Weak Biofilm Formation Research Articles

Virulence genes and antibiotic susceptibility patterns of Escherichia coli isolated from nosocomial urinary tract infections in the northwest of Iran during 2022-2023: A cross-sectional study.

Urinary tract infections (UTIs) are prevalent among hospitalized patients, constituting the most frequent health-care infections. Uropathogenic Escherichia coli (UPEC) is leading causative agent of UTIs. The present study was aimed to examine the susceptibility of UPEC isolates obtained from nosocomial cases to antibiotics, as well as their biofilm formation capability and frequency of virulence genes. A total of 100 UPEC isolates were collected from nosocomial UTIs at Imam Reza Hospitals in Tabriz, Iran, spanning from April 2022 to January 2023. The antimicrobial susceptibility patterns were evaluated using the disk diffusion method, along with the detection of broad-spectrum β-lactam enzymes (ESBLs) and carbapenemases. The ability of isolates to form biofilms was assessed using the microtiter-plate method, while the PCR method was employed to identify the presence of virulence genes. The highest resistance was observed toward piperacillin (82%), followed by aztreonam and ciprofloxacin (81%), while the lowest resistance was found against piperacillin/tazobactam (12%) and meropenem (9%). ESBLs were detected in 62% of the isolates. The microtiter-plate results revealed strong, moderate, and weak biofilm formation abilities in 32%, 33%, and 24% of the isolates, respectively. The most prevalent virulence gene was fimA (74%) followed by hlyF (68%), papA (44%), papC (32%), iroN (26%), and cnf (20%). The elevated levels of resistance to multiple antimicrobial agents, coupled with the co-presence of virulence genes and biofilm formation abilities, contribute to the persistence of UPEC-related infections, particularly in hospitalized patients. These findings underscore the necessity of implementing an effective program to control nosocomial UTIs caused by UPEC in the healthcare centers.

Characterization and Prevalence of Antibiotic Resistance Staphylococcus aureus in Street Food: A Public Health Concern

Food samples containing Staphylococcus aureus pose a serious health risk. This study aimed to examine the prevalence of S. aureus strains in various food samples sourced from Mailsi and Multan. Many food samples including yogurt, bakery products, and raw and cooked food were examined for S. aureus. The isolated strains were confirmed through Mannitol agar fermentation, catalase, coagulase, and urease tests. Hemolysis on blood agar and biofilm formation were also assessed to determine toxin production. Antibiotic sensitivity testing was conducted using the Kirby Bauer method on MH agar, and multiple antibiotics were tested. Out of all samples, a total of 50 S. aureus strains were obtained, mainly from milk and milk-based products. Yellow colonies on mannitol salt agar confirmed S. aureus presence, with all isolates testing positive for coagulase, catalase, and urease. The presence of hemolysins: beta, gamma, and alpha were revealed by hemolysis assays. Biofilm assay results showed variation among the strains, with some categorized as strong, moderate, or weak biofilm formers. Regarding antibiotic sensitivity, most strains exhibited multidrug resistance, particularly against certain antibiotics. Vancomycin showed varying susceptibility patterns, some strains showed susceptibility and intermediate resistance, whereas only milk samples showed resistance. This study emphasizes the prevalence of MDR S. aureus strains in food samples. The study underscores the significance of antimicrobial stewardship programs and stringent food safety measures in preventing the spread of antibiotic-resistant strains and reducing foodborne illnesses. Further research is needed to explore the mechanisms behind antibiotic resistance and toxin production in S. aureus strains from food samples.

Antibacterial Properties of Cinamaldehyde and its Impact on Biofilm Formation by Respiratory Isolates of Corynebacterium Propinquum

Objective: Analyze the antibacterial properties of cinnamaldehyde (CN) and its effect on biofilm formation by Corynebacterium propinquum. Theoretical Framework: C. propinquum can form biofilm and be multiresistant. In the context of advancing resistance and the need for new and more effective drugs, constituents extracted from cinnamon, such as CN, stand out as they have proven antimicrobial activity. Method: It was investigated whether CN essential oil (EO) was hemolytic for sheep erythrocytes. The antibacterial activity against nasopharyngeal C. propinquum strains was evaluated by determining the minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC), and by analyzing the combinatorial effect with chloramphenicol (CL). Biofilm formation on a hydrophobic surface (polystyrene), in the presence or absence of subinhibitory concentrations (SubMIC) of CN, was also evaluated. Results and Discussion: The results demonstrated that CN was not hemolytic. Despite the MIC being higher than that of CL, the antibacterial action was strong for both strains, but it was impossible to determine the MBC. The combinatorial antibacterial effect of CN+CL demonstrated partial synergy or indifference. Weak biofilm formation was observed for the strains and the presence of CN SubMIC, although it did not change the pattern, increased its production. Research Implications: This research's practical and theoretical implications suggest promising antibacterial properties of CN on emerging pathogen C. propinquum. Originality/Value: This study is relevant not only because it demonstrates aspects of virulence of a pathogen that has been little studied, but also because it shows the antibacterial action of CN, a constituent of an easily accessible spice. It highlighted the possibility of future research into the treatment of infections caused by a microorganism that is often multi-resistant.

Isolation and characterization of cefotaxime resistant Escherichia coli from household floors in rural Bangladesh

Antimicrobial resistance (AMR) is a rising health concern worldwide. As an indicator organism, E. coli, specifically extended-spectrum β-lactamase (ESBL) producing E. coli, can be used to detect AMR in the environment and estimate the risk of transmitting resistance among humans, animals and the environment. This study focused on detecting cefotaxime resistant E. coli in floor swab samples from 49 households in rural villages in Bangladesh. Following isolation of cefotaxime resistant E. coli, DNA extracted from isolates was subjected to molecular characterization for virulence and resistance genes, determination of resistance to multiple classes of antibiotics to define multidrug resistant (MDR) and extensively drug resistant (XDR) strains, and the biofilm forming capacity of the isolates. Among 49 households, floor swabs from 35 (71 %) households tested positive for cefotaxime resistant E. coli. Notably, all of the 91 representative isolates were ESBL producers, with the majority (84.6 %) containing the blaCTX-M gene, followed by the blaTEM and blaSHV genes detected in 22.0 % and 6.6 % of the isolates, respectively. All isolates were MDR, and one isolate was XDR. In terms of pathogenic strains, 8.8 % of the isolates were diarrheagenic and 5.5 % were extraintestinal pathogenic E. coli (ExPEC). At 25 °C, 45 % of the isolates formed strong biofilm, whereas 43 % and 12 % formed moderate and weak biofilm, respectively. On the other hand, at 37 °C, 1.1 %, 4.4 % and 93.4 % of the isolates were strong, moderate and weak biofilm formers, respectively, and 1.1 % showed no biofilm formation. The study emphasizes the importance of screening and characterizing cefotaxime resistant E. coli from household floors in a developing country setting to understand AMR exposure associated with floors.

Targeted metabolomics analysis approach to unravel the biofilm formation pathways of Enterococcus faecalis clinical isolates.

(i) To characterize Enterococcus faecalis biofilm formation pathways by semi-targeted metabolomics and targeted nitrogen panel analysis of strong (Ef63) and weak (Ef 64) biofilm forming E. faecalis clinical isolates and (ii) to validate the identified metabolic markers using targeted inhibitors. Previous proteomics profiling of E. faecalis clinical isolates with strong and weak biofilm formation revealed that differences in metabolic activity levels of small molecule, nucleotide and nitrogen compound metabolic processes and biosynthetic pathways, cofactor metabolic process, cellular amino acid and derivative metabolic process and lyase activity were associated with differences in biofilm formation. Hence, semi-targeted analysis of Ef 63, Ef 64 and ATC control strain Ef 29212 was performed by selecting metabolites that were part of both the previously identified pathways and a curated library with confirmed physical and chemical identity, followed by confirmatory targeted nitrogen panel analysis. Significantly regulated metabolites (p < .05) were selected based on fold change cut-offs of 1.2 and 0.8 for upregulation and downregulation, respectively, and subjected to pathway enrichment analysis. The identified metabolites and pathways were validated by minimum biofilm inhibitory concentration (MBIC) and colony forming unit (CFU) assays with targeted inhibitors. Metabolomics analysis showed upregulation of betaine, hypoxanthine, glycerophosphorylcholine, tyrosine, inosine, allantoin and citrulline in Ef 63 w.r.t Ef 64 and Ef 29212, and thesemetabolites mapped to purinemetabolism, urea cycle and aspartate metabolism pathways. MBIC and CFU assays using compounds against selected metabolites and metabolic pathways, namely glutathione against hypoxanthine and hydroxylamine against aspartate metabolism showed inhibitory effects against E. faecalis biofilm formation. The study demonstrated the importance of oxidative stress inducers such as hypoxanthine and aspartate metabolism pathway in E. faecalis biofilm formation. Targeted therapeutics against these metabolic markers can reduce the healthcare burden associated with E. faecalis infections.

Biofilm generation and antibiotic resistant profile of extensive and multidrug resistant Pseudomonas aeruginosa from burn patients in Ahvaz: A cross-sectional study.

Multidrug and extensive drug-resistant Pseudomonas aeruginosa was extracted from burn patients referring to burn centers in southwest Iran so that biofilm generation and antibiotic resistance could be investigated. A specific primer was used to confirm all our considered 110 P. aeruginosa culture-positive reports on 345 burn patients. The resistance of P. aeruginosa to seven antibiotics and Colistin with minimum inhibitory concentration (MIC) was assessed. Biofilm formation was assessed by the phenotypic study of specimens under Congo red agar and microtiter plate assays. One hundred and 10 clinical P. aeruginosa isolates taken from burn wound infections were validated. Among P. aeruginosa isolates, Piperacillin, Ceftazidime, Maeropenem, Gentamycin, and Gatifloacin had the highest resistance to antibiotics, while Ticarcillin-Clavulanic acid and Ceftolozane-Tazobactam showed the least resistance. MICs were then evaluated via the E test. Seven isolates were resistant to colistin. Colistin reference MICs for multidrug-resistant P. aeruginosa prevalence was 38%, while it was 22% for extensively drug-resistant (XDR) P. aeruginosa. One P. aeruginosa was pandrug-resistant (PDR). Under Congo red agar test, 66 isolates (67%) formed biofilms and black colonies, whereas 44 isolates (50%) had red colonies. In MTP, 76% formed biofilm. 40%, 32%, 21% of the isolates were strong, moderate, and weak biofilm formers, respectively, while 43% did not form biofilms. The P. aeruginosa resistance to antimicrobial agents has largely challenged the control of the infection. Accordingly, a higher resistance occurred when the isolates were transferred to the patients. Less than 50% P. aeruginosa samples generated strong biofilms. Consequently, hygienic measurements are essential to inhibit P. aeruginosa transmission to hospitalized patients.

Prevalence of antimicrobial resistance, biofilm formation, efflux pump activity, and virulence capabilities in multi-drug-resistant Klebsiella pneumoniae isolated from freshwater fish farms.

The present study aimed to determine the antibiotic resistance, underlying mechanisms, antibiotic residues, and virulence genes involved in 32 multi-drug-resistant Klebsiella pneumoniae isolates from freshwater fishes in Andhra Pradesh, India. Antibiogram studies revealed that all isolates were multi-drug-resistant, harbored tetA (96.8%), tetC (59.3%), tetD (71.9%), nfsA (59.3%), nfsB (53.1%), sul2 (68.7%), qnrC (43.7%), qnrD (50%), blaSHV (75%), blaTEM (68.7%), and blaCTX-M (93.7%) genes. Multiple antibiotic resistance index was calculated as 0.54. Sixteen isolates were confirmed to be hyper-virulent and harbored magA and rmpA genes. In total, 46.9, 31.2, and 21.9% of the isolates were categorized as strong, moderate, or weak biofilm formers, respectively. All isolates possessed an active efflux pump and harbored acrA, acrB, acrAB, and tolC genes in 94% of the isolates, followed by mdtK (56.2%). Porins such as ompK35 and ompK36 were detected in 59.3 and 62.5% of the isolates, respectively. Virulence genes fimH-1, mrkD, and entB were present in 84.3, 81.2, 87.5% of the isolates, respectively. These findings imply a potential threat that multi-drug-resistant bacterial pathogens could transmit to surrounding environments and humans through contaminated water and the aquaculture food chain.

Genotypic and phenotypic characterization of determinants that mediate antimicrobial resistance in Escherichia coli strains of clinical origin in South-Western Nigeria.

Multidrug resistant bacterial pathogens employ different mechanisms in evading the action of antibiotics. Multidrug resistance is wide spread among strains of Escherichia coli implicated in several infections including urinary tract infections, gastrointestinal infections, meningitis and bacteraemia. This study investigates the antibiotic resistance profile, efflux pump activity and biofilm formation ability of E. coli strains isolated from clinical samples. A total of 32E. coli strains isolated from clinical samples were characterized and subjected to antibiotic susceptibility testing using standard methods. Isolates were screened phenotypically for biofilm formation and efflux pump activity. While molecular detection of genes encoding curli fimbriae and efflux pump activity was done by PCR. All 32 (100%) E. coli isolates were resistant to ceftazidime, cefuroxime, cefixime, amoxicillin-clavulanate, ofloxacin and ciprofloxacin. While 30 (93.8%) were resistant to gentamicin, 27 (84.4%) were resistant to cefepime and the least resistance of 15.6% was to imipenem. Efflux pump encoding gene tolC was detected in 13(40.6%) of the isolates, while 1(3.1%) harboured acrA gene. acrB gene was not detected in any of the isolates. Seven (21.9%) of the isolates were strong biofilm formers, while 5 (15.6%) and 20 (62.5%) were moderate and weak biofilm formers respectively. csgA gene was detected in all E. coli isolates. High antibiotic resistance of E. coli strains observed in this study is of public health significance. . It is therefore important to scale up efforts in regular monitoring of antibiotic resistance in both community and hospital settings.

Evaluation of the antibacterial activity of CuO and ZnO nanoparticles against uropathogenic Escherichia coli

E. coli responsible for urinary tract infections (UTI) became resistant to obtainable therapies. So, nanotechnology appears currently as promising innovation among various strategies under development. In the present study we evaluate the antimicrobial effects of CuO and ZnO nanoparticles against E. coli isolated from UTI. Biofilm production and expression of quorum sensing genes luxS and motA were performed on E. coli treated with the nanoparticles. luxS and motA genes were detected in the tested E. coli. All isolates produced strong, moderate and weak biofilm. All isolates had a weak biofilm formation and decreased gene expression for luxS and motA genes after treatment with CuO, ZnO and their mixture. Besides, CuO and ZnO had non-toxic effects on Human Dermal Fibroblasts and neonatal normal cells. In conclusion, the mixture of CuO and ZnO had a role in decreasing the gene expression of luxS and motA genes more than each alone.

Molecular characterization of antimicrobial resistance and virulence factors in coagulase – negative staphylococci nasal isolates among adult patients in a tertiary hospital in North-Western Nigeria

Coagulase-negative staphylococci are harmless microorganisms that can become pathogenic when the host resistance is impaired. Their ability to exhibit multidrug-resistance and form biofilm limit treatment options and contributes to the health and financial burden on health care systems worldwide. This study aimed to provide data on the acquisition and expression of genetic elements associated with resistance and virulence in the organisms from nasal colonization of adults admitted to a University Teaching Hospital in Nigeria. One hundred and twenty-three presumptive staphylococci isolates were obtained from the medical microbiology laboratory of Ahmadu Bello University Teaching Hospital (ABUTH), Zaria. The isolates were characterized, and the antibiotics susceptibility pattern assessed. Biofilm formation was evaluated by the micro-titre plate method, and polymerase chain reaction was used to detect resistance genes (mecA and ermB), virulence gene (psm-mec) and adhesin genes, (icaAB, icaC and icaD) in the isolates. Out of the 60 coagulase-negative staphylococci isolates, 50 (83.3%) were strong biofilm formers, 5 (8.3%) were moderate biofilm formers and 5 (8.3%) were weak biofilm formers. For the 14 isolates that were characterized by PCR, mecA and ermB resistance genes were detected in 78% and 71% of the isolates respectively. Twenty-one percent had psm-mec virulence gene while biofilm adhesin genes, icaAB and icaD were detected in 36% and 57% respectively, icaC gene was not detected. This study confirmed formation of biofilm, carriage of resistance and virulence genes which have been shown to play a role in pathogenesis. This study shows the necessity of the periodical monitoring of the drug resistance pattern and virulence factors of coagulase-negative staphylococci.

Detection of biofilm formation and antibiotics resistance of Staphylococcus spp. isolated from humans’ and birds’ oral cavities

Background: Staphylococcus spp. are widely distributed in nature and can cause nosocomial, skin infections, and foodborne illness, and it may lead to severe financial losses in birds by causing systemic infection in numerous organs. Aim: This study was conducted to determine the prevalence of Staphylococcus spp. in humans and birds in Baghdad city. Methods: Seventy-six oral cavity swabs were collected, including 41 from birds and 35 from breeders. All samples were examined by bacteriological methods and identified by using the VITEK technique, the samples were then further studied to test the ability of biofilm formation, and MDR factors and MAR index were tested with the use of seven antibiotics. Results: Among the 76 oral swabs, 37 samples were positive (48.68%) for Staphylococcus spp.: 7 human samples (20%) and 30 bird samples (73.17%). In humans, S. lentus was the most prevalent (42.85%) followed by S. aureus (28.57%), S. hominis and S. sciuri were at (14.29%) to each. In birds, S. pseudintermedius, S. gallinarum, S. lentus, S. haemolyticus, Staphylococcus spp, S. sciuri and S. xylosus were detected in 36.67%, 16.67%, 10%, 10%, 13.33%, 3.33% and 3.33% respectively. Staphylococcus isolates from the human samples demonstrated that only S. lentus was resistant 33.33% to ME, OX, and SXT. Furthermore, one of them was MDR and high MAR index value. The antimicrobial pattern of Staphylococcus spp. isolated from birds was as follows: S. pseudintermedius isolates demonstrated 100% resistance to CN, CIP, SXT, and MDR (100%) and high MAR indices value; S. xylosus was resistant 100% against ME, CN, SXT and Do and it was MDR with high MAR index;S. lentus was resistant 25% against ME, OX, C, and SXT, whereas, S. gallinarum was resistant 33.33% against ME and OX. The results demonstrated that biofilm formation of the Staphylococcus spp. isolated from human samples were weak biofilm formers: S. lentus, S. hominis, and S. aureus, while other S. aureus (50%) was moderate. In birds, the majority of the isolates had non biofilm-producing capabilities, while 80% of S. lentus and 100% of S. xylosus showed moderate biofilm formation. Conclusion: Healthcare problem was observed in this study due to high MDR and MAR index among Staphylococcus spp. isolated from pet birds to their owners and vice versa.

Prevalence of Gram-Positive Bacteria in Hospital Cafeteria Fast Foods in Dhaka, Bangladesh and Their Resistance to Current Antibacterial Agents

Emerging antibiotic resistance of Gram-positive bacteria leading to strikethrough infections are one of the major threats to global public health. This research aimed to determine different circulating Gram-positive bacterial species as well as their antibiotic resistance pattern along with their phylogenetic analysis. Fifty (50) Gram-positive isolates from different bacterial species were isolated from eight different hospital cafeteria fast foods of Dhaka, Bangladesh of which 58% of the isolates were from sandwiches and 42% of the isolates were from burger samples. The isolates were divided into six different groups based on different biochemical tests belonging to Bacillus spp. followed by Planococcus spp., Micrococus spp., Streptococcus spp., Clostridium spp. and Staphylococcus spp. Distribution of isolates varied greatly keeping the highest number of isolates from Bacillus spp. and lowest in Staphylococcus spp. BLAST search and phylogenetic analysis revealed that all sequenced isolates were Gram-positive bacteria under firmicutes and shared their identity with Chryseomicrobium spp., Sporosarcina psychrophila, Bacillus licheniformis. About 6% of the isolates were strong biofilm former whereas 4%, 24% and 66% of the isolates were moderate biofilm formers, weak biofilm formers and non-biofilm formers respectively. About 6% of the isolates showed β-hemolytic properties. About 72% of the isolates were multi-drug resistant. Among the antibiotics, ampicillin and penicillin showed the highest level of resistance followed by cephalexin, nitrofurantoin, meropenem and no resistance to imipenem. A higher proportion of multi-drug resistant (MDR) Gram-positive isolates were revealed from the hospital cafeteria fast foods demonstrating the risky situation regarding hospital associated infections caused by Gram-positive bacteria. Bioresearch Commu. 10(1): 1361-1370, 2024 (January)

BIOFILM FORMATION AND VIRULENCE FACTORS DISTRIBUTION AMONG CLINICAL ISOLATES OF Pseudomonas aeruginosa

Introduction: Pseudomonas aeruginosa is an opportunistic microorganism frequently isolated in the hospital environment and has been classified by the World Health Organization as a critical pathogen due to its resistance to antibiotics and its varied virulence factors. Objective: To evaluate biofilm formation and the expression of virulence factors of 30 clinical isolates and 2 standard strains of P. aeruginosa. In addition, this work aims to analyze the distribution of virulence factors in the different categories of biofilm formation. Methodology: Biofilm formation assays were performed in polystyrene microplate with crystal violet coloring and the strains were classified in groups. Other virulence factors were studied with specific phenotypic detection methods. Results: All P. aeruginosa were able to form biofilms, with 46.9% classified as strong formers, 25% as moderate and 28.12% as weak biofilm formers. Most isolates expressed the virulence factors analyzed and among them, pyoverdine was the most prevalent, followed by the production of lipases B, hemolysin, and protease. Lipases A showed the lowest incidence. Conclusion: Although no significant correlation was detected between biofilm formation of P. aeruginosa clinical isolates and virulence factors, it was observed that in the strong formers group there was expression of alkaline protease and lipases B in all strains. The results suggest that the pathogenicity of P. aeruginosa is a multifactorial phenomenon and that virulence factors, although present in most isolates, occur independently of the biofilm formation category of the studied strains.

Isolation and identification of antimicrobial susceptibility, biofilm formation, efflux pump activity, and virulence determinants in multi-drug resistant Pseudomonas aeruginosa isolated from freshwater fishes.

The present study was undertaken to evaluate the prevalence, underlying resistance mechanism, and virulence involved in Pseudomonas aeruginosa (n = 35) isolated from freshwater fishes in Andhra Pradesh, India. Antibiogram studies revealed that 68.5, 62.8, 37.1, 11.4, 8.5, 57.1, 54.2, and 48.5% of isolates had resistance to oxytetracycline, co-trimoxazole, doxycycline, enrofloxacin, ciprofloxacin, cefotaxime, ceftazidime, and ampicillin, respectively. The resistant isolates harboured the tetA (85.7%), tetD (71.4%), tetM (91.4%), sul1 (80%), blaCTX-M (57.1%), blaTEM (42.8%), and blaSHV (48.5%) genes. In total, 50% of the isolates were altered as multi-drug resistant, and the multiple antibiotic resistance index was calculated as 0.4. Furthermore, 37.3, 48.5, and 14.2% of isolates were categorized as strong, moderate, and weak biofilm formers, possessing pslA (91.5%) and pslD (88.6%) biofilm encoding genes. In total, 82.8% of the isolates exhibited efflux pump activity and harboured the mexA (74.2%), mexB (77.1%), and oprM (37.1%) genes. Virulent genes oprL, toxA, exoS, and phzM were detected in 68.5, 68.5, 100, and 17.1% of isolates, respectively. The data suggested that P. aeruginosa harbours multiple resistance mechanisms and virulence factors that may contribute to antibiotic resistance and pathogenicity, and their distribution in fish culture facilities highlights the public health hazards of the food chain.

Detection and characterization of putative hypervirulent Klebsiella pneumoniae isolates in microbiological diagnostics

Hypervirulent Klebsiella pneumoniae strains (hvKp) can cause invasive community-acquired infections in healthy patients of all ages. In this study, the prevalence of putative hvKp in a German tertiary center was investigated and hvKp were characterized by phenotypic and molecular assays. All K. pneumoniae isolates in routine microbiological diagnostics from a single center were screened by string-testing over a period of 6 months. String-test positive (≥ 0.5 mm) isolates were re-evaluated on different media and under various conditions (aerobe, anaerobe). For string-test positive isolates, genes (magA, iutA, rmpA and rmpA2) associated with hypermucoviscosity and hypervirulence were amplified by multiplex PCR. PCR-positive isolates were subjected to whole-genome sequencing and sedimentation and biofilm formation assays. From 1310 screened K. pneumoniae isolates in clinical routine 100 isolates (7.6%) were string test positive. From these, 9% (n = 9) were defined as putative hvKp (string-test+/PCR+). Highest rate of string-test-positive isolates was observed on MacConkey agar under aerobic conditions. Amongst these nine putative hvKp isolates, the international lineage ST23 carrying hvKp-plasmid pKpVP-1 was the most common, but also a rare ST86 with pKpVP-2 was identified. All nine isolates showed hypermucoviscosity and weak biofilm formation. In conclusion, 9% of string-positive, respectively 0.69% of all K. pneumoniae isolates from routine were defined as putative hypervirulent. MacConkey agar was the best medium for hvKp screening.

Multidrug-resistant Escherichia coli isolated from patients and surrounding hospital environments in Bangladesh: A molecular approach for the determination of pathogenicity and resistance

Extended spectrum β-lactamase producing Escherichia coli (ESBL E. coli) is a primary concern for hospital and community healthcare settings, often linked to an increased incidence of nosocomial infections. This study investigated the characteristics of ESBL E. coli isolated from hospital environments and clinical samples. In total, 117 ESBL E. coli isolates were obtained. The isolates were subjected to molecular analysis for the presence of resistance and virulence genes, antibiotic susceptibility testing, quantitative adherence assay, ERIC-PCR for phylogenetic analysis and whole genome sequencing of four highly drug resistant isolates. Out of the 117 isolates, 68.4% were positive for blaCTX-M, 39.3% for blaTEM, 30.8% for blaNDM-1, 13.7% for blaOXA and 1.7% for blaSHV gene. Upon screening for diarrheagenic genes, no isolates were found to harbour any of the tested genes. In the case of extraintestinal pathogenic E. coli (ExPEC) virulence factors, 7.6%, 11%, 5.9%, 4.3% and 21.2% of isolates harbored the focG, kpsMII, sfaS, afa and iutA genes, respectively. At a temperature of 25°C, 14.5% of isolates exhibited strong biofilm formation with 21.4% and 28.2% exhibiting moderate and weak biofilm formation respectively, whereas 35.9% were non-biofilm formers. On the other hand at 37°C, 2.6% of isolates showed strong biofilm formation with 3.4% and 31.6% showing moderate and weak biofilm formation respectively, whereas, 62.4% were non-biofilm formers. Regarding antibiotic susceptibility testing, all isolates were found to be multidrug-resistant (MDR), with 30 isolates being highly drug resistant. ERIC-PCR resulted in 12 clusters, with cluster E−10 containing the maximum number of isolates. Hierarchical clustering and correlation analysis revealed associations between environmental and clinical isolates, indicating likely transmission and dissemination from the hospital environment to the patients. The whole genome sequencing of four highly drug resistant ExPEC isolates showed the presence of various antimicrobial resistance genes, virulence factors and mobile genetic elements, with isolates harbouring the plasmid incompatibility group IncF (FII, FIB, FIA). The sequenced isolates were identified as human pathogens with a 93.3% average score. This study suggests that ESBL producing E. coli are prevalent in the healthcare settings of Bangladesh, acting as a potential reservoir for AMR bacteria. This information may have a profound effect on treatment, and improvements in public healthcare policies are a necessity to combat the increased incidences of hospital-acquired infections in the country.

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

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

Sub-MIC streptomycin and tetracycline enhanced Staphylococcus aureus Guangzhou-SAU749 biofilm formation, an in-depth study on transcriptomics

Staphylococcus aureus is a major human pathogen, a potential “Super-bug” and a typical biofilm forming bacteria. With usage of large amount of antibiotics, the residual antibiotics in clinical settings further complicate the colonization, pathogenesis and resistance of S. aureus. This study aimed at investigating the phenotypical and global gene expression changes on biofilm formation of a clinical S. aureus isolate treated under different types of antibiotics. Firstly, an isolate Guangzhou-SAU749 was selected from a large sale of previously identified S. aureus isolates, which exhibited weak biofilm formation in terms of biomass and viability. Secondly, 9 commonly prescribed antibiotics for S. aureus infections treatment, together with 10 concentrations ranging from 1/128 to 4 minimum inhibitory concentration (MIC) with 2-fold serial dilution, were used as different antibiotic stress conditions. Then, biofilm formation of S. aureus Guangzhou-SAU749 at different stages including 8 h, 16 h, 24 h, and 48 h, was tested by crystal violet and MTS assays. Thirdly, the whole genome of S. aureus Guangzhou-SAU749 was investigated by genome sequencing on PacBio platform. Fourthly, since enhancement of biofilm formation occurred when treated with 1/2 MIC tetracycline (TCY) and 1/4 MIC streptomycin (STR) since 5 h, the relevant biofilm samples were selected and subjected to RNA-seq and bioinformatics analysis. Last, expression of two component system (TCS) and biofilm associated genes in 4 h, 8 h, 16 h, 24 h, and 48 h sub-MIC TCY and STR treated biofilm samples were performed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Although most antibiotics lowered the biomass and cell viability of Guangzhou-SAU749 biofilm at concentrations higher than MIC, certain antibiotics including TCY and STR promoted biofilm formation at sub-MICs. Additionally, upon genome sequencing, RNA-seq and RT-qPCR on biofilm samples treated with sub-MIC of TCY and STR at key time points, genes lytR, arlR, hssR, tagA, clfB, atlA and cidA related to TCS and biofilm formation were identified to contribute to the enhanced biofilm formation, providing a theoretical basis for further controlling on S. aureus biofilm formation.

Synergistic Effects of Gentamicin, Cefepime, and Ciprofloxacin on Biofilm of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen involved in number of hospital-acquired infections such as catheter-associated urinary tract infections, bacteremia, septicemia, skin infections, and ventilator-associated pneumoniae. Biofilm formation is an important trait implicated in chronic infections, such as cystic fibrosis and chronic pulmonary obstruction. We evaluated effects of gentamicin, cefepime, and ciprofloxacin on biofilm of P. aeruginosa. A total of 266 isolates were collected from the Armed Forces Institute of Pathology (AFIP). Antibiotic susceptibility was assessed by double disk synergy testing. ESBL and carbapenemase detection was performed by phenotypic testing. Molecular screening of the genes was done by PCR. Micro-dilution broth method was used to determine minimum inhibitory concentrations of antibiotics. Biofilm formation was done by micro-titer plate assay. Overall, 20% of the P. aeruginosa isolates were extensively drug-resistant (XDR-PA), and 25% were multi-drug-resistant (MDR-PA). Likewise, 43% of the isolates were ESBL producers, and carbapenemase production was detected in 40% of the isolates. Molecular analysis confirmed occurrence of different resistant factors in ESBL-positive isolates; 67% carried blaTEM, 62% blaCTXM-15, 41% blaSHV, 34% blaCTXM-14, and 33% blaOXA-1. In addition, 68% of the carbapenem-resistant isolates were positive for blaNDM-1, 25% for blaOXA-48, and 22% for blaKPC-2. Biofilm formation was assessed for 234 isolates, out of which 28% were strong biofilm formers. Moderate and weak biofilm formers constituted 46% and 23%, respectively. Overall, ciprofloxacin, levofloxacin, and cefepime showed inhibitory effects on P. aeruginosa biofilms. Antibiotics in combination showed strong synergistic effects (ciprofloxacin and cefepime), while gentamicin and cefepime resulted in complete eradication of P. aeruginosa biofilm. We confirm strong synergistic effects of gentamicin and cefepime that completely eradicated P. aeruginosa biofilm. We further confirm inhibitory effects of ciprofloxacin, levofloxacin, and cefepime on P. aeruginosa biofilms. Hence, combination therapy can be more effective against biofilm-associated infections.

Biofilm stimulating activity of solanidine and Solasodine in Pseudomonas aeruginosa

BackgroundBiofilm formation has reported as an important virulence associated properties of Pseudomonas aeruginosa that is regulated by quorum-sensing associated genes. Biofilm and quorum-sensing interfering properties of steroidal alkaloids, Solanidine and Solasodine were investigated in the present study.ResultsBiofilm formation capacity and relative expression level of five studied genes(lasI, lasR, rhlI, rhlR and algD) were significantly increased dose-dependently after treatment with sub-inhibitory concentrations (32 and 512 µg/ml) of the both Solanidine and Solasodine. Biofilm formation capacity was more stimulated in weak biofilm formers(9 iaolates) in comparison to the strong biofilm producers(11 isolates). The lasI gene was the most induced QS-associated gene among five investigated genes.ConclusionBiofilm inducing properties of the plants alkaloids and probably medicines derived from them has to be considered for revision of therapeutic guidelines. Investigating the biofilm stimulating properties of corticosteroids and other medicines that comes from plant alkaloids also strongly proposed.