细菌生物膜与细菌耐药性研究进展

细菌生物膜是一种包裹于细胞外多聚物基质中的黏附于非生物或生物表面的微生物菌落。作为一种生存策略,绝大多数细菌在合适的条件下都会产生生物膜,生物膜状态下的细菌相对其游离状态有着更强的耐药性,是导致临床上出现难治性感染的重要原因之一。主要综述了生物膜的形成、耐药机制及抗生物膜的策略,以便寻找有效控制生物膜相关感染的手段,指导临床合理用药和新药开发。     

假单胞菌生物膜形成调控机制研究进展

假单胞菌是原料乳中常见的污染微生物,其会在乳品生产设备表面形成生物膜,导致设备腐蚀,且假单胞菌难以清除。同时假单胞菌形成的生物膜能促进假单胞菌耐热酶的分泌,还可以作为致病菌的生长基质,从而给乳品安全带来风险。本文总结了假单胞菌生物膜调控机制的研究进展和影响生物膜形成的环境因素,为假单胞菌生物膜的防控提供依据。     

链球菌生物膜形成中的群感效应

链球菌是一群种类多样的革兰氏阳性细菌,定殖在人的表皮、粘膜及牙齿表面。许多链球菌由感受态刺激肽（competence-stimulating peptide,CSP）介导的群感效应（quorum sensing,QS）与形成遗传转化的感受态有关。近期研究表明许多细菌的CSP与细胞密度适应性反应（如感受态与生物膜的形成）直接相关。本文综述了由群感效应介导生物膜形成的链球菌,充分阐明CSP功能,从感受态的形成到其它与细胞密度有关的表型,包括对生物膜形成的影响,以便对CSP的作用有新的理解,为研发新型抗生物膜感染药物、提出有效的生物膜感染防制策略提供理论基础。     

Biofilms: A role in recurrent mastitis infections?

Mastitis remains one the most important diseases in dairy cattle despite the progress made in improving general udder health in recent years. Epidemiological studies have revealed that following treatment with antimicrobials, bacteriological cure rates vary between 0% and 80% but with no evidence of a significant loss of activity of the major classes of antibiotics licensed for the treatment of bovine mastitis. Recurrent infections are often attributable to biofilm growth of bacteria and this review provides an overview of those mechanisms related to bacterial biofilm growth in mastitis. Biofilm formation is accompanied by significant genetic and subsequent physiological changes in the microorganisms resulting, inter alia, in a loss of sensitivity to virtually all classes of antibiotics.     

Nosocomial infections and bacterial antibiotic resistance in a university equine hospital

A base-line study of bacteria isolated from horses admitted to the Veterinary Medical Teaching Hospital during a 6-month period was performed to determine the extent of multiresistant nosocomial infections caused by gram-negative aerobic bacteria other than Salmonella spp. Results of this study indicated that 21.9% of the 105 horses from which cultures and sensitivities were available had developed nosocomial gram-negative aerobic infections, with high rates of resistance to gentamicin, kanamycin, and trimethoprim sulfadiazine, three of the most often prescribed antibiotics in this hospital. In addition, a prospective study of antibiotic-resistant bacteria of fecal origin was performed to determine whether there was a change in the degree of antibiotic resistance of a horse's intestinal flora while the horse was hospitalized. Bacterial culturing for gram-negative lactose fermenting bacteria was done on fecal specimens collected directly from the rectum on day 1 and day 7 of a horse's hospitalization. Susceptibility testing was done on each isolant. Of the 24 paired fecal specimens obtained, Escherichia coli and Klebsiella sp isolated on day 7 were resistant to a significantly higher number of antibiotics than day 1 isolants (P = 0.003, P = 0.043, respectively).     

ANTIMICROBIAL SUSCEPTIBILITY PATTERNS OF BIOFILM FORMING STAPHYLOCOCCUS AUREUS ISOLATED FROM PIGEON EXTERNAL OCULAR INFECTIONS

Pathogenic microorganisms are commonly associated with external ocular infections in birds. Pathogen virulence factors as well as reduced host defenses resulting from poor living conditions, nutrition, genetics, physiology, hygiene, fever, and age may increase host susceptibility. Staphylococcus species are bacteria known to serve as opportunistic pathogens in eye infections. The changing profile of microorganisms involved in ocular infections and the emergence of acquired microbial resistance dictate the need for investigative studies regarding bacterial profiles and antimicrobial susceptibility patterns for external ocular infections. The aim of this study was to determine the prevalence of Staphylococcus aureus ocular infections in pigeons and to evaluate their biofilm production ability and antibiotic resistance patterns. Twenty pigeons with confirmed eye infections were included in this project. Conjunctival specimens were collected with swabs presoaked in sterile normal saline. Bacterial growth was identified by standard laboratory procedures and susceptibility testing of the isolates was performed by the Kirby-Bauer method. The ability of the isolates to form a biofilm was also assessed using the microtiter plate method. Of the 20 specimens processed, 20% of the pigeons had staphylococcal eye infections. The resistance pattern of these isolates showed that Staphylococcus spp. from pigeon samples were resistant to tetracycline (100%), erythromycin (100%), azithromycin (100%), nalidixic acid (100%), and cefazolin (50%). All of the Staphylococcus spp. isolated from the pigeons were susceptible to gentamycin and furazolidone. The results of the biofilm detection test showed that 75% of the isolates were biofilm producers. In conclusion, biofilm forming S. aureus with multidrug resistance patterns were the most prevalent bacteria isolated from the pigeons examined in this research study.     

Les biofilms bactériens : leur importance en santé animale et en santé publique

Bacterial biofilms are structured communities of bacterial cells enclosed in a self-produced polymer matrix that is attached to a surface. Biofilms protect and allow bacteria to survive and thrive in hostile environments. Bacteria within biofilms can withstand host immune responses, and are much less susceptible to antibiotics and disinfectants when compared to their planktonic counterparts. The ability to form biofilms is now considered an attribute of many microorganisms. Diseases associated with biofilms require novel methods for their prevention, diagnosis and treatment; this is largely due to the properties of biofilms. Furthermore, the presence of biofilms on surfaces found at farms, slaughterhouses or food processing plants will have an impact on the efficacy of disinfection protocols. Surprisingly, biofilm formation by bacterial pathogens of veterinary or zoonotic importance has received relatively little attention. The objective of this brief Review article is to bring awareness about the importance of biofilms to animal health stakeholders.(Translated by the authors)     

In vitro evaluation of the impact of silver coating on Escherichia coli adherence to urinary catheters

A silver-coated urinary catheter was compared to a non-silver-coated urinary catheter for the ability to reduce adherence of 6 isolates of Escherichia coli. Catheters were incubated with E. coli strains for 0, 24, 48, and 72 h. Broth was sampled at all time points to determine CFU/mL. Catheters were subjected to sonication to determine adhered bacteria at all time points, and scanning electron microscopy (SEM) to semi-quantitatively assess biofilm formation. Silver-coated catheters had significantly less adhered bacteria than non-silver-coated catheters at times 24, 48, and 72 h. Subjectively, silver-coated urinary catheters had less biofilm formation than non-silver-coated urinary catheters as assessed by SEM. Silver coating of catheters was associated with reduced adherence of E. coli in an in vitro evaluation. Testing of catheters in dogs in vivo is required to determine if there is a reduction in catheter-associated urinary tract infections.     

金黄色葡萄球菌在生物被膜态与浮游态的转录组差异表达分析

金黄色葡萄球菌是引起奶牛细菌性乳腺炎的主要原因，生物被膜的形成是金黄色葡萄球菌在不利环境条件下持久性存在的关键因素。探索同一株菌在生物被膜态与浮游态生长状态下的耐药性与其生长状态的相关性，可为进一步探究金黄色葡萄球菌的耐药性机制奠定基础。本研究培养了金黄色葡萄球菌的生物被膜，使用光学显微镜和扫描电镜观察其形成过程。测定并比较了9种抗菌药物对32株金黄色葡萄球菌在生物被膜态和浮游态的最小抑菌浓度，并对两种状态下的金黄色葡萄球菌进行转录组学测序，筛选出具有显著性差异的细胞信号通路和表达基因，同时对主要差异表达的基因进行RT-qPCR验证。结果发现，在生物被膜形成前期，随着培养时间的延长，显微镜下观察到的生物被膜态菌聚集面积越来越大，结构也越来越紧密，培养至72 h后，生物被膜逐渐开始分散。MIC测定结果显示浮游态菌的抑菌浓度低于生物被膜态菌。转录组结果显示两种状态菌的差异表达基因共1 512个，其中，生物被膜态菌中上调基因760个，下调752个。GO与KEGG富集分析显示，相比于浮游态菌，生物被膜态菌中与代谢相关的通路显著富集，其次为氨基酸的生物合成和ABC转运蛋白通路。与生物被膜形成相关的基因，如编码ABC转运蛋白的基因表达上调，而与代谢途径相关的基因下调。RT-qPCR验证了10个主要差异基因，其表达差异趋势与转录组测序结果一致。这些差异可能对金黄色葡萄球菌生物被膜态的高耐药性和细菌毒力的研究有所帮助。     

Biofilm formation in Haemophilus parasuis: relationship with antibiotic resistance,serotype and genetic typing

Biofilms are surface-associated microbial communities, which are encased in self-synthesized extracellular environment. Biofilm formation may trigger drug resistance and inflammation, resulting in persistent infections. Haemophilus parasuis is the etiological agent of a systemic disease, Glässer's disease, characterized by fibrinous polyserositis, arthritis and meningitis in pigs. The purpose of this study was to examine the correlation between biofilm and antibiotic resistance among the clinical isolates of H. parasuis. In the present study, we tested biofilm-forming ability of 110 H. parasuis isolates from various farms using polystyrene microtiter plate assays. Seventy-three isolates of H. parasuis (66.4%) showed biofilm formation and most of them performed weak biofilm-forming ability (38/73). All isolates were tested for antimicrobial susceptibility to 18 antimicrobial agents by the broth microdilution method. H. parasuis isolates showed very high resistance (>90%) to sulfanilamide, nalidixic acid, and trimethoprim. Resistance to eight antibiotics such as penicillin (41.1% vs 8.1%), ampicillin (31.5% vs 8.1%), amoxicillin (28.8% vs 5.4%), gentamicin (46.6% vs 24.3%), cefazolin (19.2% vs 2.7%), doxycycline (19.2% vs 8.1%), cefotaxime (11% vs 2.7%), and cefaclor (13.7% vs 5.4%) was comparatively higher among biofilm producers than non-biofilm producers. Pulsed-field gel electrophoresis (PFGE) analyses could distinguish various isolates. Our data indicated that H. parasuis field isolates were able to form biofilms in vitro. In addition, biofilm positive strains had positive correlation with resistance to β-lactams antibiotics. Thus, biofilm formation may play important roles during H. parasuis infections.     

Biofilm formation in bacterial pathogens of veterinary importance

Bacterial biofilms are structured communities of bacterial cells enclosed in a self-produced polymer matrix that is attached to a surface. Biofilms protect and allow bacteria to survive and thrive in hostile environments. Bacteria within biofilms can withstand host immune responses, and are much less susceptible to antibiotics and disinfectants when compared with their planktonic counterparts. The ability to form biofilms is now considered a universal attribute of micro-organisms. Diseases associated with biofilms require novel methods for their prevention, diagnosis and treatment; this is largely due to the properties of biofilms. Surprisingly, biofilm formation by bacterial pathogens of veterinary importance has received relatively little attention. Here, we review the current knowledge of bacterial biofilms as well as studies performed on animal pathogens.     

抗菌肽与抗生素的体外抗菌效果比较

应用由 Hancock实验室改进的微量肉汤稀释法测定了 cecropin P1、cecropin A、magainin 2、defensin 1、bactenecin、lactoferricin和 indolicidin等 7种抗菌肽和盐酸金霉素、去甲万古霉素、土霉素、强力霉素等 4种抗生素的体外抗菌活性 ,并且使用薄层琼脂糖孔穴扩散法比较了抗菌肽中 cecropin P1和 cecropin A与土霉素和呋喃唑酮对大肠杆菌 K88的抑菌效果。结果发现 ,抗菌肽与抗生素一样对几种革兰氏阳性菌和阴性菌都有不同程度的抗菌效果。其中 cecropin A、cecropin P1和 defensin1对大肠杆菌的 2个菌株 ATCC2 5 92 2和 K88的抗菌活性高于抗生素 ,defensin1是各种抗菌肽中对金黄色葡萄球菌抗菌效果最好的。抗生素对试验用金黄色葡萄球菌的抑制活性总体上要好于抗菌肽。与抗生素相比 ,cecropin A、cecropin P1和 indolicidin对猪霍乱沙门氏菌和鼠伤寒沙门氏菌具有更好的抗菌效果。另外 ,抗菌肽的抑菌圈边缘十分清晰 ,而抗生素的整个抑菌圈都比较模糊且界线不明。因此 ,从抗菌效果方面考虑 ,抗菌肽可以代替抗生素用于疾病的预防和治疗     

大肠杆菌生物被膜与耐药性的关系

生物被膜的形成是大肠杆菌引起消化道反复难治性感染的重要因素。大肠杆菌形成生物被膜后使感染易于慢性化、控制困难,具有高度耐药性的同时还能逃避免疫系统的攻击和抗菌药物的杀伤作用。生物被膜的耐药机制主要包括营养限制、渗透障碍、表型结构学说等。现就大肠杆菌生物被膜的形成、耐药机制及其防治策略等研究现状做一综述。     

大肠埃希菌生物被膜研究进展

细菌生物被膜指多个细菌黏附于机体或物体表面,分泌胞外多聚物将其自身包裹其中而形成的结构。研究表明人类许多细菌感染与生物被膜有关,生物被膜具有极高的抗药性和免疫逃逸能力,这也是许多细菌感染难以根除的重要原因之一,近年来已成为医学界关注的热点。大肠埃希菌是最重要的条件致病菌之一,论文从大肠埃希菌生物被膜的形态结构、检测方法、耐药机制、应对策略4个方面综述了大肠埃希菌生物被膜研究的进展。     

Biofilm formation by field isolates and reference strains of Haemophilus parasuis

The ability to form biofilms for a total of 80 field isolates and 15 reference strains of Haemophilus parasuis, the etiological agent of Glasser's disease, was tested by glass tube and polystyrene microtiter plate assays. A total 43% of field isolates, including strains representing 13 serovars (except serovars 3 and 8) and non-typable strains, exhibited the ability to form biofilms at different levels via polystyrene microtiter plate assays. Among the reference strains representing 15 serovars, only serovars 2, 9, 12, 13 and 15 could not form biofilms on the polystyrene surface. A total of 85% of the strains forming biofilms at air-liquid interfaces in glass tubes also formed biofilms on polystyrene surfaces. Generally, non-virulent serovars showed a higher degree of biofilm formation than virulent serovars. The biofilm formation phenotype of most strains was maintained when cultures were passaged on agar and in broth. H. parasuis from the nasal cavities of pigs experimentally infected with biofilm-positive bacteria maintained the biofilm formation phenotype, whereas bacteria recovered from the lung and brain lost the ability to form biofilms. The biofilm-negative strains did not recover the ability to form biofilms via experimental infection. Our data indicate that most serovars of H. parasuis could form biofilms in vitro, and the biofilm formation phenotype is associated with the recovery site of the strains and is maintained when bacteria are passaged in vitro and in the upper respiratory tract.     

Biofilm bacteria: formation and comparative susceptibility to antibiotics

The Calgary Biofilm Device (CBD) was used to form bacterial biofilms of selected veterinary gram-negative and gram-positive pathogenic bacteria from cattle, sheep, pigs, chicken, and turkeys. The minimum inhibitory concentration (MIC) and minimum biofilm eradication concentration (MBEC) of ampicillin, ceftiofur, cloxacillin, oxytetracycline, penicillin G, streptomycin, tetracycline, enrofloxacin, erythromycin, gentamicin, tilmicosin, and trimethoprim-sulfadoxine for gram-positive and -negative bacteria were determined. Bacterial biofilms were readily formed on the CBD under selected conditions. The biofilms consisted of microcolonies encased in extracellular polysaccharide material. Biofilms composed of Arcanobacterium (Actinomyces) pyogenes, Staphylococcus aureus, Staphylococcus hyicus, Streptococcus agalactiae, Corynebacterium renale, or Corynebacterium pseudotuberculosis were not killed by the antibiotics tested but as planktonic bacteria they were sensitive at low concentrations. Biofilm and planktonic Streptococcus dysgalactiae and Streptococcus suis were sensitive to penicillin, ceftiofur, cloxacillin, ampicillin, and oxytetracycline. Planktonic Escherichia coli were sensitive to enrofloxacin, gentamicin, oxytetracycline and trimethoprim/ sulfadoxine. Enrofloxacin and gentamicin were the most effective antibiotics against E. coli growing as a biofilm. Salmonella spp. and Pseudomonas aeruginosa isolates growing as planktonic populations were sensitive to enrofloxacin, gentamicin, ampicillin, oxytetracycline, and trimethoprim/sulfadoxine, but as a biofilm, these bacteria were only sensitive to enrofloxacin. Planktonic and biofilm Pasteurella multocida and Mannheimia haemolytica had similar antibiotic sensitivity profiles and were sensitive to most of the antibiotics tested. The CBD provides a valuable new technology that can be used to select antibiotics that are able to kill bacteria growing as biofilms.     

Human health aspects of antibiotic use in food animals: a review

Antibiotic use not only selects for resistance in pathogenic bacteria, but also in the commensal flora of exposed individuals. Veterinary surgeons regularly prescribe antibiotics for food animals to treat bacterial infections just as doctors do for human patients. In addition, however, animal feeds contain added antibiotics not for therapy but for economic reasons: to enhance the growth rate of these animals. Several of the antibiotics used as growth promoters are analogues of and fully cross resistant with important antibiotics used in human medicine. As a result of this high exposure to antibiotics, the prevalence of resistant bacteria in the faecal flora of these animals is high. These resistant bacteria can be directly and indirectly, via foods of animal origin, transferred to humans and either colonize the human intestinal tract or exchange their resistance genes with commensal bacteria of humans. As the intestinal flora functions as a reservoir of resistance genes for pathogenic bacteria and because many bacterial species of the intestinal flora are potential pathogens, the efficacy of antibiotic therapy in human medicine may be jeopardized.     

Surface behaviour of S. Typhimurium,S. Derby,S. Brandenburg and S. Infantis

Cross-contamination due to Salmonella on the surface of processing equipment greatly contributes to contamination of pork products. Therefore, a clear understanding of surface and survival behaviour of relevant Salmonella serovars in pork processing environments is needed to develop better strategies for Salmonella control. Within this study the biofilm forming behaviour of S. Typhimurium, S. Derby, S. Brandenburg and S. Infantis isolates was analysed using the crystal violet assay. This assay, commonly used to analyse total biofilm formation, revealed variation in biofilm forming capacity between and within serovars. This has not been shown before for S. Derby, S. Brandenburg and S. Infantis. From each serovar, isolates with different biofilm forming capacity were selected to analyse biofilm formation on stainless steel. This revealed no significant differences between biofilm formation on polystyrene compared to stainless steel. Furthermore a relation was observed between biofilm forming capacity of an isolate and survival on stainless steel surfaces. On such surfaces, biofilms showed greater and longer survival than planktonic cells, and they were less susceptible to peracetic acid disinfection treatments. However, the latter effect was marginal and only observed in the presence of organic material, which drastically decreased the activity of peracetic acid. With the obtained results a hierarchical cluster was also performed to identify differences and similarities between the four different serovars. This indicated that the surface behaviour of S. Typhimurium was more comparable to S. Infantis than to S. Derby or S. Brandenburg.     

噬菌体在防控畜禽细菌性感染中的应用

近年来,因持续饲喂或滥用抗生素而引起的耐药性、药物残留和食品安全等问题日益严重,尤其是多重耐药和泛耐药菌的出现严重威胁着人类的健康。因此,寻找一种安全、高效的抗生素替代品是保障动物源食品安全、提高动物健康水平,及促进社会发展的必然需求。噬菌体是一类能够侵染细菌、真菌、放线菌等微生物的病毒总称,与传统抗生素相比,噬菌体具有存在广泛、特异性强、安全和研发成本低等优势。人类关于噬菌体的研究报道由来已久,但是随着抗生素和化学抗菌药物的大量出现,噬菌体在欧美等发达国家的研究一度中断,直至20世纪80年代,由于多重耐药菌的出现,人类又重新重视噬菌体的研究,随着现代生物技术的发展,噬菌体已广泛应用到人类疫病防控、动植物病原菌清除及保障食品安全等方面,已成为人类对抗细菌性感染的利器。作者简述了噬菌体生物学特性、噬菌体疗法优缺点,以及防控猪、鸡的大肠杆菌、沙门氏菌、弯曲杆菌等常见细菌性感染方面的研究,以期为动物生产应用提供理论依据。