Biofilm-positive Strains Research Articles

Process optimization of malachite green degradation by mixed biofilm positive bacteria: Application of Box–Behnken designs, RSM and whole genome sequences analysis

Bacterial biofilm positive strains recovered from engine oil polluted-soil was investigated for its capacity to remove malachite green (MG) in mineral salt solution. Box–Behnken designs combining four factors (glucose, yeast extract, temperature and agitation) with three levels (−1, 0, +1) was used to found the best biotic and abiotic conditions for MG removal. The biodegradation potential of the selected microbial consortium was also confirmed using analytical techniques. Then, whole genome sequencing (WGS) analysis were performed to detect genes related to biofilm formation and chemicals biodegradation. Our results showed that the selected strains (G5, G13 and G15) were able to form biofilm on congo red agar plates developing almost black, very black and almost black colonies respectively. Experimental results using Box-Behnken designs showed that decolorization rate of MG reach 89.35% in these optimized conditions: glucose (2%), yeast extract (1%), temperature (40 °C), and agitation (150 rpm). Molecular investigation revealed that the isolated strains were identified as Cronobacter sakazakii G5, Enterobacter hormaechei G13, and Klebsiella pneumoniae G15 based on the whole genome sequences . Whole genome sequences analysis demonstrated the presence of genes involved in biofilm formation as well as dyes and chemical detoxification. The findings presented here provide useful information for development an efficient microbial consortium, formed by adherent bacteria, able to remove dye from coloured wastewater.

Effect of Long-Term Use of Alcohol-Containing Handwashing Gels on the Biofilm-Forming Capacity of Staphylococcus epidermidis.

The spread of coronavirus disease 2019 (COVID-19) has promoted the use of hand sanitizers among the general population as recommended by health authorities. Alcohols, which are used in many hand sanitizers, have been shown to promotes the formation of biofilms by certain bacteria and to increase bacterial resistance to disinfection. We investigated the effect of continued use of alcohol-based gel hand sanitizer on biofilm formation by the Staphylococcus epidermidis resident strain isolated from the hands of health science students. Hand microbes were counted before and after handwashing, and the ability to produce biofilms was investigated. We found that 179 (84.8%) strains of S. epidermidis isolated from hands had the ability to form biofilm (biofilm-positive strains) in an alcohol-free culture medium. Furthermore, the presence of alcohol in the culture medium induced biofilm formation in 13 (40.6%) of the biofilm-negative strains and increased biofilm production in 111 (76.6%) strains, which were classified as low-grade biofilm-producing. Based on our findings, there is no clear evidence that the continued use of alcohol-based gels results in the selection of strains with the capacity to form biofilms. However, other disinfectant formulations that are more commonly used in clinical settings, such as alcohol-based hand-rub solutions, should be tested for their long-term effects.

Multi-Strain and -Species Investigation of Volatile Metabolites Emitted from Planktonic and Biofilm Candida Cultures.

Candida parapsiliosis is a prevalent neonatal pathogen that attains its virulence through its strain-specific ability to form biofilms. The use of volatilomics, the profiling of volatile metabolites from microbes is a non-invasive, simple way to identify and classify microbes; it has shown great potential for pathogen identification. Although C. parapsiliosis is one of the most common clinical fungal pathogens, its volatilome has never been characterised. In this study, planktonic volatilomes of ten clinical strains of C. parapsilosis were analysed, along with a single strain of Candida albicans. Headspace-solid-phase microextraction coupled with gas chromatography-mass spectrometry were employed to analyse the samples. Species-, strain-, and media- influences on the fungal volatilomes were investigated. Twenty-four unique metabolites from the examined Candida spp. (22 from C. albicans; 18 from C. parapsilosis) were included in this study. Chemical classes detected across the samples included alcohols, fatty acid esters, acetates, thiols, sesquiterpenes, and nitrogen-containing compounds. C. albicans volatilomes were most clearly discriminated from C. parapsilosis based on the detection of unique sesquiterpene compounds. The effect of biofilm formation on the C. parapsilosis volatilomes was investigated for the first time by comparing volatilomes of a biofilm-positive strain and a biofilm-negative strain over time (0–48 h) using a novel sampling approach. Volatilomic shifts in the profiles of alcohols, ketones, acids, and acetates were observed specifically in the biofilm-forming samples and attributed to biofilm maturation. This study highlights species-specificity of Candida volatilomes, and also marks the clinical potential for volatilomics for non-invasively detecting fungal pathogens. Additionally, the range of biofilm-specificity across microbial volatilomes is potentially far-reaching, and therefore characterising these volatilomic changes in pathogenic fungal and bacterial biofilms could lead to novel opportunities for detecting severe infections early.

S-alkylated thiosemicarbazone derivatives: Synthesis, crystal structure determination, antimicrobial activity evaluation and molecular docking studies

Increasing antimicrobial resistance is one of the most serious threats to human health worldwide. Therefore, there is an urgent need for the discovery of novel antimicrobial agents. Herein, we presented the synthesis of ten thiosemicarbazone derivatives (T1-T10) obtained by the reaction of S-alkylthiosemicarbazide with various dicarbonyl derivatives. The compounds were characterized by IR, 1H NMR, ESI-MS and X-ray crystallography. Reaction with the dicarbonyl compound bearing the 4-fluorobenzoyl group unexpectedly gave a pyrazole derivative (T8) containing the entire S-methylthiosemicarbazone backbone. We extensively screened these derivatives for their antimicrobial activities against Mycobacterium tuberculosis and various bacterial and Candida strains. Additionally, the biofilm inhibition capacity of T8 was evaluated on Staphylococcus epidermidis and Pseudomonas aeruginosa biofilm positive strains. To find out the potential mechanism of anti-biofilm activity against PAO1, the docking studies of T8 were carried out into the binding site of LasR, which is the main regulator of bacterial cell-to-cell communication system known as quorum sensing.

Quercetin Inhibits Biofilm Formation by Decreasing the Production of EPS and Altering the Composition of EPS in Staphylococcus epidermidis

Staphylococcus epidermidis is an opportunistic pathogen, and its biofilm formation ability is an important virulent factor. Quercetin, a typical flavonoid ubiquitously used in dietary supplementation, is known for its antioxidant property, but its anti-biofilm activity against S. epidermidis remains unknown. In this study, the anti-biofilm activity of quercetin was investigated using S. epidermidis ATCC35984, a strong biofilm-positive strain. An attempt was made to disclose the mechanisms of the anti-biofilm activity of quercetin. S. epidermidis exhibited a less cell surface hydrophobicity after quercetin treatment. Also, quercetin effectively inhibited S. epidermidis cells from adhering to the glass slides. Quercetin downregulated the intercellular adhesion (ica) locus and then polysaccharide intercellular adhesin (PIA) production was reduced. Therefore, S. epidermidis cells became less hydrophobic, which supported quercetin’s anti-biofilm effect. Our study suggests that quercetin from plants be given further attention as a potential anti-biofilm agent against the biofilm formation of S. epidermidis, even biofilm infections of other bacteria.

Inhibitory effect of trisodium citrate on biofilms formed by Klebsiella pneumoniae

ObjectivesKlebsiella pneumoniae is a significant nosocomial pathogen related to ventilator-associated pneumonia owing to biofilm formation. Trisodium citrate (TSC) has antibacterial activity, but there is little research on the effect of TSC on biofilm formed by K. pneumoniae. The aims of this study were to evaluate the inhibitory effect of 4% TSC on K. pneumoniae biofilm formation and to determine the best time of TSC addition for biofilm inhibition. MethodsA total of 45 K. pneumoniae strains isolated from tracheal tip and sputum specimens were included. Modified Congo red agar was used to screen for biofilm production. Biofilm-positive strains were cultured for 4 days. TSC (4%) was added either initially or 3 days later. Crystal violet staining was used to quantify biofilm mass by measuring the optical density at 570 nm (OD570). Scanning electron microscopy (SEM) was used to observe biofilm morphology. ResultsThe OD570 was significantly lower in the 4% TSC group than that in the no-TSC group during the 4-day experiment. Compared with addition of TSC after 3 days, initial TSC addition resulted in a significant absorbance decrease (Day 4, 0.63 ± 0.11 later-TSC group vs. 0.41 ± 0.16 initial-TSC group). As observed by SEM, bacteria were stacked most densely in the no-TSC group on Day 4. In contrast, few bacteria were observed when TSC was added initially, whilst bacteria were obviously dispersed when TSC was added after 3 days. ConclusionTSC can inhibit K. pneumoniae biofilm formation and has the best effect when added initially.

Identification of ability to form biofilm in Candida parapsilosis and Staphylococcus epidermidis by Raman spectroscopy.

Aim: Finding rapid, reliable diagnostic methods is a big challenge in clinical microbiology. Raman spectroscopy is an optical method used for multiple applications in scientific fields including microbiology. This work reports its potential in identifying biofilm positive strains of Candida parapsilosis and Staphylococcus epidermidis. Materials & methods: We tested 54S. epidermidis strains (23 biofilm positive, 31 negative) and 51C. parapsilosis strains (27 biofilm positive, 24 negative) from colonies on Mueller-Hinton agar plates, using Raman spectroscopy. Results: The accuracy was 98.9% forC. parapsilosis and 96.1% forS. epidermidis. Conclusion: The method showed great potential for identifying biofilm positive bacterial and yeast strains. We suggest that Raman spectroscopy might become a useful aid in clinical diagnostics.

Dynamic observation and antibiotic susceptibility analysis of biofilm of Staphylococcus aureus isolated from clinical specimens

Objective To analyze the correlation between antibiotic susceptibility andbiofilm formation of Staphylococcus aureus. Methods According to Standardized Operational Guidance for Clinical Microbiological Testing, fifty-eight non-repetitive pathogenic Staphylococcus aureus isolates were collected from blood, urine, sputum and purulent secretions of inpatients in the Laboratory Department of the First Affiliated Hospital of Guangxi Medical University in January 2018. The antibiotics susceptibility of strains was characterized by disk diffusion method and VITEK-2.96-hole culture. The biofilm formation ability was detected bythe crystal violet assay and Congo redplate methods.The in vitro dynamic forming process of S.aureus′sbiofilm was analyzed by crystal violet staining combined with bacterial culture in 96 wells.Besides, the rate differences of drug resistance between biofilm producers and non-producers was performed by Chi-square test. The diameter of bacteriostasis zone of biofilm producers and non-producers was compared by t test. The drug resistance among strains with different biofilm formation ability was analyzed withnonparametric rank sum test. Results The positive ratio of biofilm producing S.aureus was higher in MRSA (68.42%) than in MSSA(20.00%) (χ2=12.304,P=0.001. Antibiotic resistance rates of biofilm producers were higher than non-producers.The resistance rates of biofilm positive strains to oxacillin and clindamycin were 73.33% and 53.33%, respectively. The antibiotic resistance of the strain was higher along with the biofilm forming ability was increasing.(χ2=9.099,P=0.008). Depths of the S.aureus′s biofilm on the 96-well plates increased significantly over time and reached biofilm maturation after 72 hours′ incubation. Light microscopic observation revealed that the mature biofilm was compact and growing with many layers. Conclusions For the clinical isolates of S.aureus, the antibiotic resistance of biofilm producers is significantly higher than non-producers. Strains with stronger biofilm forming ability had higher antibiotics resistance. Depths of the S.aureus′s biofilm on the 96-well plates increased significantly over time and reached biofilm maturation after 72 hours′ incubation. Key words: Staphylococcus aureus; Biofilms; Microbial sensitivity tests; Methicillin-resistant staphylococcus aureus

Comparison of biofilm-producing Enterococcus faecalis, Enterococcus faecium, and unusual Enterococcus strains

The present study focused on determining the prevalence of biofilm-forming ability in Enterococcus faecalis , E. faecium , and unusual Enterococcus clinical isolates, and comparison of resistance and the prevalence of selected virulence factors among biofilm-positive strains. The ability to form biofilm was detected in 13.3% of E. faecalis , 90% of E. faecium , and 57.1% of unusual Enterococcus strains ( p =0.026). All E. faecalis strains were susceptible to β-lactams, while 37.5% of unusual and all E. faecium isolates were resistant to these antibiotics. Resistance to gentamicin was detected in 75% of E. faecalis , 55.5% of E. faecium , and 25% of other strains; resistance to streptomycin in 25%, 83.3%, and 50%, respectively. Analysis of the virulence revealed that the enterococcal surface protein ( esp ) gene was found in all E. faecium , 75.0% of E. faecalis , and 37.5% of other strains; collagen adhesin gene ( ace ) in 100%, 25.0%, and 37.5%; and hyaluronidase gene ( hyl ) in 83.3%, 0%, and 37.5%, respectively. Analysis of the resistance and virulence patterns showed that E. faecium isolates had the greatest variety of virulence and resistance determinants, while the lowest variety was exhibited by unusual strains. These findings indicate that unusual biofilm-producing Enterococcus strains have lower resistance and virulence potency than E. faecalis and E. faecium .

In vitro ability of mastitis causing pathogens to form biofilms.

This Research Communication describes the study of in vitro biofilm formation of mastitis causing pathogens. Biofilms are communities of bacteria that are attached to a surface and to each other and are embedded in a self-produced matrix of extracellular polymeric substances. Biofilm formation is an important virulence factor that may result in recurrent or persistent udder infections and treatment failure through increased resistance to antibiotics and protection against host defences. In the present study 252 bacterial isolates from milk samples from bovine udder quarters with intramammary infections were examined with Congo Red agar (CRA) method and tube method (TM) for their ability to form biofilms. Both tests revealed a high number of biofilm-positive strains. Literature reports that the cure rates for Staphylococcus aureus infected udders are lower (27%) in comparison to cure rates of Streptococcus uberis (64-81%) or coagulase-negative staphylococci (CNS) mastitis (80-90%). The findings of the present study suggest that biofilm formation is not the main factor for the differences in cure rates of the various bacteria genera, because all tested pathogen groups showed a similarly high proportion of biofilm formation. Further research is needed to detect microbial biofilms on bovine udder epithelia.

Interaction of nanostructured TiO2 biointerfaces with stem cells and biofilm-forming bacteria

Nanostructured TiO2 nanotubes (NTs) of diameters from 15 to 100nm were fabricated by an electrochemical anodization process. Biofilm-positive strains of Bacillus cereus and Pseudomonas aeruginosa behaved similarly on all TiO2 NTs as well as on native titanium (Ti) foil. The adhesion and growth of mesenchymal stem cells (MSc), embryonic stem cells (ESc), and pure cardiomyocytes derived from ESc exhibited significant differences. MSc as well as ESc were, in contrast to cardiomyocytes, able to adhere, and grow on TiO2 NTs. A correlation between NTs diameter and cell behaviour was however observed in the case of MSc and ESc. MSc were not in a physiological state in the case of 100nm TiO2 NTs, while ESc were not able to grow on 15nm TiO2 NTs. It can be stated that these differences can be assigned to different diameters of the NTs but not to the chemistry of the surface. This is the first study describing the comprehensive behaviour of both eukaryotic and prokaryotic cells on TiO2 NTs. On the basis of obtained results, it can be concluded that new generation of medical devices providing selective cell behaviour can be fabricated by optimizing the nanoscale morphology of TiO2.

Prevalence and leukotoxic profile of biofilm forming Staphylococcal strains isolated from a tertiary care hospital in Mysore

Background: Staphylococci causes chronic skin infections, device associated infections, ear infections, bone infections and post-operative infections. It secretes toxins such as Pantone-Valentine Leukocidin which prolongs the infection in hospitalized patients. Aim: To investigate the prevalence of Staphylococcal biofilm associated strains and to understand the correlation between biofilm production, multi-drug resistance and leukotoxicity. Methods: 280 Staphylococcal clinical isolates obtained from biofilm associated infections are collected from a tertiary care hospital in Mysore. They were screened for the production of biofilm, multi-drug resistance and leukotoxicity. They were also screened for icaAB and PVL luk F/S genes by PCR. Correlation between leuktoxicity and biofilm production was established by finding the leukotoxic potential of biofilm positive strains. Findings: Among 280 Staphylococcal clinical isolates, 34.6% showed biofilm production. Out of these isolates, 25% showed the presence of icaAB genes and 29% showed the presence of PVL luk F/S genes. Presence of icaAB gene was found to be higher in isolates obtained from implant associated infections. But the prevalence of PVL luk F/S genes was higher in chronic skin infections. 78% of the isolates displayed multi-drug resistance and methicillin resistant strains were comparitively high in device associated infections (77%). Strong biofilm producing PVL positive isolates from chronic skin infections showed increased leucotoxicity compared to PVL negative, weak and moderate biofilm producers. Conclusion: Statistical analysis revealed that PVL in association with biofilms can promote higher leukotoxicity compared to non biofilm producers. This study foresee the essential role of biofilm production, multi-drug resistance and toxins for the increased Staphylococcal pathogenesis.

Coagulase-negative staphylococci: pathogenesis, occurrence of antibiotic resistance genes and in vitro effects of antimicrobial agents on biofilm-growing bacteria.

Coagulase-negative staphylococci (CoNS) are opportunistic pathogens that particularly cause infections in patients with implanted medical devices. The present research was performed to study the virulence potential of 53 clinical isolates of Staphylococcus capitis, Staphylococcus auricularis, Staphylococcus lugdunensis, Staphylococcus simulans, Staphylococcus cohnii and Staphylococcus caprae. All clinical strains were clonally unrelated. Isolates carried genes encoding resistance to β-lactam (mecA) (15 %), aminoglycoside [aac(6')/aph(2″)(11 %), aph (3')-IIIa (15 %), ant(4')-Ia (19 %)] and macrolide, lincosamide and streptogramin B (MLSB) [erm(A) (4 %), erm(B) (13 %), erm(C) (41 %), msr(A) (11 %)] antibiotics. CoNS isolates (64 %) were able to form biofilms. Confocal laser scanning microscopy revealed that these biofilms formed a three-dimensional structure composed mainly of living cells. All biofilm-positive strains carried the ica operon. In vitro studies demonstrated that a combination treatment with tigecycline and rifampicin was more effective against biofilms than one with ciprofloxacin and rifampicin. The minimum biofilm eradication concentration values were 0.062-0.5 µg ml-1 for tigecycline/rifampicin and 0.250-2 µg ml-1 for ciprofloxacin/rifampicin. All CoNS strains adhered to the human epithelial cell line HeLa, and more than half of the isolates were able to invade the HeLa cells, although most invaded relatively poorly. The virulence of CoNS is also attributed to their cytotoxic effects on HeLa cells. Incubation of HeLa cells with culture supernatant of the CoNS isolates resulted in cell death. The results indicate that the pathogenicity of S. capitis, S. auricularis, S. lugdunensis, S. cohnii and S. caprae is multi-factorial, involving the ability of these bacteria to adhere to human epithelial cells, form biofilms and invade and destroy human cells.

Biofilm Producing Staphylococcus epidermidis Strains Isolated From Clinical Samples in Tehran, Iran

Background: Staphylococcus epidermidis is well documented as an opportunistic pathogen causing biofilm in patients and healthy individuals. Objectives: The aim of this experimental study was to describe the antibiotic resistance patterns of biofilm producing S. epidermidis strains isolated from clinical samples in Tehran, Iran. Moreover, the role of different genes in biofilm formation was also described. Patients and Methods: A total of 250 S. epidermidis strains were isolated from patients in a private hospital of Tehran, Iran from February to December 2014. The biofilm formation of each strain was determined using combination of qualitative Congo-Red agar and quantitative microtiter plate assay, and presence of different genes involved in control and formation of biofilm was detected by the polymerase chain reaction (PCR). Susceptibility of S. epidermidis strains to 19 antibiotics was examined. Results: The results of the biofilm assay revealed that 82% of strains produced black colonies on Congo red agar plates and 68% were able to attach strongly to polystyrene microplates. One hundred percent, 88%, 84%, 64% and 60% of biofilm-producing strains were resistant to penicillin, cefoxitin, erythromycin, trimethoprim-sulfamethoxazole and kanamycin, respectively. On the other hand, none of the strains showed resistance to vancomycin, linezolid, quinupristin/dalfopristin. The icaA, icaD, aap and atlE genes were detected in all biofilm-producing strains and presence of IS256 transposon was limited to 84% of biofilm positive strains. Conclusions: The results of this study illustrated the high prevalence of antibiotic resistant biofilm-producing S. epidermidis strains in this hospital, which could be a reservoir for antibiotic resistance genes.

Therapeutic effects of a combined antibiotic-enzyme treatment on subclinical mastitis in lactating dairy cows

The objective of this study was to evaluate a combined antibiotic-enzyme therapy for Staphylococcus aureus mastitis and biofilm formation. A total of 141 cases of S. aureus chronic mastitis from three farms were divided into two groups: the control group (n = 54) were treated with Nafpenzal® ointment; the enzyme + antibiotic group (n = 87) were treated with Nafpenzal® plus an enzymatic ointment (MastiVeyxym®). Quantitative determination of biofilm formation was determined using a colorimetric microplate assay and the detection of ica genes by PCR. Enzyme + antibiotic therapy did not significantly improve cure rates compared to control (48.3 vs 38.9%). The cure rate for infections caused by biofilm-positive strains was 42.6 and 46.6% for control and enzyme + antibiotic groups, respectively (P > 0.05). Comparison of cure rates between farms showed a relationship with somatic cell count (SCC), parity and oxacillin resistance. 79.4% of the isolates produced biofilm and antibacterial resistance rates for oxacillin, penicillin and streptomycin were 25.5, 71.6 and 95.7%, respectively. These results indicate that while the increase in mastitis cure rate using enzymatic therapy was not significant, this treatment could be useful in some situations.

Biofilm formation by blood stream staphylococcal isolates from febrile neutropenic cancer patients

Objectives: The aim of this study was to define the main causative organisms of bacteremia in febrile neutropenic patients and to study the prevalence of biofilm forming capability of staphylococci in blood isolates of these patients. Background: Blood stream infections remain the main cause of morbidity and death in patients undergoing treatment for cancer. Researchers showed that the first step of staphylococcal infection is the attachment to surfaces of various materials and biofilm formation. Biofilms render the cells less accessible to the defense system of the host, thus impairing the action of antibiotics. Therefore, methods identifying strains with a capacity for biofilm formation are necessary to develop effective strategies for biofilm control and improve patient care. Patients and methods: The study included 185 participants divided into two groups; group I included 155 cancer patients and group II included 30 healthy participants as control. The participants were subjected to the following: medical history, blood cultures, antimicrobial susceptibilities of the staphylococcal isolates, Congo red agar (CRA) test, and microtiter plate (MTP) test. Results: Positive blood culture represented 33% of cases; of them, gram-positive bacteria represented 78.5% (40 cases staphylococci): 17 cases were of Staphylococcus aureus and 23 cases were of Staphylococcus epidermidis . Biofilm-positive strains of S. aureus were 4/17 (23.6%) when assessed with CRA and 6/17 (35.3%) when assessed with MTP. Biofilm-positive strains of S. epidermidis were 7/23 (30.4%) when assessed with CRA and 10/23 (43.5%) when assessed with MTP. Most strains that form biofilm were more resistant to most antibiotics compared with other strains. Conclusion: There is an association between biofilm production with persistent infection and antibiotic therapy failure. The MTP is the test of choice in detecting biofilm formation among staphylococci.

Enterococcus--virulence and susceptibility to photodynamic therapy of clinical isolates from Lower Silesia, Poland.

A collection of 400 enterococcal isolates from clinical samples of hospitalized patients were studied for their virulence traits according to the isolation site. Formation of biofilm and production of DNase, hemolysin, lipase, and gelatinase were characterized. Biofilms of selected strains were examined for their susceptibility to antimicrobial photodynamic therapy (aPDT). All strains were tested for biofilm production by microtiter method and the activity of hemolysin, gelatinase, lipase, and DNase by plate method with an adequate substrate. Photodynamic therapy with Photolon and red laser light was performed towards 48 h biofilms of eight representative strains. The viability of biofilms was tested by the BactLight assay and visualized under a fluorescent microscope. Among the 400 isolates, 69.8% strains of Enterococcus faecalis, 30% of Enterococcus faecium, and 0.2% of Enterococcus casseliflavus were identified. In vitro, production of biofilm was found in 65.7% of enterococci. Biofilm-positive strains were isolated from urinary tract infections (81%), wound infections (71%), respiratory tract infections (62%), and gastrointestinal tract (47%) (colonization). Hemolysin activity was observed in 28.5%, gelatinase in 24.5%, lipase in 23%, and DNase in 3.5% of all, mostly biofilm-positive, isolates. Photodynamic therapy with Photolon efficiently reduced the enterococcal biofilms. The study demonstrated the high prevalence of biofilm-producing clinical enterococci, their virulence potential being higher than for biofilm-negative strains, and the susceptibility to aPDT of mature biofilms produced by strains, regardless of their species and site of isolation.

Catheter-related infections caused by Pseudomonas aeruginosa: virulence factors involved and their relationships.

The nosocomial pathogen Pseudomonas aeruginosa is equipped with a large arsenal of cell-associated and secreted virulence factors which enhance its invasive potential. The complex relationships among virulence determinants have hitherto not been fully elucidated. In the present study, 175 catheter-related isolates were observed for the presence of selected virulence factors, namely extracellular enzymes and siderophore production, biofilm formation, resistance to antibiotics, and motility. A high percentage of the strains produced most of the tested virulence factors. A positive correlation was identified between the production of several exoproducts, and also between the formation of both types of biofilm. An opposite trend was observed between the two types of biofilm and the production of siderophores. Whereas the relationship between the submerged biofilm production (i.e. the biofilm formed on the solid surface below the water level) and the siderophore secretion was negative, the production of air-liquid interface (A-L) biofilm (i.e. the biofilm floating on the surface of the cultivation medium) and the siderophore secretion were positively correlated. All correlations were statistically significant at the level P = 0.05 with the correlation coefficient γ ≥ 0.50. Our results suggest that: (1) the co-production of the lytic enzymes and siderophores can play an important role in the pathogenesis of the catheter-related infections and should be taken into account when the virulence potential is assessed; (2) biofilm-positive strains are capable of forming both submerged and non-attached A-L biofilms; and (3) the different micro-environment in the submerged biofilm and A-L biofilm layers have opposite consequences for the production of other virulence factors.

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.

Comparison of various methods used to detect biofilm production of Candida species.

The biofilm of organisms can be considered as a virulence factor because of the resistance towards antimicrobial agents. Formation of Candida biofilms are observed in blood, mucosal surface and most medical devices i.e. nonliving objects in patient's body. The study was undertaken to conclude the most sensitive and specific test for detection of biofilm formation. Quantitative measurement of biofilm formation was assessed by three methods- 1) XTT reductionassay, 2) Percentage Transmission (%T) and 3) Crystal Violet assay in microtitre plate for 425 Candida isolates. Out of 425 Candida strains, XTT reduction assay gave 72 Biofilm positive strains accounting for 16.94 %. The percentage transmission (% T) method gave 56 strains Biofilm positive (13.17 %) and Crystal violet assay gave 50 positive strains (11.76 %). Compared to Percentage Transmission (% T) and Crystal violet method, XTT reduction assay gave maximum percentage of Biofilm positivity. In comparison of 3 methods used for detection of biofilm formation, XTT reduction assay was the most sensitive and specific method.