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Laboratory approaches to the identification of Enterobacteriaceae.

There are numerous approaches to the identification of the Enterobacteriaceae in the clinical laboratory. Accuracy in identifying organisms is related directly to the skill and experience of the user and to the number of tests inoculated. Many of the commercially available kits offer reproducibility, convenience, accuracy, and ease of storage, particularly for smaller hospital laboratories that have neither the facilities nor the expertise to prepare and exert quality control over microbiologic media. However, none of the kits offers the accuracy and few offer the flexibility available through the use of conventional media, the variety and duration of incubation of which are virtually unlimited. The proper use of any system requires training because accurate identification requires the concurrent examination of colonial morphology and biochemical reactions-manufacturer's flow charts, reaction tables, or printed recognition systems notwithstanding. Microbiologists should be wary of new systems before impartial evaluations have been performed and published in scientific journals; manufacturers' technical literature and salesmen's presentations are, for obvious reasons, seldom objective.

Bacteriological Techniques↗

Species differences in plasmid carriage in the Enterobacteriaceae.

Modern concerns about the spread of antibiotic resistance raise questions about the effect of bacterial species on plasmid evolution and maintenance. We studied 223 Enterobacteriaceae isolated from wild mammals and determined the number of plasmids per isolate, the size of those plasmids, and the distribution of plasmid incompatibility groups N, P, W, FII, and A/C. All of these variables were non-randomly distributed with respect to bacterial species, suggesting that host-cell factors constrain the plasmids that a strain will carry. The implication for the evolution of multiple-resistance plasmids in a clinical setting is that although inter-generic plasmid transfer may introduce a novel resistance plasmid into a bacterial genus, it is likely to be modified to suit the requirements of the new host cell. This then further suggests that resistance plasmids will evolve independent lineages within bacterial species although the genes incorporated in them may have come from the same original source.

Animals↗

Enterobacteriaceae found in high numbers in fish, minced meat and pasteurised milk or cream and the presence of toxin encoding genes.

Enterobacteriaceae were found in high numbers after storage at 7 degrees C in 6% of consumers packs of pasteurised milk or cream, in 31% of retailed fish and in 100% of retail packs of minced meat. Seventy two fresh-water fishes, 40 packs of minced meat and 430 milk packs were sampled. One hundred and eighty four isolates were randomly picked from Tryptone glucose extract (TGE) agar (30 degrees C for 3d) or Violet red bile glucose (VRBG) agar (37 degrees C for 1d). In minced meat, Serratia liquefaciens, Hafnia alvei, Rahnella aquatilis were frequently encountered. On fish, the most frequently found species were R. aquatilis, and in milk, the dominating species were S. liquefaciens, H. alvei and R. aquatilis. One to three isolates of Citrobacter freundii were found in all three food categories. Using a polymerase chain reaction (PCR) technique, the gene of Escherichia coli heat-labile toxin (lt) was indicated in one fish isolate of R. aquatilis whereas heat-stable toxin genes (s.t.) were indicated in four H. alvei isolates, two originating from fish and two from minced meat. Positive PCR-reaction for vero cytotoxin genes were found in one H. alvei strain originating from fish (vt1), in two S. liquefaciens strains from minced meat (vt2), and in a C. freundii reference strain. One of the st-positive H. alvei strains from meat harboured the eaeA gene involved in the attaching phenotype of enteropathogenic E. coli.

Animals↗

A sensitive method for demonstration of decarboxylase activities amongst Enterobacteriaceae without use of pH indicators.

Lysine decarboxylase and ornithine decarboxylase activities have been demonstrated in a range of Enterobacteriaceae and related taxa by methods not involving pH indicators. Detection of the diamines produced, cadaverine and putrescine, was achieved by use of reagents based on 5-nitrosalicylaldehyde in the presence of triethanolamine and nickel ions. The method has been shown to be a simple and sensitive means for demonstration of decarboxylase activities when organisms were grown in the presence of the appropriate amino acid. Formation of highly coloured insoluble metal chelates was indicative of enzymatic activity. Results correlated well with existing indicator methods while being superior to the latter with respect to interpretation.

Cadaverine↗

Detection of specific bacterial enzymes by high contrast metal chelate formation. Part I. 8-Hydroxyquinoline-beta-D-glucoside, an alternative to aesculin in the differentiation of members of the family Enterobacteriaceae.

8-Hydroxyquinoline-beta-D-glucoside has been shown to be a useful alternative substrate for the differentiation of members of the family Enterobacteriaceae based on the presence or absence of beta-glucosidase. In an identification protocol based on multipoint-inoculated plates 8-hydroxyquinoline-beta-D-glucoside when incorporated into an agar based medium supplemented with a ferric salt produced intense black pigmentation localised solely in the immediate vicinity of beta-glucosidase positive organisms. In contrast, using conventional aesculin plates, positive organisms produced a brown ferric-aesculetin complex, the diffusibility of which limited interpretation especially where large numbers of colonies were involved. Almost complete agreement was found when 1964 routine 'coliform' organisms were tested by both methods. The method described allows use of a 36 place 'minipin' inoculator without any danger of 'nearest neighbour' interference and constitutes a cost-effective method suitable for either manual or automatic reading.

Chemical Phenomena↗

Dissemination of gentamicin-resistant plasmids among strains of Enterobacteriaceae.

The frequency of gentamicin-resistance among strains of many species of Enterobacteriaceae isolated from in-patients in our hospital has increased considerably since 1979. To clarify the reason for this increase, we tried to detect R-plasmids conferring gentamicin-resistance in all of the isolates in 1981 and found that most of them had conjugative R-plasmids. For epidemiological purposes we classified these into types I to X and 'others' by the following criteria: the nature of the resistances conferred; their incompatibility group; their molecular weight and the pattern of the DNA fragments after digestion with restriction endonucleasis. In the urology department, type-I plasmids conferring resistance to gentamicin, kanamycin, tobramycin and amikacin, and type-II plasmids conferring resistance to gentamicin, kanamycin and tobramycin were detected in the isolates belonging respectively to six species and twelve biotypes, and four species and six biotypes. In the second surgery ward, isolates belonging to six species and twelve biotypes were found to have type-III plasmids conferring resistance to gentamicin, kanamycin, tobramycin and chloramphenicol. Our investigation shows that not only antibiotic-resistant strains but also R-plasmids may be disseminated in hospitals.

Conjugation, Genetic↗

Phylogenetic position of phytopathogens within the Enterobacteriaceae.

The almost complete 16S rDNA sequences of twenty nine plant-associated strains, representing species of the genera Erwinia, Pantoea and Enterobacter were determined and compared with those of other members of the Enterobacteriaceae. The species of the genus Erwinia may be divided into three phylogenetic groups. Cluster I represents the true erwinias and comprises E. amylovora, E. mallotivora, E. persicinus, E. psidii, E. rhapontici and E. tracheiphila. We propose to unite the species of cluster II, E. carotovora subsp. atroseptica, E. carotovora subsp. betavasculorum, E. carotovora subsp. carotovora, E. carotovora subsp. odorifera, E. carotovora subsp. wasabiae, E. cacticida, E. chrysanthemi and E. cypripedii in the genus Pectobacterium respectively as P. carotovorum subsp. atrosepticum comb. nov., P. carotovorum subsp. betavasculorum comb. nov., P. carotovorum subsp. carotovorum comb. nov., P. carotovorum subsp. odoriferum comb. nov., P. carotovorum subsp. wasabiae comb. nov., P. cacticidum comb. nov., P. chrysanthemi and P. cypripedii. The species E. alni, E. nigrifluens, E. paradisiaca, E. quercina, E. rubrifaciens and E. salicis, comprising cluster III, are being classified into a new genus Brenneria gen. nov. respectively as B. alni comb. nov., B. nigrifluens comb. nov., B. paradisiaca comb. nov., B. quercina comb. nov., B. rubrifaciens comb. nov. and B. salicis comb. nov. The species of the genus Pantoea, included in this study, form a monophyletic unit (cluster IV), closely related with Erwinia, whereas the three phytopathogenic species of the genus Enterobacter are scattered among the genera Citrobacter and Klebsiella.

Bacterial Typing Techniques↗

Evaluation of MicroScan ESBL confirmation panel for Enterobacteriaceae-producing, extended-spectrum beta-lactamases isolated in Japan.

We assessed use of the MicroScan ESBL confirmation panel (Dade Behring, Tokyo, Japan) for the detection of eight Enterobacteriaceae-producing extended-spectrum beta-lactamases (ESBL) species. Of 137 bacterial strains isolated from patients in 32 hospitals in the Kinki area of Japan, 91 produced ESBL and comprised 60 bacteria (of E. coli, K. oxytoca, and K. pneumoniae) targeted by the NCCLS ESBL test and 31 non-target bacteria such as chromosomal AmpC-producing bacteria (e.g., Serratia marcescens, Enterobacter spp.). Sensitivity and specificity of the MicroScan panel for the target bacteria were 92% and 93%, respectively; sensitivity and specificity for non-target bacteria were 52% and 100%, respectively. There were 20 ESBL-positive strains that were not inhibited by clavulanic acid in the MicroScan panel (3 of 32 ESBL-producing E. coli strains, 1 of 24 K. pneumoniae, 1 of 4 K. oxytoca, 8 of 13 E. cloacae, and 7 of 14 S. marcescens), and most of them were bacteria not targeted by the NCCLS test. In 19 of the 20 strains, the synergy effect of clavulanic acid was observed in the modified-double-disk synergy test using only the cefepime-disk. Because these strains had high MICs of > or = 16 microg/ml for cephamycins such as cefoxitin and cefmetazole, these strains might produce high levels of AmpC in addition to ESBL. The MicroScan ESBL confirmation panel showed excellent performance in detecting target, but not other bacteria. Addition of cefepime and clavulanic acid to the MicroScan panel may significantly improve detection of non-target bacteria.

Anti-Bacterial Agents↗

A putative insect intracellular endosymbiont stem clade, within the Enterobacteriaceae, infered from phylogenetic analysis based on a heterogeneous model of DNA evolution.

Insect intracellular symbiotic bacteria (intracellular endosymbionts, or endocytobionts) were positioned within the gamma 3-Proteobacteria using a non-homogeneous model of DNA evolution, allowing for rate variability among sites, for GC content heterogeneity among sequences, and applied to a maximum likelihood framework. Most of them were found to be closely related within the Enterobacteriaceae family, located between Proteus and Yersinia. These results suggest that such a bacterial group might possess several traits allowing for insect infection and the stable establishment of symbiotic relationships and that this could represent a stem clade for numerous insect endocytobionts. Based on the estimations of the equilibrium GC content and branch lengths in the phylogenetic tree, we have made comparisons of the relative ages of these different symbioses.

Animals↗

Detection of aminopeptidases in Enterobacteriaceae by three simple chromogenic tests.

The action of 907 strains belonging to the various genera and species of the family Enterobacteriaceae were assayed on three chromogenic substrates used for the detection of aminopeptidases. The observed patterns of reactions may be useful for the biochemical identification of some serovars and biovars, particularly in the genus Shigella.

Aminopeptidases↗

In vitro efficacy of six cephalosporins tested against Enterobacteriaceae isolated at 38 North American medical centres participating in the SENTRY Antimicrobial Surveillance Program, 1997-1998.

The SENTRY Antimicrobial Surveillance Program is an ongoing international collaboration that monitors the predominant bacterial and fungal pathogens and antimicrobial susceptibility patterns associated with community-acquired and nosocomial infections. SENTRY data on the current cephalosporin susceptibility patterns (1997-98) of North American isolates of clinically important Enterobacteriaceae were analyzed. Susceptibility to a selection of cephalosporins was assessed at a central laboratory using reference broth microdilution methods and interpretive criteria specified by the National Committee for Clinical Laboratory Standards. The third- and fourth-generation cephalosporins tested demonstrated excellent activity against Escherichia coli and Klebsiella pneumoniae, whereas some of the older agents maintained good efficacy. Extended spectrum beta-lactamases were detected in all regions of the United States and Canada (1.8-10.7%). Cefepime was the most active agent tested against pathogens with the potential for enzyme-mediated resistance due to Amp C. The third-generation agents maintained acceptable efficacy against Serratia marcescens, but were less effective against Citrobacter and Enterobacter species. The older cephalosporins were generally inadequate against these pathogens, in contrast to cefepime, which was the widest spectrum cephalosporin overall. Some significant regional variations in spectrum were detected.

Cefepime↗

Comparative antimicrobial spectrum and activity of BMS284756 (T-3811, a desfluoroquinolone) tested against 656 Enterobacteriaceae, including preliminary in vitro susceptibility test comparisons and development.

BMS284756 (T-3811), a novel des-F(6)-quinolone, was evaluated using isolates of Enterobacteriaceae from the SENTRY Antimicrobial Surveillance Program tested by Etest (AB BIODISK, Solna, Sweden), reference broth microdilution and disk diffusion (5-microg) methods. Ciprofloxacin, levofloxacin, gemifloxacin and gatifloxacin were also tested by broth microdilution as comparator antimicrobial agents within the same drug class. The 656 isolate collection included species from the genera Citrobacter, Enterobacter, Escherichia, Hafnia, Klebsiella, Morganella, Pantoea, Proteus, Providencia, Salmonella, and Serratia. BMS284756 was slightly less active than comparison fluoroquinolones against these isolates (MIC(90), 4 mg/l versus 0.06-2 mg/l). However, at a proposed susceptible breakpoint of < or =4 mg/l, 90.7% of the isolates processed were susceptible to BMS284756, demonstrating an equivalent spectrum of activity to all other agents except gemifloxacin (86.6%). In general, isolates requiring >4 mg/l of BMS284756 for inhibition of growth were also less susceptible to the comparators suggesting cross-resistance is common between des-F(6)- and fluoro-quinolones. Excellent correlation was observed between broth microdilution MIC results and 5-microg disk zone diameters (r=0.94), and between broth microdilution dilution and Etest MIC values (r=0.96). In conclusion, BMS284756 has an activity and spectrum similar to contemporary fluoroquinolones and in vitro test methods (NCCLS, Etest) appear accurate and reproducible

Anti-Infective Agents↗

Study of beta-lactam resistance in ceftazidime-resistant clinical isolates of Enterobacteriaceae.

Mechanisms and transferability of beta-lactam resistance in 50 ceftazidime resistant strains of Enterobacteriaceae was studied. These strains were selected from 1991 E. coli, 1035 Enterobacter spp., 168 Citrobacter spp. and 1371 Klebsiella spp., isolated from patients hospitalized in ICUs and in the pediatric and urology departments of six hospitals in Bratislava during the years 1994-1996. The selected strains expressed the resistance not only to ceftazidime (50/50) but also to ampicillin (50/50), ceftriaxone (50/50), cefotaxime (49/50) and cefoxitin (45/50). The mechanism of resistance in all 50 strains was the production of beta-lactamases by conjugation, using either ceftazidime or cefotaxime for the selection of transconjugants and by isolation of R-plasmids ranging from to 55-87 kb from donor strains and from transconjugants. A total of 21 isolates possessed chromosomally encoded resistance and 25 clinical isolates and their transconjugants expressed ESBL sensitive to clavulanate. Selected E. coli and Klebsiella pneumoniae isolates expressed the presence of TEM and SHV enzymes determined by isoelectric focusing. The possible trends in the development of antimicrobial resistance in Slovakia in the future are indicated.

Anti-Bacterial Agents↗

Evaluation of the Titertek-Enterobac-Automated System (TTE-AS) for identification of members of the family Enterobacteriaceae.

The Titertek-Enterobac-Automated System (TTE-AS, Flow Laboratories, Meckenheim, FRG) was evaluated for identification of members of the family of Enterobacteriaceae using a total of 816 bacterial strains obtained from stock cultures and clinical specimens. A comparison of the results of the TTE-AS kit with conventional methods was conducted. Identifications were classified as correct, not identified (two or more taxa possible, identification score less than 80%, supplementary tests for final identification were not performed), and incorrect. Correct identification results were further subdivided according to the level of identification by species or biotype: (a) greater than or equal to 98%, (b) 90% to 97%, (c) 80% to 89%. When compared with conventional identification results, the TTE-AS system correctly identified 95.3% (778 of 816 strains), 90.0% (734 strains) belonging to category (a), 3.4% (28 strains) belonging to category (b), and 2.0% (16 strains) belonging to category (c). For 18 (2.2%) of the remaining strains, the system generated the correct identification result, but with an identification level less than 80% (low discrimination results). 11 strains (1.3%) were misidentified and the system failed to generate identification results with 9 strains (1.1%). Reactions within TTE-AS were reproducible with an estimated probability of erroneous test results of 1.7%.

Enterobacteriaceae↗

A note on some Enterobacteriaceae from the faeces of small wild British mammals.

Over a four-year period 878 samples of faeces were collected from five species of small wild rodents and two species of shrews at sites in England, Scotland and Wales. A search was made for Salmonella in these samples but no isolations of this genus were made. Several other genera of Enterobacteriaceae were identified.

Animals↗

Restriction endonuclease characterization of resistant plasmids in Enterobacteriaceae isolated from children in the Sudan.

The investigation of plasmid similarity is an important component in the surveillance of antimicrobial resistance and in the detection of epidemic plasmids. The use of restriction endonucleases in the classification of transferable, multiply-resistant plasmids from faecal Enterobacteriaceae isolated at the Children's Emergency Hospital, Khartoum was investigated. Twenty-four transconjugant plasmids, coding for 11 different resistance patterns, each of molecular weight 62 MDa, were studied using four restriction enzymes; Pst I, EcoR I, Hind III and Ava II. Fifteen different digest profiles were obtained. Restriction profiles discriminated between plasmids with differing resistance patterns and demonstrated homology of plasmids with common resistance patterns. Restriction endonuclease digest patterns provide a potentially rapid and reproducible method of plasmid classification, that could contribute towards surveillance systems in tropical countries with a high prevalence of antimicrobial resistance.

Child↗

Study of colonization resistance for Enterobacteriaceae in man by experimental contamination and biotyping as well as the possible role of antibodies in the clearance of these bacteria from the intestines.

The colonization resistance (CR) of the digestive tract was determined in 10 healthy volunteers by oral contamination with a neomycin resistant Escherichia coli (NR-E. coli) strain and measurement of the faecal concentration of this strain during 14 days after the contamination. This 'gold standard' was compared with another parameter of CR; the determination of the mean number of different biotypes of Enterobacteriaceae isolated from four faecal samples per volunteer. Both measures are significantly correlated (P less than 0.01). The NR-E. coli strain could be cultured from faecal samples of 4/10 volunteers as long as 300 days after contamination. Serum antibody titres against endogenous E. coli strains and the NR-E. coli strain used for experimental oral contamination were measured by an indirect immunofluorescence (IIF) assay. The assay was read by a video camera connected to an image processing system. The 95% confidence limits of antibody titres (log2) against endogenous E. coli strains ranged between less than 3 and 7.1 for IgA, between less than 3 and 8.7 for IgG and between less than 3 and 7.4 for IgM. Antibody titres against the NR-E. coli4 strain were within this (normal) range. The serum antibody titres against the NR-E. coli strain increased slowly after oral contamination, especially IgG and IgM. Little increase in IgA titres could be observed. An increase of serum antibody titres did not correlate with the elimination of the oral contaminant from the intestines. Therefore, we conclude that the CR is not IgG nor IgM antibody mediated.

Adult↗

Plasmids mediating resistance to gentamicin and other antibiotics in Enterobacteriaceae from four hospitals in Melbourne.

In twenty-nine gentamicin-resistant strains (mostly Klebsiella species) isolated from four Melbourne hospitals over a six month period, the genes for gentamicin resistance were carried on conjugative plasmids belonging to the rare incompatibility group L. These plasmids conferred resistance to gentamicin and sisomycin but not to amikacin or tobramycin, and were inferred to specify the enzyme aminoglycoside 3-N-acetyltransferase (AAC(3)I or GAT I). In addition, they specified resistance to a number of other antibiotics, including ampicillin and carbenicillin, chloramphenicol, kanamycin and neomycin, spectinomycin and streptomycin, sulphonamides and tetracyclines. Although they had a wide host range amongst the Enterobacteriaceae, none of these plasmids appeared able to transfer to Pseudomonas.

Anti-Bacterial Agents↗