Quantitative analysis of viable,stressed and dead cells of Campylobacter jejuni strain 81-176

Campylobacter jejuni is an important foodborne gastrointestinal pathogen and highly sensitive to environmental stresses. Research has shown that changes in culturability, cell morphology, and viability occur when C. jejuni cells are subjected to stresses. In this study, real-time PCR, ethidium monoazide (EMA) in combination with real-time PCR (EMA-PCR), BacLight bacterial viability staining, and agar plate counting methods were used to quantitatively analyze viable, stressed, and dead C. jejuni strain 81-176. The real-time PCR assay provides highly sensitive and specific quantification of total genome copies of C. jejuni culture in different growth phases. Our results also reveal that real-time PCR can be used for direct quantification of Campylobacter genome release into Phosphate Buffered Saline (PBS) as an indicator of cell lysis. Using EMA-PCR, we obtained a dynamic range of greater than 3 logs for differentiating viable vs. dead cells. The viability and morphological characteristics of the stressed cells after one-week incubation at 25 °C, in air, and under nutrient-poor conditions were investigated. Our results indicated that, over 99% of the stressed cells were converted from the spiral to the coccoid form and became non-culturable. However, more than 96% of the coccoid cells retained their membrane integrity as suggested by both the BacLight staining and EMA-PCR analyses. Thus, to detect C. jejuni under stress conditions, conventional culturing method in conjunction with EMA-PCR or BacLight staining might be a more appropriate approach.     

食源性空肠弯曲菌的分子分型及其遗传多样性分析

为了解在2012~2014年从中国不同城市分离到的食源性空肠弯曲菌的遗传多样性特征,本研究采用多序列位点分型(multilocus sequencing typing,MLST)的方法对分离到的33株食源性空肠弯曲菌进行分子分型。研究中,对33株菌株的管家基因使用7对不同的引物进行PCR扩增,并对扩增的产物使用凝胶电泳鉴定后进行测序。将测序结果同Pub MLST中Campylobacter数据库进行比对,以获得对应菌株ST型,并提交新的ST型数据。利用其MLST数据构建进化树和最小生成树。结果显示33株食源性空肠弯曲菌可以分为27个ST型,其中有14种为新的ST型,可形成11种克隆复合体,优势克隆复合体为CC22和CC45,部分菌株的CC型也曾在临床上发现。共存在91种核苷酸多态性位点,部分等位基因之间存在重组。这表明在2012-2014年间分离得到的菌株具有丰富的遗传多样性,并且有潜在致病风险。     

Crystal Structure of the Recombination Mediator Protein RecO from Campylobacter jejuni and Its Interaction with DNA and a Zinc Ion

Homologous recombination is involved in repairing DNA damage, contributing to maintaining the integrity and stability of viral and cellular genomes. In bacteria, the recombination mediator proteins RecO and RecR are required to load the RecA recombinase on ssDNA for homologous recombination. To structurally and functionally characterize RecO, we determined the crystal structure of RecO from Campylobacter jejuni (cjRecO) at a 1.8 Å resolution and biochemically assessed its capacity to interact with DNA and a metal ion. cjRecO folds into a curved rod-like structure that consists of an N-terminal domain (NTD), C-terminal domain (CTD), and Zn²⁺-binding domain (ZnD). The ZnD at the end of the rod-like structure coordinates three cysteine residues and one histidine residue to accommodate a Zn²⁺ ion. Based on an extensive comparative analysis of RecO structures and sequences, we propose that the Zn²⁺-binding consensus sequence of RecO is CxxC…C/HxxC/H/D. The interaction with Zn²⁺ is indispensable for the protein stability of cjRecO but does not seem to be required for the recombination mediator function. cjRecO also interacts with ssDNA as part of its biological function, potentially using the positively charged patch in the NTD and CTD. However, cjRecO displays a low ssDNA-binding affinity, suggesting that cjRecO requires RecR to efficiently recognize ssDNA for homologous recombination.     

2种检测方法对弯曲菌检出率的影响

目的比较国标法和双孔滤膜法在生禽肉中弯曲菌的检测率,探索生禽肉中弯曲菌分离鉴定的有效检测方法。方法随机采集冷冻和新鲜禽肉产品共102份,通过国标法和双孔滤膜法2种方法检测,观察2种方法从样品处理、增菌和分离培养过程中的不同,并比较2种方法检测弯曲菌检出率的差异。结果102份样品中国标法检出弯曲菌的检出率为28.43%,其中空肠弯曲菌和结肠弯曲菌的检出率分别为13.725%和14.705%;双孔滤膜法检出弯曲菌的检出率为53.92%,其中空肠弯曲菌和结肠弯曲菌的检出率分别为26.47%和27.45%;双孔滤膜法检出率显著高于国标法,差异有统计学意义(P<0.05)。结论应用双孔滤膜法能显著提高生禽肉食品中弯曲菌的检出率。     

Control of campylobacter in poultry industry from farm to poultry processing unit: A review

Campylobacter is an emerging zoonotic bacterial threat in the poultry industry. Most of the human cases of campylobacteriosis recorded have revealed their poultry origins. Various control measures have been employed both at the farm and processing levels to combat with it. The antibiotic treatment, phage therapy, competitive exclusion, and vaccination have been adapted at the farm level to reduce colonization of Campylobacter in poultry gut. While prevention of intestinal spillage, scheduled slaughter, logistic slaughter, chemical decontamination of carcasses are recommended to reduce contamination during processing. The postharvest interventions such as heat treatment, freezing, irradiation of contaminated carcass can effectively reduce Campylobacter contamination. Thus, integrated approaches are required to tackle infection of Campylobacter in humans.     

Effect of crust freezing applied alone and in combination with ultraviolet light on the survival of Campylobacter on raw chicken

The application of crust freezing (CF) applied as a stand-alone treatment or in combination with ultraviolet (UV) light for reducing the level of artificially inoculated Campylobacter jejuni on raw chicken was investigated. CF air temperatures of −5, −15 and −27 °C (±3 °C) with freezing times of 70, 15 and 6 min, respectively, were used. The level of C. jejuni on chicken was also examined following subsequent refrigerated (0–4 °C) storage at 3 and 7 days. All CF treatments resulted in significant reductions compared to untreated controls (P < 0.05). Although combining CF with UV also resulted in significant reductions for C. jejuni, the combined treatments were generally no more effective than treatment by CF alone. Overall, the color of chicken drumsticks was not affected by CF treatments (P ≥ 0.05). In general, CF resulted in increased drip loss (P < 0.05), which increased over storage time and was greater at higher CF temperatures. The current study indicates that CF has potential for reducing the levels of C. jejuni by between 0.5 and 1.5 log₁₀ CFU/g and impacts minimally on the color of treated skin.     

Biofilm formation by Campylobacter jejuni in controlled mixed-microbial populations

This study was to screen the ability of biofilm formation by Campylobacter jejuni strains found in New Zealand, and investigate the biofilm growth of C. jejuni in a controlled mixed-microbial population that includes five different bacteria. The ability of C. jejuni to form a biofilm in monoculture and mixed-microbial populations was measured in a laboratory assay using a microtiter plate screening assay. The optical density of the biofilm and cell growth from mixed-microbial populations was converted to a Biofilm Formation Index (BFI). This index was used to standardize the biofilm formation in the mixed-microbial populations. High BFI was observed for Enterococcus faecalis (2.30) and Staphylococcus simulans (3.75) when they were grown with C. jejuni multilocus sequence type ST-474: a dominant poultry and human-associated type in New Zealand. C. jejuni cells were recovered from most of the biofilms containing E. faecalis and/or S. simulans. These results suggest that E. faecalis and S. simulans may play a role in biofilm formation in the poultry environment as both of these microorganisms are found in poultry processing environments and were able to form a biofilm in association with C. jejuni under microaerobic conditions. Understanding the relationships among C. jejuni, E. faecalis and S. simulans in poultry processing plants and farms may help in the design of strategies to reduce the reservoir of contamination of these bacteria and reduce the incidence of campylobacteriosis.     

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.     

2016—2018年北京市腹泻患者弯曲菌耐药性与脉冲场凝胶电泳分子分型的研究

目的分析北京市腹泻患者弯曲菌(Campylobacter spp.)分离株的抗生素敏感性和脉冲场凝胶电泳(pulsed field gel electrophoresis,PFGE)分子分型特征。方法2016—2018年,对北京市19家医院肠道门诊分离的弯曲菌采用琼脂稀释法进行抗生素敏感性检测,按照PulseNet的方法进行PFGE实验和聚类分析。结果所有空肠弯曲菌至少对一种抗生素有耐药性。空肠弯曲菌对环丙沙星(95.30%)的耐药率最高,其次为四环素(94.02%)和萘啶酸(92.31%),结肠弯曲菌对环丙沙星(95.00%)、四环素(95.00%)的耐药率最高,其次为萘啶酸(90.00%)。空肠弯曲菌菌株和结肠弯曲菌菌株的多耐药率分别是55.56%和75.00%。228株空肠弯曲菌共产生135种PFGE带型,40株结肠弯曲菌共产生34种PFGE带型,带型分布较为分散。结论2016—2018年北京市腹泻患者弯曲菌分离株的耐药率较为严重,耐药模式复杂,PFGE带型呈多态性分布。     

肉鸡屠宰加工过程中空肠弯曲菌污染的监测与分析

目的 调查分析浙江金华地区肉鸡屠宰加工过程中空肠弯曲菌的污染现状及规律。方法 选择金华地区6家肉鸡屠宰加工企业, 通过直接计数法对空肠弯曲菌定性定量检测, 分析其流行病学规律。结果 采集的2139份样品中, 泄殖腔、脱毛、取内脏、消毒预冷、包装和速冻等环节样品的空肠弯曲菌阳性率分别为92.48%、83.39%、98.12%、79.31%、86.21%和77.45%, 空肠弯曲菌阳性数分别为12926.14±1821.84 CFU/g、379.01±67.44、856.66±206.27、110.57±19.52、138.01±76.5、67.90±46.79 CFU/100 cm2。总体上, 空肠弯曲菌阳性率和阳性数均呈现出先升高、后降低、再升高的变化规律, 大型企业的空肠弯曲菌污染情况较中小型企业要好。结论 所检测的6家肉鸡屠宰加工企业均被空肠弯曲菌污染, 大型企业应重视包装环节的生产工艺和卫生消毒, 中小型企业更要严格把控包装环节的操作规范, 提高脱毛和消毒预冷工艺。