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[Consensus for antimicrobial susceptibility testing for Enterobacteriaceae. Subcommittee on Antimicrobials, SADEBAC (Argentinian Society of Clinical Bacteriology), Argentinian Association of Microbiology].

Taking into account previous recommendations from the National Committee for Clinical Laboratory Standards (NCCLS), the Antimicrobial Committee, Sociedad Argentina de Bacteriología Clínica (SADEBAC), Asociación Argentina de Microbiología (AAM), and the experience from its members and some invited microbiologists, a consensus was obtained for antimicrobial susceptibility testing and interpretation in most frequent enterobacterial species isolated from clinical samples in our region. This document describes the natural antimicrobial resistance of some Enterobacteriaceae family members, including the resistance profiles due to their own chromosomal encoded beta-lactamases. A list of the antimicrobial agents that should be tested, their position on the agar plates, in order to detect the most frequent antimicrobial resistance mechanisms, and considerations on which antimicrobial agents should be reported regarding to the infection site and patient characteristics are included. Also, a description on appropriate phenotypic screening and confirmatory test for detection of prevalent extended spectrum beta-lactamases in our region are presented. Finally, a summary on frequent antimicrobial susceptibility profiles and their probably associated resistance mechanisms, and some infrequent antimicrobial resistance profiles that deserve confirmation are outlined.

Anti-Bacterial Agents↗

Extended-spectrum beta-lactamase-producing Enterobacteriaceae at Middlemore Hospital.

AIMS: To review patients colonised or infected with extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBLPE) at Middlemore Hospital, Auckland, New Zealand. METHODS: All patients who had an ESBLPE isolated at the Middlemore Hospital Microbiology Laboratory from January 2001 to June 2004 were included in this review. RESULTS: ESBLPE were isolated from 132 patients during the review period. There were 12 patients colonised or infected with an ESBLPE in 2001, 34 in 2002, 43 in 2003, and 43 in the first 6 months of 2004. The isolates were Escherichia coli (n=56), Enterobacter spp. (n=55), and Klebsiella pneumoniae (n=21). ESBLPE were isolated from a wide range of specimens including peripheral blood in 18 patients. Thirty-three (25%) patients had an ESBLPE isolated within 48 hours of admission; seven of these patients were neither long-term care facility (LTCF) residents nor had hospital admissions in the previous 6 months. Thirty-one patients (23%) resided in a LTCF before their admission; four patients from the same LTCF had indistinguishable isolates. All isolates tested were susceptible to meropenem and imipenem. All but one isolate tested was susceptible to ertapenem, and all but two were susceptible to amikacin. CONCLUSIONS: Colonisation and infection due to ESBLPE are increasing at Middlemore Hospital and in the Auckland community. We expect this trend to continue. There is evidence to suggest transmission of ESBLPE both in the Auckland community and LTCFs. Antibiotics useful for treatment of patients with proven ESBLPE infection at Middlemore Hospital include amikacin or a carbapenem. Careful infection control practices and antibiotic prescribing will be necessary to reduce the rate of increase of ESBLPE colonisation and infection at Middlemore Hospital and in the Auckland community.

Adolescent↗

[Experience and problems with biochemical differentiation of Enterobacteriaceae with microtiter plates].

A self-made miniaturized test system for the biochemical identification of enterobacteriaceae in microtitre plates has been examined. Special emphasis was laid on easy and unequivocal interpretation of the reactions. This was achieved among other things by including further sugar fermentations. Applying a corresponding reaction matrix it is possible to get clear identifications, even in the case of missing test results. The performed tests show the usefulness of our test system.

Bacteriological Techniques↗

[Application of a computer program for identifying different genera and species of the Enterobacteriaceae family in clinical bacteriology].

Computer programs were developed for the selection of a minimum set of biochemical tests that allow the identification of the species of Enterobacteriaceae with major clinical significance. The system proposed consists of nine conventional biochemical tests, the results of which are interpreted with the help of a numeric code. This selects the most probable species for each result and, when necessary, additional tests can be performed to confirm the identification proposed. The system (SYS9E) was used in the identification of 66 strains of nosocomial origin. The results were compared with those of commercial systems.

Bacteriological Techniques↗

Enterobacteriaceae associated with animals in health and disease.

Members of at least eight of the 14 general of the Enterobacteriaceae are found associated with animals. A large number of these are present as members of the microbiota of the gut of many animals without causing harmful effects. Indeed, their presence as part of the stable flora contributes to the defences of the animal by excluding enteric pathogens by competitive exclusion which contributes to the phenomenon of [colonization resistance' (van der Waaij 1983). some of these strains can cause disease if they contaminate a normally 'sterile' site such as the body cavities. In contrast, particular strains within each bacterial species are overt pathogens and are regularly associated with disease. Nevertheless, even these may colonize the animal without producing clinical illness. In this paper E. coli and Salmonella spp. have been used to illustrate the various aspect of host-parasite relationships.

Animals↗

[Evaluation of two miniaturized systems widely used in the laboratories for the identification of the "Enterobacteriaceae" (author's transl)].

We have attended to a comparative research between two commercial microsystems: Enterotube and Minitek in order identify the Enterobacteriaceae and a reference system given by the combination of the usual macromethods already used in our laboratory. We have examined 401 bacterial cultures of Gram bacillus which we trought to belong to Enterobacteriums, coming from clinical material (excrements, urine, pharyngeal swabs, vaginal swabs, urethral swabs and espectoration) we have received for the bacteriological diagnosis. 390 of 401 cultures have shown to be Enterobacteriums. The biochemical reactions they have given show that the Enterotube and the Minitek have, with the usual system a good accordance for the following tests: dextrose (acid and gaz) lysin and ornithine decarboxylase, production of H2S and indole, phenylalanine deaminase and urease; while we have some statistically significant discordances for the fermenting of lactose and the use of citrate. We have also significant discordances E/C for the fermenting of dulcitole while the ones of Minitek are acceptables. The notes recommend the use, in the specialized bacteriological laboratories, of the conventional tests.

Bacteriological Techniques↗

[Use of the API 20E system for rapid identification of Enterobacteriaceae (six hours) (author's transl)].

The authors propose a modification of the method of use of the API 20E system permitting more rapid identification of Enterobacteriaceae within six hours (3 hours preculture and 3 hours incubation on an API 20E plate) it was possible to identify correctly 67% of 192 strains studied at species level and 75.5% studied at generic level. One may note four mistakes (2.1%) of which 3 were minor, (species within the same genus). The construction of a base of numerical data adapted to the technic within six hours would no doubt permit us to reduce the percentage undetermined.

Enterobacteriaceae↗

Combination effect of cefmenoxime with four aminoglycosides on Enterobacteriaceae susceptible and moderately susceptible to this new cephalosporin.

The inhibitory combination effect of cefmenoxime with gentamicin, tobramycin, netilmicin and amikacin against 148 Enterobacteriaceae was compared using the checkerboard agar dilution technique. On average 41% of cefmenoxime-moderately susceptible (MIC between 2 and 32 micrograms/ml) or cefmenoxime-resistant (MIC greater than 32 micrograms/ml) strains were inhibited by synergistic, and 23,5% by partial synergistic cefmenoxime-aminoglycosides combinations. Against cefmenoxime-susceptible strains, these rates of synergy or partial synergy were twofold lower, but only one isolate was not susceptible to these combinations. The amikacin-cefmenoxime interaction was the most performant.

Amikacin↗

Incidence of enteritis of Enterobacteriaceae isolates possessing human colonization factor antigen. New human colonization factors.

Of 462 Enterobacteriaceae strains including 435 Escherichia coli isolated from 250 patients, 298 haemagglutinating (HA) cultures were classified into 36 different HA groups. Sixteen of them belonged to Evan's I or II groups, although none possessed CF I or CF II antigen detectable by slide agglutination. Seventy-seven strains showed 4+ mannose resistant (MR) HA with human (53), bovine (2), chicken (6), guinea pig (7) or human and guinea pig (9) erythrocytes. These strains were significantly more frequent in patients under one year of age. Eighty-eight percent of the typable strains belonged to E. coli serogroups O1, O2, O4, O6, O18. HA positivity and fimbrial structures were correlated in 2 isolates (15/1, O18a, c:-K77: H-; 12/2/1 O1: K1: H .). Fimbriae of the two strains exhibited adhesive properties. Their fimbrial antigens differed serologically from each other and from those of the reference strains H 10407 and PB 176. Forty-nine of 4+ human MRHA strains showed variable reactions in the two sera for the new fimbrial antigens.

Antigens, Bacterial↗

Comparison of six systems for the identification of Enterobacteriaceae.

Six widely used commercial systems for Enterobacteriaceae identification (API 20 E, API 10, Enterotube, Enterotube II, Micro-ID and Minitek) were evaluated at the same time by testing 611 organisms. Conventional media were used for comparison. API 20 E, Enterotube II and Micro-ID were highly accurate and identified about 91% of the strains to the species level. The remaining organisms required additional tests and only a small minority was misidentified. Percentages of correct identification to the species level were 87.2 for Enterotube, 74.9 for API 10 and 67.1 for Minitek. Enterotube, Enterotube II and Micro-ID were, in our hands, the easiest systems to operate.

Bacteriological Techniques↗

Evaluation of API 20E System and Encise Enterotube for the identification of Enterobacteriaceae of animal origin.

The API 20E System and the Encise Enterotube were evaluated for the identification of the Enterobacteriaceae isolated from clinical specimens of animal origin at a veterinary diagnostic laboratory. Compared to conventional tubed media, the API 20E System identified 235 of 240 isolates (97.9%) correctly. The Encise Enterotube correctly identified 229 of the 240 isolates (95.4%). Thus, both these identification systems could be used to replace conventional methods for identifying members of this family isolated from animal origin.

Animals↗

[An oral enteritis-vaccine composed of twelve heat-inactivated Enterobacteriaceae 3. Communication: studies on efficacy tests in mice protection tests (author's transl)].

The polyvalent vaccine consists of twelve heat-inactivated species of Enterobacteriaceae (six strains of Salmonellae, two strains of Shigellae, four strains of Dyspepsia coli). The above vaccine is administered orally (6) to man for prophylactic purposes against local infections. The present communication describes the efficacy results of the vaccine obtained for different parameters by the mouse protection test. For this purpose, seven different infection models were used: oral infection with a strain of S. typhimurium and a strain of S. enteritidis, respectively, and intraperitoneal infection with the following five strains: S.l typhimurium, S. panama, S. enteritidis, Sh. flexneri 2a, or E. coli 2380. For 10 days the mice were daily immunized with the twelve-fold vaccine orally administered by means of a probang. On the 10th day after the last orally applied antigen, the animals were challenged with the seven strains mentioned above. The success of vaccination was determined by the difference of mortality between vaccinated and non-vaccinated mice. The results show (Table 1) following the vaccination that a significant effect could be observed and statistically be evaluated for 6 models of infection. Optimal values showing the highest consistency rate were found for the model of intraperitoneal infection with the strain of E. coli 2380. Which is why, studies were made for the following parameters by using the latter mode of infection. The efficacy of the oral immunization depends on the dosage of the vaccine (Table 2). Even at a dilution of 1 : 1000, the effect of the vaccine was still sufficient. Only a dilution of 1 : 10 000 made the vaccination almost ineffective. - The content of humidity of the lyophilized vaccine in a range of 4% to 12% did not influence the immunogenicity (Table 3). - The protection obtained by vaccination was found to last unexpectedly long. The vaccinated mice were still well protected even one year after the oral vaccination (Table 4), which means - when referred to the life-span of mice - that protection is effected for almost a life-time. The lyophilized vaccine does not even loose its immunogenicity after storage at 22 degrees C and 40 degrees C over a period of 3 years (Table 5). These polyvalent lyophilized vaccine are, therefore, storable even under tropical conditions without cooling. This is a further great advantage of this vaccine.

Administration, Oral↗

[Taxonomic structure of Gram-negative bacteria from the family Enterobacteriaceae isolated from oncological patients with infectious complications].

The flora isolated from cancer patients in cases of infectious complications has been found to consist of Gram-negative bacteria. Of 414 isolated Gram-negative bacteria strains, 369 strains belonged to the family Enterobacteriaceae, 38 strains to the family Pseudomonaceae, and 7 strains to other families. Enterobacteria were mainly represented by bacteria of the tribes Escherichieae, Klebsielleae, Proteeae. The prominence of individual species in different infectious complication has proved to depend on their localization. Thus, Kl. pneumoniae show a distinct prevalence in purulent septic complications of the respiratory organs, in most of other infections E. coli have been found to play the dominating role. This study demonstrates the etiological role of enterobacteria in hospital infections in patients with malignant tumors.

Cross Infection↗

[An evaluation of the PATHOTEC system for identification of Enterobacteriaceae (author's tranls)].

One hundred species of Enterobacteriaceae were examined according to the PathoTec Rapid I-D system, and the results were compared to the results obtained by conventional bacteriological methods. The results of the examinations are given in Tables II, III, and IV. It is concluded that 97% agreement between the results of biochemical reactions is excellent, whereas 81% agreement between the diagnoses is not quite satisfactory; but it can be improved to 90% fairly easily, which would be a satisfactory result.

Bacteriological Techniques↗

Ceftriaxone. A new cephalosporin with aqueous humor levels effective against enterobacteriaceae.

One- or two-gram doses of ceftriaxone were administered intravenously to 30 patients before cataract extraction. With the 1-g dose, mean aqueous humor concentrations of 0.93 and 0.88 microgram/mL were found at approximately 2 and 12 hours after administration, respectively. With the 2-g dose, a mean level of 2.47 micrograms/mL was observed at two hours; levels of more than 2 micrograms/mL were found in two patients 13 hours after administration. Both the 1- and 2-g doses thus produce aqueous humor levels many times higher than the minimum inhibitory concentration of ceftriaxone for 90% of most Enterobacteriaceae, excluding Pseudomonas. Concentrations adequate for Staphylococcus aureus and Staphylococcus epidermidis were not, however, obtained.

Adult↗

The rfb genes in Azotobacter vinelandii are arranged in a rfbFGC gene cluster: a significant deviation to the arrangement of the rfb genes in Enterobacteriaceae.

We report the identification of rfbF and rfbC located adjacent to the previously identified rfbG (Gavini et. al. Biochem. Biophys. Res. Commun. 1997, 240, 153-161) from the non-symbiotic, non-pathogenic soil bacterium Azotobacter vinelandii. The rfbF open reading frame encodes a putative polypeptide of 256 amino acids. This polypeptide shares a homology of 74% with the RfbF of Synechocystis sp. and a 70% homology with the AscA of Yersinia pseudotuberculosis which function as alpha-D-glucose-1-phosphate cytidylyltransferases in the biosynthesis of the O-antigen. The rfbC encodes a putative polypeptide of 186 amino acids. It shows strongest homology to the RfbC of Synechocystis sp. (64%) and Salmonella typhimurium (40%). RfbC functions as a dTDP-4-Dehydrorhamnose 3,5-Epimerase. The genes identified here have a low G + C content (approximately 56%) as compared to the A. vinelandii chromosome (approximately 63%) which is characteristic of the rfb clusters identified in other bacteria and may be indicative of the acquisition of the rfb genes by interspecific gene transfer. Despite the high level of sequence conservation, the organization of the rfb genes in A. vinelandii deviates from the arrangement of the most thoroughly studied rfb gene clusters of Enterobacteriaceae.

Amino Acid Sequence↗

Occurrence of aminoglycoside phosphotransferase subclass I and II structural genes among Enterobacteriaceae spp. isolated from meat samples.

3'-Aminoglycoside phosphotransferase [APH(3')] enzymes are a group responsible for resistance to the antibiotics kanamycin (Km) and neomycin (Nm) in bacteria. Escherichia coli ECT24, originally isolated from a meat sample, harboured an 83-kb conjugative R-plasmid (pRPJ24) that carries transferable resistance to Km and Nm. Plasmid pRPJ24 was transferred by conjugation to Enterobacter cloacae 94R, which was used as the source of plasmid DNA in development of a probe for the Km-resistance determinant. Random cloning of BamHI and HindIII double-digest restriction fragments of pRPJ24 in the pUC18 vector plasmid produced clones resistant to both Nm and Km carrying a 1.9-kb DNA insert. Southern hybridization of pRPJ24 cloned chimeric plasmid DNA (pKPJ94) showed homology with the APH(3')II gene from transposon Tn5. A PstI digest of pKPJ94 produced a 920-bp fragment which hybridized with the APH(3')II structural gene, and was used as a DNA probe for the APH(3')II subclass gene. A 980-bp BamHI fragment from plasmid pGH54 carrying the APH(3')I gene from transposon Tn903 was used as a subclass I probe. Total DNA from 206 randomly screened Km-resistant Enterobacteriaceae isolates from raw ground beef and chicken meat samples were examined for the occurrence of APH(3') subclass I and II using non-radioactively-labelled DNA probes. Thirty-six percent and 60% of the isolates examined carried subclass I and II resistances, respectively, in the isolates from chicken meat samples. The corresponding values for bacterial strains from raw ground beef samples were 51% and 72%, respectively. Four percent of the resistant bacterial isolates from chicken samples did not display homology to either probe.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Objective quantitation of serum antibody titres against Enterobacteriaceae using indirect immunofluorescence, read by videocamera and image processing system.

A new way of measuring indirect immunofluorescence (IIF) of microscopic bacterial slide preparations by videocamera and an image processing system is presented. This method is compared with the conventional method of reading the slides by eye. The advantages of this new approach are objective reading, greater accuracy and easier performance. We have applied the method to measure serum antibody titres against endogenous Enterobacteriaceae. The method offers the opportunity to combine IIF with automatic morphological analysis, thereby maximally exploiting the possibilities of the immunofluorescence technique.

Antibodies, Bacterial↗