Antimicrobial resistance of Campylobacter jejuni and Campylobacter coli with special reference to plasmid profiles of Japanese clinical isolates.

A total of 111 clinical isolates of Campylobacter jejuni and 10 clinical isolates of Campylobacter coli were characterized by their susceptibility to nine antimicrobial agents and by their plasmid profiles on agarose gel electrophoresis. All of the C. jejuni isolates were susceptible to chloramphenicol, ciprofloxacin, erythromycin, kanamycin, and nalidixic acid, but 55% were tetracycline resistant. In the 10 C. coli isolates, a high prevalence of multiple-antibiotic resistance was noted. Plasmids were found in 82% of the tetracycline-resistant and 15% of the tetracycline-susceptible C. jejuni isolates. Tetracycline resistance in six randomly selected C. jejuni isolates, which contained 50- or 135-kilobase (kb) plasmids, was transferred by conjugation to a Campylobacter fetus subsp. fetus recipient with recovery of a 50- or a 45-kb plasmid from transconjugants. From one multiple-antibiotic-resistant C. coli isolate, resistance to tetracycline, kanamycin, and chloramphenicol was transferred concomitantly with a 58-kb plasmid, pNR9589. Nonconjugative 98-kb plasmids, pNR9131 and pNR9581, from C. coli isolates with resistance to tetracycline, kanamycin, and erythromycin were shown by cloning experiments to code for at least kanamycin resistance. Restriction digests revealed that 50-kb plasmids from tetracycline-resistant C. jejuni isolates were identical, although plasmids from multiple-antibiotic-resistant C. coli isolates shared partial DNA homology to each other. Cloning of the kanamycin and chloramphenicol resistance genes of pNR9589 into Escherichia coli showed that the two genes are closely linked or clustered. Double-digestion analysis of the fragments encoding the kanamycin resistance of pNR9131, pNR9581, and pNR9589 showed that these three plasmids contain a common fragment related to kanamycin resistance.     

Incidence of antibiotic resistance in Campylobacter jejuni isolated in Alberta, Canada, from 1999 to 2002, with special reference to tet(O)-mediated tetracycline resistance

Of 203 human clinical isolates of Campylobacter jejuni from Alberta, Canada (1999 to 2002), 101 isolates (50%) were resistant to at least 64 microg of tetracycline/ml, with four isolates exhibiting higher levels of tetracycline resistance (512 microg/ml). In total, the MICs for 37% of tetracycline-resistant isolates (256 to 512 microg/ml) were higher than those previously reported in C. jejuni (64 to 128 microg/ml). In the tetracycline-resistant clinical isolates, 67% contained plasmids and all contained the tet(O) gene. Four isolates resistant to high levels of tetracycline (MIC = 512 microg/ml) contained plasmids carrying the tet(O) gene, which could be transferred to other isolates of C. jejuni. The tetracycline MICs for transconjugants were comparable to those of the donors. Cloning of tet(O) from the four high-level tetracycline-resistant isolates conferred an MIC of 32 microg/ml for Escherichia coli DH5alpha. In contrast, transfer to a strain of C. jejuni by using mobilization conferred an MIC of 128 microg/ml. DNA sequence analysis determined that the tet(O) genes encoding lower MICs (64 to 128 microg/ml) were identical to one other, although the tet(O) genes encoding a 512-microg/ml MIC demonstrated several nucleotide substitutions. The quinolone resistance determining region of four ciprofloxacin-resistant isolates (2%) was analyzed, and resistance was associated with a chromosomal mutation in the gyrA gene resulting in a Thr-86-Ile substitution. In addition, six kanamycin-resistant isolates contained large plasmids that carry the aphA-3 marker coding for 3'-aminoglycoside phosphotransferase. Resistance to erythromycin was not detected in 203 isolates. In general, resistance to most antibiotics in C. jejuni remains low, except for resistance to tetracycline, which has increased from about 8 to 50% over the past 20 years.     

Cloning and expression of a tetracycline resistance determinant from Campylobacter jejuni in Escherichia coli.

The tetracycline resistance gene (tet) from the Campylobacter jejuni plasmid pFKT1025 was cloned into both pUC18 and pBR322 and was expressed when the chimeric plasmids were introduced into Escherichia coli. The location of the tet determinant on the chimeric plasmids was determined by BAL 31 deletion mapping within a 2.25-kilobase (kb) RsaI-HincII fragment. A protein of approximately 70 kilodaltons was consistently produced by E. coli maxicells harboring the cloned tet determinant. A 500-base-pair restriction fragment from within the 2.25-kb tet region was shown to hybridize only to DNA from tetracycline-resistant strains of C. jejuni and C. coli, but not to the DNA of organisms known to carry the streptococcal tetM determinant. No homology was noted between the DNA of 10 tetracycline-resistant isolates of campylobacter and the streptococcal tetL, tetM, or tetN determinants when tested under conditions of high stringency. However, homology was noted between a 5.0-kb HincII restriction fragment containing the tetM determinant and two C. jejuni tet R factors under conditions of reduced stringency.     

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.     

Antibiotic resistance of Campylobacter jejuni and Campylobacter coli clinical isolates from Poland

We tested 102 Campylobacter jejuni and 6 Campylobacter coli clinical isolates from Poland. All were susceptible to erythromycin. Among the tested C. jejuni isolates 55.9% and 13.7% were resistant to ciprofloxacin and tetracycline, respectively. Replacement of Thr86 with Ile in GyrA and a plasmid-borne tet(O) gene were the main resistance mechanisms for fluoroquinolones and tetracycline, respectively.     

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.     

Multiple genetic elements carry the tetracycline resistance gene tet(W) in the animal pathogen Arcanobacterium pyogenes

The tet(W) gene is associated with tetracycline resistance in a wide range of bacterial species, including obligately anaerobic rumen bacteria and isolates from the human gut and oral mucosa. However, little is known about how this gene is disseminated and the types of genetic elements it is carried on. We examined tetracycline-resistant isolates of the animal commensal and opportunistic pathogen Arcanobacterium pyogenes, all of which carried tet(W), and identified three genetic elements designated ATE-1, ATE-2, and ATE-3. These elements were found in 25%, 35%, and 60% of tetracycline-resistant isolates, respectively, with some strains carrying both ATE-2 and ATE-3. ATE-1 shows characteristics of a mobilizable transposon, and the tet(W) genes from strains carrying this element can be transferred at low frequencies between A. pyogenes strains. ATE-2 has characteristics of a simple transposon, carrying only the resistance gene and a transposase, while in ATE-3, the tet(W) gene is associated with a streptomycin resistance gene that is 100% identical at the DNA level with the aadE gene from the Campylobacter jejuni plasmid pCG8245. Both ATE-2 and ATE-3 show evidence of being carried on larger genetic elements, but conjugation to other strains was not observed under the conditions tested. ATE-1 was preferentially associated with A. pyogenes strains of bovine origin, while ATE-2 and ATE-3 elements were primarily found in porcine isolates, suggesting that these elements may circulate in different environments. In addition, four alleles of the tet(W) gene, primarily associated with different elements, were detected among A. pyogenes isolates.     

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 two plasmids from Campylobacter jejuni isolates that carry the aphA-7 kanamycin resistance determinant.

Two small plasmids of 11.5 and 9.5 kb, each carrying an aphA-7 kanamycin phosphotransferase gene, were studied. The MICs of kanamycin for the two human Campylobacter jejuni isolates harboring the plasmids were 10,000 and 5,000 micrograms/ml, while the MICs of amikacin were 32 and 8 micrograms/ml, respectively. The MICs of gentamicin and tobramycin were less than or equal to 2 micrograms/ml for both isolates. The restriction endonuclease maps of the plasmids were similar, with the larger plasmid showing two discrete regions of additional DNA. When the aphA-7 gene from each plasmid was cloned into pBR322, the aphA-7 gene expressed the kanamycin resistance phenotype in Escherichia coli. For transformants containing the cloned aphA-7 gene, kanamycin MICs were greater than or equal to 128 micrograms/ml. The aphA-7 gene was also subcloned from the plasmid pFKT4420 into the E. coli-Streptococcus shuttle vector pDL278 and was transformed into Streptococcus gordonii Challis. For streptococcal transformants containing the novel plasmid, kanamycin MICs were 4,000 micrograms/ml. In the presence of a tetracycline resistance plasmid, both small plasmids could be mobilized during conjugal matings to Campylobacter coli recipients.     

Detection of two different kanamycin resistance genes in naturally occurring isolates of Campylobacter jejuni and Campylobacter coli.

A total of 225 isolates of Campylobacter jejuni and 54 isolates of Campylobacter coli were screened for resistance to kanamycin. Among these, five resistant isolates of C. jejuni and six resistant isolates of C. coli, all with different plasmid patterns, were identified. Each contained at least one plasmid greater than or equal to 41 kilobases in size. The MIC of kanamycin for all 11 strains was determined to be greater than or equal to 256 micrograms/ml by an agar dilution method. In addition, all of the strains exhibited resistance to tetracycline (greater than or equal to 16 micrograms/ml). Eight of the 11 strains transferred the kanamycin resistance phenotype to other Campylobacter strains by conjugation. DNA from 9 of the 11 strains hybridized to a DNA probe specific for the 3'-O-aminoglycoside phosphotransferase type III gene. The remaining two strains also failed to show homology with DNA probes specific for the genes encoding 3'-O-aminoglycoside phosphotransferase types I, II, and III. The novel kanamycin resistance gene was cloned into the vector pBR322 and was expressed in Escherichia coli. Phosphocellulose paper binding assays on sonicates of the E. coli strain carrying the cloned kanamycin determinant demonstrated significant activity against kanamycin, neomycin, and amikacin but not against butirosin, gentamicin, tobramycin, or lividomycin, suggesting that the enzyme is the product of a 3'-O-aminoglycoside phosphotransferase type of aminoglycoside resistance gene.     

tet(L)-mediated tetracycline resistance in bovine Mannheimia and Pasteurella isolates

OBJECTIVES: Tetracycline-resistant Mannheimia and Pasteurella isolates, which were negative for the tetracycline resistance genes (tet) commonly detected among these bacteria, were investigated for other tet genes present and their location. METHODS: Mannheimia and Pasteurella isolates were investigated for their MICs of tetracycline and their plasmid content. Identification of tet genes was achieved by PCR. Plasmids mediating tetracycline resistance were identified by transformation and hybridization experiments. Plasmid pCCK3259 from Mannheimia haemolytica was sequenced completely and analysed for its structure and organization. RESULTS: All tetracycline-resistant isolates carried the gene tet(L) either on plasmids or on the chromosome. Two M. haemolytica isolates and one Mannheimia glucosida isolate harboured a common 5.3 kb tet(L) plasmid, designated pCCK3259. This plasmid was similar to the tet(B)-carrying tetracycline resistance plasmid pHS-Tet from Haemophilus parasuis and the streptomycin/spectinomycin resistance plasmid pCCK647 from Pasteurella multocida in the parts coding for mobilization functions. The tet(L) gene was closely related to that of the Geobacillus stearothermophilus plasmid pTB19. However, the translational attenuator responsible for the tetracycline-inducible expression of tet(L) was missing in plasmid pCCK3259. A recombination site was identified downstream of tet(L), which might explain the integration of the tet(L) gene region into a basic pCCK3259 replicon. CONCLUSION: A tet(L) gene was shown for the first time to be responsible for tetracycline resistance in Mannheimia and Pasteurella isolates. This report demonstrates a lateral transfer of a tetracycline efflux gene in Gram-negative bovine respiratory tract pathogens, probably originating from Gram-positive bacteria.     

DNA probes for identification of tetracycline resistance genes in Campylobacter species isolated from swine and cattle

Tetracycline-resistant strains of Campylobacter jejuni and Campylobacter coli from swine and cattle colons were isolated and characterized by hybridization with DNA probes. A probe consisting of the 1.8-kilobase (kb) HincII fragment from pUA466 was highly specific for the detection of tetracycline resistance (Tcr) in C. jejuni and C. coli. The 5-kb tetM DNA probe from Streptococcus agalactiae plasmid pJI3 which has homology with the 1.8-kb HincII fragment from pUA466 could also be used to detect Tcr Campylobacter strains. However, the tetM probe had a much lower sensitivity and required a lower stringency of hybridization. Therefore, the 1.8-kb HincII fragment appeared to be more appropriate for the classification of Tcr in Campylobacter spp. No homology was detected between the Tcr determinant from Campylobacter spp. and the tetL and tetN probes from Streptococcus spp. DNA homology was demonstrated between pUA649, a derivative of plasmid pUA466 which had lost most of the Tcr region, and Tcr plasmids from C. jejuni and C. coli isolated from animal and human sources. There was also homology between pUA649 and the chromosomes of C. jejuni and C. coli strains. In this study, all but one of the tetracycline-resistant C. coli and C. jejuni strains contained plasmids of approximately 50 kb which hybridized with the 1.8-kb HincII probe. In one C. coli strain (UA703), Tcr appeared to be chromosomally mediated.     

Plasmid-encoded Tet B tetracycline resistance in Haemophilus parasuis

The complete sequence of two plasmids, pHS-Tet (5.1 kb) and pHS-Rec (9.5 kb), isolated from Haemophilus parasuis strain HS1543 has been obtained. Plasmid pHS-Tet contains four open reading frames including a tet(B) tetracycline resistance gene which unusually did not have an associated tetR repressor gene. From a total of 45 H. parasuis isolates surveyed (15 international reference strains, 15 field isolates selected for their genetic diversity, and 15 recent Australian field isolates), 2 tetracycline-resistant field isolates (HS226 and HS1859) were identified. Analysis of three additional isolates from the same disease outbreak as strain HS1859 revealed a further tetracycline-resistant H. parasuis strain (HS1857, serovar 8) and a tetracycline-resistant Actinobacillus pleuropneumoniae strain (HS1861). An approximately 10.6-kb plasmid was identified in field isolate HS226 and outbreak strains HS1857, HS1859, and HS1861. Southern hybridization analysis of these plasmids showed that the Tet B determinant was present, and restriction digest comparisons suggest that these plasmids are related. This is believed to be the first report of native H. parasuis plasmids and Tet B-mediated tetracycline resistance in this microorganism.     

Widespread distribution of a tet W determinant among tetracycline-resistant isolates of the animal pathogen Arcanobacterium pyogenes

Tetracycline resistance is common among isolates of the animal commensal and opportunistic pathogen Arcanobacterium pyogenes. The tetracycline resistance determinant cloned from two bovine isolates of A. pyogenes was highly similar at the DNA level (92% identity) to the tet(W) gene, encoding a ribosomal protection tetracycline resistance protein, from the rumen bacterium Butyrivibrio fibrisolvens. The tet(W) gene was found in all 20 tetracycline-resistant isolates tested, indicating that it is a widely distributed determinant of tetracycline resistance in this organism. In 25% of tetracycline-resistant isolates, the tet(W) gene was associated with a mob gene, encoding a functional mobilization protein, and an origin of transfer, suggesting that the determinant may be transferable to other bacteria. In fact, low-frequency transfer of tet(W) was detected from mob+ A. pyogenes isolates to a tetracycline-sensitive A. pyogenes recipient. The mobile nature of this determinant and the presence of A. pyogenes in the gastrointestinal tract of cattle and pigs suggest that A. pyogenes may have inherited this determinant within the gastrointestinal tracts of these animals.     

Identification of the tetracycline resistance gene, tet(O), in Streptococcus pneumoniae.

Five isolates of Streptococcus pneumoniae resistant to tetracycline but lacking tet(M) were studied. The tetracycline resistance gene, tet(O), was detected for the first time in the pneumococcus. The gene was amplified and sequenced and found to share 99% nucleotide sequence identity and 99, 99, and 98% deduced amino acid sequence identity with the tet(O) resistance genes of Streptococcus mutans, Campylobacter coli, and Campylobacter jejuni, respectively.     

Antimicrobial sensitivity and plasmid-mediated tetracycline resistance in Campylobacter jejuni isolated in Bangladesh

The antimicrobial sensitivity of Campylobacter jejuni isolated in Bangladesh from patients with diarrhoea, asymptomatic carriers and domestic animals was performed. All isolates were sensitive to erythromycin, gentamicin, furazolidone and kanamycin. Seven percent isolates were resistant to tetracycline, 8% to nalidixic acid, 37% to ampicillin and 100% to sulfamethoxazole-trimethoprim and cephalothin. Tetracycline resistance was observed to be plasmid mediated. No plasmid band(s) coding for ampicillin, sulfamethoxazole-trimethoprim or cephalothin resistance were observed, possibly indicating chromosomally located resistance. No significant differences in the susceptibility patterns of C. jejuni isolated from the different sources was observed. However, 10 patients' isolates showed low molecular weight (2-3, 7 Mdaltons) plasmid band(s) which were completely absent in isolates from asymptomatic carriers and animals.     

Presence of the tet(O) gene in erythromycin- and tetracycline-resistant strains of Streptococcus pyogenes and linkage with either the mef(A) or the erm(A) gene

Sixty-three recent Italian clinical isolates of Streptococcus pyogenes resistant to both erythromycin (MICs >or=1 microg/ml) and tetracycline (MICs >or= 8 microg/ml) were genotyped for macrolide and tetracycline resistance genes. We found 19 isolates carrying the mef(A) and the tet(O) genes; 25 isolates carrying the erm(A) and tet(O) genes; and 2 isolates carrying the erm(A), tet(M), and tet(O) genes. The resistance of all erm(A)-containing isolates was inducible, but the isolates could be divided into two groups on the basis of erythromycin MICs of either >128 or 1 to 4 microg/ml. The remaining 17 isolates included 15 isolates carrying the erm(B) gene and 2 isolates carrying both the erm(B) and the mef(A) genes, with all 17 carrying the tet(M) gene. Of these, 12 carried Tn916-Tn1545-like conjugative transposons. Conjugal transfer experiments demonstrated that the tet(O) gene moved with and without the erm(A) gene and with the mef(A) gene. These studies, together with the results of pulsed-field gel electrophoresis experiments and hybridization assays with DNA probes specific for the tet(O), erm(A), and mef(A) genes, suggested a linkage of tet(O) with either erm(A) or mef(A) in erythromycin- and tetracycline-resistant S. pyogenes isolates. By amplification and sequencing experiments, we detected the tet(O) gene ca. 5.5 kb upstream from the mef(A) gene. This is the first report demonstrating the presence of the tet(O) gene in S. pyogenes and showing that it may be linked with another gene and can be moved by conjugation from one chromosome to another.     

Prevalence of tetracycline resistance genes in oral bacteria

Tetracycline is a broad-spectrum antibiotic used in humans, animals, and aquaculture; therefore, many bacteria from different ecosystems are exposed to this antibiotic. In order to determine the genetic basis for resistance to tetracycline in bacteria from the oral cavity, saliva and dental plaque samples were obtained from 20 healthy adults who had not taken antibiotics during the previous 3 months. The samples were screened for the presence of bacteria resistant to tetracycline, and the tetracycline resistance genes in these isolates were identified by multiplex PCR and DNA sequencing. Tetracycline-resistant bacteria constituted an average of 11% of the total cultivable oral microflora. A representative 105 tetracycline-resistant isolates from the 20 samples were investigated; most of the isolates carried tetracycline resistance genes encoding a ribosomal protection protein. The most common tet gene identified was tet(M), which was found in 79% of all the isolates. The second most common gene identified was tet(W), which was found in 21% of all the isolates, followed by tet(O) and tet(Q) (10.5 and 9.5% of the isolates, respectively) and then tet(S) (2.8% of the isolates). Tetracycline resistance genes encoding an efflux protein were detected in 4.8% of all the tetracycline-resistant isolates; 2.8% of the isolates had tet(L) and 1% carried tet(A) and tet(K) each. The results have shown that a variety of tetracycline resistance genes are present in the oral microflora of healthy adults. This is the first report of tet(W) in oral bacteria and the first report to show that tet(O), tet(Q), tet(A), and tet(S) can be found in some oral species.     

Antimicrobial resistance in Campylobacter strains isolated from French broilers before and after antimicrobial growth promoter bans

OBJECTIVES: The antimicrobial susceptibility of Campylobacter strains isolated from standard and free-range broilers in 1992-1996 and 2001-2002 was studied. METHODS: Strains were isolated from caeca or skin samples collected from standard or free-range broilers arriving in slaughterhouses. The MICs of ampicillin, nalidixic acid, enrofloxacin, tetracycline, erythromycin and gentamicin were determined by agar dilution and compared according to species (Campylobacter jejuni or Campylobacter coli), production system and sampling period. RESULTS: Results showed that all chickens harboured Campylobacter. An increase over time of the C. coli/C. jejuni ratio for standard chickens occurred. A wide range of MICs was observed among isolates from the same broiler or from the same farm. Strains collected on entry to the slaughterhouse and after storage showed no significant difference in their antibiotic resistance. C. coli was more resistant than C. jejuni to tetracycline and erythromycin during the first period and to all tested molecules (except gentamicin) during the second period. Strains isolated from standard chickens were also more often resistant than those isolated from free-range broilers. The percentage of C. jejuni strains resistant to ampicillin decreased from 1992-1996 to 2001-2002, whereas no change could be observed for the other antimicrobial agents. However, for C. coli the resistance to ampicillin, nalidixic acid, enrofloxacin, tetracycline and erythromycin significantly increased. CONCLUSION: There was an increase in the incidence of antibiotic resistance of C. coli between 1992-1996 and 2001-2002.