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PubMed2003

Influence of food preservation parameters and associated microbiota on production rate, profile and stability of acylated homoserine lactones from food-derived Enterobacteriaceae.

Quorum-dependent regulation is mediated by N-acyl-L-homoserine lactones (AHLs) in several Gram-negative bacteria. The production of AHLs has typically been studied using pure bacteria cultures grown in nutrient-rich media at optimal temperature. AHLs are produced in several chill-stored foods by Gram-negative bacteria participating in spoilage. As part of our investigation of the role of AHLs in food quality, we studied the AHL production in two Enterobacteriaceae isolated from cold-smoked salmon under growth conditions typical of those found in cold-smoked salmon. We tested the influence of carbon source (glucose, sucrose, xylose, arabinose, mannose, mannitol and sorbitol), temperature (5 and 25 degrees C), salt concentration (0-7%), pH (6, 7 and 8) and co-existing lactic acid bacteria microflora on the AHL profile and production rate from Serratia proteamaculans strain B5a and Enterobacter agglomerans strain B6a. The two strains produced the same types of AHLs under all conditions tested. The specific AHL concentrations (moles/liter/OD(450)) changed slightly for both strains at the various conditions. S. proteamaculans strain B5a produced approximately 150 nM/OD(450) N-3-oxo-hexanoyl homoserine lactone (OHHL) and E. agglomerans strain B6a produced two major signals, OHHL and N-3-oxo-octanoyl homoserine lactone (OOHL) in a 1:9 ratio with a total concentration of approximately 3000 nM/OD(450). The AHL signal molecules became unstable with increasing pH (>7.5). In cold-smoked salmon, pH is approximately 6 and therefore only a low degree of pH-induced turnover is expected to occur in this product. Overall, our study demonstrates that food-derived Enterobacteriaceae produce AHLs of the same type and in the same magnitude when grown under food-relevant conditions as when grown in laboratory media at high temperature. Also, the AHLs produced in foods will be relatively stable and their regulatory impact lasting during storage.

4-Butyrolactone↗
PubMed1998

Update on media for isolation of Enterobacteriaceae from foods.

Testing for 'total' Enterobacteriaceae, coliforms and Escherichia coli as marker organisms in foods and detection of specific pathogens of the family Enterobacteriaceae, including pathogenic E. coli, Salmonella, Shigella and Yersinia spp. is widely applied in many food control laboratories. This review describes some recent developments in culture media for these organisms. Methods for enumeration of E. coli include the standard MPN technique, a membrane-filter method and the use of media containing chromogenic and fluorogenic indicators for beta-D-glucuronidase (GUD) activity. Shiga toxin-producing E. coli O157 strains usually do not ferment sorbitol and are GUD-negative. These characteristics are used in selective media for these organisms, such as cefixime tellurite sorbitol MacConkey agar. For the detection of salmonellae, motility enrichment in Modified Semisolid Rappaport-Vassiliadis (MSRV) medium shows equal or better results than the use of standard Rappaport-Vassiliadis broth. Addition of nitrofurantoin to diagnostic semisolid salmonella agar and to xylose lysine desoxycholate agar favours the isolation of S. enteritidis. Recently developed salmonella media use different selective and diagnostic properties, such as acid formation from propylene glycol, glucuronate fermentation, fermentation of glycerol and addition of Tergitol 4 as selective agent. The isolation of Shigella spp. from foods is rather difficult and further evaluation of suggested isolation systems and the development of more effective methods for the isolation of this pathogen are needed. Yersinia enterocolitica includes both pathogenic and nonpathogenic biotypes and serogroups. As no single procedure will recover all pathogenic strains of Y. enterocolitica, the use of two isolation procedures in parallel is recommended.

Culture Media↗
PubMed1984

Differentiation of Enterobacteriaceae and Vibrionaceae by a micromethod for determination of carbon substrate assimilation.

The assimilation techniques described for taxonomic study are unsuitable for routine diagnosis because of the need for purification and standardization of substrates, the great quantity of medium consumed and difficulties in interpreting the results. A standardized micromethod (API strip) for the study of carbon substrate assimilation by bacteria has recently been described (Zbl. Bakt. Hyg., I. Abt. Orig. A 255 (1983) 479-488). The prototype gallery used consisted of two strips of 32 microtubes each containing dehydrated carbon substrate. Each strip contained 30 tests plus positive and negative controls. The suspension medium was a synthetic semi-gel. A total of 914 strains of Gram negative rods representing 44 species of Enterobacteriaceae and Vibrionaceae were tested (Table 1). Automatic reading was performed after incubation at 32 degrees C for one day, or two days for slow growing bacteria using an ATB - 1500 reader (API System) linked to a HP 85 microcomputer (Hewlett-Packard). Most species showed typical carbon substrate assimilation patterns allowing their differentiation from other species within each genus (Table 2). The results obtained with the micromethod agreed in large measure with the nutritional patterns reported by other workers. It should be possible to use these results to construct a coherent framework of tests suitable for identifying species of Enterobacteriaceae and Vibrionaceae of clinical significance.

Bacteriological Techniques↗
PubMed1988

Further differentiation of Enterobacteriaceae by means of siderophore-pattern analysis.

By means of a combination of 5 siderophore bioassays using several indicator strains, genera, species and subspecies of Enterobacteriaceae can be further differentiated. Enterobactin, aerobactin and other siderophores produced can be detected. Each strain shows specific pattern which we called siderophore-pattern. It is easy to separate Morganella, Proteus, Providencia, Yersinia strains from the genera Salmonella, Shigella, Escherichia coli, Enterobacter, Citrobacter, Klebsiella, Serratia. Enterobacter agglomerans strains differ from other Enterobacter species with respect to their siderophore pattern. In Salmonella strains there are differences between the subspecies I and IV. Additionally the most strains of Salmonella subspecies I from nosocomial infections produced aerobactin, in the most cases determined by plasmids. Among Shigella strains different siderophore pattern exist according to other epidemiological markers. S. flexneri strains of serovar 6 produced contrary to the strains of other serovars enterobactin. By means of the siderophore-pattern analysis E. coli strains of serovars 01; 02; 018 can be further differentiated. E. coli 01:K1 strains containing the fimbrial antigen F11 produced aerobactin whereas the F9 strains did not. All Hafnia alvei strains produced a uniform siderophore pattern, different from all other members of the enterobacteriaceae family. With this aim Hafnia alvei strains can be easily separated under practical conditions.

Enterobacteriaceae↗
PubMedDate not supplied

Validation of the VITEK2 and the Advance Expert System with a collection of Enterobacteriaceae harboring extended spectrum or inhibitor resistant beta-lactamases.

The susceptibility testing accuracy of the VITEK2 system and the ability of the Advance Expert System (AES) to provide interpretive readings were evaluated against 86 extended spectrum (ESBL) and 6 inhibitor-resistant-TEM (IRT) beta-lactamases producing Enterobacteriaceae clinical isolates. VITEK2 MICs of 12 beta-lactams were compared with those obtained by the standard NCCLS microdilution technique. The overall essential agreement ( +/- 1 log dilution) was 87.8%. Discrepancies were mainly observed with cefepime (30.3% of total number of discrepancies), ceftazidime (21.2%), and cefotaxime (15.1%). MIC discrepancies were slightly higher in CTX-M- (14.4%) than in TEM- (12.5%) or SHV- (11.9%) type ESBL producers and were rare in IRT producers (1.4%). Overall interpretive agreement was 92.5% and minor, major, and very major errors were 5.4%, 1.7%, and 2.1%, respectively. The AES was able to identify an ESBL phenotype in 85 out of 86 isolates (98.8%) and an IRT phenotype in all 6 isolates harboring these enzymes, thus reducing very major errors to 0.9%. The VITEK2 system, in conjunction with the AES software, is a reliable tool for detection of ESBL or IRT producing Enterobacteriaceae isolates.

Anti-Bacterial Agents↗
PubMed1996

Accurate characterization of ofloxacin susceptibility with Enterobacteriaceae using a modified GNS F6 card and the bioMerieux Vitek System.

Routine antimicrobial susceptibility testing of Enterobacteriaceae using the Vitek System (bioMerieux Vitek, Hazelwood, MO) and the GNS-F6 card revealed discrepancies between the activity of ofloxacin and ciprofloxacin, primarily with isolates of Klebsiella pneumoniae. Specifically, during a one-year period, 12% of 618 ciprofloxacin-susceptible isolates were determined to be ofloxacin resistant with the GNS-F6 card. A similar problem, but one of lower magnitude, was observed with Serratia marsescens. That these represented false ofloxacin resistance results was confirmed by comparison of broth microdilution determinations of ofloxacin MICs with F6 results on a collection of 203 fresh clinical isolates of K. pneumoniae and 39 isolates of S. marsescens. The GNS-F6 card was then modified by the manufacturer to include a new formulation of ofloxacin and assessed using a collection of 224 recent clinical isolates of Enterobacteriaceae and Pseudomonas aeruginosa, and 78 stock cultures of enteric Gram-negative bacilli selected specifically because of disproportionately high rates of fluoroquinolone resistance. No ofloxacin false resistant results were observed with this collection when the modified GNS-F6 card was evaluated in comparison to a standardized broth microdilution MIC test. The current clinical version of the Vitek System appears to accurately assess ofloxacin susceptibility.

Anti-Infective Agents↗
PubMed1997

Accuracy of the Vitek system for antimicrobial susceptibility testing Enterobacteriaceae bloodstream infection isolates: use of "direct" inoculation from Bactec 9240 blood culture bottles.

A recent investigation indicates that rapid antimicrobial susceptibility tests (AST) can affect patient therapy leading to reductions in health-care costs for some patient populations. However, there is little information relative to the often performed direct inoculation of positive blood culture bottles into rapid AST systems. AST results of direct inoculated Vitek (bioMerieux Vitek, Hazelwood, MO, USA) GNS cards were compared to those inoculated per package insert recommendations and a reference broth microdilution test using 50 consecutive Enterobacteriaceae bloodstream infection isolates. Escherichia coli (44% of isolates), Klebsiella ssp. (30%), and six other members of this family were tested against 15 antimicrobial agents. The direct inoculation method produced only two false-susceptible (0.3%), seven false-resistant (0.9%; six different drugs), and 48 minor errors (6.4%). The GNS cards inoculated in the usual, recommended manner had no very major error, and 7.5% combined major and minor errors. If the results of the urinary infection-specific drugs (nitrofurantoin, trimethoprim/sulfamethoxazole; not appropriate for bacteremia therapy) and ampicillin/sulbactam were deleted, both Vitek inoculation methods yielded results well within acceptable limits (< or = 4.5% overall error). These results indicate that the direct inoculation method of Vitek GNS cards from Enterobacteriaceae bloodstream infections (detected by Bactec 9240, Becton-Dickinson, Cockeysville, MD, USA) performed as well as the NCCLS broth microdilution test. Thus, a procedural modification of this type could further accelerate rapid access to accurate AST data.

Bacteremia↗
PubMed2001

Performance of the VITEK2 system for identification and susceptibility testing of routine Enterobacteriaceae clinical isolates.

The VITEK2 system was evaluated with 138 fresh consecutive routine clinical Enterobacteriaceae isolates. Susceptibility results to 10 beta-lactams, three aminoglycosides and a quinolone were compared with those obtained following the NCCLS standard microdilution. API20E was used as reference method for identification. All but three isolates were correctly identified in 3 h at species level (97.8%), two isolates (1.4%) at genus level and only one isolate was misidentified. Overall essential agreement for susceptibility testing was 97.1%. Discrepancies were mainly observed with piperacillin (1.1%), cefuroxime (0.6%) and amoxycillin/clavulanate (0.3%). Discrepancies for aminoglycosides and ciprofloxacin were low (<0.1%). Minor, major and very major errors (NCCLS categories) were 4.1%, 0.2% and 6.1%, respectively. Very major errors were due to piperacillin (4.5%), ampicillin (0.8%) and amoxycillin/clavulanate (0.8%). The VITEK2 system gave accurate identification and susceptibility testing results of routine Enterobacteriaceae clinical isolates.

4-Quinolones↗
PubMed2002

Prevalence of extended spectrum beta-lactamases among Enterobacteriaceae: an Italian survey.

The prevalence of extended-spectrum beta-lactamase (ESBL) production by consecutive non-repeated isolates of Enterobacteriaceae was determined over a 6-month period. A total of 8015 strains were isolated from ten Italian laboratories and 509 (6.3%) of these were designated ESBL producers from the results of a double-disk synergy test. Escherichia coli was the most isolated microrganism, followed by Klebsiella pneumoniae and Proteus mirabilis. Providencia stuartii (28.1%) was the most frequently isolated ESBL producer, followed by K. pneumoniae and Enterobacter aerogenes (20.5%). However, amongst all ESBL producers, K. pneumoniae (38.2%) was the most represented followed by P. mirabilis (25.7%). All the strains positive to DD tests were confirmed for the carriage of TEM and SHV genes using colony-blot hybridisation (CH). A total of 447 strains (88.0%) were CH-positive, of which 42.3% hybridised with the TEM-type probe, 30.1% with the SHV-type probe and 15.6% with both probes. In conclusion, our findings indicate that 6.3% of all Enterobacteriaceae tested produced ESBLs, 42.3% of which were TEM-derived enzymes. More than 20% of P. stuartii, K. pneumoniae and E. aerogenes harbour these enzymes. The double-disk test seems to be a useful test to identify ESBL producing strains.

Anti-Bacterial Agents↗
PubMed1972

Biotyping of Enterobacteriaceae as a test for the evaluation of isolation systems.

Arguments in favour of biotyping of Enterobacteriaceae excreted in the faeces of isolated patients, as a method of investigating the efficiency of the isolation procedures, are presented as well as a technical outline of the procedure. The study included three kidney transplantation patients, five acute myeloid leukaemia patients and four healthy persons as controls.The results show, apart from new colonizations during isolation, a difference in the mean number of contaminations and colonizations with different Enterobacteriaceae biotypes. It is concluded from these results, that the isolation procedures were not completely effective and that the AML patients studied had a decreased colonization resistance of their digestive tract. This was less evident in the kidney transplant group.

Anti-Bacterial Agents↗
PubMed1990

Determination of colonization resistance of the digestive tract by biotyping of Enterobacteriaceae.

In studies concerning the effect of antibiotics on faecal microflora, Colonization Resistance is an important parameter. Colonization Resistance correlates inversely with the number of different biotypes of Enterobacteriaceae isolated from faecal samples. Nine healthy volunteers were studied during 6 weeks, in order to determine the natural variation in the number of different biotypes of Enterobacteriaceae per faecal sample. The numbers of biotypes ranged from 1-15 per faecal sample, the mean number of biotypes varied between 2.6 and 7.3 different biotypes per faecal sample per healthy volunteer. Inter-individual variations of five biotypes in the mean number of biotypes per faecal sample are normal. We assessed the minimal number of faecal samples that should be taken for comprehensive biotyping so as to determine reliably the mean number of different biotypes representative for the Colonization Resistance of an individual. It was found that a minimum of four faecal samples was required.

Adult↗
PubMed1988

Conservation of the D-mannose-adhesion protein among type 1 fimbriated members of the family Enterobacteriaceae.

A variety of genera and species of the family Enterobacteriaceae bear surface fimbriae that enable them to bind to D-mannose residues on eukaryotic cells. Until recently, it was thought that the D-mannose binding site was located in the major structural subunit (FimA), of relative molecular mass (Mr) 17,000 (17 K), of these organelles in Escherichia coli. New evidence indicates that this binding site resides instead in a minor protein Mr 28-31 K (FimH) located at the tips and at long intervals along the length of the fimbriae, and is reminiscent of the minor tip adhesion proteins of pyelonephritis-associated pili (Pap) and S fimbriae. In contrast to the antigenic heterogeneity of the major FimA subunit, the antigenic structure of FimH is conserved among different strains of E. coli. Here, we report an even broader conservation of this minor adhesion protein extending to other genera and species of type 1 fimbriated Enterobacteriaceae. Our results may have implications for the development of broadly protective vaccines against Gram-negative bacillary infections in animals and perhaps in man.

Antibodies, Monoclonal↗
PubMed2003

Transfer of antibiotic resistance between commensal and pathogenic members of the Enterobacteriaceae under ileal conditions.

AIM: To determine the rate of antibiotic resistance transmission between commensal and pathogenic representatives of the Enterobacteriaceae. METHODS AND RESULTS: Through the use of a validated in vitro simulation of the porcine ileum, the transmission of antibiotic resistance was detected between commensal Escherichia coli, E. coli O157 and Salmonella spp. Countable transconjugant populations arose readily and, in one example, proved capable of indefinite persistence. CONCLUSIONS: Genetic material conferring antibiotic resistance is readily transmissible between members of the Enterobacteriaceae under ileal conditions. Recipient phenotype influences the persistence of multi-resistant transconjugants. SIGNIFICANCE AND IMPACT OF THE STUDY: The observation that the conjugal transmission of antibiotic resistance is commonplace under ileal conditions impacts primarily on the risk of food contamination by multi-resistant bacteria. The establishment of a multi-resistant transconjugant population as a dominant member of the microflora maintains a genetic reservoir of antimicrobial resistance.

Animals↗
PubMed1995

FimH adhesin of type 1 pili is assembled into a fibrillar tip structure in the Enterobacteriaceae.

Type 1 pili are heteropolymeric mannosebinding fibers produced by all members of the Enterobacteriaceae family. The bulk of the fiber is composed of FimA. Two macromolecular complexes responsible for mediating an interaction with mannose-containing receptors were purified from fimA- Escherichia coli by mannose affinity chromatography and ion-exchange chromatography. One complex contained only the mannose-binding adhesin, FimH, associated with FimG, a minor component of the type 1 pilus. In the other complex the FimG-FimH moiety was loosely associated with a chaperone-minor subunit complex (FimC-FimF), possibly representing an intermediate in tip fibrilla assembly. The FimC chaperone has also been shown to form a preassembly complex with FimH that has been purified and characterized previously. Purified FimC did not bind to the FimG-FimH complex but did recognize FimH dissociated from the FimG-FimH complex. Quick-freeze deep-etch electron microscopy revealed that the FimG-FimH complex had a thin fibrillar architecture. High-resolution electron microscopy of type 1 pili revealed that a 16-nm fibrillar tip structure with an architecture identical to that of the FimG-FimH complex was joined end-to-end to the pilus rod. In a fimH- deletion mutant, the tip fibrillae joined to pilus rods were approximately 3 nm in length. The full-length tip fibrilla was restored by complementation with the fimH gene in trans. The bipartite nature of the type 1 pilus was also demonstrated on pili purified from clinical isolates of members of the Enterobacteriaceae family arguing that it is a conserved feature of the type 1 pilus.

Adhesins, Escherichia coli↗
PubMed1990

Epidemiology of antibiotic resistance in human isolates of Enterobacteriaceae in Sicily.

The authors have studied the antimicrobial susceptibility of 1073 clinical isolates of various genera of Enterobacteriaceae (collected during the period July-December 1988) to ampicillin, piperacillin, cefotaxime, ceftazidime, ceftriaxone, aztreonam, imipenem, gentamicin, amikacin, netilmicin, norfloxacin, and ciprofloxacin. Antimicrobial susceptibility was determined by Bauer-Kirby disk diffusion method. Of 1073 tested bacteria, 704 (65.6%) produced beta-lactamase detectable by nitrocefin test. The highest percentage of resistant strains occurred with ampicillin (70%) followed by piperacillin (24%) and cefotaxime (19%). Lower percentages of resistant strains were found for gentamicin (10%), aztreonam (8%), netilmicin (7%), norfloxacin (5%) and amikacin (4%). Two percent of the strains were resistant to ciprofloxacin and 0.5% to imipenem. The incidence of resistance in Klebsiella sp., Enterobacter sp., E. coli and Proteus sp. was compared to that found among 872 strains isolated during July-Dec. 1984. In all the Enterobacteriaceae, mainly Enterobacter sp., the increase in the resistance was high for ampicillin, piperacillin and cefotaxime and lower for gentamicin.

Drug Resistance, Microbial↗
PubMed1963

SEPARATION, CHARACTERIZATION, AND BIOLOGICAL SIGNIFICANCE OF A COMMON ANTIGEN IN ENTEROBACTERIACEAE.

An antigen common to Enterobacteriaceae and closely associated with endotoxin fractions has been separated by chromatography on DEAE cellulose employing elution with a NaCl gradient. The purified common antigen fails to coat erythrocytes, is poorly, if at all antigenic, it is non-dialyzable and excluded from sephadex G-100 gel. It is composed of polysaccharide and polypeptide. The most important property of this antigen thus far determined appears to be its interference with the specificity of the hemagglutination test commonly employed to measure antibody to O antigen of Enterobacteriaceae. It may also have taxonomic significance in classification of this family of bacteria.

Antibodies↗
PubMed2002

Multidrug resistance among Enterobacteriaceae is strongly associated with the presence of integrons and is independent of species or isolate origin.

This study investigated the extent to which multidrug resistance (MDR) among Enterobacteriaceae is related to DNA elements called "integrons," whether the relationship is species dependent or origin dependent, and which resistance patterns are associated with integrons. Analysis of 867 nonrepeat isolates comprising 8 species and originating from the community and 23 European hospitals showed a significant relation between MDR and integrons, independent of species or origin. Although resistance to each tested antimicrobial agent was significantly associated with integrons, only resistance to sulfamethoxazole, cotrimoxazole, gentamicin, tobramycin, ampicillin, piperacillin, and cefuroxime predicted the presence of integrons. Combined resistance to both ampicillin and sulfamethoxazole-trimethoprim was the starting point for the development of resistance to additional beta-lactams, aminoglycosides, cephalosporins, and ciprofloxacin, a development paralleled by an increasing prevalence of integrons. The acquisition of resistance genes is not random, and the transfer of integron-carrying elements plays a dominant role in the development of MDR by Enterobacteriaceae.

Anti-Bacterial Agents↗
PubMed1992

Molecular epidemiology of endemic ciprofloxacin-susceptible and -resistant Enterobacteriaceae.

OBJECTIVES: To elucidate the epidemiology of widespread ciprofloxacin resistance in our Veterans Affairs medical center using whole cell DNA analysis. DESIGN: In vitro study of ciprofloxacin resistant and susceptible Enterobacteriaceae isolated during the course of a clinical epidemiologic study of quinolone resistance. SETTING: Veterans Affairs Medical Center with acute care and long-term care divisions. RESULTS: We examined 40 ciprofloxacin-resistant strains of Serratia marcescens, Proteus mirabilis, and Providencia stuartii using restriction endonuclease analysis of whole cell DNA and compared them with concomitantly isolated ciprofloxacin sensitive strains. We sought to determine whether resistant strains were identical to susceptible strains, indicating in vivo emergence of resistant strains from susceptible strains, and whether resistant strains were shared among patients. All 26 ciprofloxacin-resistant S marcescens isolates shared a single ecoRI restriction pattern. Multiple patterns were seen in the ciprofloxacin-susceptible S marcescens isolates; however, several isolates had a pattern matching that of the resistant isolates. Similar results were seen among the P mirabilis isolates. Three different ecoRI patterns were found among the ciprofloxacin-resistant P stuartii isolates; none matched those found among the susceptible isolates. The frequency of spontaneous emergence of ciprofloxacin resistance in susceptible S marcescens strains with restriction pattern matching that of the resistant strains was significantly higher than that of nonmatching strains. CONCLUSIONS: Ciprofloxacin-resistant strains of Enterobacteriaceae became widespread within a short period of time. Resistant strains of S marcescens and P mirabilis arose from endemic susceptible strains. The resistant strain of S marcescens appeared to arise from a susceptible strain with a relatively high frequency of spontaneous ciprofloxacin resistance.

Case-Control Studies↗