Crystal Violet Staining Method Research Articles

Assessment of biofilm formation by Campylobacter spp. isolates mimicking poultry slaughterhouse conditions

This research aimed to assess the biofilm formation ability of Campylobacter strains under temperature and oxygen stress conditions, similar to those found in the industrial environment, to explain the persistence of this pathogen on the poultry slaughter line. A collection of C. jejuni and C. coli isolates (n = 143) obtained from poultry samples (cecal content and neck skin), collected at slaughterhouse level, from diverse flocks, on different working days, was genotyped by flaA-restriction fragment length polymorphism (RFLP) typing method. A clustering analysis resulted in the assignment of 10 main clusters, from which 15 strains with different flaA-RFLP genotypes were selected for the assessment of biofilm formation ability and antimicrobial susceptibility. Biofilm assays, performed by crystal violet staining method, were conducted with the goal of mimicking some conditions present at the slaughterhouse environment, based on temperature, atmosphere, and contamination levels. Results indicated that many C. jejuni strains with similar flaA-RFLP profiles were present at the slaughterhouse on different processing days. All the strains tested (n = 15) were multidrug-resistant except for one. Biofilm formation ability was strain-dependent, and it appeared to have been affected by inoculum concentration, temperature, and tolerance to oxygen levels. At 10°C, adherence levels were significantly lower than at 42°C. Under microaerobic and aerobic atmospheres, at 42°C, 3 strains (C. jejuni 46E, C. jejuni 61C, and C. coli 65B) stood out, exhibiting significant levels of biofilm formation. C. jejuni strains 46E and 61C were inserted in clusters with evidence of persistence at the slaughterhouse for a long period of time. This study demonstrated that Campylobacter strains from broilers are capable of forming biofilms under conditions resembling the slaughterhouse environment. These results should be seen as a cue to improve the programs of hygiene implemented, particularly in those zones that can promote biofilm formation.

Biofilm formation by Non-O157 Shiga toxin-producing Escherichia coli in monocultures and co-cultures with meat processing surface bacteria

This study investigated the impact of meat processing surface bacteria (MPB) on biofilm formation by non-O157 Shiga toxin-producing Escherichia coli (STEC), and potential links between biofilm formation by STEC and biofilm-related genes in their genomes. Biofilm development by 50 MPB and 6 STEC strains in mono- and co-cultures was assessed by the crystal violet staining method, and their expression of curli and cellulose was determined using the Congo red agar method. Genes (n = 141) associated with biofilm formation in the STEC strains were profiled. Biofilm formation in general correlated with cellulose and curli expression in both mono- and co-cultures. Most MPB strains had antagonistic effects on the biofilm formation of the STEC strains. Of the genes investigated, 81% were common among the STEC strains and there seems to be a gene-redundancy in biofilm formation. The inability of the O26 strain to form biofilms could be due to mutations in the rpoS gene. Truncation in the mlrA gene in the O145 strain seems not affecting its biofilm formation alone or with MPB. The O45 strain, despite having the greatest number of biofilm-related genes, did not form measurable biofilms. Overall, biofilm formation of STEC was affected by curli-cellulose expression and companion strains.

Conditions of In Vitro Biofilm Formation by Serogroups of Listeria monocytogenes Isolated from Hass Avocados Sold at Markets in Mexico.

Listeria monocytogenes is an important pathogen that has been implicated in foodborne illnesses and the recall of products such as fruit and vegetables. This study determines the prevalence of virulence-associated genes and serogroups and evaluates the effects of different growth media and environmental conditions on biofilm formation by L. monocytogenes. Eighteen L. monocytogenes isolates from Hass avocados sold at markets in Guadalajara, Mexico, were characterized by virulence-associated genes and serogroup detection with PCR. All isolates harbored 88.8% actA, 88.8% plcA, 83.3% mpl, 77.7% inlB, 77.7% hly, 66.6% prfA, 55.5% plcB, and 33.3% inlA. The results showed that 38.8% of isolates harbored virulence genes belonging to Listeria pathogenicity island 1 (LIPI-1). PCR revealed that the most prevalent serogroup was serogroup III (1/2b, 3b, and 7 (n = 18, 66.65%)), followed by serogroup IV (4b, 4d–4e (n = 5, 27.7%)) and serogroup I (1/2a–3a (n = 1, 5.5%)). The assessment of the ability to develop biofilms using a crystal violet staining method revealed that L. monocytogenes responded to supplement medium TSBA, 1/10 diluted TSBA, and TSB in comparison with 1/10 diluted TSB (p < 0.05) on polystyrene at 240 h (p < 0.05). In particular, the biofilm formation by L. monocytogenes (7.78 ± 0.03–8.82 ± 0.03 log10 CFU/cm2) was significantly different in terms of TSBA on polypropylene type B (PP) (p < 0.05). In addition, visualization by epifluorescence microscopy, scanning electron microscopy (SEM), and treatment (DNase I and proteinase K) revealed the metabolically active cells and extracellular polymeric substances of biofilms on PP. L. monocytogenes has the ability to develop biofilms that harbor virulence-associated genes, which represent a serious threat to human health and food safety.

Suppressive effects of gecko cathelicidin on biofilm formation and cariogenic virulence factors of Streptococcus mutans

ObjectiveThe aims of this study were to investigate the effectivity of gecko cathelicidin Gj-CATH2 on biofilm formation and cariogenic virulence factors of S. mutans, and preliminary explore its function mechanisms. DesignMinimum inhibitory concentration and bacterial killing kinetics assays were performed to assess the antimicrobial effect of Gj-CATH2.The influence of Gj-CATH2 on S. mutans biofilm formation was determined by crystal violet staining method and observed by SEM. The effects of Gj-CATH2 on exopolysaccharides (EPS) synthesis, bacterial aggregation, acidogenicity and aciduricity of S. mutans were also investigated. Quantitative real-time PCR was conducted to acquire the expression profile of related genes. ResultsGj-CATH2 showed strong bactericidal and anti-biofilm effects on S. mutans. SEM confirmed the reduction of the dense structure in S. mutans biofilm in Gj-CATH2-treated groups. Gj-CATH2 significantly inhibited EPS synthesis, cell aggregation, acid production of S. mutans, but showed no influence on its acid proof. Furthermore, the expression of genes related to biofilm formation (gtfB/C/D, gbpB/D), quorum sensing system (luxS and comD/E) and acidogenicity (ldh) was significantly suppressed by Gj-CATH2. Gj-CATH2 also displayed advantageous resistance in human saliva and exhibited negligible toxicity against mammalian cells. ConclusionsGj-CATH2 inhibited S. mutans biofilm formation by targeting the bacterial adhesion and the biofilm maturation stages. Gj-CATH2 significantly suppressed virulence factors production of S. mutans, resulting in decreased EPS synthesis and reduced acidogenicity of bacteria. These findings suggest Gj-CATH2 might be a promising agent for clinical application in prevention of dental caries.

Phenotypic and genotypic characterization of mcr-1-positive multidrug-resistant Escherichia coli ST93, ST117, ST156, ST10, and ST744 isolated from poultry in Poland

BackgroundA plasmid-mediated mechanism of bacterial resistance to polymyxin is a serious threat to public health worldwide. The present study aimed to determine the occurrence of plasmid-mediated colistin resistance genes and to conduct the molecular characterization of mcr-positive Escherichia coli strains isolated from Polish poultry.MethodsIn this study, 318 E. coli strains were characterized by the prevalence of mcr1–mcr5 genes, antimicrobial susceptibility testing by minimal inhibitory concentration method, the presence of antimicrobial resistance genes was screened by PCR, and the biofilm formation ability was tested using the crystal violet staining method. Genetic relatedness of mcr-1-positive E. coli strains was evaluated by multilocus sequence typing method.ResultsAmong the 318 E. coli isolates, 17 (5.35%) harbored the mcr-1 gene. High antimicrobial resistance rates were observed for ampicillin (100%), tetracycline (88.24%), and chloramphenicol (82.35%). All mcr-1-positive E. coli strains were multidrug-resistant, and as many as 88.24% of the isolates contained the blaTEM gene, tetracycline (tetA and tetB), and sulfonamide (sul1, sul2, and sul3) resistance genes. Additionally, 41.18% of multidrug-resistant, mcr-1-positive E. coli isolates were moderate biofilm producers, while the rest of the strains showed weak biofilm production. Nine different sequence types were identified, and the dominant ST was ST93 (29.41%), followed by ST117 (17.65%), ST156 (11.76%), ST 8979 (11.76%), ST744 (5.88%), and ST10 (5.88%). Moreover, the new ST was identified in this study.ConclusionsOur results showed a low occurrence of mcr-1-positive E. coli strains isolated from Polish poultry; however, all the isolated strains were resistant to multiple antimicrobial agents and were able to form biofilms at low or medium level.

Study of the impact of cultivation conditions and peg surface modification on the in vitro biofilm formation of Staphylococcus aureus and Staphylococcus epidermidis in a system analogous to the Calgary biofilm device.

Introduction. Staphylococcus aureus (SA) and Staphylococcus epidermidis (SE) are the most common pathogens from the genus Staphylococcus causing biofilm-associated infections. Generally, biofilm-associated infections represent a clinical challenge. Bacteria in biofilms are difficult to eradicate due to their resistance and serve as a reservoir for recurring persistent infections.Gap Statement. A variety of protocols for in vitro drug activity testing against staphylococcal biofilms have been introduced. However, there are often fundamental differences. All these differences in methodical approaches can then be reflected in the form of discrepancies between results.Aim. In this study, we aimed to develop optimal conditions for staphylococcal biofilm formation on pegs. The impact of peg surface modification was also studied.Methodology. The impact of tryptic soy broth alone or supplemented with foetal bovine serum (FBS) or human plasma (HP), together with the impact of the inoculum density of bacterial suspensions and the shaking versus the static mode of cultivation, on total biofilm biomass production in SA and SE reference strains was studied. The surface of pegs was modified with FBS, HP, or poly-l-lysine (PLL). The impact on total biofilm biomass was evaluated using the crystal violet staining method and statistical data analysis.Results. Tryptic soy broth supplemented with HP together with the shaking mode led to crucial potentiation of biofilm formation on pegs in SA strains. The SE strain did not produce biofilm biomass under the same conditions on pegs. Preconditioning of peg surfaces with FBS and HP led to a statistically significant increase in biofilm biomass formation in the SE strain.Conclusion. Optimal cultivation conditions for robust staphylococcal biofilm formation in vitro might differ among different bacterial strains and methodical approaches. The shaking mode and supplementation of cultivation medium with HP was beneficial for biofilm formation on pegs for SA (ATCC 29213) and methicillin-resistant SA (ATCC 43300). Peg conditioning with HP and PLL had no impact on biofilm formation in either of these strains. Peg coating with FBS showed an adverse effect on the biofilm formation of these strains. By contrast, there was a statistically significant increase in biofilm biomass production on pegs coated with FBS and HP for SE (ATCC 35983).

Molecular characteristics of antibiotic-resistant Escherichia coli and Klebsiella pneumoniae strains isolated from hospitalized patients in Tehran, Iran

BackgroundWe evaluated the distribution of carbapenem and colistin resistance mechanisms of clinical E. coli and K. pneumoniae isolates from Iran.Methods165 non-duplicate non-consecutive isolates of K. pneumoniae and E. coli were collected from hospitalized patients admitted to Iran's tertiary care hospitals from September 2016 to August 2018. The isolates were cultured from different clinical specimens, including wound, urine, blood, and tracheal aspirates. Antibiotic susceptibility testing was performed by disc diffusion and microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) guideline. The presence of extended spectrum β-lactamases (ESBLs) genes, carbapenemase genes, as well as fosfomycin resistance genes, and colistin resistance genes was also examined by PCR-sequencing. The ability of biofilm formation was assessed with crystal violet staining method. The expression of colistin resistance genes were measured by quantitative reverse transcription-PCR (RT-qPCR) analysis to evaluate the association between gene upregulation and colistin resistance. Genotyping was performed using the multi-locus sequencing typing (MLST).ResultsColistin and tigecycline were the most effective antimicrobial agents with 90.3% and 82.4% susceptibility. Notably, 16 (9.7%) isolates showed resistance to colistin. Overall, 33 (20%), 31 (18.8%), and 95 (57.6%) isolates were categorized as strong, moderate, and weak biofilm-producer, respectively. Additionally, blaTEM, blaSHV, blaCTX-M, blaNDM-1, blaOXA-48-like and blaNDM-6 resistance genes were detected in 98 (59.4%), 54 (32.7%), 77 (46.7%), 3 (1.8%), 17 (10.30%) and 3 (1.8%) isolates, respectively. Inactivation of mgrB gene due to nonsense mutations and insertion of IS elements was observed in 6 colistin resistant isolates. Colistin resistance was found to be linked to upregulation of pmrA-C, pmrK, phoP, and phoQ genes. Three of blaNDM-1 and 3 of blaNDM-6 variants were found to be carried by IncL/M and IncF plasmid, respectively. MLST revealed that blaNDM positive isolates were clonally related and belonged to three distinct clonal complexes, including ST147, ST15 and ST3299.ConclusionsThe large-scale surveillance and effective infection control measures are also urgently needed to prevent the outbreak of diverse carbapenem- and colistin-resistant isolates in the future.

Biofilm Formation by Multidrug-Resistant Serotypes of Salmonella Isolated from Fresh Products: Effects of Nutritional and Environmental Conditions

Salmonella serotypes can develop biofilms in fresh food products. This study focused on determining the antimicrobial resistance profile and the effects of different growth media and environmental conditions on biofilm formation by multidrug-resistant serotypes of Salmonella. All 49.4% of the Salmonella strains (five serotypes) were multidrug resistant. Assessment of the ability to form biofilms using the crystal violet staining method revealed that 95.6% of the strains of Salmonella were strong biofilm producers in 96-well polystyrene microtiter plates. Overall, 59.3% of the Salmonella strains showed the rdar (red dry and rough colony) morphotype, 2.1% pdar (pink dry and rough colony), 27.4% bdar (brown dry and rough colony) and 10.9% saw (smooth and white colony), at two temperatures (22 and 35 °C). Mono-species biofilms of Salmonella serotypes showed a mean cell density of 8.78 log10 CFU/cm2 ± 0.053 in TSBS (1/20 diluted TSB (tryptic soy broth) + 1% strawberry residues) and 8.43 log10 CFU/cm2 ± 0.050 in TSBA (1/20 diluted TSB + 1% avocado residues) on polypropylene type B (PP) (p &lt; 0.05). In addition, epifluorescence microscopy and scanning electron microscopy (SEM) enabled visualizing the bacteria and extracellular polymeric substances of biofilms on PP. Salmonella form biofilms depending on the serotype of the strains and the environmental conditions. Mono-species biofilms formed by Salmonella serotypes respond to nutrient limitation with the use of simplified culture media such as TSBA and TSBS.

Adaptation of Congo Red Agar Method and Microtiter Plate Assay to Study Biofilm Formation in Streptomyces

Streptomyces has many advantages for exploration in biotechnological applications because of their ability to elaborate a multitude of bioactive molecules and secondary metabolites. Despite the importance of this genus in biotechnology, biofilm formation in Streptomyces is under-investigated. The objective of this research is to adapt two assays for the assessment of biofilm formation in Streptomyces. In the present investigation, we assess and follow biofilm formation in eight Streptomyces strains using quantitative and qualitative methods. The quantitative study based on a staining of the retained biomass in the microtiter plate with crystal violet “5%” and destaining using ethanol/acetone mixture, the concentration of crystal violet in the alcoholic solution reflect the intensity of the attached biofilm. On the other hand, the qualitative one consists of using modified freeman’s method a modified congo red agar method based on the color of colonies. Quantification of biomass by crystal violet staining method confirmed that Streptomyces bellus A43 and Streptomyces bellus A61 are biofilm-forming and this ability increase with the period of incubation. Our results showed that sixStreptomyces strains arenon-slime producing/non-biofilm forming. Two Streptomyces strains are slime producing/biofilm forming; this character vanishes at five days. Further research on genes responsible for biofilm formation in Streptomyces is highly recommended for better understanding of the phenomenon.

Study on the Inhibitory Activity and Possible Mechanism of Myriocin on Clinically Relevant Drug-Resistant Candida albicans and Its Biofilms.

In order to prevent and control the infection of Candida albicans, the antifungal activity, possible mechanism of myriocin against C. albicans and its biofilm were studied. The antifungal activity of myriocin was investigated by microdilution method. The effect of myriocin on fungal cell wall or membrane was evaluated by adding sorbitol, ergosterol or phytosphingosine (PHS). The damage to the cell membrane was investigated with propidium iodide (PI) staining and visualized by scanning electron microscope (SEM). The effects on biofilms and extracellular polysaccharides (EPS) were observed by crystal violet staining method and phenol-sulfuric acid method respectively. The adhesion of C. albicans cells to hydrocarbons was tested to evaluate cell surface hydrophobic (CSH). The combined effects of myriocin and antifungal drugs commonly used in clinical practice were investigated by using the checkerboard microdilution method. Minimal inhibitory concentrations (MICs) were found to be 0.125-4 µg/mL. Myriocin was found to affect both cell wall and cell membrane. After exposure to myriocin, biofilm and EPS were found to be inhibited and removed, and the CSH was decreased. The combined fungistasis of myriocin and voriconazole (VCZ) or amphotericin B (AMB) were additive. Myriocin had significant antifungal activity against C. albicans, and the antifungal mechanisms might be cell wall and membrane damage. Myriocin effectively inhibited and eliminated biofilms, and its mechanism may be related to the inhibition of EPS and CSH.

Difference in biofilm formation between carbapenem-resistant and carbapenem-sensitive Klebsiella pneumoniae based on analysis of mrkH distribution

ObjectTo analyze the difference in biofilm formation between carbapenem-resistant and carbapenem-sensitive Klebsiella pneumoniae based on analysis of mrkH distribution and to further explore the function of mrkH for biofilm formation from the perspective of gene regulation. Methods40 imipenem-resistant strains and 40 imipenem-sensitive strains were selected to conduct experiments. Carbapenem (imipenem) susceptibility test was performed by the agar-dilution method. blaKPC resistance gene, type 3 fimbriae-related coding genes (mrkA and mrkD) and regulation gene (mrkH) were screened by PCR. Biofilm formation assay was performed using crystal violet staining method in MHB. The relative expression of genes that critically involved in biofilm formation (mrkA, luxS, pgaA) and carbapenem resistance (ompk35, ompk36, acrB) were measured by quantitative real-time PCR (qRT-PCR). Furthermore, the mrkH cassette was cloned into pGEM-T Easy plasmid to yield pGEM:pmrkH and expressed in Escherichia coli DH5α and K. pneumoniae FK1911, and the biofilm formation assay after transformation was further tested. ResultsThe MICs of imipenem were all more than 16 μg/mL in 40 imipenem-resistant strains and ranged from 0.125 μg/mL to 0.5 μg/mL in 40 imipenem-sensitive strains. Moreover, the blaKPC was identified in the 40 imipenem-resistant K. pneumoniae strains. All 80 K. pneumoniae strains were found to carry mrkA and mrkD genes. Interestingly, the mrkH gene was detected in 43 strains, of which 32 were carbapenem-sensitive strains. The biofilm formation capacity of strains carried mrkH cassette was significantly higher than other 37 strains in MHB media. The relative expression of mrkA in K. pneumoniae carrying mrkH gene was significantly up-regulated. Importantly, the biofilm formation ability of FK1911-pGEM:pmrkH strain was more higher than the strain of FK1911 in MHB medium. ConclusionsOur data demonstrated that MrkH played a crucial role in the regulation of biofilm formation by K. pneumoniae. In contrast to carbapenem-sensitive K. pneumoniae, carbapenem-resistant K. pneumoniae was less likely to have strong biofilm-forming capacity because it does not carry the mrkH gene.

Bone and Joint Infection Involving Corynebacterium spp.: From Clinical Features to Pathophysiological Pathways.

Introduction: Corynebacteria represent often-neglected etiological agents of post-traumatic and/or post-operative bone and joint infection (BJI). We describe here clinical characteristics and bacteriological determinants of this condition.Methods: A retrospective cohort study described characteristics, outcome and determinants of treatment failure of all patients with proven Corynebacterium spp. BJI (i.e., ≥2 culture-positive gold-standard samples). Available strains were further characterized regarding their antibiotic susceptibilies, abilities to form early (BioFilm Ring Test®) and mature (crystal violet staining method) biofilms and to invade osteoblasts (gentamicin protection assay).Results: The 51 included BJI were mostly chronic (88.2%), orthopedic device-related (74.5%) and polymicrobial (78.4%). After a follow-up of 60.7 weeks (IQR, 30.1–115.1), 20 (39.2%) treatment failures were observed, including 4 Corynebacterium-documented relapses, mostly associated with non-optimal surgical management (OR 7.291; p = 0.039). Internalization rate within MG63 human osteoblasts was higher for strains isolated from delayed (>3 months) BJI (p < 0.001). Infection of murine osteoblasts deleted for the β1-integrin resulted in a drastic reduction in the internalization rate. No difference was observed regarding biofilm formation.Conclusions: Surgical management plays a crucial role in outcome of BJI involving corynebacteria, as often chronic and device-associated infections. Sanctuarisation within osteoblasts, implicating the β1 cellular integrin, may represent a pivotal virulence factor associated with BJI chronicity.

Decreased Biofilm Formation Ability of a Multidrug-Resistant Pseudomonas aeruginosa Strain After Exposure to a Simulated Microgravity Environment

Background: The refractory infection induced by multidrug-resistant (MDR) Pseudomonas aeruginosa has become one of the most urgent problems in hospitals. The biofilms formed by P. aeruginosa increase its resistance to antibiotics. A simulated microgravity (SMG) environment provides a platform to understand the factors affecting biofilm formation in bacteria. Objectives: This study aimed to investigate the SMG effects on MDR P. aeruginosa biofilm formation and explore the relevant mechanisms. Methods: In this study, a clinostat was used to simulate a microgravity (MG) environment. The motility and biofilm formation ability of MDR P. aeruginosa were observed using the swimming test and the crystal violet staining method, respectively. The underlying mechanism of phenotypic changes was further investigated by comparative transcriptomic analysis. Results: Multidrug-resistant P. aeruginosa grown under the SMG condition exhibited decreased swimming motility and biofilm formation ability compared to those under the normal gravity (NG) condition. Further analysis revealed that the decreased swimming motility and biofilm formation ability could be attributed to the downregulated expression of genes responsible for flagellar synthesis (flhB, fliQ, and fliR) and type IV pili biogenesis (pilDEXY1Y2VW). Conclusions: This is the first study to perform experiments on MDR P. aeruginosa under the SMG condition. It will be beneficial to understand the mechanism of MDR P. aeruginosa biofilm formation and develop new treatment strategies for infectious diseases induced by MDR P. aeruginosa in the future.

Study of Biofilm Formation and Antibiotic Resistance Pattern of Bacteria Isolated from Diabetic Foot Ulcers in H&amp;#244;pital de R&amp;#233;f&amp;#233;rence 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.

Assessment of Chlorine Resistance Enterobacter cloacae Isolated from Water Storage Tanks in Sulaimaniyah City-Iraq

This study was designed to determine the extent of contamination of water storage tanks by non-lactose fermenter Enterobacter spp, and to characterize the chlorine and antibiotic resistance status. Moreover, to find the correlation between biofilm formation and resistance to chlorine. For this purpose, a total of 60 water samples were collected from residential and restaurant water storage tanks. Bacterial analysis and antibiotic susceptibility profiles of the samples were assessed by the Most Probable Number (MPN) and Vitek 2 compact tests, respectively. The biofilm formation was quantified by crystal violet staining method and chlorine resistance test by microdilution technique. Obtained results indicated that water samples were contaminated by Escherichia coli (35%). Additionally, water samples that were assessed to be potable by the MPN test showed that (44%) positive for Enterobacter cloacae. Results of chlorine resistance test revealed variation in resistance of E. cloacae to different concentrations of chlorine, and relatively similar antibiotic susceptibility profiles. Moreover, biofilm analysis showed the isolates that were resistant to concentration of chlorine 400 mg L-1, have formed significantly more biofilm than those that were resistant to other concentrations. A positive non-linear correlation (r = 0.72) was found between the degree of biofilm formation and the ability of isolates to resist different chlorine concentrations, and no correlation has been detected between antibiotic and chlorine resistance. It can be concluded that the presence of chlorine resistant E. cloacae in drinking water can pose a real public health threat. The routine microbial water analysis should be modified to include detection of non-lactosefermenter Enterobacter.

Investigation of the association of virulence genes and biofilm production with infection and bacterial colonization processes in multidrug-resistant Acinetobacter spp.

The aim of this study was to evaluate the phenotypic and molecular patterns of biofilm formation in infection and colonization isolates of Acinetobacter spp. from patients who were admitted in a public hospital of Recife-PE-Brazil in 2018-2019. For the biofilm phenotypic analysis, Acinetobacter spp. isolates were evaluated by the crystal violet staining method; the search of virulence genes (bap, ompA, epsA, csuE and bfmS) was performed by PCR; and the ERIC-PCR was performed for molecular typing. Amongst the 38 Acinetobacter spp. isolates, 20 were isolated from infections and 18 from colonization. The resistance profile pointed that 86.85% (33/38) of the isolates were multidrug-resistant, being three infection isolates, and two colonization isolates resistant to polymyxin B. All the isolates were able to produce biofilm and they had at least one of the investigated virulence genes on their molecular profile, but the bap gene was found in 100% of them. No clones were detected by ERIC-PCR. There was no correlation between biofilm formation and the resistance profile of the bacteria, neither to the molecular profile of the virulence genes. Thus, the ability of Acinetobacter spp. to form biofilm is probably related to the high frequency of virulence genes.

Honey Potential as Antibiofilm, Antiquorum Sensing and Dispersal Agent against Multispecies Bacterial Biofilm.

This study is first to test Pakistani honey bees, Apis dorsata and A. cerana honey samples as anti biofilm, anti quorum sensing (QS) and biofilm dispersal agents honey against multispecies biofilm of bacteria (obtained from obese patients). Briefly, five previously identified isolates Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Morganella morganii and Klebsiella pneumoniae (MT448672-MT448676) were selected. Antibiogram study of all five isolates was tested against three antibiotics viz., erythromycin (20 µg/mL), lincomycin (100 µg/mL) and rifampicin (100 µg/mL). In order to form multispecies biofilm, identified bacteria were grown in batch culture by mixing equal volumes (OD590nm = 0.1) of 2, 3 and 5 bacterial isolates. In total 11 groups (g1-g11) were made. Crystal violet (CV) staining method was used to evaluate the antibiofilm potential and biofilm dispersal potential of both honey samples. QS inhibition in P. aeruginosa was measured following culture supernatant method. Antibiogram study showed significant (p < 0.05) resistance by P. aeruginosa against tested antibiotics. E. coli, M. morganii and K. pneumoniae were significantly susceptible to erythromycin and S. aureus to lincomycin. Both honey samples at 2% and 5% concentrations showed significant (p < 0.05) inhibition potential of multispecies biofilm by all test groups (g1-g11). Though A. dorsata honey significantly inhibited biofilm formation at 2 and 5% against all groups but 2% concentration was highly significant against g2-g4 groups. Regarding A. cerana honey, 2% concentration was significantly effective against g1, g4-g7 and g9-g11 groups. Both honey samples significantly inhibited QS at 2 and 5%. The 5% concentration of A. dorsata honey significantly dispersed biofilm by all groups compared to 2% which showed dispersal potential only by g2 and g3 groups. Accordingly, honey samples showed significant antibiofilm, anti-QS and biofilm dispersal potentials thus can be considered as good alternative to antibiotics.

Honokiol inhibits the growth of SKBR3 cells.

BackgroundBreast cancer is one of the most malignant tumors in the reproductive system and has a poor prognosis. Finding drugs with high efficiency, low side-effects, and low cost has become a research hotspot.MethodsIn the present study, we treated SK-BR-3 cells with different doses of honokiol. Crystal violet staining method was used to detect changes in the total number of living cells; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to detect the effect of honokiol on SK-BR-3 cell proliferation. Cell migration ability change was determined by wound healing assay. Cell invasion ability change was determined by Transwell migration assay. Flow cytometry was used to detect the apoptotic rate of SK-BR-3 cells, and Western blot was used to detect the expression levels of proliferation-associated protein (PCNA); migration- and invasion-related protein matrix metalloproteinase-2 (MMP-2); vimentin; apoptosis-related proteins Bcl-xl, caspase 3, and cleaved caspase 3 (CC3); and β-catenin and its downstream target molecule c-Myc.ResultsCompared with the control group, different doses of honokiol have different degrees of inhibitory effects on cells, including proliferation and invasion and migration (P<0.01). After treatment with 50 or 60 µmol·L–1 honokiol, the apoptotic rate of SK-BR-3 cells increased (both P<0.01); PCNA expression was significantly downregulated (P<0.01). Intracellular accumulation of apoptosis-related proteins Bcl-xl and caspase-3 decreased but C-C3 increased. We also found downregulation of MMP-2 expression, a protein related to invasion and migration (P<0.01), and a decrease in the expression levels of the Wnt/β-catenin signaling pathway-related proteins β-catenin and c-Myc (P<0.01).ConclusionsHonokiol can promote the apoptosis of SK-BR-3 cells and can inhibit the proliferation, migration, and invasion of human breast cancer SK-BR-3 cells. The underlying mechanism may be through inhibiting the activation of the Wnt signaling pathway.

Artesunate inhibits Staphylococcus aureus biofilm formation by reducing alpha-toxin synthesis

Staphylococcus aureus is one of the most common pathogens in bacterial biofilm infections. Antibiotic treatment for infection caused by S. aureus biofilms is challenging, and few effective strategies have been developed to combat these infections. The aim of this study was to investigate the effect and possible mechanisms of artesunate on the biofilm formation of S. aureus. Bacterial growth curves were determined by a microtiter plate. Biofilm formation was determined by the crystal violet staining method and confocal laser scanning microscopy. Bacterial adhesion was assayed by the colony-counting method. The expression of virulence and adhesion genes was determined by real-time PCR. The hemolytic activity and expression of ɑ-hemolysin were analyzed using rabbit erythrocytes and Western blotting. The results showed that artesunate could significantly inhibit the biofilm formation of S. aureus in a dose-dependent manner. Artesunate could also inhibit bacterial adhesion and the expression of hla, RNAIII and agrA as well as ɑ-hemolysin production. The effect of artesunate on adhesion genes (clfA, clfB, fnbA, fnbB) had strain specificity, but it did not affect the expression of ica genes. The results indicated that artesunate might inhibit ɑ-hemolysin synthesis by the agr system, which inhibits biofilm formation.

Characterization of carbapenem-resistant hypervirulent Acinetobacter baumannii strains isolated from hospitalized patients in the mid-south region of China

BackgroundAcinetobacter baumannii has traditionally been considered an opportunistic pathogen with low virulence. In this study, we characterized the carbapenem-resistant hypervirulent A. baumannii (CR-hvAB) stains isolated from our hospital in mid-south region of China.ResultsBlood samples collected between January 2017 and May 2019 were used for virulence experiments and biofilm assays of individual carbapenem-resistant A. baumannii (CR-AB) strains, performed using a Galleria mellonella infection model and crystal violet staining method, respectively. CR-AB isolates that induced high mortality in the G. mellonella infection model were subjected to genotyping, susceptibility testing, and clinical data analysis, and the genetic characterization of these isolates was performed by whole-genome sequencing (WGS). Among the 109 CR-AB clinical strains, the survival rate of G. mellonella larvae infected with 7 (6.4%) CR-AB isolates (number of strains with mortality of 0, 10 and 20% was 4, 1, and 2, respectively), was significantly lower than that of A. baumannii ATCC 19606 (100.0%) and the remaining CR-AB isolates (> 80.0%). Consistent with these results, patients infected with these seven isolates had an average 7-day mortality rate of 42.9%, suggesting that the isolates were CR-hvAB. These seven isolates belonged to four sequence types (STs): ST457, ST195, ST369, and ST2088 (a new ST), and mainly ST457 (n = 4). The results of the biofilm study showed that eight strains had powerful biofilm ability (strong [n = 1] and moderate [n = 7] biofilm producers) including these seven CR-hvAB isolates.ConclusionsCR-hvAB isolates that induced a high mortality rate were cloned in our hospital, most of which belonged to ST457; thus, monitoring of these strains, particularly ST457, should be strengthened in the future. Meanwhile, A. baumannii, which was isolated from blood specimens and found to powerful biofilm-forming ability, is a probable hvAB isolate.