Biofilm Production Research Articles

Listeria monocytogenes in Fruits and Vegetables: Antimicrobial Resistance, Biofilm, and Genomic Insights

Background/Objectives: Listeria monocytogenes is a foodborne pathogen that can infect both humans and animals and cause noninvasive gastrointestinal listeriosis or invasive listeriosis. The objectives of this study were to determine the genetic diversity of L. monocytogenes; the genes associated with its resistance to antibiotics, benzalkonium chloride (BC), and cadmium chloride (CdCl2); and its biofilm formation. Methods: A total of 132 fresh fruits (44 samples) and vegetables (88 samples) were selected for this study. The genetic diversity of the isolates and the genes associated with their antibiotic resistance were determined using PCR amplification; meanwhile, their levels of susceptibility to antibiotics were determined using the agar diffusion method. Their levels of resistance to BC and CdCl2 were determined using the minimum inhibitory concentration method, and their capacity for biofilm formation was evaluated using the crystal violet staining method. Results: A total of 17 L. monocytogenes strains were collected: 12.8% (17/132) from fresh fruits and vegetables in this study. The isolates of L. monocytogenes belonged to phylogenetic groups I.1 (29.4% (5/17); serotype 1/2a) and II.2 (70.5% (12/17); serotype 1/2b); strains containing Listeria pathogenicity islands (LIPIs) were also identified at prevalence rates of 100% for LIPI-1 and LIPI-2 (17/17), 29.4% for LIPI-3 (5/17), and 11.7% for LIPI-4 (2/17). The antibiotic susceptibility tests showed that the L. monocytogenes isolates exhibited six different multiresistant patterns, with multiple antibiotic resistance (MAR) index of ≥0.46 (70.5%; 12/17); additionally, the genes Ide, tetM, and msrA, associated with efflux pump Lde, tetracycline, and ciprofloxacin resistance, were detected at 52.9% (9/17), 29.4% (5/17), and 17.6% (3/17), respectively. The phenotypic tests showed that 58.8% (10/17) of cadmium-resistant L. monocytogenes isolates had a co-resistance of 23.5% (4/17) to BC. Finally, all strains of L. monocytogenes exhibited moderate biofilm production. Conclusions: The results of this study contribute to our understanding of the persistence and genetic diversity of L. monocytogenes strains isolated from fresh fruits and vegetables; in addition, their resistance to CdCl2, which is correlated with co-resistance to BC disinfectant, is helpful for the food industry.

Biofilm Production in Intensive Care Units: Challenges and Implications

The prevalence of infections amongst intensive care unit (ICU) patients is inevitably high, and the ICU is considered the epicenter for the spread of multidrug-resistant bacteria. Multiple studies have focused on the microbial diversity largely inhabiting ICUs that continues to flourish despite treatment with various antibiotics, investigating the factors that influence the spread of these pathogens, with the aim of implementing sufficient monitoring and infection control methods. Despite joint efforts from healthcare providers and policymakers, ICUs remain a hub for healthcare-associated infections. While persistence is a unique strategy used by these pathogens, multiple other factors can lead to persistent infections and antimicrobial tolerance in the ICU. Despite the recognition of the detrimental effects biofilm-producing pathogens have on ICU patients, overcoming biofilm formation in ICUs continues to be a challenge. This review focuses on various facets of ICUs that may contribute to and/or enhance biofilm production. A comprehensive survey of the literature reveals the apparent need for additional molecular studies to assist in understanding the relationship between biofilm regulation and the adaptive behavior of pathogens in the ICU environment. A better understanding of the interplay between biofilm production and antibiotic resistance within the environmental cues exhibited particularly by the ICU may also reveal ways to limit biofilm production and indivertibly control the spread of antibiotic-resistant pathogens in ICUs.

Investigation of EpsA, OmpA, and Bap Genes among MDR and XDR Acinetobacter baumannii Isolates in Khorramabad, Iran.

Acinetobacter baumannii is an opportunistic hospital pathogen with high antibiotic resistance, and the ability to produce biofilm. This study aimed to investigate epsA, ompA, and bap genes involved in biofilm formation in MDR and XDR clinical isolates of Acinetobacter baumannii in Khorramabad, Iran. In this study, 79 A. baumannii isolates were collected from various samples of the patients admitted to tertiary hospitals in Khorramabad city, Iran, between January and August 2019. After performing the semi-quantitative evaluation of biofilm production by microtiter plate assay, screening of isolates carrying epsA, ompA, and bap genes was done by PCR method. Finally, statistical analyses were conducted using SPSS 22. Among 79 A. baumannii isolates, 52% XDR, 40% MDR, and 16% non-XDRMDR isolates were found to be biofilm producers. All XDR and 94% MDR isolates had ompA and epsA genes, and bap genes were detected among > 80% of these isolates. Moreover, the presence of biofilm-related genes and biofilm production among non-XDRMDR isolates were less than among resistant isolates (p≤ 0.01). Based on the results, biofilm production and simultaneous presence of epsA, ompA, and bap genes among MDR, and XDR A. baumannii isolates have been found to be significantly more than non-XDR-MDR isolates.

Antibiotic Resistance, Biofilm Formation, and Identification of FimH and FimA Adhesion Genes in Uropathogenic Escherichia Coli (UPEC) Isolated from Patients in Baghdad Province

Urinary tract infections (UTIs) are one of the most common infectious disorders worldwide. The most frequent cause of UTIs is uropathogenic Escherichia coli (UPEC). The current study is designed to assess the resistance of antibiotics, the formation of biofilm, and the detection of adhesion genes in E. coli isolated from patients with UTIs. A hundred and fifty samples were collected from patients with confirmed UTIs in Baghdad province during the period of October 2022 to February 2023. Isolation and identification of E. coli were performed using cultural characteristics, gram staining, and biochemical examination, and the results indicated that 52 (35%) isolates were identified as E. coli. Eleven antibiotic discs were utilized to estimate the sensitivity of E. coli isolates. 98% of isolates were resistant to ampicillin, followed by 75.5% of isolates resistant to both trimethoprim/sulfamethoxazole and fosfomycin, followed by 73.5, 71.4, 67.3, 53.1, and 6.1% of isolates resistant to imipenem, ceftazidime, nitrofurantoin, cefepime, and amikacin. All isolates were sensitive to piperacillin-tazobactam, ciprofloxacin, and aztreonam at 100%, 100%, and 98%, respectively. The microtiter plate method was utilized to estimate the ability of E. coli to form biofilm. The majority of isolates (69.2%; n = 36) were moderate biofilm producers, while 19.2% (n = 10) and 11.5% (n = 6) of isolates were strong and weak biofilm producers, respectively. Polymerase Chain Reactions (PCR) were applied to determine the gene expression level of adhesion genes (fimH and fimA genes). Forty isolates harbored both fimH and fimA genes.

Targeting virulence of resistant Escherichia coli by the FDA-approved drugs sitagliptin and nitazoxanide as an alternative antimicrobial approach.

The spread of multidrug-resistant Escherichia coli in healthcare facilities is a global challenge. Hospital-acquired infections produced by Escherichia coli include gastrointestinal, blood-borne, urinary tract, surgical sites, and neonatal infections. Therefore, novel approaches are needed to deal with this pathogen and its rising resistance. The concept of attenuating virulence factors is an alternative strategy that might lead to low levels of resistance and combat this pathogen. A sub-inhibitory concentration (¼ MIC) of sitagliptin and nitazoxanide was used for phenotypic assessments of Escherichia coli virulence factors such as biofilm production, swimming motility, serum resistance, and protease production. Moreover, qRT-PCR was used to determine the impact of sub-MIC of the tested drugs on the relative expression levels of papC, fimH, fliC, kpsMTII, ompT_m, and stcE genes encoding virulence factors in Escherichia coli. Also, an in vivo model was conducted as a confirmatory test. Phenotypically, our findings demonstrated that the tested strains showed a significant decrease in all the tested virulence factors. Moreover, the genotypic results showed a significant downregulation in the relative expression levels of all the tested genes. Besides, the examined drugs were found to be effective in protecting mice against Escherichia coli pathogenesis. Sitagliptin and nitazoxanide exhibited strong anti-virulence activities against Escherichia coli. In addition, it is recommended that they might function as adjuvant in the management of Escherichia coli infections with either conventional antimicrobial agents or alone as alternative treatment measures.

Synergistic potential of Leu10-teixobactin and cefepime against multidrug-resistant Staphylococcus aureus

Staphylococcus aureus (S. aureus) is a significant Gram-positive opportunistic pathogen behind many debilitating infections. β-lactam antibiotics are conventionally prescribed for treating S. aureus infections. However, the adaptability of S. aureus in evolving resistance to multiple β-lactams contributed to the persistence and spread of infections, exemplified in the emergence of methicillin-resistant S. aureus (MRSA). In the present study, we investigated the efficacies of the synthetic teixobactin analogue, Leu10-teixobactin, combined with the penicillinase-resistant cephalosporin cefepime against MRSA strains. The Leu10-teixobactin and cefepime combination exerted synergism against most strains tested in broth microdilution assay. Time-kill profiles showed that both Leu10-teixobactin and cefepime predominantly exhibited synergistic activity, with > 2.0-log10CFU decrease compared to monotherapy at 24 h. Moreover, biofilm assays revealed a significant inhibition of biofilm production in ATCC™43300 cells treated with sub-MICs of Leu10-teixobactin and cefepime. Subsequent electron microscopy studies showed more extensive damage with the combination therapy compared to monotherapies, including aberrant bacterial morphology, vesicle formation and substantial lysis, indicating combined damage to the cell wall. Quantitative real-time PCR revealed marked perturbation of genes mecA, sarA, atlA, and icaA, substantiating the apparent mode of combined antibacterial action of both antibiotics against peptidoglycan synthesis and initial biofilm production. Hence, the study highlights the prospective utility of the Leu10-teixobactin-cefepime combination in treating MRSA infections via β-lactam potentiation.

Virulence characterization of Staphylococcus spp. isolated from mozzarella cheeses and cheese-slicers

Different species of the Staphylococcus genus can be found as contaminants in foods and may carry virulence factors that could potentially make them The enterotoxigenic potential ofpathogenic or transfer genes to other bacteria. Therefore, the aim of the present study was to isolate and identify species of Staphylococcus genus isolated from sliced mozzarella cheeses and cold-cut slicers and characterize biofilm formation in vitro, presence of enterotoxins and antibiotic resistance genes, and β-lactamase production. Sampling was carried out in 44 retail markets and isolates identified on species level. Resistance genes blaZ and mecA, as well as enterotoxin genes sea, seb, sec, and sed, were identified using PCR. Phenotypic evaluation of biofilm formation and detection of β-lactamase enzyme were also analyzed. Nine species of coagulase-negative Staphylococcus (CNS) were identified from 85 bacterial isolates. Of this total, 67.0% were positive for the presence of one or both antimicrobial resistance genes blaZ and mecA, while the β-lactamase enzyme was detected in 52.9% isolates. Regarding biofilm production, 48.2% of isolates were positive and 51.8% were negative. However, none of the isolates were identified with the enterotoxin genes. The results demonstrate the potential risk of cross-contamination, as biofilm-producing CNS could persist in the cheese retail environment, highlighting the danger of resistance genes transferring to other commensal or pathogenic bacteria.

THE PREVALENCE OF (OMPA, CSUE) GENES AMONG BIOFILM PRODUCER ACINETOBACTER BAUMANNII ISOLATES

Acinetobacter baumannii is a significant public health problem because it is capable of forming biofilms that may be responsible for the survival of this pathogen in the hospital environment. The aim of this study is to determine the role of (OmpA, CsuE) genes in biofilm production among A. baumannii isolates. A total of 100 clinical isolates of bacteria were collected from a different sources. All isolates were identified by biochemical test, specific selective media (CHROMagar) and polymerase chain reaction by detection presence of (blaOXA51) gene. only 60 isolates were diagnosed as A. baumannii, and to determine the biofilm production capacity two different methods were used Congo red agar method(CRA) and Microtiter plate method (MTP). MTP method result showed 85% of isolates were able to form biofilms, on the other hand, 33.33% of isolates had a biofilm production ability by showing bright black colonies on CRA. Conventional PCR was used to detect the presence of (OmpA, CsuE) genes and the result showed the prevalence of both genes in all isolates were 97%(58/60). It also shows a strong correlation between the presence of these genes and biofilm formation.

Synthesis, Antibacterial, Anti-Biofilm Properties, and Docking Study of Indeno[1,2-b]Pyridin-5-One Derivatives

In this context, we designed and synthesized a series of hydrazonal and their related indeno[1,2-b]pyridin-5-one derivatives to investigate their antibacterial and anti-biofilm properties. Several of the synthesized compounds exhibited significant efficacy against all microorganism species tested. Most of the compounds demonstrated favorable results when tested against Gram-positive bacteria. The derivatives 4a, 4f, 6c, and 6f exhibit the highest antimicrobial efficacy, as indicated by their minimum inhibitory concentration (MIC) values ranging from 4 to 512 μg/mL. We conducted additional investigations on 4a, 6c, and 6f for the purpose of examining their synergy using Checkerboard assay. Compound 6c demonstrated a synergistic impact against methicillin-sensitive Staphylococcus aureus (MSSA) and Pseudomonas aeruginosa, while exhibiting moderate synergistic activity against methicillin-resistant Staphylococcus aureus (MRSA). In addition, the simultaneous use of gentamycin with compounds 4a and 6f demonstrates a synergistic impact in fighting against methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa, respectively. Three chosen compounds were evaluated for their antibiofilm efficacy. Application of 4a, 6c, and 6f effectively reduced the production of biofilms in MRSA, MSSA, and Pseudomonas aeruginosa, resulting in a significant decrease compared to the untreated samples. In addition, ADME and pharmacokinetic analyses were conducted for the three most potent derivatives, namely 4a, 6c, and 6f. Compounds 4a and 6c were subjected to docking in the LasR Quorum-Sensing Receptor, whereas compounds 6c and 6f were docked in the sortase enzyme.

Virulome and phylogenomic profiling of a novel Burkholderia pseudomallei strain from an Indian clinical isolate.

Highly pathogenic Burkholderia pseudomallei is the causative agent of melioidosis, a neglected tropical disease endemic in Southeast Asian tropical region. This bacterium encompasses diverse virulence factors which further undergo dynamic gene-expression flux as it transits through distinct environmental niches within the host which may lead to manifestation of differential clinical symptoms. B. pseudomallei, is classified as a Tier 1 select agent in the United States and regarded as a risk group 3 organism in India with the potential to be used as bioweapon. Considering these facts, it is vital to uncover both physiological and genetic heterogeneity of B. pseudomallei, particularly to identify any novel virulence factors that may contribute to pathogenicity. B. pseudomallei strain CM000113 was isolated from a clinical case in India, characterized it for its physiological, biochemical, and prominently genetic traits through WGS. It has a type 2 morphotype with faster doubling time and high biofilm producing capacity as compared to Pseudomonas aeruginosa. The genome size is 7.3 Mbp and it is phylogenetically close to B. pseudomallei strain Mahidol 1106a and Burkholderia mallei Turkey 2. We observed genetic heterogeneity, as key virulence factors that were identified shows sequence dissimilarity with reference strains. Additionally, presence of genomic islands, harbouring two virulence factors, GmhA and GmhB2, associated with pathogenesis indicates possibility of horizontal gene transfer. These results emphasize the need for an extensive study focusing the genome of B. pseudomallei and its associated heterogeneity, to identify molecular biomarkers aiding to develop point-of-care diagnostic kits for early diagnosis of melioidosis.

Characterization of Yeast Isolated from the Gut Microbiota of Tunisian Children with Autism Spectrum Disorder

Research on the microbiota–gut–brain axis in autism has primarily focused on bacteria, with limited attention to fungi. There is a growing interest in understanding the involvement of fungi, particularly Candida, in patients with autism spectrum disorder. The aim of this study was to assess the prevalence, antifungal susceptibility profiles and virulence factors of Candida isolates from the guts of Tunisian children with autism. Twenty-eight children with autism and forty-six controls were enrolled. Candida isolates from the faecal samples were identified using biochemical and molecular methods; antifungal susceptibility testing was determined by the EUCAST broth microdilution method and virulence factors, including biofilm formation, cell surface hydrophobicity and phospholipase and proteinase activities, were assessed in vitro. As a result, Candida was detected in 13 children with autism (46.4%) and 14 control children (30.4%). Candida albicans was found to be the most common species isolate in the faeces of both groups of children. Antifungal susceptibility profiles showed that one Candida isolate was resistant to amphotericin B and anidulafungin (3.7%), six were resistant to micafungin (22.2%) and five were resistant to fluconazole (18.5%). All Candida isolates were biofilm producers. Of the twenty-seven isolates, only four showed phospholipase activity (14.8%), eight showed aspartyl-proteinase activity (29.6%) and nine were hydrophobic (33.3%). These results highlight the presence of Candida in the guts of children with autism, as well as the ability to express multiple virulence factors and the antifungal resistance, and they emphasize the need for further studies to confirm intestinal Candida colonization and its potential role in autism.

Characterization and genomic analysis of phage vB_SmaP_c9-N, a novel Stenotrophomonas maltophilia podophage with antibiofilm activity.

Stenotrophomonas maltophilia strains are increasingly emerging as multidrug-resistant pathogens. Moreover, S. maltophilia commonly produces biofilms that enhance antibiotic resistance in bacteria. Phages are effective alternative drugs for treating S. maltophilia infections. In this study, the lytic phage vB_SmaP_c9-N (abbreviated as Φc9-N), which is specific for S. maltophilia, was isolated from Nanhu Lake, Wuhan, China. Electron microscopy observation revealed that Φc9-N is a podophage. Φc9-N is stable over a wide pH range, from pH 4 to 10, and its activity did not change after storage at 4°C for 2months. The latency period of Φc9-N is 5 min, and its outbreak period is 35min. Antibacterial tests showed that Φc9-N could effectively inhibit the growth of S. maltophilia c24. Moreover, the biofilm production of S. maltophilia c24 decreased when Φc9-N was administered either to the forming biofilm or to the mature biofilm. These results suggest that Φc9-N has application potential in clinical treatment. The genome of Φc9-N is a dsDNA of 43,170 bp with 55 putative unidirectional genes, 18 of which were assigned putative functions, while other genes encoded hypothetical proteins. Genome sequence comparisons and phylogenetic analysis indicated that Φc9-N represents a new species, and, together with the Stenotrophomonas phages BUCT700, BUCT703, BUCT598, and vB_SmaS_P15, can be included in the newly proposed genus "Maltovirus" in the family Autographiviridae.

Critical resistance to carbapenem and aminoglycosides in Pseudomonas aeruginosa: spread of blaNDM/16S methylase armA harboring isolates with intrinsic resistance mechanisms in Kerman, Iran

BackgroundCarbapenem-resistant Pseudomonas aeruginosa (CRPA) is one of the main Gram-negative bacterium causes of infections in hospital settings, and the spread of them is a significant challenge to public health.MethodsA total of 30 non-duplicate isolates of CRPA were collected. Antibacterial susceptibility of isolates to antibiotic agents, AmpC β-lactamase production, and biofilm formation were determined. Minimum biofilm inhibitory concentrations (MBIC) of isolates to cefepime (FEP), imipenem (IPM), ceftazidime (CAZ), and meropenem (MEM) were evaluated with/without cloxacillin (CLX). The carbapenemase and 16 S rRNA methylase genes were identified by PCR, and the transcription levels of oprD, ampC, and mexA genes were determined by quantitative real-time PCR (qPCR). ERIC-PCR was used to detect genetic relationships among isolates.ResultsAll isolates were multidrug resistant (MDR) and strong biofilm producers. The resistance genes including blaNDM, blaIMP, blaVIM, blaSIM, blaGES, and armA were detected in 21 (70%), 6 (20%), 3 (10%), 2 (6.6%), 1 (3.3%), and 17 (56.6%) of the isolates, respectively. CLX at 500 and 1000 µg/mL significantly reduced the level of MIC to MEM, IPM, CAZ, and FEP, also at 2000 µg/mL significantly reduced the level of MBIC to MEM, IPM, CAZ, and FEP. In all isolates, the transcription levels of oprD were significantly downregulated as well as significantly increased for ampC and mexA. ERIC-PCR typing results divided 30 isolates into four clusters A to D.ConclusionIn this study, we reported the spread of different clones of CRPA harboring co-existence of various carbapenemase genes with armA 16 S rRNA methylase for the first time in Kerman, Iran. Also, our isolates had several mechanisms of resistance to carbapenems as well as ability biofilm formation along with resistance to aminoglycosides, the further spread of which could cause serious challenges in our hospital settings. Therefore, serious monitoring is necessary to reduce their prevalence.

Characterization of novel phage pK3–24 targeting multidrug-resistant Klebsiella pneumoniae and its therapeutic efficacy in Galleria mellonella larvae

Klebsiella pneumoniae is a common, conditionally pathogenic bacterium that often has a multidrug-resistant phenotype, leading to failure of antibiotic therapies. It can therefore induce serious diseases, including community-acquired pneumonia and bloodstream infections. As an emerging alternative to antibiotics, phages are considered key to solving the problem of drug-resistant bacterial infections. Here, we report a novel phage, pK3–24, that mainly targets ST447 K. pneumoniae. Phage pK3–24 is a T7-like short-tailed phage with a fast adsorption capacity that forms translucent plaques with halos on bacterial lawns. The optimal multiplicity of infection (MOI) is 0.01, and the average burst size is 50 PFU/mL. Phage pK3–24 shows environmental stability, surviving at below 50 °C and at pH values of 6–10. It has a double-stranded DNA genome of 40,327 bp and carries no antibiotic-resistance, virulence, or lysogeny genes. Phylogenetic analysis assigned phage pK3–24 to the genus Przondovirus as a new species. Phage pK3–24 inhibited the production of biofilm. Moreover, treatment with pK3–24 at doses with an MOI > 1 effectively reduced the mortality of Galleria mellonella larvae infected with ST447 K. pneumoniae.

Quinolone and Tetracycline-Resistant Biofilm-Forming Escherichia coli Isolates from Slovak Broiler Chicken Farms and Chicken Meat

Escherichia coli isolates from intensive poultry production are associated with antimicrobial resistance and worldwide health problems. The aim of the study was to detect and evaluate the phenotypic and genotypic antimicrobial resistance, biofilm formation, phylogenetic typing, and virulence factors in E. coli isolates from the rectal swabs of chickens from two farms and swabs of chicken meat purchased from Slovakian food markets. Interpretative readings of minimal inhibitory concentration (MIC) revealed dominant resistance to ampicillin (>50%) in both groups. We also detected higher resistance to ciprofloxacin (45%), tetracycline, ampicillin + sulbactam, and trimethoprim + sulfonamide (each >30%). Here, 28.57% of the strains studied were multidrug-resistant (MDR). The formation of weak biofilms was confirmed in 8.8% of E. coli, while one of the strains obtained from chicken cloacal swabs was classified as a strong biofilm producer. The most frequently confirmed phylogenetic groups in E. coli were B1 and A1 in all groups. PCR detection revealed the presence of genes encoding tetracycline resistance (tetAB) and plasmid-mediated quinolone resistance (qnrABS), and Int1 (52.9%), Tn3 (76.5%), kpsMT II (8.8%), fimA (97.1%), cvaC (38.2%), and iutA (76.5%) genes in the strains studied. Our results demonstrate that chickens and chicken meat were the source of antibiotic-resistant, biofilm-forming, and virulent E. coli, representing a potential risk from the point of view of the One Health concept.

Suppression of virulence factors of uropathogenic Escherichia coli by Trans-resveratrol and design of nanoemulgel

BackgroundDevelopment of multidrug resistance in Uropathogenic Escherichia coli (UPEC) makes treatment of Urinary Tract Infections (UTIs) a major challenge. This study was conducted to investigate the effect of trans-resveratrol (t-RSV) at a subinhibitory concentration (sub-MIC-t-RSV) on phenotypic and genotypic expression of virulence factors of clinical isolates of UPEC and develop a nanoformulation of t-RSV. Fifty-five clinical UPEC strains were investigated for the presence of virulence factors by phenotypic methods and PCR detection of virulence genes. The effect of sub-MIC-t-RSV was studied on the phenotypic and genotypic expression of virulence factors. t-RSV-loaded nanoemulgel formulation was prepared and characterized.ResultsOut of the 55 tested isolates, 50.9% were biofilm producers, 23.6% showed both mannose-sensitive and mannose-resistant hemagglutination, 21.8% were serum-resistant, 18.2% were hemolysin producers, while 36.4% showed cytotoxic effect on HEp-2 cells. A total of 25.5% of the isolates harbor one or more of hly-A, cnf-1 and papC genes, while 54.5% were positive for one or more of fimH, iss and BssS genes. A concentration of 100 µg/mL of t-RSV effectively downregulates the phenotypic and genotypic expression of the virulence factors in positive isolates. A stable t-RSV-nanaoemulgel with droplet size of 180.3 nm and Zetapotential of -46.9 mV was obtained.ConclusionThe study proves the effective role of t-RSV as an antivirulence agent against clinical UPEC isolates in vitro and develops a stable t-RSV-nanoemulgel formulation to be assessed in vivo. The promising antibacterial and antivirulence properties of t-RSV place this natural compound to be a better alternative in the treatment of persistent UTIs.

2,4-Di-Tert-Butylphenol of Streptomyces luridiscabiei MMS-10 Inhibits Biofilm Forming Cariogenic Streptococcus mutans ULSP-2.

Dental caries is a common chronic infectious disease of the oral cavity that affects the overall oral health of the individual. Cariogenic bacteria have long been recognized for their role in developing chronic dental infections. Drug-resistant bacteria represent a global challenge to effective pathogen control in caries. The present study aimed to isolate and identify soil actinomycetes for their antibacterial and anti-biofilm activities against antibiotic-resistant and biofilm-forming cariogenic bacteria. Thirteen caries bacteria isolated from infected tooth samples were evaluated for antibiotic resistance and biofilm formation. The isolate ULSP-2 showed the highest antibiotic resistance score (0.714) and was found to be a strong biofilm producer when tested by congo red agar and microtiter plate assays. The bacterium was identified as Streptococcus mutans based on morphological, biochemical, and molecular characterization. The effect of ethyl acetate extracts from 20 soil actinomycetes on the growth and biofilm formation ability of S. mutans was evaluated. The MMS-10 extract strongly inhibited growth (18.5 ± 0.5mm) and biofilm formation (56.46 ± 0.32%) of S. mutans at 100µg/mL. The isolate MMS-10 was identified at the molecular level as Streptomyces luridiscabiei. Based on FTIR, NMR, and GC-MS analysis, the purified MMS-10 extract was characterized and identified as 2,4-Di-tert-butylphenol. The metabolite's physiological, physicochemical, and pharmacokinetic properties were analyzed using the Swiss ADME web server and found to satisfy the criteria of drug-likenessof a molecule. The study revealed the significance of soil actinomycetes in controlling growth and biofilm formation in cariogenic S. mutans.

MALDI-TOF MS profiling to predict resistance or biofilm production in gram-positive ESKAPE pathogens from healthcare-associated infections

Antimicrobial resistance and biofilm production in healthcare-associated infections is a health issue worldwide. This study aimed to identify potential biomarker peaks for resistance or biofilm production in ESKAPE pathogens using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Antimicrobial susceptibility and biofilm production were assessed on selected isolates. Biomarker peaks were identified using MALDI Biotyper and ClinProTools software. Among resistant strains, 90.0 % were carbapenem-resistant Acinetobacter baumannii (CRAB), 39.0 % were methicillin-resistant Staphylococcus aureus (MRSA), 17.98 % were multidrug-resistant (MDR) Pseudomonas aeruginosa, 21.6 % were vancomycinresistant Enterococcus (VRE), and 2.55 % were carbapenem-resistant Enterobacterales (CRE). Biofilm production was 40.0 % in VRE and 45.8 % in MRSA. Although no potential biomarker peaks for biofilm production were detected, several potential biomarker peaks for drug resistance in VRE (n=5), MRSA (n=4), and MDR P. aeruginosa (n=4) were detected, suggesting avenues for the development of rapid diagnostic tools.

Signaling Molecules in Pseudomonas aeruginosa Response to Antibiotics at Sub-Inhibitory Concentrations

Signaling Molecules are N-acylhomoserine lactones (AHLs) that form the Pseudomonas aeruginosa cell information system which determines gene expressions in a population dependent manner called quorum sensing (QS). Signal molecules, which are chemically varied, control genes expressions to antibiotics resistance, pathogenicity, motility, biofilm development, bioluminescence, secondary metabolite production, and plasmid transfer. This research was aimed to identify the signaling molecules in Pseudomonas aeruginosa response to antibiotics at sub-inhibitory concentrations. One hundred and fifty (150) clinical swab specimens were collected from urinary catheter wound and ear infection of patients and the swabs inoculated using standard microbiology method. The isolates were characterized based on the bacteriological methods such as morphology and biochemical tests. The isolates were further confirmed by species specific PCR by amplification of 16S rRNA and the amplicons were analyzed by gel electrophoresis; and further genomic sequencing was done and blast with NCBI database mining. Disc diffusion method was applied for the antibiotics susceptibility pattern. The isolates cell suspensions were analyzed by Gas Chromatography-Mass Spectrometry (GC-MS). The result of the isolation showed 22(59.46%) from wound infections, 12(32.43%) from ear infections and 3(8.11%) from urinary catheter. The isolates were Gram negative, produced β-hemolysis on blood agar and the morphology is small pigmented circular in form. The isolates showed positive result to catalase, oxidase, citrate, nitrate and indole tests. The amplification of 16S rRNA gene region resulted in the band size of 1500bp PCR product and the BLAST analysis gave 99% similarity. The resistant antibiotics susceptibility showed that Pseudomonas aeruginosa is multidrug resistant bacteria. The GC-MS results obtained from urinary catheter isolates showed four (4) signaling molecules such as N-Octanoyl-L-homoserine Lactone, N-Decanoyl-DL-Homoserine Lactone, N-Dodecanoyl-DL-Homoserine Lactone and N-Tetradecanoyl-L-Homoserine Lactone. Three (3) signaling molecules such as N-Octanoyl-L-homoserine Lactone, N-Decanoyl-DL-Homoserine Lactone and N-Tetradecanoyl-L-Homoserine Lactone were obtained from wound isolates. N-Tetradecanoyl-L-Homoserine Lactone was the only signaling molecule obtained from ear isolates. Further research should be done to develop rapid tests that could be possible to detect acyl homoserine specific to Pseudomonsa aeruginosa present to human samples and deliver results in real time. Signaling molecules inhibitors (SMI) are possible potential therapeutics that could produce the subsequent class of antibacterial drugs. The fundamental role of each signal molecule in the production of biofilms and virulent factors should also be investigated, as well as whether Pseudomonas aeruginosa needs one or more signaling molecules to form quorum sensing. Identification of the signals molecules (AHLs) of P. aeruginosa and developing signaling molecules inhibitors (SMI) can have an important prognostic, diagnostic and therapeutic value in human medicine for the treatment of P. aeruginosa infections.

Interference Between S. Aureus and P. Aeruginosa Clinical Isolates

General Background: Understanding microbial interactions between Pseudomonas aeruginosa and Staphylococcus aureus is crucial for clinical infections, as they often coexist and influence each other's growth and virulence. Specific Background: Both organisms are known for their ability to form biofilms and exhibit multidrug resistance (MDR), complicating treatment strategies in hospitalized patients. Knowledge Gap: Previous studies have explored the virulence of P. aeruginosa and S. aureus, but there is limited understanding of their direct in vitro interactions, particularly in protease and lipase production. Aims: The study examined the interaction between P. aeruginosa and S. aureus isolates in hospitalized patients' sputum, urine, and blood samples, focusing on virulence factors, biofilm formation, and antibiotic resistance patterns. Results: In Diyala, Iraq, 50 clinical isolates showed P. aeruginosa as protease producers, lipase producers, and biofilm producers, with significant MDR phenotypes in both species. Novelty: This study highlights the ability of P. aeruginosa to produce staphylolysin, offering novel insights into the antagonistic mechanisms that may suppress S. aureus in co-infections. Implications: The study emphasizes the significance of understanding microbial interactions in clinical infections, particularly in biofilm-associated MDR infections, to improve treatment outcomes and guide more effective therapeutic approaches. Highlights: P. aeruginosa staphylolysin inhibits S. aureus growth in vitro. Both bacteria produce biofilms and exhibit multidrug resistance. Microbial interactions impact infection severity and treatment strategies. Keywords: Microbial interactions, Pseudomonas aeruginosa, Staphylococcus aureus, Biofilm, Multidrug resistance