Insights into Cronobacter sakazakii Biofilm Formation and Control Strategies in the Food Industry

Cronobacter sakazakii(C.sakazakii)is a foodborne opportunistic pathogen that can cause life-threatening invasive diseases,such as necrotizing enterocolitis,meningitis,and sepsis in infants.The potential risk of C.sakazakii contamination of powdered infant formula(PIF)is an issue that has attracted considerable attention from manufacturers,regulators,and consumers.C.sakazakii biofilms on the surfaces of equipment and in diverse food-production environments constitute a mode of cell growth that protects the pathogen from hostile environments,and are an important source of persistent contamination of food products.Bacterial biofilms are difficult to remove due to their resistant properties.Conventional cleaning and sterilizing procedures may be insufficient for biofilm control,and may lead to further biofilm development and dispersal.Consequently,novel control strategies are being developed,such as nanotechnology-based delivery systems,interspecies interactions,antimicrobial molecules of microbial origin,natural extracts,and phages.This review focuses on describing the mechanisms underlying the biofilm formation and behavior of C.sakazakii and discussing potential control strategies.

ToxR Is Required for Biofilm Formation and Motility of Vibrio Parahaemolyticus

Vibrio parahaemolyticus, the leading cause of seafood-borne gastroenteritis, has the ability to form biofilms on biotic and abiotic surfaces. Biofilm formation is a complicated process involving many specific structures and regulatory processes. The most significant of the structures and processes include polar and lateral flagella, mannose-sensitive hemagglutinin typeⅣpili, chitin-regulated pili,capsular polysaccharide (CPS), exopolysaccharide

In vitro interference of cefotaxime at subinhibitory concentrations on biofilm formation by nontypeable Haemophilus influenzae

Objective:To investigate the in vitro interference of cefotaxime at subinhibitory concentrations [sub-minimal inhibitory concentrations(MIC)] on biofilm formation by nontypeable Haemophilus influenzae(NTHi).Methods:The interference of subinhibitory concentrations of cefotaxime on biofilm formation of the clinical strong-biofilm forming isolates of NTHi was evaluated by a microtiter plate biofilm formation assay.The effect of sub-MIC cefotaxime on bacterial cell-surface hydrophobicity was determined using a standard microbial adhesion to n-hexadecane test.Additionally,the effects on bacterial adherence to human fibronectin and expression of bacterial adhesins were also investigated.Results:Subinhibitory concentrations of cefotaxime,both at 0.1× and 0.5× MIC levels,efficiently reduced the NTHi biofilm formation,and this effect was independent of decreasing bacterial viability.Sub-MIC cefotaxime also decreased bacterial cell-surface hydrophobicity and reduced adherence to human fibronectin.Inhibition in the P2 and P6 gene expressions upon exposure to sub-MIC cefotaxime was also noted.Conclusions:Taken together,our results indicate that sub-MIC cefotaxime interferes with the formation of NTHi biofilm,and this effect is feasibly related to the interference with cell-surface hydrophobicity,fibronectin-binding activity as well as alteration of the P2 and P6 gene expression.The findings of the present study therefore provide a rationale for the use of subinhibitory concentrations of cefotaxime for treatment of NTHi-related diseases.

Anti-quorum sensing and anti-biofilm formation activities of plant extracts from South Korea

Objective: To investigate anti-quorum sensing(anti-QS) and anti-biofilm formation(antiBF) activities of the ethanol extracts of 388 plants. Methods: The anti-QS activity of the plant extracts was evaluated by disc-diffusion assays using the bio-reporter strain, Chromobacterium violaceum CV017. Pseudomonas aeruginosa PAO1, Yersinia enterocolitica ATCC 9610, and Agrobacterium tumefaciens C58, which possess QS systems, were used to evaluate the antiBF activity of the plant extracts. Results: Among 388 plant extracts, the Cornus controversa(C. controversa) and Cynanchum wilfordii extracts exhibited the strongest anti-QS activity. The C. controversa extract exhibited anti-BF activity against Pseudomonas aeruginosa, Yersinia enterocolitica and Agrobacterium tumefaciens, whereas the Cynanchum wilfordii extract exhibited no anti-BF activity against Pseudomonas aeruginosa. In addition, the C. controversa extract suppressed soft rot of cabbage. Conclusions: The C. controversa extract inhibits bacterial QS and BF, and is capable of controlling soft rot. Therefore, this extract has potential for the prevention and treatment of bacterial infections and for the development of alternatives to antibiotics.

Effects of Scutellaria Baicalensis on Activity and Biofilm Formation of Klebsiella Pneumoniae

Objective To explore the effects of Scutellaria baicalensis on activity and biofilm formation of Klebsiella pneumonia(Kp).Methods The broth and agar dilution methods were carried out to determine minimum inhibitory concentration and minimum bactericidal concentration of Scutellaria baicalensis for TW518.VITEK-32 system was used to assay TW518 susceptibility to antibiotics.Kp biofilms were formed in vitro and stained with Bac Light Live/Dead stain.The class integron geneⅠ1 m RNA expression was analyzed with RT-PCR.Results The minimum inhibitory concentration of Scutellaria baicalensis on TW518 identified as a Kp colony was 32 mg/ml,and minimum bactericidal concentration was 64 mg/ml.Scutellaria baicalensis and broad-spectrum penicillin,cephalosporin,quinolones,or beta-lactamase had synergistic bactericidal effects.Biofilm formation activity of Kp treated with Scutellaria baicalensis was significantly lower than that of the control group.And class integron geneⅠ1 m RNA expression of TW518 was significantly inhibited by Scutellaria baicalensis.Conclusions Scutellaria baicalensis has sterilization effect on Kp,and Scutellaria baicalensis could effectively inhibit Kp biofilm formation with prolonged treatment.Scutellaria baicalensis might inhibit Kp biofilm formation through down-regulating integron geneⅠ1 expression.

Identification of the nitrogen-fixing Pseudomonas stutzeri major flagellar gene regulator FleQ and its role in biofilm formation and root colonization

Flagellar biosynthesis and motility are subject to a four-tiered transcriptional regulatory circuit in Pseudomonas,and the master regulator FleQ appears to be the highest-level regulator in this hierarchical regulatory cascade.Pseudomonas stutzeri A1501 is motile by a polar flagellum;however,the motility and regulatory mechanisms involved in this process are unknown.Here,we searched the A1501 genome for flagella and motility genes and found that approximately 50 genes,which were distributed in three non-contiguous chromosomal regions,contribute to the formation,regulation and function of the flagella.The non-polar mutation of fleQ impaired flagellar biosynthesis,motility and root colonization but enhanced biofilm formation.FleQ positively regulates the expression of flagellar class Ⅱ-Ⅳ genes,suggesting a regulatory cascade that is coordinated similar to that of the well-known P.aeruginosa.Based on our results,we propose that flagellar genes in P.stutzeri A1501 are regulated in a cascade regulated by FleQ and that flagellum-driven motility properties may be necessary for competitive rhizosphere colonization.

Orostachys japonicus ethyl acetate fraction suppresses MRSA biofilm formation

Objective:To investigate the effect of Orostachys(O.)japonicus,a perennial herbaceous plant of the Family Crassulaceae,on biofilm formed by methicillin-resistant Staphylococcus aureus(MRSA).Methods:Powdered O.japonicus was extracted by 95%methanol,concentrated,and then,systematically fractionated with n-hexane,dichloromethane(DCM),ethyl acetate(EtOAc),n-butanol,and H2 O according to polarity.Among them,the flavonoid-rich EtOAc fraction demonstrated the highest antibacterial activity and was used in this study.Using the biofilm inhibition assay,cell-surface attachment assay,confocal laser scanning microscopy,latex agglutination assay,and real time qRT-PCR,we examined whether the EtOAc fraction inhibited the formation of MRSA biofilm.Results:The EtOAc fraction exhibited distinct activity against biofilm formation and cell-surface attachment of MRSA up to 1 mg/m L through down-regulating the expression of mec A gene and the production and agglutination of penicillin-binding protein 2 a as solidly observed in biofilm inhibition assay,cell-suface attachment assay,confocal laser scanning microscopy,latex agglutination assay,and real time qRT-PCR analysis.Conclusions:These results suggest that O.japonicus could be utilized as a potential resource for the development of new antibiofilm formation of MRSA and antibacterial agents in the future.

Biofilm formation in trimethoprim/sulfamethoxazole-susceptible and trimethoprim/sulfamethoxazoleresistant uropathogenic Escherichia coli

Objective: To compare bioi lm formation in trimethoprim/sulfamethoxazole(SXT)-susceptible Escherichia coli(E. coli)(SSEC) and SXT-resistant E. coli(SREC) isolated from patients with urinary tract infections, and study the motile ability and physical characteristics of the isolates.Methods: A total of 74 E. coli isolates were tested for antimicrobial susceptibility with the disc diffusion assay. Based on the SXT-susceptibility test, the E. coli isolates were divided into SSEC(N = 30) and SREC(N = 44) groups. All E. coli isolates were examined for motile ability by using a motility test medium, and for checking bioi lm formation a scanning electron microscope was used. Bacterial colony size was measured with a vernier caliper and bacterial cell length was measured under a light microscope. The bacterial growth rate was studied by plotting the cell growth(absorbance) versus the incubation time. Results: The frequencies of non-motility and biofilm formation in the SREC group were signii cantly higher than that in the SSEC group(P < 0.01). The SREC bacterial cell length was shorter than that in the SSEC group [(1.35 ± 0.05) vs.(1.53 ± 0.05) μm, P < 0.05)], whereas the bacterial colony size and mid-log phase of the growth curve were not signii cantly dif erent. Conclusions: The present study indicated that bioi lm formation and phenotypic change of uropathogenic E. coli can be attributed to the mechanism of E. coli SXT resistance.

In vitro biofilm formation in ESBL producing Escherichia coli isolates from cage birds

Objective:To determine biofilm and hydrophobicity formation ratios in extended spectrum beta lactamases(ESBL) synthesizing Escherichia coli isolates which were isolated from feces samples of 150 cage bird species randomly taken from pet shops in Hatay province,Turkey.Methods:In vitro biofilm production of 4 ESBL positive isolates were performed by Congo Red Agar(CRA),Standard Tube(ST) and Microtitre Plate(MP) methods while their hydrophobicity were examined by bacterial adhesion to hydrocarbon(BATH) test.Results:In the examined isolates,while biofilm production was found to be negative by CRA method,highest biofilm producing strain,among 4 bacteria was determined to be A42 by ST and MP methods.The Scanning Electron Microscopy(SEM) also displayed these confirmed findings.The hydrophobicity values of strains were determined to be between 22.45%and 26.42%.Conclusions:As a result,biofilm formation in cage bird feces originated ESBL positive Escherichia coli isolates was performed for the first time in Turkey.In order to present the relation between pathogenicity and biofilm production in animal originated ESBL positive isolates,further studies are required.

Study on Formation and Inhibition Mechanism of Biofilm of Pig Actinobacillus pleuropneumoniae

Pig Actinobacillus pleuropneumoniae(App) could induce chronic respiratory tract infection in pigs, which causes major economic losses on pig industry. Bacterial biofilm(BBF) is bacterial community adsorbed on the surface of biomaterials or body cavity, to protect bacteria escape, and recurrent outbreaks of related infectious diseases and chronic infections resulting therefrom are called bacterial biofilm diseases. App BF belongs to polymers with spatial structure in vitro, and its formation is regulated by multiple genes. Among them, gene deletion of the key component TolC of multidrug efflux pumps and type I secretion systems causes that App BF adhesion weakens; gene deletion of catalytic core ClpP of Clp proteolytic complex induces the inhibition of BF formation; outer membrane lipoprotein VacJ of App promotes BF formation; gene deletion of active enzyme LuxS enhances the formation of App BF and decreases bacterial adhesion ability; gene deletion of Adh obviously declines bacterial accumulation, BF formation and adhesion to host cells. In this paper, BF formation or inhibition mechanism in App is elaborated from molecular level, which could provide reference basis for exploring the prevention of its biofilm diseases.

Virulence determinants and biofilm formation in clinical isolates of Enterococcus: A cross-sectional study

Objective:To evaluate the relationship between biofilm formation and incidence of virulence determinants in clinical isolates of Enterococcus.Methods:In this cross-sectional study,the clinical isolates of Enterococcus strains were collected from the university teaching hospitals in Ahvaz,Iran from June 2017 to June 2018.Then,the prevalence of Enterococcus species,antibiotic resistance,virulence factors,and biofilm-producing ability were determined.Results:Of the 119 tested isolates,17(14.3%)were Enterococcus faecalis,72(60.5%)were Enterococcus faecium and 30(25.2%)were other Enterococcus spp.Gelatinase was detected in 97(81.5%)isolates,enterococcal surface protein in 41(34.5%)isolates,serine protease in 39(32.8%)cases,accessory colonization factor in 111(93.3%)cases and pathogenicity islands in 17(14.3%)cases.The biofilm formation ability was observed in 75(63.0%)of all isolates and the association between the presence of enterococcal surface protein gene and biofilm formation was statistically significant.Higher resistance to vancomycin,gentamycin,and teicoplanin was indicated in Enterococcus faecium with 81.8%,58.4%,and 85.7%resistance rate,respectively.All Enterococcus faecalis isolates were sensitive to teicoplanin and vancomycin.Conclusions:The presence of antibiotic-resistance with several virulence factors in Enterococcus spp has become a concern.High prevalence of enterococcal surface protein gene among biofilm-producing isolates suggests a potential relation between biofilm formation and the enterococcal surface protein gene,and further studies are needed to identify the mechanism of biofilm inhibition.

Study of Biofilm Formation and Antibiotic Resistance Pattern of Bacteria Isolated from Diabetic Foot Ulcers in Hôpital de Référence Saint Joseph, Kinshasa, Democratic Republic of Congo

Foot infections resulting from biofilm producers and multi-drug resistant organisms is one of the most important complications of diabetes mellitus, as it can impede the wound healing process. This study was carried out in order to determine the antibiotic resistance pattern and the biofilm production in diabetic foot ulcers isolates. Clinical samples were collected from patients suffering from diabetic foot ulcers by using sterile swabs. Antibiotic susceptibility test was done using disk diffusion method on Mueller Hinton Agar. Biofilm formation was assessed by Crystal Violet Staining Method. Staphylococcus aureus isolates were resistant to ofloxacin (83.3%), ciprofloxacin (75.0%), trimethoprim-sulphamethoxazole (75.0%), and gentamicin (58.8%) but very sensitive to oxacillin (100.0%) and vancomycin (91.7%). Pseudomonas aeruginosa isolates showed resistance to the commonly used antibiotics such as ofloxacin, cefotaxime, ampicillin (81.8%), ceftazidime and imipenem (72.7%). The majority of bacteria studied were biofilm producers. This study showed that bacteria isolated from diabetic foot ulcers were biofilm producers and presented resistance to commonly used antibiotics. Knowledge on antibiotic sensitivity pattern and biofilm phenotype of the isolates will be helpful in determining the drugs for the treatment of diabetic ulcers.

Effect of Ethanol Extract of Venenum Bufonis on Biofilm Formation of <i>Staphylococcus aureus</i>

Ethanol extract of Venenum Bufonis has shown many valuable bioactivities, but little is known about its effect on biofilm formation of Staphylococcus aureus. In this study, biofilm formation of S. aureus NCTC8325 with or without ethanol extract of Venenum Bufonis was tested using microtitre plate assay and Confocal Laser Scanning Microscope (CLSM) system. Results show that the biofilm formation of S. aureus with ethanol extract of Venenum Bufonis was significantly lower than that of the control group without ethanol extract of Venenum Bufonis. Meanwhile, the reduction degree was correlated to the concentration of ethanol extract of Venenum Bufonis positively. With CLSM system we can observe that looser and less biomass of the biofilm structure of the experimental group appeared than that of the control group. These results suggested that ethanol extract of Venenum Bufonis has inhibitory effect on biofilm formation of S. aureus.

A Novel Cellular Autoaggregative Developmentally CRP Regulated Behaviour Generates Massively Chondrule-Like Formations over Surface of Old <i>Escherichia coli</i>K-12 Macrocolony Biofilms

How Escherichia coli bacteria develop a particular colonial, 3-D biofilm morphological pattern is still a poorly understood process. Recently, we reported a new E. coli K-12 morphotype exhibited by old macrocolonies described as volcano-like. The formative developmental process of this morphotype has been presented as a suitable experimental model for the study of 3D patterning in macrocolony biofilms. Here, we report the optical microscopy observations and genetic analysis that have unveiled the existence of a novel autoaggregative behaviour which generates massive lumpiness over the surface of the volcano-like macrocolonies. These lumpy formations are generated by the autoaggregation and strong interaction of tightly packed bacterial cells in structures with a chondrule-like appearance which give the colony’s surface its characteristic microscopic lumpy phenotype. Furthermore, they exhibit different levels of maturation from the edge to the center of the colony. Hence, its generation appears to follow a spatiotemporal program of development during the macrocolony’s morphogenesis. Interestingly, the agar’s hardness influences the morphology exhibited by these formations, with high agar concentration (1.5%, 15 g/L) suppressing its development. This new auto-aggregative E. coli’s behaviour does not require the activity of the biofilm master regulator CsgD, the adhesiveness of flagella, pili type 1, adhesin Ag43, β-1,6-N-acetyl-D-glucosamine polymer-PGA, cellulose or colanic acid, but it is under glucose repression and the control of cAMP receptor protein (CRP). The possible physiological role of these chondrule-like formations in the adaptability of the colony to different stressful environmental conditions is discussed.

不同改性方法对生物砂滤池生物膜特性及处理受污染原水效果的影响

通过对普通石英砂(QS)进行氨基及铝钙双氢氧化物改性制得氨基改性砂(AMS)和铝钙双氢氧化物改性砂(CAS),分别以QS、AMS、CAS为滤料填充滤池进行挂膜,研究了不同石英砂表面的生物膜量、生物活性、胞外聚合物(EPS)的特性及对受污染原水的处理效果。结果表明,挂膜成功后CAS表面的生物活性最高,为42.00 mgO_(2)/(g·h),生物膜量和胞外聚合物的含量最大,分别为12.43 mg/g、105.09 mg/(g·SS),对COD_(Cr)、NH^(+)_(4)-N的去除率分别稳定在58.20%、89.50%;AMS表面的生物活性、生物膜量、EPS平均含量分别为32.97 mgO_(2)/(g·h)、9.07 mg/g、93.41 mg/(g·SS),对COD_(Cr)、NH^(+)_(4)-N的去除率分别为54.60%、85.10%。三种石英砂表面生物量分布均沿水流方向递减,EPS的含量从溶解性胞外聚合物(SL-EPS)到紧密附着性胞外聚合物(TB-EPS)呈上升趋势。

sRNA GcvB对鼠伤寒沙门菌毒力的调控作用研究

小RNA(sRNA)是一种调节分子,可以在转录后水平调节基因表达,从而改变细菌的生理特征。为了研究sRNA GcvB在鼠伤寒沙门菌毒力中的调控作用,本研究构建了GcvB基因缺失株、互补株及示踪菌,检测其对鼠伤寒沙门菌生物被膜形成及泳动能力的影响;分析亲本株与缺失株在毒力及感染动力学上的差异,预测并分析了GcvB调控的潜在靶基因,探究了GcvB对ST生物被膜、氧化应激和毒力的调控作用。结果显示,与亲本株和回补株相比,GcvB缺失后生物被膜形成能力升高、抗氧化应激能力显著增强;半数致死量(LD50)上升至3.98倍。实时荧光定量PCR(qRT-PCR)检测靶基因gcvA、bapA、hilA、igaA及sitD,结果显示gcvA、bapA、igaA及sitD的转录水平表达量上调,而hilA下降。本研究证实sRNA GcvB参与了鼠伤寒沙门菌生物被膜形成、氧化应激和毒力作用,为揭示sRNA GcvB在ST生物被膜、氧化应激和毒力中的调控机制提供了研究基础。

亚抑菌浓度抗菌药物影响金黄色葡萄球菌生物膜形成的研究进展

金黄色葡萄球菌(SA)是引起医院获得性感染的常见病原体之一,极易黏附在导管或植入物表面形成生物膜导致耐药性增加,给临床治疗带来极大挑战。近年研究表明,抗菌药物处于亚抑菌浓度时可影响SA生物膜形成。因此,本文阐述SA生物膜的形成过程及基因调控,不同抗菌药物在亚抑菌浓度下对SA生物膜形成的影响及其作用机制,以期为有效控制及治疗SA生物膜相关感染提供一定依据。

3种鸡源乳酸菌抑制大肠杆菌生物被膜形成的比较研究

试验旨在研究鼠李糖乳杆菌、戊糖片球菌和口乳杆菌3种鸡源乳酸菌对大肠杆菌生物被膜生成的影响。利用置片法对供试菌株培养48 h,定性观察细菌生长与生物被膜的形成;利用96孔微板法定量检测4种细菌生长的OD_(590)值,评价抑制生物被膜生成的效果。结果表明,大肠杆菌培养12 h开始形成生物被膜,24 h形成典型的生物被膜,并伴有交织链接的生物被膜骨架;3种乳酸菌培养36 h均未见生物被膜骨架,OD_(590)值显示鼠李糖乳杆菌生物被膜厚度优于戊糖片球菌、口乳杆菌。与大肠杆菌共培养发现,3种乳酸菌全菌液和上清液均能显著抑制大肠杆菌生物被膜的生成,抑制效果鼠李糖乳杆菌>口乳杆菌>戊糖片球菌。结果可为探索乳酸菌拮抗大肠杆菌感染机制提供参考。

生物慢滤系统处理微污染水体的试验研究:两种滤料的对比

滤料的特性是决定生物慢滤系统对各类污染物去除效果的主要因素之一,然而国内外在不同滤料的生物慢滤系统净化效能比较和微生物机制方面的研究十分有限。本研究选用石英砂和活性炭作为生物慢滤系统的滤料,比较了它们在污染物去除和微生物群落方面的异同,并在滤料堵塞时开展了反冲洗实验。结果表明,两柱均需要26 d完成挂膜。挂膜完成后,活性炭柱对浊度、CODMn和氨氮的去除率分别为92.18%、65.53%和99.37%,而石英砂柱的去除率分别为91.89%、42.91%和98.87%,活性炭柱的去除率优于石英砂柱。在挂膜期间,石英砂柱和活性炭柱都出现了亚硝酸盐氮和硝酸盐氮的积累现象。石英砂柱主要的优势菌群为A0839和Rhodobacter,而活性炭柱主要的优势菌群为Ramlibacter、Pseudomonas和Leptospirillum,从微生物群落的角度解释了活性炭柱对有机物和总氮的去除效果优于石英砂柱。活性炭滤料的穿透深度大于石英砂滤料,石英砂柱的滤料主要是前40 cm发生堵塞,而活性炭柱的滤料是前60 cm发生堵塞。在滤料膨胀系数为30%时,石英砂柱和活性炭柱的反冲洗强度分别为6.83 L·m^(-2)·s^(-1)、3.16 L·m^(-2)·s^(-1),活性炭柱的反冲洗水量比石英砂柱少了53.7%。本研究为农村地区慢滤系统滤料的选择提供了技术依据,为滤料的堵塞问题提供了经济可行的解决方案,为该技术在农村供水技术中的应用提供了技术支持。

竹炭/陶粒及组合填料BAF处理高氨氮沼液的挂膜启动效应

为研究竹炭/陶粒及组合填料曝气生物滤池(BAF)处理高氨氮沼液的启动特性,在水力停留时间为6 h,水温23.2~26.5℃,气水比5:1(DO 3~4 mg/L),进水NH_(4)^(+)-N浓度为33.1~222.3 mg/L,COD 42.5~242.3 mg/L的运行条件下,对比考察了竹炭BAF(Z-BAF)、陶粒BAF(T-BAF)和组合填料BAF(ZT-BAF)的生化特性、沿程变化和挂膜启动效应。结果表明,Z-BAF、T-BAF及ZT-BAF分别在26、18和13 d完成挂膜启动。挂膜期间,平均出水NH_(4)^(+)-N浓度分别为24.4、15.9及8.5 mg/L;平均出水COD分别为78.6、73.02和47.2 mg/L。通过检测不同运行天数BAF的沿程生化特性变化,发现挂膜24 d时,去除COD的异养菌主要集中在反应器下部,负责转化NH_(4)^(+)-N的自养菌主要集中在反应器中下部;在第24~40天,自养菌在与异养菌的竞争中逐步取得优势,逐步向反应器下部生长繁殖。电镜扫描分析确认了各反应器中的填料上附着生长良好的生物膜,且观察到相较于陶粒填料,竹炭填料表面具有更好生物膜生长特性。综上,ZT-BAF挂膜时间最短且对污染物去除效率最高,在高氨氮沼液负荷下,竹炭和陶粒作为组合填料更有利于提升BAF的挂膜启动性能。