fimA Gene Research Articles

Qualitative and Quantitative Detection of Virulence-Associated Genes in Escherichia Coli Isolates from Women with Urinary Tract Infections

Escherichia coli is the most common type of Enterobacteriaceae family that causes urinary tract infections. This bacterium causes diseases due to its many virulence factors. During the study, (150) urine samples were collected for women with symptoms of UTIs aged between (20-50) years from 1/9/2021 to 1/4/2022. Forty-five isolates of E. coli were diagnosed using a biochemical test, a VITEK2 system, and molecular detection using traditional PCR. A molecular study was conducted on 25 of these isolates using conventional PCR and qRT-PCR to investigate the presence of genes included (16S rRNA, uidA, fimA and kpsMTII). The traditional PCR results showed the presence of both 16S rRNA and uidA gene in all 25 isolates of E. coli. In contrast, the results showed that 23 (92%) and 18 (72%) isolates possessed fimA and kpsMTII genes, respectively. Following the extraction of RNA from twenty-five pathogenic isolates of E. coli and ten non-pathogenic isolates, gene expression of fimA and kpsMTII was quantified using quantitative real-time reverse transcription PCR (qRT-PCR). The observed fold change indicates a potential overexpression of the KSPMII and FimA genes in uropathogenic E. coli. The expression of the kpsMTII gene showed the highest level of folding with an average of 22.89 in the case of uropathogenic E.coli isolates compared with non-pathogenic isolates (control) with an average of 4.11. While the expression of the fimA gene with the highest level of fold change with an average of 90.52 in the case of uropathogenic E.coli isolates compared with non-pathogenic isolates (control) with an average of 0.94, Significant differences were observed among the isolates, as indicated by P values less than 0.05. This finding demonstrated that qRT-PCR detects genes that were expressed in comparison with traditional PCR. Also, qRT-PCR is a more sensitive and accurate assay than conventional PCR for determining the quantitative prevalence of E. coli in urine samples.

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.

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.

Prevalence, Virulence Genes, Drug Resistance and Genetic Evolution of Trueperella pyogenes in Small Ruminants in Western China.

Trueperella pyogenes is a significant opportunistic pathogen that causes substantial economic losses in animal agriculture due to its ability to infect various animal tissues and organs. Limited research has been conducted on the prevalence and biological characteristics of T. pyogenes isolated from sheep and goats. This study aimed to isolate T. pyogenes from clinical samples of sheep and goats in western China, examining genetic evolutionary relationships, antibiotic resistance, and virulence genes. Between 2021 and 2023, standard bacteriological methods were used to isolate and identify T. pyogenes from 316 samples (209 from goats and 107 from sheep) collected from 39 farms. Susceptibility to 14 antibiotics was tested using broth microdilution per CLSI guidelines, and PCR detected eight virulence genes. Whole-genome sequencing analyzed genetic relationships and gene carriage status in 39 isolates. The results indicated that 86 strains of T. pyogenes were isolated from 316 samples, yielding an isolation rate of 27.2% (goats n = 47, 22.5%; sheep n = 39, 36.4%). The virulence genes plo, cbpA, nanH, nanP, fimA, fimC, and fimE were present in 100%, 66.7%, 64.1%, 71.8%, 69.2%, 59.0%, and 82.1% of isolates, respectively, with none carrying the fimG gene. The dominant virulence genotype was plo/nanH/nanP/fimA/fimC/fimE. The isolates exhibited resistance to erythromycin (44.2%, 38/86), gentamicin (38.4%, 33/86), sulfamethoxazole/trimethoprim (37.2%, 32/86), tetracycline (32.6%, 28/86), and streptomycin (32.6%, 28/86), and low resistance to chloramphenicol (14.0%, 12/86), ciprofloxacin (7.0%, 6/86), penicillin (5.8%, 5/86), and clindamycin (4.7%, 4/86). All isolates were susceptible to cefotaxime, vancomycin, and linezolid. Among the 86 isolates, 37 (43.0%) displayed multidrug resistance (MDR) characteristics. The whole genome sequencing of 39 isolates identified eight types of resistance genes, including ant(2″)-Ia, ant(3″)-Ia, cmlA1, cmx, erm(X), lnu(A), sul1, and tet(W). Except for tet(W), erm(X), and sul1, the other resistance genes were reported for the first time in T. pyogenes isolated in China. The drug susceptibility test results and resistance gene detection for the isolated strains were consistent for tetracycline, erythromycin, gentamicin, and sulfisoxazole. Similar allelic profiles and genetic evolutionary relationships were found among isolates from different farms. This study highlights the antibiotic resistance status and virulence gene-carrying rate of Trueperella pyogenes, providing a basis for clinical medication.

ISOLATION AND CHARACTERIZATION OF BIOFILM FORMING ESCHERICHIA COLI FROM CHICKEN MEAT SAMPLES

The present study was aimed to investigate the presence of Escherichia coli (E.coli) in raw chicken meat samples collected from retail shops, as well as the biofilm-forming ability of field isolates, and to characterize different adhesion genes. Out of 20 chicken meat samples, 17 (85%) were positive for E. coli. Fifteen E. coli strains were characterized by PCR using the 16S rDNA primers and all the isolates were positive which confirmed that all the isolates were E.coli. Out of the 15 confirmed E.coli field isolates which were subjected to biofilm-forming assay, 46% of them were found to be strong biofilm producers. While all the isolates were screened for the presence of adhesion genes viz. luxS, csgA, fimH, fimA, and papC, the adhesion gene luxS was detected in all the strains (100%). The other adhesion genes csgA, fimH and fimA were detected in 93%, 93%, and 73% of the isolates, respectively. The E. coli field isolates were screened blaTEM gene was detected in only four strains, which was categorized under strong biofilm producers. This study demonstrated the presence of biofilm forming E. coli in the raw chicken meat samples as contaminants, causing spoilage and potentially posing risk to consumer’s health and safety.

Expression of CD14+CD282+, CD14+CD284+, CD14+HLA-DR+, CD14+CD11b+ receptors and sIgA level in premature infants with K. pneumoniae

K. pneumoniae is one of the leading microorganisms causing nosocomial infections among premature newborns. The ineffectiveness of immune defense, morphofunctional immaturity, length of hospitalization and invasive procedures create the prerequisites for the implementation of the infectious process in a hospital setting. The question of the reasons for the development of infection, the etiological agent of which colonizes the intestines, remains open. Purpose of the study: to evaluate the expression of CD14+CD282+, CD14+CD284+, CD14+HLA-DR+, and CD14+CD11b+ receptors on blood monocytes and the level of sIgA in coprofiltrates in premature infants with intestinal colonization by K. pneumonia with different genetic profiles. We examined 11 children with the uge gene (group 1), 20 newborns with the uge + fim genes (group 2), and 12 children with the kfu + uge + fim genes. Microbiological examination of feces included identification and antibiotic sensitivity of microorganisms. Detection of the uge, fim and kfu genes in K. pneumoniae strains was carried out by PCR. The expression level of monocyte activation markers was determined by flow cytometry. Gestational age and anthropometric parameters did not differ between newborns. Children identified with the fim gene in combination with other genes were more often discharged home with K. pneumoniae than with the uge gene. In these children, a decrease in the level of expression of CD14+CD282+, CD14+CD284+, CD14+CD11b+, CD14+HLA-DR+ receptors at birth and upon reaching postconceptional age, and a low sIgA content in coprofiltrates during 24 days of life were recorded. Thus, a decrease in the expression of CD14+CD282+, CD14+CD284+, CD14+CD282+ and CD14+HLA-DR+ receptors by blood monocytes and insufficiency of sIgA production in the large intestine determine a long period of colonization of K. pneumoniae strains with the presence of the fim gene in combination with other genes (from 15 to 180 days), as well as the possibility of Klebsiella infection occurring in subsequent periods of the child’s life. Much more often, children with the combination of genes uge + fim and kfu + uge + fim were discharged from the hospital with a diagnosis of anemia; only in these groups of children was the development of bronchopulmonary dysplasia recorded.

Association of antibiotic resistance and biofilm formation in Escherichia coli ST131/O25b.

Urinary tract infections are becoming difficult to treat every year due to antibiotic resistance. Uropathogenic Escherichia coli (UPEC) isolates pose a threat with a combined expression of multidrug-resistance and biofilm formation. ST131 clone is a high-risk pandemic clone due to its strong association with antimicrobial resistance, which has been reported frequently in recent years. This study aims to define risk factors, clinical outcomes, and bacterial genetics associated with ST131/O25b UPEC. In this study, antibiotic susceptibility and species-level identification of 61 clinical E. coli strains were determined by automated systems. Detection of extended-spectrum beta-lactamases was assessed by double-disk synergy test. Biofilm formation was quantified by spectrophotometric method. Virulence genes (iutA, sfa cnf-1, iroN, afa, papA, fimA), antibiotic resistance genes (blaCTX-M, blaTEM, blaSHV, blaOXA, qnrA, qnrB, qnrS, ant(2')-Ia, ant(3)-Ia, aac(3)-IIa, mcr-1, mcr-2, mcr-3, mcr-4) were investigated by PCR. The following beta-lactamase genes were identified, blaTEM (n = 53, 86.8%), blaCTX-M (n = 59, 96.7%), blaSHV (n = 47, 77.0%), and blaOXA-1 (n = 27, 44.2%). Our data revealed that 93.4% of (57/61) E. coli isolates were biofilm-producers. O25pabBspe and trpA2 were investigated for the presence of ST131/O25b clone. Among multidrug resistant isolates, co-existence of O25pabBspe and trpA2 was detected in 29 isolates (47.5%). The fimH30 and H30Rx subclones were detected in four isolates that are strong biofilm-producers. These results suggest that clinical E. coli strains may become reservoirs of virulence and antibiotic resistance genes. This study demonstrates a significant difference in biofilm formation between E. coli ST131 and non-ST131 isolates. Moreover, 86.21% (n = 25) of ST131 isolates produced strong to moderate biofilms, while only 43.75% (n = 14) of non-ST131 isolates showed the ability to form strong biofilms. Presence of iutA and fimA genes in the majority of ST131 strains showed an important role in biofilm formation. These findings suggest application of iutA and fimA gene suppressors in treatment of infections caused by biofilm-producing drug-resistant ST131 strains.

Investigating the interaction of zno nanoparticles with flagellum and fimbriae in multi-drug resistant uropathogenic bacteria encoding fli and fim genes.

Due to the increasing occurrence of drug resistant urinary tract infections (UTI) among children, there is a need to investigate alternative effective treatment protocols such as nanoparticles. Flagella and fimbriae are primary factors contributing the virulence of urinary tract infecting bacteria. The aim of this study was to assess the antibacterial effects of zinc oxide nanoparticles which have been synthesized using both chemical and green methods on multi-drug resistant (MDR) uropathogenic bacteria encoding fli and fim genes and investigating their binding ability to bacterial appendage proteins. A total of 30 urine culture samples were collected from children under 2 years old diagnosed with urinary tract infection. The isolates underwent antibiotic suseptibility assessment and the isolates demonstrating MDR were subjected to molecular amplification of fimG (fimbrial) and fliD and fliT (flagellal) genes. The confirmation of cellular appendages was achieved through silver nitrate staining. The antibacterial efficacy of the synthetized nanoparticles was assessed using the micro and macrodilution methods. The successful binding of nanoparticles to bacterial appendage proteins was confirmed through mobility shift and membrane filter assays. The dimensions of chemically synthesized ZnO nanoparticles and green nanoparticles were measured at 30nm and 85nm, respectively, with the exhibition of hexagonal geometries. The nanoparticles synthesized through chemical and green methods exhibited minimum inhibitory concentrations (MIC) of 0.0062-0.025g/L and 0.3g/L, respectively. The ability of ZnO nanoparticles to bind bacterial appendage proteins and to combat MDR uropathogenic bacteria are promising for new treatment protocols against UTI in children in future.

Discovery of β-nitrostyrene derivatives as potential quorum sensing inhibitors for biofilm inhibition and antivirulence factor therapeutics against Serratia marcescens.

Quorum sensing (QS) inhibition has emerged as a promising target for directed drug design, providing an appealing strategy for developing antimicrobials, particularly against infections caused by drug-resistant pathogens. In this study, we designed and synthesized a total of 33 β-nitrostyrene derivatives using 1-nitro-2-phenylethane (NPe) as the lead compound, to target the facultative anaerobic bacterial pathogen Serratia marcescens. The QS-inhibitory effects of these compounds were evaluated using S. marcescens NJ01 and the reporter strain Chromobacterium violaceum CV026. Among the 33 new β-nitrostyrene derivatives, (E)-1-methyl-4-(2-nitrovinyl)benzene (m-NPe, compound 28) was proven to be a potent inhibitor that reduced biofilm formation of S. marcescens NJ01 by 79%. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) results revealed that treatment with m-NPe (50 μg/ml) not only enhanced the susceptibility of the formed biofilms but also disrupted the architecture of biofilms by 84%. m-NPe (50 μg/ml) decreased virulence factors in S. marcescens NJ01, reducing the activity of protease, prodigiosin, and extracellular polysaccharide (EPS) by 36%, 72%, and 52%, respectively. In S. marcescens 4547, the activities of hemolysin and EPS were reduced by 28% and 40%, respectively, outperforming the positive control, vanillic acid (VAN). The study also found that the expression levels of QS- and biofilm-related genes (flhD, fimA, fimC, sodB, bsmB, pigA, pigC, and shlA) were downregulated by 1.21- to 2.32-fold. Molecular dynamics analysis showed that m-NPe could bind stably to SmaR, RhlI, RhlR, LasR, and CviR proteins in a 0.1 M sodium chloride solution. Importantly, a microscale thermophoresis (MST) test revealed that SmaR could be a target protein for the screening of a quorum sensing inhibitor (QSI) against S. marcescens. Overall, this study highlights the efficacy of m-NPe in suppressing the virulence factors of S. marcescens, identifying it as a new potential QSI and antibiofilm agent capable of restoring or improving antimicrobial drug sensitivity.

Assessment of mrkD and T1 fim Genes of K. pneumoniae and Their Association with Biofilm Formation in Community-Acquired Infections

Background: Klebsiella pneumoniae is a pathogenic bacteria that can form biofilms on abiotic and biotic surfaces. The constraints of the therapeutic choices against K. pneumoniae arise from its innate capacity to develop biofilm and harboring determinants of multidrug resistance. Objective: The current study aimed to assess the prevalence of some fimbrial genes (type 1 and type 3) of K. pneumoniae strains in community-acquired infections obtained from various clinical sources on their ability to produce biofilm. Methods: A total of 256 samples (106 male and 150 females) were taken from various clinical sources including Urine, wound swabs, sputum, burn swabs, bronchial wash, diabetic foot ulcer, and higher vaginal swabs) from outpatients in Kirkuk City hospitals. The isolates were identified on the Blood agar, MacConkey agar and Eosin Methylene Blue agar based on the cultural, morphological, in addition to biochemical assays, and confirmed using the automated VITEK 2 system. The Microtiter plate (MTP) method was employed to detect Biofilm formation. Subsequently, a polymerase chain reaction was performed to determine the presence of the T1 fim and mrkD genes. Result: Out of 256 only 38 (14.8%) isolates belonged to Klebsiella pneumoniae. Of all isolates, 38(100%) were biofilm producers. The PCR results revealed that all 38 isolates showed positive (100%) for T1 fim and 37 isolates (97.4%) for mrkD.

1H, 13C and 15N assignment of self-complemented MrkA protein antigen from Klebsiella pneumoniae

Klebsiella pneumoniae (Kp) poses an escalating threat to public health, particularly given its association with nosocomial infections and its emergence as a leading cause of neonatal sepsis, particularly in low- and middle-income countries (LMICs). Host cell adherence and biofilm formation of Kp is mediated by type 1 and type 3 fimbriae whose major fimbrial subunits are encoded by the fimA and mrkA genes, respectively. In this study, we focus on MrkA subunit, which is a 20 KDa protein whose 3D molecular structure remains elusive. We applied solution NMR to characterize a recombinant version of MrkA in which the donor strand segment situated at the protein’s N-terminus is relocated to the C-terminus, preceded by a hexaglycine linker. This construct yields a self-complemented variant of MrkA. Remarkably, the self-complemented MrkA monomer loses its capacity to interact with other monomers and to extend into fimbriae structures. Here, we report the nearly complete assignment of the 13C,15N labelled self-complemented MrkA monomer. Furthermore, an examination of its internal mobility unveiled that relaxation parameters are predominantly uniform across the polypeptide sequence, except for the glycine-rich region within loop 176–181. These data pave the way to a comprehensive structural elucidation of the MrkA monomer and to structurally map the molecular interaction regions between MrkA and antigen-induced antibodies.

Frequency of Fimbrial Gene Types I, Ib, and II in Clinical Strains of Porphyromonas gingivalis Characterized From Periodontitis Patients.

Objective Porphyromonas gingivalis (P. gingivalis) is considered the predominant pathogen in association with different stages of periodontitis, and fim genes play a vital role in adherence and colonization. This study is thus aimed to detect the prevalence of P. gingivalis and the frequency of fim gene types among the clinical strains isolated from periodontitis patients. Methods Plaque samples (N = 45) were collected from patients with three different stages of periodontitis (n = 15 in each group). All the samples were inoculated onto sterile anaerobic blood agar and were processed anaerobically using a GasPak system at 37°C for five to sevendays. Standard microbiological techniques were used to identify P. gingivalis. Genomic DNA was extracted, and polymerase chain reaction (PCR) was carried out to detect the frequency of three fim gene types, using specific primers. Results P. gingivalis was more prevalent in Group III (93.3%), followed by 26.7% in Group II, and 13.3% in Group I. Maximum isolates were seen in the age group of 40-50, with no significance within the genders. fim type I was frequent in Group III (78.5% (n = 11)), followed by 0.25% (n = 1) under Group II, with no other fim types in the other groups. Conclusion Prevalence of P. gingivalis and frequency of fim genes, in association with its virulence, were observed. Periodical monitoring of such virulence genes would aid in the theranostic approach to combat the complications caused by P. gingivalis in periodontitis cases.

Biogenic amine tryptamine in human vaginal probiotic isolates mediates matrix inhibition and thwarts uropathogenic E. coli biofilm

Probiotics offer a promising prophylactic approach against various pathogens and represent an alternative strategy to combat biofilm-related infections. In this study, we isolated vaginal commensal microbiota from 54 healthy Indian women to investigate their probiotic traits. We primarily explored the ability of cell-free supernatant (CFS) from Lactobacilli to prevent Uropathogenic Escherichia coli (UPEC) colonization and biofilm formation. Our findings revealed that CFS effectively reduced UPEC’s swimming and swarming motility, decreased cell surface hydrophobicity, and hindered matrix production by downregulating specific genes (fimA, fimH, papG, and csgA). Subsequent GC–MS analysis identified Tryptamine, a monoamine compound, as the potent bioactive substance from Lactobacilli CFS, inhibiting UPEC biofilms with an MBIC of 4 µg/ml and an MBEC of 8 µg/ml. Tryptamine induced significant changes in E. coli colony biofilm morphology, transitioning from the Red, Dry, and Rough (RDAR) to the Smooth and White phenotype, indicating reduced extracellular matrix production. Biofilm time-kill assays demonstrated a four-log reduction in UPEC viability when treated with Tryptamine, highlighting its potent antibacterial properties, comparable to CFS treatment. Biofilm ROS assays indicated a significant elevation in ROS generation within UPEC biofilms, suggesting a potential antibacterial mechanism. Gene expression studies with Tryptamine-treated samples showed a reduction in expression of curli gene (csgA), consistent with CFS treatment. This study underscores the potential of Tryptamine from probiotic Lactobacilli CFS as a promising antibiofilm agent against UPEC biofilms.

Subminimum Inhibitory Concentrations Tetracycline Antibiotics Induce Biofilm Formation in Minocycline-Resistant Klebsiella pneumonia by Affecting Bacterial Physical and Chemical Properties and Associated Genes Expression.

Biofilm formation of Klebsiella pneumoniae can protect bacteria from antibiotics and is difficult to eradicate. Thus, the influence of subinhibitory concentrations of antibiotics on bacteria is becoming increasingly important. Our study showed that subminimum inhibitory concentrations (sub-MICs) of tetracycline antibiotics can increase biofilm formation in minocycline-resistant Klebsiella pneumoniae clinical strains. However, in the bacterial adhesion and invasion experiments, the adhesion and invasion ability decreased and the survival rate of Galleria mellonella increased. Under sub-MICs of tetracycline antibiotics treatment, abnormal stretching of bacteria was observed by scanning electron microscopy. Treatment with sub-MICs of tetracyclines leads to increased surface hydrophobicity and eDNA content and decreased outer membrane permeability. The expression levels of the fimA, luxS, qseB, and qseC genes decreased, the expression level of mrkA increased, and the expression level of acrA was inconsistent under different tetracycline antibiotics treatments. Together, our results suggested that the increase in Klebsiella pneumoniae biofilm formation caused by sub-MICs of tetracycline antibiotics may occur by affecting bacterial physical and chemical properties and associated genes expression.

Exploring Genetic and Phenotypic Factors Contributing to Urethral Catheter Biofilm Formation in Hospitalised Patients in Jordan

Background The biofilm phenomenon represents a prevalent mode of microbial life in nature which is characterized by cells irreversibly attaching to surfaces or each other and getting embedded in a matrix of extracellular polymeric substances. Object This study aimed to identify and characterize the genes associated with the common bacterial species responsible for biofilm formation in the catheters of hospitalized patients. Method Different bacterial strains were collected from catheterised patients at three local Jordanian hospitals for biofilm formation. The isolates were identified using Gram stain and Remel Rapid test. Biofilm formation was detected using the Tube method and tissue culture plate method. Result The presence of fimA and csgD genes was detected by polymerase chain reaction (PCR). Gram-negative bacteria species were isolated on the urethral catheters and the result shows the majority of the isolates were E. coli (40%), followed by K. pneumonia (27%). In comparison, the least was Citrobacter sp (2.6%). Similarly, some Gram-positive bacteria were also identified such as Staphylococcus aureus (4%) and Staphylococcus epidermidis (2.6%). K. pneumonia is strongly associated with biofilm formation (45%) followed by E. coli (33%). Conclusion Biofilm-associated genes, fimA and csgD were detected in all biofilm-positive samples containing the F plasmid, while csgD was detected in all biofilm-negative samples. Biofilm formation tends to be a prevalent process in patients included in this study and may cause dangerous complications in the patients especially in the elderly due to prolonged catheterization periods.

Prevalence and Molecular Detection of Virulence Genes among Multidrug-Resistant Escherichia coli from Human Clinical Samples and Poultry in Duhok City, Iraq

Abstract Background: Extended-spectrum beta-lactamase (ESBL)-producing, carbapenem-resistant Escherichia coli has increased virulence and multidrug resistance (MDR). Objectives: This study was designed to ascertain the frequency of some virulence factor genes, antibiotic susceptibility patterns, ESBLs, and MDR, focused on colistin-resistant E. coli strains of human and animal origin in Duhok city, Iraq. Materials and Methods: Between December 2020 and April 2021, a total of 150 E. coli isolates (110 from human clinical specimens and 40 from poultry cloacal swabs) were included in this study. The isolates underwent screening for antibiotic susceptibility, MDR, ESBL, and molecular detection of four virulence genes (fimA, cnfL, crL, and hlyA) was conducted using the polymerase chain reaction. Results: Urine specimens (77.2%) compared to blood, wound, vaginal swab, sputum, and semen from outpatients (71.8%). All strains from humans and poultry showed high resistance to ampicillin (86%–97%), ceftriaxone (74%–47%), tetracycline (72%–85%), ciprofloxacin (48%–97%), and colistin (17%–12%). The lowest levels of resistance were found for carbapenems (4%–4%), and the MDR for the isolates was 63%–93%. Apart from carbapenems, colistin-resistant isolates, especially those from poultry, exhibited significant resistance to other antibiotics, and 57% of these isolates being ESBL producers. Three virulence genes (fimA, cnfL, and crL) were highly prevalent (92%) in human isolates, with the crL gene being predominant (100%). Among poultry isolates, fimA was more prevalent (94%) while crL was less common (6%). Conclusion: The predominance of isolates of colistin-resistant poultry origin and the virulence of isolates of human E. coli origin indicate that both strains are currently experiencing an increase in antibiotic resistance.

Antimicrobial efficiency of bromhexine hydrochloride against endometritis-causing Escherichia coli and Trueperella pyogenes in bovines.

In this study, the main agents associated with endometritis in cows in the state of Santa Catarina, Brazil, were identified and the resistance profile and virulence mechanisms of the bacterial isolates were evaluated. Isolates of Escherichia coli and Trueperella pyogenes were tested for their biofilm forming ability and the antimicrobial action of bromhexine hydrochloride in combination with other antimicrobials. A total of 37 uterine lavage samples were collected from cows with endometritis. Of the 55 bacteria isolated, 25.4% were identified as T. pyogenes and 16.3% as E. coli. The bacterial isolates showed greater resistance to sulfamethoxazole + trimethoprim (58.2%) and tetracycline (56.3%). Among the species, E. coli showed the highest resistance rates, with 100% of isolates showing resistance to amoxicillin, streptomycin, and gentamicin. The results of the minimum inhibitory concentration for the T. pyogenes isolates showed that 91.6% of the isolates were resistant to enrofloxacin and tetracycline, and 75% were resistant to ceftiofur and sulfamethoxazole + trimethoprim. All E. coli and T. pyogenes isolates showed biofilm forming ability. The plo, fimA, and nanH genes were identified in 100% of T. pyogenes isolates. In parallel, 100% of E. coli isolates had the fimH gene, and 11.1% had the csgD gene. Bromhexine hydrochloride showed antimicrobial activity against 100% of E. coli isolates and 66.6% of T. pyogenes isolates. Furthermore, when associated with antimicrobials, bromhexine hydrochloride has a synergistic and additive effect, proving to be an option in the treatment of endometritis in cows and an alternative for reducing the use of antimicrobials.

Development and Evaluation of a Multiplex Fluorescence PCR for Salmonella Virulence Genes Analysis

Background: Salmonellosis, a disease caused by Salmonella, is a significant public health concern and economic burden worldwide. The ability of various Salmonella serovars to cause disease is closely linked to the virulence genes they possess. Objectives: The aim of this study was to develop a multiplex fluorescence PCR for detecting ten major virulence genes (ssaR, spvC, pefA, sipA, fimA, sifA, sopE2, sopB, prgH, and stn) in Salmonella. Methods: Primer pairs specific to ten target virulence genes were designed using Primer Premier 5.0 and distributed across two reaction tubes. The multiplex fluorescence PCR was optimized by adjusting one factor at a time. Results: A total of sixty Salmonella strains were analyzed using the newly developed multiplex fluorescence PCR. All strains contained seven or more of the tested virulence genes. The positive rates of virulence genes ssaR, sipA, sopE2, sopB, prgH, and stn were high, each at 100%. The positive rate of sifA was also relatively high at 81.67%. However, the positive rates of spvC at 5% and pefA at 3.33% were relatively low. Conclusions: The newly developed multiplex fluorescence PCR provides a straightforward, cost-effective, and high-throughput solution for detecting virulence genes in Salmonella. It has the potential to become a routine method for analyzing Salmonella virulence genes.

Molecular Characterization of Resistance and Virulence Factors of Trueperella pyogenes Isolated from Clinical Bovine Mastitis Cases in China.

The present study was designed to investigate the resistance determinants and virulence factors of 45 Trueperella pyogenes isolates from clinical bovine mastitis in Hexi Corridor of Gansu, China. Minimum inhibitory concentrations (MICs) was tested by E-test method. Gene of antimicrobial resistance, virulence integrase and integron gene cassettes were determined by PCR and DNA sequencing. The T. pyogenes isolates exhibited high resistance to streptomycin (88.9%) and tetracycline (64.4%), followed by erythromycin (15.6%) and gentamicin (13.3%). Resistance to streptomycin was most commonly encoded by aadA9 (88.9%); and to tetracycline, by tetW (64.4%). Importantly, all streptomycin-resistant isolates carried aadA9 alone or in combination with aadA1, aadA11 and strA-strB. Similarly, all tetracycline-resistant isolates harbored tetW alone or in combination with tetA33. Meanwhile, ermX was detected in 13.3% isolates, only one erythromycin-resistant isolate was not identified for this gene. Moreover, all T. pyogenes isolates carried class 1 integrons, and 17.8% of them contained gene cassettes, including arrays aadA1-aadB (4.4%), aad A24-dfrA1-ORF1 (2.2%) and aadA1 (2.2%). Furthermore, all tested isolates harbored virulent genes plo and fimA, followed by fimC (88.9%), fimE (86.6%) nanP (75.6%), nanH (40.0%), cbpA (35.6%) and fimG (6.7%). To our knowledge, this is the first report of integron gene cassettes of T. pyogenes isolates from bovine mastitis cases in China. These findings are useful for developing the prevention and the virulence factors of T. pyogenes could be promising candidates for vaccine antigens for bovine mastitis caused by T. pyogenes in China.

Type 1 fimbrial phase variation in multidrug-resistant asymptomatic uropathogenic Escherichia coli clinical isolates upon adherence to HTB-4 cells.

The adherence of bladder uroepithelial cells, subsequent expression, and regulation of type 1 fimbrial genes (key mediator of attachment) in clinical multidrug-resistant uropathogenic Escherichia coli (MDR-UPECs) isolated from individuals with asymptomatic bacteriuria (ABU) remain unexplored till date. Therefore, this study aimed to investigate the underlying molecular mechanisms associated with the adherence of clinical MDR-ABU-UPECs to human a uroepithelial cell line (HTB-4), both in the absence and presence of D-Mannose. These investigations focused on phase variation, expression, and regulation of type 1 fimbriae and were compared to a prototype ABU-strain (E. coli 83972) and symptomatic MDR-UPECs. Discordant to the ABU prototype strain, MDR-ABU-UPECs exhibited remarkable adhesive capacity that was significantly reduced after D-mannose exposure, fairly like the MDR symptomatic UPECs. The type 1 fimbrial phase variation, determined by the fim switch analysis, asserted the statistically significant incidence of "both OFF and ON" orientation among the adherent MDR-ABU-UPECs with a significant reduction in phase-ON colonies post-D-mannose exposure, akin to the symptomatic ones. This was indicative of an operative and alternating type 1 fimbrial phase switch. The q-PCR assay revealed a coordinated action of the regulatory factors; H-NS, IHF, and Lrp on the expression of FimB and FimE recombinases, which further controlled the function of fimH and fimA genes in ABU-UPECs, similar to symptomatic strains. Therefore, this study is the first of its kind to provide an insight into the regulatory crosstalk of different cellular factors guiding the adhesion of ABU-UPECs to the host. Additionally, it also advocated for the need to accurately characterize ABU-UPECs.