Congo Red Agar Plate Research Articles

Identification and detection of pathogenic bacteria from patients with hospital-acquired pneumonia in southwestern Iran; evaluation of biofilm production and molecular typing of bacterial isolates

BackgroundHospital-acquired pneumonia (HAP) is the second most common nosocomial infection in intensive care units (ICUs). The present study aims to determine the prevalence of pathogenic bacteria, their biofilm formation, and molecular typing from patients with HAP in southwestern Iran.MethodsFifty-eight patients with HAP participated in this cross-sectional study. Sputum and endotracheal aspirate were collected from each patient for isolation and detection of bacteria. Biofilm formation was evaluated using Congo red agar or Microtiter plate assay. The antimicrobial susceptibility patterns of the isolates were investigated. The multiplex polymerase chain reaction (M-PCR) technique was used to determine the Staphylococcal Cassette Chromosome mec (SCCmec) types of methicillin-resistant Staphylococcus aureus (MRSA) strains. All S. aureus isolates were typed using the agr typing method. A repetitive element sequence-based PCR (rep-PCR) typing method was used for typing of Gram-negative bacteria. Data were analyzed using the Statistical Package for the Social Sciences (SPSS) software version 15 and the chi-square test.ResultsBacteria were isolated in 52 (89.7%) of patients. Acinetobacter baumannii (A. baumannii) was the most prevalent organism (37%), followed by S. aureus, Pseudomonas aeruginosa (P. aeruginosa), and Escherichia coli (E. coli). Using the PCR method, 56 bacteria were detected. A. baumannii was the most prevalent (35.7%) organism. A. baumannii and P. aeruginosa were biofilm-producing. All Gram-negative isolates were colistin-sensitive, and most of the A. baumannii isolates were multidrug-resistant (MDR). MRSA was identified in 12 (80%) S. aureus isolates, and 91.6% of MRSA were SCCmec type III. The agr type III was the most predominant. The rep-PCR analysis showed seven different patterns in 20 A. baumannii, six patterns in 13 P. aeruginosa, and four patterns in 6 E. coli.ConclusionA. baumannii was more prevalent than S. aureus in ventilator-associated pneumonia (VAP), while S. aureus is a major pathogen in non-ventilator hospital-acquired pneumonia (NV-HAP), possibly due to the tendency of the former to aquatic environments. Based on the rep-PCR typing method, it was concluded that bacteria were transmitted from patients or healthcare workers among different wards. Colistin can be used as a treatment in Gram-negative MDR isolates.

Antibiofilm activity of silver nanoparticles against biofilm forming Staphylococcus pseudintermedius isolated from dogs with otitis externa.

BackgroundSilver nanoparticles (AgNPs) are known to possess antimicrobial properties. Although the antibiofilm activity of AgNPs has been demonstrated in humans, this activity has not yet been elucidated in veterinary medicine.ObjectivesThe purpose of this study was to evaluate the antibiofilm activity of silver nanoparticles against Staphylococcus pseudintermedius.MethodsTen isolates of S. pseudintermedius obtained from dogs with otitis externa were treated with AgNPs, and the antibiofilm activity was measured using a modified microtiter plate and Congo red agar (CRA) method and scanning electron microscopy.ResultsAgNPs displayed a significant dose‐dependent antibiofilm activity and reduced biofilm formation at concentrations of 20 and 10 µg/ml (p < 0.05). S. pseudintermedius exposed to 20 µg/ml of AgNPs formed less bacterial slime compared to the controls on CRA plates. Scanning electron micrographs showed that the biofilm had few individually scattered cells along its surface when treated with AgNP concentrations of 20 and 10 µg/ml. Untreated surfaces showed an aggregated biofilm.ConclusionsOur results suggested that AgNP may be a valuable alternative antibiofilm agent for canine otitis externa.

Quercetin mediated inhibition of Staphylococcus aureus biofilms and the impact of the isolate phenotype and genotype

Aim: This study was designed to assess the antibiofilm activity of quercetin on characterized S. aureus isolates. Methodology: This study evaluated 36 S. aureus isolates, each of which was identified using Gram staining, culture, biochemical, and PCR assays. Isolates were cultured and their biofilm production was evaluated using Congo red agar (CRA) plates, microtiter plate tests and PCR, and the effects of quercetin were examined. Results: The CRA results revealed that eight (22.3%) S. aureus isolates were strongly positive for biofilm production and an additional 18 isolates (50%) showed moderate biofilm capacity. The remaining 10 isolates were negative (27.7%) for biofilm production. S. aureus isolates were divided into strong positive, intermediate, and negative groups, 27.8%, 44.5%, and 27.7%, respectively. Scanning electron microscopy showed that the biofilm-producing isolates appeared as aggregates of cells within a heavy matrix. In addition, PCR assay identified IcaA and IcaD (66.6% for both) biofilm production genes in most isolates and IcaC (61.1%), IcaB, FnbB (33.3% for both), and Fib (22.2%) in several other strains. Quercetin significantly inhibited biofilm activity in biofilm producing S. aureus isolates in a dose-dependent manner, with an inhibition rate of 29.6-87.7%. Interpretation: Biofilm production is dependent on Ica gene phenotype and strains with an IcaABCD or IcaABD phenotype produce more biofilm than strains with IcaAD phenotype. Quercetin significantly inhibited S. aureus biofilm production, irrespective of Ica phenotype.

Correlation Between Biofilm-Formation and the Antibiotic Resistant Phenotype in Staphylococcus aureus Isolates: A Laboratory-Based Study in Hungary and a Review of the Literature.

IntroductionStaphylococcus aureus (S. aureus) is an important causative pathogen in human infections. The production of biofilms by bacteria is an important factor, leading to treatment failures. There has been significant interest in assessing the possible relationship between the multidrug-resistant (MDR) status and the biofilm-producer phenotype in bacteria. The aim of our present study was to assess the biofilm-production rates in clinical methicillin-susceptible S. aureus [MSSA] and methicillin-resistant S. aureus [MRSA] isolates from Hungarian hospitals and the correlation between resistance characteristics and their biofilm-forming capacity.MethodsA total of three hundred (n=300) S. aureus isolates (corresponding to MSSA and MRSA isolates in equal measure) were included in this study. Identification of the isolates was carried out using the VITEK 2 ID/AST system and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Antimicrobial susceptibility testing was performed using the Kirby–Bauer disk diffusion method and E-tests, confirmation of MRSA status was carried out using PBP2a agglutination assay. Biofilm-production was assessed using the crystal violet (CV) tube-adherence method and the Congo red agar (CRA) plate method.ResultsThere were significant differences among MSSA and MRSA isolates regarding susceptibility-levels to commonly used antibiotics (in case of erythromycin, clindamycin and ciprofloxacin: p<0.001, gentamicin: p=0.023, sulfamethoxazole/trimethoprim: p=0.027, rifampin: p=0.037). In the CV tube adherence-assay, 37% (n=56) of MSSA and 39% (n=58) of MRSA isolates were positive for biofilm-production, while during the use of CRA plates, 41% (n=61) of MSSA and 44% (n=66) of MRSA were positive; no associations were found between methicillin-resistance and biofilm-production. On the other hand, erythromycin, clindamycin and rifampin resistance was associated with biofilm-positivity (p=0.004, p<0.001 and p<0.001, respectively). Biofilm-positive isolates were most common from catheter-associated infections.DiscussionOur study emphasizes the need for additional experiments to assess the role biofilms have in the pathogenesis of implant-associated and chronic S. aureus infections.

The Effect of Antimicrobial Resistance Plasmids Carrying blaCMY-2 on Biofilm Formation by Escherichia coli from the Broiler Production Chain.

Extended-spectrum cephalosporin-resistant Escherichia coli (ESCR E. coli) with plasmids carrying the blaCMY-2 resistance gene have been isolated from the Norwegian broiler production chain through the Norwegian monitoring program for antimicrobial resistance in animals, food and feed, NORM-VET. The aim of the present study was to investigate the biofilm forming abilities of these strains, and in particular to see whether these might be influenced by the carriage of blaCMY-2 plasmids. The ESCR E. coli from the broiler production chain displayed relatively low biofilm forming abilities in the crystal violet biofilm assay as compared to quinolone-resistant E. coli (QREC) from the same population (mean ± SD = 0.686 ± 0.686 vs. 1.439 ± 0.933, respectively). Acquisition of two different blaCMY-2 plasmids by QREC strains reduced their biofilm production in microtiter plates, but not their biofilm production on Congo Red agar plates. Furthermore, motility was reduced, but not planktonic growth. We hypothesize that genes carried by these plasmids may have caused the observed reduction in biofilm formation, possibly mediated through changes in flagellar expression or function. Furthermore, this may help explain the different biofilm forming abilities observed between ESCR E. coli and QREC. The results also indicate that the risk of biofilm reservoirs of antimicrobial resistant E. coli on in the broiler production is lower for ESCR E. coli than for QREC.

In-Vitro Evaluation of Biofilm and Hemolysis activity of Candida albicans Isolated from Oral Cavity

Candida albicans is a member of the healthy human microflora, colonizing several niches in the body and can cause opportunistic infection under host debilitated and immunocompromised condition. The present study aimed to investigate the in-vitro hemolytic activity of C. albicans isolated from oral cavity and screen biofilm through three different methods. During the study, 200 oral rinse samples from general human population were analyzed in microbiology laboratory of Central Campus of Technology, Tribhuvan University, Hattisar, Dharan. Nepal. Candida albicans were isolated and identified by conventional microbiological procedures. The hemolytic activity was evaluated through two different Sabouraud dextrose broth media (SDB) containing 7% defibrinated human blood, one supplemented with 3% glucose (SDBwG) and the other without glucose (SDBwoG). The biofilm formation was screened through congo red agar, tube method and tissue culture plate method. In this present study, 42 (21%) isolates of Candida albicans were isolated from 200 oral rinse samples. Isolated Candida albicans exhibited mean hemolysis activity of 28.66% on human blood SDB without glucose and 43.55% on human blood SDB with 3% glucose. Tissue culture plate method was considered sensitive, specific and accurate method for quantitative screening of biofilm in comparison to tube method and congo red agar method. This research concluded that Candida albicans exhibited greater hemolytic activity in human blood with glucose (SDBwG) than in human blood without glucose (SDBwoG). This finding explains that an increased blood glucose concentration may contribute to increased hemolysis activity of Candida albicans that could play pathogenic role for inducing infection like oral candidiasis in debilitated host like diabetic patients. Tissue culture plate method can accurately screen biofilms than tube and congo red agar method.&#x0D; Int. J. Appl. Sci. Biotechnol. Vol 8(4): 394-399

Detection and Control of Biofilm Formation by Staphylococcus aureus from Febrile Neutropenic Patient.

IntroductionFebrile neutropenia (FN) is the evolution of fever in a patient with neutropenia over 38.0°C. Neutropenia is diagnosed when absolute neutrophil count (ANC) <1500 cells/µL. FN represents a common complication of cancer treatment. Hence, it is featured to be a major cause of morbidity and mortality in cancer patients. Staphylococcus aureus is one of the most important microorganisms isolated from the blood of febrile neutropenic patients. Infections caused by S. aureus range from mild to life-threatening diseases. Biofilm production by S. aureus is one of the most significant virulence factors of the bacterium as it prevents the penetration of antibiotics. Recently, it has been shown that S. aureus carries the ica operon responsible for biofilm production. The aim of the work is to determine a genotypic characterization that includes not only the detection of icaA and icaD genes in S. aureus but also the determination of their relation to clinical and microbiological features. Empiric antibacterial treatment was recommended for cancer patients receiving chemotherapy.Materials and MethodsThe relation between the presence of icaA and icaD and biofilm production was determined in a collection of 66 S. aureus samples from febrile neutropenic patients. Biofilm-forming ability was tested on Congo Red agar plates. Also, the effect of the most sensitive antibiotics on the bacterial cells was determined by an electron microscope.ResultsOf the bacterial samples, 48 were biofilm-productive and 18 were non-biofilm productive. For the biofilm productive bacteria, 37.5% were positive for icaA, 22.9% were positive for icaD and 10.4% were positive for both. Linezolid was the most effective antibiotic and it is highly recommended for the treatment of febrile neutropenia caused by biofilm-productive S. aureus. Severe changes were found on the bacterial cell after being treated with Linezolid. The icaA and icaD genes were present in only 50% of biofilm-productive bacteria.ConclusionThe ica operon is present in only 50% of biofilm-productive S. aureus and Linezolid is the best antibiotic against these bacteria.

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.

Biofilm formation and pathogenicity of marine-derived Acinetobacter sp. VITRSA1 in Paratelphusa hydrodromous and its toluene remediation

Abstract The Acinetobacter sp. VITRSA1 strain was isolated from the Pichavaram (mangrove), Tamil Nadu, India; and confirmed as gram-negative, aerobic, and non-motile bacteria. The 16S rRNA gene sequence analysis showed 99% similarity with Acinetobacter calcoaceticus JO1 and the Antibiotic Susceptibility Test (AST) showed; resistant to ampicillin, bacitracin, and trimethoprim; intermediate resistant to methicillin, and sensitive to streptomycin. The Acinetobacter sp. VITRSA1 strain was able to form biofilm at 25 °C and 37 °C, on Congo-Red agar plate with a metallic sheen and also on SLI (solid-liquid interface) and ALI (air-liquid interface) – confirmed using microtitre plate, and coverslip assays, respectively. The biofilm formation on Paratelphusa hydrodromous (freshwater crab) showed strong adhesion of bacteria to the crab shell and produced lesions, eventually causing mortality to crabs - indicating shell disease. On contrary to the pathogenicity, the Acinetobacter sp. VITRSA1 was able to degrade toluene and use it as a sole carbon source, which can be useful in recycling industries. This study demonstrates the biofilm formation and antibiotic resistance property of newly isolated Acinetobacter sp., from the mangrove ecosystem and its ability to cause mortality among crabs along with its bioremediation potentials.

Biofilm formation of Candida Spp. isolated from the vagina and antibiofilm activities of lactic acid bacteria on the these Candida Isolates.

In this study, it was aimed to investigate the effects of bacterial cells and cell-free filtrates of Lactobacillus acidophilus 8MR7 and Lactobacillus paracasei subspecies paracasei 10MR8 on the biofilm formation of 3 Candida tropicalis, 3 C. glabrata and 12 C. albicans isolated from the vagina and identified their virulence factors. Haemolytic activities esterase activities, and phospholipase activities as virulence factors of Candida strains were determined. Biofilm formations of these isolates were determined by Congo Red agar and microtitration plate method. Antibiofilm activities of bacterial cells and cell-free filtrates of L. acidophilus 8MR7 and L. paracasei subspecies paracasei 10MR8 on Candida isolates were determined by the microtitration plate method. Bacterial cells of L. acidophilus 8MR7 and L. paracasei subspecies paracasei 10MR8 were not very effective in the inhibition of biofilm, whereas it has been observed that the cell-free filtrates of these bacteria inhibit the formation of biofilms of Candida strains. Although the main mechanism for inhibiting the formation of Candida spp. biofilm is the competition for adhesion, it is concluded that the substances contained in the cell-free filtrates of lactic acid bacteria are also important. These isolates promise hope as potential bacteria that can be used for anti-adhesion purposes in health-care materials.

Biofilm forming properties of quinolone resistant Escherichia coli from the broiler production chain and their dynamics in mixed biofilms

BackgroundQuinolone resistant Escherichia coli (QREC) have been found in samples from Norwegian broiler chicken, despite quinolones not being administered to poultry in Norway. Biofilm production may be one factor contributing to the observed persistence in the broiler production chain. In the present study, 158 QREC strains from chicken caecal and retail meat samples were screened for biofilm production in microtiter plates, biofilm morphotype on Congo Red (CR) agar plates and phylotype by multiplex PCR. Furthermore, the dynamics in mixed biofilms with strains of different morphotypes were studied on glass slides and on CR agar plates.ResultsAll strains but one produced biofilm in microtiter plates and/or on CR agar plates at room temperature. There were no differences between strains from chicken caecum and chicken retail meat in the mean amount of biofilm produced in microtiter plates. Furthermore, no differences in biofilm production were observed between phylotypes. However, significant differences in biofilm production were found between biofilm morphotypes. The morphotype RDAR (red dry and rough, which has both curli and cellulose in the matrix, was displayed by 70% of the strains. Mean biofilm production by these strains were significantly higher than by strains with the morphotypes PDAR (pink dry and rough) with only cellulose or BDAR (brown dry and rough) with only curli. Interestingly, the two latter morphotypes produced biofilms with the morphotype RDAR when grown together. None of the strains achieved significantly higher numbers of colony forming units (cfu) in mixed biofilms than in single strain biofilms on glass slides.ConclusionsThe results indicate that QREC can form biofilm reservoirs on both inert and organic surfaces in production environments, as well as on meat. This may contribute to persistence and dissemination of the strains. Strains with both curli and cellulose in the biofilm matrix were significantly better biofilm formers than strains lacking one of these components. However, strains with only one of the components could compensate for this by producing mixed biofilms with strains having the other component, and thereby most likely enhance their probabilities of persistence in the production environment.

The determination of antimicrobial and antibiofilm activities of foodborne lactic acid bacteria against Enterobacter cloacae isolates

The aim of this study was to investigate the antimicrobial and antibiofilm activities of 10 different lactic acid bacteria (LAB) strains isolated from local food sources of animal origin against 4 Enterobacter cloacae isolates obtained from clinical cases and determine their adhesion potentials to intestinal epithelial cells. In this study, all Enterobacter cloacae isolates (P3, P4, P5, P7) identified with the BD Phoenix automation system were detected to form biofilm with both Congo red agar and Microtiter plate methods. Amoxicillinclavulanate, cefuroxime, and ampicillin resistance was determined in all isolates. It was determined that LAB strains producing exopolysaccharide (EPS) were able to colonize intestinal epithelial cells. It is noteworthy that LAB extracts were effective to inhibit the biofilm formation of P3Ec, which had higher antibiotic resistance than those of other isolates. Antimicrobial effect of LAB extracts on Enterobacter cloacae were also detected by both agar disc diffusion and well diffusion tests. In this study, all of the isolated LAB strains (especially L. lactis, L. fermentum, and L. casei) are good candidates for controlling Enterobacter cloacae biofilm formation. These findings indicate that L. lactis, L. fermentum, and L. casei can potentially be developed as novel antibiofilm agents.

A Cyclic-di-GMP signalling network regulates biofilm formation and surface associated motility of Acinetobacter baumannii 17978

Acinetobacter baumannii has emerged as an increasing multidrug-resistant threat in hospitals and a common opportunistic nosocomial pathogen worldwide. However, molecular details of the pathogenesis and physiology of this bacterium largely remain to be elucidated. Here we identify and characterize the c-di-GMP signalling network and assess its role in biofilm formation and surface associated motility. Bioinformatic analysis revealed eleven candidate genes for c-di-GMP metabolizing proteins (GGDEF/EAL domain proteins) in the genome of A. baumannii strain 17978. Enzymatic activity of the encoded proteins was assessed by molecular cloning and expression in the model organisms Salmonella typhimurium and Vibrio cholerae. Ten of the eleven GGDEF/EAL proteins altered the rdar morphotype of S. typhimurium and the rugose morphotype of V. cholerae. The over expression of three GGDEF proteins exerted a pronounced effect on colony formation of A. baumannii on Congo Red agar plates. Distinct panels of GGDEF/EAL proteins were found to alter biofilm formation and surface associated motility of A. baumannii upon over expression. The GGDEF protein A1S_3296 appeared as a major diguanylate cyclase regulating macro-colony formation, biofilm formation and the surface associated motility. AIS_3296 promotes Csu pili mediated biofilm formation. We conclude that a functional c-di-GMP signalling network in A. baumannii regulates biofilm formation and surface associated motility of this increasingly important opportunistic bacterial pathogen.

Virulence factors and antimicrobial resistance of Staphylococcus aureus isolated from the production process of Minas artisanal cheese from the region of Campo das Vertentes, Brazil

Staphylococcus aureus is one of the main pathogens found in cheeses produced with raw milk, including Minas artisanal cheese from Brazil. However, information about S. aureus isolated from artisanal cheeses and its sources of production in small-scale dairies is very limited. We aimed to characterize the virulence factors of S. aureus isolated from raw milk, endogenous starter culture, Minas artisanal cheese, and cheese handlers from the region of Campo das Vertentes, Minas Gerais, Brazil. We identified the staphylococcal isolates by MALDI-TOF mass spectrometry. We evaluated biofilm production on Congo red agar and polystyrene plates. We used PCR to detect icaA, icaB, icaC, sea, seb, sec, sed, see, tsst-1, agr, and mecA. We evaluated the expression of staphylococcal toxin genes in PCR-positive staphylococcal isolates using quantitative reverse-transcription PCR, and we evaluated the production of these toxins and their hemolytic activity in vitro. We also evaluated the antimicrobial resistance profile of the staphylococcal isolates. For statistical analysis, we used cluster analysis, χ2 tests, and correspondence tests. We analyzed 76 staphylococcal isolates. According to PCR, 18.42, 18.42, 2.63, and 77.63% were positive for sea, tsst-1, sec, and agr, respectively. We found low expression of staphylococcal toxin genes according to quantitative reverse-transcription PCR, and only 2 staphylococcal isolates produced toxic shock syndrome toxins. A total of 43 staphylococcal isolates (56.58%) had hemolytic activity; 53 were biofilm-forming on Congo red agar (69.73%), and 62 on polystyrene plates (81.58%). None of the staphylococcal isolates expressed the mecA gene, and none presented a multi-drug resistance pattern. The highest resistance was observed for penicillin G (67.11%) in 51 isolates and for tetracycline (27.63%) in 21 isolates. The staphylococcal isolates we evaluated had toxigenic potential, with a higher prevalence of sea and tsst-1. Biofilm production was the main virulence factor of the studied bacteria. Six clusters were formed whose distribution frequencies differed for hemolytic activity, biofilm formation (qualitative and quantitative analyses), and resistance to penicillin, tetracycline, and erythromycin. These findings emphasize the need for effective measures to prevent staphylococcal food poisoning by limiting S. aureus growth and enterotoxin formation throughout the food production chain and the final product.

Environmental conditions steer phenotypic switching in acute hepatopancreatic necrosis disease-causing Vibrio parahaemolyticus, affecting PirAVP /PirBVP toxins production.

Bacteria in nature are widely exposed to differential fluid shears which are often a trigger for phenotypic switches. The latter mediates transcriptional and translation remodelling of cellular metabolism impacting among others virulence, antimicrobial resistance and stress resistance. In this study, we evaluated the role of fluid shear on phenotypic switch in an acute hepatopancreatic necrosis disease (AHPND)-causing Vibrio parahaemolyticus M0904 strain under both in vitro and in vivo conditions. The results showed that V. parahaemolyticus M0904 grown at lower shaking speed (110 rpm constant agitation, M0904/110), causing low fluid shear, develop cellular aggregates or floccules. These cells increased levan production (as verified by concanavalin binding) and developed differentially stained colonies on Congo red agar plates and resistance to antibiotics. In addition, the phenotypic switch causes a major shift in the protein secretome. At 120 rpm (M0904/120), PirAVP /PirBVP toxins are mainly produced, while at 110 rpm PirAVP /PirBVP toxins production is stopped and an alkaline phosphatase (ALP) PhoX becomes the dominant protein in the protein secretome. These observations are matched with a very strong reduction in virulence of M0904/110 towards two crustacean larvae, namely, Artemia and Macrobrachium. Taken together, our study provides substantial evidence for the existence of two phenotypic forms in AHPND V. parahaemolyticus strain displaying differential phenotypes. Moreover, as aerators and pumping devices are frequently used in shrimp aquaculture facilities, they can inflict fluid shear to the standing microbial agents. Hence, our study could provide a basis to understand the behaviour of AHPND-causing V. parahaemolyticus in aquaculture settings and open the possibility to monitor and control AHPND by steering phenotypes.

Biofilm Formation and Detection of Fluoroquinolone- and Carbapenem-Resistant Genes in Multidrug-Resistant Acinetobacter baumannii.

Acinetobacter baumannii is an important opportunistic pathogen that shows resistance to cephalosporins, penicillins, carbapenems, fluoroquinolones, and aminoglycosides, the multiresistance being associated with its ability to form biofilms in clinical environments. The aim of this study was to determine biofilm formation and its potential association with genes involved in antibiotic resistance mechanisms of A. baumannii isolates of different clinical specimens. We demonstrated 100% of the A. baumannii isolates examined to be multidrug resistant (MDR), presenting a 73.3% susceptibility to cefepime and a 53.3% susceptibility to ciprofloxacin. All A. baumannii isolates were positive for blaOXA-51, 33.3% being positive for blaOXA-23 and ISAba1, and 73.3% being positive for gyrA. We found 86.6% of A. baumannii strains to be low-grade biofilm formers and 13.3% to be biofilm negative; culturing on Congo red agar (CRA) plates revealed that 73.3% of the A. baumannii isolates to be biofilm producers, while 26.6% were not. These properties, combined with the role of A. baumannii as a nosocomial pathogen, increase the probability of A. baumannii causing nosocomial infections and outbreaks as a complication during therapeutic treatments and emphasize the need to control A. baumannii biofilms in hospital environments.

Correlation of biofilm formation and antibiotic resistance among clinical and soil isolates of Acinetobacter baumannii in Iraq.

Acinetobacter baumannii is an opportunistic pathogen that is reported as a major cause of nosocomial infections. The aim of this study was to investigate the biofilm formation by A. baumannii clinical and soil isolates, to display their susceptibility to 11 antibiotics and to study a possible relationship between formation of biofilm and multidrug resistance. During 8months period, from June 2016 to January 2017, a total of 52 clinical and 22 soil isolates of A. baumannii were collected and identified through conventional phenotypic, chromo agar, biochemical tests, API 20E system, and confirmed genotypically by PCR for blaOXA-51-like gene. Antibiotic susceptibility of isolates was determined by standard disk diffusion method according to Clinical and Laboratory Standard Institute. The biofilm formation was studied using Congo red agar, test tube, and microtiter plate methods. The clinical isolates were 100% resistance to ciprofloxacin, ceftazidime, piperacillin, 96.15% to gentamicin, 96.15% to imipenem, 92.31% to meropenem, and 78.85% to amikacin. The soil A. baumannii isolates were 100% sensitive to imipenem, meropenem, and gentamicin, and 90.1% to ciprofloxacin. All A. baumannii isolates (clinical and soil) were susceptible to polymyxin B. The percentage of biofilm formation in Congo red agar, test tube, and microtiter plate assays was 10.81%, 63.51%, and 86.48%, respectively. More robust biofilm former population was mainly among non-MDR isolates. Isolates with a higher level of resistance tended to form weaker biofilms. The soil isolates exhibited less resistance to antibiotics than clinical isolates. However, the soil isolates produce stronger biofilms than clinical isolates.

The phenotypic and genotypic characteristics of biofilm formation and SCCmec typing of Staphylococcus epidermidis isolated from different sources

IntroductionStaphylococcus epidermidis is a main cause of healthcare-associated infections, which known as a common pathogen mostly infecting critically ill or immunocompromised patients carrying medical devices. To evaluate the biofilm formation and staphylococcal chromosomal cassette mec (SCCmec) typing of S. epidermidis from different parts of two tertiary hospitals in Tehran, Iran. MethodsIn this study, 103 clinical and non-clinical isolates of S. epidermidis were assessed for biofilm production on Congo red agar (CRA) and microtiter plate (MTP) methods. The icaABCD genes in S. epidermidis and SCCmec were also investigated. ResultsOut of 103, 20(19.4%) isolates formed black colonies (biofilm-forming strains) on CRA. The MTP method showed that19 (18.4%) isolates was strongly positive; the reaction of 25(24.2%) isolates was moderately positive, and in the remaining 27(26.2%) isolates, reaction was weakly positive. Additionally, the results of biofilm production on CRA agreed with microtiter plate assay and PCR assay. Fourteen SCCmec type-I were identified among the total 54Methicillin-resistant S. epidermidis isolates. ConclusionThe main factor involved in the pathogenesis of S. epidermidis is expression of ica genes, that mediate biofilm formation. This study indicates a high prevalence of the icaABCD genes among S. epidermidis isolates, and their presence is not always associated with in vitro formation of biofilm.

Biofilm Inhibitory Potential of Westiellopsis prolifica Extract Against Some Pathogenic Microorganisms

Resistance of microorganisms to many kinds of antibiotics push towards using natural products to eliminate the biofilm. In this study, 7 different species of algae were identified, 3 species of Chlorophyta and 3 of Cyanophyta. Westiellopsis prolifica is considered a potent organism. Fourier transform infrared spectroscopy (FTIR) was determined 13 clear bands, and the main bands were carbohydrate (1200- 900 cm−1), protein (1660 and around 1540 cm−1) and lipid bands (1740 cm−1). Extracellular crude acetone extract from W. prolifica better than hexane extract and more efficient on negative gram bacteria than positive gram bacteria. Antibiofilm was conducted by Congo red Agar and Microtiter plate against bacterial isolates (Staphylococcus aureus, Bacillus subtilis, Streptococcus sp., Shigella sp., Proteus sp. and Pseudomonas aeruginosa) and two fungi (Aspergillus niger and Candida albicans). The highest inhibition was against Streptococcus sp. and the remaining ratio of biofilm was 0.7 %, while lower inhibition 33 % against C.albicans. The GC-MS analysis of the purified extract has identified many active compounds, mainly were 4-Trimethyl-1-hexene, Octadecane, Ethylene-14-Pentadecane, n-Hexadecanoic acid, Octadecadienoic acid, Octadecadienoyl chloride, and Phytol.

Phenotypic and genotypic characterization of Staphylococcus spp. isolated from mastitis milk and cheese processing: Study of adherence and biofilm formation.

The aim of this study was to identify the phenotypic and genotypic profiles of Staphylococcus spp. isolated from mastitis milk and cheese processing plant.To evaluate the biofilm production of wild-type strains on contact surfaces by testing different factors through adhered cells and biofilm quantifications, finally, these biofilms were observed by Scanning Electron Microscopy (SEM). Congo red agar (CRA) plate method was used to identify slime production by strains. Screening of genes encoding adhesion factors and biofilm formation was carried out using PCR. After strains selection, adhesion and biofilm assays were designed testing different times (12, 48, 96 h), strains (n = 13), contact surfaces (stainless steel and polypropylene), and temperatures (5 °C and 25 °C); and then, bacterial count and crystal violet staining were conducted. Relative frequencies of positive on CRA and genes presence were determined, and Friedman test was applied for bacterial counts and OD values. Additionally, significant factors (P ≤ .05) were subjected to multiple comparisons using the Nemenyi test. The slime production in CRA was observed by visual inspection in 38.7% of strains. A large distribution of genes was described among strains, implying a high variability of genotypic profiles. Moreover, relative frequencies of CRA positive and gene presence were described. The developed assay showed that the strain, temperature, contact surface, were significant for both variables. The SEM corroborated the findings, showing greater biofilm formation on stainless steel at 25 °C. Thus, it is essential to highlight the importance of temperature control and material with low superficial energy to avoid biofilm formation by staphylococci.