环介导等温扩增法快速检测乳中阪崎肠杆菌

利用环介导等温扩增方法的快速、简便等优点,建立一种检测乳中阪崎肠杆菌的方法。以阪崎肠杆菌的OmpA序列为靶基因,设计特异性引物。优化并建立LAMP检测乳中阪崎肠杆菌的方法。结果表明,LAMP检测阪崎肠杆菌纯培养物的灵敏度为3.7×101cfu/mL,其灵敏度是PCR方法的10倍。人工污染阪崎肠杆菌灭菌乳的检测限为4.3×101cfu/mL。对23株致病菌进行特异性实验,特异性良好。该方法具有特异性强、灵敏度高、设备简便、耗时短等优点,在食品检测中具有良好的应用前景。      

基于全基因组测序的阪崎克罗诺杆菌婴幼儿食品分离株分子特征研究

目的 研究婴幼儿食品中分离的阪崎克罗诺杆菌(C.sakazakii)分子生物学特征。方法 对婴幼儿食品中C.sakazakii进行分离鉴定、药物敏感性试验和全基因组测序,利用BioNumerics软件对全基因组数据进行拼接组装,对组装基因组开展多位点序列分型(MLST)、核心基因组多位点序列分型(cgMLST)、毒力基因和耐药基因分析,并与PubMLST数据库中ST型进行比较分析。结果 本研究中分离的9株C.sakazakii共分为5个ST型,ST1和ST64型为主要型别,同时也是PubMLST数据库中C.sakazakii的主要型别。3株C.sakazakii携带mcr-9耐药基因,但药敏结果显示所有菌株对包括多粘菌素B和多粘菌素E在内的12种抗生素均敏感。除携带共同毒力基因谱外,ST1、ST64和ST458型携带脂多糖基因gtrB。cgMLST聚类分析显示,9株C.sakazakii呈高度多样性。结论 与临床分离株相关的ST1和ST64型是本研究食品分离株中的主要型别,提示C.sakazakii具有潜在的致病性,有必要对婴幼儿食品中C.sakazakii开展连续监测,为预防控制由其引起的食源性疾病提供依据。     

免疫磁分离和可视化金纳米杂交探针法快速检测婴幼儿配方乳粉中的阪崎克罗诺杆菌

利用免疫磁分离和金纳米杂交探针策略,开发一种快速和灵敏的可视化检测婴幼儿配方乳粉（powdered infant formula,PIF）中阪崎克罗诺杆菌（Cronobacter sakazakii）方法。抗体功能化的磁颗粒捕获C. sakazakii,金纳米探针对聚合酶链式反应的产物进行分析,这种探针法可以替代传统的电泳。免疫磁颗粒是由100?μL磁颗粒、120?μL碳化二亚胺盐酸盐、80?μL?C.?sakazakii单克隆抗体和体积分数0.02%的吐温-20制备。特异性检测中仅有C.?sakazakii是阳性结果。在纯培养基和不经预富集的人工污染PIF中,分别能检测到102?CFU/mL和103?CFU/g的C.?sakazakii。经过3?h的预富集,此方法可以在PIF中检测到低至4.5×101 CFU/g的C. sakazakii。可视化检测、电泳和紫外扫描光谱结果一致。因此,免疫磁分离和金纳米探针的方法可以替代凝胶电泳,对于快速检测C. sakazakii具有重要的意义。此方法将有助于检测PIF样本的C. sakazakii。     

Prevalence and Antimicrobial Resistance of Cronobacter sakazakii Isolated from Domestic Kitchens in Middle Tennessee, United States

Cronobacter sakazakii is an emerging opportunistic pathogen that has been implicated in cases of severe meningitis, sepsis, and necrotizing enterocolitis in premature and full-term infants. In this study, the prevalence of C. sakazakii was estimated in selected domestic kitchens in middle Tennessee. Antimicrobial resistance patterns of these C. sakazakii isolates were examined for points of public health significance. A total of 234 contact sites in 78 domestic kitchens were tested for C. sakazakii. Consumers' used dishcloths and cleaning sponges were also tested. Antimicrobial susceptibility of the identified C. sakazakii isolates was determined for 10 antimicrobial drugs by means of the disk diffusion method. C. sakazakii was recovered from 26.9% of domestic kitchens visited. Multidrug resistance was observed; the highest resistance was to penicillin (76.1% of isolates) followed by tetracycline (66.6%), ciprofloxacin (57.1%), and nalidixic acid (47.6%). None of the C. sakazakii isolates were resistant to gentamicin. These results suggest that antibiotic-resistant C. sakazakii could be present at various sites in domestic kitchens.     

Detection of Enterobacter sakazakii strains by real-time PCR

A precise 5' nuclease (TaqMan) real-time PCR was developed and validated in house for the specific detection of Enterobacter sakazakii isolates. Specifically designed nonpatented primers and a hydrolysis (TaqMan) probe were used to target the 16S rRNA gene. All 27 E. sakazakii and 141 non-E. sakazakii strains tested with the real-time PCR were identified correctly. To monitor false-negative results, an internal amplification control was coamplified with the same primers used for the E. sakazakii DNA. The detection probability of the assay was 56% when an E. sakazakii cell suspension containing 10(2) CFU/ml was used as template in the PCR (0.5 CFU per reaction) and 100% with a 10(3) CFU/ml suspension. This PCR assay should be very useful for the diagnostic detection of E. sakazakii in foods, especially powdered infant formula, after cultural enrichment.     

婴幼儿奶粉中阪崎克罗诺杆菌隐性污染状况分析及针对性检测技术研究进展

婴幼儿奶粉中存在的阪崎克罗诺杆菌对婴幼儿危害性极强,卫生标准中对该微生物有严格限制,但检出现象仍然时有发生。存在许多因素导致阪崎克罗诺杆菌在婴幼儿奶粉生产线中的潜伏,本文阐述了其在生产空间中的暴露特征,重点论述逆境胁迫下该菌耐受表型和活的非可培养表型形成的隐性污染对监测和清除污染残留的不利影响。讨论了各类检测技术的优缺点,强调检测灵敏度、回收率和区分活的非可培养状态菌对于控制隐性污染的重要性。以期为阪崎克罗诺杆菌的监测和防控提供新思路,为保障婴幼儿奶粉生产安全提供参考和建议。     

牛奶发酵过程中阪崎克罗诺杆菌可形成活的非可培养状态

该研究以保加利亚乳杆菌为牛奶发酵剂,研究了牛奶发酵过程中阪崎克罗诺杆菌与保加利亚乳杆菌的相互作用以及阪崎克罗诺杆菌存活量的变化情况,并结合16S rDNA高通量测序技术和PMA-qPCR方法分析阪崎克罗诺杆菌在牛奶发酵过程中是否形成“活的非可培养”状态。结果显示,保加利亚乳杆菌在牛奶中发酵48 h后pH值可达到3.40,酸度值可达212.00。在发酵前期接种阪崎克罗诺杆菌,48 h后pH值和酸度值分别为3.50和193.30;在发酵中期接种阪崎克罗诺杆菌,48 h后pH值和酸度值分别为3.47和214.30。发酵前期阪崎克罗诺杆菌的污染对保加利亚乳杆菌发酵结果的影响更大。阪崎克罗诺杆菌在牛奶中生长48 h后菌浓度可稳定在9.08 lg(CFU/mL);不论在牛奶经发酵前期或中期接种阪崎克罗诺杆菌,发酵后期平板计数法均检测不到阪崎克罗诺杆菌,但16s rDNA和PMA-qPCR技术均可检测到阪崎克罗诺杆菌活菌的存在。该研究结果说明,阪崎克罗诺杆菌在牛奶发酵后进入了“活的非可培养”状态,该状态的存在会导致该菌检测时活菌数量的低估,从而引发乳及乳制品的安全隐患。     

Evaluation of different methods for the detection and identification of Enterobacter sakazakii isolated from South African infant formula milks and the processing environment

Enterobactersakazakii is an emerging pathogen associated with life-threatening neonatal infections resulting from the consumption of contaminated powdered infant formula milk (IFM). Recent taxonomic analyses have determined that E. sakazakii comprises a number of genomospecies, and it has been proposed that E. sakazakii be reclassified as a novel genus, "Cronobacter". Accurate methods are required for the rapid detection and identification of this group of micro-organisms, since even low cell numbers have been reported to cause disease. The aim of this study was to evaluate various E. sakazakii detection methods in order to ascertain the most suitable method for detection and identification of these pathogenic agents. Samples from IFM and the environment were evaluated for the presence of E. sakazakii using the isolation steps (pre-enrichment, enrichment and selection) described in the Food and Drug Administration (FDA) method for E. sakazakii detection. Sixty-four isolates (50 from IFM and 14 from the environment) were selected from tryptone soy agar (TSA), regardless of colony appearance, and these isolates were identified by 16S ribosomal DNA (rDNA) sequencing. Thereafter, different culture-dependent and culture-independent methods were evaluated to accurately detect and identify the E. sakazakii isolates. These methods included the assessment of yellow pigment production on TSA, typical colonies on chromogenic Druggan-Forsythe-Iversen (DFI) and Chromocult(R) Enterobacter sakazakii (CES) media and polymerase chain reaction (PCR) using six different species-specific primer pairs described in the literature. Identification of E. sakazakii using yellow pigment production was demonstrated to have a low sensitivity, specificity and accuracy (87%, 71% and 74%, respectively), which lowers the suitability of the FDA method. Chromogenic DFI and CES media were sensitive, specific and accurate (100%, 98% and 98%, respectively) for the detection of E. sakazakii. The specificity of the PCR amplifications ranged from 8% to 92%, emphasising the need for rigorous primer testing against closely related species. Of the primer pairs evaluated, Esakf/Esakr were the most suitable for E. sakazakii detection and identification. The detection limit of Esakf/Esakr was found to be 10(4) CFU/ml. This study demonstrated that no single method was capable of unambiguously confirming the presence and identity of E. sakazakii isolates, that each method had inherent advantages and disadvantages, and that in most cases several methods were required for accurate detection and identification. Further, it was demonstrated that the current FDA method for E. sakazakii detection should be revised in the light of the availability of more sensitive, specific and accurate detection methods.     

Characterization of a collection of Enterobacter sakazakii isolates from environmental and food sources

Enterobacter sakazakii has emerged as a rare cause of neonatal meningitis, septicemia and enterocolitis. Contaminated infant milk formula (IMF) has been identified as one infection route. A small number of clinical outbreaks have been epidemiologically linked to IMF contaminated post-pasteurization during manufacture and/or mishandled when reconstituted. Currently no agreed standardized typing protocol has been developed to trace E. sakazakii. The objectives of this study were to apply biochemical and genetic methods to characterize 51 environmental and food E. sakazakii isolates and 6 E. sakazakii type strains. Isolates were presumptively identified using biochemical profiles based on API 20E and ID32E methods and by culture on differential selective Druggan Forsythe Iversen (DFI) agar. Identification was subsequently confirmed by real time polymerase chain reaction (PCR). All but one of the isolates was identified as E. sakazakii by biochemical profiling. One isolate was identified as Escherichia vulneris by ID 32E and as Pantoea agglomerans by API 20E. All isolates produced green/blue colonies on DFI medium characteristic of this organism. Real time PCR could differentiate between E. sakazakii, Enterobacter spp. and other Enterobacteriacae. Analysis of RAPD banding patterns revealed 3 major clusters of E. sakazakii. There was a large degree of diversity noted amongst the remaining isolates. Our findings indicate that RAPD may be applied as a useful and reliable tool for direct comparison of E. sakazakii isolates providing traceability through the infant formula food chain.     

THERMAL RESISTANCE, SURVIVAL AND INACTIVATION OF ENTEROBACTER SAKAZAKII (CRONOBACTER SPP.) IN POWDERED AND RECONSTITUTED INFANT FORMULA

Enterobacter sakazakii has recently been recognized as an opportunistic foodborne pathogen, and dry infant formula serves as the mode of transmission. The objectives of this study were to investigate the heat resistance, survival and inactivation under room and refrigeration temperatures storage of dry and reconstituted infant formula milk (IFM). E. sakazakii strains (eight strains) showed a wide variability in heat resistance at different temperatures (55, 60 and 63C). The D-values at 55C ranged from 1.51 to 14.83 min, at 60C from 0.17 to 2.71 min and at 63C from 0.05 to 0.88 min. The calculated z values for the studied E. sakazakii strains ranged from 3.76–10.11C. Microwave oven heating of 60-mL portions of reconstituted IFM for 40–50 s was effective in eradicating inoculated E. sakazakii. Storing powdered IFM for 15 days at 4C resulted in at least a 1-log reduction in E. sakazakii strains, whereas storing reconstituted IFM at 4C for 2 weeks resulted in more than a 2-log reduction in E. sakazakii.

PRACTICAL APPLICATIONS

This study shows that E. sakazakii strains differ widely in their heat resistance. No differences were observed between biofilm formers and nonformers in terms of heat-resistance in thermal inactivation kinetics experiments. Conventional high temperature short-time pasteurization processes are considered sufficient to inactivate all E. sakazakii strains, and a household microwave oven (40–50 s for 60-mL portions) can be used to inactivate E. sakazakii if present in reconstituted infant formula milk (IFM). Growth of E. sakazakii can be inhibited in powdered and reconstituted IFM by refrigeration. Also, it is recommended that reconstituted IFM be discarded or refrigerated if not immediately consumed. The probiotic L. acidophilus ATCC 4356 was not effective in inhibiting E. sakazakii in powdered or reconstituted IFM.     

Characterization of the gene encoding the 16S rRNA of Enterobacter sakazakii and development of a species-specific PCR method

The gene encoding the 16S rRNA of Enterobacter (E.) sakazakii (ATCC 29544, plus four strains isolated from powdered infant formula) was studied, and the sequence compared with those of other Enterobacteriaceae in aspects of genetic variability. Sequence differences between E. sakazakii and other Enterobacteriaceae within the hypervariable regions V1, V2, and V3, respectively, were used to develop two PCR methods for E. sakazakii. PCR1 employed a primer pair located in V1/V2, while PCR2 utilized a primer pair located in V1/V3, respectively. The two PCR methods were tested against a set of 57 E. sakazakii and 148 non-E. sakazakii isolates. PCR1 gave an amplicon with a size of 406 bp and resulted in 100% positive results for E. sakazakii, but also detected Citrobacter koseri/amalonaticus and all nine tested Salmonella enterica serovars. In contrast, PCR2 (amplicon size of 952 bp) gave positive results only for E. sakazakii, thus allowing specific identification of this species.     

原儿茶醛对阪崎克罗诺肠杆菌毒力因子的抑制作用

探究植物源活性物质原儿茶醛对阪崎克罗诺肠杆菌泳动运动能力、生物被膜形成能力、黏附和侵入Caco-2细胞能力及9 种与菌体相关毒力基因转录的影响。结果表明,原儿茶醛对6 株阪崎克罗诺肠杆菌实验菌株的最小抑菌质量浓度（minimal inhibitory concentrations,MIC）和最小杀菌质量浓度均为2 mg/mL,对阪崎肠杆菌ATCC29544亚抑制浓度分别为1/200、1/100 MIC和1/50 MIC。亚抑制浓度的原儿茶醛能够抑制阪崎克罗诺肠杆菌在琼脂软平板中泳动运动能力;减弱菌体在12 ℃和25 ℃下不同时间段（24、48、72 h）生物被膜的形成能力;降低细菌黏附及侵入Caco-2细胞能力;下调9 种与菌体相关毒力基因的转录量。研究结果表明原儿茶醛能够抑制阪崎克罗诺肠杆菌多种毒力因子,其有潜力作为抗生素补充剂或抗毒性物质预防及控制阪崎克罗诺肠杆菌引起的相关感染。     

免疫富集联合MALDI-TOF MS检测奶粉中阪崎克罗诺杆菌的方法建立

目的:制备高效且特异性好的阪崎克罗诺杆菌抗体及其免疫磁珠,建立免疫富集联合基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption/ionization time of flight mass spectrometry, MALDI-TOF MS)检测奶粉中的阪崎克罗诺杆菌的方法。方法:制备阪崎克罗诺杆菌混菌抗体及其免疫磁珠,对免疫磁珠分别在纯培养及奶粉基质中的捕获率进行研究,利用MALDI-TOF MS对不同奶粉基质中不同杂菌污染条件下的检测样本进行鉴定并验证免疫磁珠的特异性。结果:阪崎克罗诺杆菌免疫磁珠在纯培养条件及奶粉基质中对4株阪崎克罗诺杆菌及其混菌的捕获率均>80%,联合MALDI-TOF MS鉴定结果显示在奶粉基质中不同杂菌污染条件下具有较好的鉴定结果,即使在高比例（1:100）杂菌污染条件下,仍能准确鉴定奶粉中阪崎克罗诺杆菌,此时检测样品中阪崎克罗诺杆菌浓度仅为20 CFU/mL。结论:本研究建立了一种操作简便、鉴定结果准确的免疫富集联合MALDI-TOF MS检测奶粉中阪崎克罗诺杆菌的方法,为奶粉中阪崎克罗诺杆菌的快速准确鉴定提供了新的方法参考。     

Effects of extended dry storage of powdered infant milk formula on susceptibility of Enterobacter sakazakii to hot water and ionizing radiation

Infant milk formula has been identified as a potential source of Enterobacter sakazakii, which has been implicated in neonatal meningitis and necrotizing enterocolitis. This study was undertaken to determine whether the length of E. sakazakii storage in powdered infant milk formula (PIMF) affected the ability of the pathogen to survive subsequent reconstitution of the powder with hot water or treatment with gamma radiation. Five E. sakazakii strains were mixed individually with PIMF and kept for up to 12 months at 25 degrees C. After storage PIMF was reconstituted with water at 60 to 100 degrees C or was exposed to < or = 5 kGy of gamma radiation. Without any treatment secondary to drying, E. sakazakii counts decreased < 1 log/g after 1 month but decreased about 4 log/g during storage for 8 to 12 months. Dry storage decreased thermal resistance but increased resistance of E. sakazakii to ionizing radiation in PIMF. Reconstitution of contaminated powder with water at 70 degrees C after 1 month of dry storage reduced E. sakazakii viability slightly, > 2 log/g, and after powder was stored for 12 months all E. sakazakii strains were eliminated. In contrast, desiccation substantially increased the resistance of E. sakazakii strains to ionizing radiation. Although the D-value for E. sakazakii IMF1 following overnight storage in PIMF was 0.98 kGy, > 4 kGy was required to kill 1.5 log/g of the same strain that had survived 12 months in dry PIMF. Results suggested that low-dose irradiation will more effectively eliminate E. sakazakii from PIMF if the treatment is applied shortly after PIMF manufacture.     

Growth Kinetics and Model Comparison of Cronobacter sakazakii in Reconstituted Powdered Infant Formula

Cronobacter sakazakii is a life-threatening bacterium, infrequently implicated in illnesses associated with the consumption of powdered infant formula (PIF). It can cause rare but invasive infections in neonatal infants who consume contaminated PIF. The objective of this research was to investigate the growth kinetics and develop mathematical models to predict the growth of heat-injured C. sakazakii in reconstituted PIF (RPIF). RPIF, inoculated with a 6-strain cocktail of non-heat-treated (uninjured) or heat-injured C. sakazakii, was incubated at different temperatures to develop growth models. Except for storage at 6 °C, C. sakazakii grew well at all test temperatures (10 to 48 °C). Uninjured C. sakazakii exhibited no observable lag phase, while a lag phase was apparent in heat-treated cells. A simple 3-parameter logistic equation was used to fit growth curves for non-heat-treated cells, while both Baranyi and Huang models were suitable for heat-treated C. sakazakii. Calculated minimum and maximum growth temperatures were 6.5 and 51.4 °C for non-heat-treated cells, and 6.9 and 50.1 °C for heat-treated cells of C. sakazakii in RPIF, respectively. There was no significant difference between growth rates of non-heat-treated and heat-injured cells in RPIF. For heat-treated cells of C. sakazakii, the lag phase was temperature-dependent and very short (between 25 °C and 48 °C). These results suggest that both non-heat-treated and heat-injured C. sakazakii cells may present a risk to infants if the pathogens are not completely destroyed by heat in RPIF and then exposed to subsequent temperature abuse. Practical Application: C. sakazakii is a life-threatening bacterium found in powdered infant formula (PIF). This study shows that the uninjured bacterium exhibits very short or no lag phase if not refrigerated and can grow well in reconstituted PIF (RPIF), while the heat-injured cells can multiply at an equivalent rate following metabolic recovery. Temperature abuse may allow C. sakazakii to grow and endanger infants fed with RPIF. Predictive models developed in this study can be used to estimate the growth and conduct risk assessments of this pathogen.     

奶粉中坂崎肠杆菌检测方法研究进展

坂崎肠杆菌为肠杆菌科肠杆菌属，奶粉（尤其是婴幼儿配方奶粉）中坂崎肠杆菌的污染可引发严重的公共卫生问题，其主要检测方法包括传统的细菌学培养方法及各种基于聚合酶链式反应（PcR）的分子生物学检测方法，改进的培养方法和快速特异的分子生物学方法已成为坂崎肠杆菌检测的重要发展方向。     

Enterobacter sakazakii: infectivity and enterotoxin production in vitro and in vivo

Enterobacter sakazakii has been implicated as the causal organism in a severe form of neonatal meningitis, with reported mortality rates of 40 to 80%. Dried infant formula has been identified as a potential source of the organism in both outbreaks and sporadic cases. In this study, clinical and foodborne isolates of E. sakazakii were evaluated for enterotoxin production by the suckling mouse assay. In addition, suckling mice were challenged both orally and by intraperitoneal injection. Of 18 E. sakazakii strains evaluated, four were found to test positive for enterotoxin production. All strains of E. sakazakii were lethal to suckling mice at 10(8) CFU per mouse by intraperitoneal injection, while two strains caused death by the peroral route. In in vitro assays, CHO, Vero, and Y-1 cells demonstrated both cell lysis and rounding when exposed to E. sakazakii strain LA filtrates. This is the first report describing any putative virulence factors of E. sakazakii.     

Development of an immobilization and detection method of Enterobacter sakazakii from powdered infant formula

Enterobacter sakazakii is an emerging opportunistic pathogen that is associated with rare but life-threatening cases of meningitis, necrotizing enterocolitis, and sepsis in premature and full-term infants. In the present study, a procedure was developed for immobilization of E. sakazakii with zirconium hydroxide coupled with detection by a species-specific duplex PCR, based on 16s-23s rDNA internal transcribed spacer (ITS) and ompA gene. Specificity of duplex PCR was tested against two-type strains, six isolates of E. sakazakii and other eight non-E. sakazakii species. When pure culture of E. sakazakii was used for immobilization, total recovery rate ranged from 79.4% to 99.6% of input bacteria, and the detection limit of duplex PCR was 3x10(5)CFU/ml. Different levels of E. sakazakii were inoculated into 90ml reconstituted powdered infant formula (PIF), and detection limit of duplex PCR was 3x10(0)CFU/ml with 24-30h enrichment after immobilization. When the experiment was performed in the presence of 10(2)CFU/ml Salmonella typhimurium, the detection limit of duplex PCR was not affected after enrichment. Seven out of 13 commercial PIF were detected positive by duplex PCR after immobilization, while only three were positive by biological methods. This study demonstrates that the combination of immobilization method with duplex PCR is easy, rapid, and efficient, and may have applications for the detection of E. sakazakii in more PIF samples.     

百里酚和香芹酚对阪崎克罗诺肠杆菌的抑制作用

为探究百里酚和香芹酚对阪崎克罗诺肠杆菌的抑制作用及机理。测定百里酚、香芹酚对阪崎克罗诺肠杆菌最小抑菌浓度(MIC),研究两种物质对阪崎克罗诺肠杆菌生长曲线、膜电位、胞内pH、胞内ATP、细胞膜完整性的影响,利用场发射扫描电镜观测细胞形态的变化。结果表明:百里酚和香芹酚对阪崎克罗诺肠杆菌具有良好的抑制作用,两者对9株阪崎克罗诺肠杆菌的最小抑菌浓度均在0.1~0.2 mg/mL之间。百里酚和香芹酚均可使阪崎克罗诺肠杆菌细胞膜出现超极化,浓度为4MIC的百里酚和香芹酚分别使菌株的胞内pH由5.45降低至4.96和4.97,胞内ATP浓度由0.584降低为0.048和0.027 μmol/L,细胞膜完整性分别下降85.0%和88.1%。场发射扫描电镜结果显示,百里酚和香芹酚均能够导致菌体出现干瘪皱缩,细胞膜出现孔洞甚至裂解。结果显示,百里酚和香芹酚对阪崎克罗诺肠杆菌均具有良好的抑制效果,其作用机理可能与细胞膜的通透性及细胞形态有关。百里酚和香芹酚虽然互为同分异构体,但二者对阪崎克罗诺肠杆菌的抑制作用并未表现出明显的规律性差异。