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Activity of antiseptics against biofilms of mixed bacterial species growing on silicone surfaces.

As part of a programme to establish the relative merits of antiseptics that are used as bladder instillations to control urinary tract infections in patients with indwelling catheters, the activity of five such formulations were tested against dense (10(9) cfu/cm2) mixed biofilms composed of Citrobacter diversus, Pseudomonas aeruginosa and Enterococcus faecalis growing on silicone discs. All three species were resistant to chlorhexidine (200 mg/l) and povidone-iodine (1% v/v) in the biofilm mode of growth, whereas these agents rapidly eliminated viable cells from urine suspensions of the mixed community. Lactic acid (1% v/v) produced a 1 log reduction of the biofilm population within 30 min of exposure. The mandelic acid (1% w/v) and mandelic acid (0.5% w/v)/lactic acid (0.5% v/v) mixture proved to be the most effective in eliminating the biofilm organisms. It is suggested that these latter solutions should now be tested for efficacy in bladder washouts against urinary tract infections in catheterized patients.

Anti-Infective Agents, Local↗

Clinical consequences of development of resistance to third generation cephalosporins.

Eighteen patients are described in whom initially sensitive microorganisms were replaced by resistant isolates during administration of ceftriaxone (n = 8), cefoperazone (n = 5), moxalactam (n = 4), cefotaxime (n = 2) or ceftazidime (n = 1), despite combination with aminoglycosides. All patients had documented gram-negative infections; in 12 patients underlying haematological diseases were present. Resistant strains of Enterobacter cloacae (14), Serratia marcescens (4), Klebsiella oxytoca (3), Pseudomonas aeruginosa (2) and Citrobacter freundii (2) emerged within 2 to 19 (mean 9) days after the beginning of treatment. In 12 patients relapse or secondary infections occurred. Seven of the patients with haematological disorders died. Resistance development was seen in 8 of 29 patients on ceftriaxone and 4 of 10 patients on moxalactam during prospective evaluations; the other drugs were used sporadically. Thus, selection of resistant bacteria is relatively frequent and may have serious clinical consequences in patients with impaired host-defense mechanisms.

Adolescent↗

Incidence of inducible beta-lactamases in gram-negative septicemia isolates from twenty-nine European laboratories. European Study Group on Antibiotic Resistance.

In 1984 the European Study Group on Antibiotic Resistance (ESGAR), which is made up of 29 laboratories in 12 European countries, consecutively collected gram-negative bacilli and staphylococci isolates from blood and using the microdilution method performed susceptibility testing with 11 beta-lactam antibiotics. A total of 2,578 isolates were collected; 68% were gram-negatives and 32% staphylococci. Pseudomonas spp. accounted for 12% of the strains, Enterobacter spp. 7%, Serratia spp. 3%, indole-positive Proteus spp. 1%, Citrobacter spp. and Morganella spp. 0.9% each. Strains with inducible beta-lactamases were detected by the cefoxitin disc diffusion method in 11% of all gram-negatives and in 67% of the relevant species. The production of inducible beta-lactamase was confirmed by elevated MICs to and decreased killing by piperacillin, cefotaxime and ceftazidime after induction of enzyme production with low concentrations of cefoxitin. This phenomenon was not observed with mecillinam or the new penem Sch 34343.

Amdinocillin↗

In vitro antibacterial activity of norfloxacin compared with eight other antimicrobial agents.

The antibacterial activity of norfloxacin, an organic acid structurally related to nalidixic acid, was compared with that of the oral cephalosporins cefaclor and cephalexin, and with that of nalidixic acid, cinoxacin, amikacin, ampicillin, trimethoprim alone and the combination of trimethoprim and sulfamethoxazole. Agar dilution studies were performed with a total of 398 clinical isolates of gram-negative bacteria. Norfloxacin was found to be the most active drug studied against each of the different groups of organisms tested. MIC90 values for norfloxacin were as follows: Citrobacter spp., 2 micrograms/ml; Enterobacter spp., 0.13 micrograms/ml; Escherichia coli, 0.06 micrograms/ml; Klebsiella spp., 0.13 micrograms/ml; Proteus spp., 0.06 micrograms/ml; Salmonella spp., 1 microgram/ml; Serratia spp., 0.13 micrograms/ml; and Pseudomonas spp., 2 micrograms/ml. MIC90 values for the other drugs were 4 micrograms/ml or greater and many organisms were totally resistant to one or more of the other drugs (MIC greater than 128 micrograms/ml). Cross resistance between norfloxacin and the related drugs nalidixic acid and cinoxacin was not observed.

Anti-Bacterial Agents↗

Induction of beta-lactamase by cefoxitin in anaerobic intestinal microflora.

Beta-lactamases produced by two anaerobic bacterial strains, Bacteroides ovatus Ax34:1 and Clostridium butyricum NBL3, were shown to be significantly inducible under anaerobic conditions in subinhibitory concentrations of cefoxitin. The induction ratio of beta-lactamase production for Bacteroides ovatus was 2.6 and for Clostridium butyricum 1.6. Incubation of faecal samples with different concentrations of cefoxitin did not result in any induction of beta-lactamase production. When adding a highly inducible aerobic strain (Citrobacter freundii F72:6, induction ratio of 26.5 in broth culture) to faecal samples, an induction ratio of 4.5 was reached. Faeces seem to inhibit beta-lactamase induction in aerobic and anaerobic bacteria. The inducible enzymes produced by the anaerobic strains did not have the same properties as beta-lactamases from aerobic inducible strains, according to substrate profiles and inhibition studies. The results of the present study indicate that increased levels of beta-lactamases in the normal intestinal microflora, which often are observed after administration of beta-lactam agents, are probably due to selection of stably derepressed mutants rather than to induction of beta-lactamase production.

Anti-Bacterial Agents↗

Effect of glucose on the biochemical properties of the bacterial cytoplasmic membrane.

The cytoplasmic membrane isolated from cells of Citrobacter freundii growing in a cultivation medium with glucose was found to exhibit a decreased ability to oxidize formate and succinate and a decreased activity of formate, succinate and lactate dehydrogenase as compared with the cytoplasmic membrane of cells growing on galactose. The activation energy for dehydrogenation and oxidation of succinate simultaneously increases. The quantitative content of cytochromes and quinones in both types of membranes does not differ considerably. The two types of membranes also do not mutually differ in the qualitative and quantitative representation of fatty acids in lipids and in the sensitivity to the effect of low concentrations of detergents. The mass ratio of proteins and lipids is lower in the membranes of cells grown on glucose.

Cell Membrane↗

Mercuric reductase gene transfer from soil to rumen bacteria.

Conjugal transfer between soil bacterial population and microorganisms isolated from the rumen of herbivores from mercury-polluted area was investigated. The transfer of merA encoding mercury-resistance plasmids from soil bacteria Enterobacter cloacae and Enterococcus durans into two ruminal isolates Citrobacter freundii and Bacillus subtilis was observed. Approximately the same frequency of mobilization in mating experiments was observed for both Gram-negative (approximately 2.5 x 10(-8), transconjugants-to-recipient ratio) and Gram-positive (approximately 1.3 x 10(-8)) bacteria.

Animals↗

There is a low incidence of recurrent bacteriuria in painful bladder syndrome/interstitial cystitis patients followed longitudinally.

The objective of this paper was to establish whether patients with confirmed painful bladder syndrome/interstitial cystitis (PBS/IC) presenting with symptoms of UTI have actual bacteriuria vs a flare of their PBS/IC symptoms. One hundred and six (n = 106) consecutive female patients (mean age 39.8 +/- 14 years) with newly diagnosed IC were identified and followed longitudinally for 24 months. At the initial visit and at all subsequent visits, urinary specimens were obtained by sterile catheterization (Bard 14Fr female) and cultured for bacteria. Eight patients had an initially positive urine culture, and repeat cultures 8 weeks after treatment were all negative. Once sterile urine was established, the diagnosis of PBS/IC was confirmed. A pelvic pain/urgency/frequency (PUF) questionnaire score was obtained from 89 patients. After the diagnosis of PBS/IC, all patients received multimodal treatment. Patients were instructed to present to the office whenever they developed symptoms of UTI, at which time a sterile catheter specimen was obtained and sent for culture. Greater than 10(3) colonies were considered positive. Patients who did not report flares were contacted to establish whether unreported treatments were given. Seventy-two patients (68%) had no UTI episodes or flares. The remaining 34 patients (32%) presented with 54 flares, of which 44 were culture-negative and 10 were culture-positive. A single flare was reported by 21 patients during the 24 months, with three positive cultures (14.3%). Recurrent UTI symptoms (two to four flares) were seen in a small group (n = 13) for a total of 33 flares. Of these, seven had two flares each (12 negative, 2 positive), five had three flares each (12 negative, 3 positive), and one patient had four flares (two negative, two positive). Therefore, within the group with recurrent symptoms, seven positive cultures were obtained for a rate of recurrent bacteriuria of 6.6% (7/106). Nine of the 10 positive bacterial cultures were due to gram-negative bacteria: Escherichia coli (n = 6), Proteus mirabilis (n = 1), Klebsiella pneumonia (n = 1), and Citrobacter sp. (n = 1). One grew Streptococcus sp. There was no difference between the flare group and nonflares in regards to age or PUF scores between groups. This study is the first to report on the low incidence of confirmed UTIs in a large group of PBS/IC patients followed longitudinally. These data suggest that only a small number of PBS/IC patients with UTI symptoms have positive urine cultures (9.4%; 10/106). Although the symptoms of recurrent UTI are prevalent in IC patients, the incidence of confirmed recurrent UTIs is only 6.6%. Because the flares of IC are usually self-limiting, treatment response to antibiotics may be misleading in light of the low incidence of positive urine cultures. These data suggest that the symptom flares of IC are not usually associated with recurrent UTI and, therefore, are likely due to a triggering of the other painful mechanisms involved in IC patients who are culture-negative.

Adult↗

3-Methylaspartate ammonia-lyase as a marker enzyme of the mesaconate pathway for (S)-glutamate fermentation in Enterobacteriaceae.

The enzyme 3-methylaspartase (3-methylaspartate ammonia-lyase, EC 4. 3.1.2) was found in the cells of enteric bacteria, especially in the genera Citrobacter and Morganella, that were grown under anoxic and oxygen-limited conditions. The enzymes were purified to homogeneity from the cell-free extracts of 18 active strains and had similar enzymological properties such as action on columns, specific activity, molecular weight, subunit structure, and N-terminal amino acid sequence similarity. The production of the enzyme was dependent on the limitation of oxygen during growth and was arrested by aeration. The addition of external electron acceptors such as dimethylsulfoxide could support cell growth and production of the enzyme. Activities of glutamate mutase (EC 5.4.99.1) and (S)-citramalate hydrolyase (EC 4.2.1.34), key enzymes of the mesaconate pathway of (S)-glutamate fermentation in the genus Clostridium, were detected in the cells of the active strains grown under oxygen-limited conditions. Based on the results, the mesaconate pathway is proposed to explain the (S)-glutamate fermentation process observed in Enterobacteriaceae, and 3-methylaspartase could be a marker enzyme for this pathway.

Aerobiosis↗

Isolation and characterisation of bacterial strains containing enantioselective DMSO reductase activity: application to the kinetic resolution of racemic sulfoxides.

The kinetic resolution of racemic sulfoxides by dimethyl sulfoxide (DMSO) reductases was investigated with a range of microorganisms. Three bacterial isolates (provisionally identified as Citrobacter braakii, Klebsiella sp. and Serratia sp.) expressing DMSO reductase activity were isolated from environmental samples by anaerobic enrichment with DMSO as terminal electron acceptor. The organisms reduced a diverse range of racemic sulfoxides to yield either residual enantiomer depending upon the strain used. C. braakii DMSO-11 exhibited wide substrate specificity that included dialkyl, diaryl and alkylaryl sulfoxides, and was unique in its ability to reduce the thiosulfinate 1,4-dihydrobenzo-2, 3-dithian-2-oxide. DMSO reductase was purified from the periplasmic fraction of C. braakii DMSO-11 and was used to demonstrate unequivocally that the DMSO reductase was responsible for enantiospecific reductive resolution of racemic sulfoxides.

Citrobacter↗

Detection of bacterial DNA in painful degenerated spinal discs in patients without signs of clinical infection.

A local inflammatory and potentially painful response, of which the ultimate cause is unknown, has been described in nervous tissues in contact with degenerated disc material in patients with low back and leg pain. With the rationale that a possible cause of such inflammation could be bacterial infection, we utilized PCR (polymerase chain reaction) amplification of the 16S rRNA (ribosomal RNA) gene followed by gene sequencing, to investigate whether bacterial DNA might be detected in the degenerative discs of 10 patients operated for disc herniation or post-discectomy syndrome. One patient with disc hernia harbored DNA homologous to Bacillus cereus, and in one patient suffering from post-discectomy syndrome, Citrobacter braaki/freundii DNA was detected. The finding demonstrates that 16S rRNA PCR can be a useful tool in search of bacterial DNA in degenerated discs, which in turn may be indicative of low-grade infection, manifesting itself only as pain rather than as clinical infection.

Adult↗

Crystallization and crystal data on tyrosine phenol-lyase.

Crystals of the apoenzyme of tyrosine phenol-lyase (EC 4.1.99.2), a pyridoxal 5'-phosphate-dependent enzyme from Citrobacter intermedius, have been grown by vapor diffusion of an ammonium sulfate solution to a protein solution. The crystals belong to space group P2(1)2(1)2, with dimensions of a = 75.5 A, b = 138.4 A and c = 94.1 A and diffract up to 2.7 A resolution. The asymmetric unit contains one half of the enzyme tetrameric molecule. Two heavy-atom derivatives of the crystals have been obtained.

Ammonium Sulfate↗

The polypeptide chain fold in tyrosine phenol-lyase, a pyridoxal-5'-phosphate-dependent enzyme.

The tyrosine phenol lyase (EC 4.1.99.2) from Citrobacter intermedius has been crystallised in the apo form by vapour diffusion. The space group is P2(1)2(1)2. The unit cell has dimensions a = 76.0 A, b = 138.3 A, c = 93.5 A and it contains two subunits of the tetrameric molecule in the asymmetric unit. Diffraction data for the native enzyme and two heavy atom derivatives have been collected with synchrotron radiation and an image plate scanner. The structure has been solved at 2.7 A resolution by isomorphous replacement with subsequent modification of the phases by averaging the density around the non-crystallographic symmetry axis. The electron density maps clearly show the relative orientation of the subunits and most of the trace of the polypeptide chain. Each subunit consists of two domains. The topology of the large domain appears to be similar to that of the aminotransferases.

Chemical Phenomena↗

Conservation of organization in the specificity polypeptides of two families of type I restriction enzymes.

We have identified the recognition sequence for the Citrobacter freundii restriction endonuclease CfrA, a member of the A-family of type I R-M enzymes. This bipartite target sequence differs in both its components from those of other type I enzymes. We determined the nucleotide sequence of its specificity gene (hsdS) and a comparison of this with its relative EcoA identifies two extensive variable regions, an organization analogous to that found in the K-family of type I R-M enzymes. The specificity polypeptides of the A-family, unlike those of K, have an N-terminal conserved region, and this includes a sequence repeated within the central conserved region. A second repeat sequence, identified at the amino acid level, coincides with the only sequence similarity common to all type I S polypeptides. Sequences immediately downstream from the hsdS genes of EcoA, CfrA, EcoK, B and D are almost identical, consistent with an allelic chromosomal location.

Amino Acid Sequence↗

The mutagenic action of nitroimidazoles. II. Tinidazole, ipronidazole, panidazole and ornidazole.

The 5-nitroimidazoles tinidazole (Fasigyn), ipronidazole (Ro-7-1554), panidazole and ornidazole (Tiberal, Ro-7-0207) in concentrations of 0.02--1 mM per liter increased the mutation frequency of Klebsiella pneumoniae. Escherichia coli K12 and Citrobacter freundii to streptomycin resistance, including streptomycin dependence, in Luria and Delbrück's fluctuation test. At low concentration (0.1 mM), the increase of the mutation frequency caused by each compound was nearly the same, i.e. 3--4 times the spontaneous mutation frequency. At higher concentrations, considerable differences between the mutagenic activities of the compounds occurred.

Citrobacter↗

Plasmid encoded beta-lactamases resistant to inhibition by clavulanic acid produced by calf faecal coliforms.

Two new plasmid encoded beta-lactamase enzymes produced by a strain of Escherichia coli and a strain of Citrobacter freundii isolated from calf faeces have been characterised. Both enzymes were similar to TEM-1 in terms of substrate and inhibition profiles and physical properties but differed from TEM-1 in being far less susceptible to the beta-lactamase inhibitors clavulanic acid or tazobactam. In each case transfer of the plasmid E coli K12 rendered it clinically resistant to the combination of amoxycillin and clavulanic acid. The beta-lactamase from the E coli had an iso-electric point (pI) of 5.4 and was encoded on a plasmid of 95 Kbp which also mediated resistance to tetracycline, sulphonamides, apramycin, streptomycin and gentamicin. The beta-lactamase from the C freundii had a pI of 5.2 and was encoded on a 75 Kbp plasmid which also mediated resistance to trimethoprim, chloramphenicol, apramycin, gentamicin and tobramycin.

Amoxicillin↗

Microbial pollution indicators in Brazilian tropical and subtropical marine surface waters.

The specificity of indicators that depend on elevated incubation temperature as a selective factor for their enumeration was questioned because of the possibility of interference from autochthonous microorganisms adapted to high ambient temperatures. Lactose-fermenting cultures isolated from fecal coliform tests of tropical marine surface waters were identified as consisting of about 70% Escherichia coli, and most of the remaining cultures being Klebsiella, Enterobacter or Citrobacter species. This confirmed the taxonomic specificity of fecal coliform tests for these waters. Fecal and total coliforms, fecal streptococcus, heterotrophic bacteria and yeast counts had correlations of above 99% confidence levels with most other microbial and chemical parameters studied. Waters with fecal coliform counts above 1000 per 100 ml had increased incidence of presumptive pathogenic yeasts, Pseudomonas aeruginosa and Salmonella. Our data support the use of coliforms or fecal streptococci as indicators of recent fecal pollution in tropical marine waters and yeast or heterotrophic bacteria counts as complementary indicator methods for these waters.

Brazil↗

Antibiotic resistance among coliform bacteria isolated from carcasses of commercially slaughtered chickens.

A total of 322 coliform bacteria Escherichia coli, Enterobacter spp., Citrobacter spp., Klebsiella spp. and Serratia spp., were isolated from 50 carcasses of commercially slaughtered chickens. Their resistance to ampicillin, tetracycline, gentamicin, chloramphenicol, cephalotine, cotrimoxazole, nalidixic acid and nitrofurantoin, were determined. The most commonly found resistance was to tetracycline followed by cephalotine, cotrimoxazole and nalidixic acid. A large percentage of E. coli (41%) and Klebsiella spp. (38%) showed multiple antibiotic resistance.

Animals↗