Congo Red Agar Medium Research Articles

Association Between Biofilm Formation by U.P.E.C. and Serum Level of Several Cytokines

One hundred and eighty-nine subjects from Baghdad enrolled in this study (110 female and 79 male) and gathered into two investigated groups; the first group consisted of 149 patients, and the second group consisted of 40 healthy individuals. Results revealed after clinical laboratory diagnosis of urine samples 12 (8.1%) gave a negative bacterial culture, 137 (91.9%) were positive culture, while all urine samples of healthy control were negative. Gram staining and microscopic examination of bacterial colonies showed that 11(8.03%) out of 137 isolates were identified as Gram-positive and 126 (91.97%) as Gram-negative. After biochemical analysis and diagnosis by the Vitik system, the data demonstrated that a single infectious agent caused all U.T.I. cases. U.P.E.C. represented the most common bacterial agent because of several virulence factors responsible for its pathogenicity. The test tube method and Congo red agar medium have been used to detect biofilm formation. Results demonstrate that 129 (94.16 %) of bacterial isolates were producers, while just 8 (5.84 %) were non-producers. The results of the microtiter plate method revealed that the isolates were categorized into four groups: Strong, moderate, weak, and harmful. 22 (63.5%) were strong biofilm producers, 28 (20.449%) were moderate producers, 14 (10.22%) were weak producers, and 8 (5.84%) were unable to form biofilm. Serum levels of IL-1β, IL-6 and IL-8 were estimated by Sandwich ELISA, which were significantly higher in patients with different types of U.T.I.s than the healthy group. This study concluded that the U.P.E.C. represented the most common prevalent agent of U.T.I.s and more efficient biofilm-producer bacteria. The test tube method is the best qualitative, quick, and easy detection method of biofilm formation, while the microtiter plate is the best quantitative and sensitive method. A positive correlation was found between biofilm formation and elevated serum levels of proinflammatory cytokines, proportionally increased with advanced and severe, especially in old persons. Keywords: UTIs; ELISA; IL-1β; IL-6; IL-8; Iraq

Antibiotics Resistance and Adhesive Properties of Clinical Staphylococcus aureus Isolated from Wound Infections.

Staphylococcus aureus (S. aureus) is a ubiquitous pathogen responsible for several severe infections. This study aimed to investigate the adhesive properties and antibiotic resistance among clinical S. aureus isolated from Hail Hospital Province, Kingdom of Saudi Arabia (KSA), using molecular approaches. This study was conducted according to the ethical committee at Hail's guidelines on twenty-four S. aureus isolates. A polymerase chain reaction (PCR) was performed to identify genes encoding the β-lactamase resistance (blaZ), methicillin resistance (mecA), fluoroquinolone resistance (norA), nitric oxide reductase (norB), fibronectin (fnbA and fnbB), clumping factor (clfA) and intracellular adhesion factors (icaA and icaD). This qualitative study tested adhesion based on exopolysaccharide production on Congo red agar (CRA) medium and biofilm formation on polystyrene by S. aureus strains. Among 24 isolates, the cna and blaz were the most prevalent (70.8%), followed by norB (54.1%), clfA (50.0%), norA (41.6%), mecA and fnbB (37.5%) and fnbA (33.3%). The presence of icaA/icaD genes was demonstrated in almost all tested strains in comparison to the reference strain, S. aureus ATCC 43300. The phenotypic study of adhesion showed that all tested strains had moderate biofilm-forming capacity on polystyrene and represented different morphotypes on a CRA medium. Five strains among the twenty-four harbored the four genes of resistance to antibiotics (mecA, norA, norB and blaz). Considering the genes of adhesion (cna, clfA, fnbA and fnbB), these genes were present in 25% of the tested isolates. Regarding the adhesive properties, the clinical isolates of S. aureus formed biofilm on polystyrene, and only one strain (S17) produced exopolysaccharides on Congo red agar. All these results contribute to an understanding that the pathogenesis of clinical S. aureus isolates is due to their antibiotic resistance and adhesion to medical material.

Biofilm-Forming Heavy Metal Resistance Bacteria From Bungus Ocean Fisheries Port (PPS) West Sumatra as a Waters Bioremediation Agent.

<b>Background and Objective:</b> Heavy metals are one of the most worrisome pollutants due to their toxicity. Prolonged exposure to heavy metals and their accumulation and biomagnification properties adversely affect aquatic biota and human health. The ability of microorganisms to bioremediate heavy metals into non-toxic forms is one solution. The research aims of the study were to find biofilm-forming heavy metal-resistant bacteria isolated from the waters of the Bungus Samudra Fishery Port (PPS), Padang City. <b>Materials and Methods:</b> This study used a marine agar medium modified with the addition of K<sub>2</sub>Cr<sub>2</sub>O<sub>7</sub>, Pb(NO<sub>3</sub>)<sub>2</sub> and CdSO<sub>4</sub>•H<sub>2</sub>O, Marine Broth medium and Congo Red Agar medium. The research methods include, the isolation of bacteria, isolate resistance test to heavy metals, testing the ability of isolates to form biofilms and determine the ability of isolates to reduce heavy metals. Furthermore, molecular identification of bacterial isolates was carried out to determine the type of species. <b>Results:</b> Five heavy metal-resistant bacterial isolates were found that were able to form biofilms, namely isolates B3Cd, B5Cr, B7Pb, B6Pb and B3Pb. The five isolates were able to reduce heavy metal content by 38.67-61.191%. Identification of the best bacterial isolates on each heavy metal tested, namely B3Cd, B5Cr and B7Pb, respectively, showed the type of <i>Acinetobacter schindleri</i>, <i>Acinetobacter</i> sp. and <i>Bacillus</i> sp. <b>Conclusion:</b> These three selected potential isolates can be used as bioremediation agents in metal-polluted waters in the future.

Characterization of fungi isolated from cattle dung with potential lignocellulolytic activities

Agricultural wastes not only serve as sources of environmental pollutions, but also serve as magnificent source of plant nutrients and natural repositories of bio-tools especially fungi of industrial interest for lignocellulolytic enzymes production. However, residues management particularly in rice–wheat system is a tedious phenomenon due to narrow gap between harvesting and sowing of these two crops. Hence, microbial intervention is one of the key options that may use successfully for their management. Thus, cellulolytic fungi were isolated from the cattle dung collected from various locations for cellulose manufacturing. The cellulolytic property of fungal isolates was confirmed by plate screening assay based on yellow zone formation surrounding of fungal culture on Congo red agar medium plate owing to production of cellulase enzyme. The colony diameter, clear zone diameter and cellulolytic index showing in descending order of CRD3, CRD5, CRD6 and CRD10 isolates, but –glucosidase activity was greater in CRD3 and CRD5 while, the lowest was in CRD9. However, isolates CRD7, CRD8 and CRD10 exhibited greater biochemical and enzymatic activity than rest of the promising fungal isolates. Similarly, isolates CRD1 and CRD2 utilized most of carbon sources as carbon substrate, but CRD7, CRD8 and CRD10 could not utilized mannitol and cellulose as carbon substrate. Overall, isolate CRD10 showed as a promising isolate in respect of biochemical, carbon utilization pattern and enzymatic activity than rest of the promising isolates.

Phenotypic and Genomic Insights into Biofilm Formation in Antibiotic-Resistant Clinical Coagulase-Negative Staphylococcus Species from South Africa.

The work aims to investigate biofilm formation and biofilm/adhesion-encoding genes in coagulase-negative staphylococci (CoNS) species recovered from blood culture isolates. Eighty-nine clinical CoNS were confirmed using the VITEK 2 system, and antibiotic susceptibility testing of isolates was conducted using the Kirby-Bauer disk diffusion method against a panel of 20 antibiotics. Isolates were qualitatively screened using the Congo red agar medium. Quantitative assays were performed on microtiter plates, where the absorbances of the solubilised biofilms were recorded as optical densities and quantified. In all, 12.4% of the isolates were strong biofilm formers, 68.5% had moderate biofilm capacity, and 17.9% showed weak capacity. A subset of 18 isolates, mainly methicillin-resistant S. epidermidis, were investigated for adherence-related genes using whole-genome sequencing and bioinformatics analysis. The highest antibiotic resistance rates for strongly adherent isolates were observed against penicillin (100%) and cefoxitin (81.8%), but the isolates showed no resistance to linezolid (0.0%) and tigecycline (0.0%). The icaABC genes involved in biofilm formation were detected in 50% of the screened isolates. Other adherence-related genes, including autolysin gene atl (88.8%), elastin binding protein gene ebp (94.4%), cell wall-associated fibronectin-binding protein gene ebh (66.7%), clumping factor A gene clfA (5.5%), and pili gene ebpC (22.2%) were also found. The insertion sequence IS256, involved in biofilm formation, was found in 10/18 (55.5%) screened isolates. We demonstrate a high prevalence of biofilm-forming coagulase-negative staphylococci associated with various resistance phenotypes and a substantial agreement between the possession of biofilm-associated genes and the biofilm phenotype.

ANTIFUNGAL DRUG RESISTANCE PATTERN AMONG BIOFILM PRODUCING STRAINS OF CANDIDA SPECIES ISOLATED FROM BLOODSTREAM INFECTIONS

Background & objectives: The emergence of drug resistant Candida species has become an important cause of health care associated infections. Antimicrobial resistance is very high in biolm-producing Candida strains. This study was carried out with an aim to study species distribution of Candida isolates among candidemia patients, biolm formation as a virulence factor and their antifungal susceptibility pattern. A total ofMethods: 100 consecutive Candida isolates obtained from blood culture samples were included in the study. Identication of the isolates were done by conventional method and VITEK 2. The antifungal susceptibility were done by VITEK 2 and E test method. Biolm formation was detected using Congo red agar medium, Test tube method and Microtitre plate method. Of the total 100 Candida isolates evaluated, Candida albicansResults: (n=34 isolates) was most common followed by C. tropicalis (n=20 isolates). A larger number of Candida spp. (71%) produced biolm. Biolm production by NAC was signicantly more frequent (75.75%) than that by C. albicans (61.76%). Antifungal resistance rates for Fluconazole (30.98%), Amphotericin B (15.49%) and Flucytosine (11.26%) were higher among the biolm producing strains. All the strains of C. krusei were resistant to Fluconazole and Flucytosine and all strains of C. auris were found resistant to uconazole. Interpretation & Conclusion: Speciation of Candida becomes important as the prevalence of NAC is increasing. Biolm formation exhibits increased resistance to commonly available antifungal therapies. These data has become necessary to reduce the net effect of the increasing severity of Candida infections, drug resistance, and economic burden.

Evaluation of Biofilm Formation in Coagulase Negative Staphylococci on Various Congo Red Agar Medium

Evaluation of Biofilm Formation in Coagulase Negative Staphylococci on Various Congo Red Agar Medium

KOAGÜLAZ-NEGATİF STAPHYLOCOCCUS (KNS) VE MACROCOCCUS CASEOLYTICUS SUŞLARININ BİYOFİLM OLUŞTURMA ÖZELLİKLERİNİN VE ADEZYON FAKTÖRLERİNİN ARAŞTIRILMASI

Bu çalışmada geleneksel fermente Türk sucuklarından izole edilmiş toplam 50 adet koagulaz-negatif Staphylococcus (KNS) ve 10 adet Macrococcus caseolyticus suşlarının biyofilm oluşturma özellikleri fenotipik olarak belirlenmiş ve aynı zamanda suşlarda biyofilm oluşumu ile ilişkili genler ve adezyon genlerinin varlığı multipleks polimeraz zincir reaksiyonu (mPZR) ile araştırılmıştır. Fenotipik olarak slaym (slime) üretimi Congo Red agar (CRA), biyofilm üretim düzeyi mikrotitre plaka yöntemi ile belirlenmiştir. CRA besiyerinde slaym üreticisi olduğu belirlenen 33 suşun tamamının mikrotitre plaka yöntemi ile biyofilm üreticisi olduğu tespit edilmiştir. KNS ve M. caseolyticus suşlarının 28’inin orta, 23’ünün zayıf ve 1’nin güçlü biyofilm üreticisi olduğu saptanmıştır. Sekiz suşta ise biyofilm üretiminin olmadığı tespit edilmiştir. mPZR denemeleri sonucu biyofilm üreticisi olduğu belirlenen KNS ve M. caseolyticus suşlarının %84.62’sinin araştırılan genlerden en az birini içerdiği bulunmuştur. Suşların %66.67’sinde eno geni, %15.67’sinde icaA geni varlığı belirlenmiştir. Diğer taraftan biyofilm üreticisi olduğu belirlenen 8 suşun ise çalışma kapsamında araştırılan biyofilm genlerinin hiçbirini içermediği tespit edilmiştir.

Characterization of a biosurfactant producing electroactive Bacillus sp. for enhanced Microbial Fuel Cell dye decolourisation

A biosurfactant producing Gram positive bacterium isolated from anodic biofilm of textile wastewater fed MFC was identified as Bacillus sp. MFC (Accession number: MT322244). Scanning Electron Microscopy of the bacterium showed appendages, the bacterium forms biofilm on Congo red agar medium. The obtained results showed that the addition of 5 mg/l endogenous biosurfactant to the bacterial cells resulted in 19-fold increase in bacterial surface-bound exopolysaccharides (EPS) and 1.94-fold increase in biofilm. However, when the biosurfactant concentration increased to 20 and 40 mg/l, EPS and biofilm decreased and the cells lost their colony forming ability. The dielectric properties of the bacterial cells showed increase in conductivity and relative permittivity with increasing biosurfactant concentrations. The shape of the voltammogram currents peak, their location and Electrochemical impedance spectroscopy (EIS) suggest the involvement of biofilm as direct electron transfer pathway. The average voltage obtained was 0.65 V as compared to 0.45 V for the control MFC. Decolourization was tested for Congo red in a double chamber Microbial Fuel Cell (MFC), the results showed 2-fold increase in decolourization when biosurfactant is added post biofilm formation. The results confirm that Bacillus sp. MFC possess electrogenic properties and that adding low concentrations of endogenous biosurfactant to 24 h biofilm accelerates electron transfer by inducing perforations in the cell wall and increasing EPS as an electron transfer transient medium. Therefore, MFC performance can be enhanced.

Correlation between biofilm formation and antimicrobial susceptibility pattern toward extended spectrum β-lactamase (ESBL)- and non-ESBL-producing uropathogenic bacteria.

Urinary tract infections (UTIs) are the most common bacterial infection encountered worldwide and are associated with significant morbidity and mortality. The present study was undertaken to investigate the biofilm-forming ability, antibiotic susceptibility patterns and extended spectrum β-lactamase (ESBL) production of seven uropathogenic isolates comprising both Escherichia coli and Klebsiella pneumoniae. The morphological, cultural and biochemical tests for the identification of the isolates, antibiotic susceptibility test, detection of ESBL production, biofilm formation on 96-well microtiter plate and Congo red agar (CRA) media are performed. The antimicrobial susceptibility profiles obtained in this study showed that the most active drugs gentamicin, amikacin and imipenem (100% sensitivity) were followed by amoxicillin-clavulanic acid (85% sensitivity), co-trimoxazole, ciprofloxacin (57% sensitivity) ceftazidime and kanamycin (50% sensitivity). All the isolates showed resistance to amoxicillin followed by ceftriaxone and cefotaxime (71% resistance), and the scenario gets more complicated because of the production of ESBL by five isolates (three E. coli isolates and two K. pneumoniae). The strains were also able to form biofilm as tested on CRA medium and by microtiter plate assay. The correlation between ESBL, non-ESBL and biofilm-producing E. coli and K. pneumonia was determined along with the multiple drug resistance patterns of E. coli and K. pneumonia. The findings of the study indicate that the emergence and rapid spread of such multidrug-resistant pathogens are of great concern. Early detection of ESBL-producing pathogen is of paramount clinical importance; therefore, strict infection control practices as well as therapeutic guidance for confirmed infection can be rapidly initiated.

Comparison of genotypic and phenotypic characteristics in biofilm production of Staphylococcus aureus isolates

Staphylococcus aureus which is an important pathogen, is known to have several virulence factors. The pathogenicity of S.aureus isolates are related with features like adherence, various toxins, enzymes, structural and extracellular factors. Slime factor and biofilm formation are also the pathogenicity factors of the bacteria. Biofilm formation is usually associated with chronic infections and has become a subject of interest in a wide area of research. Biofilm is an adherent structure formed by bacteria encased within a matrix produced on natural body surfaces or medical devices. As the biofilm producing S.aureus isolates are more resistant to antibiotics and and their biofilms prevent phagocytosis, the treatment of infections caused by biofilm positive S.aureus isolates become difficult. The icaADBC locus and some proteins have provento be responsible for the formation of S.aureus type biofilm. The aim of this study was to investigate the relationship between the genotypic and phenotypic detection methods of biofilm formation with icaA, icaD and bap genes and phenotype expressions which are responsible for the formation of biofilm in S.aureus isolates. One hundred seventy five S.aureus isolates from various clinical specimens were included in the study. For the phenotypic detection of biofilm producing isolates Congo red agar (CRA) medium and microplate method were used. The biofilm-producing strain Staphylococcus epidermidis ATCC 35984 and S. epidermidis ATCC 12228 were used as positive and negative controls, respectively. One hundred fifty two S.aureus biofilm producing isolates were detected at least by either Congo Red agar or microplate method and all isolates were screened for icaA, icaD and bap genes by PCR. The production of polysaccharide intracellular adhesins /poly-N-acetyl-beta-1-6-glucosamine (PIA/PNAG) was also investigated in S.aureus isolates. For the detection of PIA/PNAG chemiluminescence dot-blot method was used. According to the phenotypic evaluations based on colony morphologies in CRA medium, biofilm formation were found as negative in 101 of 175 isolates (57.7%), while biofilm formation were positive in 74 (42.3%) of the isolates. As a result of quantitative evaluation by microplate method based on absorbance measurement, biofilm production was determined as negative in 34 (19.4%) specimens, moderate in 112 (64.0%) specimens and strong in 29 (16.6%) specimens. Microplate and CRA methods were incompatible with each other when compared for their biofilm production determination (p< 0.001). Among the 152 clinical isolates used to determine the presence of icaA and icaD genes responsible for the formation of biofilms, the genes were detected in 136 (89.5%) of the isolates and in the S.epidermidis ATCC 35984 strain used as a positive control. icaA and icaD genes were not detected in sixteen of the 152 (10.5%) clinical isolates and in the S.epidermidis ATCC 12228 strain used as a negative control. A weak-moderate correlation was found between icaA and icaD genes and biofilm production determined in CRA medium. A good correlation was found between icaA and icaD genes and biofilm production detected in microplate method. bap gene was determined in only one of the 152 clinical S.aureus isolates studied and in S.aureus V329 strain used as positive control. For the detection of PIA/PNAG which was synthesized by icaADBC genes, 50 clinical S.aureus isolates were used. PNAG production was determined in 42 isolates with positive icaA and icaD genes by the chemiluminescence dot-blot method, and no PNAG production was determined among eight isolates with negative ica genes. As a result, it was found that the microplate method was more sensitive and reliable for the phenotypic determination of biofilm formation in S.aureus isolates, high level of biofilm formation among clinical S.aureus isolates (about 80%), the role of icaA and icaD genes and products (PIA/PNAG) in biofilm production was determined.

Cellulase from Bacillus licheniformis and Brucella sp. isolated from mangrove soils of Mahanadi river delta, Odisha, India

In the present study, two cellulose-degrading bacteria (CDB-5 and CDB-12) were isolated from mangrove soils of Mahanadi river delta, based on halo zone formation in Congo red agar medium and evaluation for cellulase production in CMC broth medium. Based on morphological, biochemical and 16S rRNA gene sequencing, the two strains, CDB-5 and CDB-12, were identified as Brucella sp. and Bacillus licheniformis, respectively. The gene bank accession number of the strains CDB-5 and CDB-12 are KR632646 and KR632645, respectively. The strain Brucella sp. and B. licheniformis showed an enzyme activity of 96.37 U/ml and 98.25 U/ml, respectively, after 72 h of incubation period. Enzyme production was optimized under different growth conditions such as pH, temperature, agitation rate, carbon source, sodium chloride (NaCl), and nitrogen sources. Maximum cellulase production by both the strains was obtained in the same parameter condition such as pH (7.0), rpm (150), and NaCl (2%, w/v) which varies for other parameters. The strain, CDB-5, produced maximum cellulase at 35 °C temperature, maltose as a carbon source, and yeast extract as a nitrogen source where as the strain CDB-12 produces maximum cellulase at 45 °C temperature, carboxyl methyl cellulose (CMC) as carbon source and trypton as a nitrogen source. The bacterial crude enzyme was purified by ammonium sulfate precipitation followed by overnight dialysis. SDS-PAGE analysis of the partially purified cellulase enzyme exhibited band sizes of approximately 55 and 72 kDa.

Association between toxin-antitoxin systems and biofilm formation.

Background:Toxin-antitoxin (TA) systems are found on the chromosomes and plasmids of many Bacteria such as Escherichia coli. The roles of TA systems in bacteria are enigmatic. Multiple biological functions of TA systems are proposed including growth modulation, persistence, and biofilm formation. Biofilms of E. coli are cause of urinary tract infections, as well as bacteraemia.Objectives:The current study aimed to find the association between biofilm formation and toxin-antitoxin systems in clinical isolates of E. coli.Materials and Methods:A total of 150 E. coli isolates were evaluated for biofilm formation by Congo red agar medium (CRA) and microtiter plate assay and the presence of different TA systems including MazEF, RelBE, hipBA, ccdAB and MqsRA.Results:The results of the analysis revealed that 107 E. coli isolates were potent for biofilm formation by CRA. The findings by microtiter plates showed that 102 E. coli isolates were biofilm producers. The results indicated that 80%, 85%, 70%, 91% and 82% of the isolates possessed MazEF, RelBE, hipBA, ccdAB and MqsRA TA loci, respectively.Conclusions:The analysis recommended that TA genes are prevalent in clinical isolates of E. coli strains. The analysis revealed that hipBA TA system is associated with biofilm formation.

INFLUENCE OF PAPER AND PULP INDUSTRY EFFLUENTS ON PHYSICO-CHEMICAL & BIOLOGICAL PROPERTIES OF SOIL

Effect of Paper and Pulp Industry effluents on soil was assessed for Physico-chemical & Biological properties of soil in the present study. Discharge of Paper &Pulp Industry effluents on to the soil caused changes in Physico-chemical &biological properties of soil. These changes include increase in clay and silt percentages, Electrical conductivity, Water Holding capacity, and Organic matter, total N, available P, and K in test over control sample. However, there was a less sand, higher bacterial and fungal populations were recorded in test soil. Also, cellulose degrading bacteria were isolated from paper and pulp Industry effluent contaminated soil by Cellulose Congo Red Agar Medium.

Isolation and Identification of Cellulose Degrading Bacteria from Mangrove Soil of Mahanadi River Delta and Their Cellulase Production Ability

This study was conducted to isolate cellulose degrading bacteria from mangrove soil of Mahanadi river delta, Odisha, India and also to evaluate their cellulase production ability. Results showed that in total fifteen cellulose degrading bacteria were isolated based on their halo zone formation on congored agar medium. Their maximum carboxymethylcellulose hydrolysis capacities (HC value) were ranged from 1.18 to 2.5 cm. A CMCase test of these fifteen isolates showed that their enzyme activity ranged from 2.471 to 98.253 U/ml/min. From the morphological, aand biochemical characterisation, the isolates were identified as Micrococcus spp., Bacillus spp., Pseudomonas spp., Xanthomonas spp. and Brucella spp.

Virulence, Speciation and Antibiotic Susceptibility of Ocular Coagualase Negative Staphylococci (CoNS).

Coagulase negative Staphylococci (CoNS) are common inhabitants of human skin and mucous membranes. With the emergence of these organisms as prominent pathogens in patients with ocular infections, investigation has intensified in an effort to identify important virulence factors and to inform new approaches to treatment and prevention. To isolate CoNS from ocular specimens; to study the possible virulence factors; speciation of coagulase negative staphylococci (CoNS) which were isolated from ocular complications; antibiotic susceptibility testing of ocular CoNS. The specimens were collected from the target patients who attended the Microbiology Laboratory of a tertiary care eye hospital in Coimbatore, Tamilnadu state, India. The isolates were subjected to tube and slide coagulase tests for the identification of CoNS. All the isolates were subjected to screening for lipase and protease activities. Screening for other virulence factors viz., slime production on Congo red agar medium and haemagglutination assay with use of 96-well microtitre plates. These isolates were identified upto species level by performing biochemical tests such as phosphatase test, arginine test, maltose and trehalose fermentation tests and novobiocin sensitivity test. The isolates were subjected to antibiotic susceptibility studies, based on the revised standards of Clinical and Laboratory Standards Institutes (CLSI). During the one year of study, among the total 260 individuals who were screened, 100 isolates of CoNS were obtained. Lipolytic activity was seen in all the isolates, whereas 38 isolates showed a positive result for protease. A total of 63 isolates showed slime production. Of 100 isolates, 30 isolates were analyzed for haemagglutination, where 4 isolates showed the capacity to agglutinate the erythrocytes. The results of the biochemical analysis revealed that of the 100 isolates of CoNS, 43% were Staphylococcus epidermidis. The other isolates were identified as S. xylosus (n=8), S. captis (n=16), S. haemolyticus (n=10), S. saccharolyticus (n=2), S. hominis (n=5), S. saprophyticus (n=6) and S. intermedius (n=1). On the other hand, 9 isolates were not identified. In the antibiotic susceptibility analysis, it was found that most of the isolates were sensitive to vancomycin, amikacin and linczolid and resistant to cefatoxime, oxacillin, bacitracin and nalidixic acid. S. epidermidis was found to be predominant in causing the ocular complications. Slime production, heamagglutination, protease and lipase activities could be the putative virulence factors of CoNS. Antibiotic susceptibility patterns of CoNS against antibacterial agents revealed maximum resistance to beta lactam groups, and the resistance was found to be higher to oxacillin, and lowest to vancomycin.

Investigating the Ability of some Bacterial Species to Produce Slime Layer

This study includes the investigation of the ability of some gram positive and gram negative bacterial species to produce slime layer which play a role in biofilm formation and complication of pathogenic infections and resistance to antibiotic therapy. Thirty five bacterial isolates were used in this study, they include: Staphylococcus aureus (14 isolates), Pseudomonas aeruginosa (7 isolates), Escherichia coli (6 isolates), Proteus mirabilis (3 isolates), Salmonella spp. (2 isolates), Enterobacter aerogenes, Klebsiella pneumoniae, Serratia spp. Three methods were used to detect slime layer production which include: Congo red agar medium (CRA), modified congo red agar method (mCRA), and Microtiter (MTP) method. The results showed that there is no differences between Congo red agar and modified Congo red agar methods. Microtiter plate method was more sensitive for slime layer detection as the rate of production was 51.429% from all isolates as compared with 31.429% positive results in Congo red agar method. There are clear differences in moderate and negative results for both methods.

Determination the titer antibodies against LPS extracted from Pseudomonas aeruginosa isolated from eye infection

rom September 2008 to May 2009, 122 specimens were collected by sterile cotton swaps from patients suffering from eye infection at Ibn Al-Haitham Teaching Eye Hospital, 12 of them (9.83% isolation percentage) were diagnosed as Pseudomonas aeruginosa isolates by using biochemical tests, API 20 E system: they were named (P(1), P(2),……..P(12)). The agglutination sera for the grouping of P. aeruginosa test show that all the isolates belong to the P. aeruginosa strains (6, 9, 12, 16). The result show that P10 isolate have a greater ability to adhesion when all P. aeruginosa isolates were tested on Congo red agar medium and adherence to smooth surfaces. The chemical analysis of crude LPS that was extracted from P10 by using digestive enzyme and hot phenol water method showed that the percentage of carbohydrates was 7.3 and the percentage of protein was 1.3. No nucleic acids were found while the chemical analysis of partially purified LPS that was made by gel-filtration chromatography by using Sephacryl 200 S showed that the percentage of carbohydrates in the partial purified LPS was 19.5% and the percentage of binding proteins was 0.006%. No nucleic acids were found. When the molecular weight of LPS was measured by gel-filtration chromatography by using Sepharose C1- 6B- 200 it was found to be 630957 Dalton. The sera were prepared by using two wild type rabbits (weight 2-2.5 kg). They were injected with five doses over 70 days of different concentrations of partially purified LPS that have been extracted from P. aeruginosa P (10) isolate. One rabbit was used as a control that has been injected with PBS. The titer of antibodies in sera was determined by passive haemagglutination and it was in the immunized rabbit’s sera 160, and the best concentration of LPS was 10µg.

Soil Diffusion System Enriches the Growth of Diverse and Previously Uncultivated Bacterial Taxa

Recent studies have demonstrated that culturing microorganisms in association with their native habitat promoted the growth of diverse and previously uncultivable bacteria. We developed a soil diffusion system (SDS) where soil microorganisms were inoculated and allowed to grow on regenerated cellulose filters (RCF) that were tightly coupled with the soil habitat. The objective of the study was to assess the influence of the native habitat and the biotic (microbe–microbe) and abiotic soil effects on the selective growth of microbial communities. Regenerated cellulose filters were used as support for the growing microbial community. A polycarbonate membrane (0.003-μm pore size) was inserted between the RCF and the soil to prevent the movement of bacteria and larger organisms. Three different SDS treatments were used to differentiate biotic and abiotic growth effects along with a control-SDS treatment and a traditional culturing medium. The treatments were: (i) inoculated RCF on unsterilized soil (BioticRCF), (ii) inoculated RCF on autoclave-sterilized soil (Abiotic-HNRCF), (iii) inoculated RCF without soil but amended with sterilized soil extracts (Abiotic-LNRCF), and (iv) sterilized cellulose–Congo red agar medium (CCRA). A fifth uninoculated RCF (UninocRCF) treatment on unsterilized soil was included as control to check for contamination from the soil, and all the treatments were replicated thrice. Following 20 d of incubation, the developing communities from all treatments were characterized using 16S rRNA gene clone libraries. Our results showed that nutrient levels had a small effect on the growing communities, and as hypothesized, the community growing in association with the unsterilized soil (BioticRCF) was the richest and most unique among all treatments. Previously uncultured members of the phyla Proteobacteria, Bacteroidetes, and few members of Verrucomicrobia, Planctomycetes, and OP10 were detected on the BioticRCF treatment. Members of the phylum Firmicutes were the dominating bacteria in abiotic treatments, followed by the members of phylum Planctomycetes. The CCRA medium supported the growth of a less diverse community, with ∼91% of the sequences closely related to isolates of the phyla Proteobacteria, Bacteroidetes, and Firmicutes. The presence or absence of a living microbial community appears to have a significant impact on the richness and structure of bacterial community growth and development.

In vitro biofilm formation by uropathogenic Escherichia coli and their antimicrobial susceptibility pattern

ObjectiveTo detect in vitro biofilm formation of uropathogenic Escherichia coli (E. coli) (UPEC) strains isolated from urine specimens and also to determine their antimicrobial susceptibility pattern using 13 commonly used antibiotics. MethodsThe present study comprised of 166 urine specimens collected from tertiary care hospitals in and around Coimbatore, South India. All the specimens were subjected to gram staining, bacterial culture and the E. coli strains were screened for biofilm formation using Tube Method (TM), Congo Red Agar (CRA) and Tissue Culture Plate method (TCP) respectively. Subsequently, the antimicrobial susceptibility test was performed by Kirby Bauer-disk diffusion method for the biofilm and non-biofilm producing E. coli strains. ResultsOf the 100 (60.2 %) E. coli strains, 72 strains displayed a biofilm positive phenotype under the optimized conditions in the Tube Method and the strains were classified as highly positive (17, 23.6%), moderate positive (19, 26.3 %) and weakly positive (36, 50.0 %), similarly under the optimized conditions on Congo Red agar medium, biofilm positive phenotype strains were classified as highly positive (23, 23 %), moderate positive (37, 37 %) and weakly positive (40, 40%). While in TCP method, the biofilm positive phenotype strains were also classified as highly positive (6, 6 %), moderate positive (80, 80 %) and weakly positive (14, 14 %), it didn't not correlate well with the tube method for detecting biofilm formation in E. coli. The rates of antibiotic resistance of biofilm producing E. coli were found to be 100 % for chloramphenicol and amoxyclav (amoxicillin and clavulanic acid), 86% for gentamicin and cefotaxime, 84% for ceftazidime, 83% for cotrimoxazole and piperacillin/tazobactam, 75% for tetracycline and 70% for amikacin. ConclusionsThis study reveals the prevalence and antimicrobial susceptibility pattern of biofilm and non-biofilm producing uropathogenic E. coli strains.