Qijing Zhang

Prevalence and antimicrobial resistance of Campylobacter isolates in broilers from China.

The prevalence and antimicrobial resistance of Campylobacter spp. in broiler chickens were determined in Shandong Province, China. In total, 275 Campylobacter isolates were obtained from 767 broiler cecal samples, including 208 Campylobacter jejuni, 53 Campylobacter coli, and 14 unidentified Campylobacter isolates. Minimal inhibitory concentrations of 11 antimicrobial agents were determined using the agar dilution method recommended by CLSI. More than 98% of the tested Campylobacter isolates were resistant to quinolones (nalidixic acid, ciprofloxacin and enrofloxacin) and tetracyclines (tetracycline and doxycycline). The C. jejuni isolates also exhibited a high rate of resistance to phenicol antibiotics and a moderate rate of resistance to macrolides and gentamicin. On the contrary, the C. coli isolates showed a high-level resistance to macrolides and gentamicin and little resistance to phenicol antibiotics. The vast majority of the Campylobacter isolates were classified as multidrug resistant. These findings reveal a broad extent of antimicrobial resistance in Campylobacter isolates from poultry in China and underline the need for prudent use of antibiotics in poultry production to minimize the spread of antibiotic resistant Campylobacter.

Phenotypic and Genotypic Evidence for l-Fucose Utilization by Campylobacter jejuni

Campylobacter jejuni remains among the leading causes of bacterial food-borne illness. The current understanding of Campylobacter physiology suggests that it is asaccharolytic and is unable to catabolize exogenous carbohydrates. Contrary to this paradigm, we provide evidence for l-fucose utilization by C. jejuni. The fucose phenotype, shown in chemically defined medium, is strain specific and linked to an 11-open reading frame (ORF) plasticity region of the bacterial chromosome. By constructing a mutation in fucP (encoding a putative fucose permease), one of the genes in the plasticity region, we found that this locus is required for fucose utilization. Consistent with their function in fucose utilization, transcription of the genes in the locus is highly inducible by fucose. PCR screening revealed a broad distribution of this genetic locus in strains derived from various host species, and the presence of this locus was consistently associated with fucose utilization. Birds inoculated with the fucP mutant strain alone were colonized at a level comparable to that by the wild-type strain; however, in cocolonization experiments, the mutant was significantly outcompeted by the wild-type strain when birds were inoculated with a low dose (10⁵ CFU per bird). This advantage was not observed when birds were inoculated at a higher inoculum dose (10⁸ CFU per bird). These results demonstrated a previously undescribed substrate that supports growth of C. jejuni and identified the genetic locus associated with the utilization of this substrate. These findings substantially enhance our understanding of the metabolic repertoire of C. jejuni and the role of metabolic diversity in Campylobacter pathobiology.

Molecular Evidence for Zoonotic Transmission of an Emergent Highly Pathogenic Campylobacter jejuni Clone in the United States

ABSTRACT Campylobacter jejuni is a major zoonotic pathogen. A highly virulent, tetracycline-resistant C. jejuni clone (clone SA) has recently emerged in ruminant reservoirs and has become the predominant cause of sheep abortion in the United States. To determine whether clone SA is associated with human disease, we compared the clinical isolates of clone SA from sheep abortions with the human isolates of the PulseNet National Campylobacter databases at the CDC and the FDA using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and serotyping. The combined SmaI and KpnI PFGE pattern designations of clone SA from sheep were indistinguishable from those of 123 (9.03%) human C. jejuni isolates (total, 1,361) in the CDC database, among which 56 were associated with sporadic infections and 67 were associated with outbreaks that occurred in multiple states from 2003 to 2010. Most of the outbreaks were attributed to raw milk, while the sources for most of the sporadic cases were unknown. All clone SA isolates examined, including PFGE-matched human isolates, belong to sequence type 8 (ST-8) by MLST and serotype HS:1,8, further indicating the clonality of the related isolates from different host species. Additionally, C. jejuni clone SA was identified in raw milk, cattle feces, the feces and bile of healthy sheep, and abortion cases of cattle and goats, indicating the broad distribution of this pathogenic clone in ruminants. These results provide strong molecular and epidemiological evidence for zoonotic transmission of this emergent clone from ruminants to humans and indicate that C. jejuni clone SA is an important threat to public health.

Detection of the staphylococcal multiresistance gene cfr in Proteus vulgaris of food animal origin

OBJECTIVESnTo investigate the presence and the genetic environment of the multiresistance gene cfr in naturally occurring Gram-negative bacteria of pigs.nnnMETHODSnA total of 391 bacterial isolates with florfenicol MICs of ≥16 mg/L, obtained from 557 nasal swabs of individual pigs, were screened by PCR for the known florfenicol resistance genes. The species assignment of the cfr-carrying isolate was based on the results of Grams staining, colony morphology, 16S rDNA sequencing and biochemical profiling. The location of the cfr and floR genes was determined by Southern blotting and the regions flanking the cfr gene were sequenced by a modified random primer walking strategy.nnnRESULTSnA single Proteus vulgaris isolate, which carried the genes floR and cfr, was detected in this study. A cfr-carrying segment of 7 kb with homology to a staphylococcal plasmid was found to be inserted into the chromosomal fimD gene of P. vulgaris. This segment was flanked by two IS26 elements located in the same orientation, which are believed to have played a role in this integration process. Stability testing via inverse PCR approaches showed that this integrate is not entirely stable, but the cfr-carrying centre region plus one IS26 copy can be looped out via IS26-mediated recombination.nnnCONCLUSIONSnTo the best of our knowledge, this is the first report of the cfr gene in a naturally occurring Gram-negative bacterium. Surveillance and monitoring of the cfr gene in Gram-negative bacteria are warranted with respect to food safety and consumer protection.

Detection of the staphylococcal multiresistance gene cfr in Escherichia coli of domestic-animal origin

OBJECTIVESnTo investigate the presence and the genetic environment of the multiresistance gene cfr in Escherichia coli found in domestic animals.nnnMETHODSnA total of 1230 E. coli isolates, collected from pigs, chickens and ducks, were screened by PCR for the cfr gene. The location of the cfr gene was determined by Southern blotting, the transferability of cfr gene was tested by conjugation and transformation, and the regions flanking the cfr gene were sequenced by a modified random primer walking strategy. The location of the cfr promoter sequence was analysed by mapping the cfr transcription start site using rapid amplification of 5 cDNA ends (5 RACE).nnnRESULTSnOnly a single strain from the nasal swab of a pig harboured the cfr gene. Southern blotting indicated that the cfr gene was located on a ~110 kb plasmid, designated pEC-01. A cfr-carrying segment of 1545 bp with a sequence identical to that of the cfr-harbouring plasmid pSCFS1 was flanked by two IS26 elements in the same orientation. The IS26 transposition created a new hybrid promoter in which the -35 region was part of the left inverted repeat of IS26 while the -10-like sequence was part of the original cfr upstream region.nnnCONCLUSIONSnTo the best of our knowledge, this is the first report of the cfr gene in a naturally occurring E. coli strain. Continued surveillance of the presence of the cfr gene in Gram-negative bacteria of domestic-animal origin is warranted.

Antimicrobial resistance in Campylobacter coli isolated from pigs in two provinces of China

The aim of this study was to determine the prevalence, antimicrobial resistance and molecular epidemiology of Campylobacter coli isolated from swine in China. A total of 190 C. coli isolates obtained from two slaughter houses and ten conventional pig farms in Shandong (SD, n=95) and Ningxia (NX, n=95) provinces were tested for their susceptibility to 14 antimicrobials. A high prevalence (>95%) of ciprofloxacin and tetracycline-resistant strains was observed in both SD and NX. The erythromycin and clindamycin resistance rates of C. coli from NX (ERY: 54.7% CLI: 43.2%) were higher than those from SD (ERY: 37.9%, CLI: 35.8%). A significant difference (P<0.05) was observed in erythromycin resistance rate, but not (P>0.05) in clindamycin resistance rate. while the resistance rates of ampicillin and kanamycin in NX (AMP: 34.7%, KAN: 43.2%) were significantly lower (P<0.05) than those in SD (AMP: 51.6%, KAN: 71.6%). None of the tested isolates were resistant to phenicols. The majority of the isolates from both provinces (SD: 80% and NX: 73.7%) showed multi-drug resistance profiles. The point mutations of A2075G in the 23S rRNA and C257T in the gyrA gene were detected in 98% (87/89) of macrolide resistant isolates and all ciprofloxacin resistant isolates, respectively. In addition, all tetracycline-resistant isolates harbored the tet(O) gene. The high prevalence of antimicrobial resistance in C. coli strains derived from pigs in China was observed and was likely due to the extensive use of various antimicrobials. Prudent use of antimicrobial agents on farms should be further emphasized to control the dissemination of antimicrobial resistant C. coli.

Report of ribosomal RNA methylase gene erm(B) in multidrug-resistant Campylobacter coli

OBJECTIVESnCampylobacter is a major foodborne enteric pathogen and macrolides are the drug of choice for the clinical therapy of campylobacteriosis. Macrolide resistance among Campylobacter compromises clinical treatment, is associated with adverse health events and is a significant public health concern. Here, we report the first identification of a horizontally transferrable macrolide resistance mechanism in porcine Campylobacter coli ZC113 that is mediated by a ribosomal RNA methylase, Erm(B).nnnMETHODSnHorizontal transfer of a macrolide resistance determinant between C. coli and Campylobacter jejuni was performed by natural transformation. Whole-genome sequencing was initially used to identify the ribosomal methylase-encoding gene erm(B) in Campylobacter. Cloning of erm(B) into C. jejuni NCTC 11168 was performed to evaluate whether the erm(B) gene is responsible for high-level macrolide resistance in Campylobacter.nnnRESULTSnThe erm(B) gene was identified in ZC113, conferred high-level resistance to macrolides and was associated with a chromosomal multidrug-resistant genomic island (MDRGI). The MDRGI probably originated from Gram-positive bacteria and was horizontally transferred between C. coli and C. jejuni via natural transformation. Furthermore, the erm(B)-positive isolate ZC113 was resistant to all clinically important antibiotics used for treating campylobacteriosis and is essentially multidrug-resistant Campylobacter.nnnCONCLUSIONSnTo the best of our knowledge, this is the first report of a horizontally transferable macrolide resistance mechanism in thermophilic Campylobacter. Surveillance of erm(B) and its associated MDRGI in both C. coli and C. jejuni is urgently warranted.

Roles of lipooligosaccharide and capsular polysaccharide in antimicrobial resistance and natural transformation of Campylobacter jejuni

OBJECTIVESnThis study is aimed to determine the role of capsular polysaccharide (CPS) and lipooligosaccharide (LOS) in modulating antimicrobial resistance and natural transformation of Campylobacter jejuni, an important food-borne human pathogen.nnnMETHODSnA series of C. jejuni mutants, which are defective in either CPS or LOS or both, were constructed. The antimicrobial susceptibility, bacterial surface hydrophobicity, natural transformation frequency and DNA binding and uptake were measured and compared between the mutants and the wild-type strain.nnnRESULTSnTruncation of LOS greatly reduced (8-fold) the intrinsic resistance of C. jejuni to erythromycin, a key antibiotic used for treating human campylobacteriosis, while the loss of CPS did not result in significant changes in the susceptibility to antimicrobial agents. Notably, mutation of LOS also significantly increased (>16-fold) the susceptibility to erythromycin in C. jejuni mutants carrying the A2074G mutation in 23S rRNA. The increased susceptibility to erythromycin in the LOS mutant was probably due to enhanced permeability to this antibiotic, because the LOS mutation rendered the surface of C. jejuni more hydrophobic. Loss of CPS and truncation of LOS increased the transformation frequency by 4- and 25-fold, respectively, and mutation of both CPS and LOS resulted in a 97-fold increase in the transformation frequency. Consistent with the increased transformation frequencies, the CPS and LOS mutants showed enhanced rates of DNA uptake.nnnCONCLUSIONSnThese results demonstrate that the surface polysaccharides in C. jejuni contribute to the resistance to erythromycin, a clinically important antibiotic, but restrict natural transformation.

The new genetic environment of cfr on plasmid pBS-02 in a Bacillus strain

Sir, The gene cfr, encoding a 23S rRNA methyltransferase, confers resistance to five chemically unrelated antimicrobial classes, including phenicols, lincosamides, oxazolidinones, pleuromutilins and streptogramin A (PhLOPSA), and has been observed mainly in staphylococcal isolates over the past decade.1–3 Our previous study reported a cfr-carrying plasmid, pBS-01, in a novel strain (BS-01) of a Bacillus species isolated from a pig farm in China.4 In our routine surveillance study on antimicrobial resistance in farm animals in 2010, another florfenicol and chloramphenicol-resistant Bacillus species strain (named BS-02) from swine faeces was identified. Gram staining, sequence analysis of the 16S rRNA and API 50CH testing associated with the API 20E system (bioMérieux, France) showed that BS-02 had an identical profile to that of strain BS-01, which suggested that BS-02 also belongs to a novel Bacillus species. Despite their identical profiles, more than six PFGE band differences were observed between BS-01 and BS-02 (data not shown), indicating that they belong to different clonal types of the same species.

Sensitization of Campylobacter jejuni to fluoroquinolone and macrolide antibiotics by antisense inhibition of the CmeABC multidrug efflux transporter

OBJECTIVESnThe aim of this study was to investigate the feasibility and efficacy of antisense-mediated gene silencing by peptide nucleic acid (PNA) for specific inactivation of the CmeABC multidrug efflux transporter in Campylobacter jejuni.nnnMETHODSnPNA was designed to bind to the cmeA transcript and to inhibit the translation of CmeA, the periplasmic component of the RND-type CmeABC efflux transporter of C. jejuni. Inhibition of CmeA production was determined by western blotting. MICs of clinically important antibiotics, including ciprofloxacin and erythromycin, were measured in the presence of the CmeA-specific PNA (CmeA-PNA).nnnRESULTSnCmeA-PNA greatly reduced the expression level of CmeA. Consistent with the reduced CmeA production, CmeA-PNA rendered C. jejuni strains more susceptible to ciprofloxacin and erythromycin. At a concentration of 2 microM, CmeA-PNA resulted in 8- and 4-fold reductions in the MICs of ciprofloxacin and erythromycin, respectively, in C. jejuni NCTC 11168. CmeA-PNA also increased the susceptibility to the antibiotics in C. jejuni strains that were resistant to ciprofloxacin or erythromycin.nnnCONCLUSIONSnAntisense technology is a feasible method to suppress the function of the CmeABC multidrug efflux transporter, which may be further exploited to control antibiotic-resistant Campylobacter.