Phenotypic changes contributing to Enterobacter gergoviae biocide resistance.

UNLABELLED: Enterobacter gergoviae is a recurrent contaminant of cosmetic and hygiene products. To understand how this bacterium adapts to biocides, we studied Ent. gergoviae CIP 76.01 and its triclosan and Methylisothiazolinone-chloromethylisothiazolinone (MIT-CMIT) tolerant isogenic mutants. They were compared with others also isolated from contaminated cosmetics. Phenotypic differences were noted and these included changes in the bacterial envelope and flagella along with differences in motility, and biofilm growth rates. Triclosan and MIT-CMIT derivatives expressed flagella and other MIT-CMIT derivatives exhibited some external appendages. Those bacteria expressing a high-level minimal inhibitory concentration to MIT-CMIT, expressed a strong biofilm formation. No differential phenotypes were noted for carbon source utilisation. Enterobacter gergoviae demonstrated a diverse response to both of these preservatives contained in cosmetic preparations, depending on their concentrations. Interestingly, this adaptive response is associated with modifications of filament structure-related proteins contributing to increase the organism motility and the production of biofilm. SIGNIFICANCE AND IMPACT OF THE STUDY: Recurrent contaminations of cosmetics products by Ent. gergoviae, needed a better understanding concerning the bacterial adaptation to preservative agents, with particular concern to triclosan and MIT-CMIT. We demonstrated that bacteria response is associated to various mechanisms represented by expression of external appendages (pili or fimbriae) that control cell motility and biofilm formation and evolving as the concentration of biocides adaptation increased. Such mechanisms which are not chemical specific can also promote a cross-resistance to other biocidal agents. The characterization of Ent. gergoviae adaptability to biocides allows industry to adjust the ranges of concentrations and composition of preservatives in formula.

Faecal carriage of carbapenemase-producing Gram-negative bacilli in hospital settings in southern France.

The emergence of carbapenemase-producing Gram-negative bacilli is a worldwide problem. To date, no study has evaluated the prevalence of faecal carriage of carbapenemase-producing and carbapenem-resistant Gram-negative bacilli (CR GNB) in France. From 1 February to 30 April 2012, we conducted a prospective, multicentre study in three University Hospitals and four General Hospitals in the south of France. The carriage of carbapenemase-producing Enterobacteriaceae (CPE) and other CR GNB was screened by both cultivation on chromID® CARBA and chromID® OXA-48 media (bioMérieux) and molecular tools [multiplex polymerase chain reaction (PCR) and NucliSENS EasyQ® KPC (bioMérieux)]. The genetic relationship between isolates was assessed by rep-PCR (DiversiLab, bioMérieux) or multilocus sequence typing (MLST). The prevalences of CR GNB and carbapenemase-producing bacteria were 2.4 % (27/1,135) and 0.4 % (n = 5), respectively. Two strains corresponded to OXA-23-producing Acinetobacter baumannii and belonged to the widespread sequence type (ST) 2/international clone II, whereas one strain was an ST15 OXA-48-producing Klebsiella pneumoniae. Two OXA-48-producers were detected exclusively by PCR. This first French study revealed the very low dissemination of carbapenemase-producing bacteria in patients attending hospitals in southern France during a non-outbreak situation. However, the increasing description of epidemic cases in this area must reinforce the use of hygiene procedures to prevent diffusion of these multidrug-resistant microorganisms.

Enterobacter gergoviae adaptation to preservatives commonly used in cosmetic industry.

OBJECTIVE: The aim of this study was to obtain a better understanding regarding the origin of recurrent contamination by Enterobacter gergoviae in diverse cosmetic formula. We studied 65 isolates collected from various sources (clinical, food, cosmetics). METHODS: RAPD analysis using AP12H, REP and ERIC-PCR was carried out for epidemiological typing. Evaluation of susceptibility to preservatives currently used in cosmetics for a representative panel of collection strains was measured. Preservative efficacy was evaluated by minimum inhibitory concentrations and minimum bactericidal concentrations (MBCs). RESULTS: Eighty per cent of isolates was unrelated. E. gergoviae showed significant levels of resistance to preservatives. MBC was higher than maximum permitted concentrations imposed by European Commission (EC). Association of preservatives showed in rare case additive effects, and no synergic effects were observed. CONCLUSION: Most of the cosmetic formulations are contaminated with unrelated E. gergoviae strains. Maximum allowed concentrations for sodium benzoate are inefficient to limit proliferation and control adaptability to this bacterium in cosmetic products. Efflux mechanisms should be involved in methylisothiazolinone-chloromethylisothiazolinone and triclosan adaptation.

Faecal carriage of oxyiminocephalosporin-resistant Enterobacteriaceae among paediatric units in different hospitals in the south of France.

The aim of this study was to determine the presence of oxyiminocephalosporin-resistant (OCR) Gram-negative bacilli and extended-spectrum ß-lactamase (ESBL)-producing isolates in stool specimens obtained from paediatric patients hospitalised for acute diarrhoea. We conducted a prospective, multicentre study over a period of 6 months in seven hospitals in the south of France. Samplings were carried out from infants admitted for acute diarrhoea with no previous antibiotic treatment in the last week. Bacteria in stool specimens were screened for the presence of OCR Gram-negative bacilli on Drigalski agar supplemented with ceftazidime and ESBL CHROMagar® media, and confirmed by the Rosco tablets test. Genetic detection was performed by the Check MDR® microarray and by polymerase chain reaction (PCR) and sequencing with bacterial DNA extracted from isolates. The presence of OCR enterobacteria was markedly high (177/1,118 patients, 15.2 %), with an important community origin (66.1 %). The majority of multidrug-resistant (MDR) bacteria were Enterobacter cloacae (106, 59.9 %) and Escherichia coli (61, 34.5 %). The prevalence of ESBL and CTX-M producers represented 5.2 and 4.3 % of the isolates, respectively. The main proportion of these ESBL carriers was found in children less than 1 year of age (53.4 %). One carbapenemase (IMP-1) was detected. The study revealed the wide dissemination of MDR bacteria in infants attending hospitals in the south of France during a non-outbreak situation, in particular, the spread of cefotaximase and the detection of a carbapenemase. This worrisome situation must reinforce the use of hygiene procedures and appropriate antibiotics to control the emergence and spread of OCR organisms.

Cross-resistance between biocides and antimicrobials: an emerging question.

The widespread use of biocides, and their resulting dissemination in the environment, can contribute to adaptations in bacteria leading to the development of low-level susceptibility to antibacterial agents. The mechanisms of resistance in bacteria are similar for both antimicrobials and biocides, and exposure to biocides can result in cross-resistance to antibacterial agents. Resistance mechanisms altering the activity of biocide and antibiotic molecules are discussed with regard to regulation and mode of action in the light of laboratory studies of induced resistance. It is clear that in order to preserve their activity and avoid the development of possible cross-resistance, prudent use of antibacterial agents is to be strongly recommended, not only in clinical settings but also in veterinary and agricultural and other applications.

Membrane permeability, a pivotal function involved in antibiotic resistance and virulence in Enterobacter aerogenes clinical isolates.

Imipenem-susceptible E. aerogenes isolates exhibiting extended spectrum ß-lactamases, target mutations and a basal efflux expression, were identified in five patients. After imipenem treatment, imipenem-intermediate susceptible (IMI-I) or resistant (IMI-R) isolates emerged in these patients. Alteration in porin synthesis and increase in efflux expression were observed in the IMI-I isolates whereas complete loss of the porins, LPS alteration and efflux overexpression were observed in the IMI-R isolates. Bacterial virulence of the strains was investigated by the Caenorhabditis elegans model. The IMI-R isolates were shown to be significantly less virulent than the IMI-susceptible or IMI-I isolates. The pleiotropic membrane alteration and its associated fitness burden exhibited by E. aerogenes isolates influence their antibiotic resistance and their virulence behaviour. These findings highlight the balance between the low permeability-related resistance and virulence and their relationships with the treatment of resistant pathogens.

Physiological characterisation of the efflux pump system of antibiotic-susceptible and multidrug-resistant Enterobacter aerogenes.

Enterobacter aerogenes predominates amongst Enterobacteriaceae species that are increasingly reported as producers of extended-spectrum beta-lactamases. Although this mechanism of resistance to beta-lactams is important, other mechanisms bestowing a multidrug-resistant (MDR) phenotype in this species are now well documented. Amongst these mechanisms is the overexpression of efflux pumps that extrude structurally unrelated antibiotics prior to their reaching their targets. Interestingly, although knowledge of the genetic background behind efflux pumps is rapidly advancing, few studies assess the physiological nature of the overall efflux pump system of this, or for that matter any other, bacterium. The study reported here evaluates physiologically the efflux pump system of an E. aerogenes ATCC reference as well as two strains whose MDR phenotypes are mediated by overexpressed efflux pumps. The activities of the efflux pumps in these strains are modulated by pH and glucose, although the effects of the latter are essentially restricted to pH 8, suggesting the presence of two general efflux pump systems, i.e. proton-motive force-dependent and ABC transporter types, respectively.

Enterobacter gergoviae and the prevalence of efflux in parabens resistance.

OBJECTIVES: In order to characterize the mechanism involved in parabens resistance, we studied 13 Enterobacter gergoviae collected from diverse cosmetic formulations containing parabens as preservatives and 10 isolates from clinical or industrial sources. METHODS: RAPD and ERIC-PCR were employed and compared for the epidemiological typing. To study antibiotic and paraben susceptibility, the standard disc diffusion method and the 2-fold dilution method in Luria-Bertani medium were used. Characterization of porins was performed using immunodetection with polyclonal antibodies. Resistance mechanisms against parabens membrane permeabilization were evaluated by measuring K(+) efflux using a specific electrode. mar regulon identification and comparison were carried out. RESULTS: Epidemiological typing confirmed that most of the cosmetic formulations were contaminated by unrelated strains. All of the E. gergoviae strains presented high methylparaben MICs, ranging from 1 to 3.8 g/L, values that were 2-5 times higher than for Escherichia coli or Enterobacter aerogenes, even in strains overexpressing MarA. These MICs decreased in the presence of phenylalanine arginine beta-naphthylamide, pinpointing efflux as a major mechanism of parabens resistance even in E. gergoviae clinical strains. CONCLUSIONS: This is the first report showing the role of active efflux in the parabens resistance in E. gergoviae, a mechanism that may explain its frequent isolation in parabens-containing cosmetics compared with other enterobacterial species. Paraben efflux seems to be regulated by a mar-independent process in E. gergoviae.

Imipenem resistance of enterobacter aerogenes mediated by outer membrane permeability.

Multidrug-resistant Enterobacter aerogenes strains are increasingly isolated in Europe and especially in France. Treatment leads to imipenem resistance, because of a lack of porin. We studied the evolution of resistance in 29 strains isolated from four patients during their clinical course. These strains belonged to the prevalent epidemiological type observed in France in previous studies (C. Bosi, et al., J. Clin. Microbiol. 37:2165-2169, 1999; A. Davin-Regli et al., J. Clin. Microbiol. 34:1474-1480, 1996). They also harbored a TEM-24 extended-spectrum beta-lactamase-coding gene. Thirteen strains were susceptible to gentamicin and resistant to imipenem and cefepime. All of the patients showed E. aerogenes strains with this resistance after an imipenem treatment. One patient showed resistance to imipenem after a treatment with cefpirome. Twelve of these 13 strains showed a lack of porin. Cessation of treatment with imipenem for three patients was followed by reversion of susceptibility to this antibiotic and the reappearance of porins, except in one case. For one patient, we observed three times in the same day the coexistence of resistant strains lacking porin and susceptible strains possessing porin. The emergence of multidrug-resistant E. aerogenes strains is very disquieting. In our study, infection by E. aerogenes increased the severity of the patients' illnesses, causing a 100% fatality rate.

Most Enterobacter aerogenes strains in France belong to a prevalent clone.

The aim of this study was to determine the distribution in France of the Enterobacter aerogenes prevalent clone isolated in the hospitals of the Marseille area (A. Davin-Regli, D. Monnet, P. Saux, C. Bosi, R. Charrel, A. Barthelemy, and C. Bollet, J. Clin. Microbiol. 34:1474-1480, 1996). A total of 123 E. aerogenes isolates were collected from 23 hospital laboratories and analyzed by random amplification of polymorphic DNA and enterobacterial repetitive intergenic consensus-PCR to determine their epidemiological relatedness. Molecular typing revealed that 21 of the 23 laboratories had isolated this prevalent clone harboring the plasmid encoding for extended-spectrum beta-lactamase of the TEM-24 type. Most isolates were susceptible only to imipenem and gentamicin. Their dissemination seems to be clonal and was probably the result of the general use of broad-spectrum cephalosporins and quinolones. Four isolates showed an alteration of their outer membrane proteins, causing decrease of susceptibility to third-generation cephalosporins and imipenem and leading to the critical situation of having no alternative therapeutic. The large dissemination of the E. aerogenes prevalent clone probably results from its good adaptation to the antibiotics administered in France and the hospital environment, particularly in intensive care units.

A cluster of cases of infections due to Aeromonas hydrophila revealed by combined RAPD and ERIC-PCR.

Two polymerase chain reaction (PCR)-based methods were used for epidemiological typing of Aeromonas hydrophila. Random amplification of polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) were applied to an outbreak involving seven patients. The epidemiological situation appeared complex; with the exception of two clinical isolates, all gave unique patterns with both techniques. These methods demonstrated nosocomial transmission in one unit and permitted the study to exclude a common environmental source in the hospital. The coincidental clustering of patients infected with A. hydrophila probably resulted from an increased prevalence of aeromonads in waters during summer, although no single RAPD or ERIC-PCR pattern was found among both clinical and environmental samples. RAPD and ERIC-PCR proved to be effective for the epidemiological study of A. hydrophila strains.

A nosocomial outbreak due to Enterobacter cloacae strains with the E. hormaechei genotype in patients treated with fluoroquinolones.

During a 7-month period, we isolated 21 highly fluoroquinolone-resistant Enterobacter cloaecae strains in units from two hospitals in Marseille, France. Random amplification of polymorphic DNA showed clonal identity between isolates which, furthermore, presented the Enterobacter hormaechei genotype on DNA-DNA hybridization. The emergence of this clone was observed only in patients treated with fluoroquinolones.

Epidemiological investigation of Pseudomonas aeruginosa nosocomial bacteraemia isolates by PCR-based DNA fingerprinting analysis.

Between July 1994 and March 1995, 64 isolates of Pseudomonas aeruginosa were implicated in bacteraemia in 25 cancer patients in five wards of two hospitals. These, together with 24 environmental isolates and one isolate from a bacteraemia in a non-cancer patient were examined by three PCR-based DNA fingerprinting methods: random amplified polymorphic DNA (RAPD), enterobacterial-repetitive intergenic consensus (ERIC)-PCR, and 16S-23S spacer region-based RAPD. These methods were reproducible, discriminatory and showed close agreement; all indicated that 47 isolates that had caused bacteraemia in 19 cancer patients were indistinguishable. Seventeen other isolates that had caused bacteraemia in 10 cancer patients were discriminated into eight further groups, and the 24 environmental and non-cancer patient isolates into further distinct groups. No environmental source of the epidemic strain was found, but it was suspected that the outbreak was related to infusion implants.

Serratia marcescens nosocomial outbreak due to contamination of hexetidine solution.

During a 10-week period, 16 patients in a neurosurgery intensive care unit were involved in an outbreak of Serratia marcescens. The epidemic strain was found in several flasks of 1:4 diluted hexetidine solution, an antiseptic used for patient mouth washing. Testing of the bactericidal activity of the diluted antiseptic revealed that all the epidemic strains were able to grow in the diluted antiseptic solution. Strains isolated from clinical samples and from the antiseptic solution were compared by random amplification of polymorphic DNA. Epidemiologic typing data implicated the diluted antiseptic solution as the single source of this S. marcescens outbreak.

Molecular epidemiology of Enterobacter aerogenes acquisition: one-year prospective study in two intensive care units.

To evaluate the respective contributions of patient-to-patient transmission and endogenous acquisition of Enterobacter aerogenes isolates, we conducted a prospective epidemiologic study in two intensive care units (ICUs) between May 1994 and April 1995. We collected a total of 185 E. aerogenes isolates: 130 from 51 patients in a surgical ICU (SICU), 45 from 26 patients in a medical ICU (MICU), and 10 from the environments in these two ICUs. All isolates were typed by random amplification of polymorphic DNA and enterobacterial repetitive intergenic consensus PCR. Among the 175 clinical isolate, we observed 40 different profiles by random amplification of polymorphic DNA and 36 different profiles by enterobacterial repetitive intergenic consensus PCR. We identified a ubiquitous and prevalent clone, corresponding to 58% of SICU and 41% of MICU clinical isolates. Three epidemiologically related strains were specific to each ICU and represented 17% of SICU and 24% of MICU clinical isolates; unique type strains represented 17 and 29% of SICU and MICU clinical isolates, respectively, and E. aerogenes strains which were spread to a limited degree and which were isolated less than five times during the 1-year study period represented 8 and 6% of SICU and MICU clinical isolates, respectively. Our results show that E. aerogenes is acquired in the ICU in three different ways: patient-to-patient spread of a prevalent or an epidemiologically related strain, acquisition de novo of a strain from patients' own flora, and acquisition of a nonendemic strain followed by occasional patient-to-patient transmission. The findings point out the importance of patient-to-patient transmission in E. aerogenes acquisition and suggest that changes in E. aerogenes ecology in the hospital have taken place during the past decade.

Investigation of outbreaks of Enterobacter aerogenes colonisation and infection in intensive care units by random amplification of polymorphic DNA.

During a 4-month period, 41 isolates of Enterobactor aerogenes were cultured from different specimens from a 14-bed intensive care unit (ICU1). These were obtained from 12 patients out of a total of 187 patients admitted to the ICU. Sixteen E. aerogenes isolates were cultured from another ICU (ICU2) 6 months later. Six non-outbreaks associated strains were included as controls and all the isolates were compared by random amplification of polymorphic DNA (RAPD), with three different 10-mer oligonucleotide primers. RAPD fingerprinting with primer AP12h was as discriminatory as the combined results from all three primers and defined 22 different patterns for the 41 isolates from the ICU1. In nine instances, isolates with indistinguishable RAPD patterns were detected in two-to-five patients over a 3-15-day period, suggesting patient-to-patient transmission. During their stay in ICU1, patients harboured one-to-12 distinguishable isolates. Isolates from ICU2 were indistinguishable by RAPD analysis with the three different primers. These findings suggest that the cluster of colonisations and infections in ICU1 was a 'false outbreak', consisting of successive patient-to-patient transmission of different E. aerogenes strains. In contrast, the outbreak on ICU2 probably involved the extensive spread of a single strain.

Use of random amplified polymorphic DNA for epidemiological typing of Stenotrophomonas maltophilia.

We used the technique of random amplification of polymorphic DNA (RAPD) to type 130 isolates of Stenotrophomonas (Xanthomonas) maltophilia, using four arbitrary short primers. Of the 130 isolates, 51 were from the hospital environment, 48 from clinical specimens and 31 were geographically diverse environmental isolates. DNA amplification with the four sets of primers generated 112 RAPD patterns that differed by two or more bands in one of the four primers. Sixteen pairs of isolates were of the same RAPD pattern and some of these pairs represented clinical strains obtained from patients hospitalized at the same time in the same ward. In three patients, two to three strains of S. maltophilia which gave different RAPD fingerprints were isolated on the same day from different specimens. RAPD fingerprinting demonstrated great genomic diversity within the species S. maltophilia and provided an effective method for the study of the epidemiology of both clinical and environmental strains.

Variations in DNA concentrations significantly affect the reproducibility of RAPD fingerprint patterns.

The influence of the DNA concentration was tested using two different primers and nine DNA samples. Major modifications in the DNA banding pattern were apparent between successive dilutions. Such differences could be explained by concomitant changes in three different molecular conditions: the presence of perfect priming sites, the amplification of rare sites and the existence of mismatch annealing events. At low DNA concentrations (less than 1 pg/microliter), molecular events occurred at random and had a direct consequence on the reproducibility of RAPD profiles. At the appropriate DNA concentration (between 100 ng/microliters and 10 pg/microliters), reproducibility was adequate at a given concentration, but RAPD profiles differed from one dilution to another. These observations demonstrate the usefulness of the bis-benzimide method for quantification of DNA extracts.

Efficient discrimination of Mycobacterium tuberculosis strains by 16S-23S spacer region-based random amplified polymorphic DNA analysis.

Amplification of the region separating the genes coding for 16S and 23S rRNA was performed with 15 Mycobacterium tuberculosis isolates and the type strain, ATCC 27294. Reproducible amplification patterns were obtained. PCR products were then used as target DNA for random amplified polymorphic DNA (RAPD) analysis. The discriminatory power was higher than when whole genomic DNA was used as a RAPD template. 16S-23S spacer region-based RAPD analysis was a simple and efficient method of differentiation. Consequently, it may be a useful tool for epidemiologic studies of tuberculosis.

A Simple Method for Selective Isolation of Stenotrophomonas maltophilia from Environmental Samples.

Stenotrophomonas maltophilia is a commonly found environmental bacterium that is associated with the plant rhizosphere. It shows increasing prevalence in immunocompromised patients. We report a simple method for selective isolation of S. maltophilia from soils which makes use of both its resistance to imipenem and its requirement for methionine.