Role for tandem duplication and lon protease in AcrAB-TolC- dependent multiple antibiotic resistance (Mar) in an Escherichia coli mutant without mutations in marRAB or acrRAB

A spontaneous mutant (M113) of Escherichia coli AG100 with an unstable multiple antibiotic resistance (Mar) phenotype was isolated in the presence of tetracycline. Two mutations were found: an insertion in the promoter of lon (lon3::IS186) that occurred first and a subsequent large tandem duplication, dupIS186, bearing the genes acrAB and extending from the lon3::IS186 to another IS186 present 149 kb away from lon. The decreased amount of Lon protease increased the amount of MarA by stabilization of the basal quantities of MarA produced, which in turn increased the amount of multidrug effux pump AcrAB-TolC. However, in a mutant carrying only a lon mutation, the overproduced pump mediated little, if any, increased multidrug resistance, indicating that the Lon protease was required for the function of the pump. This requirement was only partial since resistance was mediated when amounts of AcrAB in a lon mutant were further increased by a second mutation. In M113, amplification of acrAB on the duplication led to increased amounts of AcrAB and multidrug resistance. Spontaneous gene duplication represents a new mechanism for mediating multidrug resistance in E. coli through AcrAB-TolC.

Fast DNA serotyping of Escherichia coli by use of an oligonucleotide microarray

Classical antibody-based serotyping of Escherichia coli is an important method in diagnostic microbiology for epidemiological purposes, as well as for a rough virulence assessment. However, serotyping is so tedious that its use is restricted to a few reference laboratories. To improve this situation we developed and validated a genetic approach for serotyping based on the microarray technology. The genes encoding the O-antigen flippase (wzx) and the O-antigen polymerase (wzy) were selected as target sequences for the O antigen, whereas fliC and related genes, which code for the flagellar monomer, were chosen as representatives for the H phenotype. Starting with a detailed bioinformatic analysis and oligonucleotide design, an ArrayTube-based assay was established: a fast and robust DNA extraction method was coupled with a site-specific, linear multiplex labeling procedure and hybridization analysis of the biotinylated amplicons. The microarray contained oligonucleotide DNA probes, each in duplicate, representing 24 of the epidemiologically most relevant of the over 180 known O antigens (O antigens 4, 6 to 9, 15, 26, 52, 53, 55, 79, 86, 91, 101, 103, 104, 111, 113, 114, 121, 128, 145, 157, and 172) as well as 47 of the 53 different H antigens (H antigens 1 to 12, 14 to 16, 18 to 21, 23 to 34, 37 to 43, 45, 46, 48, 49, 51 to 54, and 56). Evaluation of the microarray with a set of defined strains representing all O and H serotypes covered revealed that it has a high sensitivity and a high specificity. All of the conventionally typed 24 O groups and all of the 47 H serotypes were correctly identified. Moreover, strains which were nonmotile or nontypeable by previous serotyping assays yielded unequivocal results with the novel ArrayTube assay, which proved to be a valuable alternative to classical serotyping, allowing processing of single colonies within a single working day.

Pathotyping Escherichia coli by using miniaturized DNA microarrays

The detection of virulence determinants harbored by pathogenic Escherichia coli is important for establishing the pathotype responsible for infection. A sensitive and specific miniaturized virulence microarray containing 60 oligonucleotide probes was developed. It detected six E. coli pathotypes and will be suitable in the future for high-throughput use.

Increased genome instability in Escherichia coli lon mutants: relation to emergence of multiple-antibiotic-resistant (Mar) mutants caused by insertion sequence elements and large tandem genomic amplifications

Thirteen spontaneous multiple-antibiotic-resistant (Mar) mutants of Escherichia coli AG100 were isolated on Luria-Bertani (LB) agar in the presence of tetracycline (4 microg/ml). The phenotype was linked to insertion sequence (IS) insertions in marR or acrR or unstable large tandem genomic amplifications which included acrAB and which were bordered by IS3 or IS5 sequences. Five different lon mutations, not related to the Mar phenotype, were also found in 12 of the 13 mutants. Under specific selective conditions, most drug-resistant mutants appearing late on the selective plates evolved from a subpopulation of AG100 with lon mutations. That the lon locus was involved in the evolution to low levels of multidrug resistance was supported by the following findings: (i) AG100 grown in LB broth had an important spontaneous subpopulation (about 3.7x10(-4)) of lon::IS186 mutants, (ii) new lon mutants appeared during the selection on antibiotic-containing agar plates, (iii) lon mutants could slowly grow in the presence of low amounts (about 2x MIC of the wild type) of chloramphenicol or tetracycline, and (iv) a lon mutation conferred a mutator phenotype which increased IS transposition and genome rearrangements. The association between lon mutations and mutations causing the Mar phenotype was dependent on the medium (LB versus MacConkey medium) and the antibiotic used for the selection. A previously reported unstable amplifiable high-level resistance observed after the prolonged growth of Mar mutants in a low concentration of tetracycline or chloramphenicol can be explained by genomic amplification.

Fluid administration, brain edema, and cerebrospinal fluid lactate and glucose concentrations in experimental Escherichia coli meningitis

The effect of no fluids versus liberal fluid supplementation on brain edema and cerebrospinal fluid (CSF) lactate and glucose concentrations was compared in rabbits with experimental Escherichia coli meningitis. Fluid restriction for the duration of the experiment (19 h) led to a decrease in body weight by approximately 5%, while the high fluid regimen increased body weight by approximately 5%. Infected animals developed brain edema compared with controls, but the fluid regimen had no measurable effect on the degree of edema. In contrast, fluid-restricted animals had significantly higher CSF lactate and lower CSF glucose concentrations than fluid-supplemented animals (lactate, 13.5 +/- 3.5 vs. 10.1 +/- 3.3 mmol/L; glucose, 1.89 +/- 1.39 vs. 4.11 +/- 1.39 mmol/L). These results fail to support the hypothesis that administration of large amounts of fluid in this model of gram-negative bacterial meningitis aggravates brain edema.

Use of ampicillin-sulbactam for treatment of experimental meningitis caused by a beta-lactamase-producing strain of Escherichia coli K-1

We evaluated the pharmacokinetics and therapeutic efficacy of ampicillin combined with sulbactam in a rabbit model of meningitis due to a beta-lactamase-producing strain of Escherichia coli K-1. Ceftriaxone was used as a comparison drug. The MIC and MBC were 32 and greater than 64 micrograms/ml (ampicillin), greater than 256 and greater than 256 micrograms/ml (sulbactam), 2.0 and 4.0 micrograms/ml (ampicillin-sulbactam [2:1 ratio, ampicillin concentration]) and 0.125 and 0.25 micrograms/ml (ceftriaxone). All antibiotics were given by intravenous bolus injection in a number of dosing regimens. Ampicillin and sulbactam achieved high concentrations in cerebrospinal fluid (CSF) with higher dose regimens, but only moderate bactericidal activity compared with that of ceftriaxone was obtained. CSF bacterial titers were reduced by 0.6 +/- 0.3 log10 CFU/ml/h with the highest ampicillin-sulbactam dose used (500 and 500 mg/kg of body weight, two doses). This was similar to the bactericidal activity achieved by low-dose ceftriaxone (10 mg/kg), while a higher ceftriaxone dose (100 mg/kg) produced a significant increase in bactericidal activity (1.1 +/- 0.4 log10 CFU/ml/h). It appears that ampicillin-sulbactam, despite favorable CSF pharmacokinetics in animals with meningitis, may be of limited value in the treatment of difficult-to-treat beta-lactamase-producing bacteria, against which the combination shows only moderate in vitro activity.

Evaluation of piperacillin-tazobactam in experimental meningitis caused by a beta-lactamase-producing strain of K1-positive Escherichia coli

We evaluated the pharmacokinetics and therapeutic efficacy of piperacillin combined with tazobactam, a novel beta-lactamase inhibitor, in experimental meningitis due to a beta-lactamase-producing strain of K1-positive Escherichia coli. Different doses of piperacillin and tazobactam, as single agents and combined (8:1 ratio; dosage range, 40/5 to 200/25 mg/kg per h), and of ceftriaxone were given to experimentally infected rabbits by intravenous bolus injection followed by a 5-h constant infusion. The mean (+/- standard deviation) rates for penetration into the cerebrospinal fluid of infected animals after coadministration of both drugs were 16.6 +/- 8.4% for piperacillin and 32.5 +/- 12.6% for tazobactam. Compared with either agent alone, combination treatment resulted in significantly better bactericidal activity in the cerebrospinal fluid. The bactericidal activity of piperacillin-tazobactam was dose dependent: cerebrospinal fluid bacterial titers were reduced by 0.37 +/- 0.19 log10 CFU/ml per h with the lowest dose versus 0.96 +/- 0.25 log10 CFU/ml per h with the highest dose (P less than 0.001). At the relatively high doses of 160/20 and 200/25 mg of piperacillin-tazobactam per kg per h, the bactericidal activity of the combination was comparable to that of 10 and 25 mg of ceftriaxone per kg per h, respectively.

Antibiotic therapy, endotoxin concentration in cerebrospinal fluid, and brain edema in experimental Escherichia coli meningitis in rabbits

We investigated the effect of cefotaxime and chloramphenicol on endotoxin concentrations in cerebrospinal fluid (CSF) and on the development of brain edema in rabbits with Escherichia coli meningitis. Both antibiotics were similarly effective in reducing bacterial titers. Cefotaxime, but not chloramphenicol, induced a marked increase of endotoxin in CSF, from log10 1.5 +/- 0.8 to log10 2.8 +/- 0.7 ng/ml (P less than .01). This result was associated with an increase in brain water content (405 +/- 12 g of water/100 g of dry weight compared with 389 +/- 8 g in untreated controls; P less than .01), whereas in animals treated with chloramphenicol, brain water content was identical to controls. The cefotaxime-induced increase in endotoxin concentration and brain edema were both neutralized by polymyxin B, which binds to the lipid A moiety of endotoxin, or by a monoclonal antibody to lipid A. These results indicate that treating gram-negative bacillary meningitis with selected antibiotics induces increased endotoxin concentrations in CSF that are associated with brain edema.

Immune response of horses to vaccination with the recombinant Hc domain of botulinum neurotoxin types C and D

Botulinum neurotoxins, predominantly serotypes C and D, cause equine botulism through forage poisoning. The C-terminal part of the heavy chain of botulinum neurotoxin types C and D (HcBoNT/C and D) was expressed in Escherichia coli and evaluated as a recombinant mono- and bivalent vaccine in twelve horses in comparison to a commercially available toxoid vaccine. A three-dose subcutaneous immunization of adult horses elicited robust serum antibody response in an ELISA using the immunogen as a capture antigen. Immune sera showed dose-dependent high potency in neutralizing specifically the active BoNT/C and D in the mouse protection assay. The aluminium hydroxide based mono- and bivalent recombinant HcBoNT/C and D vaccines were characterized by good compatibility and the ability to elicit protective antibody titers similar or superior to the commercially available toxoid vaccine.

Comparison of genotypes and antibiotic resistances of Campylobacter jejuni and Campylobacter coli on chicken retail meat and at slaughter

Multilocus sequence typing (MLST) and antibiotic resistance patterns of Campylobacter jejuni and Campylobacter coli from retail chicken meat showed high overlap with isolates collected at slaughterhouses, indicating little selection along the production chain. They also showed significant common sequence types with human clinical isolates, revealing chicken meat as a likely source for human infection.

Genotypes and antibiotic resistances of Campylobacter jejuni and Campylobacter coli isolates from domestic and travel-associated human cases

Multilocus sequence typing (MLST) extended with flaB typing of 425 Campylobacter jejuni isolates and 42 Campylobacter coli isolates revealed quite a low overlap between human isolates from travel-associated and domestic cases in Switzerland. Men were more frequently affected by Campylobacter than women, but strains from women and, overall, from travel-associated cases showed mutations conferring quinolone resistance more frequently than strains from men and domestic cases, respectively.

Use of diagnostic microarrays for determination of virulence gene patterns of Escherichia coli K1, a major cause of neonatal meningitis

Forty Escherichia coli strains isolated primarily from neonatal meningitis, urinary tract infections and feces were screened for the presence of virulence genes with a newly developed microarray on the array tube format. A total of 32 gene probes specific for extraintestinal as well as intestinal E. coli pathotypes were included. Eighty-eight percent of the analyzed strains were positive for the K1-specific probe on the microarray and could be confirmed with a specific antiserum against the K1 capsular polysaccharide. The gene for the hemin receptor ChuA was predominantly found in 95% of strains. Other virulence genes associated with K1 and related strains were P, S, and F1C fimbriae specific for extraintestinal E. coli, the genes for aerobactin, the alpha-hemolysin and the cytotoxic necrotizing factor. In two strains, the O157-specific catalase gene and the gene for the low-molecular-weight heat-stable toxin AstA were detected, respectively. A total of 19 different virulence gene patterns were observed. No correlation was observed between specific virulence gene patterns and a clinical outcome. The data indicate that virulence genes typical of extraintestinal E. coli are predominantly present in K1 strains. Nevertheless, some of them can carry virulence genes known to be characteristic of intestinal E. coli. The distribution and combination of virulence genes show that K1 isolates constitute a heterogeneous group of E. coli.

Virulence typing of Escherichia coli using microarrays

We describe a microarray based broad-range screening technique for Escherichia coli virulence typing. Gene probes were amplified by PCR from a plasmid bank of characterised E. coli virulence genes and were spotted onto a glass slide to form an array of capture probes. Genomic DNA from E. coli strains which were to be tested for the presence of these virulence gene sequences was labelled with fluorescent cyanine dyes by random amplification and then hybridised against the array of probes. The hybridisation, washing and data analysis conditions were optimised for glass slides, and the applicability of the method for identifying the presence of the virulence genes was determined using reference strains and clinical isolates. It was found to be a sensitive screening method for detecting virulence genes, and a powerful tool for determining the pathotype of E. coli. It will be possible to expand and automate this microarray technique to make it suitable for rapid and reliable diagnostic screening of bacterial isolates.

Antimicrobial resistance in Escherichia coli strains isolated from Swiss weaned pigs and sows

Based on Directive (EC) No 99/2003, monitoring programs on the development of antimicrobial resistance in bacteria from livestock are implemented in many European countries. The aim of the present study was (i) to establish comprehensive baseline data on the antimicrobial resistance situation in Escherichia coli isolates obtained from healthy pigs (pooled fecal samples) originating from 60 Swiss pig-breeding farms, and (ii) to analyze differences in the resistance frequency between Escherichia coli isolates from weaned pigs and sows. Susceptibility testing (disc diffusion method) was performed on 429 isolates from weaned pigs and 431 isolates from sows. Overall, 17.7% of the isolates from weaned pigs and 22.5% of the Escherichia coli isolates from sows were susceptible to all antibiotics tested. Low resistance prevalence was found for amoxicillin, amoxicillin/clavulanic acid, ampicillin, cefquinome, ciprofloxacin, colistin, florfenicol, and gentamicin. The most frequently found resistances were against streptomycin (60.6% of the isolates from weaners and 64.3% of the isolates from sows), sulfonamide (51.5% and 26.9%), tetracycline (35.2% and 22.0%), and trimethoprim (27.5% and 11.1%). With exception of colistin, most resistances were found for those antibiotics commonly used on the farms. Except for ciprofloxacin and streptomycin, isolates from weaned pigs showed higher resistance prevalence than those from sows. This difference was significant for cefquinome, florfenicol, sulfonamide, tetracycline, and trimethoprim (p<0.05).

Mdt(A), a new efflux protein conferring multiple antibiotic resistance in Lactococcus lactis and Escherichia coli

The mdt(A) gene, previously designated mef214, from Lactococcus lactis subsp. lactis plasmid pK214 encodes a protein [Mdt(A) (multiple drug transporter)] with 12 putative transmembrane segments (TMS) that contain typical motifs conserved among the efflux proteins of the major facilitator superfamily. However, it also has two C-motifs (conserved in the fifth TMS of the antiporters) and a putative ATP-binding site. Expression of the cloned mdt(A) gene decreased susceptibility to macrolides, lincosamides, streptogramins, and tetracyclines in L. lactis and Escherichia coli, but not in Enterococcus faecalis or in Staphylococcus aureus. Glucose-dependent efflux of erythromycin and tetracycline was demonstrated in L. lactis and in E. coli.