山东省鸡源空肠弯曲菌喹诺酮类药物耐药机制研究

通过对采集于山东省肉鸡屠宰场的202株空肠弯曲菌进行喹诺酮类耐药分子机制的研究,包括7种可以移动耐药基因筛查,喹诺酮耐药决定区(QRDR)突变检测和parC的筛查,确定了我国山东省鸡源空肠弯曲菌对喹诺酮类药物的耐药表型主要为gyrA中QRDR C-257-T突变所造成.并发现在空弯中存在于gyrA和gyrB基因上的部分沉默突变有着地域流行性特点.这些观察结果为解释我国食品动物源空肠弯曲菌耐药性现状,防控耐药空肠弯曲菌传播和流行提供基础数据.     

Genotype analysis of Campylobacter jejuni isolated from broilers in Poland

Twenty-six Campylobacter jejuni strains isolated from poultry were analyzed by genotypic typing including ITS-profiling, REP- and ERIC-PCR. ITS-profiling revealed the presence of 8 different genotypes. Amplification of REP sequences by PCR gave similar results with 10 different genotypes. ERIC-PCR was found to be the most discriminatory for typing C. jejuni. As many as 13 different DNA patterns were obtained with this technique. Based on data obtained it was found that C. jejuni isolates recovered from broilers at the slaughterhouses in southwest Poland are characterized by a high degree of genetic heterogeneity.     

空肠弯曲杆菌对喹诺酮类药物产生耐药性的作用机制和发展趋势

从 2 0世纪 70年代起 ,空肠弯曲杆菌 (Campy-lobacter jejuni)在全世界就被普遍认为是肠道感染的主要原因。主要是通过被污染的食物造成人类的感染。大部分空肠弯曲杆菌肠炎的病例并不需要抗生素治疗 ,因为大多数空肠弯曲杆菌肠炎是自限性感染 ,临床症状较轻。但是严重的感染和被拖延了的肠炎病例、败血症都需要抗生素治疗。喹诺酮类药物是治疗空肠弯曲杆菌肠炎中使用最普遍的药。随着喹诺酮类抗性菌种造成感染数量的增多 ,人类感染空肠弯曲杆菌病例也越来越多。人们在肉用动物生产中使用了大量的喹诺酮类药物 ,使喹诺酮类药物在食物链中出…     

Genotyping Campylobacter jejuni strains isolated from the gut and oviduct of laying hens

Campylobacter jejuni frequently colonizes the avian intestine. Recent evidence suggests that this organism can also colonize the oviduct of laying hens. However, the source and role of this colonization are unknown. Isolates from the ceca, cloacae, and oviducts of 11 laying hens in three intensive egg-producing flocks were genotyped by Fla typing with the restriction fragment length polymorphism of the polymerase chain reaction product of the flaA and flaB genes (fla typing) and pulsed-field gel electrophoresis (PFGE). A diversity in fla types and PFGE types was observed within and between flocks. Individual birds could be colonized by different genotypes at various intestinal and oviduct sites. However, the oviduct of individual birds appeared to be colonized by only one genotype at the time of sampling. In two birds, matching isolates investigated from the intestinal and reproductive tracts were genotypically identical but different from those oviduct isolates found in other birds in the same flock. Interestingly, not all cecal isolates appeared to be equally able to colonize the oviduct. These results suggest that oviduct colonization may result from ascending infection via the cloaca and that some strains of C. jejuni may be better adapted than others to oviduct colonization.     

Multilocus sequence typing of Campylobacter jejuni from broilers

Campylobacter jejuni isolates from a national Swedish Campylobacter monitoring in broilers were characterized by multilocus sequencing typing (MLST) in order to study the genetic diversity of this bacterial population. Isolates were initially characterized by pulsed-field gel electrophoresis (PFGE). One hundred were chosen for MLST genotyping. PFGE identified 69 distinct types compared to 44 different sequence types (STs) identified with MLST. Eighteen STs had not been described previously, while the remaining 26 STs were assigned to previously known clonal complexes. The majority of isolates were of genotypes noted in broilers and in humans in earlier studies. However, three clonal complexes, ST-206 complex, ST-677 complex and ST-1034 complex, previously associated with wild bird and environmental samples, were among the genotypes found. This study shows that most of the Swedish broiler isolates were of genotypes noted as common in broilers. However, it also highlights the potential influence of environmental sources on the broiler C. jejuni genotypes.     

Suitability of SSCP-PCR analysis for molecular detection of quinolone resistance in Campylobacter jejuni

Foodborne infections with Campylobacter spp. are increasing, especially antibiotic resistant strains are emerging. Quinolone resistant isolates can cause failure of therapy in severe clinical infections. Molecular characterisation is needed for the detection of resistant variants of C. jejuni. Therefore 23 isolates from poultry and human medicine as well as three control strains were tested for their minimal inhibitory concentration, their Single-Strand-Conformation-Polymorphism (SSCP)-PCR pattern (a method for the detection of resistance determining point mutations), and their sequence of the quinolone resistance determining region (QRDR). Six different SSCP types could be identified: two types for quinolone resistant isolates and other types containing so called silent mutations without influence on the resistance. A genotypic monitoring of the quinolone resistance in C. jejuni can be useful for the early detection of new resistance variants. As a screening method for detection of point mutations in the QRDR the SSCP-PCR can be applied. Compared to other genotypic methods the SSCP-PCR is less time and cost consuming and needs only standard technical equipment.     

Identification of nine sequence types of the 16S rRNA genes of Campylobacter jejuni subsp. jejuni isolated from broilers

Background

Campylobacter is the most commonly reported bacterial cause of enteritis in humans in the EU Member States and other industrialized countries. One significant source of infection is broilers and consumption of undercooked broiler meat. Campylobacter jejuni is the Campylobacter sp. predominantly found in infected humans and colonized broilers. Sequence analysis of the 16S rRNA gene is very useful for identification of bacteria to genus and species level. The objectives in this study were to determine the degree of intraspecific variation in the 16S rRNA genes of C. jejuni and C. coli and to determine whether the 16S rRNA sequence types correlated with genotypes generated by PFGE analysis of SmaI restricted genomic DNA of the strains.

Methods

The 16S rRNA genes of 45 strains of C. jejuni and two C. coli strains isolated from broilers were sequenced and compared with 16S rRNA sequences retrieved from the Ribosomal Database Project or GenBank. The strains were also genotyped by PFGE after digestion with SmaI.

Results

Sequence analyses of the 16S rRNA genes revealed nine sequence types of the Campylobacter strains and the similarities between the different sequence types were in the range 99.6–99.9%. The number of nucleotide substitutions varied between one and six among the nine 16S rRNA sequence types. One of the nine 16S rRNA sequence profiles was common to 12 of the strains from our study and two of these were identified as Campylobacter coli by PCR/REA. The other 10 strains were identified as Campylobacter jejuni. Five of the nine sequence types were also found among the Campylobacter sequences deposited in GenBank. The three 16S rRNA genes in the analysed strains were identical within each individual strain for all 47 strains.

Conclusion

C. jejuni and C. coli seem to lack polymorphisms in their 16S rRNA gene, but phylogenetic analysis based on 16S rRNA sequences was not always sufficient for differentiation between C. jejuni and C. coli. The strains were grouped in two major clusters according to 16S rRNA, one cluster with only C. jejuni and the other with both C. jejuni and C. coli. Genotyping of the 47 strains by PFGE after digestion with SmaI resulted in 22 subtypes. A potential correlation was found between the SmaI profiles and the 16S rRNA sequences, as a certain SmaI type only appeared in one of the two major phylogenetic groups.     

Heterogeneity of Campylobacter jejuni and Campylobacter coli strains from healthy sheep

The objectives of this study were to identify, at species level, thermophilic campylobacters isolated from clinically healthy sheep by a multiplex polymerase chain reaction (mPCR). The heterogeneity among Campylobacter jejuni and C. coli isolates was also investigated using a restriction fragment length polymorphism (RFLP) analysis of the flagellin (flaA) gene. Samples of intestinal contents, gall bladders and faeces were collected from 610 healthy sheep. While gall bladder samples were plated directly onto Preston agar, an enrichment stage was applied for intestinal and faecal samples. Of the 610 samples, 302 (49.5%) were positive for Campylobacter spp. Using a mPCR assay for species identification, 103 (34.1%) were positive with C. jejuni-specific primers, while 100 (33.1%) were positive with C. coli-specific primers. Additionally, 16 (11.9%) of the intestinal content samples were positive for both species by mPCR. All the isolates identified as C. jejuni and C. coli were successfully subtyped by flaA typing. Of 203 isolates tested, 48 different flaA types were found. Twenty-six flaA types were identified among C. jejuni isolates and the remaining 22 from C. coli isolates.     

Dissemination of antimicrobial resistant strains of Campylobacter coli and Campylobacter jejuni among cattle in Washington State and California

The purpose of this study was to investigate the genetic similarity of Campylobacter jejuni and Campylobacter coli with similar antimicrobial resistance phenotypes, isolated from cattle on different farms and at different times, in order to evaluate the possible existence of disseminated antimicrobial resistant clones. PFGE after SmaI and KpnI restriction identified 23 and 16 distinct PFGE patterns among 29 C. jejuni and 66 C. coli isolates, respectively. In C. coli, 51 (77%) of the resistant isolates demonstrated one of the four indistinguishable PFGE patterns, whereas only 24% doxycycline resistant C. jejuni shared one of the two indistinguishable PFGE patterns. The genetic mechanisms of resistance were homogeneous within and between these clonal types. Genetically indistinguishable (clonal) groups of C. coli accounted for most Campylobacter sp. with multiple antimicrobial resistance observed in this study, consistent with a role for clonal dissemination in the epidemiology of resistance in this species.     

Antimicrobial susceptibilities of Campylobacter strains isolated from broilers in the southern part of Japan from 1995 to 1999

Cecal contents (16 samples/each flock) of broilers derived from 212 flocks were investigated for colonization of Campylobacter from 1995 to 1999 in the southern part of Japan, and the isolates were tested for antimicrobial susceptibilities. C. jejuni-positive flocks numbered 42 (19.8%) and C. coli-positive ones 26 (12.3%); Campylobacter spp. were recovered from 68 flocks (32.1%) in total. MICs of ampicillin, erythromycin (EM), tetracycline, nalidixic acid (NA), norfloxacin (NFLX), and ofloxacin (OFLX) to these 68 Campylobacter isolates were determined. Quinolone-resistant Campylobacter isolates numbered 22 (32.4%). All the isolates except one were cross-resistant to NA, OFLX, and NFLX. A high frequency of quinolone-resistance was found in both C. jejuni and C. coli, whereas a high level of EM-resistance was found in only C. coli strains. All C. jejuni isolates were sensitive to EM.     

Serotypes of Campylobacter jejuni and C coli isolated from dogs

The serotypes of Campylobacter jejuni and C coli isolated from 56 dogs were established by the Penner serotyping scheme. A total of 37 C jejuni and 19 C coli were typed. Only two of the C coli strains were typable by the Penner method compared to 29 of 37 C jejuni strains. Pen 2 and 4 were the most predominant serotypes, constituting 41-4 per cent of the typable C jejuni strains. All but one of the C jejuni strains belonging to serotypes pen 1 and 2 were isolated from dogs with diarrhoea.     

Fla-DGGE analysis of Campylobacter jejuni and Campylobacter coli in cecal samples of broilers without cultivation

In a commercial broiler flock during rearing multiple genotypes of Campylobacter jejuni may be present as well as in gastrointestinal tracts of individual birds. The aim of this study was to optimize and apply a denaturing gradient gel electrophoresis assay of the flagellin gene (fla-DGGE) for analysis of C. jejuni and Campylobacter coli in cecal samples of broilers without prior cultivation. One C. coli and 21 C. jejuni strains isolated from broiler flocks, of which 14 typed as unique by restriction fragment length polymorphism of flaA and two undefined strains, were clustered into 9 groups when applying fla-DGGE. Spiking of cecal samples revealed that fla-DGGE is able to detect at least 4.55-5.96logCFUCampylobacter/mlcecal material. The presence of 3 strains spiked in cecal material was demonstrated by fla-DGGE as the corresponding bands were visible on the DGGE gel. Naturally contaminated cecal samples were shown to contain different types of C. jejuni and C. coli. Fla-DGGE has some potential as a cultivation-independent fast primary subtyping method for C. jejuni and C. coli in cecal samples of broilers.     

Pathogenicity of Campylobacter jejuni and Campylobacter coli strains in the pregnant guinea pig model

Pathogenicity of 17 Campylobacter isolates for pregnant guinea pigs was investigated. Of 14 isolates, 12 (86%) produced rates of abortion ranging from 13% to 87%. Two isolates did not produce abortion. Reference strains of C fetus subsp venerealis produced abortion in 60% to 87% and C fetus subsp fetus produced abortion in 60% of the guinea pigs. Inoculated organisms were recovered from uterus, blood, liver, kidney, spleen, and gallbladder of the guinea pigs at rates as high as 83% for 2 ovine isolates and as low as 13% for 2 bovine and 1 human isolates. Most isolations were from the uterus. Two avian isolates were not recovered. Within the C jejuni and C coli group, the ovine and the human isolates appear to be more pathogenic. Swine, bovine, and avian isolates were less pathogenic. Seemingly, the pregnant guinea pig was a suitable and practical model for evaluating the pathogenicity of Campylobacter organisms, regardless of their host of origin.     

Feed can be a source of Campylobacter jejuni infection in broilers

1. The aim was to determine the importance of a contaminated diet as a possible cause of Campylobacter jejuni infection in broilers.

2. This study evaluated the viability of C. jejuni in both starter and finisher diets and the interference from other mesophilic bacteria in this viability.

3. Starter and finisher samples of broiler diet were deliberately contaminated with 3 or 5 log CFU·g^?1 of C. jejuni (NCTC 11351) and then maintained at two different storage temperatures (25°C or 37°C) for 3 or 5 d.

4. C. jejuni survived during this period and, when inoculated at 10³ CFU·g^?1, multiplied with greater proliferation at a storage temperature of 37°C. There was no relationship between the amount of mesophilic bacteria and C. jejuni viability.

5. This study highlights the importance of the diet in the epidemiology of C. jejuni in broilers.     

猪源链球菌对环丙沙星耐药性与耐药基因突变的相关性研究

通过微量稀释法测定28株猪源链球菌对环丙沙星的MIC值,研究东北地区猪源链球菌对环丙沙星耐药性与parC、gyrA基因突变的相关性.通过PCR方法扩增parC和gyrA基因喹诺酮耐药决定区(QRDR)并测序分析;18株耐药菌在parC基因80位的突变(AGC→ATT)导致氨基酸Ser→Ile突变,11株高度耐药菌在gyrA基因81位的突变(CAG→)CAT、CTT或CTA)导致氨基酸Ser→Ile、Phe或Tyr的突变.当菌株对环丙沙星的MIC值≤1μ/mL时,parC和gyrA基因的QRDR区均未有突变;而当MIC ≥2μg/mL时,ParC的氨基酸发生了Ser80→Ile的突变,同时发生GyrA氨基酸Ser81突变的菌株,耐药水平很高.研究表明,环丙沙星低水平类耐药是由于拓扑异构酶Ⅳ改变引起,而高水平耐药是由拓扑异构酶Ⅳ、DNA旋转酶共同改变引起的.实验结果证明,在一定条件下,耐药性的高低与突变位点的多少成正比.     

Genetic comparison of Campylobacter jejuni isolated from different cattle farms

To compare the genotypes of Campylobacter jejuni, isolates of cattle origin were collected from 9 Polish farms and genotyped by ERIC-PCR. We identified 28 genotypes among the 43 C. jejuni isolates, and demonstrated high genomic diversity. The highest level of diversity was observed in strains isolated from stanchion-barn animals in opposition to those from the loose-housing system.     

PFGE and PCR/RFLP typing of Campylobacter jejuni strains from poultry

1. Pulsed-field gel electrophoresis (PFGE) and PCR-restriction fragment length polymorphisms of the flagellin gene (fla-RFLP) were used to analyse 92 poultry and 110 human strains of Campylobacter jejuni. 2. Among poultry strains, 11 fla-RFLP and 11 PFGE subtypes were found, while human strains could be divided into 23 fla-RFLP and 32 PFGE subtypes. Altogether, 31 fla-RFLP and 32 PFGE subtypes were found. 3. The results show that individual flocks in farms are mostly infected with a single C. jejuni clone, while during subsequent colonisation their genotypes altered. fla-RFLP and PFGE profiles in poultry and humans were identical in less than 6% of cases. The results found so far confirm previous findings that chicken meat does not represent as important a source of campylobacteriosis as was previously believed. 4. The typing of Campylobacter sp. forms the basis for an evaluation of the current state and risk assessment of various Campylobacter sp. sources in relation to humans. Examination of samples with only one method is insufficient for epidemiology studies, because apparently different clones identified with one method could originate from a single clone, which could be proved with the other method.