Biofilm formation by Salmonella spp. and Listeria monocytogenes on plastic surface

AIMS: To investigate the biofilm formation by 122 Salmonella spp. and 48 Listeria monocytogenes strains on a plastic surface. METHODS: Quantification of biofilm formation was performed in brain heart infusion (BHI), trypcase soya broth (TSB), meat broth (MB) and 1/20 diluted trypcase soya broth (1/20-TSB) in plastic microtitre plates. RESULTS: All tested Salmonella spp. and L. monocytogenes strains produced biofilm in a suitable medium. However, the quantities of biofilm produced by Salmonella spp. were greater than those produced by tested L. monocytogenes strains. The nutrient content of the medium significantly influenced the quantity of produced biofilm. Diluted TSB was the most effective in promoting biofilm production by Salmonella spp., followed by TSB, while the least quantity of biofilm was formed in BHI and MB. L. monocytogenes produced the highest quantities of biofilm in BHI, followed by TSA, then MB, and the least quantities of biofilm were produced in 1/20-TSB. CONCLUSIONS: Salmonella spp. produces more biofilm in nutrient-poor medium, while L. monocytogenes produce more biofilm in nutrient-rich medium.     

鼠伤寒沙门菌pStSR100质粒对生物膜形成的影响

目的:鼠伤寒沙门菌在多种表面形成的生物膜对其致病性和引起食物中毒等方面起着重要作用,本研究探讨鼠伤寒沙门菌pStSR100质粒对细菌在不同材质表面生物膜形成的影响。方法:用LB(Luria-Bertani,LB)培养基和TSB(Tryptose Soya Broth,TSB)培养基分别将携带pStSR100质粒的野生株在96孔板与放置无菌小圆玻片的24孔板中静态培养48 h,用结晶紫半定量法确定生物膜形成的适宜培养基。将野生株与消除质粒的突变株,用结晶紫半定量法和激光共聚焦显微镜(Confocal Laser scanning microscopy,CLSM)观察其在聚苯乙烯培养板和小圆玻片表面形成生物膜的差异。结果:用LB培养时细菌生物膜的形成能力高于用TSB培养,LB培养基更适宜生物膜形成;结晶紫半定量法结果表明野生株比突变株在小圆玻片表面形成生物膜的能力明显增强,而在聚苯乙烯培养板表面两者则无明显差异;CLSM观察发现,野生株在小圆玻片表面形成融合成片的大克隆,突变株仅形成较小克隆。结论:鼠伤寒沙门菌pStSR100质粒能促进该菌在亲水性材质表面生物膜的形成,但其对该菌在疏水性材质表面生物膜的形成未见明显影响,这一新发现为进一步研究鼠伤寒沙门菌生物膜形成的调控机制,研制抗感染材料提供了理论和实验依据。     

鼠伤寒沙门菌pStSR100质粒对生物膜形成的影响

目的：鼠伤寒沙门菌在多种表面形成的生物膜对其致病性和引起食物中毒等方面起着重要作用,本研究探讨鼠伤寒沙门菌pStSR100质粒对细菌在不同材质表面生物膜形成的影响。方法：用LB（Lufia—Bertani,LB）培养基和TSB（TryptoseSoyaBroth,TSB）培养基分别将携带pStSR100质粒的野生株在96孔板与放置无菌小圆玻片的24孔板中静态培养48h,用结晶紫半定量法确定生物膜形成的适宜培养基。将野生株与消除质粒的突变株,用结晶紫半定量法和激光共聚焦显微镜（ConfocalLaserscanningmicroscopy,CLSM）观察其在聚苯乙烯培养板和小圆玻片表面形成生物膜的差异。结果：用LB培养时细菌生物膜的形成能力高于用TSB培养,LB培养基更适宜生物膜形成;结晶紫半定量法结果表明野生株比突变株在小圆玻片表面形成生物膜的能力明显增强,而在聚苯乙烯培养板表面两者则无明显差异;CLSM观察发现,野生株在小圆玻片表面形成融合成片的大克隆,突变株仅形成较小克隆。结论：鼠伤寒沙门菌pStSR100质粒能促进该茵在亲水性材质表面生物膜的形成,但其对该菌在疏水性材质表面生物膜的形成未见明显影响,这一新发现为进一步研究鼠伤寒沙门菌生物膜形成的调控机制,研制抗感染材料提供了理论和实验依据。     

Influence of Culture Medium pH on Growth, Aggregation, and Biofilm Formation of Xylella fastidiosa

The major feature of Xylella fastidiosa growing in its hosts, as well as in culture media, is its cellular aggregation and biofilm formation, leading to partial obstruction of the xylem causing water stress in the plant. We report that growth, aggregation, and biofilm formation of X. fastidiosa are influenced by the medium pH. We have verified that X. fastidiosa cell aggregation is reversibly inhibited by decreasing the medium pH from 6.6 to 6.4. Biofilm formation on glass walls was affected as well, and a concomitant decrease in cell multiplication was observed below pH 6.4. The manipulation of culture medium pH can be used as a tool for the cloning of X. fastidiosa strains isolated from plant hosts, because different strains can inhabit the same plant. Also, X. fastidiosa mutants produced by gene manipulation can be isolated from cell aggregates containing transformed and untransformed cells.     

Biofilm growth and detachment of Actinobacillus actinomycetemcomitans

The gram-negative, oral bacterium Actinobacillus actinomycetemcomitans has been implicated as the causative agent of several forms of periodontal disease in humans. When cultured in broth, fresh clinical isolates of A. actinomycetemcomitans form tenacious biofilms on surfaces such as glass, plastic, and saliva-coated hydroxyapatite, a property that probably plays an important role in the ability of this bacterium to colonize the oral cavity and cause disease. We examined the morphology of A. actinomycetemcomitans biofilm colonies grown on glass slides and in polystyrene petri dishes by using light microscopy and scanning and transmission electron microscopy. We found that A. actinomycetemcomitans developed asymmetric, lobed biofilm colonies that displayed complex architectural features, including a layer of densely packed cells on the outside of the colony and nonaggregated cells and large, transparent cavities on the inside of the colony. Mature biofilm colonies released single cells or small clusters of cells into the medium. These released cells adhered to the surface of the culture vessel and formed new colonies, enabling the biofilm to spread. We isolated three transposon insertion mutants which produced biofilm colonies that lacked internal, nonaggregated cells and were unable to release cells into the medium. All three transposon insertions mapped to genes required for the synthesis of the O polysaccharide (O-PS) component of lipopolysaccharide. Plasmids carrying the complementary wild-type genes restored the ability of mutant strains to synthesize O-PS and release cells into the medium. Our findings suggest that A. actinomycetemcomitans biofilm growth and detachment are discrete processes and that biofilm cell detachment evidently involves the formation of nonaggregated cells inside the biofilm colony that are destined for release from the colony.     

Probiotic Lactobacilli Interfere with <Emphasis Type="Italic">Streptococcus mutans</Emphasis> Biofilm Formation In Vitro

In clinical studies, probiotic bacteria have decreased the counts of salivary mutans streptococci (MS). We compared the effects of probiotic Lactobacillus strains on the biofilm formation of Streptococcus mutans. The bacterial strains used included four S. mutans strains (reference strains NCTC 10449 and Ingbritt and clinical isolates 2366 and 195) and probiotic strains Lactobacillus rhamnosus GG, L. plantarum 299v, and L. reuteri strains PTA 5289 and SD2112. The ability of MS to adhere and grow on a glass surface, reflecting biofilm formation, was studied in the presence of the lactobacilli (LB). The effect of LB culture supernatants on the viability of the MS was studied as well. All of the LB inhibited the biofilm formation of the clinical isolates of MS (P < 0.001). The biofilm formation of the reference strains of MS was also inhibited by the LB, but L. plantarum and L. reuteri PTA 5289 showed a weaker inhibition when compared to L. reuteri SD2112 and L. rhamnosus GG. Viable S. mutans cells could be detected in the biofilms and culture media only when the experiments were performed with the L. reuteri strains. The L. reuteri strains were less efficient in killing the MS also in the tests performed with the culture supernatants. The pHs of the supernatants of L. reuteri were higher compared to those of L. rhamnosus GG and L. plantarum; P < 0.001. In conclusion, our results demonstrated that four commonly used probiotics interfered with S. mutans biofilm formation in vitro, and that the antimicrobial activity against S. mutans was pH-dependent.     

Temperature-regulated formation of mycelial mat-like biofilms by Legionella pneumophila

Fifty strains representing 38 species of the genus Legionella were examined for biofilm formation on glass, polystyrene, and polypropylene surfaces in static cultures at 25 degrees C, 37 degrees C, and 42 degrees C. Strains of Legionella pneumophila, the most common causative agent of Legionnaires' disease, were found to have the highest ability to form biofilms among the test strains. The quantity, rate of formation, and adherence stability of L. pneumophila biofilms showed considerable dependence on both temperature and surface material. Glass and polystyrene surfaces gave between two- to sevenfold-higher yields of biofilms at 37 degrees C or 42 degrees C than at 25 degrees C; conversely, polypropylene surface had between 2 to 16 times higher yields at 25 degrees C than at 37 degrees C or 42 degrees C. On glass surfaces, the biofilms were formed faster but attached less stably at 37 degrees C or 42 degrees C than at 25 degrees C. Both scanning electron microscopy and confocal laser scanning microscopy revealed that biofilms formed at 37 degrees C or 42 degrees C were mycelial mat like and were composed of filamentous cells, while at 25 degrees C, cells were rod shaped. Planktonic cells outside of biofilms or in shaken liquid cultures were rod shaped. Notably, the filamentous cells were found to be multinucleate and lacking septa, but a recA null mutant of L. pneumophila was unaffected in its temperature-regulated filamentation within biofilms. Our data also showed that filamentous cells were able to rapidly give rise to a large number of short rods in a fresh liquid culture at 37 degrees C. The possibility of this biofilm to represent a novel strategy by L. pneumophila to compete for proliferation among the environmental microbiota is discussed.     

肺炎克雷伯菌KP1＿4563基因突变株的构建及其生物膜表型分析

KP1_4563基因是肺炎克雷伯菌NTUH-K2044中假设的蛋白编码基因,与Ⅲ型菌毛的功能有关。本实验首先采用同源重组基因敲除方法构建肺炎克雷伯菌KP1_4563基因缺失的突变株(Kp-△4563),然后PCR扩增KP1_4563基因片段,克隆到质粒p BAD33上,将重组质粒导入Kp-△4563获得回补株(Kpc-△4563)。分别测定野生株、突变株、回补株用普通LB培养基,改良Minka培养基以及含胆汁盐的LB培养基培养时生物膜形成能力,以此来探讨KP1_4563基因以及不同培养基对肺炎克雷伯菌体外生物膜形成的影响。我们成功构建KP1_4563基因缺失的突变株和回补株Kpc-△4563。与野生株相比,突变株Kp-△4563生物膜形成能力减弱,回补株介于野生株和突变株之间。使用改良Minka培养基使菌株菌毛化以及加入胆汁盐可以增加生物膜的形成能力。这些分析表明肺炎克雷伯菌KP1_4563基因能正调控细菌生物膜的形成。体外培养使细菌菌毛化以及加入胆汁盐可以促进生物膜的形成。     

A comparative study of biofilm formation by Shiga toxigenic Escherichia coli using epifluorescence microscopy on stainless steel and a microtitre plate method

Attachment of Shiga toxigenic Escherichia coli (STEC) to surfaces and the formation of biofilms may enhance persistence in a food processing environment and present a risk of contaminating products. Seven strains of STEC and three non-STEC strains were selected to compare two biofilm quantification methods; epifluorescence microscopy on stainless steel (SS) and a microtitre plate assay. The influence of prior growth in planktonic (nutrient broth) and sessile (nutrient agar) culture on biofilm production, as well as expression of surface structures and the possession of antigen 43 (encoded by agn43) on biofilm formation were also investigated. Biofilms were produced in diluted nutrient broth at 25 degrees C for 24 and 48 h. Curli expression was determined using congo red indicator agar, while the presence of agn43 was determined using polymerase chain reaction. No correlation was found between counts for epifluorescence microscopy on SS and the absorbance values obtained with the microtitre plate method for planktonic and sessile grown cultures. Different abilities of individual STEC strains to attach to SS and microtitre plates were found with some strains attaching better to each surface following growth in either planktonic or sessile culture. All O157 STEC strains had low biofilm counts on SS for planktonic and sessile grown cultures; however, one STEC O157:H- strain (EC516) had significantly greater (p<0.05) biofilm production on microtitre plates compared to the other O157 STEC strains. EC516 and other STEC (O174:H21 and O91:H21) strains expressing curli fimbriae were found to produce significantly greater (p<0.05) biofilms on microtitre plates compared to the non-curli expressing strains. No relationship was found between the production of type-I fimbriae, motility, agn43 and bacterial physicochemical properties (previously determined) and biofilm formation on SS or microtitre plates. Variations between the two biofilm determination methods may suggest that the biofilm production on microtitre plates may not be appropriate to represent other surfaces such as SS and that caution should be taken when selecting a method to quantify biofilm production on a surface.     

Effect of environmental factors on biofilm formation by clinical<Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> isolates

The influence of environmental factors (temperature, aerobiosis-anaerobiosis, static-dynamic conditions, pH) was determined on biofilm formation by 51 S. maltophilia clinical isolates. The strains produced more biofilm at 32 degrees C than at 37 or 18 degrees C. Aerobic and 6% CO2 atmosphere yielded comparable biofilm amounts, higher than under anaerobic conditions. Biofilm production was not affected by static vs. agitated culture conditions. Biofilm production at pH 7.5 and 8.5 was comparable but significantly higher than at pH 5.5. The capacity of individual strains to form biofilm and thus contribute to the severity of some diseases is influenced by host traits and environmental conditions at the site of infection, and play an important role in the pathogenesis of biomaterial-related disease caused by S. maltophilia.     

阿萨希毛孢子菌的溶血活性及生物膜形成能力与其基因型关系研究

目的 分析临床分离自尿路感染患者的阿萨希毛孢子菌的体外溶血活性及生物膜形成能力与其基因型的关系,为临床诊治提供依据.方法 玻璃珠法提取总DNA,并采用PCR技术利用IGS1区特异性引物确定其基因型别;同时,平板法和XTT还原比色法分别检测阿萨希毛孢子菌的溶血活性及生物膜形成能力,并分析其与基因型的关系.结果 10株分离自尿路感染的阿萨希毛孢子菌基因型分别属于Ⅰ、Ⅲ、Ⅳ,其中以Ⅳ型为主;所分离菌株均具有不同程度的溶血活性;除1例分离株外,其余各分离株均具有在聚苯乙烯表面形成生物膜的能力;基因型Ⅲ型菌株具有较强的溶血活性和生物膜形成能力.结论 分离自尿路感染患者的10株阿萨希毛孢子菌以Ⅳ型为主,而其中Ⅲ型菌株表现出较强的溶血活性和生物膜形成能力.     

Production of antibacterial compounds and biofilm formation by Roseobacter species are influenced by culture conditions

Bacterial communities associated with marine algae are often dominated by members of the Roseobacter clade, and in the present study, we describe Roseobacter phenotypes that may provide this group of bacteria with selective advantages when colonizing this niche. Nine of 14 members of the Roseobacter clade, of which half were isolated from cultures of the dinoflagellate Pfiesteria piscicida, produced antibacterial compounds. Many non-Roseobacter marine bacteria were inhibited by sterile filtered supernatants of Silicibacter sp. TM1040 and Phaeobacter (formerly Roseobacter) strain 27-4, which had the highest production of antibacterial compound. In contrast, Roseobacter strains were susceptible only when exposed to concentrated compound. The production of antibacterial compound was influenced by the growth conditions, as production was most pronounced when bacteria were grown in liquid medium under static conditions. Under these conditions, Silicibacter sp. TM1040 cells attached to one another, forming rosettes, as has previously been reported for Phaeobacter 27-4. A spontaneous Phaeobacter 27-4 mutant unable to form rosettes was also defective in biofilm formation and the production of antibacterial compound, indicating a possible link between these phenotypes. Rosette formation was observed in 8 of 14 Roseobacter clade strains examined and was very pronounced under static growth in 5 of these strains. Attachment to surfaces and biofilm formation at the air-liquid interface by these five strains was greatly facilitated by growth conditions that favored rosette formation, and rosette-forming strains were 13 to 30 times more efficient in attaching to glass compared to strains under conditions where rosette formation was not pronounced. We hypothesize that the ability to produce antibacterial compounds that principally inhibit non-Roseobacter species, combined with an enhancement in biofilm formation, may give members of the Roseobacter clade a selective advantage and help to explain the dominance of members of this clade in association with marine algal microbiota.     

一株工业腐败物中魏氏柠檬酸杆菌的分离鉴定及产生物膜特性分析

【目的】分离和鉴定工业腐败物中高产细菌生物膜菌株,并明确该菌的部分产膜特性。【方法】通过微孔板结晶紫染色法对分离的菌株进行产膜能力评价,根据菌落形态、生理生化特性和16S rRNA序列的系统进化树分析进行菌株鉴定;同时利用扫描电子显微镜(SEM)和结晶紫染色法分别研究材料及温度对该菌产膜特性和能力的影响。【结果】筛选出一株高产细菌生物膜菌株,经鉴定该菌为魏氏柠檬酸杆菌;其在玻璃、不锈钢和聚氯乙烯(PVC)材料表面均能形成生物膜;温度条件显著影响产膜能力,在30°C时,菌株在PVC材料表面形成生物膜能力最强。【结论】工业腐败物中含有高产细菌生物膜菌株,并且产膜受附着物和温度影响。     

Estimation of the biofilm formation ability of Pseudomonas putida in discrete samples from continuous culture

Summary Test systems were set up in order to evaluate the ability of biomass from a continuous culture to form biofilms. A film-forming strain of Pseudomonas putida was used as the test organism. The adsorption of resting cells onto glass surfaces was measured in specially designed chambers containing 1 ml of cell suspension. Both the quantity and the physiological activity of the adsorbed cells, in terms of optical density after detachment and pH change of a substrate exposed to the adsorbed cells, were measured. The analysis of biomass from continuous cultures of Pseudomonas putida verified the suitability of the methods. Furthermore, other properties of importance to biofilm formation such as hydrophobicity and flocculation capacity of the cells were investigated.It was shown for samples deriving from different dilution rates that the cell adsorption rate drastically increased at dilution rates higher than the _max of the culture. Simultaneously, higher values of hydrophobicity and flocculation capacity were observed.It was also shown that the age and thickness of the biofilm subsequently produced in the continuous culture influenced the metabolic activity per unit of biomass attached to the surface. The methods described in this investigation may facilitate the study of parameters important to biofilm formation as well as the metabolic activity of the attached biomass.     

Biofilm formation on human airway epithelia by encapsulated Neisseria meningitidis serogroup B

Neisseria meningitidis is the etiologic agent of meningococcal meningitis. We compared 48-h biofilm formation by N. meningitidis serogroup B strains NMB, MC58, C311 and isogenic mutants defective in capsule formation on SV-40 transformed human bronchial epithelial (HBE) cells in a flow cell. We demonstrated that strains NMB and NMB siaA-D were defective in biofilm formation over glass, and there was a partial rescue of biofilm growth for strain NMB on collagen-coated coverslips at 48 h. We demonstrated all three serogroup B strains form biofilms of statistically equivalent average height on HBE cells as their isogenic capsular mutants. Strain NMB also formed a biofilm of statistically equivalent biomass as the NMB siaA-D mutant on HBE cells at 6 and 48 h. These biofilms are significantly larger than biofilms formed over glass or collagen. Verification that strain NMB expressed capsule in biofilms on HBE cells was demonstrated by staining with 2.2.B, a monoclonal antibody with specificity for the serogroup B capsule. ELISA analysis demonstrated that strains MC58 and C311 also produced capsules during biofilm growth. These findings suggest that encapsulated meningococci can form biofilms on epithelial cells suggesting that biofilm formation may play a role in nasopharyngeal colonization.     

Influence of temperature and plumbing material selection on biofilm formation and growth of Legionella pneumophila in a model potable water system containing complex microbial flora.

Survival and growth of Legionella pneumophila in both biofilm and planktonic phases were determined with a two-stage model system. The model used filter-sterilized tap water as the sole source of nutrient to culture a naturally occurring mixed population of microorganisms including virulent L. pneumophila. At 20 degrees C, L. pneumophila accounted for a low proportion of biofilm flora on polybutylene and chlorinated polyvinyl chloride, but was absent from copper surfaces. The pathogen was most abundant on biofilms on plastics at 40 degrees C, where it accounted for up to 50% of the total biofilm flora. Copper surfaces were inhibitory to total biofouling and included only low numbers of L. pneumophila organisms. The pathogen was able to survive in biofilms on the surface of the plastic materials at 50 degrees C, but was absent from the copper surfaces at the same temperature. L. pneumophila could not be detected in the model system at 60 degrees C. In the presence of copper surfaces, biofilms forming on adjacent control glass surfaces were found to incorporate copper ions which subsequently inhibited colonization of their surfaces. This work suggests that the use of copper tubing in water systems may help to limit the colonization of water systems by L. pneumophila.     

Biofilm formation and interactions of bacterial strains found in wastewater treatment systems

Biofilm formation and adherence properties of 13 bacterial strains commonly found in wastewater treatment systems were studied in pure and mixed cultures using a crystal violet microtiter plate assay. Four different culture media were used, wastewater, acetate medium, glucose medium and diluted nutrient broth. The medium composition strongly affected biofilm formation. All strains were able to form pure culture biofilms within 24 h in at least one of the tested culture media and three strains were able to form biofilm in all four culture media, namely Acinetobacter calcoaceticus ATCC 23055, Comamonas denitrificans 123 and Pseudomonas aeruginosa MBL 0199. The adherence properties assessed were initial adherence, cell surface hydrophobicity, and production of amyloid fibers and extracellular polymeric substances. The growth of dual-strain biofilms showed that five organisms formed biofilm with all 13 strains while seven formed no or only weak biofilm when cocultured. In dual-strain cultures, strains with different properties were able to complement each other, giving synergistic effects. Strongest biofilm formation was observed when a mixture of all 13 bacteria were grown together. These results on attachment and biofilm formation can serve as a tool for the design of tailored systems for the degradation of municipal and industrial wastewater.     

Application of a multiple surface tissue culture propagator for the production of cell monolayers,virus, and biochemicals

A new concept of tissue culture equipment and procedures was developed for the mass-scale growth of several types of animal tissue cells in monolayers on multiple glass surfaces. Continuous, cell lines, primary and diploid cell strains were grown in this equipment. Cells studied include primary bovine kidney, human diploid WI-38, human foreskin, and mouse CCL1 cells. Photomicrographic comparisons of cells grown by these techniques indicate they are morphologically identical to tissue culture cells grown in glass bottles or tubes. The growth of the tissue culture cells in the propagator was monitored by carbohydrate Utilization and acid production. Large-scale production of viruses and biochemicals on cells grown in the multiple-plate tissue culture propagator was accomplished. Virus titers were equal to those obtained from conventional bottle or tube cultures for several strains of influenza, parainfluenza, and respiratory syneytial viruses. High-titred mouse interferon was also produced in this system. In addition to tissue culture cell production, Eaton agent, Mycoplasma pneumoniae was grown on the multiple glass surfaces on a mass scale.     

Influence of culture heterogeneity in cell surface charge on adhesion and biofilm formation by Enterococcus faecalis

Biofilm formation is an increasing problem in medicine, due to the intrinsic resistance of microorganisms in the biofilm mode of growth against the host immune system and antimicrobial therapy. Adhesion is an important step in biofilm formation, influenced, among other factors, by the surface hydrophobicities and charges of both the substratum and the adhering microorganisms. Enterococcus faecalis strains generally display subpopulations with different surface charges, expressed as bimodal zeta potential distributions. Two-thirds of E. faecalis strains isolated from clogged biliary stents displayed such heterogeneity of surface charges in culture. In this study, the influence of this culture heterogeneity on initial adhesion and subsequent biofilm formation was investigated. Heterogeneous strains were retained in higher numbers on polystyrene than homogeneous strains. Also, biofilm formation was much more pronounced for heterogeneous strains than for homogeneous strains. In a population enriched to display only one subpopulation, fewer bacteria were retained than in its original heterogeneous culture. Also, the enriched subpopulation formed less biofilm than its original heterogeneous culture. The presence of ox bile during adhesion resulted in fewer retained bacteria, although heterogeneous strains were still retained in significantly higher numbers than were homogeneous strains, and, in general, the presence of ox bile reduced biofilm formation. The initial adhesion and biofilm formation were independent of the presence of the gene encoding the enterococcal surface protein (esp) or the expression of gelatinase (GelE). It is concluded that heterogeneity in cell surface charge represents an advantage for bacteria in the colonization of surfaces.