Strong Biofilm Formation Research Articles

Investigation of Pseudomonas aeruginosa Biofilm Formation and Quorum Sensing Genes in Piperacillin/Tazobactam and Ciprofloxacin Sub-minimal Inhibitory Concentrations

Pseudomonas aeruginosa is a non-fermentative, oxidase-positive, motile gram-negative bacillus widespread in nature. The virulence factors of P.aeruginosa including the ability to grow under minimal growth conditions, the widespread presence in nature, and the ability to form biofilms make P.aeruginosa a highly important bacterium along with its resistance mechanisms against many antibiotics. The ability to form biofilms increases the symptom severity in diseases caused by P.aeruginosa and causes difficulties in the treatment. The aim of this study was to investigate the effects of sub-minimal inhibitory concentrations (sub-MIC) of piperacillin/tazobactam (TZP) and ciprofloxacin (CIP) which are used for the treatment of P.aeruginosa infections on biofilm formation and to investigate the relationship between the severity of biofilm formation and Quorum Sensing (QS) genes. The study included 24 P.aeruginosa isolates from the culture collection of Medical Microbiology Laboratory of Gazi University Faculty of Medicine. MIC values of TZP and CIP antibiotics were determined by the microdilution method. The biofilm layers in the antibiotic-free medium and in the sub-MIC (MIC/2, MIC/4 ve MIC/8) concentrations of antibiotics were visualized by using a scanning electron microscope (SEM). The QS genes (lasI, lasR, rhlI, and rhlR) of the 24 isolates with known biofilm characteristics were identified via the amplification of chromosomal DNA by using PCR method. In the study, it was foundthat both antibiotics reduced biofilm formation in a dose-dependent manner in sub-MIC concentrations compared to the antibiotic-free condition and that MIC/2 was the concentration, which reduced the biofilm formation most. These results were further confirmed by viewing the SEM images. The QS genes lasI, lasR, and rhlI were detected in a total of 19 isolates with moderately strong and strong biofilm formation, the rhlR gene was detected in six of the strong biofilm-forming isolates, in four of the moderately strong biofilm-forming isolates, and in three of the weak biofilm-forming isolates, respectively. The investigation of the effects of sub-MIC concentrations of antimicrobials, used for the treatment of P.aeruginosa infections, on the biofilm formation of P.aeruginosa and the investigation and better understanding of the QS systems associated with biofilm production will allow for finding out new treatment approaches and offer different options in combating infections with high morbidity and mortality.

Microbiological Analysis of Punch Biopsies of Tonsillar Crypts of Patients with Chronic Tonsillitis

Statement of the Problem: Medicine identified that recurrent and chronic infections of palatine tonsils are exacerbated by the creation of biofilms by microorganisms in the wet and warm folds of the tonsils which act as a repository of infection (Abu Bakar et al., 2018). Although 99% of all bacteria exist within biofilms, it is the planktonic state and swab samples from tonsillar surface that historically have received the attention. This is likely due to the fact that disease - causing bacteria is grown more rapidly in the planktonic form and it is much easier to sample fluids as opposed to surfaces (Snyder et al., 2009). Clinical Practice: At Pauls Stradiņs Clinical University Hospital was decided to microbiologically analyze the punch biopsy samples of tonsillar crypts of patients with chronic tonsillitis. Patients who met Paradise criteria were admitted to the Department of Otorhinolaryngology for tonsillectomies. Biopsies were taken before operations. Pathogenic bacteria of biopsy samples were tested for antibacterial susceptibility and biofilm producing ability. Results of the microbiological analysis revealed variety of pathogenic bacteria with resistance to common antibiotics un strong biofilm formation ability. Conclusion & Significance: Isolated pathogens from tonsillar crypts were resistant to antibiotics which are used to treat tonsillitis. Isolated pathogens from tonsillar crypts were weak, moderate or strong biofilm producers. Only 20 % of strains did not produce biofilm. During further studies the role of strong biofilm producers has to be evaluated as biofilm production plays a key role in the pathogenesis of chronic tonsillitis.

Pathogenic characteristics of Staphylococcus aureus isolates from arthroplasty infections.

Staphylococcus aureus has a great ability to form biofilms on implant-related biomaterials. This study aimed to investigate the resistance, biofilm and molecular characteristics of S. aureus strains isolated from patients with postoperative infections after arthroplasty in two Chinese tertiary care hospitals during 2017 to 2018. Antimicrobial susceptibility was determined by the agar dilution method. Bacterial biofilm formation was determined by crystal violet staining. The genes related to biofilm formation and molecular typing were analyzed by PCR amplification. A total of 33 isolates were collected, 21 of which were from Henan. The strains were completely sensitive to vancomycin, linezolid, and nitrofurantoin. All the isolates had adhesion ability and could produce biofilms. Of the isolates, 75.0% from Chongqing and 85.7% from Henan had stronger biofilm formation abilities. The strains from Henan had slightly higher resistance, adhesion and biofilm-forming abilities than those from Chongqing. The strains in both hospitals carried at least two genes related to biofilm formation, and the ica and fnb genes were the most frequently detected genes. Three SCCmec types and seven sequence types (STs) were found in Henan, and two SCCmec types and six STs were found in Chongqing. ST239-SCCmec III was the main epidemic clone in the two hospitals. The resistance phenotype and molecular characteristics of S. aureus strains varied in different hospitals. The results reflect the potential risks of S. aureus infection in postoperative arthroplasty patients. Our study provides a powerful basis for the clinical treatment, infection control and monitoring of outbreaks of epidemic strains.

Clinical and Molecular Epidemiology of Stenotrophomonas maltophilia in Pediatric Patients From a Chinese Teaching Hospital.

Objective: To study the molecular epidemiological characteristics of Stenotrophomonas maltophilia (SMA) isolated from patients in a pediatric teaching hospital in Shanghai so as to provide data for the prevention and treatment of SMA.Methods: Non-repetitive SMA strains were isolated from patients from January 2013 to December 2014. The cloning characteristics were analyzed using multilocus sequence typing (MLST) and pulsed field gel electrophoresis (PFGE), and the drug resistance was determined using the Kirby-Bauer disk method. Virulence genes and biofilm genes were detected using polymerase chain reaction (PCR). The biofilm forming ability was analyzed using the semi-quantitative biofilm formation test.Results: A total of 104 strains were collected, primarily from the pediatric intensive care unit and thoracic surgery, and these strains were isolated from sputum sources (n = 82). A majority of the patients were male (67/104), and the age range was between 6 days and 12 years old. A total of 95 patients had 1–3 baseline diseases. All of the patients had prior use of 1–4 antimicrobial agents. A total of 59 STs were detected using the MLST analysis, of which 45 were new. The sequence types of the SMA were scattered, with no trend in the clonal spread. The PFGE showed that the 104 strains could be divided into 93 clusters, with no obvious cluster aggregations. All of the strains were susceptible to levofloxacin, trimethoprim/sulfamethoxazole, and minocycline. The positive rates of the virulence genes stmPr1, stmPr2, smf-1, and smlt3773 locus were 98.1, 86.5, 100, and 91.3%, respectively. All of the strains had biofilm formation, and most of the strains had strong biofilm formation abilities. The positive rates of the three biofilm genes rmlA, spgM, and rpfF were 83.7, 100, and 45.2%, respectively. However, the point mutations of rmlA and spgM with strong biofilm formation abilities were significantly different from those with weak biofilm formation abilities.Conclusion: Most infected patients had prior use of antibiotics and underlying diseases, and the positive rate of the virulence gene was high. The strains were susceptible to three kinds of antibiotics and had strong biofilm formation abilities. The mutations of rmlA and spgM may be related to the biofilm formation ability, and no obvious clonal transmissions were found in the same clinical department.

Antimicrobial effect of spore-forming probiotics Bacillus laterosporus and Bacillus megaterium against Listeria monocytogenes.

There are several reports on the detection of Listeria monocytogenes in Iran, which has been identified as a major public health problem. In addition, due to drug resistance and strong biofilm formation of this pathogen, new approaches such as using probiotics are needed to combat this bacterium. The aim of this study was to investigate the effect of spore-forming probiotics on the expression of virulence genes hly, plc, inlA, and invasion of L. monocytogenes. Spore-forming probiotics were co-cultured with L. monocytogenes at different time points and following cell count, the expression level of virulence genes of L. monocytogenes was assessed by real-time PCR. To investigate the effect of the isolated strains on the invasiveness of L. monocytogenes at different time points, HT-29 cell line was used. Sporulated probiotics reduced the growth of L. monocytogenes in broth medium at different time periods. The number of L. monocytogenes co-cultured with Bacillus laterosporus decreased by 1, 3, 3, and 6 log at 2, 4, 8 and 24h after treatment, respectively. In addition, the expression of virulence genes was decreased at different time points and the expression of hly and plcA genes was more affected. The invasion rate of L. monocytogenes on HT-29 cells was decreased by 1 to 6 log at different time points. Both probiotic strains inhibited bacterial growth at different time points; however, more sensitive methods should be established for an accurate assessment.

In-vitro Investigation of Antibiotics Efficacy Against Uropathogenic Escherichia coli Biofilms and Antibiotic Induced Biofilm Formation at Sub-Minimum Inhibitory Concentration of Ciprofloxacin.

BackgroundCommunity-acquired urinary tract infections are associated with significant morbidity, and uropathogenic Escherichia coli (UPEC) alone causes 90% of urinary tract infections. This bacterium retains a diverse armament of virulence factors including fimbria, hemolysins, and siderophores production. In a post invasion scenario, formation of intracellular communities mimic biofilm-like characteristics and are linked to recurrent urinary tract infections. We investigated the effects of different frontline antibiotics on the formation, inhibition, and eradication of biofilms of virulent UPEC strains.Materials and MethodsA total of 155 UPEC strains were scrutinized for various virulence factors including gelatinase, cell surface hydrophobicity, hemagglutination, and serum bactericidal activity. Biofilm formation was confirmed by three different methods: Congo red agar, test tube, and tissue culture plate method. Biofilm inhibition and eradication assays were performed according to the standard protocols. Topographical analysis of biofilms was done by scanning electronic microscopy (SEM).ResultsOut of 155 strains, 113 (73%) were strong biofilm formesr, while 37 (24%) produced biofilms at moderate level. Significant differences were observed between MICs of planktonic cells (MIC-p) and MICs of UPEC biofilms (MIC-b). Among tested frontline antibiotics, levofloxacin successfully inhibited biofilms at a concentration of 32 µg/mL, while trimethoprim eradicated biofilms at higher concentrations (512–1024 µg/mL). Ciprofloxacin treatment at sub-MIC level significantly enhanced biofilm formation (P<0.05).ConclusionThe majority of UPEC strains are strong biofilm formers and show higher tolerance towards frontline antibiotics in biofilm form. We observed significant inhibitory effects of levofloxacin (32 µg/mL) on UPEC biofilms, while treatment with sub-minimal concentrations of ciprofloxacin significantly enhanced biofilm formation. Out of all tested antibiotics, trimethoprim (512–1024 µg/mL) eradicated UPEC biofilms.

Phenotypic characterization of Staphylococcus aureus isolated from foods of animal origin and other related sources

The present study aimed to phenotypically characterize S. aureus isolates originated from bovine raw milk and different steps of the pig slaughterhouse flowchart to evaluate the biofilm formation ability of these bacterial isolates, the factors that may influence this ability, the behavior of these bacterial pathogens in the food environment, and the sensitivity of these microorganisms to methicillin. In this survey, 28 S. aureus isolates were analyzed, seven of which originated from bovine raw milk, and 21 from swine samples. Biofilm production ability was observed in 57.1% (16/28) of the isolates tested corresponding to 85.7% (6/7) of bovine milk samples and 47.6% (10/21) of pig samples. Most of the isolates previously classified as strong biofilm formers maintained this ability after being exposed to different types of sublethal stress. Regarding the sensitivity of the isolates to methicillin, 42.9% (12/28) were resistant. Practical applications The practical applications are the phenotypic characterization of S. aureus, verification of biofilm formation by gifts present in the food industry, and of antimicrobial resistance of isolates.

Outbreak of KPC-2 Carbapenem-resistant Klebsiella pneumoniae ST76 and Carbapenem-resistant K2 Hypervirulent Klebsiella pneumoniae ST375 strains in Northeast China: molecular and virulent characteristics

BackgroundCarbapenem-resistant hypervirulent Klebsiella pneumoniae strains have recently come into existence worldwide; however, researchers in northeast China are not aware of their clinical features and molecular characteristics.MethodsHere, the molecular and virulent characteristics of 44 carbapenem-resistant K. pneumoniae (CRKP) isolates collected from January 2015 to December 2017 were studied. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were carried out to define the clonal relatedness among the isolates. PCR and capsular serotyping of the virulence-associated genes, as well as biofilm formation and serum complement-mediated killing assays, were employed to determine the virulent potential. The genomic features and associated mobile genetic elements of JmsCRE57 were detected by whole genome sequencing.ResultsThe only positive isolate was JmsCRE57, which belonged to the ST375 serotype K2 that expressed uge, mrkD, fimH, kpn, aerobactin and rmpA virulence-associated genes and showed strong biofilm formation and serum sensitivity. Sequencing results showed that the JmsCRE57 genome mainly consisted of a circular chromosome, three antimicrobial resistant plasmids and a virulent plasmid. The antimicrobial resistant plasmid expressing blaKPC-2, blaCTX-M-15, aph(3″)-Ib, aph(6)-Id, qnrB1, aac(3)-IIa, aac(6′)-Ib-cr, blaOXA-1, blaTEM-1B, catB4, sul2, dfrA14 and blaSHV-99. The virulent plasmid belonged to the IncHI1B group, which is mainly composed of mucoid phenotype genes and siderophore-associated genes. The remaining CRKP strains that expressed uge, fimH, mrkD and kpn virulence-associated genes were not successfully typed.ConclusionOur results provide new insights on the epidemiology of carbapenem-resistant K2 hypervirulent K. pneumoniae ST375 and CRKP ST76 strains in northeast China, which may help control their future outbreaks.

Molecular Characterization of Carbapenem-Resistant Acinetobacter baumannii Associated with Nosocomial Infection in the Pelotas, RS, Brazil

Acinetobacter calcoaceticus-Acinetobacter baumannii complex (ACB) comprises some opportunistic pathogens associated with infectious outbreaks in hospital settings. A. baumannii is the most relevant species owing to its capacity to develop resistance to the different classes of antimicrobials. The aim of this study was to identify the species, establish the genetic patterns, resistance and biofilm profiles in ACB isolates associated with nosocomial infection in a hospital of Pelotas, Rio Grande do Sul, Brazil. Twenty-two clinical isolates were characterized at the species level through multiplex polymerase chain reaction (PCR) for the gyrB and blaOXA51-like genes, and the genetic relationship was determined through pulsed-field gel electrophoresis (PFGE). Their antibiotic resistance profiles and carbapenemases synthesis were evaluated following CLSI guidelines. PCR was carried out to evaluate the presence of carbapenemases genes and the isolates were classified for their biofilm-forming ability. All isolates obtained in the study were identified as A. baumannii and 72.7% of the isolates were classified as strong biofilm formers. In the class carbapenems, 95.4% and 77.3% of the isolates were resistant to meropenem and imipenem, respectively. The blaVIM gene was identified in 90.9% of isolates and carbapenemases synthesis were confirmed in 95.4% of the isolates. Fourteen genetic patterns were confirmed through PFGE analyses. The isolates collected within a time gap of 2 years demonstrated a genetic relationship, and the same clone was identified in different departments in the hospital. To the best of our knowledge, this is the first report of identification and characterization of A. baumannii nosocomial isolates in Pelotas, RS, Brazil.

Hindering of Cariogenic Streptococcus mutans Biofilm by Fatty Acid Array Derived from an Endophytic Arthrographis kalrae Strain.

Streptococcus mutans has been considered as the major etiological agent of dental caries, mostly due to its arsenal of virulence factors, including strong biofilm formation, exopolysaccharides production, and high acid production. Here, we present the antivirulence activity of fatty acids derived from the endophytic fungus Arthrographis kalrae isolated from Coriandrum sativum against Streptococcus mutans. The chemical composition of the fatty acids was analyzed by gas chromatography–mass spectrometry GC-MS and revealed nine compounds representing 99.6% of fatty acids, where unsaturated and saturated fatty acids formed 93.8% and 5.8 % respectively. Oleic and linoleic acids were the major unsaturated fatty acids. Noteworthy, the fatty acids at the concentration of 31.3 mg L–1 completely inhibited Streptococcus mutans biofilm, and water insoluble extracellular polysaccharide production in both polystyrene plates, and tooth model assay using saliva-coated hydroxyapatite discs. Inhibition of biofilm correlated significantly and positively with the inhibition of water insoluble extracellular polysaccharide (R = 1, p < 0.0001). Furthermore, Arthrographis kalrae fatty acids at a concentration of 7.8 mg L–1 exhibited acidogenesis-mitigation activity. They did not show bactericidal activity against Streptococcus mutans and cytotoxic activity against human oral fibroblast cells at the concentration used. On the other hand, saliva-coated hydroxyapatite discs treated with sub-minimum biofilm inhibitory concentration of fatty acids showed disturbed biofilm architecture with a few unequally distributed clumped matrices using fluorescence microscopy. Our findings revealed that the intracellular fatty acid arrays derived from endophytic Arthrographis kalrae could contribute to the biofilm-preventing alternatives, specifically Streptococcus mutans biofilms.

Antibiofilm Activity of a Broad-Range Recombinant Endolysin LysECD7: In Vitro and In Vivo Study.

Surfaces of implanted medical devices are highly susceptible to biofilm formation. Bacteria in biofilms are embedded in a self-produced extracellular matrix that inhibits the penetration of antibiotics and significantly contributes to the mechanical stability of the colonizing community which leads to an increase in morbidity and mortality rate in clinical settings. Therefore, new antibiofilm approaches and substances are urgently needed. In this paper, we test the efficacy of a broad-range recombinant endolysin of the coliphage LysECD7 against forming and mature biofilms. We used a strong biofilm producer—Klebsiella pneumoniae Ts 141-14 clinical isolate. In vitro investigation of the antibacterial activity was performed using the standard biofilm assay in microtiter plates. We optimized the implantable diffusion chamber approach in order to reach strong biofilm formation in vivo avoiding severe consequences of the pathogen for the animals and to obtain a well-reproducible model of implant-associated infection. Endolysin LysECD7 significantly reduced the biofilm formation and was capable of degrading the preformed biofilm in vitro. The animal trials on the preformed biofilms confirmed these results. Overall, our results show that LysECD7 is a promising substance against clinically relevant biofilms.

Synergistic effects of pomegranate and rosemary extracts in combination with antibiotics against antibiotic resistance and biofilm formation of Pseudomonas aeruginosa.

The combination of plant extract and antibiotic represents a template for developing of antibiofilm drugs. This study investigated the synergistic effects of pomegranate/rosemary/antibiotic combinations against antibiotic resistance and biofilm formation of Pseudomonas aeruginosa. The results showed that 17 (85%) of total P. aeruginosa isolates were biofilm producers; however, 5 (25%) isolates were demonstrated as a strong biofilm producer. The highest MIC level (1024μg/ml) of tested antibiotics against strong biofilm producer isolates was observed with piperacillin, however the MIC ranges of ceftazidime, gentamycin, imipenem, and levofloxacin against these isolates were reached to (256-1024μg/ml), (32-1024μg/ml), (8-1024μg/ml), and (8-512μg/ml), respectively. PS-1 was the representative isolate for strong biofilm formation and high antibiotic resistance. 16S rRNA gene analysis suggested that PS-1 (accession No. MN619678) was identified as a strain of P. aeruginosa POA1. Pomegranate and rosemary extracts were the most effective extracts in biofilm inhibition, which significantly inhibited 91.93 and 90.83% of PS-1 biofilm, respectively. Notably, the synergism between both plant extracts and antibiotics has significantly reduced the MICs of used antibiotics at the level lower than the susceptibility breakpoints. Pomegranate/rosemary/antibiotic combinations achieved the highest biofilm eradication, which ranging from 90.0 to 99.6%, followed by the eradication ranges of pomegranate/rosemary combination, rosemary, and pomegranate extracts, which reached to (76.5-85.4%), (53.1-73.7%), and (41.2-71.5%), respectively. The findings suggest that pomegranate/rosemary/antibiotic combinations may be an effective therapeutic agent for antibiotic resistance and biofilm formation of P. aeruginosa.

Genotyping Diversity of Echerichia coli isolated from UTI in Iraqi Patients

Background Escherichia coli (E. coli) is the predominant uropathogen isolated in acute, community-acquired uncomplicated UTIs in adults and children. Pateints and Methods: A total of (150) clinical samples (Urine) where collected from the patients at admission to the hospital in a sterile container. A loopful of uncentrifuged urine samples was cultured onto blood agar, MacConkey agar, HiCrome UTI Agar and EMB media,the diagnosis of E.coli bacteria was confirmed by using VITEK 2 system. Antimicrobial susceptibility testing of E. coli isolates was carried out against the antibiotics by Disc Diffusion Method ,ERIC PCR used for determined genotyping diversity. Results: E.coli showed antibiotic resistant to Garamycine (86%), Ciproflaxcin (66%), Ceftriaxone (46%) and Rifampcine (100%). The biofilm formation result showed out of 50 E. coli isolate (12%) were weak biofilm formation, (72%) were moderate biofilm formation and (16%) were strong biofilm formation. Genotyping by ERIC-PCR (Enterobacterial Repetitive Intergenic Consensus) of 50 E.coli isolates subjected to ERIC-PCR, 47 isolates have amplications ranging from 700 to < 3000bp The generated banding profile showed highly polymorphic DNA fragments in these 47 isolates Conclusion : : E.coli resistance to Garamycine (86%), Ciproflaxcin (66%), Ceftriaxone (46%) and Rifampcine (100%). The biofilm formation result 50 E. coli isolate (12%) were weak biofilm formation, (72%) were moderate biofilm formation. Genotyping by ERIC-PCR (Enterobacterial Repetitive Intergenic Consensus) of E.coli isolates subjected to ERIC-PCR, 47 isolates have amplications ranging from 700 to < 3000bp The generated banding profile was highly polymorphic DNA fragments in these isolates.

Genotypic and Phenotypic Evaluation of Biofilm Production and Antimicrobial Resistance in Staphylococcus aureus Isolated from Milk, North West Province, South Africa.

Background: Biofilm formation in S. aureus may reduce the rate of penetration of antibiotics, thereby complicating treatment of infections caused by these bacteria. The aim of this study was to correlate biofilm-forming potentials, antimicrobial resistance, and genes in S. aureus isolates. Methods: A total of 64 milk samples were analysed, and 77 S. aureus were isolated. Results: Seventy (90.9%) isolates were biofilm producers. The ica biofilm-forming genes were detected among 75.3% of the isolates, with icaA being the most prevalent (49, 63.6%). The icaB gene was significantly (P = 0.027) higher in isolates with strong biofilm formation potentials. High resistance (60%–90%) of the isolates was observed against ceftriaxone, vancomycin, and penicillin, and 25 (32.5%) of S. aureus showed multidrug resistance (MDR) to at least three antibiotics. Five resistance genes, namely blaZ (29, 37.7%), vanC (29, 37.7%), tetK (24, 31.2%), tetL (21, 27.3%), and msrA/B (16, 20.8%) were detected. Most MDR phenotypes possessed at least one resistance gene alongside the biofilm genes. However, no distinct pattern was identified among the resistance and biofilm phenotypes. Conclusions: The high frequency of potentially pathogenic MDR S. aureus in milk samples intended for human consumption, demonstrates the public health relevance of this pathogen in the region.

Colistin Susceptibility in Carbapenem Resistant Klebsiella Pneumoniae and their Ability of Biofilm Formation

A total of 157 clinical samples were collected from different clinical specimens (urine, sputum, blood, swabs, and cannula) from several hospitals in Iraq. Among the samples, 51 isolates (32.48%) of Klebsiella pneumoniae were identified according to morphologicaland cultural characteristics as well as the Enterosystem 18R test. Higher numbers of K. pneumoniae isolates were observed in urine samples (26, 52%) than the other samples, and in females (70.6%) than males (29.4%) (female: male ratio of about 2.4:1). Antibiotic susceptibility of K. pneumoniae against 12 commonly used antibiotics was determined through the disc-diffusion method. The results revealed a higher resistance rate in 51 isolates (100%) against Cephalexin, followed by Ceftazidime (50, 98%), while the lowest resistance rate (24, 47%) was against each of Imipenem and Meropenem. Also, the investigation of the minimum inhibitory concentration (MIC) of Colistin using E-test (strips) demonstrated that 33 isolates were resistance, as compared to 31 using the disk diffusion assay. DNA was extracted from K. pneumoniae isolates and molecularly tested using polymerase chain technique (PCR) with a specific primer and 108 bp product to detect the rpoB gene that represents this bacteria . Also, all of the 51 isolates of K. pneumoniae identified by the rpoB gene were detected for the expression of the Colistin drug resistance gene mgr-B , which was amplified (347 bp) using a specific primer. Colistin resistance gene mgr-B was amplified and sequenced from the twenty isolates. Only 6 isolates appeared with a single nucleotide substitution; G instead A, A instead G, C instead G and G instead C. Also, this study tested biofilm formation from K. pneumoniae isolates , using the microtiter plate method, in association with Colistin and Carbapenem resistant. The Colistin and Carbapenem resistance pattern was compared to the ability of biofilm-formation as weak formation versus strong and also, Multi-drug resistant isolates were more common among weak versus strong biofilm formers.

Robust approach leading to novel densely functionalized four-cyclic benzo[e]pyrazolo[5′,1′:2,3]pyrimido[4,5-b][1,4]diazepines with antibacterial activity toward resistant strains

A straightforward approach for the regioselective synthesis of various derivatives of benzo[e]pyrazolo[5′,1′:2,3]pyrimido[4,5-b] [1, 4] diazepine as a novel heterocyclic system from the annulation of 6-bromo-7-chloro-3-cyano-2-(ethylthio)-5-methylpyrazolo[1,5-a]pyrimidine with several 2-amino-N-substituted benzamides as bident nucleophiles in the presence of K2CO3 and DMF has been disclosed. The right regioisomer was elucidated by 2D-NOESY NMR spectroscopy, as well. Most of the novel synthetic compounds exhibited antimicrobial activity though relatively at high concentrations against multi-drug-resistant Klebsiella pneumoniae and Escherichia coli clinical isolates possessed of strong biofilm formation ability. N-Cyclohexyl-substituted pyrazolopyrimidobenzodiazepine (3 h) as the most potent compound represented 100% growth inhibitory on both types of bacterial strains, which brings promises for further changes to develop potential antimicrobial drugs.

Rapid biodegradation and biofilm-mediated bioremoval of organophosphorus pesticides using an indigenous Kosakonia oryzae strain -VITPSCQ3 in a Vertical-flow Packed Bed Biofilm Bioreactor.

The widespread use of pesticides has been one of the major anthropogenic sources of environmental pollution. Organophosphorus (OP) pesticides are predominantly used in agriculture due to their broad-spectrum insecticidal activity and chemical stability. The study was focused on the biodegradation of OP pesticides, Profenofos (PF) and Quinalphos (QP) in culture media using bacterium isolated from wetland paddy rhizosphere. The strain VITPSCQ3 showed higher pesticide tolerance, efficient biofilm formation and was capable of synthesizing organophosphate degrading enzymes. Based on the 16S rRNA gene sequencing the isolate exhibited maximum sequence similarity with Kosakinia oryzae (GenBank accession number: KR149275). Biodegradation assay with various concentrations of PF and QP (200, 400, 600 and 800mgL-1) showed maximum degradation up to 82% and 92% within 48h. The kinetic studies revealed the biodegradation rates (k) to be 0.0844 min-1 and 0.107 min-1 with half-lives (h) of 18h and 14.8h for PF and QP. The degradation products were identified by GCMS and possible degradation pathways were proposed using Insilico techniques. To the best of our knowledge, this is the first report on the biodegradation of PF and QP using Kosakonia oryzae. Bioremoval of PF and QP from aqueous solution was performed using the biofilm of VITPSCQ3 developed on selected substrates in a circulating Vertical-flow packed-bed biofilm (VFPBB) bioreactor. Charcoal, gravel and mushroom (Agaricus bisporus) were used as biofilm carriers. Mushroom showed strong biofilm formation with optimum biodegradation capacity of up to 96% for PF and 92% for QP within 120min reaction time.

Assessment of Biofilm Formation among the Clinical Isolates of Escherichia coli in a Tertiary Care Hospital

Aims: Identification and grading of the Escherichia coli according to their biofilm production capability.&#x0D; Study Design: Cross-sectional study.&#x0D; Place and Duration of Study: This was conducted in Department Microbiology at M.S. Ramaiah Medical college and Hospital, Bengaluru from March 2017 to August 2017.&#x0D; Methodology: A total of 55 non repetitive Escherichia coli isolates were identified from various clinical samples like urine, pus ,tissue and peritoneal fluids .All the organisms were isolated in pure culture and biofilm formation was detected in vitro by Gold standard TCP (Tissue culture plate) method. Organisms were incubated for an extended period of 48 hours and the biofilms were detected by acetone alcohol elution method. Organisms were categorized as strong, moderate, weak and no biofilm producers based on the obtained OD value of the elute.&#x0D; Results: Majority of the isolates of Escherichia coli were obtained from catheterized urine culture (67.03%) followed by pus (25.50%).Most of the isolates were capable of forming biofilm in vitro by tissue culture plate method except a few (9.1%). 40% of the isolates were strong biofilm formers which had &gt;4 ODC. 25.5% showed medium biofilm-forming capability and rest 25.5% showed weak biofilm formations in vitro.&#x0D; Conclusion: The ability to form biofilm from a species can give us a better understanding of the biofilm-related infections pertaining to the particular group. Detection of biofilms remains a most important determinant to approximate the incidence of such infections. Categorization of organisms according to their biofilm formation may help us understand the frequency of biofilm-associated infections, and thus take necessary precautions to avoid the problem. Further studies involving the detection of biofilm may be conducted and the tests can be implemented in routine diagnostic microbiology to assess the usefulness of the methods in detection of biofilm-related infections.

Type VI secretion system of Pseudomonas aeruginosa is associated with biofilm formation but not environmental adaptation.

Pseudomonas aeruginosa encodes three type VI secretion systems (T6SSs), namely H1‐, H2‐, and H3‐T6SS. P. aeruginosa hemolysin‐coregulated protein (Hcp) is the effector protein and the hallmark of T6SS. Although T6SS is ubiquitous and affects ecology and human health, its general mechanism and physiological role are still not fully understood. Therefore, in this study, we investigated the impact of the P. aeruginosa T6SS on biofilm formation and environmental adaptation. To this end, we collected P. aeruginosa clinical isolates, divided them into strong biofilm formation (SBF) and nonbiofilm formation (NBF) groups based on their biofilm‐forming ability, and compared their associated clinical characteristics. The duration of hospitalization was longer in patients infected with SBF than those infected with NBF strains. The expression levels of T6SS‐related genes (hcp1 and hcp3) and a quorum‐sensing gene (lasR) were higher in the SBF group as compared to those in the NBF group. In addition, the expression level of lasR was negatively associated with that of hcp1, but was positively associated with those of hcp2 and hcp3. Moreover, we evaluated the expression of T6SS‐ and biofilm‐associated genes in planktonic and biofilm cells of the P. aeruginosa strain PAO1, and constructed strain PAO1△clpV1 to study the adaptation characteristics of H1‐T6SS. The expression levels of hcp1, hcp2, hcp3, lasR, and other biofilm‐associated genes were significantly higher in PAO1 biofilm cells as compared to those of planktonic cells. However, except for swarming ability as a vital feature for biofilm formation, there were no significant differences in the biofilm‐forming ability and expression of biofilm‐associated genes, adherence ability, growth characteristics, resistance to acid and osmotic pressure, surface structure, and morphology between the PAO1△clpV1 and PAO1 wild‐type strains. Collectively, our results suggest that T6SS might play a role in biofilm formation and that H1‐T6SS does not contribute to environmental adaptation in P. aeruginosa.

Prevalence of multidrug-resistant Staphylococcus aureus isolates with strong biofilm formation ability among animal-based food in Shanghai

Antimicrobial resistance has gradually become a serious problem threatening public health and food safety throughout the world. Biofilm is one of the important factors affecting the antimicrobial resistance of bacteria. Staphylococcus aureus usually has strong biofilm formation ability, and it is widely found in animal-based food. The purpose of this study is to determine the prevalence, antimicrobial resistance and biofilm formation of S. aureus in animal-based food. Total 959 samples representing eight types of animal-based foods were collected from randomly selected locations (21 supermarkets and 18 wet markets) throughout the Shanghai city. The overall isolation rate of S. aureus was 17.2% (165/959). For each food category, the isolation rate was 21.8% for chicken (45/206), 21.5% for pork (71/331), 15.2% for beef (16/105), 13.8% for duck (9/65), 12.1% for aquatic products (17/141), 8.6% for egg (5/58), and 7.1% for lamb (2/28), respectively. No isolate was found from pasteurized milk (n = 25). Antimicrobial susceptibility test showed that among all the S. aureus isolates, 90.3% were resistant to at least one antimicrobial, 39.4% were multi-drug resistant, and 23 isolates were methicillin-resistant S. aureus (MRSA). Comparing the resistance rates to different antimicrobials, S. aureus had the highest resistance rate to penicillin, up to 82.4% (136/165); followed by erythromycin (57.6%, 95/165) and tetracycline (27.9%, 46/165). All isolates were sensitive to vancomycin. With the microtiter plate and crystal violet staining assay, 64.8% of all the 165 isolates had strong biofilm formation ability and 20.0% were moderate producers. Remarkably, significant difference was found in biofilm formation ability between those isolates from supermarkets and wet markets (p < 0.01). According to WGS analysis of 19 multi-drug resistant isolates with strong biofilm formation ability, ST7 was the dominant sequence type. Combined analysis showed that S. aureus isolates with drug resistance phenotypes usually had stronger ability to form biofilm.