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PubMed1986

Antibiotic resistance and conjugative R plasmids in clinical isolates of Enterobacteriaceae in Peninsular Malaysia.

Twenty-five strains of enterobacteria, isolated from man in Peninsular Malaysia and consisting of seven Enterobacter spp., five Escherichia coli, five Salmonella spp., four Klebsiella spp., two Shigella spp., one Proteus sp. and and one Providencia sp., were tested for antibiotic resistance and conjugative R plasmids. They were all sensitive to nalidixic acid and resistant to at least three antibiotics. The number of resistances ranged from 3 to 11 antibiotics, including cefoperazone and sisomicin (two) newly released antibiotics), in addition to common drugs of current use. Of the 25 isolates, 19 (76%) conjugally transferred, at varied frequencies, at least two resistance determinants. Results from equilibrium density gradient centrifugation, agarose gel electrophoresis and transformation experiments provided proof that the transferable resistances were plasmid-mediated. Restriction endonuclease cleavage patterns showed that the plasmids from Proteus strain K005 and Providencia strain K001 may be identical.

Anti-Bacterial Agents↗
PubMed1987

Effects of infection with Enterobacteriaceae enteropathogens on subsequent infection with Ascaris suum in the laboratory mouse.

Prior to infection with Ascaris suum, ICR strain mice were inoculated with Salmonella typhisuis intraperitoneally or via gastric gavage. Similarly, Salmonella cholerae-suis var. kunzendorf, Salmonella typhimurium and enterotoxigenic Escherichia coli were administered to mice via gastric gavage 2 weeks prior to A. suum inoculation. Previous inoculation with S. typhisuis, via the intraperitoneal or gastric gavage routes and S. cholerae-suis var. kunzendorf decreased recovery of ascarid larvae from mice lungs. This effect appeared to be due to entrapment of migrating larvae by inflammatory reactions in the liver. This reaction was suspected to be due to non-specific resistance stimulated by the prior exposure to the bacterial pathogen. The number of A. suum larvae in the lungs of mice previously inoculated with S. typhimurium or enterotoxigenic E. coli (ETEC) was variable and in some cases greater in mice which had received the bacterial inoculation.

Animals↗
PubMed1992

Distribution of insertion sequence IS1 in multiple-antibiotic resistant clinical Enterobacteriaceae strains.

The presence of insertion sequence IS1 in 70 multiple-antibiotic resistant clinical strains was determined. This 70-strain collection comprised 46 Escherichia coli, 18 Salmonella and 6 Shigella strains. The presence of IS1 was detected in the chromosome and plasmids of 73% and 63% of the strains, respectively, and 51% of the strains carried IS1 in both. The frequency of IS1 was higher in Salmonella than in E. coli and Shigella strains. A total of 31 strains carried large plasmids with IS1; 10 of these strains (32.3%) were able to transfer all or some of the antibiotic resistance markers to E. coli K12 or S. typhimurium recipient strains. Resistance markers of all clinical strains were maintained stably after several generations of growth. The presence of IS1 in a relatively high percentage of plasmids of multiple-antibiotic resistant clinical isolates, suggests a role for this sequence in the dissemination of genes which code for antibiotic resistance.

DNA Transposable Elements↗
PubMed1992

Does high level production of SHV-type penicillinase confer resistance to ceftazidime in Enterobacteriaceae?

We report the isolation of a clinical isolate of Klebsiella pneumoniae that showed resistance to ceftazidime (MIC: 8 micrograms/ml), susceptibility to aztreonam (MIC: 2 micrograms/ml) and cefotaxime (MIC: 0.015 micrograms/ml). A synergistic effect between clavulanic acid and ceftazidime or aztreonam against this strain was also observed. The strain hyperproduced SHV-1 penicillinase (990 U/g) which is encoded by a self-transferrable plasmid of at least 150 kb. That the ceftazidime-resistance phenotype could be due to hyperproduction of SHV-1 penicillinase is supported by the study of a spontaneous ceftazidime-resistant mutant in vitro obtained from an Escherichia coli strain containing plasmid p453 encoding the SHV-1. Indeed, this mutant hyperproducing SHV-1 (2200 U/g) was resistant to ceftazidime (MIC: 16 micrograms/ml) and aztreonam (MIC: 8 micrograms/ml) but susceptible to cefotaxime (MIC: 0.03 ng/ml). Clavulanic acid showed a synergistic effect when associated with ceftazidime or aztreonam. In contrast, the hyperproduction of TEM-1 (790 U/g) did not confer a ceftazidime- and aztreonam-resistant phenotype while hyperproduction of both TEM-1 and SHV-1 increased the resistance to amoxycillin/clavulanic acid and to cephalothin.

Ceftazidime↗
PubMed1989

Characterization of the plasmid genes blaT-4 and blaT-5 which encode the broad-spectrum beta-lactamases TEM-4 and TEM-5 in enterobacteriaceae.

We have determined the nucleotide sequence of the plasmid genes blaT-4 and blaT-5 which encode the broad-substrate-range beta-lactamases TEM-4 and TEM-5, respectively. The TEM-4 enzyme, which confers high-level resistance to cefotaxime (Ctx) and ceftazidime (Caz), differed from the TEM-1 penicillinase by four amino acid substitutions. Two of the mutations are identical to those responsible for the wide substrate range of the TEM-3 beta-lactamase which hydrolyses Ctx and Caz. The amino acid sequence of TEM-5, which confers higher levels of resistance to Caz than to other recently developed cephalosporins, differed from that of TEM-1 by three mutations distinct from those of TEM-4. Analysis of the location of the mutations in the primary and tertiary structures of class A beta-lactamases suggests that interactions between the substituted residues and beta-lactam antibiotics non-hydrolysable by TEM-1 and TEM-2 allow TEM-4 and TEM-5 to hydrolyse efficiently novel broad-spectrum cephalosporins such as Ctx and Caz.

Amino Acid Sequence↗
PubMed1993

Distribution of the Shigella sonnei insertion elements in Enterobacteriaceae.

The distribution of IS1, IS600, IS629, IS630 and IS640, present in an Shigella sonnei strain, was examined in strains belonging to various species of enteric bacteria. Four Shigella species including Sh. sonnei contained all IS elements, several of which were in large numbers, and showed species-specific distribution patterns. The other strains contained some of the IS elements in a few copies or none at all, except for some clinical isolates in the Escherichia coli strains, which showed similar distribution patterns to those of the Shigella species, suggesting that the E. coli isolates are closely related to those in Shigella. The IS elements examined may be used to classify various bacterial strains and to identify the Shigella strains and some of the E. coli strains to be isolated from various sources.

DNA Transposable Elements↗
PubMed1995

Conservation of cis-acting elements within the tor regulatory region among different Enterobacteriaceae.

The Escherichia coli (Ec) torCAD operon encoding the trimethyl amine N-oxide (TMAO) reductase system is induced by both TMAO and anaerobiosis. The tor regulatory regions from bacteria related to Ec have been amplified by the polymerase chain reaction (PCR) using degenerate oligodeoxyribonucleotide primers based on conserved sequences of the tor products. The amplified regions from Salmonella enteritidis and Sa. typhimurium (St) were the same size as that from Ec and showed 82% identity with it. Interestingly, four boxes of a 10-nucleotide motif (5'-CTGTTCATAT) were found in direct repeat at the same location in the tor regulatory region of the three species. Although the amplified fragment from Shigella sonneï (Ss) was highly homologous to the Ec corresponding segment, the first tor box was missing. In Ec, the St and Ss tor promoters were still regulated by both TMAO and anaerobiosis, but their transcriptional activities were significantly lower than that of the Ec tor promoter. Deletion of the two first boxes of the Ec tor regulatory region inactivated the tor promoter while deletion of the region just upstream from the tor boxes led to a significant decrease in tor expression. Our results strongly suggest that the tor boxes, as well as specific sequences outside the tor boxes, play an important role in the expression of the tor operon.

Base Sequence↗
PubMed1984

Enzyme immunoassay for the quantitation of human immunoglobulin G specific for the glycolipid of Enterobacteriaceae.

An enzyme immunoassay (EIA) is reported that quantitates human immunoglobulin (Ig)G specific for the glycolipid (Re-GL) of Salmonella minnesota Re 595. Re-GL was adsorbed to polystyrene microcuvettes, and 100 microliters of appropriately diluted lots of human plasma or affinity-purified rabbit anti-Re-Gl IgG was added to the cuvettes. The cuvettes were incubated, washed, and then reacted with Staphylococcus aureus protein A-peroxidase. After a final wash the substrate 2,2'-azino-di(3-ethylbenzthiazoline)sulfonic acid (ABTS) was added to yield a blue-green color with a maximum absorption at 405 nm. Using the affinity-purified rabbit anti-Re-IgG as standard, it was possible to quantitate specific levels of anti-Re-GL. The EIA was rapid (2 hr), with an absolute sensitivity of 5 ng of specific anti-Re-GL IgG and an interassay coefficient of variation of 24%. On a unit-mass basis, rabbit anti-Re-GL IgG had 10-fold greater activity than comparable affinity-purified human anti-Re-GL IgG from normal donor plasma. When a rabbit standard was used, the mean level of specific anti-Re-GL IgG in lots of pooled human plasma, which consisted of 18,330 liters, was 1.1 +/- 0.28 micrograms/ml. Of 237 individual donors tested, 24 (10%) had levels of anti-Re-GL IgG that were 10-fold or greater than the mean of 1.1 micrograms/ml. The EIA provided a rapid, reproducible method for accurate quantitation of specific anti-Re-GL IgG in the screening of large volumes of human plasma.

Animals↗
PubMed1987

Bactericidal activities of ten different fluoroquinolones against selected Enterobacteriaceae.

Ten fluoroquinolone compounds and two other 4-quinolones were tested in a microdilution system. Minimal inhibitory concentrations and minimal bactericidal concentrations were determined with 1 X 10(7) and 5 X 10(5) colony forming units per milliliter. All 12 compounds were bactericidal. Continued inhibition by drug carried over in the subcultured samples was documented and that represents a potential source of technical error that needs to be controlled when determining minimal bactericidal concentrations for these extremely potent drugs.

Anti-Bacterial Agents↗
PubMedDate not supplied

Differentiation of Enterobacteriaceae, Pseudomonas aeruginosa, and Bacteroides and Haemophilus species in gram-stained direct smears.

The accuracy of examination of the Gram-stained direct smear to classify presumptively Gram-negative rods into three morphotype groups, that is, (a) Enteric bacteria, (b) Pseudomonas, and (c) Bacteroides or Haemophilus, was evaluated. Randomly selected clinical strains (4-9) each of Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Ps. aeruginosa, B. fragilis, and H. influenzae were used to produce peritonitis or subcutaneous abscesses in mice. A Gram-stained direct smear was prepared from exudate collected from each animal. The direct smears were examined to classify bacteria observed into one of the three morphotype groups. The percent accuracy was 82, 56, and 95, respectively, and 76 overall. The assumption was made that classification was based primarily on differences in length and width of the organisms. To test this hypothesis, we prepared scanning electron photomicrographs from each specimen of exudate and measured the lengths and widths of bacteria. Examination of the Gram-stained direct smear was more accurate for classification of enteric bacteria, H. influenzae, or B. fragilis. Electron microscopy was more accurate for classification of Ps. aeruginosa. The higher length-width radio should be helpful in recognizing Ps. aeruginosa in direct smears.

Abscess↗
PubMed1992

The E-Test applied to susceptibility tests of gonococci, multiply-resistant enterococci, and Enterobacteriaceae producing potent beta-lactamases.

The E-Test (AB Biodisk, Solna, Sweden), a new and novel approach developed to test antimicrobial susceptibility, was compared with the agar dilution method. A collection of 57 organisms, including those with known resistances to various antimicrobial agents was tested against 15 drugs using the National Committee for Clinical Laboratory Standard (NCCLS) methods. The E-Test quantitative accuracy (+/- 2 log2 dilutions) compared with agar dilution results was 99% for Neisseria gonorrhoeae, 95% for Escherichia coli strains with extended-spectrum beta-lactamases, 100% for Enterobacter-Citrobacter spp. with type-1 stably derepressed enzymes, and 95% for Enterococcus spp. with various drug resistances (penicillins, ciprofloxacin, gentamicin, and vancomycin). The E-Test procedures with these species were easy to perform, reproducible, and quantitatively and qualitatively accurate (89%-100%). The E-Test appears to be an acceptable option for susceptibility testing of these difficult-to-treat, drug-resistant bacterial strains.

Anti-Bacterial Agents↗
PubMed1992

Detection of point mutation in bla T genes of Enterobacteriaceae by biotinylated oligonucleotide probes using microwell hybridization and enzymofluorometric method.

Point mutation in the nucleotide sequence of the structural genes for the TEM-type penicillinases can broaden their substrate spectrum towards all beta-lactams except cephamicins and imipenem. We describe here hybridization techniques for the detection of point mutations by non-radioactive oligonucleotide probes with plasmid DNA carrying bla T genes immobilized in polystyrene microwells. After hybridization in discriminating conditions with corresponding biotinylated oligonucleotide probes, the hybrids were detected by using a streptavidin-alkaline phosphatase conjugate and a fluorogenic substrate, 4-methylumbelliferyl-phosphate. The adsorption of DNA to microwells used in the present work was found to be independent of Mg2+ and Na+ concentrations. By this method, less than 3 fmols of target DNA were sufficient for the detection of point mutation.

Adsorption↗
PubMed2004

A novel glutamate-dependent acid resistance among strains belonging to the Proteeae tribe of Enterobacteriaceae.

Morganella, Providencia and Proteus strains were capable of surviving pH 2.0 for 1 h if glutamate was present. These strains did not have glutamic acid decarboxylase activity and the gadAB genes were not detected in any of these bacteria. When exposed to pH 2.0 acid shocks, the survival rate of these bacteria was significantly increased with glutamate concentrations as low as 0.3 mM in the acid media. Escherichia coli cells incubated at pH 3.4 consumed four times more glutamate and produced at least 7-fold more gamma-amino butyric acid than Morganella, Providencia and Proteus strains. These results indicate that strains belonging to the Proteeae tribe might have novel glutamate dependent acid-resistance mechanisms.

Enterobacteriaceae↗