plasmid-mediated resistance mechanism, whereas in K. pneumoniae qnr genes were found mostly. In K. pneumoniae isolates, qnr and aac(6')-Ib-cr co-occurred with ESBL genes (n = 13; bla(CTX-M) and bla(SHV)) and/or bla(AmpC) (n = 3; bla(DHA-1)). E. coli ST131 was the predominant clone, which accumulated a high number of chromosomal mutations. The I529L SNP in parE was a signature of most, but not all, ST131 strains. In contrast to E. coli, fluoroquinolone resistance mechanisms were predominantly plasmid-encoded in K. pneumoniae.

Current Status of Antibiograms of Listeria ivanovii and Enterobacter cloacae Isolated from Ready-To-Eat Foods in Alice, South Africa

This study assessed the antimicrobial susceptibility of 51 Listeria ivanovii and 33 Enterobacter cloacae strains isolated from various ready-to-eat foods sold in Alice, South Africa. Isolates were identified using standard microbiological tests and further confirmed using API 20E and API Listeria kits. The disc diffusion technique was used to screen for antimicrobial susceptibility against 15 antimicrobials; minimum inhibitory concentration of five antibiotics was determined by the broth dilution method. All the strains of E. cloacae (100%) and 96% of L. ivanovii isolates were resistant to at least four or more of the antibiotics; nineteen antibiotypes were obtained based on the antibiotics used in the study. Antibiotype A5: AR PGR VAR ER APR was predominant in both L. ivanovii (23.5%) and E. cloacae (57.5%) isolates. Marked susceptibility of Listeriaivanovii was observed against chloramphenicol, ciprofloxacin, streptomycin and trimethoprim/sulfamethoxazole (100%) each while E. cloacae registered 100% susceptibility to ciprofloxacin only. Various percentages of susceptibility was reported to chloramphenicol and gentamicin (91%) each, nalidixic acid (97%) and streptomycin (94%). The MIC90 ranged from 0.004–7.5 µg/mL with E. cloacae being the most susceptible organism. The study demonstrated the presence of multi-resistant strains of bacteria in ready-to-eat-foods and speculates that these foods could serve as important vehicles transmitting multi-resistant bacteria to humans.

Phenotypic and Genotypic Characterizations of Campylobacter jejuni Isolated from the Broiler Meat Production Process

A set of C. jejuni isolates of different origins and flaA-genotypes obtained throughout the broiler meat production chain was tested in this study for a possible correlation of their origin, phylogenetic relationship, and phenotypic properties. Interestingly, the results showed a correlation of the origin and the phylogenetic relationship between the C. jejuni isolates and their ability to form biofilm, but not in their ability to survive at -18, 5, 20, and 48°C. Two strains, a broiler cloacae isolate and a broiler fillet isolate, were unable to develop biofilm, while most of the C. jejuni isolates originating from meat and surfaces of the slaughterhouse readily formed biofilms after both 24, 48, and 72h. Interestingly, these biofilm-forming strains were closely related. Furthermore, two strains that were isolated after disinfection developed significantly more biofilms after 24h of incubation than the remaining strains. A comparative genomic analysis using DNA microarrays showed that the gene contents of strains that efficiently formed biofilms were different from those that did not. The study suggests that biofilm formation might be a lineage specific property, allowing C. jejuni to both survive environmental stress at the slaughterhouse and to attach to the surface of meat.

Emergence of VIM-4- and SHV-12-producing Enterobacter cloacae in a neonatal intensive care unit

In order to reveal colonization with multidrug-resistant bacteria early, routine screening is done on samples of all patients of the neonatal intensive care units at Semmelweis University, Hungary. Due to the extended-spectrum β-lactamase (ESBL) screening examinations, emergence of multidrug-resistant Enterobacter cloacae isolates was found with suspicion of clonal transmission, therefore active microbiological surveillance was initiated. The aim of our study was to characterize 60 E. cloacae isolates recovered in a 7-month period in 2010. MIC values of antibiotics were determined and ESBL and carbapenemase production was tested. Metallo-β-lactamase (MBL) genes, ESBL genes, and class-1 integrons were characterized, and the possible clonal relationship between isolates was investigated. The isolates showed increased MIC values for carbapenems and cephalosporins. All 60 E. cloacae strains recovered from 16 neonates proved to be VIM-4 MBL producers. Fifty-three strains were SHV-12 ESBL producers also. In all cases, the blaVIM-4 gene was a part of class-1 integron, In238a. XbaI-macrorestriction analysis by pulsed-field gel electrophoresis (PFGE) revealed identical patterns for the isolates. Our study supports the importance of active microbiological surveillance as well as molecular epidemiology at the NICUs as a part of infection control.

Prevalences of the Enterobacter cloacae complex and its phylogenetic derivatives in the nosocomial environment

Despite the importance of the Enterobacter cloacae complex as a nosocomial pathogen, little is known about the relative contribution of its single species and genotypes to its pathogenicity. We studied here the relationship between phylogenetically related strains and clinical infection sites. A total of 196 prospectively collected isolates of the E. cloacae complex were included in the study, comprising 67 blood culture isolates. Isolates were analyzed for clonality by pulsed-field gel electrophoresis (PFGE) and genetically assigned to their genotypes by partial hsp60 sequencing. Most isolates belonged in phylogenetic clade 1, with E. hormaechei being the most prevalent species. Notably, some genotypes and species, indeed, showed predilections to certain clinical specimens, whereas clinical outbreaks with members of the E. cloacae complex were rare. Our study stresses the need to identify alleged E. cloacae isolates on the subspecies level in clinical routine in order to obtain more insight into their distinct infectious potentials.

Characterization of drug-resistance and molecular epidemiology of Enterobacter cloacae among patients with clinical infections in a teaching hospital, China

A total of 114 non-repetitive Enterobacter cloacae isolates were collected from patients with clinical infections and tested for susceptibility to 15 antimicrobials. Enterobacterrepetitive intergenic consensus (ERIC) analysis was used to determine the genetic relatedness between isolates with enzymes. Touchdown polymerase chain reaction (PCR) and single PCR were used to detecting the genes encoding beta-lactamase (single PCR rechecked) and integron, aac-(6’)-Ib, respectively. Transferability of drug-resistant was studied by conjugation experiments. 41.2% strains possessed one or two AmpC β-lactamase genes, and 29.8% isolates harbored one or more broad-spectrum beta-lactamase genes. While aac(6’)-Ib was detected in six isolates. 26 isolates were detected with class1 integron and four different gene cassettes were found in these strains. None of isolate was carbapenemase producer. Patients with these bacterial infections suffered a failure of different cephalosporin treatment in clinical. The presence of producing ESBLs, AmpC β-lactamase was correlated with the reduced susceptibility to carbapenems among E. cloacae. In addition, these genes could transmit between different species by plasmids and enhanced by insertion sequences. ERIC revealed 11 unrelated profiles, which indicated that there was scattered transmission among E. cloacae with enzymes in the hospital environment, even acrossed more than three years. Key words: Enterobacter repetitive intergenic consensus, integron, plasmid, resistance.

Role of IncHI2 plasmids harbouring blaVIM-1, blaCTX-M-9, aac(6′)-Ib and qnrA genes in the spread of multiresistant Enterobacter cloacae and Klebsiella pneumoniae strains in different units at Hospital Vall d’Hebron, Barcelona, Spain

Seven Enterobacter cloacae isolates and seven Klebsiella pneumoniae isolates harbouring a phenotype compatible with the production of a metallo-β-lactamase were recovered between 2009 and 2011 in three Intensive Care Units of Hospital Vall d’Hebron (Barcelona, Spain). The presence of blaVIM, blaIMP, blaNDM, blaCTX-M, aac(6′)-Ib, qnrA, qnrB and qnrS genes was screened by polymerase chain reaction (PCR) and sequencing. Clonal relatedness of the isolates was assessed by pulsed-field gel electrophoresis (PFGE) and, in the case of K. pneumoniae isolates, by multilocus sequence typing (MLST). PCR-based replicon typing, Southern hybridisation, plasmid double-locus sequence typing and MOB relaxase classification methods were used to identify and characterise the plasmids carrying the resistance genes. Transferability of the identified plasmids was tested by conjugation assays. All 14 isolates were found to carry blaVIM-1, blaCTX-M-9 (except one isolate), aac(6′)-Ib and qnrA genes. Clonality assessment demonstrated that E. cloacae isolates were distributed in three clonal clusters, whereas all of the K. pneumoniae isolates belonged to one unique clone, identified as sequence type ST252. All studied isolates harboured a large conjugative IncHI2 MOBH11 plasmid carrying all of the detected resistance genes. Plasmid DNA analysis showed that all of them belonged to the ST1 IncHI2 plasmid cluster and shared the same relaxase partial sequence. In conclusion, the present study describes the dissemination within a hospital of multiresistant E. cloacae and K. pneumoniae isolates producing VIM-1. A complex clonal epidemiology of the E. cloacae isolates was observed and plasmid DNA analysis strongly supports horizontal exchanges of the same IncHI2 plasmid between different strains and species.

Epidemiological study of goats infected with diarrhia caused by some bacteria of enterobacteriaceae

This study was conducted to isolate of Enterobacteriace family and designed to determine the prevalence of this bacteria in goats in 6 residance area in Baghdad Governorate ( Yousifia, Mahmodia, Shoulla, Sader city, Khadmia and farm of College of Veterinary Medicine ). 300 fecal samples were collacted from diarrheaic goats aged between (1 day to 2 year) from male and female for many month ,Diagnostic study depending upon the morphological and cultural properties of the isolates on some selective media in addItion to different biochemical tests and API-20E and serological tests isolates of Salmonella. The results showed Isolation of 111bacterial Isolates which included 6 different species of enterobacteriace, Citrobacte braackii )48)isolates, Protues mirabilis(36) isolates, Protues vulgaris ( 4 ) isolates , Enterobacter cloacae (14)isolates , Klebsiella pneumonia isolates ( 5 ) , Salmonella enteritidis (2) isolates , Salmonella Newport(1)isolate and Providenica rettgeri(1)isolate. Results showed significant difference at p<0.05 in distribution of infection according to age of animals and months of year and didn’t showed significant difference at p<0.05 to all of bacteria isolated. In Salmonella infection the animals suffered from increase body temperature, pulse and respiratory rates, diarrhea and sever dehydration, the feces was white to green in colour containing blood , while the animals infected with other bacteria suffering from similar sings but less in severity than occurred in Salmonella.

Occurrence and characteristics of extended-spectrum-β-lactamase- and AmpC-producing clinical isolates derived from companion animals and horses

To investigate the occurrence and characteristics of extended-spectrum β-lactamase (ESBL)- and AmpC-producing Enterobacteriaceae isolates in clinical samples of companion animals and horses and compare the results with ESBL/AmpC-producing isolates described in humans. Between October 2007 and August 2009, 2700 Enterobacteriaceae derived from clinical infections in companion animals and horses were collected. Isolates displaying inhibition zones of ≤ 25 mm for ceftiofur and/or cefquinome by disc diffusion were included. ESBL/AmpC production was confirmed by combination disc tests. The presence of resistance genes was identified by microarray, PCR and sequencing, Escherichia coli genotypes by multilocus sequence typing and antimicrobial susceptibility by broth microdilution. Sixty-five isolates from dogs (n = 38), cats (n = 14), horses (n = 12) and a turtle were included. Six Enterobacteriaceae species were observed, mostly derived from urinary tract infections (n = 32). All except 10 isolates tested resistant to cefotaxime and ceftazidime by broth microdilution using clinical breakpoints. ESBL/AmpC genes observed were bla(CTX-M-1, -2, -9, -14, -15,) bla(TEM-52), bla(CMY-2) and bla(CMY-)(39). bla(CTX-M-1) was predominant (n = 17). bla(CTX-M-9) occurred in combination with qnrA1 in 3 of the 11 Enterobacter cloacae isolates. Twenty-eight different E. coli sequence types (STs) were found. E. coli carrying bla(CTX-M-1) belonged to 13 STs of which 3 were previously described in Dutch poultry and patients. This is the first study among a large collection of Dutch companion animals and horses characterizing ESBL/AmpC-producing isolates. A similarity in resistance genes and E. coli STs among these isolates and isolates from Dutch poultry and humans may suggest exchange of resistance between different reservoirs.

In vitro susceptibilities of clinical isolates of ertapenem-non-susceptible Enterobacteriaceae to cefotaxime, ceftazidime, cefepime and aztreonam

To investigate the in vitro susceptibility of ertapenem-non-susceptible Enterobacteriaceae (ENSE) isolates to cefotaxime, ceftazidime, cefepime and aztreonam. Clinical isolates of ENSE tested in this study were obtained from 10 major teaching hospitals in Taiwan during the period January 2008 to October 2010. MICs of ertapenem, cefotaxime, ceftazidime, cefepime and aztreonam were determined by the agar dilution method and were interpreted based on 2011 MIC interpretive criteria recommended by the CLSI and EUCAST. A total of 412 non-duplicate ENSE isolates (with ertapenem MIC values ≥ 0.5 mg/L) were tested. These comprised 72 isolates of Escherichia coli [28 (38.9%) were extended-spectrum β-lactamase (ESBL)-producing isolates], 167 isolates of Klebsiella pneumoniae [74 (44.3%) were ESBL-producing isolates], 115 isolates of Enterobacter cloacae, 13 isolates of Enterobacter aerogenes, 20 isolates of Citrobacter freundii and 25 isolates of Serratia marcescens. According to 2011 CLSI (EUCAST) MIC interpretive breakpoints for Enterobacteriaceae, 64% (31%) of all ENSE isolates, 45.8% (16.7%) of E. coli isolates, 53% (29.3%) of K. pneumoniae isolates and 86.1% (35.7%) of E. cloacae isolates were susceptible to cefepime. As for ESBL-producing ENSE isolates, 25% and 14.3% of E. coli isolates and 36.5% and 10.8% of K. pneumoniae isolates were susceptible to cefepime based on CLSI and EUCAST criteria, respectively. Cefepime exerts in vitro antimicrobial activity against a significant portion of clinical isolates of ENSE, although there are discrepancies between results obtained using the CLSI-2011 and EUCAST-2011 guidelines.

Molecular characterization of NDM-1 producing Enterobacteriaceae isolates in Singapore hospitals

In this study, we molecularly characterized 12 NDM-1 producing clinical Enterobacteriaceae (Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae) isolates that were part of a collection of non-carbapenem susceptible isolates obtained during a one-year period. These isolates were obtained from four local general hospitals in Singapore. Polymerase chain reaction (PCR) assays and sequencing was used to determine the presence of β-lactamase encoding genes (bla) including bla NDM-1 and plasmid-mediated quinolone and aminoglycoside resistance determinants. Conjugation experiments were performed to determine the transferability of bla NDM-1. Isolate relatedness was determined by multilocus sequence typing (MLST). The isolates were completely resistant to the second- and third-generation cephalosporins tested as well as carbapenems. Susceptibility profiling of the isolates indicated that 100% retained susceptibility to tigecycline while 11/12 (91.7%) were susceptible to colistin. The bla NDM-1 gene was encoded on plasmids that were easily transferable. None of the patients had a travel history to countries where NDM-1 has been reported. The isolates appear clonally unrelated with MLST, revealing a diversity of clonal types among the Klebsiella pneumoniae and Escherichia coli isolates. The ease of NDM-1 plasmid transmissibility may help their dissemination among the Enterobacteriaceae. Although it appears that the isolates are clonally unrelated, epidemiological links cannot be fully excluded without further research.

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.

Nosocomial outbreak of carbapenem-resistant Enterobacter cloacae highlighting the interspecies transferability of the blaOXA-48 gene in the gut flora

Nosocomial outbreak of carbapenem-resistant Enterobacter cloacae highlighting the interspecies transferability of the blaOXA-48 gene in the gut flora

Chromate-resistance genes in plasmids from antibiotic-resistant nosocomial enterobacterial isolates

The presence of chromate-resistance genes in enterobacteria was evaluated in a collection of 109 antibiotic-resistant nosocomial isolates from nine major cities in México. Results were compared with the presence of mercury-resistance genes. Susceptibility tests showed that 21% of the isolates were resistant to chromate (Cr(R)), whereas 36% were resistant to mercury (Hg(R)). Cr(R) levels were high in Klebsiella pneumoniae (61%), low in Enterobacter cloacae (12%) and Escherichia coli (4%), and null in Salmonella sp. isolates. Colony hybridization demonstrated that the majority of metal-resistant isolates hybridized with chrA gene (87% of Cr(R) isolates), encoding a CHR transporter homologue, and merA gene (74% of Hg(R) isolates), encoding MerA mercuric reductase, suggesting that most isolates expressed these widespread metal-resistance systems. Southern blot hybridization of Cr(R) isolates showed that plasmids of 80, 85, and 95 kb from K. pneumoniae isolates, and of 100 kb from an E. cloacae isolate, contained chrA-related sequences. These plasmids belonged to IncN or IncP incompatibility groups, and conferred Cr(R), as well as multiple antibiotic resistance, when transferred by conjugation to an E. coli standard strain. These data indicated that Cr(R) genes may be distributed among clinical enterobacteria via conjugative plasmids, probably by coselection with antibiotic-resistant genes.

Aminoglycoside Susceptibility Profiles of Enterobacter cloacae Isolates Harboring the aac(6´)-Ib Gene

The aminoglycoside 6'-N-acetyltransferases of type Ib (aac(6')-Ib) gene confers resistance to amikacin, tobramycin, kanamycin, and netilmicin but not gentamicin. However, some isolates harboring this gene show reduced susceptibility to amikacin. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommends a revision of the phenotypic description for isolates harboring the aac(6')-Ib gene. In this study, we determined the aminoglycoside susceptibility profiles of 58 AAC(6')-Ib-producing Enterobacter cloacae isolates. On the basis of the CLSI and EUCAST breakpoints, a large proportion (84.5% and 55.2%, respectively) of these 58 isolates were found to be susceptible to amikacin. However, among the isolates that were shown to be anikacin-susceptible according to the CLSI and EUCAST breakpoints, only 30.6% and 18.8% isolates, respectively, could be considered to have intermediate resistance on the basis of the EUCAST expert rules. Further studies should be conducted to determine the aminoglycoside susceptibility profiles of aac(6')-Ib-harboring isolates from various geographic regions and to monitor the therapeutic efficacy of amikacin in infections caused by these isolates.

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.

Metallo-β-lactamase-producing Enterobacteriaceae isolates at a Taiwanese hospital: lack of distinctive phenotypes for screening

We investigated the prevalence of metallo-β-lactamases (MBLs) among 1,827 Enterobacteriaceae isolates collected in 2006 and evaluated the VITEK 2 microbiology system, modified Hodge test, and 2 combined disk tests as the screening tools for MBLs by using these isolates and 77 previously characterized IMP-8 producers. The IMP-8 MBL was identified in 18 isolates of 2006, and the IMP-8-positive isolates represented 0.2%, 1.1%, and 5.0% of all Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae isolates, respectively. Only one-third of all MBL producers could be recognized by either VITEK 2 or the Hodge test. MBL production could be identified in 38 (40%) of the 95 IMP-8-producing isolates by the combined disk test using meropenem disks supplemented by phenylboronic acid and EDTA, and only 2 (2.1%) isolates gave positive results in the combined disk test using meropenem disks supplemented with dipicolinic acid. Of all IMP-8 producers, 37.9%, 50.5%, and 32.6% were nonsusceptible to tigecycline, fluoroquinolones, and both, respectively. In conclusion, this study demonstrated the lack of distinct phenotypes that could be easily identified among the IMP-8-producing Enterobacteriaceae isolates at a Taiwanese hospital. Continuous surveillance and monitoring are needed because the widespread of tigecycline- and fluoroquinolone-coresistant MBL producers may become a serious therapeutic problem.

IMP metallo- -lactamase-producing clinical isolates of Enterobacter cloacae in the UK

Sir, Carbapenems, such as imipenem, meropenem and ertapenem, are often the main option for treatment of infections caused by multiresistant Gram-negative bacteria, particularly those that produce extended-spectrum or AmpC b-lactamase enzymes. However, the clinical utility of these antimicrobials is under threat with the emergence and spread of acquired genes coding for carbapenem-hydrolysing b-lactamases. These enzymes are broadly classified as serine carbapenemases (e.g. KPC) or metallo-carbapenemases (e.g. IMP, VIM and NDM), based on the hydrolytic mechanism at the active site. Ertapenem resistance in Enterobacter spp. is not uncommon and is mostly mediated by a combination of AmpC enzyme production and porin loss; imipenem and meropenem resistance is rarer, and more likely to be due to acquired carbapenemases. We report our experience with the first IMP metallo-b-lactamaseproducing Enterobacter cloacae isolated from patients admitted to Addenbrooke’s Hospital. In 2008–09, three isolates of meropenem-resistant E. cloacae were referred to the HPA Microbiology Services, Colindale, for confirmation. Two of three isolates were from blood cultures of haematology patients in an intensive care unit at the same time and one was from the urine of a renal transplant recipient. These isolates were resistant to gentamicin, tobramycin and tigecycline, and retained susceptibility only to amikacin and colistin. At the reference laboratory, the isolates were tested for carbapenem resistance by the BSAC agar dilution method and screened for carbapenemase production by the modified Hodge test. Carbapenemase genes were sought by PCR. PFGE of total DNA, digested with XbaI, was performed for strain comparison. All three meropenem-resistant E. cloacae isolates showed potentiation of the activity of imipenem in the presence of EDTA, indicating that they produced metallo-carbapenemases, and gave positive modified Hodge test results. PCR revealed the carbapenemases to be IMP types. A 651 bp intragenic fragment, from nucleotides 47 to 697 of the 741 bp blaIMP allele, was sequenced from all three isolates. The predicted amino acid sequences were identical to IMP-1 carbapenemase and, hence, different from other known IMP variants. PFGE showed that the three isolates belonged to a single strain; nevertheless, no epidemiological link was identified between the renal and haematology patients, or with hospitals elsewhere in the UK or overseas. This is the first identification of E. cloacae with an IMP carbapenemase in the UK based on submissions to HPA Microbiology Services, Colindale. It is part of a pattern whereby carbapenemase production is becoming more widespread in the Enterobacteriaceae, though, generally, KPC, NDM and VIM enzymes are more frequent than IMP types. – 6 The emergence of carbapenemases, which is often linked, as here, to resistance to multiple other drug classes, is a great public health concern. Producer strains should be actively sought to prevent their transmission among patients.