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gyrA mutations associated with fluoroquinolone resistance in eight species of Enterobacteriaceae.

Fluoroquinolone resistance (FQ-R) in clinical isolates of Enterobacteriaceae species has been reported with increasing frequency in recent years. Two mechanisms of FQ-R have been identified in gram-negative organisms: mutations in DNA gyrase and reduced intracellular drug accumulation. A single point mutation in gyrA has been shown to reduce susceptibility to fluoroquinolones. To determine the extent of gyrA mutations associated with FQ-R in enteric bacteria, one set of oligonucleotide primers was selected from conserved sequences in the flanking regions of the quinolone resistance-determining regions (QRDR) of Escherichia coli and Klebsiella pneumoniae. This set of primers was used to amplify and sequence the QRDRs from 8 Enterobacteriaceae type strains and 60 fluoroquinolone-resistant clinical isolates of Citrobacter freundii, Enterobacter aerogenes, Enterobacter cloacae, E. coli, K. pneumoniae, Klebsiella oxytoca, Providencia stuartii, and Serratia marcescens. Although similarity of the nucleotide sequences of seven species ranged from 80.8 to 93.3%, when compared with that of E. coli, the amino acid sequences of the gyrA QRDR were highly conserved. Conservative amino acid substitutions were detected in the QRDRs of the susceptible type strains of C. freundii, E. aerogenes, K. oxytoca (Ser-83 to Thr), and P. stuartii (Asp-87 to Glu). Strains with ciprofloxacin MICs of >2 microg/ml expressed amino acid substitutions primarily at the Gly-81, Ser-83, or Asp-87 position. Fluoroquinolone MICs varied significantly for strains exhibiting identical gyrA mutations, indicating that alterations outside gyrA contribute to resistance. The type and position of amino acid alterations also differed among these six genera. High-level FQ-R frequently was associated with single gyrA mutations in all species of Enterobacteriaceae in this study except E. coli.

Amino Acid Sequence↗

Occurrence of extended-spectrum beta-lactamases in members of the family Enterobacteriaceae in Italy: implications for resistance to beta-lactams and other antimicrobial drugs.

An Italian nationwide survey was carried out to assess the prevalences and the antimicrobial susceptibilities of members of the family Enterobacteriaceae producing extended-spectrum beta-lactamases (ESBLs). Over a 6-month period, 8,015 isolates were obtained from hospitalized patients and screened for resistance to extended-spectrum cephalosporins and monobactams. On the basis of a synergistic effect between clavulanate and selected beta-lactams (ceftazidime, aztreonam, cefotaxime, cefepime, and ceftriaxone), 509 isolates were found to be ESBL positive (6.3%). Colony blot hybridization with bla(TEM) and bla(SHV) DNA probes allowed one to distinguish four different genotypes: TEM-positive, SHV-positive, TEM- and SHV-positive, and non-TEM, non-SHV ESBL types. MICs for each isolate (E-test) were obtained for widely used beta-lactams, combinations of beta-lactams with beta-lactamase inhibitors, aminoglycosides, and fluoroquinolones. Among ESBL-positive strains, Klebsiella pneumoniae, Proteus mirabilis, and Escherichia coli accounted for 73.6% of isolates. Overall, TEM-type ESBLs were more prevalent than SHV-type enzymes (234 versus 173), whereas the prevalence of strains producing both TEM- and SHV-type ESBLs was similar to that of isolates producing non-TEM, non-SHV enzymes (55 and 38, respectively). In vitro, all but one of the ESBL-producing isolates remained susceptible to imipenem. Susceptibility to other drugs varied: piperacillin-tazobactam, 91%; amoxicillin-clavulanic acid, 85%; cefoxitin, 78%; amikacin, 76%; ampicillin-sulbactam, 61%; ciprofloxacin, 58%; and gentamicin, 56%. Associated resistance to aminoglycosides and ciprofloxacin was observed most frequently among TEM-positive strains. Since therapeutic options for multiresistant Enterobacteriaceae are limited, combinations of beta-lactams and beta-lactamase inhibitors appear to represent an important alternative for treating infections caused by ESBL-producing ENTEROBACTERIACEAE:

Aminoglycosides↗

Antibacterial activity of selected beta-lactam and aminoglycoside antibiotics against cephalothin-resistant Enterobacteriaceae.

The in vitro antibacterial activity of four beta-lactam antibiotics (cefatrizine [BL-S640], cefamandole, cefoxitin, and carbenicillin) and three aminoglycosides (amikacin, gentamicin, and tobramycin) was determined against 197 strains of cephalothin-resistant Enterobacteriaceae. Eighty strains were found to be gentamicin-sensitive, and 117 were found to be gentamicin-resistant. Carbenicillin was the most active beta-lactam antibiotic against gentamicin-sensitive Serratia marcescens and Enterobacter spp. Cefoxitin was the most active beta-lactam antibiotic against the remaining gentamicin-sensitive and -resistant Enterobacteriaceae, including Providencia stuartii and indole-positive Proteus spp. Cefatrizine exhibited little activity against the organisms studied. Cefamandole was less active than cefoxitin and carbenicillin. Amikacin was the most effective agent in vitro. With the exception of S. marcescens, cefoxitin appeared to be the next most promising agent in vitro against gentamicin- and cephalothin-resistant Enterobacteriaceae.

Aminoglycosides↗

Impact of an urban effluent on antibiotic resistance of riverine Enterobacteriaceae and Aeromonas spp.

In order to evaluate the impact of an urban effluent on antibiotic resistance of freshwater bacterial populations, water samples were collected from the Arga river (Spain), upstream and downstream from the wastewater discharge of the city of Pamplona. Strains of Enterobacteriaceae (representative of the human and animal commensal flora) (110 isolates) and Aeromonas (typically waterborne bacteria) (118 isolates) were selected for antibiotic susceptibility testing. Most of the Aeromonas strains (72%) and many of the Enterobacteriaceae (20%) were resistant to nalidixic acid. Singly nalidixic acid-resistant strains were frequent regardless of the sampling site for Aeromonas, whereas they were more common upstream from the discharge for enterobacteria. The most common resistances to antibiotics other than quinolones were to tetracycline (24.3%) and beta-lactams (20.5%) for Enterobacteriaceae and to tetracycline (27.5%) and co-trimoxazole (26.6%) for Aeromonas. The rates of these antibiotic resistances increased downstream from the discharge at similar degrees for the two bacterial groups; it remained at high levels for enterobacteria but decreased along the 30-km study zone for Aeromonas. Genetic analysis of representative strains demonstrated that these resistances were mostly (enterobacteria) or exclusively (Aeromonas) chromosomally mediated. Moreover, a reference strain of Aeromonas caviae (CIP 7616) could not be transformed with conjugative R plasmids of enterobacteria. Thus, the urban effluent resulted in an increase of the rates of resistance to antibiotics other than quinolones in the riverine bacterial populations, despite limited genetic exchanges between enterobacteria and Aeromonas. Quinolone resistance probably was selected by heavy antibiotic discharges of unknown origin upstream from the urban effluent.

Aeromonas↗

Efficiency of a multitest system (Enterotube) for rapid identification of Enterobacteriaceae.

Enterotube, a multiple-test system which combines nine biochemical tests useful in the identification of members of the family Enterobacteriaceae, was tested and compared with the PathoTec test system in the identification of gram-negative bacilli isolated from clinical specimens. It was found that both the Enterotube and the PathoTec systems rapidly and accurately defined the position of the organisms in the major groups of the family Enterobacteriaceae. Discrepancies were noted between the results of citrate tests on Simmons' citrate-agar and in the Enterotube, as well as between Simmons' citrate-agar and the PathoTec citrate test. It was concluded that the Enterotube system provides a simple, reliable, and rapid method for the presumptive identification of Enterobacteriaceae. The major advantage of the Enterotube is that all tests are done simultaneously by inoculation from a single isolated colony.

Bacteriological Techniques↗

Evaluation of modified R-B system for identification of members of the family Enterobacteriaceae.

In a paired, double-blind study, the modified ("Beckford tube") R-B system was compared with conventional bacteriological procedures for the identification of members of the family Enterobacteriaceae from clinical isolates and stock cultures. The tests in the R-B system yielding positive reactions comparable to those predicted by Ewing's taxonomic classification of Enterobacteriaceae were production of hydrogen sulfide and presence of lysine and ornithine decarboxylasè activities. The test reactions in the R-B system found to be comparable to those in the conventional method were fermentation of glucose, hydrogen sulfide production, and lysine and ornithine decarboxylase activities. The production of gas from glucose was positive in the R-B system more often than in the conventional method; however, the motility test and the production of indole were positive less often in the R-B system. Adequate preliminary identification of the Enterobacteriaceae with the R-B system is enhanced if Simmons' citrate and Christensen's urea tests are used concomitantly. These findings emphasize the manufacturer's instructions that, in interpretation of results, colonial morphology and biochemical reactions must be used concurrently to make an accurate identification.

Bacteriological Techniques↗

Cross-reactivity of major outer membrane proteins of Enterobacteriaceae, studied by crossed immunoelectrophoresis.

Outer membrane fractions were prepared from 11 bacteria in the family Enterobacteriaceae: Escherichia coli serotypes O1K-, O4K2, O26K60, O75K-, and O111K58, Shigella flexneri, Salmonella typhimurium, Klebsiella pneumonia, Serratia marcescens, Proteus vulgaris, Proteus mirabilis, and Providencia stuartii. All strains studied were found to contain one non-peptidoglycan-bound, heat-modifiable outer membrane protein, and one or two peptidoglycan-associated major outer membrane proteins in the 27,000- to 40,000-dalton range. Crossed immunoelectrophoresis using sodium dodecyl sulfate-polyacarylamide gel electrophoresis for separation of the antigens in the first dimension of the procedure was shown to provide a useful model system for studying the antigenic relationships of the major outer membrane proteins in Enterobacteriaceae species. Peptidoglycan-bound major outer membrane proteins of all bacteria studied reacted with antiserum against the purified peptidogylcan-bound matrix protein I of E. coli O26K60 in this system. Non-peptidoglycan-associated proteins of all strains cross-reacted with protein II of E. coli O26K60 in both their unmodified and their heat-modified forms. These results indicate that the genes coding for the major outer membrane proteins in the family Enterobacteriaceae have been well enough conserved during the course of evolution to allow significant antigenic cross-reactivity between the corresponding proteins in different enterobacterial species.

Antigens, Bacterial↗

The 5'-nucleotidases and cyclic phosphodiesterases (3'-nucleotidases) of the Enterobacteriaceae.

All members of the Enterobacteriaceae possess distinct 5'-nucleotidases and cyclic phosphodiesterases (3'-nucleotidases) that can be differentiated from the acid and alkaline phosphatases and the acid sugar hydrolases. The nucleotidases and cyclic phosphodiesterases of the various Enterobacteriaceae are remarkably similar in properties. All of the 5'-nucleotidases hydrolyze 5'-nucleotides, adenosine triphosphate, and uridine diphosphoglucose. Their pH optimum is from 5.7 to 6.1. The cyclic phosphodiesterases hydrolyze 3'-nucleotides, cyclic phosphonucleotides, bis-(p-nitrophenyl)phosphate, and p-nitrophenylphosphate. Their pH optimum is from 7.2 to 7.8. For both enzymes, cobalt showed optimal metal stimulation. An intracellular protein inhibitor for the 5'-nucleotidase is present in all of the Enterobacteriaceae. No inhibitor of cyclic phosphodiesterase activity exists, although hydrolysis of both cyclic phosphonucleotides and 3'-nucleotides is inhibited by ribonucleic acid. Neither of the enzymes is subject to control by phosphate level or by catabolite repression. Of the other bacteria studied, only Haemophilus and Bacillus subtilis contained significant 3'- or 5'-nucleotidase activity.

Bacillus subtilis↗

Taxonomic investigations on expressed and cryptic phospho-beta-glucosidases in Enterobacteriaceae.

In the Enterobacteriaceae, beta-glucosides are catabolized by a complex system formed of three permeases, with partly overlapping substrate specificities, and two hydrolytic enzymes, phospho-beta-glucosidase A and B, which hydrolyze only phosphorylated beta-glucosides. Some Enterobacteriaceae such as Klebsiella-Aerobacter (Enterobacter) possess the complete system; others possess only parts of it or may have a cryptic phospho-beta-glucosidase activity without permease activity. A screening test applied to strains belonging to several genera of Enterobacteriaceae showed that strains of Citrobacter, Hafnia, and Serratia exhibit a degree of similarity in phospho-beta-glucosidase activity and inducibility which could be useful in their taxonomic characterization; others, such as Aerobacter aerogenes, Erwinia, and Proteus vulgaris, are more heterologous. Owing to the presence of inducible phospho-beta-glucosidases A and B in Citrobacter, the fermentation of beta-methyl glucoside and the fermentation of arbutin in mixture with cellobiose could be of diagnostic value in the differentiation of Citrobacter from Salmonella. Wild-type strains of Escherichia coli, Shigella, and Salmonella are phenotypically similar in their inability to catabolize beta-glucosides, the presence of constitutive P-beta-glucosidase A, and the lack of beta-glucoside permeases I and II. Their beta-glucoside-fermenting mutants show, however, a phospho-beta-glucosidase and beta-glucoside permease activity which is characteristic for mutants from each genus. The differences in the phenotype of the mutants reflect probable differences in the presence of cryptic genes in the wild-type strains and could be of evolutionary significance.

Bacteriological Techniques↗

Collaborative investigation of the AutoMicrobic System Enterobacteriaceae biochemical card.

The Enterobacteriaceae biochemical card used in six separate laboratories to identify 170 representatives of Enterobacteriaceae. The AutoMicrobic System (Vitek Systems, Inc.) performed with an accuracy of 97.8% as compared with 98.1% by the standard method selected and 97.6% by a commerically prepared manual system approach. During this time, 5,450 clinical isolates belonging to Enterobacteriaceae were analyzed. Compared with the routine methods used in the various laboratories, the AutoMicrobic System identified 96.4% correctly

Bacteriological Techniques↗

Comparison of the automicrobic system with API, enterotube, micro-ID, micro-media systems, and conventional methods for identification of Enterobacteriaceae.

Identification of Enterobacteriaceae by the AutoMicrobic System Enterobacteriaceae Biochemical Card was evaluated. Recent clinical isolates of enteric gram-negative bacilli (192) and glucose nonfermenters (3) were identified by the AutoMicrobic System, Micro-Media Systems, Micro-ID, API, and Enterotube II in comparison with conventional methods. The AutoMicrobic System and Micro-Media Systems correctly identified 97% of the organisms tested. Micro-ID, API, and Enterotube II correctly identified 94, 92, and 84% of the organisms, respectively. In addition to a high degree of identification accuracy, the AutoMicrobic System was convenient to operate and produced identification results in 8 h. Operation of the AutoMicrobic System also required minimal personnel time because it automatically monitored and interpreted the biochemical reactions and reported organism identifications. The AutoMicrobic System appears to be an efficient and accurate system for the identification of Enterobacteriaceae.

Bacteriological Techniques↗

Evaluation of the MS-2 system for rapid identification of Enterobacteriaceae.

The precision, accuracy, and other performance characteristics of the MS-2 (Abbott Laboratories, Diagnostic Division, Dallas, Tex.) system for the identification of Enterobacteriaceae were evaluated in a collaborative study involving three clinical laboratories. When identifying 150 unknown, coded organisms, the MS-2 system was 97%, 98%, and 93% accurate, respectively, in three laboratories. The system showed an overall accuracy of 94% when compared with conventional manual tube methods in identifying 1,154 clinical isolates of 26 species of Enterobacteriaceae. Discrepancies between automated and conventional methods were chiefly caused by biochemical variants, especially among Enterobacter species. The MS-2 system was rapid and simple to operate and produced printed results of bacterial identification in 5 h. The cost of disposable components compared favorably with commercial, visually read systems for identifying Enterobacteriaceae.

Bacteriological Techniques↗

Sensitivity, specificity, and reproducibility of the automicrobic system (with the Enterobacteriaceae-plus biochemical card) for identifying clinical isolates of Gram- negative bacilli.

Two independent laboratories tested 1,743 clinical isolates by using the Enterobacteriaceae-plus Biochemical Card in the AutoMicrobic system (AMS) and identical standard reference methods. Included were 55 isolates representing 11 species that cannot be identified by the enterobacteriaceae-plus Biochemical Card computer program; 3 or these isolates were incorrectly identified as Pseudomonas cepacia. With the other 1,688 isolates, 5% of the AMS identifications were considered to be equivocal (probability value, less than 0.80), and the remaining test were 97% accurate (sensitive), Difficulty was observed in the ability of the AMS to identify some H2S-negative Citrobacter freundii species. An AMS response of P. cepacia was also considered nonspecific, because several other organisms were misidentified at P. cepacia. Reproducibility of the system was documented by testing 125 strains on 3-separate days; only 6 strains produced significantly variable results. The AMS (with the Enterobacteriaceae-plus Biochemical Card) was a very satisfactory, automated system for accurately identifying most gram-negative bacilli within 8 to 13 h.

Aeromonas↗

Reevaluation of the API 20E identification system versus conventional biochemicals for identification of members of the family Enterobacteriaceae: a new look at an old product.

The API 20E bacterial identification system has been used for 19 years, often as the standard with which other identification systems are compared. Because the accuracy of this system compared with conventional biochemical tests has not been determined in many years, we evaluated the API 20E linear strip by using 291 typical and atypical strains of the family Enterobacteriaceae taken from a culture collection. At 24 h, the API 20E correctly identified by genus and species 229 of 291 (78.7%) of the strains, using Salmonella and Shigella serotyping where indicated. At 48 h, 95.2% were correctly identified by using additional biochemical tests as recommended by the manufacturer. The API 20E misidentified eight (2.7%) strains; these strains were not limited to any particular genus. When 81 of these Enterobacteriaceae strains were arranged into a weighted assortment correlating to the frequency with which they might be found in a clinical laboratory, the API 20E correctly identified 71 (87.7%) at 24 h and 78 (96.3%) at 48 h. This evaluation concluded that the accuracy of the identification of Enterobacteriaceae strains at 24 h (78.7%) may be significantly lower than that of earlier evaluations. However, there is no significant difference in the ability of the API 20E to correctly identify "challenge" type organisms (229 of 291) versus routine hospital isolates (71 of 81) (P greater than 0.05), but the system is not as accurate as the conventional biochemical method of identification.

Bacterial Typing Techniques↗

Occurrence and detection of extended-spectrum beta-lactamases in members of the family Enterobacteriaceae at a veterans medical center: seek and you may find.

A total of 907 consecutive isolates of members of the family Enterobacteriaceae recovered during a 20-week period were tested for production of extended-spectrum beta-lactamases (ESBLs) by the double-disk (DD) potentiation method. Of 84 DD-positive isolates, 83 (9.2%) produced ESBLs based on isoelectric focusing. SHV-derived ESBLs and several TEM-derived ESBLs were present in nine species, including the first isolate of Citrobacter koserii and Morganella morganii known to harbor an SHV-derived ESBL. Results of testing 58 nonrepeat isolates for ESBL production by several recommended methods were as follows (percent detected in parentheses): DD method with aztreonam (95), ceftazidime (79), ceftriaxone (88), or cefpodoxime (90); broth microdilution method with ceftazidime (86) or cefotaxime (91) alone or in combination with clavulanate; and the standard disk diffusion method with new breakpoints and standard concentrations of aztreonam (78), ceftazidime (79), ceftriaxone (83), or cefpodoxime (98) or a novel concentration (5 microg) of ceftazidime (88). In three instances during an extended part of the study, an ESBL-producing isolate and a non-ESBL-producing isolate of the same species were recovered from a single blood culture bottle. These data indicate that ESBLs occur in several species of Enterobacteriaceae and at a relatively high incidence at our institution and that the standard disk diffusion method with cefpodoxime and the DD method with several beta-lactams are practical and cost-effective methods for detecting ESBL-producing isolates of Enterobacteriaceae.

Aztreonam↗

BetalasEN: microdilution panel for identifying beta-lactamases present in isolates of Enterobacteriaceae.

A dried investigational use-only microdilution panel named betalasEN (a short named derived from the panel's purpose, to identify beta-lactamases in Enterobacteriaceae) containing 10 beta-lactam drugs with and without beta-lactamase inhibitors was developed to identify beta-lactamases among clinical isolates of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter koseri, Citrobacter freundii group, Enterobacter spp., and Serratia marcescens. The MICs obtained with a collection of 383 organisms containing well-characterized beta-lactamases were used to develop numeric codes and logic pathways for computerized analysis of results. The resultant logic pathways and betalasEN panel were then used to test and identify beta-lactamases among 885 isolates of Enterobacteriaceae recovered in cultures obtained at six different hospital laboratories across the United States. beta-Lactamases present in 801 (90.5%) of the 885 isolates were identified by betalasEN by using the existing logic pathways and codes or after minor modifications were made to the existing codes. The 84 strains that gave codes that betalasEN could not identify were collected, reidentified, and retested by using betalasEN. Three strains had been misidentified, 54 strains gave different codes upon repeat testing that could be identified by betalasEN, and 27 strains repeated new codes. The beta-lactamases in these strains were identified, and the new codes were added to the betalasEN logic pathways. These results indicate that betalasEN can identify clinically important beta-lactamases among most isolates of Enterobacteriaceae. The results also show that good quality control and attention to proper performance of the tests are essential to the correct performance of betalasEN.

Anti-Bacterial Agents↗

Esculin hydrolysis by Enterobacteriaceae.

Literature reports disagree concerning esculin hydrolysis in the family Enterobacteriaceae. A total of 2,490 strains of the family were investigated for esculin hydrolysis by two methods, the esculin spot test and the PathoTec incubation strip, which measures constitutive enzyme, and five growth-supporting methods, which determine both constitutive and inducible enzymes. The five growth-supporting media studied were: Vaughn-Levine, the standard esculin hydrolysis medium (P. R. Edwards and W. H. Ewing, Identification of Enterobacteriaceae, 3rd ed., 1972); Vaughn-Levine without iron; Vaughn-Levine without Andrade's indicator; and bile-esculin medium. Growth media were incubated at 35 degrees C and checked every 24 h for 120 h. On growth media, 0.3% of Escherichia coli were positive in 24 h, 34% in 48 h, and 61% in 120 h. No strains were positive on the "nongrowth" tests. It appeared that the esculin hydrolysis enzyme(s) of E. coli was inducible rather than constitutive. All esculin hydrolyzers, which yielded positive tests on "constitutive tests" and 24-h tests, were limited to the genera Klebsiella, Enterobacter, and Serratia and species of Proteus vulgaris, Proteus rettgeri, and Citrobacter diversus. When used with standardized inoculum size and incubation time, the esculin hydrolysis test is very useful for differentiation within the family Enterobacteriaceae.

Citrobacter↗

Enterobacteriaceae and neonatal necrotising enterocolitis.

A comparative study of bowel colonisation and incidence of necrotising enterocolitis in neonates admitted to an intensive care unit is reported. Neonates of less than 33 weeks gestational age requiring mechanical ventilation for respiratory distress syndrome were randomised during the first week of life to receive either vancomycin and aztreonam or vancomycin and gentamicin for episodes of suspected sepsis after the first week of life. A higher proportion of neonates who received vancomycin and gentamicin had faecal colonisation with enterobacteriaceae at the end of the second, third, and fourth weeks of life. Treatment with vancomycin and aztreonam was associated with a rapid quantitative reduction in faecal colonisation with enterobacteriaceae, whereas there was no quantitative reduction in colonisation with enterobacteriaceae associated with treatment with vancomycin and gentamicin. There were no differences between the two groups in faecal colonisation with anaerobes, Enterococcus sp, Staphylococcus sp, or yeasts. Six (14.6%) of 41 who received vancomycin and gentamicin compared with 0 of 40 who received vancomycin and aztreonam subsequently developed necrotising enterocolitis.

Aztreonam↗