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PubMed2005

Evolution of TEM-type extended-spectrum beta-lactamases in clinical Enterobacteriaceae strains in Poland.

Seventeen extended-spectrum beta-lactamase (ESBL)-producing isolates of the family Enterobacteriaceae recovered from 1998 to 2000 in hospitals of five different cities in Poland were analyzed. They expressed several TEM-type ESBLs, TEM-4, TEM-29, TEM-85, TEM-86, TEM-93, and TEM-94. TEM-85 (L21F, R164S, E240K, T265M), TEM-86 (L21F, R164S, A237T, E240K, T265M), TEM-93 (M182T, G238S, E240K), and TEM-94 (L21F, E104K, M182T, G238S, T265M) were identified for the first time. Including the enzymes described earlier, TEM-47, TEM-48, TEM-49, and TEM-68, the group of known ESBLs of the TEM family produced by enterobacteria in Polish hospitals has increased to 10 variants. Comparative sequence analysis of the genes coding for all these beta-lactamases revealed a view of their possible evolution, which, apart from the gradual acquisition of various mutations, could also have involved recombination events. Two different bla(TEM-1) gene alleles were precursors of the ESBL genes: bla(TEM-1A), which was the ancestor of bla(TEM-93), and bla(TEM-1F), from which all the remaining genes originated. The evolution of the bla(TEM-1F)-related genes most probably consisted of three major separate lineages, one of which, including bla(TEM-4), bla(TEM-47), bla(TEM-48), bla(TEM-49), bla(TEM-68), and bla(TEM-94), was highly structured itself and could have been initiated by the bla(TEM-25) gene, identified exclusively in France so far. Plasmid fingerprinting analysis revealed a high degree of diversity of plasmids carrying related bla(TEM) genes, which suggested either the intense diversification or transposition of bla(TEM) genes between different plasmids or some contribution of convergent evolution. The results of this study clearly demonstrate that the environment of Polish hospitals has been highly favorable for the rapid evolution of ESBLs.

Cloning, Molecular↗
PubMed2005

Survey of Enterobacteriaceae producing extended-spectrum beta-lactamases in a Slovak hospital: dominance of SHV-2a and characterization of TEM-132.

Eighty-five extended-spectrum beta-lactamase-producing Enterobacteriaceae from a Slovak hospital have been studied. SHV-2a was predominant, but other variants have been detected, namely, SHV-5, SHV-12, TEM-12, TEM-15, and TEM-132, which differed from TEM-1 by amino acid substitutions R164H, E240K, and I173V and had kinetic properties similar to those of TEM-28.

Anti-Bacterial Agents↗
PubMed1976

Extrachromosomal deoxyribonucleic acid in R factor-harboring Enterobacteriaceae.

Extrachromosomal deoxyribonucleic acid (DNA) from 24 different R factor-harboring Enterobacteriaceae was isolated and characterized by analytical ultracentrifugation and electron microscopy. The R factors represented 15 different patterns of transferable drug resistance found in enterobacteria from an enclosed geographic area. All of the strains contained extrachromosomal, circular DNA molecules within the range of 0.4 to 52 mum. More than one size class of circular DNA molecules was observed in the majority of the extrachromosomal DNA preparations. The buoyant density of the extrachromosomal DNA ranged from 1.700 to 1.720 g/cm3. The majority of the bacteria contained extrachromosomal DNAs of various densities. Three-fourths of the R factors were classified as fi+. The investigation illustrates the extensive variability in the physical characteristics of plasmid DNA from R factor-harboring strains.

DNA, Bacterial↗
PubMed1963

EPISOME-MEDIATED TRANSFER OF DRUG RESISTANCE IN ENTEROBACTERIACEAE. VI. HIGH-FREQUENCY RESISTANCE TRANSFER SYSTEM IN ESCHERICHIA COLI.

Watanabe, Tsutomu (Keio University, Tokyo, Japan). Episome-mediated transfer of drug resistance in Enterobacteriaceae. VI. High-frequency resistance transfer system in Escherichia coli. J. Bacteriol. 85:788-794. 1963.-R-factors (transmissible drug-resistance factors composed of the resistance-transfer factor and markers of resistance to sulfonamide, streptomycin, chloramphenicol, and tetracycline) gave rise to a high-frequency transfer system in Escherichia coli K-12 F(-), F(+), and Hfr strains that was similar to the high-frequency colicinogeny transfer system (HFCT) found by Stocker and Ozeki in Salmonella typhimurium with colicinogenic factor I. The procedure for obtaining a high-frequency transfer system of R-factors was identical to that for HFCT. The majority of cells of the high-frequency resistance transfer system were composed of cells which had just received R-factors by conjugation. These cells could act as highly competent donor cells for further transfer of the R-factors by conjugation. The frequencies of transfer of R-factors were as high as 1.5 to 7.5 per resistant donor cell introduced into mixed incubation with drug-sensitive recipient cells for 1 hr at 37 C, whereas the usual donor cells could transfer the R-factors at frequencies of, at most, 1:30 under similar conditions of mixed cultivation. It was also found that the aerobic conditions for growth of donor cells rather reduced the frequencies of transfer of R-factors in the usual systems. Possible mechanisms of the high-frequency resistance transfer system are discussed.

Anti-Bacterial Agents↗
PubMed1964

SEPARATION BY ETHANOL OF COMMON AND SOMATIC ANTIGENS OF ENTEROBACTERIACEAE.

Suzuki, T. (Children's Hospital, Buffalo, N.Y.), E. A. Gorzynski, and E. Neter. Separation by ethanol of common and somatic antigens of Enterobacteriaceae. J. Bacteriol. 88:1240-1243. 1964.-Ethanol in a concentration of 85% permits separation from crude culture supernatant fluids of the common enterobacterial antigen and the O antigen, the former being ethanol-soluble, the latter being ethanol-insoluble. The evidence was obtained in hemagglutination, hemolysis, and hemagglutination-inhibition tests. In contrast to the crude antigens obtained from enteric bacteria other than Escherichia coli O14, the ethanol-soluble fraction is immunogenic in the rabbit upon intravenous injection, and antibodies against the common antigen are produced in relatively high titers. Aqueous mixtures of ethanol-soluble and -insoluble fractions engender antibodies against the common antigen in titers significantly lower than those stimulated by the soluble fraction alone. Ethanol-treated whole antigen fails to stimulate antibody formation against this antigen. These results suggest the presence of an inhibitor in the ethanol-insoluble fraction.

Animals↗
PubMed1979

Evaluation of Micro-Media Quad Panels for identification of the Enterobacteriaceae.

The Micro-Media Quad Enteric Panel and coding system were evaluated by comparing the identifications of 193 stock and 216 clinical isolates, representing 31 species of Enterobacteriaceae, with those provided by conventional methods. The results corresponded for 91% of the organisms if only the single or most probable identification was accepted and for 93% if the correct identification was listed as one of several possible, but not the most probable, identification by the coding system. With 73% of the isolates, the single identification provided by the coding system was correct, and in 20% of instances the correct identification was listed as the first of several choices in descending order of probability but requiring additional tests for differentiation. In most of the latter instances, these additional tests consisted of serological confirmation of salmonellae or shigellae. Discrepancies consisted of incorrect identifications (4%) and organisms with code numbers not appearing in the coding system (3%).

Bacteriological Techniques↗
PubMed1981

Kluyvera, a new (redefined) genus in the family Enterobacteriaceae: identification of Kluyvera ascorbata sp. nov. and Kluyvera cryocrescens sp. nov. in clinical specimens.

Kluyvera is proposed as a new genus for the group of organisms formerly known as Enteric Group 8 (synonym = API group 1). Strains of Kluyvera share the properties of most members of the family Enterobacteriaceae: they are gram-negative rods, motile with peritrichous flagella, catalase positive, and oxidase negative; they grow on MacConkey agar, ferment D-glucose with the production of acid and gas, and are susceptible to many antibiotics. Strains are usually indole positive, methyl red positive, Voges-Proskauer negative, citrate positive, H2S (triple sugar iron) negative, urea negative, phenylalanine deaminase negative, lysine decarboxylase positive, arginine dihydrolase negative, and ornithine decarboxylase positive. Kluyvera strains ferment many of the sugars and polyhydroxyl alcohols used in identification. By deoxyribonucleic acid-deoxyribonucleic acid hybridization, strains of Kluyvera were divided into three groups. Kluyvera ascorbata is proposed as the type species for the genus. Most strains of K. ascorbata have been isolated from clinical specimens. K. cryocrescens is proposed as the second species. It was occasionally isolated from clinical specimens, but it was isolated more commonly from the environment. Kluyvera species group 3 was heterogeneous, but was distinct from the two named species by deoxyribonucleic acid hybridization. This group was rare, so no species name will be proposed at this time. K. ascorbata can be differentiated from K. cryocrescens by its positive ascorbate test, inability to grow at 5 degrees C in a refrigerator, and smaller zones of inhibition around carbenicillin and cephalothin disks. The test normally used for identification does not clearly differentiate these two species. Kluyvera species are probably infrequent opportunistic pathogens. The most common source is sputum, where they are probably not clinically significant. Five strains have been from blood cultures. More information is needed about the incidence and clinical significance of the genus Kluyvera.

Base Composition↗
PubMed1985

Rapid microbiochemical method for presumptive identification of gastroenteritis-associated members of the family Enterobacteriaceae.

A method for rapid screening of isolates of pathogenic members of the family Enterobacteriaceae is described. Flow charts are used in conjunction with triple sugar iron agar, o-nitrophenyl-beta-D-galactopyranoside-phenylalanine-motility sulfate screening media, oxidase test, and six rapid biochemical tests, namely, lysine decarboxylase, urease, indole, esculin hydrolysis, malonate, and xylose. This scheme is used to provide an inexpensive but rapid presumptive identification of Salmonella, Shigella, Edwardsiella, Aeromonas, Plesiomonas, Vibrio, and Yersinia isolates from stool cultures.

Culture Media↗
PubMed1989

Enterobacter hormaechei, a new species of the family Enterobacteriaceae formerly known as enteric group 75.

The name Enterobacter hormaechei is proposed for a new species of the family Enterobacteriaceae, formerly called Enteric Group 75, which consists of 23 strains, 22 of which were isolated from humans. DNAs from 12 E. hormaechei strains tested were highly related to the type strain (ATCC 49162) by DNA hybridization, using the hydroxyapatite method (80 to 97% in 60 degrees C reactions; 80 to 90% in 75 degrees C reactions). The strains were most closely related (50 to 63%) to Enterobacter cloacae, Enterobacter dissolvens, Enterobacter taylorae, and Enterobacter nimipressuralis. E. hormaechei strains were positive within 48 h for the following: Voges-Proskauer test; citrate utilization (Simmons and Christensen); urea hydrolysis (87%); ornithine decarboxylase; growth in potassium cyanide (KCN); malonate utilization; production of acid from D-glucose, L-arabinose, cellobiose, dulcitol (87%), D-galactose, maltose, D-mannitol, D-mannose, L-rhamnose, sucrose, trehalose, and D-xylose; acid production from mucate; nitrate reduction; and o-nitrophenyl-beta-D-galactopyranoside. Delayed positive reactions were seen in tests for arginine dihydrolase, gas from D-glucose, acid from alpha-methyl-D-glucoside, and acetate utilization. E. hormaechei was negative in tests for indole production; H2S production; phenylalanine deaminase; lysine decarboxylase; gelatin hydrolysis; acid production from D-adonitol, D-arabitol, erythritol, glycerol, i(myo)-inositol, melibiose, raffinose, and D-sorbitol; esculin hydrolysis; DNase; lipase; and tyrosine clearing. Variable reactions occurred in tests for methyl red, motility, and tartrate. All strains tested were susceptible or moderately susceptible to amikacin, azlocillin, cefotaxime, ceftazidime, ceftriaxone, chloramphenicol, gentamicin, mezlocillin, moxalactam, piperacillin, trimethoprim-sulfamethoxazole, sulfisoxazole, thienamycin, tobramycin, and trimethoprim. All strains tested were resistant to nitrofurantoin; the majority were resistant to ampicillin, cefoxitin, and cephalothin. Four isolates were from blood; most other isolates were from wounds or sputum.

Adolescent↗
PubMed1991

Fimbrial types among respiratory isolates belonging to the family Enterobacteriaceae.

Bacterial attachment is believed to be an early step in gram-negative nosocomial pneumonia. The frequency of fimbria-associated adhesins among respiratory pathogens has not been studied in detail. In this study isolates belonging to the family Enterobacteriaceae, prospectively obtained from intensive care unit patients who were suspected of having nosocomial pneumonia, were examined for fimbria-associated adhesins. Type 3, P, type 1, and other fimbrial phenotypes were identified by specific hemagglutination and electron microscopy. The Klebsiella type 3 fimbrial phenotype was further characterized by using a monoclonal antibody. Also, both type 3 and Escherichia coli P fimbrial genotypes were detected by using DNA colony blot assays. The frequencies of genera or species isolated were as follows: Enterobacter (38.6%), Klebsiella (26.8%), Serratia (17.7%), E. coli (13%), and Proteus (5.2%). Isolates of Klebsiella oxytoca, K. pneumoniae, and Enterobacter cloacae most commonly possessed the type 3 fimbrial phenotype and genotype. The phenotype and genotype for E. coli P fimbriae (46.2 and 50%, respectively), a known pathogenic determinant in the urinary tract, were detected more frequently than expected. In addition, a previously unspecified hemagglutinin that was specific for porcine erythrocytes was almost uniformly expressed among isolates of Enterobacter aerogenes. Finally, the expression of the type 1 fimbrial phenotype was widely detected among the isolates tested but notably absent among K. oxytoca and Proteus mirabilis isolates. The frequency of the various fimbrial types identified suggests a role for these bacterial organelles in adherence to respiratory epithelia.

Bacterial Adhesion↗
PubMed1976

Analysis of cost and accuracy of alternative strategies for Enterobacteriaceae identification.

Analysis of the cost of time and material required for the diagnosis of Enterobacteriacea isolates indicated that a conventional 17-tube (20-test) setup costs $7.98 per isolated identified. Using the API 20E, a similar identification cost $3.02. A conventional 7-tube (10-test) setup cost $3.60, whereas the comparable cost by 30% while increasing the number of isolate identified correctly by 3%. Other strategres using the API 20E or a deoxyribounclease test were also evaluated for cost and accuracy.

Bacteriological Techniques↗
PubMed1993

Parallel comparison of accuracy of API 20E, Vitek GNI, MicroScan Walk/Away Rapid ID, and Becton Dickinson Cobas Micro ID-E/NF for identification of members of the family Enterobacteriaceae and common gram-negative, non-glucose-fermenting bacilli.

We compared the API 20E (21 h) (API; bioMérieux Vitek, Hazelwood, Mo.), the Vitek GNI card (4 to 18 h) (Vitek; bioMérieux Vitek), the identification portion of the MicroScan Walk/Away Rapid Neg Combo 3 panel (2 h) (W/A; Baxter Diagnostics, Inc., West Sacramento, Calif.), and the Becton Dickinson Cobas Micro ID-E/NF rotor (21 h) (Cobas; Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.), versus conventional biochemicals for their abilities to identify accurately 252 strains of biochemically typical and atypical members of the family Enterobacteriaceae and common non-glucose-fermenting gram-negative bacilli. All strains used were included in the data base of each product. At the end of the initial incubation, 194 (77.0%), 213 (84.5%), 198 (78.6%), and 192 (76.2%) strains were correct to the genus and species levels with the API, Vitek, W/A, and Cobas systems, respectively. After additional biochemical tests were performed, as directed by each manufacturer's protocol, the numbers of strains correctly identified to the genus and species levels were 241 (95.6%), 234 (92.8%), 243 (96.4%), and 230 (91.3%) with the four systems, respectively. The errors were random in all systems, with the exception of two atypical Salmonella enteritidis strains, each of which was misidentified by three systems. After the initial recommended incubation period, both API and Cobas were significantly less accurate than Vitek (Yates' corrected P < 0.05). No significant differences were noted between the results of Vitek and W/A or between the results of API and W/A. After additional tests were completed, Cobas was significantly less accurate than W/A (P < 0.05) but was equal in accuracy to Vitek and API. API, Vitek, and W/A were equal in accuracy after these same additional tests. All four systems were significantly more accurate after additional biochemical testing than after the initial reporting period (194 of 252 versus 241 of 252 for API, 213 of 252 versus 234 of 252 for Vitek, 198 of 252 versus 243 or 252 for W/A, and 192 of 252 versus 230 of 252 for Cobas [P<0.05]).

Bacteriological Techniques↗
PubMed1993

Evaluation of the L-pyrrolidonyl-beta-naphthylamide hydrolysis test for the differentiation of members of the families Enterobacteriaceae and Vibrionaceae.

A simple, rapid, and inexpensive spot test incorporating the substrate pyrrolidonyl naphthylamide was used to examine pyrrolidonyl peptidase activity among 800 bacterial strains belonging to the families Enterobacteriaceae and Vibrionaceae. The pyrrolidonyl naphthylamide test was found to be particularly useful in separating Citrobacter spp. (100% positive) from Salmonella spp. (0.4% positive) and Escherichia coli (0% positive). Furthermore, it would appear to offer a safer alternative to the traditional potassium cyanide test for differentiating citrobacters from salmonellae.

Bacterial Typing Techniques↗
PubMed1997

Evaluation of Vitek GNI+ and Becton Dickinson Microbiology Systems Crystal E/NF identification systems for identification of members of the family Enterobacteriaceae and other gram-negative, glucose-fermenting and non-glucose-fermenting bacilli.

We evaluated the Vitek GNI+ and Becton Dickinson Crystal E/NF identification systems for their ability to accurately identify 619 and 626 strains, respectively, of members of the family Enterobacteriaceae and other glucose-fermenting and non-glucose-fermenting gram-negative rods. All strains tested were taken from a stock collection and passed three times on 5% sheep blood agar prior to testing. These strains represented a more rigorous challenge to both systems than one resulting from the testing of consecutive clinical isolates. Testing with both systems was done according to the manufacturers' instructions, and tests were repeated in duplicate when errors occurred. Vitek version 5.01 and Crystal version 3.0 softwares were used for identifications. The identification results from each system were compared with identifications previously determined with reference biochemicals. At the completion of the appropriate incubation period, the GNI+ and Crystal systems correctly identified 80.1 and 71.1% of the total isolates, respectively. After additional tests suggested by the software programs were completed, the GNI+ had an accuracy of 87.6% and the Crystal system's accuracy had improved to 87.9%. The error rates for the GNI+ and Crystal systems were 6.5 and 5.3%, respectively. A report of "no identification" was given for 6.0 and 6.9% of the isolates, respectively, and was associated with no particular organism group. One isolate each of Acinetobacter lwoffii and Vibrio alginolyticus would not grow in the Vitek card. The average times to detection for correct enteric identifications in the GNI+ system were 4.1 and 6.8 h for nonenteric identifications, while the Crystal results were routinely read at 18 h. We conclude that there was no significant difference (P > 0.05) between the results of the GNI+ card and those of the Crystal E/NF system after additional testing was performed with the group of organisms tested, but the overall accuracy for both systems in this study was below 90%.

Bacterial Typing Techniques↗
PubMed1999

Comparison of agar dilution, disk diffusion, MicroScan, and Vitek antimicrobial susceptibility testing methods to broth microdilution for detection of fluoroquinolone-resistant isolates of the family Enterobacteriaceae.

Fluoroquinolone resistance appears to be increasing in many species of bacteria, particularly in those causing nosocomial infections. However, the accuracy of some antimicrobial susceptibility testing methods for detecting fluoroquinolone resistance remains uncertain. Therefore, we compared the accuracy of the results of agar dilution, disk diffusion, MicroScan Walk Away Neg Combo 15 conventional panels, and Vitek GNS-F7 cards to the accuracy of the results of the broth microdilution reference method for detection of ciprofloxacin and ofloxacin resistance in 195 clinical isolates of the family Enterobacteriaceae collected from six U.S. hospitals for a national surveillance project (Project ICARE [Intensive Care Antimicrobial Resistance Epidemiology]). For ciprofloxacin, very major error rates were 0% (disk diffusion and MicroScan), 0.9% (agar dilution), and 2.7% (Vitek), while major error rates ranged from 0% (agar dilution) to 3.7% (MicroScan and Vitek). Minor error rates ranged from 12.3% (agar dilution) to 20.5% (MicroScan). For ofloxacin, no very major errors were observed, and major errors were noted only with MicroScan (3.7% major error rate). Minor error rates ranged from 8.2% (agar dilution) to 18.5% (Vitek). Minor errors for all methods were substantially reduced when results with MICs within +/-1 dilution of the broth microdilution reference MIC were excluded from analysis. However, the high number of minor errors by all test systems remains a concern.

Anti-Infective Agents↗
PubMed2000

Outbreak of nosocomial infections due to extended-spectrum beta-lactamase-producing strains of enteric group 137, a new member of the family Enterobacteriaceae closely related to Citrobacter farmeri and Citrobacter amalonaticus.

A member of the Enterobacteriaceae initially identified as Kluyvera cryocrescens by the MicroScan Gram-Negative Combo 13 panel caused an outbreak of nosocomial infections in four patients (pneumonia, n = 2; urinary tract infection, n = 1; wound infection, n = 1) and urinary tract colonization in one patient. When the strains were tested by the Enteric Reference Laboratory of the Centers for Disease Control and Prevention, biochemical results were most compatible with Yersinia intermedia, Kluyvera cryocrescens, and Citrobacter farmeri but identification scores were low and test results were discrepant. However, when the biochemical test profile was placed in the computer database as a new organism, all strains were identified as the organism with high identification scores (0. 999968 to 0.999997) and no discrepant test results. By 16S rRNA sequence analysis the organism clustered most closely with, but was distinct from, Citrobacter farmeri and Citrobacter amalonaticus. Based on its unique biochemical profile and rRNA sequence, this organism is designated Enteric Group 137. Restriction endonuclease analysis and taxonomic antibiograms of strains causing the outbreak demonstrated a single clone of Enteric Group 137, and antibiotic susceptibility testing revealed the presence of extended-spectrum beta-lactamase (ESBL) resistance. Enteric Group 137 appears to be a new opportunistic pathogen that can serve as a source of ESBL resistance in the hospital.

Aged↗
PubMed2001

Yield of vancomycin-resistant enterococci and multidrug-resistant Enterobacteriaceae from stools submitted for Clostridium difficile testing compared to results from a focused surveillance program.

It has been suggested that a method of performing surveillance for vancomycin-resistant enterococci (VRE) is to screen specimens submitted for Clostridium difficile testing. We compared this approach to our focused surveillance program of high-risk units during October 1997 to compare the yield of VRE and multidrug-resistant Enterobacteriaceae (MDRE) with both methods. Of the stools submitted for C. difficile testing, 14% were positive for VRE or MDRE, whereas rectal swabs from routine surveillance yielded 11% VRE- or MDRE-positive results. Although stools submitted for C. difficile testing resulted in a higher percentage of positive cultures, 14 VRE- and 2 MDRE-positive patients from our high-risk population were missed because many patients had no stool submitted for C. difficile testing. Therefore, while screening stools submitted for C. difficile testing cannot replace our focused surveillance program, it appears advantageous to assess these stools at various intervals to detect new patient reservoirs of drug-resistant organisms that may benefit from routine surveillance cultures.

Clostridioides difficile↗