Antibiotic resistance and resistance mechanisms in Campylobacter jejuni and Campylobacter coli

Campylobacter jejuni and Campylobacter coli are recognized as the most common causative agents of bacterial gastroenteritis in the world and infections with these organisms occur more frequently than do infections due to Salmonella species, Shigella species, or Escherichia coli 0157:H7. The incidence of human Campylobacter infections has increased markedly in both developed and developing countries worldwide and, more significantly, so has the rapid emergence of antibiotic-resistant Campylobacter strains, with evidence suggesting that the use of antibiotics, in particular the fluoroquinolones, as growth promoters in food animals and the veterinary industry is accelerating this trend. In this minireview, the patterns of emerging resistance to the antimicrobial agents useful in treatment of the disease are presented and the mechanisms of resistance to these drugs in Campylobacter spp are discussed.     

Effect of temperature on growth and chemotactic behaviour of Campylobacter jejuni

AIM: To determine the effect of two physiologically important temperatures on growth and chemotaxis in Campylobacter jejuni. METHODS AND RESULTS: Growth curves of Camp. jejuni were compared at 37 degrees C and 42 degrees C. Chemotaxis was compared at 37 degrees C and 42 degrees C by the disc and capillary assays. Student's t-test was applied to the results of the capillary assay to assess the significance in the difference between chemotaxis at the two temperatures. Both, the growth rate and chemotactic ability of the isolate, were found to be greater at 37 degrees C. CONCLUSIONS: Quorum sensing (related to population density), a regulation mechanism of virulence in micro-organisms, has been reported in Campylobacter. Chemotaxis is also a known virulence factor of Campylobacter. Both, growth (in terms of population density) and chemotaxis, being greater at 37 degrees C than at 42 degrees C, suggests that the physiological temperature of humans (37 degrees C) might be more favourable for the expression of virulence in Campylobacter than that of birds (42 degrees C). SIGNIFICANCE AND IMPACT OF THE STUDY: It is as yet not known why Campylobacter causes disease in humans but is avirulent in birds. This study suggests that the human body temperature is optimum for growth and chemotaxis in Campylobacter. There is scope for the study of temperature regulation of other virulence determinants of Campylobacter.     

139株空肠弯曲菌耐药性分析

空肠弯曲菌(Campylobacter jejuni)是最常见的食源性病原菌之一。本研究采用微量肉汤稀释法对分离得到的139株空肠弯曲菌(117株为禽源样本分离株,22株为人源样本分离株)进行耐药性检测。通过对最小抑菌浓度(MIC)的判定结果得出:120株(86. 33%)空肠弯曲菌分离株对6类9组临床常用的抗生素表现出不同程度的耐药,其中禽源空肠弯曲菌耐药率为83. 76%,22株人源空肠弯曲菌均表现出耐药性。对喹诺酮类抗生素表现出高度耐药(环丙沙星80. 58%,萘啶酸77. 70%);对四环素类表现为中等耐药(四环素53. 24%);对部分大环内酯类、氨基糖苷类、林可酰胺类表现为低耐药(庆大霉素7. 19%,阿奇霉素5. 76%,克林霉素6. 47%);对酰胺醇类、部分大环内酯类表现为敏感(氟苯尼考0%,红霉素0%、泰利霉素0%)。139株空肠弯曲菌共产生14种耐药谱型,以TET-CIP-NAL谱型最多,占比38. 13%,耐三重及以上抗生素的多重耐药菌株占比53. 24%。禽源菌株中多重耐药占比46. 15%,人源菌株中多重耐药占比90. 91%。研究结果显示空肠弯曲菌耐药现状不容乐观,尤其对喹诺酮类与四环素类抗生素耐药性较为突出,且过半数菌株为多重耐药。本研究为食源性空肠弯曲菌的防控及临床用药提供参考。     

Relationships between bacterial genotypes and in vitro virulence properties of Campylobacter jejuni and Campylobacter coli isolated from turkeys

AIMS: Campylobacter isolates from turkeys were genotyped and characterized by their in vitro virulence properties. Relationships between bacterial genotypes and virulence properties were analysed. METHODS AND RESULTS: Isolates were analysed by pulsed-field gel electrophoresis and fla typing. The toxin production was determined on the phenotypic level using a CHO-K1 cell culture model and on the genotypic level using PCR for detection of the cdtA, cdtB and cdtC genes. Although the cdtB gene was detected from 100% of the Campylobacter jejuni and Campylobacter coli isolates we observed three different morphological pictures on the cells. Cytotoxicity was associated with cell distension or cell rounding. All four Camp. coli strains and one Camp. jejuni strain did not produce any cytotoxic changes on the cells. Adhesion, invasion and survival of Campylobacter isolates were determined in a Caco-2 cell culture model. All isolates adhered to and invaded Caco-2 cells, whereas 64.7% of the strains survived for 48 h in the cells. CONCLUSION: Seventeen Campylobacter isolates from turkeys were classified into four groups with regard to their in vitro abilities. Jackknife analysis revealed a strong association between these groups and genotype clusters. SIGNIFICANCE AND IMPACT OF THE STUDY: Typing methods have generally failed to identify strains with specific virulence properties. This study suggests that a relationship between subgroups of Campylobacter with common in vitro virulence characteristics and genotypes exist.     

Conjugal transfer of antibiotic resistances and plasmids from Campylobacter jejuni clinical isolates

Six Campylobacter jejuni clinical isolates were examined for the occurrence of plasmids in association with antibiotic resistances as well as conjugal transfer. All the isolates were found to carry three similar plasmids of 78 kb, 12.6 kb and 3.3 kb in size. Multiple resistance to at least three of the antibiotics tested was observed with resistance to tetracycline most common. En bloc transfer of donor resistances at frequencies ranging from 10(-8) to 10(-4) were seen in all but one of the isolates during conjugation. The conjugal transfer of erythromycin, neomycin and streptomycin were observed to occur at frequencies similar to that of chloramphenicol, kanamycin and tetracycline. In isolate ABA94, three different antibiotic resistance phenotypes of the transconjugants were seen. In addition to en bloc transfer of the donor resistances, in approximately 10% of the transconjugants the streptomycin resistance was lost although these transconjugants carried the donor complement of three plasmids. In a further 1% of the transconjugants, resistance to kanamycin only was detected and these transconjugants did not carry any plasmids.     

Evaluation of cytotoxic activity in fecal filtrates from patients with Campylobacter jejuni or Campylobacter coli enteritis

We sought to determine the prevalence of cytotoxic activity in fecal filtrates from persons with C. jejuni or C. coli enteritis. Stool specimens were collected from 20 persons with C. jejuni or C. coli enteritis, 20 persons with acute diarrheal illnesses of other causes, and 9 healthy, asymptomatic persons. Fecal filtrates were then incubated with Chinese hamster ovary (CHO) or HeLa cells. The fecal filtrate from 1 of the 20 (5%) persons with Campylobacter enteritis was cytotoxic for HeLa cells at a titer of 1:40, and 10 (50%) were cytotoxic for CHO cells at maximum titers of 1:20. Cytotoxic activity for CHO cells at a median titer of 1:20 was also present in 40% of the fecal filtrates from persons with diarrhea due to causes other than Campylobacter enteritis, and in 33% of filtrates from healthy, asymptomatic persons. The observed low level of cytotoxicity in fecal filtrates from all patient groups studied likely resulted from non-specific factors, unrelated to the pathogenesis of Campylobacter enteritis.     

The Campylobacter jejuni glycome

Microbial cell surface glycans in the form of glycolipids and glycoproteins frequently play important roles in cell-cell interaction and host immune responses. Given the likely importance of these surface structures in the survival and pathogenesis of Campylobacter jejuni, a concerted effort has been made to characterise these determinants genetically and structurally since the genome was sequenced in 2000. We review the considerable progress made in characterising the Campylobacter glycome including the lipooligosaccharide (LOS), the capsule and O- and N-linked protein glycosylation systems, and speculate on the roles played by glycan surface structures in the life-cycle of C. jejuni.     

Speciating Campylobacter jejuni and Campylobacter coli isolates from poultry and humans using six PCR-based assays

Six previously published polymerase chain reaction (PCR) assays each targeting different genes were used to speciate 116 isolates previously identified as Campylobacter jejuni using routine microbiological techniques. Of the 116 isolates, 84 were of poultry origin and 32 of human origin. The six PCR assays confirmed the species identities of 31 of 32 (97%) human isolates and 56 of 84 (67%) poultry isolates as C. jejuni. Twenty eight of 84 (33%) poultry isolates were identified as Campylobacter coli and the remaining human isolate was tentatively identified as Campylobacter upsaliensis based on the degree of similarity of 16S rRNA gene sequences. Four of six published PCR assays showed 100% concordance in their ability to speciate 113 of the 116 (97.4%) isolates; two assays failed to generate a PCR product with four to 10 isolates. A C. coli-specific PCR identified all 28 hippuricase gene (hipO)-negative poultry isolates as C. coli although three isolates confirmed to be C. jejuni by the remaining five assays were also positive in this assay. A PCR-restriction fragment length polymorphism assay based on the 16S rRNA gene was developed, which contrary to the results of the six PCR-based assays, identified 28 of 29 hipO-negative isolates as C. jejuni. DNA sequence analysis of 16S rRNA genes from four hipO-negative poultry isolates showed they were almost identical to the C. jejuni type strain 16S rRNA sequences ATCC43431 and ATCC33560 indicating that assays reliant on 16S rRNA sequence may not be suitable for the differentiation of these two species.     

Antimicrobial resistance of Campylobacter jejuni and Campylobacter coli isolates from broiler chickens isolated at an Irish poultry processing plant

AIMS: The antibiotic susceptibility of Campylobacter jejuni and Campylobacter coli isolates from broiler chickens were determined in order to evaluate the level of antibiotic resistance of Campylobacter species in the Irish poultry industry. METHODS AND RESULTS: Seventy-eight Camp. jejuni and 22 Camp. coli strains were examined for susceptibility to eight antibiotics using the disc diffusion assay. The highest level of resistance of the Camp. jejuni isolates was recorded to ampicillin (35.9%), followed by 20.5% to tetracycline, 20.5% to naladixic acid, 17.9% to ciprofloxacin, 10.2% to erythromycin, 2.5% to streptomycin and 1.2% to kanamycin. Multidrug resistance to two or more antibiotics was seen for 30.7% of Camp. jejuni strains. Resistance of the Camp. coli isolates was shown to ampicillin (9%) and tetracycline (18.2%). CONCLUSIONS: The majority of Camp. jejuni strains were susceptible to antibiotics commonly used for human therapy. Camp. coli strains showed very low resistance levels and were susceptible to six of the eight antimicrobial agents studied. SIGNIFICANCE AND IMPACT OF THE STUDY: Levels of Camp. jejuni and Camp. coli antimicrobial resistance in Irish poultry production was assessed to determine the current situation in Ireland. The prevalence of antibiotic resistance of Campylobacter strains isolated from broiler chickens was low.     

Campylobacter jejuni: a study on environmental conditions affecting culturability and in vitro adhesion/invasion

Aims:  Nongrowing cultures of Campylobacter jejuni lose their culturability. It is unclear whether this loss in culturability also affects their ability to interact with host cells. The purpose of this study was to determine the relevance of the number of culturable cells to the ability to adhere/invade in Caco-2 cells.
Methods and Results:  For C. jejuni C356, culturability and adhesion/invasion were monitored in time (days) under different storage conditions (temperature, medium, atmosphere). Decrease rates of both culturability and adhesion/invasion were dependent on the conditions used, but the number of adhering/invading cells per culturable cell was not affected by the environmental conditions. Furthermore five strains were monitored at one condition. The culturability and adhesion/invasion decrease rates did not significantly differ per strain; however the number of adhering/invading cells per culturable cell was strain dependent.
Conclusions:  Culturability and adhesion/invasion of C. jejuni are linearly related. The number of adhering/invading cells per culturable C. jejuni cell is strain dependent, but is not affected by environmental conditions.
Significance and Impact of the Study:  It was shown that the number of culturable cells is a good measure for the in vitro adhesion/invasion of. C. jejuni.     

Resistance to quinolones in Campylobacter jejuni and Campylobacter coli from Danish broilers at farm level

AIMS: To investigate the prevalence of quinolone resistance among Campylobacter jejuni and Camp. coli isolates from Danish poultry at the farm level, as well as for the whole country. METHODS AND RESULTS: Data and isolates were collected from a national surveillance of Campylobacter in poultry. Quinolone resistance was investigated by determination of minimum inhibitory concentration (MIC) to nalidixic acid and enrofloxacin. Among Camp. jejuni and Camp. coli combined, 7.5% were resistant to nalidixic acid. Quinolone resistance varied considerably from farm to farm, with 0% on some farms and almost 100% on others, but the resistance was evenly distributed geographically. With respect to isolates from farms where resistance was detected, quinolone resistance was higher among Camp. coli (28.7%) than among Camp. jejuni (11.3%). PFGE typing of quinolone-resistant and quinolone-susceptible isolates from four farms indicated that certain resistant isolates belonged to specific clones that were able to persist on the farms during several rotations, even in the absence of selective pressure. Some clones were present and repeatedly isolated in both a quinolone-susceptible and quinolone-resistant variant. CONCLUSIONS: Overall, quinolone resistance among Campylobacter isolates from Danish broilers was 7.5% in 1998 and 1999; it was higher among Camp. coli than Camp. jejuni. Genetic diversity among resistant isolates was lower than among susceptible isolates, and certain clones existed in both a resistant and a susceptible variant. Some resistant clones appeared to persist on the farms and were repeatedly isolated from poultry flocks. SIGNIFICANCE AND IMPACT OF THE STUDY: The study is important for the understanding of persistence and dynamics of Campylobacter in broiler houses. It also highlights the extent, farm-to-farm variation and persistence of quinolone-resistant Campylobacter in broiler houses.     

Characterization of erythromycin resistance in Campylobacter coli and Campylobacter jejuni isolated from pig offal in New Zealand

AIMS: To determine the level and mechanism(s) of antimicrobial resistance in Campylobacter isolates obtained from human and environmental sources from South Canterbury, New Zealand. METHODS AND RESULTS: A total of 251 Campylobacter isolates were tested for susceptibility to ciprofloxacin, erythromycin, nalidixic acid and tetracycline using disc diffusion assays. Five pig offal isolates were observed to be highly erythromycin resistant, with minimal inhibitory concentrations determined to be >/=256 microg ml(-1). Nucleotide sequencing of the 23S ribosomal DNA (rDNA) in these resistant isolates identified an A --> G change at Escherichia coli position 2059 that has been previously implicated in erythromycin resistance in Campylobacter coli. Macrorestriction profiling using pulsed-field gel electrophoresis showed these isolates were nonclonal. CONCLUSIONS: The majority of Campylobacter isolates from South Canterbury remain sensitive to the most clinically relevant antimicrobial agents. Our results support other reports showing that specific variations in the 23S rDNA contribute to erythromycin resistance. SIGNIFICANCE AND IMPACTS OF THE STUDY: This study defines the baseline frequency of antimicrobial resistance associated with Campylobacter isolates from South Canterbury, and discusses the likely molecular mechanisms conferring erythromycin resistance in this organism. Resistance to erythromycin in these isolates is not linked to a dominant Campylobacter clone and has likely arisen independently in different genetic lines exposed to selective antimicrobial pressure.     

Campylobacter jejuni response to ox-bile stress

Campylobacter jejuni is a pathogen that colonizes the intestinal tract of humans and some animals. The in vitro responses of the bacterium to ox-bile were studied using proteomics to understand the molecular mechanisms employed by C. jejuni to survive bile stress. Its in vitro tolerance to bile was determined by growing the bacterium for 18 h in liquid cultures containing different bile concentrations. Significant growth inhibition was observed in the presence of 2.5% bile, and a decrease of 1.12 log units was measured at a bile concentration of 5%. Protein expression profiles of bacteria grown with and without bile were compared using two-dimensional polyacrylamide gel electrophoresis. Proteins with differential intensities greater than two-fold were identified using tandem mass spectrometry. Nuclear magnetic resonance spectroscopy and spectrophotometry were employed to measure enzyme activities in cell extracts from bacteria grown with and without bile. Together with proteins known to be involved in C. jejuni bile tolerance, the presence of bile modulated the expression of proteins such as elongation factors, ferritin, chaperones, ATP synthase and others, previously unknown to be implicated in the response of the bacterium to bile.     

Rapid construction of Campylobacter jejuni deletion mutants

AIMS: To develop a novel method for rapid construction of Campylobacter jejuni deletion mutants. METHODS AND RESULTS: We used overlapping extension PCR protocol to amplify a target sequence region of Camp. jejuni genomic DNA in which an internal fragment, Cj0618 coding sequence, was replaced by a chloramphenicol resistance cassette. After the resulting PCR product was introduced into electrocompetent Camp. jejuni 81-176, chloramphenicol-resistant mutants in which the wild type allele has been replaced by the deletion cassette were selected. DNA sequencing confirmed precise deletion in the Cj0618 gene. As expected from the previously reported role of Cj0618 in chick colonization, the resulting deletion mutant showed a caecal colonization defect in chick infection. CONCLUSIONS: This method can be used for rapid construction of Camp. jejuni deletion mutants. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of this method should facilitate functional characterization of various Camp. jejuni genes.     

Evidence for natural horizontal transfer of tetO gene between Campylobacter jejuni strains in chickens

AIMS: The transfer of tetO gene conferring resistance to tetracycline was studied between Campylobacter jejuni strains, in the digestive tract of chickens. METHODS AND RESULTS: In vitro conjugation experiments were first performed in order to select donor/recipient couples for further in vivo assay. Then, chickens were inoculated with a donor/recipient couple of C. jejuni strains displaying spontaneous in vitro tetracycline resistance gene transfer. The donor was a tetracycline-resistant ampicillin-susceptible strain, and the recipient was a tetracycline-susceptible ampicillin-resistant strain. Chicken droppings were streaked on antimicrobial selective media and bi-resistant Campylobacter isolates were further characterized according to their donor or recipient flaA gene RFLP profile. The acquisition of tetracycline-resistance gene by the recipient C. jejuni strain from the donor C. jejuni strain was confirmed by tetO PCR. CONCLUSIONS: The study showed that transfer of tetO gene occurs rapidly and without antimicrobial selection pressure between C. jejuni strains in the digestive tract of chickens. SIGNIFICANCE AND IMPACT OF THE STUDY: The rapid and spontaneous transfer of tetO gene may explain the high prevalence of tetracycline resistance in chicken Campylobacter strains.     

Detection and quantification of Campylobacter jejuni and Campylobacter coli using real-time multiplex PCR

Aims: We describe a real‐time quantitative multiplex polymerase chain reaction (qmPCR) assay to identify and discriminate between isolates of Campylobacter jejuni and Campylobacter coli. Methods and Results: Two novel sets of primers and hydrolysis probes were designed to amplify the unique DNA sequences within the hipO, ccoN and cadF genes that are specific to Camp. jejuni and Camp. coli. Using the designed optimized qmPCR assay conditions, the amplification efficiency is in range from 108 to 116%. These qmPCR assays are highly specific for Camp. jejuni and Camp. coli, as seen through testing of 40 Campylobacter strains and 17 non‐Campylobacter strains. In chicken juice and tap water models spiked with known quantities of Camp. jejuni, qmPCR detected 10²–10³ CFU ml^?1 within 4 h. Conclusions: The qmPCR assays developed in this study provide reliable and simultaneous detection and quantification of Camp. jejuni and Camp. coli, with good amplification reaction parameters. Significance and Impact of the Study: Following further validation, the qmPCR assay reported here has the potential to be applied to various sample types as an alternative and rapid methodology.     

空肠弯曲菌感染动物模型的研究进展

空肠弯曲菌是一种全球关注的人兽共患病原菌,感染后可引起人和动物多种疾病。动物模型是开展致病机理、疫苗评价和药物开发等研究的基础。空肠弯曲菌由于培养条件苛刻以及感染实验动物的疾病相似性、经济性和重复性等因素,仍缺乏良好的感染动物模型,其致病机理迄今尚不清楚。本文对已报道的空肠弯曲菌感染实验动物模型进行综述。     

The heterogeneity of Campylobacter jejuni and Campylobacter coli strains isolated from healthy cattle

AIMS: To identify campylobacters isolated from clinically healthy cattle at species level by a multiplex polymerase chain reaction (m-PCR). The heterogeneity among Campylobacter jejuni and Campylobacter coli isolates was also investigated by using a restriction fragment length polymorphism (RFLP) analysis of flagellin (flaA) gene. METHODS AND RESULTS: Samples of intestinal contents, gall bladders, liver and faeces were collected from a total number of 1154 healthy cattle. The samples were inoculated onto Preston enrichment broth and agar. Of 1154 samples, 301 (26.1%) were positive for Campylobacter spp. Using an m-PCR assay for species identification, 179 (59.5%) were positive with C. jejuni specific primers while 30 (10%) were positive with C. coli specific primers. None of the liver samples examined was positive for C. jejuni or C. coli by mPCR. All the isolates identified as C. jejuni and C. coli were successfully subtyped by flaA typing. Of the 209 isolates tested, 28 different flaA types were found. Twenty-three flaA types were identified among 179 C. jejuni isolates and the remaining five from C. coli isolates. CONCLUSIONS: Although the overall results suggest that the degree of heterogeneity among the flaA genes of thermophilic Campylobacter strains isolated from healthy cattle is relatively high, they should be treated cautiously as the number of band types for C. coli was low and band type 8 in C. jejuni was represented by a high percentage (%58). SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of the present study suggest that healthy cattle can play role in the contamination of environment and human food chain by Campylobacter spp.     

Inactivation of Campylobacter jejuni by high hydrostatic pressure

AIMS: To investigate the response of Campylobacter jejuni ATCC 35919 and 35921 to high pressure processing (HPP) while suspended in microbiological media and various food systems. METHODS AND RESULTS: Campylobacter jejuni 35919 and 35921 were subjected to 10-min pressure treatments between 100 and 400 MPa at 25 degrees C suspended in Bolton broth, phosphate buffer (0.2 m, pH 7.3), ultra-high temperature (UHT) whole milk, UHT skim milk, soya milk and chicken pureé. The survivability of C. jejuni was further investigated by inoculated pack studies. HPP at 300-325 MPa for 10 min at 25 degrees C was sufficient to reduce viable numbers of both strains to below detectable levels when cells were pressurized in Bolton broth or phosphate buffer. All food products examined offered a protective effect in that an additional 50-75 MPa was required to achieve similar levels of inactivation when compared with broth and buffer. Inoculated pack studies showed that the survivability of C. jejuni following pressurization improved with decreasing post-treatment storage temperature. SIGNIFICANCE AND IMPACT OF THE STUDY: These data demonstrated that HPP at levels of 相似文献   

Prevalence of antimicrobial resistance among serotypes of Campylobacter jejuni isolates from cattle and poultry in Japan

Penner serotypes of C. jejuni in a total of 601 isolates from apparently healthy cattle, layer and broiler chickens in Japan were examined between 2001 and 2006. Predominant serotypes were B (O: 2, 19.1%), D (O: 4, 13.5%), Y (O: 37, 7.3%) and G (O: 8, 5.8%), whereas the remaining serotypes made up less than 5% of the total isolates. The frequency of ampicillin resistance in serotype G (65.6%) was significantly higher than in serotypes D (12.5%), B (11.2%), and Y (0%). Our results suggest that serotype is one factor contributing to the prevalence of ampicillin resistance in C. jejuni isolates.