Alpha- and β-casein components of host milk induce biofilm formation in the mastitis bacterium Streptococcus uberis

Streptococcus uberis is an environmental udder pathogen that infects cattle and can cause persistent intramammary infection (IMI), despite the fact that isolates are mainly susceptible to antibiotics. As biofilm growth can cause persistent infection, the ability of ten S. uberis isolates from clinical and subclinical IMIs to form biofilms on the polystyrene surface of a conventional 96-microplates model was examined. Biofilm formation was judged by different staining methods (crystal violet and resazurin) and by atomic force and fluorescence microscopy. These analyses revealed that two out of ten S. uberis strains tested were able to form biofilms. Upon treatment with Proteinase K, biofilms of S. uberis were completely disintegrated, which indicates that biofilm formation is protein-mediated in these strains. Addition of trace amounts of milk, the natural growth medium of S. uberis, significantly increased biofilm formation by most of the strains initially classified as non-biofilm producers. Alpha-casein and β-casein were the primary inducers of biofilm growth, and casein degradation by serine protease activity was required to achieve maximal biofilm production. These results suggest that the extracellular proteolytic activity of S. uberis contributes to an increased biofilm formation. Such a mode of growth induced by host proteins might help to explain the persistence of IMIs caused by this pathogen.     

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 formation is prevalent among field isolates of Actinobacillus pleuropneumoniae

A total of 77 field isolates and 15 reference strains of the porcine respiratory pathogen Actinobacillus pleuropneumoniae were tested for their ability to form biofilms in a polystyrene microtiter plate assay. More than half of all field isolates, which included strains representing serotypes 1, 5 and 7, but only two reference strains (serotypes 5B and 11) exhibited biofilm formation. Strains that formed biofilms in microtiter plates also formed thick biofilms at the air-liquid interface when cultured in glass tubes with agitation. The biofilm formation phenotype was maintained indefinitely when cultures were passaged on agar but was lost after one or two passages in broth. Our findings indicate that biofilm formation is a prevalent phenotype among A. pleuropneumoniae field isolates, and that this phenotype may have been previously overlooked because of its tendency to be lost upon subculturing in broth. Biofilm formation may have relevance to the colonization, pathogenesis and transmission of this bacterium.     

不同培养条件对鸭源鸡杆菌生物被膜形成的影响

采用体外定量法检测20株鸭源鸡杆菌的被膜形成能力,并对被膜形成能力较强的1株鸭源鸡杆菌在不同环境（培养时间、培养基质、营养条件）下产生生物被膜（BF）的差异进行了研究。结果显示,大部分鸭源鸡杆菌均具有不同程度形成被膜的能力,其中3株被膜形成能力中等,2株形成能力较强;鸭源鸡杆菌在银染法、试管法和微量板定量检测法中形成BF的最佳培养时间分别是24、60、24h。其生长周期为8h开始起始黏附、20h大量黏附阶段、24h时形成了完整的生物被膜、32h生物被膜老化散播,之后起始新一轮的被膜周期。葡萄糖、蔗糖及NaCl的加入均在一定程度上抑制了被膜的形成,且随着加入量增加NaCl对被膜形成抑制更显著,而低质量浓度的MgSO4在一定程度上促进BF的形成。扫描电镜观察发现,生物被膜内鸭源鸡杆菌聚集成团状、相互黏连形成致密的三维立体结构。     

禽源肠炎沙门菌aroA基因缺失株的构建及其生物学特性研究

试验旨在研究aroA基因对肠炎沙门菌生物学特性及毒力的影响。利用λ-Red同源重组技术，将从田间分离的禽源肠炎沙门菌的aroA基因进行敲除，经连续传代检测缺失株遗传稳定性，并通过细菌生长曲线、对不同生化反应管的利用情况、生物膜形成能力、对环境应激的抵抗能力及对1日龄雏鸡的毒力比较野生株和基因缺失株之间的差异。结果显示，试验成功构建缺失株，连续传30代未发现目的基因回复突变；在相同培养条件下，缺失株与野生株在相同时间进入对数生长期和稳定期，但缺失株生长速率低于亲本株；缺失株较野生株生化特性未发生改变；缺失株的生物膜形成能力及对酸、碱、高渗和热应激的抵抗能力均显著低于野生株（P<0.05）；1日龄雏鸡分别滴口接种缺失株和野生株，观察14 d，野生株组鸡全部死亡，而缺失株组无死亡。综上所述，本试验成功构建并获得了具有良好遗传稳定性的肠炎沙门菌aroA基因缺失株，缺失株生化特性和体外生长趋势未发生明显改变，但生物膜形成能力、对环境应激抵抗能力及毒力均明显下降。本研究为进一步研究肠炎沙门菌aroA基因缺失株的免疫原性奠定了基础。     

纳米银对猪链球菌体外抗菌活性及其生物膜形成的影响

【目的】研究纳米银(silver nanoparticles, AgNPs)对多重耐药猪链球菌(Streptococcus suis,S.suis)的体外抗菌活性及其生物膜形成的影响,为解决猪链球菌的耐药问题提供参考。【方法】以5株临床分离的多重耐药猪链球菌(S1018、S894、S786、S815和S844)为研究对象,采用微量肉汤稀释法测定AgNPs对5株猪链球菌的最小抑菌浓度(minimum inhibitory concentration, MIC)和最小杀菌浓度(minimum bactericidal concentration, MBC),通过测定AgNPs作用下细菌的生长速率以及不同浓度AgNPs对猪链球菌的抑制率来评价AgNPs的抗菌活性;通过结晶紫染色法测定各菌株生物膜的形成能力和6.25、12.5、25、50μg/mL AgNPs对5株多重耐药猪链球菌菌株生物膜形成的影响。【结果】AgNPs对S1018株的MIC和MBC均为12.5μg/mL,对其余4株多重耐药猪链球菌的MIC和MBC均为25.0μg/mL;AgNPs对多重耐药猪链球菌的抗菌活性好,25～100μ...     

Identification of genes responsible for biofilm formation or virulence in Salmonella enterica serovar pullorum

Salmonella living in biofilms are more resistant to chemical and physical stresses. However, information regarding the regulation of genes involved in biofilm formation for Salmonella enterica serovar Pullorum remains limited. In this study, eight mutants with knockout of genes ompR, rpoS, rfaG, rfbH, rhlE, metE, spiA, or steB from the Salmonella enterica serovar Pullorum strain S6702 were constructed. Phenotypic analysis revealed that all mutants were similar to the wild-type strain in growth rate. Only the ompR mutant showed a complete loss of production ofcurli and biofilm formation. The other mutants showed a modified production of curli and cellulose with less effect related to biofilm formation. The results of animal experiments indicated that the deletion of genes ompR, spiA, rfaG, or metE in wild-type strains contributed to attenuation of virulence in 1-day-old chickens. This study may bring new insights into novel vaccines or therapeutic interventions against Salmonella enterica serovar Pullorum infections.     

The etiology of foul brood]

Five hundred and thirty-six samples of honeycombs were examined in a laboratory in the years 1971-1974. In all the samples clinically determined as the foul brood, B. alvei was isolated as a pure culture, and enterococci, or both microorganisms were isolated in mixed form. Twenty-five strains of the isolated streptococci were analyzed microbiologically and biochemically; on the basis of their culture and biochemical characteristics five strains were designated as Streptococcus faecalis, 14 strains as Streptococcus faecalis var. liquefaciens, five strains as Streptococcus faecium and one strain as Streptococcus durans. After checking the used taxonomic key of the culture and biochemical classification of B. alvei it may be stated that the culture and biochemical characteristics are stable. The strains of B. alvei (very dried strains), which persisted in the dried slant meat-peptone agar, were viable under the laboratory conditions, which proves the high resistance of the spores to the environment.     

Pseudomonas aeruginosa in raw and pasteurized milk

Eighteen strains of Pseudomonas aeruginosa, i.e. 8.87%, were isolated during a year from 203 samples of raw milk. Two Pseudomonas aeruginosa strains, i.e. 4%, were isolated from 50 samples of pasteurized milk. The strains were isolated using propagation techniques in meat-peptone broth with malachite green and on selective media--on centrimide agar (CEM) and on Pseudomonas F agar. All the isolated strains produced protease, whereas lipase was produced by only five strains. The strains were devitalized when exposed to pasteurization temperatures (72 degrees C) for 20 seconds. At cold store temperatures (4 degrees C), Pseudomonas aeruginosa strain cells propagated on average by two orders, inhibitory effects of low temperatures were recorded only with one strain. Inhibitory effects of milk cultures (cream, yogurt) on Pseudomonas aeruginosa were observed; their effects were more clear-cut at the temperature of 4 degrees C. The strains were markedly susceptible to gentamycin.     

Effect of temperature on the growth of Penicillium from moldy feed

178 Penicillium strains were isolated from moulded feedstuffs (mainly corn and wheat after ambient air drying, wheat after refrigeration, corn after treatment with propionic acid), and investigated for the effect of temperature on growth rate. Temperatures between 4 and 27 degrees C were used. 159 strains proved psychrotrophic, they were able to grow at 4 degrees C on malt extract agar (MA) as well as in wheat; the growth in wheat was indicated by the production of ergosterol. On MA all psychrotrophic strains were able to grow also at 10-27 degrees C. The maximum rate of growth was reached at 10-15 degrees C, 15-20 degrees C, 20-27 degrees C, or must be assumed for temperatures greater than or equal to 27 degrees C. At 4 and 10 degrees C the mean growth rate on MA was 24 and 51%, respectively, related to the mean rate at 20 degrees C; the lower temperature limit of growth was found at - 1 degree C by graphic extrapolation. The Q10 value of growth on MA is about 2, if temperatures between 20 and 10 degrees C are compared; with decreasing temperature higher Q10 values up to 3.7 are obtained. The growth rate of a strain of Penicillium aurantiogriseum on wheat was affected by temperature nearly in the same manner as on malt extract agar. The results are discussed with respect to the risk of moulding during refrigerated storage of feedstuffs.     

Characterization of Mannheimia haemolytica biofilm formation in vitro

Mannheimia haemolytica is the primary bacterial agent in the bovine respiratory disease complex. It is thought that M. haemolytica colonizes the tonsillar crypts of cattle as a commensal and subsequently descends into the lungs to cause disease. Many bacterial species persist in the host as biofilms. There is limited information about the ability of M. haemolytica to form biofilms. The aim of this study was to develop an in vitro model for M. haemolytica biofilm formation. We found that M. haemolytica required at least 36 h to form robust biofilms on plastic in vitro when incubated in RPMI-1640 tissue culture medium at 37 °C, with maximal biofilm formation being evident at 48 h. Biofilm formation was inhibited by adding the monosaccharides d(+) galactose and d(+) mannose to the growth medium. Addition of antibodies to the M. haemolytica surface protein OmpA also reduced biofilm formation. Upon evaluating the macromolecules within the biofilm extracellular polymeric substance we found it contained 9.7 μg/cm² of protein, 0.81 μg/cm² of total carbohydrate, and 0.47 μg/cm² of extracellular DNA. Furthermore, proteinase K treatment significantly decreased biofilms (P < 0.05) while α-amylase and micrococcal nuclease decreased biofilms to a lesser extent. M. haemolytica biofilm cells were more resistant than planktonic cells to the antibiotics florfenicol, gentamicin, and tulathromycin. These results provide evidence that M. haemolytica can form biofilms, which could contribute to its ability to persist as a commensal in the bovine upper respiratory tract.     

Comparison of the biological properties of various strains of Aujeszky's disease viruses

We compared three strains of Aujeszky's disease virus that had been isolated from slaughter pigs from the dormant foci of Aujeszky's disease, with the known virulent strain CVOS and TOP. No significant differences were found out in the following characters: titer value on tissue cultures from chick embryonal cells (KEB) and cellular line MDBK at the temperatures of 37 degrees C and 40 degrees C, cytopathogenic effect (CPE) in the HeLa cells, plaque formation in the KEB tissue culture, pruritus rise and the time of rabbit death after infection. Assessing the results of the comparison, the three new-isolated strains of Aujeszky's disease virus can be evaluated as strongly virulent.     

Polyacrylamide gel electrophoresis of whole-cell preparations of Rhodococcus equi.

The whole-cell proteins of ten strains of Rhodococcus equi isolated from horses, pigs, or humans, including the type strain ATCC 6939, were examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The protein profiles of seven different capsular serotypes and the type strain were very similar when bacteria were cultured under the same conditions. Protein profiles were largely unaffected by incubation at two temperatures (30 degrees C, 37 degrees C) or times (12 h, 48 h). There were generally minor differences in protein profiles between strains grown in different media (brain heart infusion, nutrient, minca broths, tryptic soy-blood agar) with the marked exception of a prominent diffuse 17.5 kd protein which was expressed in nutrient broth. This protein was not produced by the type strain and was lost on repeated passage in vitro (50th, 100th passage) in two of three other strains examined.     

Biofilms and their relevance to veterinary medicine

Bacteria are renowned for their ability to tolerate and adapt to a wide range of adverse environmental conditions. The primary mechanism that facilitates these adaptations is thought to be the capacity to form and maintain biofilms. Within a biofilm, bacteria become attached to a surface where they exist in complex communities which are able to interact with each other through intracellular communication and thus rapidly adapt to changing environments. The organisms within biofilms are notorious for their resistance towards the host immune response and antibacterial agents compared to their free-living planktonic counterparts. Consequently, biofilms are of significant importance to both clinical and veterinary science. However, although bacterial infections are widely reported in animals their association with biofilms is rarely discussed. The aim of this review is to look at the characteristics of biofilm infections in humans and to relate this knowledge to veterinary science in order to assess their relevance in this area.     

Flagellin and F4 fimbriae have opposite effects on biofilm formation and quorum sensing in F4ac+ enterotoxigenic Escherichia coli

Bacteria that form biofilms are often highly resistant to antibiotics and are capable of evading the host immune system. To evaluate the role of flagellin and F4 fimbriae on biofilm formation by enterotoxigenic Escherichia coli (ETEC), we deleted the fliC (encoding the major flagellin protein) and/or the faeG (encoding the major subunit of F4 fimbriae) genes from ETEC C83902. Biofilm formation was reduced in the fliC mutant but increased in the faeG mutant, as compared with the wild-type strain. The expression of AI-2 quorum sensing associated genes was regulated in the fliC and faeG mutants, consistent with the biofilm formation of these strains. But, deleting fliC and/or faeG also inhibited AI-2 quorum sensing activity.     

Development of a molecular diagnostic test applied to experimental abattoir surveillance on bovine tuberculosis

Biofilm formation where bacterial cells adhere to a surface and surround themselves in a polysaccharide matrix is thought to be an important factor in disease initiation and persistence for many bacterial species. We have examined biofilm formation by Mycoplasma mycoides subsp. mycoides small colony using a simple model without an air/liquid interface and have found that adherent Mmm SC was more resistant to many stresses, including heat, osmotic shock and oxidative stress. Biofilms of Mmm SC also exhibited remarkable persistence and were able to survive for up to 20 weeks in stationary phase. Significant variation was seen between Mmm SC strains in their ability to form a biofilm and the morphology of the biofilm produced with some strains unable to produce microcolonies. Proteomic analysis found that a number of proteins linked to adherence were over-expressed in biofilms compared with planktonic cells.     

致羔羊脑炎粪肠球菌生物被膜形成能力及相关基因检测研究

为观察致羔羊脑炎粪肠球菌XJ05株生物被膜（BF）的形成能力并检测致羔羊脑炎粪肠球菌BF形成相关基因的携带情况,本研究以XJ05为研究对象,运用96孔板建立不同时间BF模型,染色后酶标仪测定D_{595 nm}值,并用倒置显微镜观察BF形态。同时检测11株菌中与BF形成相关基因（gelE、esp、ace、asa1、asa373、efaA、ef0591、ef3314、ahrc、eep）的携带情况,并对相关基因片段做同源性分析。结果显示,在不同时间段XJ05株BF的生成量与空白对照组相比差异显著（P<0.05）,24 h时D_{595 nm}值最高且与其他各个时间段相比均差异显著（P<0.05）。显微镜下观察6 h时BF初具规模,可见散落的网状结构,12~24 h矩阵网格结构明显,24 h时形成高度有序的网格结构,24 h后网状结构逐渐脱落,BF开始降解。11株菌中10种相关基因的携带情况不同,有6株携带8种基因,1株携带7种基因,1株携带6种基因,1株携带2种基因,有2株未检测到10种基因的任何一种。检出的基因片段同源性均在93.3%~100.0%之间。结果表明,XJ05株能形成完整的BF,11株分离的致羔羊脑炎粪肠球菌中有9株携带部分与BF形成相关的基因。     

Detection on Biofilm and Biofilm-associated Genes of Enterococcus faecalis Inducing Lamb Encephalitis

The purpose of this study was to observe and analyze the ability of biofilm formation of the Enterococcus faecalis (E.faecalis) XJ05 and to detect if the E.faecalis which could induce lamb encephalitis carried the biofilm-associated genes. The E.faecalis XJ05 was used as the sample. The biofilm model was established by the 96-well microtiter plate and read by microplate reader in the D_{595 nm} at the same time observed by the inverted microscope. The primers were designed to examine if the 11 strains of E.faecalis carried the biofilm related genes and then the amplified products were sequenced and analyzed for homology. The results showed that the amount of BF produce in XJ05 strain was significantly different from blank control group at different time (P<0.05), and the highest D_{595 nm} value was at 24 h and significant difference compared with other time (P<0.05). The biofilm of 6 h was scattered when observed under the microscope and the time between 12 to 24 h biofilm to be more denser, after 24 h the biofilm became to degradate and loose gradually. It turned out that the 11 strains of bacteria carried different biofilm-associated genes, 8 biofilm-associated genes were found in 6 strains of bacteria, 7 biofilm-associated genes were find in 1 strain of bacterium, 6 biofilm-associated genes were found in 1 strain of bacterium, 2 biofilm-associated genes were found in 1 strain of bacterium, 2 strains of bacteria could not text out anyone of the detected genes.The homology of all detected genes were all between 93.3% to 100.0%. It concluded that the E.faecalis XJ05 could form the completed biofilm and there were 9 kinds carried the biofilm-associated genes in 11 strains of E.faecalis.