Transmissible plasmids from Campylobacter jejuni.

Tetracycline resistance in clinical isolates of Campylobacter jejuni was shown to be plasmid mediated. Intra- and interspecies transfers to C. fetus subsp. fetus were demonstrated. The frequency of transfer was increased by approximately 100-fold on a solid surface by using a plate- or filter-mating procedure, as compared with a liquid-mating method. Results of experiments in which cell-free filtrates were used to replace the donor strain in mating experiments tend to rule out bacteriophage-mediated transduction in the transfer of tetracycline resistance. The plasmid-transfer frequency was not affected when deoxyribonuclease was added to the agar used in the mating experiments, indicating that transformation was not involved. Four transmissible plasmids from different tetracycline-resistant strains of C. jejuni each had a molecular weight of 38 x 10(6). Transfer of these plasmids to Escherichia coli was not demonstrated.     

The E-Test and Campylobacter jejuni.

The E-Test is a recently introduced method for performing antimicrobial susceptibility tests. We compared the E-Test to the broth microdilution test and to the standard agar dilution test by using five antimicrobial agents tested against 55 clinical isolates of Campylobacter jejuni from 11 locations in USA (group 1). Later, we selected 30 strains (group 2), which were more resistant than the original survey isolates. Erythromycin, tetracycline, and ciprofloxacin were tested on both groups of organisms. When the three test methods were compared with each other at +/- 1 log2 dilution, the E-test gave the best overall agreement, with 85% of all strains being within acceptable limits. Category interpretation of erythromycin (drug of choice) results was a problem using current NCCLS guideline breakpoints. For the E-Test and the agar dilution method, 82% of the strains were in the intermediate category; but with the broth microdilution method, only 16.4% of the isolates were interpreted as intermediate. If the susceptible category breakpoint was raised to less than or equal to 2 micrograms/ml, then only 3% of the C. jejuni isolates would be interpreted as intermediate by any of the three methods. Our preference for antimicrobial susceptibility testing of C. jejuni is either E-Test or agar dilution at 42 degrees C for 16 hr.     

Early treatment of Campylobacter jejuni enteritis.

The bacteriologic and clinical effects of early antibiotic treatment of Campylobacter jejuni enteritis were studied. Erythromycin rapidly eliminated C. jejuni from stools, whereas trimethoprim-sulfamethoxazole did not. Despite its bacteriologic effectiveness, erythromycin did not reduce the duration or severity of diarrhea, abdominal pain, or other symptoms.     

Characterization of erythromycin resistance in Campylobacter jejuni and Campylobacter coli.

The mechanism of resistance to erythromycin, the drug of choice in the treatment of campylobacter gastroenteritis, was investigated. Erythromycin resistance (MICs, greater than 1,024 micrograms/ml) in three clinical isolates of Campylobacter jejuni and one C. coli isolate was determined to be constitutive and chromosomally mediated. In vivo protein synthesis in erythromycin-susceptible C. jejuni and C. coli strains was completely inhibited by low levels of erythromycin (5 micrograms/ml), whereas a high concentration of the antibiotic (100 micrograms/ml) had no effect on protein synthesis in erythromycin-resistant strains. Biological assays showed that extracellular degradation of erythromycin was not responsible for erythromycin resistance in strains of Campylobacter species. The rates and amounts of uptake of [14C]erythromycin by resistant and susceptible campylobacter cells were determined to be similar. Binding assays with purified campylobacter 70S ribosomes as well as 50S ribosomal subunits showed that those from erythromycin-resistant strans bound much less [14C]erythromycin than did those from susceptible strains. Genomic DNA from C. coli UA585 was used to transform erythromycin resistance to C. coli UA417. The erythromycin resistance marker was associated with a 240-kb SmaI fragment of the C. coli UA585 genome. Our results rule out erythromycin inactivation or efflux and are not consistent with the production of an RNA methylase, although they are consistent with a mutational mechanism of resistance due to a change in a ribosomal protein gene. This study constitutes a detailed biochemical and genetic characterization of erythromycin resistance in Campylobacter species.     

Antibacterial activities of nitrothiazole against Campylobacter jejuni and Campylobacter coli.

Niridazole (Ambilhar) and three other newly synthesized nitrothiazole derivatives were highly active against 19 microaerophilic campylobacters (minimum concentration required to inhibit 50% of strains [MIC50], 0.0075 to 0.015 mg/liter). There were, however, considerable differences in the susceptibility among strains tested, and one nitrothiazole derivative was rather inactive (MIC50, 2 mg/liter). Nitroimidazole derivatives, such as metronidazole and tinidazole, were less active (MIC50, 2 and 4 mg/liter, respectively). The nitrofuran derivatives, such as nitrofurazone and nitrofurantoin, were also less active (MIC50, 1 mg/liter). Niridazole and another potent nitrothiazole derivative killed the campylobacters rapidly at low concentrations. In contrast, much higher concentrations of metronidazole were required to achieve bactericidal values.     

Macrolide resistance in Campylobacter jejuni and Campylobacter coli

Infection with Campylobacter jejuni is now considered to be the most common cause of acute bacterial gastroenteritis in humans worldwide. It occurs more frequently than infections caused by Salmonella species, Shigella species, or Escherichia coli O157:H7. Although C. jejuni is also recognized for its association with serious post-infection neurological complications, most patients with C. jejuni infections have a self-limited illness. Nevertheless, a substantial proportion of these infections are treated with antibiotics. These include severe and prolonged cases of enteritis, infections in immune-suppressed patients, septicaemia and other extra-intestinal infections. Under these circumstances, erythromycin is often recommended as the drug of first choice. However, erythromycin-resistant Campylobacter have emerged during therapy with macrolides. Moreover, the widespread use of macrolides, including erythromycin, in veterinary medicine has accelerated this resistance trend. Several countries including Canada, Japan and Finland have reported C. jejuni isolates with low and stable rates of macrolide resistance. In contrast, the increasing level of macrolide resistance in C. jejuni is becoming a major public health concern in other parts of the world such as the United States, Europe and Taiwan. Macrolide resistance in Campylobacter is mainly associated with point mutation(s) occurring in the peptidyl-encoding region in domain V of the 23S rRNA gene, the target of macrolides. Several rapid and practical techniques have recently been developed for the identification of macrolide-resistant isolates of C. jejuni. The aim of this mini-review is to give an overview of the worldwide distribution of macrolide resistance in C. jejuni and Campylobacter coli as well as its possible association with the massive use of these agents in food animals. Mechanisms implicated in macrolide resistance in C. jejuni and also techniques that have been developed for the efficient detection of macrolide-associated mutation(s) will be discussed in detail.     

Susceptibility of Campylobacter jejuni and Campylobacter coli to macrolides and related compounds.

The susceptibility of 105 thermophilic campylobacters from human and swine origins to eight macrolides and related compounds was tested. Erythromycin, josamycin, clindamycin, and ASE 136 BS (a new erythromycin derivative) were the most active against the human strains. The swine strains were highly resistant, except to pristinamycin. The human Campylobacter coli strains (except for two strains) behaved like the C. jejuni strains.     

Survey of plasmids and resistance factors in Campylobacter jejuni and Campylobacter coli.

A total of 688 isolates of Campylobacter jejuni and Campylobacter coli were screened for the presence of plasmid DNA by agarose gel electrophoresis and were tested for susceptibility to ampicillin, chloramphenicol, erythromycin, streptomycin, and tetracycline. Of the isolates examined, 32% were noted to harbor plasmid DNA, ranging in size from 2.0 to 162 kilobases. Only tetracycline resistance was noted to correlate with the presence of plasmids. Plasmids capable of transferring tetracycline resistance via conjugation ranged in size from 42 to 100 kilobases. The Bg/II and Bc/I restriction endonuclease profiles of 31 plasmids examined showed marked diversity in their banding patterns. Although a high degree of DNA-DNA homology was noted among the Campylobacter spp. plasmids, no homology was noted between these plasmids and tetracycline R factors commonly found in the family Enterobacteriaceae.     

Genetic studies of kanamycin resistance in Campylobacter jejuni.

Campylobacter jejuni 3H40 and 4B20 harbored 59-kilobase (kb) self-transmissible plasmids encoding resistance to kanamycin and tetracycline. Although the two antibiotic resistances were more frequently inherited together, some transconjugants and ethidium bromide segregants which were resistant to only one of these antibiotics were recovered. The kanamycin-susceptible, tetracycline-resistant segregants carried plasmids 4 kb smaller than the 59-kb plasmids of their parents, whereas the kanamycin-resistant, tetracycline-susceptible segregants contained no detectable plasmid DNA. Restriction endonuclease maps of deleted forms of the 59-kb plasmids revealed that deletions and rearrangements of 4-kb lengths of DNA were associated with loss of kanamycin resistance. Translocation of the kanamycin resistance determinant between plasmid and chromosomal DNA was demonstrated. Such phenomena have not been previously described in C. jejuni spp. and are consistent with the interpretation that the kanamycin resistance determinant is encoded by a translocatable element.     

Characterization of high-level quinolone resistance in Campylobacter jejuni.

High-level resistance to quinolones has previously been shown to occur in Campylobacter spp. both in vitro and in patients treated with quinolones. We have selected isolates that are resistant to quinolones by plating cells from a susceptible C. jejuni strain, UA535, on medium containing nalidixic acid at 32 micrograms/ml. Fluctuation analysis indicated that resistance occurred by mutation at a frequency of 5 x 10(-8) per cell plated. Unlike what is observed with other gram-negative organisms, the nalidixic acid-resistant mutants demonstrated high-level cross-resistance (MIC, greater than or equal to 4 micrograms/ml) to newer quinolones, including ciprofloxacin, norfloxacin, and temafloxacin, yet remained susceptible to coumermycin A1 and several other unrelated antibiotics. Mutants with an identical resistance phenotype could also be selected from UA535 with ciprofloxacin and norfloxacin at a similar frequency. To study the mechanism of quinolone resistance, DNA gyrases were purified from C. jejuni UA535 and two resistant mutants by heparin-agarose and novobiocin-Sepharose chromatography. After the respective enzyme concentrations were adjusted to equivalent units of activity in the DNA supercoiling reaction, the DNA gyrases from the resistant mutants were found to be 100-fold less susceptible than the wild-type enzyme to inhibition by quinolones. Subunit switching experiments with purified A and B subunits from the wild type and one of the quinolone-resistant mutants indicated that an alteration in the A subunit was responsible for resistance. These results show that a single-step mutation can occur in vitro in the gene encoding DNA gyrase in C. jejuni, producing clinically relevant levels of resistance to the newer quinolones.     

In vitro susceptibilities of Campylobacter jejuni and Campylobacter coli to azithromycin and erythromycin.

MICs of azithromycin and erythromycin for 20 Campylobacter coli and 20 Campylobacter jejuni strains were determined. The results demonstrated that, for Campylobacter species, all high-level erythromycin-resistant strains were also resistant to azithromycin and that azithromycin did not exhibit increased potency in comparison with that of erythromycin.     

Tetracycline resistance of Australian Campylobacter jejuni and Campylobacter coli isolates

OBJECTIVES: Tetracycline resistance in Campylobacter is encoded by the tet(O) gene and is usually associated with conjugative plasmids. Little was known about tetracycline resistance in Australian Campylobacter species, therefore we investigated this resistance in 41 Campylobacter jejuni and five Campylobacter coli strains from humans and healthy chickens. METHODS: Tetracycline MICs were determined for each isolate using an agar dilution method. The distribution and localization of tet(O) on plasmid and chromosomal DNA was determined by Southern-blot experiments. The ability to transfer resistance to recipient strains was examined through conjugation studies. Identity of transconjugants was confirmed by PCR and flaA-restriction fragment length polymorphism analysis. RESULTS: High-level tetracycline resistance was observed, ranging from 32 to >256 mg/L. Plasmids were detected in 74% of isolates with plasmids between 30 and 40 kb in size most frequently isolated. tet(O) was present in all tetracycline-resistant isolates. In the majority of strains under study the tet(O) gene was chromosomally encoded. Tetracycline resistance of six C. jejuni strains in which tet(O) was plasmid mediated was transferred by conjugation to a C. jejuni recipient strain. Transfer did not occur between tetracycline-resistant C. jejuni strains and a C. coli recipient. No difference in MICs, plasmid carriage and tet(O) localization was detected between human and chicken isolates. CONCLUSIONS: These data indicate that the tet(O) gene, previously reported in Campylobacter strains throughout the world, is present in Australian Campylobacter. This study will lead to a greater understanding of antibiotic resistance distribution in Campylobacter spp. in Australia.     

Evidence for an efflux pump in multidrug-resistant Campylobacter jejuni.

Mechanisms of drug resistance in Campylobacter jejuni were investigated. Mutant strains 34PEFr, which was resistant to pefloxacin (128-fold increase in the MIC), and 34CTXr, which was resistant to cefotaxime (32-fold increase in the MIC) and which was derived from the susceptible parent 34s, were obtained by serial passages on pefloxacin and cefotaxime gradient plates, respectively. Both mutants showed cross-resistance to erythromycin, chloramphenicol, tetracycline, beta-lactams, and quinolones. While the quinolone resistance of strain PEFr could be explained by a mutation at codon 86 of the gyrA gene, the multidrug resistance phenotype of both strains was further investigated. Accumulation of pefloxacin, ciprofloxacin, and minocycline was measured by fluorometry and was found to be lower in the mutant strains than in the parent strain. Preincubation of the cells with carbonyl cyanide m-chlorophenylhydrazone, however, completely abolished this difference. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane preparations from both mutant strains showed overexpression of two proteins of 55 and 39 kDa which were absent from the outer membranes of the wild-type strain. These results indicate that in C. jejuni 34PEFr and 34CTXr, multidrug resistance is associated with an efflux system with a broad specificity.     

Emergence of fluoroquinolone resistance in Campylobacter jejuni and Campylobacter coli in subjects from Finland.

The in vitro susceptibilities of 102 human campylobacter strains isolated between 1978 and 1980 and 100 strains isolated in 1990 to ciprofloxacin, norfloxacin, erythromycin, gentamicin, and doxycycline were examined. The biotypes and heat-stable serotypes of the strains as well as antimicrobial treatments and travel history of the campylobacter-positive patients were also studied. The results indicated that susceptibility to erythromycin, gentamicin, and doxycycline has remained the same during the past 10 years. No gentamicin-resistant strains were found. Resistance to erythromycin was 3% in both groups of strains. However, the number of norfloxacin-resistant strains increased from 4 to 11% in the follow-up period, and ciprofloxacin-resistant strains, which had not occurred 10 years ago, composed 9% of the strains isolated in 1990. Thus, the increase of fluoroquinolone resistance in Campylobacter jejuni and Campylobacter coli has been significant in Finland in the past 10 years.     

Comparison of antimicrobial susceptibility patterns of Campylobacter jejuni and Campylobacter coli

To determine whether employing antibiograms is useful to separate Campylobacter jejuni and Campylobacter coli, we determined the MICs of 12 antibiotics for 104 human clinical strains and 74 swine strains. Of 74 swine strains, 5 (7%) were hippurate positive, as were 93 (89%) of 104 human strains. The 12 antimicrobial agents tested were ampicillin, amoxicillin, clindamycin, chloramphenicol, erythromycin, furazolidone, norfloxacin, nalidixic acid, rosoxacin, rosaramicin, tetracycline, and Sch 32063. Isolates from humans were significantly (P less than 0.001) more susceptible than swine strains to clindamycin, erythromycin, rosaramicin, and Sch 32063. Of 11 human hippurate-negative strains, 3 (27%) were resistant to clindamycin, erythromycin, rosaramicin, and Sch 32063, compared with 1 of 93 (1%) hippurate-positive strains. Nearly all human and swine strains were susceptible to furazolidone and nalidixic acid. Campylobacter isolates from humans and swine have different antibiograms, and the susceptibility to certain antibiotics, such as clindamycin, may be helpful for differentiation of C. jejuni from C. coli.     

Acute myocarditis secondary to Campylobacter jejuni enterocolitis

Myocarditis is a rare condition that can mimic an acute coronary syndrome (ACS). We present the case of a 24-year-old male with Noonan syndrome who presented with a diarrhoeal pro-dromal illness, acute onset chest pain, elevated cardiac biomarkers and an abnormal ECG with ST elevation in the absence of obstructive coronary artery disease. The patient had acute myocarditis secondary to Campylobacter jejuni enterocolitis. Infective myocarditis is most commonly due to a viral infection. Myocarditis is very rarely due to a bacterial infection with only isolated reports of myocarditis induced by Campylobacter jejuni infection. At follow-up he remains well. Myocarditis should be considered in all patients presenting with acute onset chest pain and elevated cardiac biomarkers.     

Campylobacter jejuni enteritis in a premature neonate

We have described the first case of Campylobacter jejuni enteritis in a premature neonate, whose clinical course was characterized by a self-limited bloody diarrhea accompanied by metabolic acidosis and poor weight gain. The importance of this organism in causing preterm labor in mothers and enteritis in premature neonates awaits systematic study.     

Characterization of the porins of Campylobacter jejuni and Campylobacter coli and implications for antibiotic susceptibility.

The major outer membrane protein was extracted from Campylobacter coli by Triton X-100/EDTA fractionation of cell envelopes. This heat-modifiable protein was shown to have pore-forming activity in black lipid bilayers. The C. coli porin formed a relatively small cation-selective pore with a mean single-channel conductance of 0.53 +/- 0.16 nS in 1.0 M KCl. There was no evidence of oligomer formation, which suggested that each protein monomer formed a pore. Pore-forming activity of the C. coli porin and similarly prepared Campylobacter jejuni porin was also measured in liposome-swelling assays. These results confirmed the cation selectivity of both pores. The C. coli porin formed a small pore, which hindered the penetration of solutes with a molecular weight of 262, and a larger pore, which hindered the penetration of solutes with a molecular weight of 340, in a protein-concentration-dependent manner. C. jejuni formed one size of pore that was slightly larger than the C. coli pore and just permitted the passage of solutes, with a molecular weight of 340. A review of the literature concerning in vitro screening of antimicrobial agents tended to confirm the low permeability of the C. jejuni outer membrane to hydrophilic antimicrobial agents except when the molecules had molecular weights of less than 360. The porins of C. jejuni and C. coli may contribute to intrinsic resistance to antimicrobial agents, whereas alternative (nonporin) routes of antimicrobial agent uptake may be more important determinants of susceptibility to antimicrobial agents.     

Development of a PCR ELISA assay for the identification of Campylobacter jejuni and Campylobacter coli.

A polymerase chain reaction (PCR) assay was developed based on a solution-hybridization colorimetric end-point detection format (PCR ELISA) for the identification of Campylobacter jejuni and Campylobacter coli. PCR primers were designed to target a gene sequence with species-specific motifs. Five biotin-labelled probes targeted to the species-specific motifs were investigated for the detection of digoxygenin-labelled PCR products from C. jejuni and C. coli using the PCR ELISA format. Two probes were identified, one which reacts with both the C. jejuni and C. coli target sequences (probe CC2) and one probe which reacts with the C. jejuni target sequence only (probe CJ2). The specificity of the assay with the CJ2 and CC2 probes was investigated with a range of Campylobacter spp., Arcobacter spp., Helicobacter spp. and a range of unrelated organisms. The PCR ELISA assay and probes were demonstrated to be specific for C. jejuni and C. coli. The sensitivity of the PCR ELISA assay was demonstrated to be 10-100-fold more sensitive than a gel-based PCR method using the same primers. This PCR ELISA assay is sensitive, specific and significantly reduces the time needed for the identification of C. jejuni and C. coli and has the potential to facilitate early detection of these important gastro-intestinal pathogens.