The Relationship between Biofilm Phenotypes and Biofilm-Associated Genes in Food-Related Listeria monocytogenes Strains.

Listeria monocytogenes is an important pathogen responsible for listeriosis, a serious foodborne illness associated with high mortality rates. Therefore, L. monocytogenes is considered a challenge for the food industry due to the ability of some strains to persist in food-associated environments. Biofilm production is presumed to contribute to increased L. monocytogenes resistance and persistence. The aims of this study were to (1) assess the biofilm formation of L. monocytogenes isolates from a meat processing facility and sheep farm previously characterized and subjected to whole-genome sequencing and (2) perform a comparative genomic analysis to compare the biofilm formation and the presence of a known set of biofilm-associated genes and related resistance or persistence markers. Among the 37 L. monocytogenes isolates of 15 sequence types and four serogroups involved in this study, 14%, 62%, and 24% resulted in the formation of weak, moderate, and strong biofilm, respectively. Increased biofilm-forming ability was associated with the presence of the stress survival islet 1 (SSI-1), inlL, and the truncated inlA genes. Combining the phenotypic and genotypic data may contribute to understanding the relationships between biofilm-associated genes and L. monocytogenes biofilm-forming ability, enabling improvement in the control of this foodborne pathogen.

Characterization of Salmonella enterica Biofilms and Antibiofilm Effect of Carvacrol and 2-Aminobenzimidazole.

Biofilm-associated foodborne Salmonella infections in poultry have become increasingly challenging for veterinarians, particularly in developing countries, and warrant thorough investigation. We assessed the biofilm-forming tendency of poultry isolates of Salmonella enterica, namely Salmonella Typhimurium (n = 23), Salmonella Infantis (n = 28), and Salmonella Heidelberg (n = 18), in nutrient-rich Rappaport-Vassiliadis Soya (RVS) peptone broth and nutrient-deficient diluted Tryptone Soya Broth (TSB). Seven of the tested isolates exhibited moderate biofilm formation in diluted TSB, whereas two showed such formation in RVS. In addition, the Congo red agar assay revealed curli and cellulose production in seven isolates. Fourteen specific biofilm-associated genes were analyzed identifying sdiA and seqA to be the most prevalent (100%), and glyA the least prevalent (69.5%). The prevalence of the genes bcsA and csgA was significantly lower in moderate and weak biofilm formers, respectively, as compared with nonbiofilm formers in RVS peptone broth. Furthermore, the compounds carvacrol and 2-aminobenzimidazole (2-ABI) effectively inhibited biofilm formation by Salmonella serovars in RVS peptone and TSB media, respectively. Whereas the antibiofilm activity of 2-ABI against Salmonella has not been reported previously, we determined its most effective concentration at 1.5 mM among tested antibiofilm treatments. These findings indicate that Salmonella strains prevalent in poultry farms have the potential to form biofilms, and the tested compounds should be further explored as supportive or alternative antimicrobials.

The frequency of adherence, biofilm-associated, Arginine Catabolic Mobile element genes, and biofilm formation in clinical and healthcare worker coagulase-negative staphylococci isolates.

BACKGROUND: Healthcare workers may pave the way for increased infections in hospitalized patients by coagulase-negative staphylococci (CoNS). Biofilm formation and antibiotic resistance are the major problems posed by CoNS in nosocomial infections. In this study, we determined biofilm production level and the distribution of biofilm-associated and virulence genes, including icaADBC, aap, bhp, atlE, embp, and fbe, as well as IS256, IS257, mecA, and ACME clusters (arc-A, opp-3AB) among 114 clinical (n = 57) and healthcare workers (n = 57) CoNS isolates in Kerman, Iran. RESULTS: In this study, more than 80% (n = 96) of isolates were methicillin-resistant CoNS (MR-CoNS). Out of 114 isolates, 33% (n = 38) were strong biofilm producers. Strong biofilm formation was found to be significantly different between clinical and healthcare workers' isolates (P < 0.050). In addition, 28% (n = 32) of isolates were positive for icaADBC simultaneously, and all were strong biofilm producers. The prevalence of icaADBC, mecA, bhp, fbe, and IS256 in clinical isolates was higher than that in healthcare workers' isolates (P < 0.050). A significant relationship was observed between clinical isolates and the presence of icaADBC, mecA, bhp, and IS256. Although these elements were detected in healthcare workers' isolates, they were more frequent in clinical isolates compared to those of healthcare workers. CONCLUSIONS: The high prevalence of ACME clusters in healthcare workers' isolates and biofilm formation of these isolates partially confirms the bacterial colonization in the skin of healthcare workers. Isolating MR-CoNS from healthcare workers' skin through similar genetic elements to clinical isolates, such as icaADBC, mecA, and IS256, calls for appropriate strategies to control and prevent hospital infections.

Comparison of biofilm production and virulence genes distribution among human and canine isolates of Staphylococcus aureus.

Background: Staphylococcus aureus is an important human and animal pathogen that can cause a wide range of infections due to numerous virulence factors. Aims: The aim of this study was to compare biofilm formation ability with different virulence factors such as bacterial motility, genes encoding biofilm associated proteins, and Panton-Valentine leukocidin (PVL) among human and canine isolates of S. aureus. Methods: A total of 60 human (30 methicillin sensitive S. aureus (MSSA) and 30 methicillin resistant S. aureus (MRSA)) and 17 canine (all MSSA) isolates of S. aureus were tested for the capability of biofilm production, motility assay, and presence of genes encoding virulence factors: ica (encoding intercellular adhesion), bap (encoding biofilm-associated protein), fnbA (encoding fibronectin-binding protein A), cna (encoding collagen-binding protein), and pvl (encoding PVL). Results: Animal isolates of S. aureus performed better biofilm production than the human strains (P=0.042), as well as human MSSA compared to the MRSA isolates (P=0.013). Our results showed that cna, fnbA, and ica genes (67.5%, 66.2%, and 42.9%, respectively) were more prevalent than bap and pvl genes (0%, and 7.8%, respectively). The ica gene was significantly more prevalent in human isolates compared to animal isolates (n=31/60 vs. n=2/17, P=0.008), whereas the cna gene was more frequent in animal isolates than in human ones (n=15/17 vs. n=37/60, P=0.0201). Significant correlations were found between the biofilm formation of animal isolates, and the presence of fnbA (P=0.029) and ica genes (P=0.001). Conclusion: This study showed a correlation between biofilm production and the presence of certain biofilm-related genes in animal isolates, as well as stronger biofilm production among MSSA human and animal isolates.

Association between ability to form biofilm and virulence factors of poultry extra-intestinal Campylobacter jejuni and Campylobacter coli.

Campylobacter species are known to be able to produce biofilm, which represents an ideal protective environment for the maintenance of such fragile bacteria. Since the genetic mechanisms promoting biofilm formation are still poorly understood, in this study we assessed the ability of C. jejuni (n = 7) and C. coli (n = 3) strains isolated from diseased poultry, and previously characterized by whole genome sequencing, to form biofilm. The in vitro analyses were carried out by using a microtiter based protocol including biofilm culturing and fixation, staining with crystal violet, and measurement of the optical density (OD570). The ability to form biofilm was categorized into four classes (no, weak, moderate, and strong producers). Potential correlations between OD570 and the presence/absence of virulence determinants were examined. The C. jejuni were classified as no (n = 3), weak (n = 2), and moderate (n = 2) biofilm producers; however, all possessed genes involved in chemotaxis, adhesion, and invasion to the host cells. No genes present exclusively in biofilm producers or in non-biofilm producers were identified. All C. coli were classified as weak producers and showed a similar set of virulence genes between each other. A trend of increased mean OD570 was observed in the presence of flaA and maf7 genes. No association between biofilm production classes and the explanatory variables considered was observed. The results of this study suggest that further investigations are needed to better identify and characterize the genetic determinants involved in extra-intestinal Campylobacter biofilm formation.

Genomic Diversity, Antimicrobial Susceptibility, and Biofilm Formation of Clinical Acinetobacter baumannii Isolates from Horses.

Acinetobacter (A.) baumannii is an opportunistic pathogen that causes severe infections in humans and animals, including horses. The occurrence of dominant international clones (ICs), frequent multidrug resistance, and the capability to form biofilms are considered major factors in the successful spread of A. baumannii in human and veterinary clinical environments. Since little is known about A. baumannii isolates from horses, we studied 78 equine A. baumannii isolates obtained from clinical samples between 2008 and 2020 for their antimicrobial resistance (AMR), clonal distribution, biofilm-associated genes (BAGs), and biofilm-forming capability. Based on whole-genome sequence analyses, ICs, multilocus (ML) and core-genome ML sequence types (STs), and AMR genes were determined. Antimicrobial susceptibility testing was performed by microbroth dilution. A crystal violet assay was used for biofilm quantification. Almost 37.2% of the isolates were assigned to IC1 (10.3%), IC2 (20.5%), and IC3 (6.4%). Overall, the isolates revealed high genomic diversity. We identified 51 different STs, including 22 novel STs (ST1723-ST1744), and 34 variants of the intrinsic oxacillinase (OXA), including 8 novel variants (OXA-970 to OXA-977). All isolates were resistant to ampicillin, amoxicillin/clavulanic acid, cephalexin, cefpodoxime, and nitrofurantoin. IC1-IC3 isolates were also resistant to gentamicin, enrofloxacin, marbofloxacin, tetracycline, and trimethoprim/sulfamethoxazole. All isolates were susceptible to imipenem. Thirty-one multidrug-resistant (MDR) isolates mainly accumulated in the IC1-IC3 groups. In general, these isolates showed less biofilm formation (IC1 = 25.0%, IC2 = 18.4%, IC3 = 15.0%) than the group of non-IC1-IC3 isolates (58.4%). Isolates belonging to the same ICs/STs revealed identical BAG patterns. BAG blp1 was absent in all isolates, whereas bfmR and pgaA were present in all isolates. At the level of the IC groups, the AMR status was negatively correlated with the isolates' ability to form a biofilm. A considerable portion of equine A. baumannii isolates revealed ICs/STs that are globally present in humans. Both an MDR phenotype and the capability to form biofilms might lead to therapeutic failures in equine medicine, particularly due to the limited availability of licensed drugs.

Analysis of biofilm formation in nosocomial Stenotrophomonas maltophilia isolates collected in Bulgaria: An 11-year study (2011-2022).

The present study aimed to explore the genotypic and phenotypic characteristics of biofilm formation in Bulgarian nosocomial Stenotrophomonas maltophilia isolates (n = 221) during the period 2011-2022, by screening for the presence of biofilm-associated genes (BAG) (spgM, rmlA and rpfF), their mutational variability, and assessment of the adherent growth on a polystyrene surface. The methodology included: PCR amplification, whole-genome sequencing (WGS) and crystal violet microtiter plate assay for biofilm quantification. The overall incidence of BAG was: spgM 98.6%, rmlA 86%, and rpfF 66.5%. The most prevalent genotype was spgM+/rmlA+/rpfF+ (56.1%), followed by spgM+/rmlA+/rpfF- (28.5%), and spgM+/rmlA-/rpfF+ (9.5%), with their significant predominance in lower respiratory tract isolates compared to those with other origin (P < 0.001). All strains examined were characterized as strong biofilm producers (OD550 from 0.224 ± 0.049 to 2.065 ± 0.023) with a single exception that showed a weak biofilm-forming ability (0.177 ± 0.024). No significant differences were observed in the biofilm formation according to the isolation source, as well as among COVID-19 and non-COVID-19 isolates (1.256 ± 0.028 vs. 1.348 ± 0.128, respectively). Also, no correlation was found between the biofilm amounts and the corresponding genotypes. WGS showed that the rmlA accumulated a larger number of variants (0.0086 per base) compared to the other BAG, suggesting no critical role of its product to the biofilm formation. Additionally, two of the isolates were found to harbour class 1 integrons (7-kb and 2.6-kb sized, respectively) containing sul1 in their 3' conservative ends, which confers sulfonamide resistance. To the best of our knowledge, this is the first study on S. maltophilia biofilm formation in Bulgaria, which also identifies novel sequence types (ST819, ST820 and ST826). It demonstrates the complex nature of this adaptive mechanism in the multifactorial pathogenesis of biofilm-associated infections.

Biofilm Formation Ability of ESBL/pAmpC-Producing Escherichia coli Isolated from the Broiler Production Pyramid.

Escherichia coli able to produce extended spectrum ß-lactamases (ESBLs) and plasmid-mediated AmpC ß-lactamases (pAmpCs) represents a serious threat to public health, since these genes confer resistance to critically important antimicrobials (i.e., third generation cephalosporins) and can be transferred to non-resistant bacteria via plasmids. E. coli are known to be able to form a biofilm, which represents a favorable environment for the exchange of resistance determinants. Here, we assessed the ability of 102 ESBL/pAmpC-producing E. coli isolated from the broiler production pyramid to form a biofilm and to identify genetic factors involved in biofilm formation. All but one of the ESBL/pAmpC-producing E. coli were able to form a biofilm, and this represents a great concern to public health. E. coli belonging to phylogroups D, E, and F, as well as strains harboring the blaCTX-M-type gene, seem to be associated with an increased biofilm capability (p < 0.05). Furthermore, virulence genes involved in adherence and invasion (i.e., csgBAC, csgDEFG, matABCDEF, and sfaX) seem to enhance biofilm formation in E. coli. Efforts should be made to reduce the presence of ESBL/pAmpC- and biofilm-producing E. coli in the broiler production pyramid and, therefore, the risk of dissemination of resistant bacteria and genes.

Carbapenem-Resistant Acinetobacter baumannii: Biofilm-Associated Genes, Biofilm-Eradication Potential of Disinfectants, and Biofilm-Inhibitory Effects of Selenium Nanoparticles.

This study aimed to investigate the biofilm-production ability of carbapenem-resistant Acinetobacter baumannii (CRAB), the biofilm-eradication potential of 70% ethanol and 0.5% sodium hypochlorite, the effects of selenium nanoparticles (SeNPs) against planktonic and biofilm-embedded CRAB, and the relationship between biofilm production and bacterial genotypes. A total of 111 CRAB isolates were tested for antimicrobial susceptibility, biofilm formation, presence of the genes encoding carbapenemases, and biofilm-associated virulence factors. The antibiofilm effects of disinfectants and SeNPs against CRAB isolates were also tested. The vast majority of the tested isolates were biofilm producers (91.9%). The bap, ompA, and csuE genes were found in 57%, 70%, and 76% of the CRAB isolates, with the csuE being significantly more common among biofilm producers (78.6%) compared to non-biofilm-producing CRAB (25%). The tested disinfectants showed a better antibiofilm effect on moderate and strong biofilm producers than on weak producers (p < 0.01). The SeNPs showed an inhibitory effect against all tested planktonic (MIC range: 0.00015 to >1.25 mg/mL) and biofilm-embedded CRAB, with a minimum biofilm inhibitory concentration of less than 0.15 mg/mL for 90% of biofilm producers. In conclusion, SeNPs might be used as promising therapeutic and medical device coating agents, thus serving as an alternative approach for the prevention of biofilm-related infections.

Biofilm formation, antibiotic resistance and genotyping of Shiga toxin-producing Escherichia coli isolated from retail chicken meats.

1. The Shiga toxin-producing Escherichia coli (STEC) is a hazardous zoonotic agent for chicken meat consumers. This study determined the serogroups and evaluated the virulence genes, antibiotic resistance, biofilm-forming profiles and genetic relationships of STEC isolates in chicken meat.2. A total of 100 samples belonging to dressed-whole chicken and different parts of the chicken (wing, breast, thigh, drumstick) were collected between September and November 2019 from different retail markets in Kayseri, Türkiye.3. Phenotypic (identification, disc diffusion test, Congo red agar and microtitre plate tests) and molecular tests (identification, serogrouping, virulence factors, biofilm, antibiotic susceptibility, 16S rRNA sequencing and enterobacterial repetitive intergenic consensus-PCR for typing of the isolates) were carried out.4. E. coli was isolated from 35% of the samples and 35% of the samples harboured at least one STEC. Among 35 STEC isolates, 3 (8.5%), 6 (17.1%), 2 (5.7%) and 3 (8.5%) were found to be positive for fliCH2, fliCH8, fliCH11, fliCH19 genes, respectively. Out of 35 STEC positive isolates, 4 (11.4%) were identified as E. coli O157, from which 2 (5.7%) were E. coli O157:H7. E. coli O157 was detected in two (10%), one (5%), one (5%) of the thigh, drumstick and whole chicken samples, respectively.5. Biofilm-forming ability was reported in 33 (94.2%) of 35 E. coli isolates, whilst the biofilm-associated genes detected among 35 STEC isolates included csgA (88.5%), fimH (88.5%), bcsA (85.7%), agn43 (14.2%) and papC (8.5%). The STEC strains showed resistance against ampicillin (88.5%) and erythromycin (88.5%), followed by tetracycline (74.2%) and gentamicin (25.7%). However, the distribution of isolates harbouring blaCMY, ere(A), tet(A) and aac(3)-IV antibiotic resistance genes was found to be 17.1%, 11.4%, 85.7% and 5.7%, respectively.6. ERIC-PCR showed that E. coli strains obtained from different parts and whole of chicken samples had genetic diversities. ERIC-PCR patterns grouped strains of 35 STEC into eight clusters designated A-H, with 73% similarity. Proper hygiene measures and staff training are essential for public health during poultry processing and in retail stores to control STEC.

Down-regulation of biofilm-associated genes in mecA-positive methicillin-resistant S. aureus treated with M. communis extract and its antibacterial activity.

Considering the prevalence of resistance to antibiotics, the discovery of effective agents against resistant pathogens is of extreme urgency. Herein, 26 mecA-positive methicillin-resistant S. aureus (MRSA) isolated from clinical samples were identified, and their resistance to 11 antibiotics was investigated. Next, the antibacterial and anti-biofilm activity of the ethanolic extract of M. communis on these strains was evaluated. Furthermore, the effect of this extract on the expression of biofilm-associated genes, icaA, icaD, bap, sarA, and agr, was studied. According to the results, all isolated strains were multidrug-resistant and showed resistance to oxacillin and tetracycline. Also, 96.15 and 88.46 % of them were resistant to gentamicin and erythromycin. However, the extract could effectively combat the strains. The minimum inhibitory concentration (MIC) against different strains ranged from 1.56 to 25 mg/ml and the minimum bactericidal concentration (MBC) was between 3.125 and 50 mg/ml. Even though most MRSA (67 %) strongly produced biofilm, the sub-MIC concentration of the extract destroyed the pre-formed biofilm and affected the bacterial cells inside the biofilm. It could also inhibit biofilm development by significantly decreasing the expression of icaA, icaD, sarA and bap genes involved in biofilm formation and development. In conclusion, the extract inhibits biofilm formation, ruins pre-formed biofilm, and kills cells living inside the biofilm. Furthermore, it down-regulates the expression of necessary genes and nips the biofilm formation in the bud.

Biofilm-Formation Ability and the Presence of Adhesion Genes in Coagulase-Negative Staphylococci Isolates from Chicken Broilers.

The aim of the study was to analyze the biofilm-production capacity of 87 coagulase-negative Staphylococcus strains (CoNS) isolated from broiler chickens and to determine the occurrence of biofilm-associated genes. The biofilm production capacity of staphylococci was assessed using the microtiter plate method (MTP), and the frequency of genes was determined by PCR. The ability to form a biofilm in vitro was shown in 79.3% of examined strains. Strong biofilm capacity was demonstrated in 26.4% of strains, moderate capacity in 25.3%, weak capacity in 27.6%, and a complete lack of biofilm production capacity in 20.7% of strains. The icaAB gene responsible for the production of extracellular polysaccharide adhesins was detected in 6.9% of strains. The other four genes, i.e., bap (encoding biofilm-associated protein), atlE (encoding cell surface protein exhibiting vitronectin-binding activity), fbe (encoding fibrinogen-binding protein), and eno (encoding laminin-binding protein) were detected in 5.7%, 19.5%, 8%, and 70.1% of strains, respectively. Demonstration of genes that play a role in bacterial biofilm formation may serve as a genetic basis to distinguish between symbiotic and potentially invasive coagulase-negative staphylococcal strains.

Detection of Acinetobacter pittii ST220 co-producing NDM-1 and OXA-820 carbapenemases from a hospital sink in a non-endemic country of NDM.

OBJECTIVES: NDM-1 is by far one of the most commonly prevalent carbapenemases in Enterobacteriaceae and Acinetobacter baumannii. This study presented an Acinetobacter pittii (A. pittii) isolate co-harboring blaNDM-1 and blaOXA-820 from a university hospital sink, where New Delhi metallo-ß-lactamase (NDM) producers have not been found in either patients or their environments. METHODS: Whole-genome sequencing was performed on the HiSeq 4000 platform, and the reads were de novo assembled using the A5-miSeq Assembly pipeline. Annotation of the resulting scaffolds were performed by using the DDBJ Fast Annotation and Submission Tool (DFAST). The blaNDM-1-carrying plasmid was determined. RESULTS: The A. pittii ST220 strain SU1805 detected from a sink strainer in the treatment room was resistant to imipenem and meropenem. Antimicrobial resistance genes blaNDM-1, blaOXA-820, blaADC-43, and aphA6 were found in this strain. The blaNDM-1 was found to be located downstream of an ISAba125 element on a plasmid pSU1805NDM with a size of 41,022 bp, and GC content of 38.3% harbouring 48 protein-coding genes. The aphA6 gene was also located upstream of the ISAba125 on the same plasmid. The A. pittii intrinsic blaOXA-213-like gene blaOXA-820 was located between fxsA and yncA genes in the chromosome. The strain also harboured biofilm-associated genes such as ompA, the csu operon and their regulating genes bfmRS. CONCLUSION: This study described the first isolation of NDM-1-producing A. pittii in Japan, and highlighted the importance of proper implementation of measures against AMR for sink drainage systems, since NDM producers may have already been hidden in such environments in a non-endemic country of NDM.

A Relationship Between O-Serotype, Antibiotic Susceptibility and Biofilm Formation in Uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) is a well-known pathogen that has perturbed the medical scenario because of its resistance to diverse therapeutic drugs and its ability to form a biofilm. Different O-serogroups are the prevalent cause of urinary tract infections (UTIs) along with their ability to form a biofilm. The present research aimed to assess antibiotic susceptibility, biofilm formation, and serotyping of UPEC isolates in conjunction with the demographic data. Antibiotic susceptibility was determined using the Kirby-Bauer method and biofilm formation was assessed phenotypically and at the molecular level. Serotyping was performed by multiplex PCR. A significant proportion of the total of 120 UPECs was isolated from women (p < 0.05). Most isolates were resistant to cefotaxime, ceftazidime, and tetracycline, but maintained their sensitivity to imipenem. O25, O15, O8, and O75 were the most commonly detected serogroups. Moreover, O25, O15, and O8 were the highest biofilm-producing serogroups among the UPEC isolates. Serogroups O75 and O21 were significantly associated with diabetic patients and subjects with renal disease, respectively (p < 0.05). Overall, our results show that UTI incidence in women should be a subject of concern. The high prevalence of the O25 serogroup associated with a specific antibiotic profile and a high percentage of biofilm formation suggests a close relation between serogroups and characteristic features of UPEC isolates.