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.     

百里香酚联合苯唑西林对耐甲氧西林金黄色葡萄球菌(MRSA)生物被膜的影响

【背景】耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus,MRSA)能以生物被膜的状态存在,从而产生多重耐药性和持续性感染。【目的】通过研究百里香酚和苯唑西林单用和联用对耐甲氧西林金黄色葡萄球菌生物被膜的形成抑制和清除作用,探究联合用药对MRSA生物被膜的影响,为临床联合应用抗MRSA药物提供理论依据。【方法】采用微量肉汤稀释法测定苯唑西林对MRSA标准菌株USA300的最低抑菌浓度;采用结晶紫染色法和菌落计数法评估百里香酚和苯唑西林单用和联用对USA300生物被膜形成抑制和清除作用。【结果】百里香酚和苯唑西林在亚抑菌浓度下对USA300生物被膜的形成具有一定的抑制作用。在较高浓度下,百里香酚对其24 h和72 h形成的生物被膜有良好的清除作用,而苯唑西林无清除作用。两药联用对生物被膜的抑制和清除作用进一步增强,在较低浓度下有较好的抑制和清除效果。【结论】百里香酚和苯唑西林联合用药与单独用药相比,对USA300的生物被膜的抑制和清除作用增强,两药联合有协同抗菌作用。     

Diversity in biofilm formation and production of curli fimbriae and cellulose of Salmonella Typhimurium strains of different origin in high and low nutrient medium

The biofilm forming behavior of 51 Salmonella Typhimurium strains was determined in Tryptone Soya Broth (TSB) and 20 times diluted TSB (1/20TSB) at 25°C and 37°C. The results indicated that biofilm forming behavior is influenced by environmental conditions and associated with the origin of the strains. Clinical, outbreak-associated and retail product isolates showed dense biofilm formation in both media at 25°C, and in TSB also at 37°C. However, industrial isolates only showed dense biofilm formation in 1/20TSB at 25°C. By enumeration of biofilm cells, LIVE/DEAD staining and SEM analysis of biofilms it was found that the ratio of cells and extracellular matrix is affected by environmental conditions. Indeed, the genes involved in curli fimbriae and cellulose production are highly induced during biofilm formation at 25°C in 1/20TSB. This indicates that these are important matrix components during biofilm formation in 1/20TSB at 25°C and that other factors contribute to biofilm formation of clinical, outbreak-associated and retail product isolates at 37°C and/or nutrient-rich conditions.     

Holo and apo-transferrins interfere with adherence to abiotic surfaces and with adhesion/invasion to HeLa cells in Staphylococcus spp.

Staphylococcus aureus and Staphylococcus epidermidis are the major cause of infections associated with implanted medical devices. Colonization on abiotic and biotic surfaces is often sustained by biofilm forming strains. Human natural defenses can interfere with this virulence factor. We investigated the effect of human apo-transferrin (apo-Tf, the iron-free form of transferrin, Tf) and holo-transferrin (holo-Tf, the iron-saturated form) on biofilm formation by CA-MRSA S. aureus USA300 type (ST8-IV) and S. epidermidis (a clinical isolate and ATCC 35984 strain). Furthermore S. aureus adhesion and invasion assays were performed in a eukaryotic cell line. A strong reduction in biofilm formation with both Tfs was obtained albeit at very different concentrations. In particular, the reduction in biofilm formation was higher with apo-Tf rather than obtained with holo-Tf. Furthermore, while S. aureus adhesion to eukaryotic cells was not appreciably affected, their invasion was highly inhibited in the presence of holo-Tf, and partially inhibited by the apo form. Our results suggest that Tfs could be used as antibacterial adjuvant therapy in infection sustained by staphylococci to strongly reduce their virulence related to adhesion and cellular invasion.     

Comparison of Biofilm Formation between Major Clonal Lineages of Methicillin Resistant Staphylococcus aureus

Objectives

Epidemic methicillin-resistant S. aureus (MRSA) clones cause infections in both hospital and community settings. As a biofilm phenotype further facilitates evasion of the host immune system and antibiotics, we compared the biofilm-forming capacities of various MRSA clones.

Methods

Seventy-six MRSA classified into 13 clones (USA300, EMRSA-15, Hungarian/Brazilian etc.), and isolated from infections or from carriers were studied for biofilm formation under static and dynamic conditions. Static biofilms in microtitre plates were quantified colorimetrically. Dynamic biofilms (Bioflux 200, Fluxion, USA) were studied by confocal laser-scanning and time-lapse microscopy, and the total volume occupied by live/dead bacteria quantified by Volocity 5.4.1 (Improvision, UK).

Results

MRSA harbouring SCCmec IV produced significantly more biomass under static conditions than SCCmec I–III (P = 0.003), and those harbouring SCCmec II significantly less than those harbouring SCCmec I or III (P<0.001). In the dynamic model, SCCmec I–III harbouring MRSA were significantly better biofilm formers than SCCmec IV (P = 0.036). Only 16 strains successfully formed biofilms under both conditions, of which 13 harboured SCCmec IV and included all tested USA300 strains (n = 3). However, USA300 demonstrated remarkably lower percentages of cell-occupied space (6.6%) compared to the other clones (EMRSA-15 = 19.0%) under dynamic conditions. Time-lapse microscopy of dynamic biofilms demonstrated that USA300 formed long viscoelastic tethers that stretched far from the point of attachment, while EMRSA-15 consisted of micro-colonies attached densely to the surface.

Conclusions

MRSA harbouring SCCmec types IV and I–III demonstrate distinct biofilm forming capacities, possibly owing to their adaptation to the community and hospital settings, respectively. USA300 demonstrated abundant biofilm formation under both conditions, which probably confers a competitive advantage, contributing to its remarkable success as a pathogen.     

Diversity in biofilm formation and production of curli fimbriae and cellulose of Salmonella Typhimurium strains of different origin in high and low nutrient medium

The biofilm forming behavior of 51 Salmonella Typhimurium strains was determined in Tryptone Soya Broth (TSB) and 20 times diluted TSB (1/20TSB) at 25°C and 37°C. The results indicated that biofilm forming behavior is influenced by environmental conditions and associated with the origin of the strains. Clinical, outbreak-associated and retail product isolates showed dense biofilm formation in both media at 25°C, and in TSB also at 37°C. However, industrial isolates only showed dense biofilm formation in 1/20TSB at 25°C. By enumeration of biofilm cells, LIVE/DEAD staining and SEM analysis of biofilms it was found that the ratio of cells and extracellular matrix is affected by environmental conditions. Indeed, the genes involved in curli fimbriae and cellulose production are highly induced during biofilm formation at 25°C in 1/20TSB. This indicates that these are important matrix components during biofilm formation in 1/20TSB at 25°C and that other factors contribute to biofilm formation of clinical, outbreak-associated and retail product isolates at 37°C and/or nutrient-rich conditions.     

Biofilm formation by Staphylococcus epidermidis depends on functional RsbU, an activator of the sigB operon: differential activation mechanisms due to ethanol and salt stress

Staphylococcus epidermidis is a common pathogen in medical device-associated infections. Its major pathogenetic factor is the ability to form adherent biofilms. The polysaccharide intercellular adhesin (PIA), which is synthesized by the products of the icaADBC gene cluster, is essential for biofilm accumulation. In the present study, we characterized the gene locus inactivated by Tn917 insertions of two isogenic, icaADBC-independent, biofilm-negative mutants, M15 and M19, of the biofilm-producing bacterium S. epidermidis 1457. The insertion site was the same in both of the mutants and was located in the first gene, rsbU, of an operon highly homologous to the sigB operons of Staphylococcus aureus and Bacillus subtilis. Supplementation of Trypticase soy broth with NaCl (TSB(NaCl)) or ethanol (TSB(EtOH)), both of which are known activators of sigB, led to increased biofilm formation and PIA synthesis by S. epidermidis 1457. Insertion of Tn917 into rsbU, a positive regulator of alternative sigma factor sigma(B), led to a biofilm-negative phenotype and almost undetectable PIA production. Interestingly, in TSB(EtOH), the mutants were enabled to form a biofilm again with phenotypes similar to those of the wild type. In TSB(NaCl), the mutants still displayed a biofilm-negative phenotype. No difference in primary attachment between the mutants and the wild type was observed. Similar phenotypic changes were observed after transfer of the Tn917 insertion of mutant M15 to the independent and biofilm-producing strain S. epidermidis 8400. In 11 clinical S. epidermidis strains, a restriction fragment length polymorphism of the sigB operon was detected which was independent of the presence of the icaADBC locus and a biofilm-positive phenotype. Obviously, different mechanisms are operative in the regulation of PIA expression in stationary phase and under stress induced by salt or ethanol.     

Inhibition of methicillin-resistant Staphylococcus aureus (MRSA) biofilm by cationic poly (D,L-lactide-co-glycolide) nanoparticles

Abstract

The emergent need for new treatment methods for multi-drug resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) has focused attention on novel potential tools like nanoparticles (NPs). In the present study, a drug-free cationic nanoparticles (CNPs) system was developed and its anti-MRSA effects were firstly investigated. The results showed that CNPs (261.7?nm, 26.1?mv) showed time- and concentration-dependent activity against MRSA growth, killing ～ 90% of planktonic bacterial cells in 3?h at 400?μg ml^?1, and completely inhibiting biofilm formation at 1000?μg ml^?1. Moreover, CNPs at 400?μg ml^?1 reduced the minimum inhibitory concentration (MIC) of vancomycin on inhibition of planktonic MRSA growth (～ 25%) and biofilm formation (～ 50%). The CNPs–bacteria interaction force was up to 22 nN. Overall, these data suggest that CNPs have a good potential in clinical applications for the prevention and treatment of MRSA infection.     

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。     

黄颡鱼暴发性出血病病原菌的分离鉴定及其生物膜形成特性

【背景】安徽省当涂县某池塘养殖黄颡鱼发生暴发性出血病,而当前对该病的病原存在争议。【目的】确定引起黄颡鱼暴发性出血病的病原菌,并明确分离菌株的生物膜形成特性,为从抗生物膜形成角度防治病原菌感染提供参考。【方法】取濒死期黄颡鱼病变脏器分别接种EPC细胞与培养基(TSB琼脂平板和血琼脂平板)分离病原,并通过人工感染回归试验确定其致病性;采用表型鉴定与16S rRNA基因序列分析相结合的方法鉴定分离菌株,并对其生物膜形成最佳条件、成膜能力及携带的生物膜形成相关基因进行研究。【结果】从病变脏器中分离纯化到一株优势菌株(HSY-2),对黄颡鱼的半数致死量为1.05×106 CFU/mL。经形态学、生化特性和细菌16S rRNA基因测序等分析确定分离株HSY-2为简达气单胞菌。其形成生物膜的最佳条件是将细菌接种TSB培养基于30 °C培养静置96 h,可形成中等强度的生物膜。同时,分离菌株携带气单胞菌甘油-3-磷酸脱氢酶D编码基因glpD、S-核糖同型半胱氨酸裂解酶基因luxS和LuxI家族蛋白同系物编码基因ahyI三种生物膜形成相关基因,但未检测到甘露糖敏感型血凝素菌毛合成蛋白Q编码基因。【结论】本实验为进一步研究简达气单胞菌生物膜形成的调控机制打下基础,并且从抗生物膜形成角度防治简达气单胞菌感染提供了参考。     

Diversity of Methicillin-Resistant Staphylococcus aureus (MRSA) Strains Isolated from Inpatients of 30 Hospitals in Orange County,California

There is a need for a regional assessment of the frequency and diversity of MRSA to determine major circulating clones and the extent to which community and healthcare MRSA reservoirs have mixed. We conducted a prospective cohort study of inpatients in Orange County, California, systematically collecting clinical MRSA isolates from 30 hospitals, to assess MRSA diversity and distribution. All isolates were characterized by spa typing, with selective PFGE and MLST to relate spa types with major MRSA clones. We collected 2,246 MRSA isolates from hospital inpatients. This translated to 91/10,000 inpatients with MRSA and an Orange County population estimate of MRSA inpatient clinical cultures of 86/100,000 people. spa type genetic diversity was heterogeneous between hospitals, and relatively high overall (72%). USA300 (t008/ST8), USA100 (t002/ST5) and a previously reported USA100 variant (t242/ST5) were the dominant clones across all Orange County hospitals, representing 83% of isolates. Fifteen hospitals isolated more t008 (USA300) isolates than t002/242 (USA100) isolates, and 12 hospitals isolated more t242 isolates than t002 isolates. The majority of isolates were imported into hospitals. Community-based infection control strategies may still be helpful in stemming the influx of traditionally community-associated strains, particularly USA300, into the healthcare setting.     

Attachment of and Biofilm Formation by Enterobacter sakazakii on Stainless Steel and Enteral Feeding Tubes

Enterobacter sakazakii has been reported to form biofilms, but environmental conditions affecting attachment to and biofilm formation on abiotic surfaces have not been described. We did a study to determine the effects of temperature and nutrient availability on attachment and biofilm formation by E. sakazakii on stainless steel and enteral feeding tubes. Five strains grown to stationary phase in tryptic soy broth (TSB), infant formula broth (IFB), or lettuce juice broth (LJB) at 12 and 25°C were examined for the extent to which they attach to these materials. Higher populations attached at 25°C than at 12°C. Stainless steel coupons and enteral feeding tubes were immersed for 24 h at 4°C in phosphate-buffered saline suspensions (7 log CFU/ml) to facilitate the attachment of 5.33 to 5.51 and 5.03 to 5.12 log CFU/cm², respectively, before they were immersed in TSB, IFB, or LJB, followed by incubation at 12 or 25°C for up to 10 days. Biofilms were not produced at 12°C. The number of cells of test strains increased by 1.42 to 1.67 log CFU/cm² and 1.16 to 1.31 log CFU/cm² in biofilms formed on stainless steel and feeding tubes, respectively, immersed in IFB at 25°C; biofilms were not formed on TSB and LJB at 25°C, indicating that nutrient availability plays a major role in processes leading to biofilm formation on the surfaces of these inert materials. These observations emphasize the importance of temperature control in reconstituted infant formula preparation and storage areas in preventing attachment and biofilm formation by E. sakazakii.     

Effect of honey on Streptococcus mutans growth and biofilm formation

Because of the tradition of using honey as an antimicrobial medicament, we investigated the effect of natural honey (NH) on Streptococcus mutans growth, viability, and biofilm formation compared to that of an artificial honey (AH). AH contained the sugars at the concentrations reported for NH. NH and AH concentrations were obtained by serial dilution with tryptic soy broth (TSB). Several concentrations of NH and AH were tested for inhibition of bacterial growth, viability, and biofilm formation after inoculation with S. mutans UA159 in 96-well microtiter plates to obtain absorbance and CFU values. Overall, NH supported significantly less (P < 0.05) bacterial growth than AH at 25 and 12.5% concentrations. At 50 and 25% concentrations, both honey groups provided significantly less bacterial growth and biofilm formation than the TSB control. For bacterial viability, the results for all honey concentrations except 50% NH were not significantly different from those for the TSB control. NH was able to decrease the maximum velocity of S. mutans growth compared to AH. In summary, NH demonstrated more inhibition of bacterial growth, viability, and biofilm formation than AH. This study highlights the potential antibacterial properties of NH and could suggest that the antimicrobial mechanism of NH is not solely due to its high sugar content.     

Peracetic acid activity on biofilm formed by Escherichia coli isolated from an industrial water system

One of the major problems in industrial water systems is the generation of biofilm, which is resistant to antimicrobial agents and causes failure of sanitization policy. This work aimed to study the anti-biofilm activity of peracetic acid (PAA) at contact times and temperatures combinations. To this end, a 96-well microtiter-based calorimetric method was applied in in vitro biofilm production using Escherichia coli, isolated from the water supply system of a pharmaceutical plant. The phenotypic and phylogenetic tests confirmed that the isolated bacteria belong to strains of Escherichia coli. The anti-biofilm activity of peracetic acid on formed biofilm was investigated at concentrations of 0·15–0·5% for a contact time of 5–15 min at 20–60°C. The maximum biofilm formation by MTP method using an Escherichia coli isolate was achieved in 96-h incubation in TSB containing wells at 37°C. Biofilm formation rate shown to be high by the environmental isolate compared with that of standard strain. PAA at concentrations above 0·25%, the temperature of 40°C and a minimum of 10 min of contact time was effective in the eradication of biofilm in an MTP-based system.     

Carbon Catabolite Control is Important for Listeria monocytogenes Biofilm Formation in Response to Nutrient Availability

The foodborne pathogen Listeria monocytogenes has the ability to develop biofilm in food-processing environment, which becomes a major concern for the food safety. The biofilm formation is strongly influenced by the availability of nutrients and environmental conditions, and particularly enhanced in poor minimal essential medium (MEM) containing glucose rather than in rich brain heart infusion (BHI) broth. To gain better insight into the conserved protein expression profile in these biofilms, the proteomes from biofilm- and planktonic-grown cells from MEM with 50?mM glucose or BHI were compared using two-dimensional polyacrylamide gel electrophoresis followed by MALDI-TOF/TOF analysis. 47 proteins were successfully identified to be either up (19 proteins) or down (28 proteins) regulated in the biofilm states. Most (30 proteins) of them were assigned to the metabolism functional category in cluster of orthologous groups of proteins. Among them, up-regulated proteins were mainly associated with the pentose phosphate pathway and glycolysis, whereas a key enzyme CitC involved in tricarboxylic acid cycle was down-regulated in biofilms compared to the planktonic states. These data implicate the importance of carbon catabolite control for L. monocytogenes biofilm formation in response to nutrient availability.     

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。     

Development of a biofilm model for Listeria monocytogenes EGD-e

The ability to form persistent biofilms makes the pathogenic bacterium Listeria monocytogenes a hazardous contaminant in food processing environments. Growth and biofilm formation of L. monocytogenes EGD-e were studied in defined medium (HTM) and in tryptic soy broth (TSB) with different supplements. TSB + 1% glucose gave optimal results. Using this medium, biofilm development on the model surface polystyrene (microtiter plate) was monitored by the standard crystal violet staining for adherent cells after bacterial cultivation for 24 and 48 h at five different temperatures (4, 18, 25, 30 and 37°C). In parallel, the matrix exopolysaccharide formed after 48 h of incubation was quantified by staining with ruthenium red. In both assays incubation at 30°C yielded the highest values. The formation of larger scale biofilms on dialysis membranes, placed on TSB agar with 1% glucose for 48 h, was studied by scanning electron microscopy. Contiguous and multilayered biofilms were observed at 18, 25, 30 and 37°C incubation temperature. The methodology is suitable for quantitative and microscopic studies and, in addition, yields sufficient cell mass for subsequent biochemical and molecular biological analyses.