Browsing by Author "Fred Breidt, Committee Member"

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Erythromycin Susceptibility and Genomic Regions Characterization of Campylobacter coli
(2007-11-01) Chan, Kamfai; Todd Klaenhammer, Committee Member; Fred Breidt, Committee Member; Sophia Kathariou, Committee Chair; Craig Altier, Committee Member
Campylobacter jejuni and Campylobacter coli are major bacterial food-borne pathogens that cause enteric diseases worldwide, resulting in significant public health and economic burden. These two closely related species colonize a wide range of farm animals including turkeys, pigs, chickens, and cattle. Consumption of meat (especially poultry) contaminated with C. jejuni or C. coli has been implicated as the major route of infection. When needed, antibiotics used for treatment are fluoroquinolones or macrolides such as erythromycin. Recent studies have shown that the percentage of C. coli that have resistance to antimicrobials, including those typically used for treatment of human campylobacteriosis, has increased, making it a top priority to investigate the mechanisms for acquisition and dissemination of antimicrobial resistance in these pathogens. Certain Campylobacter strains harbor a transcribed intervening sequence (IVS) in their 23S rRNA genes. Following transcription, the IVS is excised, leading to fragmentation of the 23S rRNA. The origin and possible functions of the IVS are unknown. Furthermore, the distribution of IVS-harboring strains within Campylobacter populations is poorly understood. In this study, strains of Campylobacter coli from turkeys, representing numerous different multilocus sequence typing (MLST)-based sequence types (STs), were characterized in terms of IVS content and erythromycin susceptibility. We identified strains that harbored IVSs in all three 23S rRNA genes, as well as strains lacked IVSs from at least one of the genes. The STs of the latter strains belonged to an unusual cluster of C. coli STs ('cluster II'), earlier found primarily in turkey strains, and characterized by presence of the C. jejuni aspA103 allele. The majority of strains harboring IVSs in all three 23S rRNA genes were resistant to erythromycin. In contrast, cluster II strains, which harbored at least one IVS-free 23rRNA gene, were susceptible to the antibiotic. Cluster II strains could be transformed to erythromycin resistance with genomic DNA from C. coli that harbored IVS and the A2075G transition in the 23S rRNA gene, associated with resistance to erythromycin in Campylobacter. Erythromycin-resistant transformants harbored both the A2075 transition and IVS. The findings suggest that absence of IVS in C. coli from turkeys is characteristic of a unique clonal group of erythromycin-susceptible strains, and that IVS can be acquired by these strains via natural transformation to erythromycin resistance. Analysis of C. coli and C. jejuni strains isolated from broilers, turkeys and swine has shown the association of the lack of IVS and erythromycin susceptibility is unique to C. coli from turkeys. The presence of the C. jejuni aspA103 allele in the chromosome of cluster II C. coli strains is a unique characteristic of this clonal group. To further characterize the genome composition in the aspA region in cluster II strains, we determined the corresponding DNA sequences from two turkey-derived cluster II C. coli strains (6979 and 7474, of ST-1150 and ST-1161, respectively). Genomic organization upstream of the aspA gene was divergent between these two cluster II strains and the reference strain C. coli RM2228, the genome sequence of which has been completed. Genes encoding a putative Crp-family transcriptional regulator (CCO0137) and a conserved hypothetical protein (CCO0138) that were present in C. coli RM2228 and C. coli 6818 were not found in the same genomic region in C. coli 6979 or C. coli 7474. Moreover, single nucleotide polymorphism (SNP) analysis revealed that genes encoding subunit II of cytochrome d ubiquinol oxidase (cydB) and a putative aspartate racemase (CJ0085c) harbored numerous C. jejuni-specific SNPs. Interestingly, genes encoding subunit I of cytochrome d ubiquinol oxidase (cydA) and uracil-DNA glycosylase (ung) harbored C. coli-specific SNPs in their 5' portions but C. jejuni-specific SNPs in their 3' portions, suggesting that these may be hybrid genes that were originated from C. jejuni and C. coli. Our data suggest the presence of recombination events in the genomic region between cydA and ung in cluster II strains of C. coli. Such genomic features may contribute to the observed prevalence of cluster II strains among C. coli from turkeys, and to the characteristic susceptibility to erythromycin exhibited by these strains.
Genetic characterization of Multidrug resistant strains of Campylobacter coli from turkeys in North Carolina
(2007-08-06) D'lima, Carol Bonnie; Dahlia Nielsen, Committee Member; Fred Breidt, Committee Member; James Brown, Committee Member; Donna Carver, Committee Member; Sophia Kathariou, Committee Chair
Commercial turkey flocks in North Carolina are frequently colonized with Campylobacter coli strains that are resistant to several antimicrobials and have been designated multidrug resistant (MDR). Multiple locus sequence typing (MLST) showed that the major sequence types (STs) were turkey-specific. Further subtyping using fla typing, pulsed field gel electrophoresis (PFGE) with SmaI and KpnI as well as plasmid profiles revealed that each of the major MDR STs contained strains of related, but distinct subtypes, providing evidence for genomic diversification within these STs. Numerous strains, with indistinguishable PFGE profiles, but different fla types suggested selection for specific flagellin sequences. The observed correlation between STs and the MDR profiles of the microbes indicates that MLST-based typing holds potential for source-tracking applications specific to the animal source (turkeys) and the antimicrobial resistance profile (MDR) of C. coli. The molecular basis for resistance of MDR strains to selected antimicrobials was investigated, and tetracycline resistant isolates were shown to harbor tet(O). Natural transformation studies were used to study the mechanism of transfer of antibiotic resistance genes in C. coli. Resistance to erythromycin and nalidixic acid/ciprofloxacin mediated by chromosomal sequences, were easily transferred from MDR strains to other C. coli, whereas resistance to tetracycline and kanamycin was not possible by transformation, suggesting that genes mediating these resistance attributes were plasmid-borne in MDR strains. Interestingly, tetracycline resistance could be readily transferred by transformation using DNA from another clonal group of C. coli strains prevalent in turkeys, suggesting chromosomal presence of the tetracycline resistance gene. MDR strains were found to be stable and maintained their MDR phenotype over 60 serial passages in vitro. The strains maintained their PFGE and plasmid profiles; and only minor differences in MICs before and after the 60 passages were observed. The findings from this study suggest that the certain strain types and clonal groups are prevalent among MDR C. coli from turkeys, and that resistance determinants to certain antibiotics can be transferred from these MDR strains to other C. coli strains.
Natural Transformation-mediated Transfer of Erythromycin Resistance in Campylobacter coli and Campylobacter jejuni
(2005-11-09) Kim, Joo-Sung; Sophia Kathariou, Committee Chair; Fred Breidt, Committee Member; Donna K. Carver, Committee Member; Paul Orndorff, Committee Member
Campylobacter is the most common bacterial agent causing human gastroenteritis and is mainly transmitted through foods. Campylobacter is a zoonotic agent, and commonly colonizes poultry and other meat animals. Whereas erythromycin resistance in Campylobacter jejuni rarely occurs, erythromycin resistance in Campylobacter coli from meat animals is frequently encountered, and could represent a substantial barrier to antibiotic treatment of human infections. Erythromycin resistance in C. coli has been associated with a point mutation (A2075G) in the 23S rRNA gene, acting synergistically with the CmeABC efflux pump. However, the mechanisms responsible for possible dissemination of erythromycin resistance in C. coli remain poorly understood. In this study we investigated transformation-mediated acquisition of erythromycin resistance by genotypically diverse C. coli strains from turkeys and swine, with total genomic DNA from erythromycin-resistant C. coli used as donor. In addition, we studied the effects of environmental factors and species (C. coli vs. C. jejuni), and fitness costs of erythromycin resistance in transformants. Overall, transformation to erythromycin resistance was significantly more frequent in C. coli from turkeys than in swine-derived strains (P<0.01) with frequency of transformation 10⁻⁻⁴ to 10⁻⁶ in turkey-derived strains, but 10⁻⁷ or less in C. coli from swine. Transformants harbored the point mutation, A2075G in the 23S rRNA gene. Erythromycin resistance was stable in transformants following serial transfers, and most transformants had high MIC values (>256μg/ml), as did the C. coli donor strains. In contrast to results obtained with transformation, spontaneous mutants had relatively low erythromycin MIC (32-64μg/ml) and lacked the A2075G mutation. Temperature profoundly affected frequency of transformation to erythromycin resistance in C. coli and transformation frequency at 42°C was significantly higher than at 25°C, 32°C and even 37°C. However, transformation to nalidixic acid resistance was not significantly affected by temperature. No significant difference in transformation frequency was detected between microaerobic (5-10% CO₂) and aerobic conditions. Starvation conditions did not affect transformation frequency to nalidixic acid resistance. Increasing incubation time from 3-4h to 15-17h significantly increased transformation frequency to erythromycin resistance (P<0.05). Transformation of C. jejuni using genomic DNA from erythromycin resistant C. coli revealed that transformation frequency of C. jejuni to erythromycin resistance was lower than C. coli, suggesting that erythromycin resistance in C. coli may not be disseminated via transformation in C. jejuni as frequently as in C. coli. Transformants derived from C. jejuni, however, had high erythromycin MIC values (>256μg/ml) and harbored the A2075G transition, similarly to C. coli transformants. When grown separately at 42°C, an erythromycin-resistant transformant derived from C. coli strain 961 had a similar growth rate as its erythromycin-sensitive parental strain, whereas an erythromycin-resistant transformant derived from C. jejuni strain SC49 had a significantly longer generation time compared to its parental strain. In competitive growth studies, however, the C. coli transformant was at competitive disadvantage in relation to its parental strain in stationary phase, whereas the C. jejuni transformant was at a slight fitness advantage after 14days. Furthermore, in the mixed culture the generation time of the C. jejuni transformant was not significantly different from that of the parental strain. In conclusion, natural transformation has the potential to contribute to dissemination of high-level resistance to erythromycin among C. coli strains colonizing meat animals and temperature can greatly affect transformation to erythromycin resistance, but not to nalidixic acid resistance. These findings suggest that ecological attributes may play an important role and exert differential impact on the potential of the organism to acquire antimicrobial resistance determinants via natural transformation. However, further study is necessary to characterize the fitness of erythromycin resistant transformants in Campylobacter and identify possible mechanisms underlying the relatively low frequency of erythromycin resistance in C. jejuni.