A real-time multiplexed PCR assay for rapid detection and differentiation of Campylobacter jejuni and Campylobacter coli

Campylobacter species are the leading agents of bacterial gastroenteritis worldwide. C. jejuni and C. coli together are responsible for more than 95% of all cases of Campylobacter-induced diarrheal disease in the United States. Detection of campylobacteria in clinical samples by conventional culture is problematic and slow due to their complex taxonomy, fastidious growth requirements, and biochemical inertness. The current study describes a rapid, sensitive, and specific real-time polymerase chain reaction (PCR) assay capable of detecting and differentiating C. jejuni (hippuricase gene, hipO) and C. coli (serine hydroxymethyltransferase gene, glyA) in a single reaction, directly from clinical isolates and human feces. The analytical specificity of the assay was demonstrated with a diverse range of Campylobacter species, related organisms, and other diarrhea-inducing bacterial pathogens. The analytical sensitivity of the multiplexed, PCR assay was 10 genome copies for both C. jejuni and C. coli. Following a rapid DNA extraction method (QIAGEN QIAamp DNA stool Mini Kit), the multiplexed PCR identified C. jejuni or C. coli in 100% of fecal samples containing 10(3) colony-forming units (CFU) per gram of feces. This assay represents the first real-time PCR method capable of detecting and differentiating C. jejuni and C. coli in a single reaction.     

PCR identification of Campylobacter coli and Campylobacter jejuni by partial sequencing of virulence genes

The objective of this study was to utilize a multiplex PCR assay for concurrent detection of Campylobacter spp. and C. coli or C. jejuni, using probes derived from genes cadF and ceuE and an undefined virulence gene. A total of 97 Campylobacter strains, isolated from turkey litter (n=74), chicken livers (n=15) and clinical (n=8) samples, were speciated using the PCR-based assay. PCR amplification of the isolates identified a 400-bp cadF gene, conserved in Campylobacter species, an 894-bp ceuE gene, specific for C. coli, and a 160-bp oxidoreductase gene, specific for C. jejuni. The approximately 35 kDa cadF adhesion proteins allow Campylobacter to bind to the intestinal epithelial cells and the 37 kDa ceuE lipoproteins are involved in siderophore transport. Sequencing of the 160-bp undefined gene yielded a 67% protein identical match with a gene encoding an oxidoreductase subunit in C. jejuni. The specificity of the assay was validated on 36 non-Campylobacter strains (11 Gram-positive and 25 Gram-negative bacteria). The PCR assay identified 59% of turkey and 47% of chicken isolates as C. jejuni, and 41% of turkey and 53% of chicken isolates as C. coli. All human isolates were identified as C. jejuni. The specificity of this assay to detect C. coli or C. jejuni was 97%.     

荧光定量PCR方法在空肠弯曲菌检测中的应用

空肠弯曲菌是引起人类食源性疾病的主要病原菌之一。快速、特异检测方法的建立对于该病原菌感染的诊断及鉴别诊断具有重要意义。荧光定量PCR方法作为一种快速诊断方法已经广泛应用于空肠弯曲菌的检测及鉴定。本文从荧光定量PCR方法在空肠弯曲菌病原检测中的建立、发展以及应用等方面进行综述。     

Development of a ceuE-based multiplex polymerase chain reaction (PCR) assay for direct detection and differentiation of Campylobacter jejuni and Campylobacter coli in Thailand.

A novel ceuE-based multiplex PCR system was developed as an efficient diagnostics test to detect and differentiate C. jejuni and C. coli. There is no cross reactivity between C. jejuni and C. coli. In addition, the assay does not produce a positive signal from other enteric bacteria including Salmonella, Shigella and Escherichia coli strains. Campylobacter detection sensitivity was determined to be equivalent to previously reported PCR for other enteric bacteria. We also noticed that silicon dioxide extraction can improve Campylobacter detection sensitivity from infected stool samples. It was demonstrated that the PCR assay developed in this study had a much better Campylobacter detection rate than the traditional culturing method (77% versus 56%). However, we also identified small numbers of culture positive stools (8%, or 16 out of 202 samples) that did not yield PCR positive results for Campylobacter. These PCR negative/culture positive stools were proven to be inhibitory to PCR amplification.     

空肠弯曲菌、单增李斯特菌和大肠杆菌O157多重PCR分子检测研究

目的建立同时检测空肠弯曲菌、单核细胞增生性李斯特菌(简称单增李斯特菌)和大肠杆菌O157的多重聚合酶链反应(PCR)检测技术。方法根椐空肠弯曲菌mapA基因序列、单增李斯特菌的编码溶血素(hly)基因序列和大肠杆菌的O157抗原(rfbE)基因序列,分别设计了3对特异引物,PCR扩增的目的基因片段为589 bp、360 bp和194bp;并对反应条件进行了优化。结果对3种致病菌检测,多重PCR检测DNA的敏感度分别为空肠弯曲菌2.264 ng、单增李斯特菌37.92 ng、大肠杆菌2.100 ng,样品检测时间6~8 h。结论该方法操作简便,检测周期短,特异性和敏感度高,为同时检测污染样品中空肠弯曲菌、单增李斯特菌和大肠杆菌O157奠定了基础。     

Development of a triplex PCR assay for the specific detection of Campylobacter jejuni,Salmonella spp., and Escherichia coli O157:H7

A triplex PCR assay was developed and evaluated for efficacy in detecting Campylobacter jejuni, Salmonella spp., and Escherichia coli O157:H7 in a variety of raw and ready-to-eat food products. Following a short enrichment period, artificially contaminated food samples were subjected to a triplex PCR assay, which incorporated published primers for each food pathogen, a protocol for sample collection, and a PCR procedure designed specifically for the assay. The selected primers amplified fragment sizes of 159 bp, 252 bp, and 360 bp for C. jejuni, E. coli O157:H7, and Salmonella spp., respectively. This assay provides specific and reliable results and allows for the cost-effective detection of all three bacterial pathogens in one reaction tube.     

空肠弯曲菌、单增李斯特菌和大肠杆菌O157多重PCR分子检测研究

目的建立同时检测空肠弯曲菌、单核细胞增生性李斯特菌（简称单增李斯特菌）和大肠杆菌O157的多重聚合酶链反应（PCR）检测技术。方法根椐空肠弯曲菌mapA基因序列、单增李斯特菌的编码溶血素（hly）基因序列和大肠杆菌的O157抗原（rfbE）基因序列,分别设计了3对特异引物,PCR扩增的目的基因片段为589 bp、360 bp和194bp;并对反应条件进行了优化。结果对3种致病菌检测,多重PCR检测DNA的敏感度分别为空肠弯曲菌2.264 ng、单增李斯特菌37.92 ng、大肠杆菌2.100 ng,样品检测时间6-8 h。结论该方法操作简便,检测周期短,特异性和敏感度高,为同时检测污染样品中空肠弯曲菌、单增李斯特菌和大肠杆菌O157奠定了基础。     

Detection and differentiation of Campylobacter jejuni and Campylobacter coli in broiler chicken samples using a PCR/DNA probe membrane based colorimetric detection assay

Campylobacter enteritis in humans has been linked to consumption of poultry meat. Surveys show that 30-100% of poultry harbour Campylobacter as normal flora of the digestive tract which indicates a need to identify prevalent organism types in flocks and trace their epidemiology. In this study we describe a Campylobacter genus specific polymerase chain reaction (PCR) assay, amplifying the 16 S-23 S rRNA intergenic spacer region with an internal Campylobacter genus specific DNA probe and species specific probes for Campylobacter jejuni and Campylobacter coli designed for confirmation of the amplified PCR products by Southern blot and colorimetric reverse hybridization assays. The specificity of this assay was established by testing a range of food pathogens. Broiler chicken samples were tested following presumptive positive identification by the Malthus System V analyser (Malthus Instruments, UK). The combined PCR and colorimetric reverse hybridization assay is easy to perform and faster than conventional methods for confirmation and identification of Campylobacter species.     

Characterization of erythromycin resistance in Campylobacter jejuni and Campylobacter coli.

The mechanism of resistance to erythromycin, the drug of choice in the treatment of campylobacter gastroenteritis, was investigated. Erythromycin resistance (MICs, greater than 1,024 micrograms/ml) in three clinical isolates of Campylobacter jejuni and one C. coli isolate was determined to be constitutive and chromosomally mediated. In vivo protein synthesis in erythromycin-susceptible C. jejuni and C. coli strains was completely inhibited by low levels of erythromycin (5 micrograms/ml), whereas a high concentration of the antibiotic (100 micrograms/ml) had no effect on protein synthesis in erythromycin-resistant strains. Biological assays showed that extracellular degradation of erythromycin was not responsible for erythromycin resistance in strains of Campylobacter species. The rates and amounts of uptake of [14C]erythromycin by resistant and susceptible campylobacter cells were determined to be similar. Binding assays with purified campylobacter 70S ribosomes as well as 50S ribosomal subunits showed that those from erythromycin-resistant strans bound much less [14C]erythromycin than did those from susceptible strains. Genomic DNA from C. coli UA585 was used to transform erythromycin resistance to C. coli UA417. The erythromycin resistance marker was associated with a 240-kb SmaI fragment of the C. coli UA585 genome. Our results rule out erythromycin inactivation or efflux and are not consistent with the production of an RNA methylase, although they are consistent with a mutational mechanism of resistance due to a change in a ribosomal protein gene. This study constitutes a detailed biochemical and genetic characterization of erythromycin resistance in Campylobacter species.     

Antibacterial activities of nitrothiazole against Campylobacter jejuni and Campylobacter coli.

Niridazole (Ambilhar) and three other newly synthesized nitrothiazole derivatives were highly active against 19 microaerophilic campylobacters (minimum concentration required to inhibit 50% of strains [MIC50], 0.0075 to 0.015 mg/liter). There were, however, considerable differences in the susceptibility among strains tested, and one nitrothiazole derivative was rather inactive (MIC50, 2 mg/liter). Nitroimidazole derivatives, such as metronidazole and tinidazole, were less active (MIC50, 2 and 4 mg/liter, respectively). The nitrofuran derivatives, such as nitrofurazone and nitrofurantoin, were also less active (MIC50, 1 mg/liter). Niridazole and another potent nitrothiazole derivative killed the campylobacters rapidly at low concentrations. In contrast, much higher concentrations of metronidazole were required to achieve bactericidal values.     

Macrolide resistance in Campylobacter jejuni and Campylobacter coli

Infection with Campylobacter jejuni is now considered to be the most common cause of acute bacterial gastroenteritis in humans worldwide. It occurs more frequently than infections caused by Salmonella species, Shigella species, or Escherichia coli O157:H7. Although C. jejuni is also recognized for its association with serious post-infection neurological complications, most patients with C. jejuni infections have a self-limited illness. Nevertheless, a substantial proportion of these infections are treated with antibiotics. These include severe and prolonged cases of enteritis, infections in immune-suppressed patients, septicaemia and other extra-intestinal infections. Under these circumstances, erythromycin is often recommended as the drug of first choice. However, erythromycin-resistant Campylobacter have emerged during therapy with macrolides. Moreover, the widespread use of macrolides, including erythromycin, in veterinary medicine has accelerated this resistance trend. Several countries including Canada, Japan and Finland have reported C. jejuni isolates with low and stable rates of macrolide resistance. In contrast, the increasing level of macrolide resistance in C. jejuni is becoming a major public health concern in other parts of the world such as the United States, Europe and Taiwan. Macrolide resistance in Campylobacter is mainly associated with point mutation(s) occurring in the peptidyl-encoding region in domain V of the 23S rRNA gene, the target of macrolides. Several rapid and practical techniques have recently been developed for the identification of macrolide-resistant isolates of C. jejuni. The aim of this mini-review is to give an overview of the worldwide distribution of macrolide resistance in C. jejuni and Campylobacter coli as well as its possible association with the massive use of these agents in food animals. Mechanisms implicated in macrolide resistance in C. jejuni and also techniques that have been developed for the efficient detection of macrolide-associated mutation(s) will be discussed in detail.     

Characterization of the porins of Campylobacter jejuni and Campylobacter coli and implications for antibiotic susceptibility.

The major outer membrane protein was extracted from Campylobacter coli by Triton X-100/EDTA fractionation of cell envelopes. This heat-modifiable protein was shown to have pore-forming activity in black lipid bilayers. The C. coli porin formed a relatively small cation-selective pore with a mean single-channel conductance of 0.53 +/- 0.16 nS in 1.0 M KCl. There was no evidence of oligomer formation, which suggested that each protein monomer formed a pore. Pore-forming activity of the C. coli porin and similarly prepared Campylobacter jejuni porin was also measured in liposome-swelling assays. These results confirmed the cation selectivity of both pores. The C. coli porin formed a small pore, which hindered the penetration of solutes with a molecular weight of 262, and a larger pore, which hindered the penetration of solutes with a molecular weight of 340, in a protein-concentration-dependent manner. C. jejuni formed one size of pore that was slightly larger than the C. coli pore and just permitted the passage of solutes, with a molecular weight of 340. A review of the literature concerning in vitro screening of antimicrobial agents tended to confirm the low permeability of the C. jejuni outer membrane to hydrophilic antimicrobial agents except when the molecules had molecular weights of less than 360. The porins of C. jejuni and C. coli may contribute to intrinsic resistance to antimicrobial agents, whereas alternative (nonporin) routes of antimicrobial agent uptake may be more important determinants of susceptibility to antimicrobial agents.     

应用TaqMan实时荧光-聚合酶链反应方法快速检测粪便标本空肠弯曲菌的研究

目的建立空肠弯曲菌TaqMan实时荧光-PCR方法,用于粪便标本的直接检测。方法根据空肠弯曲菌特异性基因hipO和mapA分别设计引物和探针,在对2组引物和探针进行灵敏度、特异性和重复性评价的基础上,对45例临床腹泻患者粪便标本提取DNA之后,荧光PCR检测,同时进行分离培养。 结果两组引物和探针能准确检测空肠弯曲菌菌株2株,检测限可达到10~20 cfu/ml,并与其他肠道致病菌无交叉反应。检测45份腹泻病例粪便标本,该方法检测到3份为阳性,同时进行的传统培养方法仅从该3份标本中的两份中分离到空肠弯曲菌。 结论本研究建立的TaqMan荧光PCR检测粪便标本中所携带的空肠弯曲菌灵敏度高,特异性好,能够提高粪便中空肠弯曲菌的阳性检出率和缩短检测时限。     

Susceptibility of Campylobacter jejuni and Campylobacter coli to macrolides and related compounds.

The susceptibility of 105 thermophilic campylobacters from human and swine origins to eight macrolides and related compounds was tested. Erythromycin, josamycin, clindamycin, and ASE 136 BS (a new erythromycin derivative) were the most active against the human strains. The swine strains were highly resistant, except to pristinamycin. The human Campylobacter coli strains (except for two strains) behaved like the C. jejuni strains.     

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.     

Tetracycline resistance of Australian Campylobacter jejuni and Campylobacter coli isolates

OBJECTIVES: Tetracycline resistance in Campylobacter is encoded by the tet(O) gene and is usually associated with conjugative plasmids. Little was known about tetracycline resistance in Australian Campylobacter species, therefore we investigated this resistance in 41 Campylobacter jejuni and five Campylobacter coli strains from humans and healthy chickens. METHODS: Tetracycline MICs were determined for each isolate using an agar dilution method. The distribution and localization of tet(O) on plasmid and chromosomal DNA was determined by Southern-blot experiments. The ability to transfer resistance to recipient strains was examined through conjugation studies. Identity of transconjugants was confirmed by PCR and flaA-restriction fragment length polymorphism analysis. RESULTS: High-level tetracycline resistance was observed, ranging from 32 to >256 mg/L. Plasmids were detected in 74% of isolates with plasmids between 30 and 40 kb in size most frequently isolated. tet(O) was present in all tetracycline-resistant isolates. In the majority of strains under study the tet(O) gene was chromosomally encoded. Tetracycline resistance of six C. jejuni strains in which tet(O) was plasmid mediated was transferred by conjugation to a C. jejuni recipient strain. Transfer did not occur between tetracycline-resistant C. jejuni strains and a C. coli recipient. No difference in MICs, plasmid carriage and tet(O) localization was detected between human and chicken isolates. CONCLUSIONS: These data indicate that the tet(O) gene, previously reported in Campylobacter strains throughout the world, is present in Australian Campylobacter. This study will lead to a greater understanding of antibiotic resistance distribution in Campylobacter spp. in Australia.     

In vitro susceptibilities of Campylobacter jejuni and Campylobacter coli to azithromycin and erythromycin.

MICs of azithromycin and erythromycin for 20 Campylobacter coli and 20 Campylobacter jejuni strains were determined. The results demonstrated that, for Campylobacter species, all high-level erythromycin-resistant strains were also resistant to azithromycin and that azithromycin did not exhibit increased potency in comparison with that of erythromycin.     

Comparison of antimicrobial susceptibility patterns of Campylobacter jejuni and Campylobacter coli

To determine whether employing antibiograms is useful to separate Campylobacter jejuni and Campylobacter coli, we determined the MICs of 12 antibiotics for 104 human clinical strains and 74 swine strains. Of 74 swine strains, 5 (7%) were hippurate positive, as were 93 (89%) of 104 human strains. The 12 antimicrobial agents tested were ampicillin, amoxicillin, clindamycin, chloramphenicol, erythromycin, furazolidone, norfloxacin, nalidixic acid, rosoxacin, rosaramicin, tetracycline, and Sch 32063. Isolates from humans were significantly (P less than 0.001) more susceptible than swine strains to clindamycin, erythromycin, rosaramicin, and Sch 32063. Of 11 human hippurate-negative strains, 3 (27%) were resistant to clindamycin, erythromycin, rosaramicin, and Sch 32063, compared with 1 of 93 (1%) hippurate-positive strains. Nearly all human and swine strains were susceptible to furazolidone and nalidixic acid. Campylobacter isolates from humans and swine have different antibiograms, and the susceptibility to certain antibiotics, such as clindamycin, may be helpful for differentiation of C. jejuni from C. coli.     

实时荧光聚合酶链反应检测空肠弯曲菌

目的 探索一种快速、敏感、特异的空肠弯曲菌检验方法.方法 根据GenBank公布的空肠弯曲菌基因序列,在其保守区域设计特异性引物与探针以建立基于TaqMan探针的实时荧光聚合酶链反应(PCR)方法,并对方法的特异性、敏感性和重复性进行研究.结果 该方法对空肠弯曲菌可产生特异扩增信号,对其他非空肠弯曲菌菌属无响应,其检测敏感性可达10cfu/mL.反应体系具有很高的稳定性.结论实时荧光PCR可快速、特异、灵敏地检测空肠弯曲菌,具有较强的实际应用价值.     

Development of a multiplex PCR assay for the identification of pathogenic genes of Escherichia coli in milk and milk products

A multiplex PCR for the simultaneous detection of some pathogenic genes of enteropathogenic, enterotoxigenic and verocytotoxin-producing Escherichia coli was developed. In this study primers found in literature as well as primers to the purpose designed were used. In this way, it was possible to generate specific fragments of 96, 170, 229, 285, 348, 414 and 510 bp for Hlya, St, EaeA, Lt, Vt1, UidA and Vt2 genes, respectively. When applied to bacterial strains experimentally inoculated in milk and milk products, the proposed PCR showed a detection limit of 5 x 10(4)CFU/ml for Hyla, St, Eaea, Vt1 primers, while for Lt and Vt2 primers the limit resulted of 10(6)CFU/ml.