Strong Biofilm Formation Research Articles

Virulence-related factors and antimicrobial resistance in Proteus mirabilis isolated from domestic and stray dogs.

Proteus mirabilis is a multi-host pathogen that causes diseases of varying severity in a wide range of mammals, including humans. Proteus mirabilis is resistant to multiple antibiotics and has acquired the ability to produce expanded spectrum of β-lactamases, leading to serious public health problems. However, the available information on P. mirabilis isolated from feces of dogs, is still poorly understood, as is the correlation between its virulence-associated genes (VAGs) and antibiotic resistance genes (ARGs). In this study, we isolated 75 strains of P. mirabilis from 241 samples, and investigated the swarming motility, biofilm formation, antimicrobial resistance (AMR), distribution of VAGs and ARGs, as well as the presence of class 1, 2, and 3 integrons in these isolates. Our findings suggest a high prevalence of intensive swarming motility and strong biofilm formation ability among P. mirabilis isolates. Isolates were primarily resistant to cefazolin (70.67%) and imipenem (70.67%). These isolates were found to carry ureC, FliL, ireA, zapA, ptA, hpmA, hpmB, pmfA, rsbA, mrpA, and ucaA with varying prevalence levels of 100.00, 100.00, 100.00, 98.67, 98.67, 90.67, 90.67, 90.67, 90.67, 89.33, and 70.67%, respectively. Additionally, the isolates were found to carry aac(6')-Ib, qnrD, floR, blaCTX-M, blaCTX-M-2, blaOXA-1, blaTEM, tetA, tetB and tetM with varying prevalence levels of 38.67, 32.00, 25.33, 17.33, 16.00, 10.67, 5.33, 2.67, 1.33, and 1.33%, respectively. Among 40 MDR strains, 14 (35.00%) were found to carry class 1 integrons, 12 (30.00%) strains carried class 2 integrons, while no class 3 integrons was detected. There was a significant positive correlation between the class 1 integrons and three ARGs: blaTEM, blaCTX-M, and blaCTX-M-2. This study revealed that P. mirabilis strains isolated from domestic dogs exhibited a higher prevalence of MDR, and carried fewer VAGs but more ARGs compared to those isolated from stay dogs. Furthermore, a negative correlation was observed between VAGs and ARGs. Given the increasing antimicrobial resistance of P. mirabilis, veterinarians should adopt a prudent approach towards antibiotics administration in dogs to mitigate the emergence and dissemination of MDR strains that pose a potential threat to public health.

Attachment characteristics and kinetics of biofilm formation by Staphylococcus aureus on ready-to-eat cooked beef contact surfaces.

Staphylococcus aureus is a food-borne pathogen that quickly forms biofilm on meat contact surfaces and thus poses a serious threat to the safety of the meat industry. This study evaluated the attachment, survival, and growth of S. aureus biofilm with exposure to environmental factors in the meat industry by simulated ready-to-eat (RTE) cooked beef product contamination scenarios. The results indicated that the meat-borne S. aureus biofilm formation dynamic could be divided into four different phases: initial adhesion (4-12h), exponential (12-24h), slow growth (1-3 days), and stationary (3-7 days). Meat-borne S. aureus has strong adhesion and biofilm formation ability, and its biofilm exhibits persistence, high-intensity metabolic activity, aerotaxis, and strain heterogeneity. This study has also demonstrated that in the long-term existence of meat-borne S. aureus biofilm on stainless steel and plexiglass surfaces(>7 days, 7.2-8.8 log CFU/cm2 ), expose to RTE cooked beef products, may cause it to become high-risk contaminated food. Meat-borne S. aureus that forms a dense and rough concave-convex in the shape of biofilm architecture was observed by scanning electron microscopy, consisting of complex components and adhesion of living and dead cells. This was further confirmed by the meat-borne S. aureus biofilm on the stainless steel surface by attenuated total reflectance Fourier transformed infrared spectroscopy, and the dominant peaks in biofilm spectra were mainly associated with proteins, polysaccharides, amino acid residues, and phospholipids(>50%). These findings may help in the identification of the main sources of contamination within the meat industry and the subsequent establishment of strategies for biofilm prevention and removal. PRACTICAL APPLICATION: This study revealed the meat-borne S. aureus biofilm formation mechanism and found that it exhibited strong colonization and biofilm-forming ability, which can persist on the contact surfaces of ready-to-eat beef products. These initial findings could provide information on the behavior of meat-borne S. aureus biofilm attached to meat contact surfaces under conditions commonly encountered in meat environments, which help to support the determination of the main sources of contamination within the meat industry and the subsequent establishment of strategies for biofilm prevention and removal. It was also helpful in controlling biofilm contamination and improving meat safety to minimize it.

Genomics, biofilm formation and infection of bladder epithelial cells in potentially uropathogenic Escherichia coli (UPEC) from animal sources and human urinary tract infections (UTIs) further support food-borne transmission

Escherichia coli is the main cause of urinary tract infections (UTI). While genomic comparison of specific clones recovered from animals, and human extraintestinal infections show high identity, studies demonstrating the uropathogenicity are lacking. In this study, comparative genomics combined with bladder-cell and biofilm formation assays, were performed for 31 E. coli of different origins: 7 from meat (poultry, beef, and pork); 2 from avian-farm environment; 12 from human uncomplicated UTI, uUTI; and 10 from human complicated UTI, cUTI. These isolates were selected based on their genetic uropathogenic (UPEC) status and phylogenetic background. In silico analysis revealed similar virulence-gene profiles, with flagella, type 1 and curli fimbriae, outer-membrane proteins (agn43, ompT, iha), and iron-uptake (iutA, entA, and fyuA) associated-traits as the most prevalent (>65%). In bladder-cell assays, moderate to strong values of association (83%, 60%, 77.8%) and invasion (0%, 70%, 55.5%) were exhibited by uUTI, cUTI, and animal-derived isolates, respectively. Of interest, uUTI isolates exhibited a significantly lower invasive capacity than cUTI isolates (p < 0.05). All isolates but one produced measurable biofilm. Notably, 1 turkey meat isolate O11:H6-F-ST457, and 2 cUTI isolates of the pandemic lineages O83:H42-F-ST1485-CC648 and O25b:H4-B2-ST131, showed strong association, invasion and biofilm formation. These isolates showed common carriage of type 1 fimbriae and csg operons, toxins (hlyF, tsh), iron uptake systems (iutA, entA, iroN), colicins, protectins (cvaC, iss, kpsM, traT), ompT, and malX. In summary, the similar in vitro behaviour found here for certain E. coli clones of animal origin would further reinforce the role of food-producing animals as a potential source of UPEC. Bladder-cell infection assays, combined with genomics, might be an alternative to in vivo virulence models to assess uropathogenicity.

Exploration of fish gut-associated actinobacteria for its antifouling activity.

An attempt has been made to screen the fish gut-associated actinobacterial cultures for antifouling compounds. Fifteen morphologically distinct bacterial cultures were recovered from the biofouling samples scraped from the boat surfaces and other maritime structures in the Kovalam beach (Lat. 12.7870°N; Long. 80.2504°E) coastal areas in Tamil Nadu, India. All the bacterial isolates were identified at generic level from which two isolates namely KB6 and KB7 as Staphylococcus sp. were found the exhibit strong biofilm formation. Four actinobacterial strains viz., IM20, PYA9, F8, and SQA4 were evaluated for inhibitory properties against biofouling bacteria using the agar plug method. Strain IM20 which strongly inhibited the biofouling bacteria was chosen as prospective strain for further studies. When compared to submerged fermentation, IM20 produced a high amount of antifouling chemicals on the agar surface fermentation. Among the solvents tested, better extraction of antifouling compounds was seen in ethyl acetate extract. Antifouling compound production by the strain IM20 was found to be influenced by a number of variables such as glucose, fructose, glutamine, malt extract, pH 7 and 9, temperature 30 and 40° C and NaCl at 2.5% and 5% concentrations. Gas chromatography-mass spectrometry (GC-MS) analysis of the strain IM20 extract revealed the presence of pyrrolo (1,2-a]pyrazine-1,4-dione, hexahydro) in significant amount. In the present study, the fish gut-associated Streptomyces sp. IM20 was identified as an unusual and newly added source for the isolation of antifouling compounds.

Stutzerimonas frequens strain TF18 with superior heterotrophic nitrification-aerobic denitrification ability for the treatment of aquaculture effluent

Currently, microbial techniques have been comprehensively exploited in aquaculture effluent treatment. However, strains for bioaugmentation face many issues, such as poor adaptability, long colonization time, and unsatisfactory treatment effect. Herein, an efficient heterotrophic nitrifying-aerobic denitrifying bacterium Stutzerimonas frequens strain TF18 was isolated from the Whiteleg shrimp (Penaeus vannamei) mariculture environment. Strain TF18 exhibited strong biofilm formation and auto-aggregation characteristics, which indicated its robust adaptability and short colonization time. Remarkably, the total ammonia nitrogen, NO2--N, and NO3--N removal efficiencies of the strain were 89.21 %, 99.76 %, and 96.16 %, respectively, in the presence of sodium acetate as carbon source and carbon-nitrogen ratio of 10. Most importantly, identification of genes related to nitrogen removal, including nirS, norB, nosZ, nasA, napA, narG, and nirB, and their relative expression further demonstrated the existence of nitrate transformation pathways in strain TF18. These results provided new guidance for practical application of S. frequens strain TF18 in aquaculture effluent treatment.

Probiotic profiling of bifidobacteria indigenous to the human intestinal mucosa shows alleviation of dysbiosis-associated pathogen biofilms

The present study was undertaken to isolate bifidobacterial probiotics and characterize the biodiversity of mucosal bacteria in the human distal gut through 16S rRNA amplicon sequencing. Bifidobacterial strains obtained by selective culturing were investigated for biofilms and probiotic characteristics. Both culture-dependent and culture-independent approaches revealed substantial microbial diversity. Bifidobacterium strains yielded robust biofilms with predominantly exopolysaccharides and eDNA matrix. Microscopy revealed species-dependent spatial arrangement of microcolonies. Following probiotic profiling and safety assessment, the inter- and intra-specific interactions in in dual strain bifidobacterial biofilms were studied. As a species, only strains of B. bifidum exhibited exclusively inductive type of interactions whereas in other species, the interactions were more varied. On the other hand, in dual species biofilms, a preponderance of inductive interactions was evident between B. adolescentis, B. thermophilum, B. bifidum, and B. longum. The strong biofilm-formers also diminished pathogenic biofilm viability, and some were proficient in cholesterol removal in vitro. None of the strains exhibited harmful enzymatic activities associated with disease pathology. Interaction between biofilm-forming bifidobacterial strains provides an understanding of their functionality and persistence in the human host, and food or medicine. Their anti-pathogenic activity represents a therapeutic strategy against drug-resistant pathogenic biofilms.

Community acquired methicillin resistant Staphylococci (CA-MRS) in fecal matter of wild birds – A ‘one health’ point of concern

BackgroundAntibiotic resistance in Staphylococci, particularly methicillin resistance is a major public health concern. While this problem has been reported from the clinical settings, its presence in non-clinical settings also needs to be investigated. The role of wildlife in carrying and disseminating the resistant strains has been established in different studies but its role in Pakistani environment has not been explored yet. To evaluate this, we investigated the carriage of antibiotic resistant Staphylococci in wild birds from Islamabad region. MethodologyBirds fecal matter were collected during September 2016-August 2017 from eight different environmental settings of Islamabad. Prevalence of Staphylococci, their susceptibility profile against eight classes of antibiotics through disc diffusion method, their SCCmec types, co-resistance of macrolide and cefoxitin through PCR assay and biofilm formation through microtitre plate assay were studied. ResultsOut of 320 birds feces collected, 394 Staphylococci were isolated, where 165 (42%) were resistant to at least one or two classes of antibiotics. High resistance was found against erythromycin (40%) and tetracycline (21%) while cefoxitin resistance was 18% and vancomycin resistance was only in 2%. One hundred and three (26%) isolates exhibited multi-drug resistance (MDR) pattern. mecA gene was detected in 45/70 (64%) cefoxitin resistant isolates. Community acquired methicillin resistant Staphylococci (CA-MRS) were 87% while Hospital acquired methicillin resistant Staphylococci (HA-MRS) were 40%. In the MRS isolates showing co-resistance to macrolides, mefA (69%) and ermC (50%) genes were more prevalent. Strong biofilm formation was observed in 90% of the MRS, of which 48% were methicillin resistant Staphylococcus aureus (MRSA) isolates while 52% were methicillin resistant coagulase negative Staphylococci (MRCoNS). ConclusionOccurrence of methicillin resistant strains of Staphylococci in wild birds suggests their role in the carriage and dissemination of resistant strains into the environment. The findings of the study strongly recommend the monitoring of resistant bacteria in wild birds and wildlife.

Situational analysis on fluoroquinolones use and characterization of high-level ciprofloxacin-resistant Enterococcus faecalis by integrated broiler operations in South Korea.

Fluoroquinolones are classified as "critically important antimicrobials for human medicine"; however, their extensive use in livestock poses a significant health risk to humans as it leads to the rapid spread of antimicrobial resistance. This study confirmed that 40.0%-71.4% of the farms in three of the five integrated broiler operations were administered ciprofloxacin (CIP). Moreover, preventive purposes (60.9%), veterinarian prescriptions (82.6%), drinking water route (100%), and 1 to 3 days (82.6%) of age were significantly highest (P < 0.05). 194 high-level ciprofloxacin-resistant (HLCR) Enterococcus faecalis (E. faecalis) were found in 65 of 74 farms, and of which, the prevalence of qnrA (63.9%), tetM (60.3%), ermB (64.9%), blaz (38.7%), and catA (34.0%) was significantly highest (P < 0.05). 154 (79.4%) isolates showed MDR, and the distribution of MDR was significantly differences among the operations (P < 0.05). All HLCR E. faecalis possessed double mutations in gyrA and parC, and S83I/S80I (90.7%) mutations were most commonly identified. Interestingly, the distribution of isolates with MICs ≥ 512 for both CIP and moxifloxacin was significantly higher in CIP-administered farms (56.5%) than in non-CIP-administered farms (41.4%) (P < 0.05). Also, the prevalence of strong or moderate biofilm formers in HLCR E. faecalis was significantly higher than that of weak and no biofilm formers (P < 0.05). HLCR E. faecalis were heavily distributed in the broiler farms in Korea; therefore, it is necessary to minimize the prevalence of resistant bacteria via structural management regulations such as cleaning and disinfection of farm environments.

Prevalence, Resistance Patterns and Biofilm Production Ability of Bacterial Uropathogens from Cases of Community-Acquired Urinary Tract Infections in South Italy.

Community-acquired urinary tract infections represent the most common infectious diseases in the community setting. Knowing the antibiotic resistance patterns of uropathogens is crucial for establishing empirical treatment. The aim of the current study is to determine the incidence of the causative agents of UTIs and their resistance profiles. Patients of all ages and both sexes were enrolled in the study, and admitted to San Ciro Diagnostic Center in Naples between January 2019 and Jun 2020. Bacterial identification and antibiotic susceptibility testing were carried out using Vitek 2 system. Among the 2741 urine samples, 1702 (62.1%) and 1309 (37.9%) were negative and positive for bacterial growth, respectively. Of 1309 patients with infection, 760 (73.1%) were females and 279 (26.9%) were males. The greatest number of positive cases were found in the in the elderly (>61 years). Regarding uropathogens, 1000 (96.2%) were Gram-negative while 39 (3.8%) were Gram-positive strains. The three most isolated pathogenic strains were Escherichia coli (72.2%), Klebsiella pneumoniae (12.4%), and Proteus mirabilis (9.0%). Strong biofilm formation ability was observed in about 30% of the tested isolates. The low resistance rates recorded against nitrofurantoin, fosfomycin, piperacillin-tazobactam, and gentamicin could suggest them as the most appropriate therapies for CA-UTIs.

Virulent potential of methicillin-resistant and methicillin-susceptible Staphylococcus pseudintermedius in dogs

Staphylococcus pseudintermedius is a zoonotic pathogen responsible for several infectious diseases in pet animals, yet its pathogenic potential is not fully understood. Thus, this study aims to unravel the virulence profile of S. pseudintermedius from canine origin. Methicillin-resistant (MRSP) and methicillin-susceptible (MSSP) strains were isolated from different infection sites and their genotypic and phenotypic features were compared to determine the clinical implications of MRSP and MSSP strains. Bacterial identification was performed using MALDI-TOF and 16S-rDNA sequencing. In addition, we used multilocus sequence typing (MLST) for strains’ sequence type (ST) determination and phylogenetic relationship. The strains were screened for toxin genes, including cytotoxins (lukS, lukF), exfoliative toxin (siet), enterotoxins (sea, seb, sec, secCanine, sel, sem, and seq) and toxic shock syndrome toxin (tst-1). In vitro phenotypic analyses assessing antimicrobial susceptibility profile, biofilm formation ability, and expression of extracellular matrix components were performed. The investigated S. pseudintermedius strains belong to 17 unique ST, most of which were classified as ST71. MSSP and MRSP strains shared siet, lukS, and lukF virulence markers. Our findings showed that some MSSP strains also harbored sel, seq, and sem enterotoxin genes, suggesting a more diverse virulence profile. All MRSP strains and 77% of MSSP strains were classified as multidrug resistant (MDR). Moreover, all investigated S. pseudintermedius strains showed strong biofilm formation ability. In summary, our findings highlight the wide spread of highly virulent and drug-resistant zoonotic S. pseudintermedius strains, being a potential concern for One Health issues.

Antimicrobial Resistance Pattern, Pathogenicity and Molecular Properties of Hypervirulent Klebsiella pneumonia (hvKp) among Hospital-Acquired Infections in the Intensive Care Unit (ICU)

Hypervirulent Klebsiella pneumoniae (hvKp) is a new emerging variant of K. pneumoniae that is increasingly reported worldwide. The variant hvKp is known to cause severe invasive community-acquired infections such as metastatic meningitis, pyogenic liver abscesses (PLA) and endophthalmitis, but its role in hospital-acquired infections (HAIs) is little known. The aim of this study was to evaluate the prevalence of hvKp among hospital-acquired (HA) K. pneumoniae infections in the intensive care unit (ICU) and to compare between hvKp and classical K. pneumoniae (cKP) regarding antimicrobial resistance pattern, virulence and molecular characteristics. The study was cross-sectional and included 120 ICU patients suffering from HA K. pneumoniae infections between January and September 2022. K. pneumoniae isolates were subjected to antimicrobial susceptibility testing and detection of extended-spectrum-β-lactamase (ESBL) production by the Phoenix 100 automated microbiology system, string test, biofilm formation, serum resistance assay, and detection of virulence-associated genes (rmpA, rmpA2, magA, iucA) and capsular serotype-specific genes (K1, K2, K5, K20, K57) by polymerase chain reaction (PCR). Of 120 K. pneumoniae isolates, 19 (15.8%) were hvKp. The hypermucoviscous phenotype was more significantly detected in the hvKp group than in the cKP group (100% vs. 7.9%, p ≤ 0.001). The rate of resistance to different antimicrobial agents was significantly higher in the cKP group than that in the hvKp group. Fifty-three strains were identified as ESBL-producing strains, which was more frequent in the cKP group than in the hvKp group (48/101 [47.5%] vs. 5/19 [26.3%], respectively, p ≤ 0.001). The hvKP isolates were highly associated with moderate and strong biofilm formation than cKP isolates (p = 0.018 and p = 0.043 respectively). Moreover, the hvKP isolates were highly associated with intermediate sensitivity and re sistance to serum in the serum resistance assay (p = 0.043 and p = 0.016 respectively). K1, K2, rmpA, rmpA2, magA and iucA genes were significantly associated with hvKp (p ≤ 0.001, 0.004, <0.001, <0.001, 0.037 and <0.001, respectively). However, K5, K20 and K57 were not associated with hvKp. The hvKp strains have emerged as a new threat to ICU patients because of their ability to cause more severe and life-threatening infections than cKP. The string test alone as a laboratory test for screening of hvKp has become insufficient. Recently, hvKp was defined as hypermucoviscous- and aerobactin-positive. It is important to improve the awareness towards the diagnosis and management of hvKp infections.

Prevalence, multiple antibiotic resistance and virulence profile of methicillin-resistant Staphylococcus aureus (MRSA) in retail poultry meat from Edo, Nigeria.

Staphylococcus aureus causes staphylococcal food poisoning and several difficult-to-treat infections. The occurrence and dissemination of methicillin-resistance S. aureus (MRSA) in Nigeria is crucial and well documented in hospitals. However, findings on MRSA from meat in the country are yet to be adequately reported. The current study determined the prevalence, virulence profile and antibiogram characteristics of MRSA from a raw chicken product from retail outlets within Edo. A total of 368 poultry meat samples were assessed for MRSA using a standard culture-based approach and characterized further using a molecular method. The antimicrobial susceptibility profile of the isolates was determined using the disc diffusion method. The biofilm profile of the isolates was assayed via the crystal violet microtitre-plate method. Virulence and antimicrobial resistance genes were screened using polymerase chain reaction via specific primers. Of the samples tested, 110 (29.9%) were positive for MRSA. All the isolates were positive for deoxyribonuclease (DNase), coagulase and beta-hemolysis production. Biofilm profile revealed 27 (24.55%) weak biofilm formers, 18 (16.36%) moderate biofilm formers, and 39 (35.45%) strong biofilm formers. The isolates harboured 2 and ≤17 virulence genes. Enterotoxin gene profiling revealed that 100 (90.9%) isolates harboured one or more genes. Resistance against the tested antibiotics followed the order: tetracycline 64(58.2%), ciprofloxacin 71(64.6%), trimethoprim 71(64.6%) and rifampin 103(93.6%). A total of 89 isolates were multidrug-resistant, while 3 isolates were resistant to all 22 antibiotics tested. The isolates harboured antimicrobial-resistant determinants such as methicillin-resistant gene (mecA), tetracycline resistance genes (tetK, tetL), erythromycin resistance genes (ermA, ermC), trimethoprim resistance gene (dfrK). All the staphylococcal cassette chromosome mec (SCCmec) IVa and SCCmec V positive isolates harboured the Panton-Valentine Leukocidin Gene (PVL). In conclusion, S. aureus was resistant to commonly used antibiotics; a concern to public health concerning the transmission of these pathogens after consuming these highlight the significance of antimicrobial and enterotoxigenic monitoring of S. aureus in food chains.

Biofilm formation and antibiotic resistance among Coagulase Negative Staphylococcus species isolated from central venous catheters of intensive care unit patients.

This study was conducted to determine the biofilm formation of coagulase negative Staphylococcus species (CoNS) isolated from patients with catheter related blood stream infection (CRBSI) and colonized central venous catheters (CVC) and their antibiotic susceptibility patterns and in situ biofilm formation of CVC tips. Eighty-two CoNS isolated from intensive care unit (ICU) patients with CRBSI (n​=​8) or colonized CVC (n​=​74) were included. Species identification and antibiotic susceptibility test were done. All isolates were screened for biofilm formation using crystal violet and 3-(4,5-dimethylthiazole-2-yl)-2-5-diphenyl-2H-tetrazolium bromide (MTT) assays and categorized as strong or moderate biofilm formers. CVC tips were subjected to crystal violet stain and scanning electron microscopy (SEM) to detect in-situ biofilm formation. Staphylococcus haemolyticus (n​=​34; 41%) was the commonest to cause both CRBSI and CVC colonization. All 82 CoNS produced biofilms. Among them 77 (93.90%) were strong biofilm formers including all from CRBSI patients and 05 (6.10%) were moderate biofilm formers as detected by both methods. SEM showed bacteria adhered to surfaces of CVC tips with microbial-aggregates embedded in extracellular matrix. Mean crystal violet absorbance of CVC from CRBSI patients (0.6628) was significantly higher than colonized CVC (mean value 0.5592) (p​=​0.030). S.haemolyticus showed higher resistance to cloxacillin compared to other CoNS (p​=​0.039). Majority of CoNS isolated were strong biofilm formers. In-situ biofilm formation on CVC tips were significantly evident in CRBSI patients compared to CVC colonized patients. S.haemolyticus is the commonest to cause both CRBSI and CVC colonization and shows significantly higher cloxacillin resistance rate.

Clinical and Bacteriological Analyses of Biofilm-Forming Staphylococci Isolated from Diabetic Foot Ulcers.

Diabetes mellitus is a chronic disease that is associated with increased morbidity and mortality. Unfortunately, foot ulcers and amputations due to diabetes are very common in developing countries. The purpose of this study was to characterize the clinical presentation of diabetic foot ulcer (DFU) infections, isolate the causative agent, and analyze the biofilm formation and distribution of biofilm-related genes among isolated Staphylococci. The study included 100 diabetic patients suffering from DFUs attending Assiut University Hospital. Swabs were collected and antimicrobial susceptibility testing of the isolates was performed. Biofilm formation was tested phenotypically among staphylococcal isolates and the frequency of different biofilm genes was analyzed by PCR. Clinical presentations of diabetic foot ulcers were correlated with bacterial genetic characteristics. Spa types were determined using DNA Gear-a software. Microbiological analysis showed that 94/100 of the DFUs were positive for bacterial growth. The majority of infections were polymicrobial (54%, n=54/100). Staphylococci were the most commonly detected organisms, of which S. aureus represented 37.5% (n=24/64), S. haemolyticus 23.4% (n=15/64), S. epidermidis 34.3% (n=22/64) and other CNS 4.7% (n=3/64). Interestingly, co-infection with more than one species of Staphylococci was observed in 17.1% (n=11/64) of samples. A high level of antibiotic resistance was observed, where 78.1% (n=50/64) of Staphylococci spp were multidrug-resistant (MDR). Phenotypic detection showed that all isolated Staphylococci were biofilm-formers with different grades. Analysis of biofilm-forming genes among Staphylococci showed that the most predominant genes were icaD, spa, and bap. Isolates with a higher number of biofilm-related genes were associated with strong biofilm formation. Sequencing of the spa gene in S. aureus showed that our isolates represent a collection of 17 different spa types. The majority of DFUs in our hospital are polymicrobial. Staphylococci other than S.aureus are major contributors to infected DFUs. MDR and biofilm formation are marked among isolates, which is paralleled by the presence of different categories of virulence-related genes. All severely infected wounds were associated with either strong or intermediate biofilm formers. The severity of DFU is directly related to the number of biofilm genes.

Curcumin-ZnO nanocomposite mediated inhibition of Pseudomonas aeruginosa biofilm and its mechanism of action

Multidrug-resistance and strong biofilm formation potential of P. aeruginosa makes it a severe risk to public health and nanoparticles/nanocomposites find potential applicability to address this crisis. Curcumin is a potential anti-biofilm/antibacterial molecule that is limited due to large sized particle, reduced water solubility and decreased bioavailability. In this study, curcumin-ZnO nanocomposite was synthesized and its anti-biofilm efficacy as well as mechanism of action against P. aeruginosa biofilm was investigated. Here, using the FTIR peaks, the novel structure of curcumin-ZnO nanocomposites was established and using that as ligand the potential anti-biofilm drug targets were identified followed by superoxide anion, lipid peroxidation, cell viability and TEM analysis to reveal its mechanism of anti-biofilm action. Here, curcumin-ZnO nanocomposites at 300 μg/mL dosage having suitable particle size (110.51 nm), improved zeta potential (−22.3 mV), reduced hydrodynamic size (253.2 nm) were more efficiently able to penetrate through biofilm matrix and bind to P. aeruginosa's cell membrane through the OprM-MexAB receptor and inhibit its growth and biofilm (efficacy = 49.36% compared to control) than ZnO nanoparticle (42.38% compared to control). Curcumin-ZnO nanocomposites could efficiently inhibit P. aeruginosa biofilm by mechanisms involving increased superoxide anion generation by 48.94% possibly due to catalase and rubredoxin-rubredoxin reductase inhibition that mediated increased bacterial membrane lipid peroxidation by 183.75% thus causing severe disintegration of cell membrane and increased cell death by 25.18% compared to control. Curcumin-ZnO nanocomposites can thus be considered as a capable anti-biofilm drug candidate against P. aeruginosa infections.

PREVALENCE, PHENOTYPIC-GENOTYPIC RESISTANCE AND BIOFILM FORMATION OF Staphylococcus aureus IN CHICKEN MEAT WITH REFERENCE TO ITS PUBLIC HEALTH HAZARD

The purpose of this study was to determine the prevalence, antimicrobial resistance in S. aureus and presence of enterotoxigenic as well as biofilm-forming genes in S. aureus bacteria isolated from broiler meat from traditional shops and supermarkets in Sharkia, Egypt. For this determination, two hundred fresh raw chicken meat cotton swabs were collected from the breast and thighs (100 each). S. aureus was highlighted through a coagulase test, and then phenotypic and genotypic characterizations were studied. Uniplex PCR was used to identify the occurrence of enterotoxin genes in the selected isolates. Finally, they were subjected to a biofilm formation test using 96-well flat bottom polystyrene microtiter plates; besides, biofilm-forming genes were investigated. Nineteen isolates out of the 200 samples tested positive for S. aureus (9.5 percent), No Vancomycin resistance strains were obtained, nor did any ciprofloxacin isolates for S. aureus while, all isolates were 100 % resistant for streptomycin, amoxicillin- clavulanic acid and sulfamethoxazole-trimethoprim S. aureus isolates were found resistant to at least one antibiotic. The multiple antibiotic resistance (MAR) index of S. aureus isolates was ranged from 0.3 to 0.7 with an average of 0.4. In The mecicillin resistant S.aureus (MRSA) strains carried mecA gene with a percentage of 5%, while the blaZ gene was distributed with a percentage of 5.5%, 11/200). The obtained isolates gave 60% strong biofilm formation and 40% were non-formers, with nil results for moderate or weak production isolates. The icaA gene was 100%, in comparison to icaD which was zero. It should be noted that strong biofilm former strains were only 100% positive for sea and seb. This study pointed out the higher prevalence of MDR isolates of S. aureus isolates in chicken meat due to inadequate handling and insufficient sanitary equipment and post microbial contamination. This finding highlighted the importance of broiler meat as a reservoir for antimicrobial-resistant strains of S. aureus and biofilm-forming of Staphylococcus aureus.

Effects of physicochemical changes (temperature, pH, and culture media) on strong biofilm formation of Acinetobacter baumannii isolated from patients with respiratory infection in Iraq

Introduction and Aim: The ability of Acinetobacter baumannii to form biofilms on biotic and abiotic surfaces is regulated by several pathogens’ virulence factors, and this is thought to be at the root of the bacteria's resistance to antibiotics. We hope to learn how temperature, pH, and iron concentrations influence the development of biofilms in A. baumannii isolated from COVID-19 and non-COVID-19 individuals, and which genes are relevant for biofilm formation and antibiotic resistance. Materials and Methods: Eight strong adherent isolates of A. baumannii from respiratory tract infection Iraqi patients (4 from COVID-19 and the other from non-COVID-19 just respiratory patients) had been used in this study which conducted from 10/1/2021 to 10/2/2022. The antibiotic sensitivity of all isolates was determined using the VITEK-2 system. The biofilm associated genes OXA-51, bap, Chaperone Usher (CsuE) and Integron-1, was detected using PCR. Isolates of A. baumainni were put through a battery of tests to determine whether they possessed the capacity to produce robust biofilms under a wide range of both physical (temperature, pH) and chemical circumstances. Results: A. baumannii showed that all isolates were multidrug resistant and positive for the biofilm genes studied. Effect on temperature on biofilm formation showed at 44ºC biofilm formation was significantly lower than that at 37ºC (mean differences of 0.178000 (t= 8.355, df:3, P=0.004) and 0.204000 (t=26.521, df:3, P=0.000) respectively). The adhesion factor value in the COVID-19 positive and negative groups decreased significantly because of the pH change. Iron concentration of 60 µM significantly lowered biofilm formation among COVID-19 group and non-COVID-19 group. Conclusion: A. baumanni are multidrug resistance isolates with a capacity to form biofilms. The ability to form biofilms by A. baumannii is strongly influenced by physical and chemical factors.

Comparative genome identification of accessory genes associated with strong biofilm formation in Vibrio parahaemolyticus

Vibrio parahaemolyticus biofilms on the seafood processing plant surfaces are a potential source of seafood contamination and subsequent food poisoning. Strains differ in their ability to form biofilm, but little is known about the genetic characteristics responsible for biofilm development. In this study, pangenome and comparative genome analysis of V. parahaemolyticus strains reveals genetic attributes and gene repertoire that contribute to robust biofilm formation. The study identified 136 accessory genes that were exclusively present in strong biofilm forming strains and these were functionally assigned to the Gene Ontology (GO) pathways of cellulose biosynthesis, rhamnose metabolic and catabolic processes, UDP-glucose processes and O antigen biosynthesis (p < 0.05). Strategies of CRISPR-Cas defence and MSHA pilus-led attachment were implicated via Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation. Higher levels of horizontal gene transfer (HGT) were inferred to confer more putatively novel properties on biofilm-forming V. parahaemolyticus. Furthermore, cellulose biosynthesis, a neglected potential virulence factor, was identified as being acquired from within the order Vibrionales. The cellulose synthase operons in V. parahaemolyticus were examined for their prevalence (22/138, 15.94 %) and were found to consist of the genes bcsG, bcsE, bcsQ, bcsA, bcsB, bcsZ, bcsC. This study provides insights into robust biofilm formation of V. parahaemolyticus at the genomic level and facilitates: identification of key attributes for robust biofilm formation, elucidation of biofilm formation mechanisms and development of potential targets for novel control strategies of persistent V. parahaemolyticus.

Biofilm formation is associated with poor outcome in prosthetic joint infections caused by Staphylococcus lugdunensis

Background Staphylococcus lugdunensis has been described as a pathogen of increasing importance in prosthetic joint infections (PJI). Our aim was to describe the clinical presentation of PJI caused by S. lugdunensis, and to correlate the biofilm-forming ability of the bacterial isolates to clinical outcome. Method S. lugdunensis isolates from PJI episodes during 2015–2019 were included and analysed for biofilm formation using a microtiter plate assay. Medical records from the corresponding patients were reviewed. Results We identified 36 patients with PJI caused by S. lugdunensis during the study period. Early postoperative PJIs were most frequent (n = 20, 56%). Surgical intervention was performed in a majority of the patients (n = 33, 92%), and the dominating type of antibiotic treatment was a combination of rifampicin and ciprofloxacin (n = 27, 75%). The treatment success-rate was 81% (n = 29). All isolates causing PJI were able to form biofilm in vitro. Biofilm formation was significantly stronger in isolates causing relapsing vs non-relapsing PJI (mean OD550 3.1 ± 0.23 vs 1.14 ± 0.73 p = .001) and strong biofilm formation was also associated with late acute hematogenic PJI (mean OD550 1.8 ± 0.93 vs. 0.93 ± 0.81, p = .01). Conclusion Strong biofilm production in S. lugdunensis isolates was associated with relapse in PJI.

Speciation of Candida isolates from vulvovaginal candidiasis cases, detection of virulence markers of Candida and antifungal susceptibility by disc diffusion method

: Species belonging to the genus are ubiquitous in nature and many of them form the normal microbial flora in human and animal bodies. A wide variety of infections on mucosal surfaces under certain conditions can be caused due to the sp: A total of 40 isolates of Candida, isolated from vaginal swabs among 476 patients with clinically suspected VVC. Antifungal susceptibility testing done using Fluconazole and Voriconazole. Virulence markers Phospholipase activity, Proteinase activity, Biofilm Productionwere detected by standard methods.: Out of 40 isolates, the following species were isolated, (52.5%) was the most common species isolated from vaginal swabs. The species isolated were (27.5%), (15%), (5%).Intense phospholipase activity was observed in 12 (30%) samples, there was no activity in 18 (45%) samples. A moderate phospholipase activity was observed in 6 (15%) isolates, poor activity in 4 (10%) samples.Proteinase activity was strongly positive in 33 isolates which was identified as and. Strong biofilm formation was observed in 9 isolates, moderate in 8, weak in 11 and negative in 12 isolates. All 40 isolates are susceptible to Fluconazole and Voriconazole.From this study it is observed that and are the major species of causing VVC. Virulence marker analysis such as that for phospholipase, proteinase and biofilm formation will help to analyse the pathogenicity and further research required to elucidate the mechanisms involved in these pathways.