High Biofilm-forming Ability Research Articles

Hypervirulent Carbapenem-Susceptible Klebsiella pneumoniae ST412/K57 with Strong Biofilm Formation: association with gas gangrene and sepsis.

Hypervirulent Klebsiella pneumoniae (hvKp) poses a serious public health threat. Gas gangrene caused by hvKp was rarely reported, potentially resulting in a poor prognosis. In this study, we described the case of a hospitalized patient with gas gangrene and sepsis by hvKP. The carbapenem-susceptible hypervirulent Klebsiella pneumoniae (CS-hvKp) strains KPLSN and KPLSX were isolated from the knee joint pus and blood specimens of the patient for further investigations. Whole genome sequencing revealed that KPLSN and KPLSX were highly homologous (SNPs<10) and belonging to ST412/K57. The minimum inhibitory concentration and minimum bactericidal concentration under biofilm values of meropenem in KPLSN and KPLSX were significantly higher than planktonic state (>128 mg/L versus 0.25 mg/L, P<0.0001). These two strains had high biofilm formation ability, and fluorescence staining experiments results showed that they were not easily killed by meropenem in the biofilm state. KPLSN and KPLSX showed high capsular production and were confirmed with high virulence through experiments with the Galleria mellonella and BALB/c mice abdominal infection model. The persistent symptoms may be due to enhanced biofilm and capsule formation. Global ST412 strains phylogenetic analysis showed their evolution towards higher virulence and resistance. It emphasizes the critical need for judicious antibiotic use and novel therapeutic approaches to combat special infections caused by these pathogens.

Characteristics of Candida albicans Derived From HIV-Positive Individuals With Oral Candidiasis: Genotyping, Phenotypic Variation, Antifungal Susceptibility, and Biofilm Formation.

Oral candidiasis (OC) is one of the most common mucosal infections in those afflicted with HIV/AIDS. This study aimed to provide detailed information on the phenotype, genotype, antifungal susceptibility, and biofilm formation ability of oral Candida albicans isolated from HIV-infected patients with OC. A total of 25 C. albicans isolates were collected from oral lesions of HIV-infected patients referred to Behavioral Diseases Counseling Center affiliated with Ahvaz Jundishapur University of Medical Sciences, Iran. The antifungal susceptibility testing was done according to CLSI M27 guideline (fourth edition). The crystal violet method was used to evaluate the biofilm formation ability of isolates. Different phenotypes were identified on yeast extract-peptone-dextrose agar medium supplemented with phloxine B. Genotyping analysis of the isolates was performed using high-resolution melting (HRM) assays and ABC genotyping. The highest and lowest susceptibility of the C. albicans isolates was found for fluconazole 24 (96%) and ITC 18 (72%), respectively. Forty-eight percent of the isolates had high biofilm formation ability and exhibited gray cell type. The most common genotype was genotype B (52%). HRM analysis of HIS3, EF3, and CDC3 markers showed three, four, and five different groups, respectively. Investigating the phenotype, antifungal susceptibility and biofilm formation ability of the C. albicans isolates obtained from oral lesions of HIV-infected patients revealed that the dominant genotypes in the current research could cause more serious infections from the oral source. We recommend further research with a larger sample size to determine the molecular epidemiology of C. albicans among HIV patients in Iran.

Genotypic and phenotypic insights into virulence factors of nosocomial Stenotrophomonas maltophilia isolates collected in Bulgaria (2011-2022).

The present study aimed to explore the virulence characteristics in 221 Bulgarian nosocomial Stenotrophomonas maltophilia isolates (2011-2022) via screening for the presence of virulence genes, their mutational variability, and the corresponding enzyme activity. PCR amplification, enzymatic assays, whole-genome sequencing (WGS), and biofilm quantification on a polystyrene plate were performed. The incidence of virulence determinants was as follows: stmPr1 (encoding for the major extracellular protease StmPr1) 87.3%, stmPr2 (minor extracellular protease StmPr2) 99.1%, Smlt3773 locus (outer membrane esterase) 98.2%, plcN1 (non-hemolytic phospholipase C) 99.1%, and smf-1 (type-1 fimbriae, biofilm-related gene) 96.4%. The 1621-bp allele of stmPr1 was most frequently found (61.1%), followed by the combined allelic variant (17.6%), stmPr1-negative genotype (12.7%), and 868-bp allele (8.6%). Protease, esterase, and lecithinase activity was observed in 95%, 98.2%, and 17.2% of the isolates, respectively. The WGS-subjected isolates (n = 9) formed two groups. Five isolates possessed only the 1621-bp variant of stmPr1, higher biofilm formation ability (Optical Density at λ = 550 nm (OD550): 1.253-1.789), as well as a low number of mutations in the protease genes and smf-1. Three other isolates had only the 868-bp variant, weaker biofilm production (OD550: 0.788-1.108), and higher number of mutations within these genes. The only weak biofilm producer (OD550 = 0.177) had no stmPr1 alleles. In conclusion, the similar PCR detection rates did not allow differentiation of the isolates. In contrast, WGS permitted stmPr1 alleles-based differentiation. To the best of our knowledge, this is the first Bulgarian study presenting genotypic and phenotypic insights into virulence factors of S. maltophilia isolates.

Weissella confusa N17 Derived from Loach (Misgurnus anguillicaudatus) Exhibits Promising for Further Applications in Loach Aquaculture.

The application of probiotics, in aquaculture, is becoming increasingly widespread and have had positive application effects. However, reports of loach-derived probiotics are quite limited. In this study, two representative strains of lactic acid bacteria with excellent traits, namely, Weissella confusa N17 and Lactobacillus saniviri N19, were screened from the intestine of healthy loaches. W. confusa N17 and L. saniviri N19 could inhibit different common various pathogenic bacteria, especially Aeromonas spp., and were sensitive to the most common antibiotics. The survival rate of the two strains exceeded 50% after 4h of incubation in 10% loach bile. Moreover, the two strains showed significant tolerance to trypsin. Their autoaggregation capacity and hydrophobicity were greater than 30%. In addition, the aggregation ability of both strains was higher than 30% for both A. veronii TH0426 and A. hydrophila TPS. The two strains had a high biofilm-forming ability and strong adhesion to epithelioma papulosum cyprini (EPC) cells. Scanning electron microscopy results showed that the culture supernatants of the two strains had a significantly destructive effect on A. veronii TH0426 and A. hydrophila TPS. Overall, the traits of W. confusa N17 were better than those of L. saniviri N19. Genome sequencing and analysis demonstrated a lack of virulence factor-related or drug resistance-related genes in genome N17. The diet supplemented with the W. confusa N17 strain significantly improved the resistance of loaches to A. veronii infection, and the protection rate reached 57.1%. Therefore, W. confusa N17 exhibits promising for further applications in loach aquaculture.

Photodynamic Therapy and Its Synergism with Melittin Against Drug-Resistant Acinetobacter baumannii Isolates with High Biofilm Formation Ability.

Drug-resistant biofilm producer A. baumannii isolates are a global concern that warns researchers about the development of new treatments. This study was designed to analyze the effect of photodynamic therapy (PDT) as monotherapy and associated with melittin on multidrug-resistant A. baumannii isolates. Sub-lethal doses of photosensitizer, LED, and PDT were determined. The PDT effect on the biofilm and expression of biofilm-associated genes was evaluated by scanning electron microscopy and quantitative real-time PCR (qRT-PCR) methods, respectively. The synergistic effect of PDT and melittin on the survival of MDR/XDR strong biofilm producer isolates was evaluated by checkerboard assay. Survival rates were significantly decreased at the lowest concentration of 12.5-50μg/ml in 4min at an energy density of 93.75J/cm2 (P < 0.05). The optimized PDT method had a bactericidal effect against all tested groups, and the mean expression levels of csu, abaI, bap, and ompA genes in the strong biofilm producers were decreased significantly compared to the control group. The combined effect of LED and melittin successfully reduced the MDR/XDR A. baumannii strong biofilm producers' growth from 3.1 logs. MB-mediated aPDT and combined treatment of PDT with melittin, which has been investigated for the first time in this study, can be an efficient strategy against MDR/XDR A. baumannii isolates with strong biofilm production capacity.

Characterization of a Helicobacter pylori strain with high biofilm-forming ability.

Introduction. Helicobacter pylori is highly polymorphic, and some strains are much more likely to cause disease than others. Biofilm formation can help bacteria to survive antibiotic treatment, immune attack and other stresses, promoting persistent infection.Hypothesis/Gap Statement. We hypothesized that H. pylori isolates from patients with more severe H. pylori-associated disease would be better at forming biofilms than isolates from patients with less severe disease.Aim. We initially aimed to determine whether or not the biofilm-forming ability of H. pylori isolates was associated with disease in the UK-based patients from whom the bacteria were isolated.Methodology. Biofilm-forming ability of H. pylori isolates was determined using a crystal violet assay on glass coverslips. The complete genome sequence of strain 444A was generated by hybrid assembly of Nanopore MinION and Illumina MiSeq data.Results. Although we found no associations between biofilm-forming ability of H. pylori and disease severity in patients, we discovered that strain 444A had particularly high biofilm-forming ability. This strain had been isolated from a patient with gastric ulcer disease and moderate to severe scores for H. pylori-induced histopathology. Analysis of the genome of the high biofilm-forming H. pylori strain 444A revealed that it possesses numerous biofilm- and virulence-associated genes and a small cryptic plasmid encoding a type II toxin-antitoxin system.Conclusion. There is substantial variation in biofilm-forming ability in H. pylori, but this was not significantly associated with disease severity in our study. We identified and characterized an interesting strain with high biofilm-forming ability, including generation and analysis of the complete genome.

Determination of the Cytotoxicity and Antibiofilm Potential Effect of Equisetum arvense Silver Nanoparticles.

This study aimed to synthesize and characterize silver nanoparticles (AgNPs) by green synthesis from Equisetum arvense (Ea) extracts and to investigate their cytotoxicity, antibiofilm activity, and α-glucosidase enzyme inhibition. Diverse characterization techniques were applied to verify the production of nanoparticles. SEM examination confirmed that the size of nanoparticles is in the range of 40-60nm. Also, interactions between silver and natural compounds of plant extract were confirmed through FT-IR and EDX analyses. It was determined that Equisetum arvense silver nanoparticles had antibiofilm activity against three different clinical strains with high biofilm-forming ability. AgNPs reduced the biofilm-forming capacity of clinical A. baumannii isolate with strong biofilm-forming capacity by approximately twofold, while the capacity of clinical K.pneumonaie and E.coli isolates decreased by 1.5 and 1.2 fold, respectively. The α-glucosidase enzyme inhibition potential of the AgNPs, which is determined as 93.50%, was higher than the plant extract with, and the α- 30.37%. MTT was performed to assess whether incubation of nanoparticles with A549 and ARPE-19 cell lines affected their viability, and a dramatic reduction in cell growth inhibition of both A549 and ARPE-19 cells was observed. It has been shown that A549 cells treated with 200 and 150µg/mL nanoparticles had less cell proliferation compared to control cells at 24-h and 48-h incubation time. According to these results, Ea-derived AgNPs appear to have potential anticancer activity against A549 cancer cells. Investigating the effects of green synthesis nanoparticles on microbial biofilm and various tumors may be important for developing new therapies. The outcomes of this study have showed that Ea-AgNPsmay have a high potential both in the treatment of pathogenic strains that form biofilms, as well as in anticancer therapy use.

BolA affects the biofilm formation ability, outer membrane permeability and virulence, thus is required for the adaptability of Salmonella enterica serotype Typhimurium to the harsh survival environment

Salmonella enterica serotype Typhimurium, an important foodborne pathogen with high adaptability to the host's internal and external survival environment, seriously threatens public health. Therefore, to understand the mechanism underlying the high adaptability, this study investigated the transcription factor BolA by constructing BolA deletion strain 269△BolA, complemented strain 269BolAR and overexpression strain 269BolA+ based on WT269. BolA significantly inhibited motility; at 6 h, the BolA overexpression strain (269BolA+) showed 91.2% and 90.7% lower motility than the wild type (WT269) and BolA deletion strain (269△BolA), respectively, by downregulating motility-related flagellar genes. BolA promoted biofilm formation; 269BolA+ showed 3.6-fold and 5.2-fold higher biofilm formation ability than WT269 and 269ΔBolA, respectively, by upregulation biofilm formation-related genes. BolA overexpression downregulated the outer membrane gene OmpF and upregulated OmpC, thereby regulating cell permeability, and reducing the antibacterial effect of vancomycin, which can destruct the outer membrane. BolA improved adaptability; 269△BolA showed higher susceptibility to eight antibiotics and 2.5- and 4-fold lower acid and oxidative stress tolerance, respectively, than WT269. In Caco-2 and HeLa cells, 269△BolA showed 2.8- and 3-fold lower cell adhesion ability, respectively, and 4- and 2-fold lower cell invasion ability, respectively, than WT269, through downregulation of the virulence genes. Thus, BolA expression promotes biofilm formation and balances the membrane permeability, thereby improving the resistance of the strains, and enhances its host cell invasion ability by upregulating bacterial virulence factors. Results of this study suggest that the BolA gene may serve as a potential target of therapeutic or preventative strategies to control Salmonella Typhimurium infections.

Prevalence and Biological Characteristics of Listeria Species Isolated from Livestock and Poultry Meat in Gansu Province, China.

Listeria monocytogenes is a widespread foodborne pathogen contaminating foods during their production or processing stages. Fresh meat is susceptible to such contamination if it is not properly preserved. Our study was conducted to reveal the level of contamination and prevalence of Listeria spp. present in livestock and poultry meat from Gansu province. A total of 1,387 samples were collected from five cities in Gansu Province according to standard sampling procedures, of which 174 samples (12.5%) were positive for Listeria species. Among them, 14 isolates of L. monocytogenes (1.0%), 150 isolates of Listeria innocua (10.8%), and ten isolates of Listeria welshimeri (0.7%) were identified by conventional bacteriological and molecular identification methods. All isolates were subjected to serological assays, antimicrobial susceptibility tests, growth curve assays, determination of biofilm-forming capacity, and cluster analysis of the 16S rRNA gene sequences. Four predominant serotypes of L. monocytogenes were identified, including 1/2a (35.7%, 5/14), 1/2b (14.3%, 2/14), 1/2c (42.9%, 6/14), and 4b (7.1%, 1/14). All L. monocytogenes isolates were resistant to tetracycline and cefoxitin. Most L. innocua isolates (63.6%, 14/22) and L. welshimeri (40%, 4/10) were resistant to tetracycline. The high biofilm-forming ability was observed among 1/2c and 1/2a serotype isolates. The cluster analysis of the 16S rRNA gene sequences revealed a close genetic relationship between the three Listeria species. This study fills the gap in the knowledge of livestock and poultry meat that carry Listeria in slaughterhouses and markets in Gansu Province.

Phenotypic and genomic comparison of dominant and nondominant sequence-type of Acinetobacter baumannii isolated in China.

A. baumannii is a common clinical pathogen that often causes pneumonia and bloodstream infections in ICU patients. Sequence types (ST) are used to investigate the distribution and spread of A. baumannii. Biological characteristics such as virulence and resistance may play a role in A. baumannii becoming a specific dominant ST(DST,ST191, ST195 and ST208) strain. To characterize the biological, genetic, and transcriptomic differences between the DST and non-dominant ST(NST,ST462 and ST547,etc.) strains in A. baumannii, we performed several biological experiments and genetic, and transcriptomic analyses. The DST group displayed more resistance ability to desiccation, oxidation, multiple antibiotics, and complement killing than the NST group. However, the latter had higher biofilm formation ability than the former. The genomic analysis showed the DST group exhibited more capsule-related and aminoglycoside-resistant genes. Besides, GO analysis indicated that functions involved in lipid biosynthetic, transport, and the metabolic process were up-regulated in the DST group, while KEGG analysis manifested that the two-component system related to potassium ion transport and pili were down-regulated. In short, resistance to desiccation, oxidation, multiple antibiotics, and serum complement killing are important reasons for the formation of DST. Genes related to capsule synthesis and lipid biosynthesis and metabolism play an important role at the molecular level in the formation of DST.

High biofilm-forming Pseudomonas strains isolated from poultry slaughterhouse surfaces: Their importance in the persistence of Salmonella enteritidis in slaughterhouses

The surfaces of poultry slaughterhouse equipment are significant sources of contamination with Pseudomonas strains, which leads to spoilage of poultry meat during subsequent refrigerated storage. In this study, Pseudomonas strains with high biofilm-forming ability were isolated from different surfaces of the poultry slaughterhouse equipment, identified based on molecular data, and characterized their biofilm-forming ability. After 24 h of incubation at 25 °C, 54 out of 58 Pseudomonas strains produced biofilm in vitro on polystyrene microplates. Seven isolates with high-ability to produce biofilm were identified as P. fragi (three strains), P. fluorescens (two strains), P. lundensis and P. cedrina. Despite their differences, these strains produced high amounts of biofilm in pure- and dual-species cultures with S. enteritidis on stainless steel surfaces. However, their ability to produce dual-species biofilms with S. enteritidis depends on whether S. enteritidis form the biofilm simultaneously with the Pseudomonas strains or whether Pseudomonas strains have already formed a biofilm. In concurrent inoculation, S. enteritidis participated in biofilm formation with all seven Pseudomonas strains with varying percent contributions. However, in delayed inoculation, S. enteritidis did not contribute in the biofilm formed by P. lundensis R26, P. fragi R39, and P. fluorescens R47. In addition to highlighting the complexity of bacterial interactions associated with Pseudomonas strains, these results showed that Pseudomonas strains can be implicated in Salmonella persistence in poultry slaughterhouses.

High Biofilm-Forming Ability and Clonal Dissemination among Colistin-Resistant Escherichia coli Isolates Recovered from Cows with Mastitis, Diarrheic Calves, and Chickens with Colibacillosis in Tunisia

Escherichia coli (E. coli) is one of the main etiological agents responsible for bovine mastitis (BM), neonatal calf diarrhea (NCD), and avian colibacillosis (AC). This study aimed to assess resistance and virulence genes content, biofilm-forming ability, phylogenetic groups, and genetic relatedness in E. coli isolates recovered from clinical cases of BM, NCD, and AC. A total of 120 samples including samples of milk (n = 70) and feces (n = 50) from cows with BM and calves with NCD, respectively, were collected from different farms in Northern Tunisia. Bacterial isolation and identification were performed. Then, E. coli isolates were examined by disk diffusion and broth microdilution method for their antimicrobial susceptibility and biofilm-forming ability. PCR was used to detect antimicrobial resistance genes (ARGs), virulence genes (VGs), phylogenetic groups, and Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) for their clonal relationship. Among the 120 samples, 67 E. coli isolates (25 from BM, 22 from AC, and 20 from NCD) were collected. Overall, 83.6% of isolates were multidrug resistant. Thirty-six (53.73%) isolates were phenotypically colistin-resistant (CREC), 28.3% (19/67) were ESBL producers (ESBL-EC), and forty-nine (73.1%) formed biofilm. The blaTEM gene was found in 73.7% (14/19) of isolates from the three diseases, whilst the blaCTXM-g-1 gene was detected in 47.3% (9/19) of isolates, all from AC. The most common VG was the fimA gene (26/36, 72.2%), followed by aer (12/36, 33.3%), cnf1 (6/36, 16.6%), papC (4/36, 11.1%), and stx1 and stx2 genes (2/36; 5.5% for each). Phylogenetic analysis showed that isolates belonged to three groups: A (20/36; 55.5%), B2 (7/36; 19.4%), and D (6/36; 16.6%). Molecular typing by ERIC-PCR showed high genetic diversity of CREC and ESBL E. coli isolates from the three animal diseases and gave evidence of their clonal dissemination within farms in Tunisia. The present study sheds new light on the biofilm-forming ability and clonality within CREC and ESBL-EC isolated from three different animal diseases in Tunisian farm animals.

Influence of disinfectants on growth of biofilm formed by &lt;i&gt;К. pneumoniae&lt;/i&gt; strains

Introduction. Currently, the problem of biofilm-associated infections is of great importance, since the classical methods of antibiotic therapy for purulent-inflammatory diseases become ineffective due to the high resistance of pathogens. An urgent task is to find ways to suppress adhesion processes or destroy an already formed biofilm using various disinfectants, enzymes, a combination of antibacterial and other drugs.Materials and methods. The object of the study is the bacteria Klebsiella pneumoniae isolated from wounds and fistulas of patients with chronic osteomyelitis. Biofilms were cultured for two days in the wells of polystyrene plates and on coverslips. In the control series of the experiment, the biofilm-forming ability of K. pneumoniae strains was studied after 48 hours. In the second series, after 24 hours, the emerging biofilm was exposed to a solution of 3 % hydrogen peroxide, in the third series, with a skin antiseptic, and after 48 hours, the intensity of biofilm formation was assessed. For statistical processing of the obtained data, the Gnumeric 1.12.17 spreadsheet program was used.Results and its discussion. K. pneumoniae strains had a high biofilm-forming ability. The impact of disinfectants on the emerging biofilm led to a decrease in the optical density in the wells of the plates and the intensity of biofilm formation. On cover slips in the second and third series of the experiment, a decrease in the total proportion of microcolonies and individual adherent cells was observed. Disinfectants (hydrogen peroxide and skin antiseptic) had a bacteriostatic effect on biofilms formed by K. pneumoniae strains. In addition, a bactericidal effect of skin antiseptic on planktonic bacterial cells was found.Conclusions. The data obtained must be taken into account for the selection of effective disinfectants used in the clinic to prevent the emergence of resistant strains of microbes.

Characterization of Probiotic Properties of Lacticaseibacillus paracasei L2 Isolated from a Traditional Fermented Food "Lben".

Lben is a dairy fermented food that is largely consumed in Tunisia for its numerous health benefits that are related to the existence of probiotics. Lactic Acid Bacteria (LAB) are well known for their beneficial probiotic properties for humans, especially when administered in adequate amounts. The aim of this study was to isolate and investigate the probiotics properties of Lacticaseibacillus paracasei L2 from Lben. The isolated strain was identified by 16S r-RNA gene sequences and MALDI- TOF MS. To evaluate the probiotic potential of the isolated bacterium, in vitro tests were performed, including adhesion ability to HCT-116 cells, survival in acid and bile salt conditions, lysozyme resistance, biofilm formation, hemolytic activity, antioxidant activity, and antimicrobial activity. Our results revealed that the selected Lacticaseibacillus paracasei L2 strain expressed a high adherence to HCT-116 cells (45.03%), survived under acidic conditions (pH3), and showed a resistance to bile salts. The strain was considered as safe (α-hemolysis). L. paracasei L2 showed a high biofilm-formation ability (OD 570 > 1.7) after 24 h of incubation. It also demonstrated an important antioxidant activity in the range of 85.31% for the intact cells. However, an antimicrobial activity against pathogens, namely Staphylococcus aureus, was detected with an IZ that was above 19 mm. In conjunction with the results obtained and the technological properties of Lacticaseibacillus paracasei L2 (proteolytic property, autolytic activity, acidifying activity, and EPS production), this strain may be used as a probiotic for manufacturing fermented foods.

Intracellular Survival of Biofilm-Forming MRSA OJ-1 by Escaping from the Lysosome and Autophagosome in J774A Cells Cultured in Overdosed Vancomycin.

We investigated the drug-resistant mechanisms of intracellular survival of methicillin-resistant S. aureus (MRSA). Our established MRSA clinical strain, OJ-1, with high biofilm-forming ability, and a macrophage cell line, J774A, were used. After ingestion of OJ-1 by J774A, the cells were incubated for ten days with vancomycin at doses 30 times higher than the minimum inhibitory concentration. The number of phagocytosed intracellular OJ-1 gradually decreased during the study but plateaued after day 7. In J774A cells with intracellular OJ-1, the expression of LysoTracker-positive lysosomes increased until day 5 and then declined from day 7. In contrast, LysoTracker-negative and OJ-1-retaining J774A cells became prominent from day 7, and intracellular OJ-1 also escaped from the autophagosome. Electron microscopy also demonstrated that OJ-1 escaped the phagosomes and was localized in the J774A cytoplasm. At the end of incubation, when vancomycin was withdrawn, OJ-1 started to grow vigorously. The present results indicate that intracellular phagocytosed biofilm-forming MRSA could survive for more than ten days by escaping the lysosomes and autophagosomes in macrophages. Intracellular MRSA may survive in macrophages, and accordingly, they could be resistant to antimicrobial drug treatments. However, the mechanisms their escape from the lysosomes are still unknown. Additional studies will be performed to clarify the lysosome-escaping mechanisms of biofilm-forming MRSA.

Correlation Between Antimicrobial Resistance, Virulence Determinants and Biofilm Formation Ability Among Extraintestinal Pathogenic Escherichia coli Strains Isolated in Catalonia, Spain.

Escherichia coli is a well-characterized bacterium highly prevalent in the human intestinal tract and the cause of many important infections. The aim of this study was to characterize 376 extraintestinal pathogenic E. coli strains collected from four hospitals in Catalonia (Spain) between 2016 and 2017 in terms of antimicrobial resistance, siderophore production, phylogroup classification, and the presence of selected virulence and antimicrobial resistance genes. In addition, the association between these characteristics and the ability to form biofilms was also analyzed. The strains studied were classified into four groups according to their biofilm formation ability: non-biofilm formers (15.7%), weak (23.1%), moderate (35.6%), and strong biofilm formers (25.6%). The strains were highly resistant to ciprofloxacin (48.7%), trimethoprim-sulfamethoxazole (47.9%), and ampicillin (38%), showing a correlation between higher resistance to ciprofloxacin and lower biofilm production. Seventy-three strains (19.4%) were ESBL-producers. However, no relationship between the presence of ESBL and biofilm formation was found. The virulence factor genes fimH (92%), pgaA (84.6%), and irp1 (77.1%) were the most prevalent in all the studied strains. A statistically significant correlation was found between biofilm formation and the presence of iroN, papA, fimH, sfa, cnf, hlyA, iutA, and colibactin-encoding genes clbA, clbB, clbN, and clbQ. Interestingly, a high prevalence of colibactin-encoding genes (19.9%) was observed. Colibactin is a virulence factor, which interferes with the eukaryotic cell cycle and has been associated with colorectal cancer in humans. Most colibactin-encoding E. coli isolates belonged to phylogroup B2, exhibited low antimicrobial resistance but moderate or high biofilm-forming ability, and were significantly associated with most of the virulence factor genes tested. Additionally, the analysis of their clonal relatedness by PFGE showed 48 different clusters, indicating a high clonal diversity among the colibactin-positive strains. Several studies have correlated the pathogenicity of E. coli and the presence of virulence factor genes; however, colibactin and its relationship to biofilm formation have been scarcely investigated. The increasing prevalence of colibactin in E. coli and other Enterobacteriaceae and the recently described correlation with biofilm formation, makes colibactin a promising therapeutic target to prevent biofilm formation and its associated adverse effects.

Biofilm formation and resistance to sanitizers by Salmonella spp. Isolated from the peanut supply chain

Pathogens such as Salmonella can be difficult to control in low-moisture food (LMF) processing plants and because of this its presence especially in biofilm mode should be prevented in such an environment. This study evaluated the capacity of Salmonellastrains isolated from the peanut supply chain (S. Muenster, S. Miami, S. Glostrup, S. Javiana, S. Oranienburg and S. Yoruba) to form biofilm as well as their resistance to sanitizers (sodium hypochlorite, peracetic acid, quaternary ammonium, alkaline chlorinated solution and biguanide). Regarding biofilm formation, there was no significant difference (p > 0.05) among the strains tested singly on AISI 304 stainless steel (SS) and polypropylene (PP) coupons at the same temperature. However, a difference (p < 0.05) between the temperatures was noted in the first hours of incubation. The sessile cells reached counts between 3 and 4 log CFU/cm2 at 25 °C whereas>5 log CFU/cm2 was observed at 37 °C after 8 h. From 24 h the counts were above 6 log CFU/cm2 for both temperatures. Nevertheless, the SEM images of the 6-strain pool showed the highest density of adhered cells after 48 h at 25 °C and 24 h at 37 °C on PP, and after 48 h at both temperatures on SS. Peracetic acid (300 mg/L) had the shortest action time against 24-h biofilm on SS and PP, with sessile cell counts below the limit of detection (0.59 log CFU/cm2) after 3 min. For 48-h and 96-h biofilm, sodium hypochlorite (200 mg/L) decreased>4 log CFU/cm2 within 5 min. Quaternary ammonium (350 mg/L) and chlorinated alkaline detergent (200 mg/L) showed intermediate performances. Only biguanide (800 mg/L) did not reduce the biofilm counts to below the limit of detection in any of the conditions evaluated. The results indicated high biofilm formation ability of the Salmonella strains isolated from the peanut supply chain. Nevertheless, in general the biofilms were sensitive to most sanitizers within 15 min of treatment. This was the first study which evaluated biofilm formation by Salmonella isolated from the peanut supply chain. Data obtained here will contribute to optimize the hygiene practices in LMF manufacturing plants.

Antibacterial and Antibiofilm Effect of Ultrasound and Mild Heat Against a Multidrug-Resistant Klebsiella pneumoniae Stain Isolated from Meat of Yellow-Feathered Chicken.

Klebsiella pneumoniae is an important foodborne pathogen with high biofilm-forming ability, which is continuously detected in food products in recent years. The antibacterial and antibiofilm activities and mechanism of ultrasonication in combination with heat treatment against K. pneumoniae were studied. K. pneumoniae planktonic and biofilm cells were treated with ultrasound (US), mild heat treatment (HT50, HT60, and HT70), and combinations of US and mild heat treatment (UH50, UH60, and UH70) for 5, 10, 20, 30, and 60 min. Results showed that the combination of US and mild heat treatment was more effective in inactivating K. pneumoniae planktonic and biofilm cells than the single treatment by counting viable bacteria. In addition, confocal laser scanning microscopy, scanning electron microscopy, and analysis of leakage of intracellular substances have revealed that the combination treatment effectively damaged the integrity of bacterial cell membrane and increased cell permeability, which led to the quick release of adenosine triphosphate (ATP) and macromolecular substances of nucleic acids and proteins. Moreover, the activities of respiratory chain dehydrogenase in planktonic and biofilm cells significantly decreased after UH treatment. The results indicated that ultrasonication and mild heat treatment had a synergistic effect on the inactivation of K. pneumoniae planktonic and biofilm cells by damaging the cell membrane and inhibiting intercellular cell respiration.

Thermonucleases Contribute to Staphylococcus aureus Biofilm Formation in Implant-Associated Infections-A Redundant and Complementary Story.

Biofilms formed by Staphylococcus aureus are one of the predominant causes of implant-associated infections (IAIs). Previous studies have found that S. aureus nucleases nuc1 and nuc2 modulate biofilm formation. In this study, we found low nuc1/nuc2 expression and high biofilm-forming ability among IAI isolates. Furthermore, in a mouse model of exogenous IAIs, Δnuc1/2 exhibited higher bacterial load on the surface of the implant than that exhibited by the other groups (WT, Δnuc1, and Δnuc2). Survival analysis of the hematogenous IAI mouse model indicated that nuc1 is a virulence factor related to mortality. We then detected the influence of nuc1 and nuc2 on biofilm formation and immune evasion in vitro. Observation of in vitro biofilm structures with scanning electron microscopy and evaluation of bacterial aggregation with flow cytometry revealed that both nuc1 and nuc2 are involved in biofilm structuring and bacterial aggregation. Unlike nuc1, which is reported to participate in immune evasion, nuc2 cannot degrade neutrophil extracellular traps. Moreover, we found that nuc1/nuc2 transcription is negatively correlated during S. aureus growth, and a possible complementary relationship has been proposed. In conclusion, nuc1/nuc2 are complementary genes involved in biofilm formation in exogenous IAIs. However, nuc2 contributes less to virulence and is not involved in immune evasion.

ВИДОВОЙ СОСТАВ АССОЦИАЦИЙ И ВЗАИМООТНОШЕНИЯ МИКРООРГАНИЗМОВ, ВЫДЕЛЕННЫХ ИЗ ОСТЕОМИЕЛИТИЧЕСКОГО ОЧАГА

Objective. To determine the species composition of the associations isolated from osteomyelitis foci and to study the character of associate relationships based on the biofilm-forming ability data. Methods. The microbiological study included clinical isolates (n=184) obtained from associations (n=88) during primary inoculations from wounds and fistulas of patients (n= 88) with chronic osteomyelitis of long tubular bones. In order to obtain an associative biofilm in vitro, the cultures of competing bacterial strains were daily mixed in 1:1 ratio. The biofilms were grown on the surface of polystyrene plates with subsequent determination of the level of biofilm formation in 24 and 48 hours. Thecoefficient of relationship (CR) was calculated to evaluate the synergistic, neutral and antagonistic relationships between bacteria in the biofilms. Results. The associations of staphylococcus with gram-negative bacteria were most frequently recovered from osteomyelitis foci. On the 1st day of the experiment, 38,6 % of associations had a moderate biofilm-forming ability, and besides, associations of gram-positive + gram-negative bacteria were observed in 36,4%; 42,1% of associations had a low biofilm-forming ability; 19,3% - had a high biofilm-forming ability. After 48 hours the percentage of mild adhesive strains remained at the same level - 38,6%, as for the low adhesive ones it decreased to 36,4%, high adhesive - increased up to 25%. Most bacterial associations manifested antagonistic relationships. Synergism in biofilm-formingby the association of S. aureus + P. aeruginosa was observed in 2 cases,while the level of film-forming was high as on the first and the second day of the study. In several associations it transformed from antagonistic to synergistic or neutral relationships. Conclusion. It has ben established that among the identified associations, the largest specific weight falls on the associations of gram-positive + gram-negative bacteria , while S. aureus is one of the most common components. These associations were noted to have high and mild activity of biofilm -forming on the surface of polystyrene plates. Relationships between the microorganisms isolated from osteomyelitis foci in associations, as a rule, are antagonistic. What this paper adds For the first time, the nature of the relationship between microorganisms in the composition of associations isolated from the osteomyelitis focus has been studied on the basis of biofilm-forming ability data. It has been established that in microbiocenosis of the osteomyelitis foci, the greatest specific weight falls on the associations of S. aureus with gram-negative bacteria. These associations have a high and moderate activity of biofilm formation on the surface of polystyrene plates. The relationships between microorganisms isolated from the osteomyelitis focus as part of associations are usually antagonistic.