Congo Red Agar Plate Research Articles

Study on E. coli and Salmonella biofilms from fresh fruits and vegetables.

Foodborne outbreaks associated with fresh fruits and vegetables are on the rise worldwide. Biofilm formation is one of the important traits of pathogens making them strongly attached to substrates as well as express virulence phenotypes. Present study investigates the biofilm forming ability of E. coli and Salmonella sp. isolated from fresh fruits and vegetables. A total of 53 strains, including 35 E. coli and 18 Salmonella sp. isolated from different fruit and vegetable samples were taken into account for the study. Initial screening for biofilm formation was done using Congo Red agar plate test. Results revealed that 22.8% E. coli and 22.2% Salmonella sp. were potential biofilm formers. However, the MTP (Micro-Titre Plate) assay suggested more isolates of both E. coli and Salmonella sp. were moderate to strong biofilm producers. Agar plate diffusion assay with Agrobacterium tumefaciens NTL-4 showed the production of quorum signaling molecules (AHLs) by three isolates of E. coli and one Salmonella sp. Two E. coli isolates showed a significant amount of EPS production indicating higher biofilm forming potential. The Presence of LUX R homologue gene (sdiA) in two of the Salmonella isolates were confirmed by PCR which demonstrated their potential pathogenicity. Results of the work underline the biofilm forming and potentially virulent capacities of isolates from the surface of fruits and vegetables.

Biofilm formation and antimicrobial susceptibility of staphylococci and enterococci from osteomyelitis associated with percutaneous orthopaedic implants.

Staphylococci and enterococci account for most deep infections associated with bone-anchored percutaneous implants for amputation treatment. Implant-associated infections are difficult to treat; therefore, it is important to investigate if these infections have a biofilm origin and to determine the biofilm antimicrobial susceptibility to improve treatment strategies. The aims were: (i) to test a novel combination of the Calgary biofilm device and a custom-made susceptibility MIC plate (Sensititre® ), (ii) to determine the biofilm formation and antimicrobial resistance in clinical isolates causing implant-associated osteomyelitis, and (iii) to describe the associated clinical outcome. Enterococci and staphylococci were characterized by microtitre plate assay, Congo Red Agar plate test, and PCR. Biofilm susceptibility to 10 antimicrobials and its relationship to treatment outcomes were determined. The majority of the strains produced biofilm in vitro showing inter- and intraspecies differences. Biofilms showed a significantly increased antimicrobial resistance compared with their planktonic counterparts. Slime-producing strains tolerated significantly higher antimicrobial concentrations compared with non-producers. All seven staphylococcal strains carried ica genes, but two did not produce slime. The degree of biofilm formation and up-regulated antibiotic resistance may translate into a variable risk of treatment failure. This new method set-up allows for the reproducible determination of minimum biofilm eradication concentration of antimicrobial agents, which may guide future antimicrobial treatment decisions in orthopaedic implant-associated infection. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2630-2640, 2017.

Biofilm and toxin profile: A phenotypic and genotypic characterization of coagulase-negative staphylococci isolated from human bloodstream infections

Coagulase-negative staphylococci (CNS) represent one of the most prevalent microorganisms in nosocomial infections worldwide, nevertheless little is known about their pathogenicity features. Thus, our aim was to characterize virulence aspects of CNS isolated from patients with bloodstream infections assisted in hospitals of Belo Horizonte, MG, Brazil. Strains were identified using bioMérieuxVitek® and for biofilm production evaluation, Congo Red Agar (CRA) and polystyrene plates were used. PCR was applied to detect icaA, icaB, icaC, atlE, sea, sec, sed, tsst-1 and agr. For statistical analyses were used hierarchical cluster, chi-square test and correspondence. 59 strains were analyzed, being S. haemolyticus the most prevalent. On CRA, 96.5% were biofilm producer, whereas on polystyrene plate, 100% showed adhesion at different times evaluated. Regarding genotypic analyses, 15.2%, 38.9%, 8.4%, 49.1%, 76.2%, 23.7%, 1.6%, 30.5% and 38.9% were positive for icaA, icaB, icaC, atlE, sea, sec, sed, tsst-1 and agr, respectively. Six clusters were formed and frequency distributions of agr, atlE, icaA, icaB, sea, sec, tsst-1 differed (P < 0.001). In conclusion, all strains were biofilm producer, with high prevalence of atlE, and had potential of toxin production, with high prevalence of sea. According to the group-analyses, icaB showed relationship with the strong adherence in samples.

Chitosanase purified from bacterial isolate Bacillus licheniformis of ruined vegetables displays broad spectrum biofilm inhibition

A number of bacterial species produces chitosanases which has variety of applications because of its high biodegradability, non-toxicity and antimicrobial assets. In the present study chitosanase is purified from new bacterial species Bacillus licheniformis from spoiled vegetable. This novel strain of Bacillus licheniformis isolated from spoilt cucumber and pepper samples has the ability to produce the chitosanase enzyme when grown on chitosan substrate. Study also examined its antibiofilm properties against diverse bacterial species with biofilm forming ability. The purified chitosanase inhibited the biofilm formation ability for all Gram-negative and Gram-positive biofilm-forming bacteria [biofilm producers] tested in this study in congo red agar and microtiter plate's methods. Highly antibiofilm activity of chitosanase was recorded against Pseudomonas aeruginosa followed by Klebsiella pneumoniae with reduction of biofilm formation upto 22 and 29%, respectively compared with [100] % of control. Biofilm formation has multiple role including ability to enhance resistance and self-protection from external stress. This chitosanase has promising benefit as antibiofilm agent against biofilm forming pathogenic bacteria and has promising application as alternative antibiofilm agents to combat the growing number of multidrug resistant pathogen-associated infections, especially in situation where biofilms are involved.

Biofilm Producing Staphylococcus epidermidis Strains Isolated From Clinical Samples in Tehran, Iran

Background: Staphylococcus epidermidis is well documented as an opportunistic pathogen causing biofilm in patients and healthy individuals. Objectives: The aim of this experimental study was to describe the antibiotic resistance patterns of biofilm producing S. epidermidis strains isolated from clinical samples in Tehran, Iran. Moreover, the role of different genes in biofilm formation was also described. Patients and Methods: A total of 250 S. epidermidis strains were isolated from patients in a private hospital of Tehran, Iran from February to December 2014. The biofilm formation of each strain was determined using combination of qualitative Congo-Red agar and quantitative microtiter plate assay, and presence of different genes involved in control and formation of biofilm was detected by the polymerase chain reaction (PCR). Susceptibility of S. epidermidis strains to 19 antibiotics was examined. Results: The results of the biofilm assay revealed that 82% of strains produced black colonies on Congo red agar plates and 68% were able to attach strongly to polystyrene microplates. One hundred percent, 88%, 84%, 64% and 60% of biofilm-producing strains were resistant to penicillin, cefoxitin, erythromycin, trimethoprim-sulfamethoxazole and kanamycin, respectively. On the other hand, none of the strains showed resistance to vancomycin, linezolid, quinupristin/dalfopristin. The icaA, icaD, aap and atlE genes were detected in all biofilm-producing strains and presence of IS256 transposon was limited to 84% of biofilm positive strains. Conclusions: The results of this study illustrated the high prevalence of antibiotic resistant biofilm-producing S. epidermidis strains in this hospital, which could be a reservoir for antibiotic resistance genes.

Detection of methicillin resistant and slime factor production of coagulase negative Staphylococcus spp. in bovine clinical mastitis by using PCR

This study aims to investigate the slime production of Coagulase negative staphylococci (CoNS) isolates by phenotypic method on Congo Red Agar plates (CRA) and Genotypic detection of icaA, icaD and mecA genes by polymerase chain reaction (PCR). Out of 105 milk samples obtained from clinical bovine mastitis, 101samples (96.2%) were positive for bacterial growth. CoNS isolates was detected in 20 isolates with a percentage of 19.8%. Their ability to form biofilm as one of the most important virulence factors of the organisms using Congo Red Agar (CRA) method was investigated in which 13 out of 17 CoNS isolates (76.47%) were found to be slime producers. By PCR, mecA gene was found in threeout of 6 CoNS isolates (50%). Also six (100%) and three (50%) isolates were positive for icaA gene and icaD gene, respectively. In addition one isolate out of the six CoNS isolates (16.67%) was positive for the presence of icaA, icaD and mecA genes and also has the ability to form biofilm. The in vitro activities of CoNS against 11 selected antimicrobial agents referred that the highest resistance rate of CoNS observed to Lincomycin (100%), followed by Cefotaxime (94.41%), Oxacillin (58.82%), Ampicillin (47.06%) and Penicillin (41.18%), while the highest rate of sensitivity observed to Enrofloxacin and Gentamicin (100%, for each), followed by Doxycycline (94.11%).Conclusion, the findings of the present study demonstrated the ability of CoNS isolated from bovine clinical mastitis to form biofilms. This must be considered as an alarming situation, and so attention must be paid toward implementation of new ways for effective prophylaxis, control, and treatment of such infections in the dairy farms. The prudent use of antibiotics and rapid and continuous screening for resistant microorganisms should be more focused to prevent the emergence and spread methicillin resistant coagulase negative staphylococci, because these strains can cause severe damage to infected sites and may be widespread in the environment.

Prospective study on the diagnostics and susceptibility testing of infections associated with osseointegrated percutaneous implants

Event Abstract Back to Event Prospective study on the diagnostics and susceptibility testing of infections associated with osseointegrated percutaneous implants Magdalena Zaborowska1, 2, Rickard Brånemark2, 3, 4, Joakim Strömberg3, Peter Thomsen1, 2 and Margarita Trobos1, 2 1 Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Department of Biomaterials, Sweden 2 University of Gothenburg, BIOMATCELL VINN Excellence Center of Biomaterials and Cell Therapy, Sweden 3 Center for Advanced Reconstruction of Extremities, Sahlgrenska University Hospital, Department of Orthopaedics, Sweden 4 Department of Orthopaedics, University of California, International Center for Osseointegration Research Education and Surgery (iCORES), United States Introduction: Biomaterial-associated infections (BAI) are among the main reasons for implant failure, often leading to long-term antimicrobial treatment and implant removal. BAI are mainly caused by biofilm producing bacteria with increased antimicrobial resistance. Proper and rapid diagnosis is important for successful treatment. Sonication is an alternative method for the diagnosis of prosthetic infections with higher sensitivity than standard tissue cultures[1]. The aims of the study were to: i) identify and quantify bacteria causing osteomyelitis associated with osseointegrated percutaneous amputation prostheses by sonication of explanted implants; ii) characterise the isolated strains by their biofilm production abilities and antimicrobial susceptibility. In parallel, the diagnostic laboratory at the hospital performed standard culturing techniques of bone tissue and blood marrow samples. Materials and Methods: A bone-anchored part (fixture) of a prosthesis was extracted due to suspicion of infection according to clinical evaluation. The retrieved fixture was placed in sterile saline, sonicated at 47 KHz for 5 min and vortexed for 1 min. The implant was removed, the remaining solution was centrifuged, pellet was re-suspended in saline and quantitative cultures were made on general purpose agar (blood agar for aerobic and brucella agar for anaerobic cultures) and selective agar plates (staphylococci agar, enterococci agar, streptococci agar, chromogenic orientation agar and pseudomonas agar). Crystal violet (CV) staining and Congo Red (CR) agar plate test was performed to evaluate the biofilm production. Identification of the staphylococcal strains was performed using API Staph. The Calgary Biofilm Device and a custom-made antimicrobial susceptibility plate were used to determine minimum biofilm eradication concentrations (MBEC) and minimum inhibitory concentrations (MIC) to 10 antimicrobial agents. Results and Discussion: We report the first results of an ongoing prospective study where the method has been tested real-time. In the first patient, Enterococcus faecalis (5x104 CFU/implant) and Escherichia coli (102 CFU/implant) were detected after sonication. These strains were both biofilm-producing strains according to CV and CR tests. Biofilms showed increased tolerance to the antimicrobials compared with the standard planktonic MIC values (Table 1). Escherichia coli was the only species detected by standard tissue cultures; in contrast, by the use of sonication, Enterococcus faecalis was further identified indicating the possibility of a polymicrobial infection with potential implications for the choice of the optimal treatment strategy for the patient. Conclusions: An increased detection of the potential causative agents by sonication of retrieved implants may improve the current diagnostics of BAI. In addition, determination of MBEC for clinically relevant antimicrobial agents has the potential to optimize the treatment choices for patients. The BIOMATCELL VINN Excellence Center of Biomaterials and Cell Therapy, the Västra Götaland Region, the Swedish Research Council (K2015-52X-09495-28-4), an LUA/ALF Research Grant (ALFGBG-138721), the IngaBritt and Arne Lundberg Foundation, the Wilhelm and Martina Lundgren Foundation, the Handlanden Hjalmar Svensson Foundation, Doctor Felix Neubergh Foundation and the Area of Advance Materials of Chalmers and GU Biomaterials within the Strategic Research Area initiative launched by the Swedish Government

Extraction and Purification of L-Asparaginase Produced by Acinetobacter Baumannii and their Antibiofilm Activity Against Some Pathogenic Bacteria

L-asparaginase is an enzyme catalyzing the hydrolysis of L-asparagine and formation of L-aspartate and ammonia and widely used as anticancer drug in pharmaceutical and food industry. This amino acid (L-asparagine) has an important role for development of cancerous; in contrast the normal cells don’t need this amino acid. Eight Acinetobacter baumannii isolates of were isolated from different blood and sputum samples and it was found that high isolation rate of Acinetobacter baumannii isolates from sputum was consisted with their association with lower respiratory tract infections. All these eight isolates were screened for higher L-asparaginase production and found that among all these isolates, Acinetobacter baumannii Sp3 gave higher asparaginase activity of 7.32 U/ml. L-asparaginase was purified to homogeneity by sequential chromatographic steps involved ammonium sulfate at 45% saturation followed by DEAE-cellulose ion exchange chromatography and sephadex G-100 gel filtration chromatography with a recovery yield of 68% and 22.65 fold of purification. L-asparaginase had antibiofilm activity against all tested biofilm forming pathogenic bacteria (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus and Acinetobacter baumannii) after using Congo red agar and Microtitration plates methods for detecting biofilm formation ability. Highly antibiofilm of L-asparaginase recorded against Klebsiella pneumoniae followed by Pseudomonas aeruginosa with reduction of biofilm formation ratio to 32 and 41% ,respectively compared with (100)% of control. Thus we can conclude that L-asparaginase has promising benefit as antibiofilm agent against biofilm forming pathogenic bacteria that have multidrug resistance.

Bacteriological study on staphylococcal bovine clinical mastitis with reference to methicillin-resistant Staph. aureus (MRSA)

This descriptive study was done on 101 milk samples obtained from clinically mastitic dairy cows in Assiut Governorate, Egypt. Staphylococcus aureus (S. aureus) was the main causative agent of clinical mastitis (34.65%) followed by S. saprophyticus (10.89%), S. intermedius and S. epidermidis (8.91%, for each). The other causative agents (non Staph. Spp.) were identified. Sensitivity test of S. aureus isolates was performed against 11 antimicrobial agents, where found that 21 S. aureus strains (60%) were methicillin resistant S. aureus (MRSA). Ten MRSA strains were subjected for: I- slime-producing factor on Congo Red Agar (CRA) plates phenotypically, as 6 isolates (60%) were positive for slime production. II- PCR which was optimized targeting mecA, icaA and icaD genes, where 5 isolates (50%) were positive for mecA gene. Six isolates (60%) and 8 isolates (80%) were positive for icaA and icaD genes, respectively. Five strains (50%) were positive for both icaA and icaD genes. Also 3 strains (30%) were positive for all mecA, icaA and icaD genes. Conclusion, it was concluded that bovine staphylococcal mastitis was the most predominant issue where S. aureus was the main cause. Detection of mecA gene in S. aureus isolates indicating that several cases suffering from S. aureus mastitis have an MRSA problem. Genotypic determination of mecA gene proved the most reliable method for detection of methicillin-resistant S. aureus. The present work paid an attention to the 3 MRSA strains (30%) were positive to all tested genes rather than slime production as the worst isolated strains all over this study (multidrug resistant, slime producing as well as carrying mecA, icaA and icaD genes). In vitro Enrofloxacin, Gentamicin and Doxycycline the most effective drugs for Staph. spp. clinical mastitis and should be recommended for treatment of such cases of bovine mastitis.

Biofilm production and antibiotic susceptibility profiles of Staphylococcus strains isolates from urinary catheter at the university hospital center of Tlemcen (Algeria)

Staphylococci are a major cause of infections associated with urinary catheterization and other medical devices. The ability of Biofilm production is an important step in the pathogenesis of these Staphylococci and depends on the expression of the icaADBC operon leading to the synthesis of a polysaccharide intercellular adhesion. In this study , 44 staphylococci isolates obtained from urinary catheter at the university hospital center (UHC) of Tlemcen (North-West Algeria) were analyzed for detecting the presence or absence of the intercellular adhesion icaA and icaD genes by polymerase chain reaction, phenotypic biofilm production was examined by tissue culture plate (TCP) and Congo red agar (CRA) methods. 17of 44 isolates were shown to carry ica -specific DNA, 18 produced slime on CRA plates but only 8 produced biofilm spontaneously on the polystyrene surfaces. Staphylococci strains isolated from urinary catheter showed high levels of resistance to penicillin (98%) and gentamicine (75%). The data reported indicate an important role of ica genes, phenotypic variability of biofilm

Relevance of biofilm formation and virulence of different species of coagulase-negative staphylococci to public health.

The ability of biofilm formation seems to play an essential role in the virulence of coagulase-negative staphylococci (CNS). The present work aimed to: (a) evaluate the biofilm-forming ability of different strains of CNS field isolates; (b) evaluate their virulence potential through the assessment of the Madin-Darby canine kidney (MDCK) cytotoxicity assay; (c) determine the frequency of biofilm-associated genes among these CNS isolates. Biofilm markers associated with biofilm formation and MDCK cells cytotoxicity were compared to find possible associations with pathogenicity. CNS isolates (n = 94) belonging to 11 different species were tested for slime production using the tube test (TA) and the Congo red agar plate test (CRA), while the presence of icaA and icaD genes were evaluated by d-PCR. Two points were addressed for the first time: (1) the specific relationship between slime phenotype and icaD gene expression; (2) the specific relationship between slime phenotype, icaAD genes, and MDCK cytotoxicity. The proportion of biofilm-positive/icaD-positive versus biofilm-positive/icaD-negative strains was 9:0 and 9:0 (81.8%) by the TA and CRA, which clearly indicates that icaD was a more reliable gene to be accounted for in the biofilm formation. MDCK recorded a higher proportion than that recorded by the CRA and TA results (MDCK-positive/icaD-positive versus MDCK-positive/icaD-negative 10:0, 90.9%). Evaluation of the ica operon, CRA plate test, TA, and MDCK can contribute to the high clinical impact in the management of antibiotic therapy, in infections associated with devices in veterinary medicine, the dairy industry, and food processing.

DETECTION OF METHICILLIN RESISTANT AND SLIME FACTOR PRODUCTION OF COAGULASE NEGATIVE STAPHYLOCOCCUS SPP. IN BOVINE CLINICAL MASTITIS BY USING PCR

The aim of this study was to determine and investigate the slime production of Coagulase negative staphylococci (CoNS) isolates by phenotypic method on Congo Red Agar plates (CRA) and Genotypic detection of icaA, icaD and mecA genes by polymerase chain reaction (PCR). The study was done on 105 milk samples obtained from bovine clinical mastitis and found that 101samples (96.2%) were positive for bacterial culture. CoNS species can be isolated from20 samples with a percentage 19.8%. Their ability to form biofilm as one of the most important virulence factors using Congo Red Agar (CRA) method was investigated, in which 13 out of 17 CoNS isolates (76.47%) were found to be slime producers. By PCR, mecA gene was found in threeout 6 CoNS isolates (50%). Also, six (100%) and three (50%) isolates were positive for icaA gene and icaD gene, respectively, and 3 isolates (50%) were positive for both icaA and icaD genes. In addition, one out of the six CoNS isolates (16.67%) was positive for the presence of icaA, icaD and mecA genes and also it has the ability to form biofilm. Conclusion, findings of the present study demonstrated the ability of CoNS isolated from bovine clinical mastitis to form biofilms. This must be considered as an alarming situation, and so attention must be paid toward implementation of new ways for effective prophylaxis, control, and treatment of such infections in the dairy farms. The prudent use of antibiotics and rapid and continuous screening for resistant microorganisms should be more focused to prevent the emergence and spread methicillin resistant coagulase negative staphylococci, because these strains can cause severe damage to infected sites and may be widespread in the environment.

Detection of Biofilm Formation in Uropathogenic Bacteria = الكشف عن تكون البيوفيلم في البكتيريا المسببة للأمراض البولية

Urinary tract infection (UTI) is the most commonly acquired bacterial infection. Biofilm formation being the prime cause for antibiotic resistance by uropathogenes. The purpose of this study was to detect biofilm formation by uropathogenes isolated from UTIs, their antibiotic susceptibility pattern and detection of genes responsible for biofilm production in some isolates. The isolated bacteria were tested for biofilm production by tube adherence (TA), congo red agar (CRA) and micro titer plate (MtP) methods. Antibiotic susceptibility pattern of uropathogens was done by Kirby-Bauer disc diffusion method and detection of genes responsible for biofilm production in some isolates by PCR. Out of 320 cultures positive cases 146 (46.5 %) isolates were biofilm producers Ecoli was the commonest uropathogen followed by Staphylococcus aureus and Enterococcus feacalis. The antibiotic resistance was higher among biofilm producers to commonly used antibiotics as compared to non biofilm producers. The detection of icaD gene of Staph. areus and cup gene ofpseudomonas by PCR was siginfiacntly related to its detection by congo red agar method, while there no significant relation between detection of ESP gene by PCR and the three used conventional methods. It was concluded that the ability of slime production, adherence and biofilm formation of uropathogenic strains had significant role in antibiotic resistance. Biofilm formation is the major virulence determinant of uropathogen, so it is necessary to screen all urinary isolates for biofilm production.

Detection of Biofilm Production and Antibiotic Resistance Pattern in Clinical Isolates from Indwelling Medical Devices

Microbial biofilms pose great threat for patients requiring indwelling medical devices (IMDs) as it is difficult to remove them. It is, therefore, crucial to follow an appropriate method for the detection of biofilms. The present study focuses on detection of biofilm formation among the isolates from IMDs. We also aimed to explore the antibiogram of biofilm producers. This prospective analysis included 65 prosthetic samples. After isolation and identification of bacteria following standard methodology, antibiogram of the isolates were produced following Kirby-Bauer disc diffusion method. Detection of biofilms was done by tube adherence (TA), Congo red agar and tissue culture plate (TCP) methods. Out of 67 clinical isolates from IMDs, TCP detected 31 (46.3 %) biofilm producers and 36 (53.7 %) biofilm non-producers. Klebsiella pneumoniae, Pseudomonas aeruginosa and Burkholderia cepacia complex were found to be the most frequent biofilm producers. The TA method correlated well with the TCP method for biofilm detection. Higher antibiotic resistance was observed in biofilm producers than in biofilm non-producers. The most effective antibiotics for biofilm producing Gram-positive isolates were Vancomycin and Tigecycline, and that for biofilm producing Gram-negative isolates were Polymyxin-B, Colistin Sulphate and Tigecycline. Nearly 46 % of the isolates were found to be biofilm producers. The antibiotic susceptibility pattern in the present study showed Amoxicillin to be an ineffective drug for isolates from the IMDs. For the detection of biofilm production, TA method can be an economical and effective alternative to TCP method.

Association of multicellular behaviour and drug resistance in Salmonella enterica serovars isolated from animals and humans in Ethiopia.

To determine the association between multicellular behaviour, integron status and antibiotic resistance among 87 Ethiopian Salmonella enterica isolates of animal and human origin. Isolates were characterized for their biofilm forming ability, antimicrobial susceptibility and the presence and characteristics of a class 1 integron and Salmonella genomic island 1 (SGI1). The majority of isolates grown at environmental temperatures (20°C) exhibited robust biofilm formation (72·4%) and displayed RDAR colony morphology on Congo red agar plates. The presence of a class 1 integron correlated with the extent of drug resistance and ability to exhibit multicellular behaviour. Although cellulose production and RDAR morphology correlated with increased multicellular behaviour, neither was required for biofilm formation. Contrary to previous reports, colony morphology was generally consistent within a serovar. No integrons were detected in isolates deficient for multicellular behaviour, indicating a potential role of bacterial community formation in transfer of genetic elements among environmental isolates. Infection by Salm. enterica is a major public health problem worldwide. The dominance of multidrug resistance and multicellular behaviour in Salmonella isolates of Ethiopian origin highlights a need for integrated surveillance and further detailed phenotypic and molecular studies of isolates from this region.

Characterization of the growth dynamics and biofilm formation of Staphylococcus epidermidis strains isolated from contaminated platelet units

Bacterial contamination of platelet concentrates (PCs) poses the highest transfusion-associated infectious risk, with Staphylococcus epidermidis being a predominant contaminant. Herein, the growth dynamics of 20 S. epidermidis strains in PCs and regular media were characterized. Strains were categorized as fast (short lag phase) or slow (long lag phase) growers in PCs. All strains were evaluated for the presence of the biofilm-associated icaAD genes by PCR, their capability to produce extracellular polysaccharide (slime) on Congo red agar plates and their ability to form surface-attached aggregates (biofilms) in glucose-supplemented trypticase soy broth (TSBg) using a crystal violet staining assay. A subset of four strains (two slow growers and two fast growers) was further examined for the ability for biofilm formation in PCs. Two of these strains carried the icAD genes, formed slime and produced biofilms in TSBg and PCs, while the other two strains, which did not carry icaAD, did not produce slime or form biofilms in TSBg. Although the two ica-negative slime-negative strains did not form biofilms in media, they displayed a biofilm-positive phenotype in PCs. Although all four strains formed biofilms in PCs, the two slow growers formed significantly more biofilms than the fast growers. Furthermore, growth experiments of the two ica-positive strains in plasma-conditioned platelet bags containing TSBg revealed that a slow grower isolate was more likely to escape culture-based screening than a fast grower strain. Therefore, this study provides novel evidence that links S. epidermidis biofilm formation with slow growth in PCs and suggests that slow-growing biofilm-positive S. epidermidis would be more likely to be missed with automate culture.

Gum arabic capped‐silver nanoparticles inhibit biofilm formation by multi‐drug resistant strains of Pseudomonas aeruginosa

Clinical isolates (n = 55) of Pseudomonas aeruginosa were screened for the extended spectrum β-lactamases and metallo-β-lactamases activities and biofilm forming capability. The aim of the study was to demonstrate the antibiofilm efficacy of gum arabic capped-silver nanoparticles (GA-AgNPs) against the multi-drug resistant (MDR) biofilm forming P. aeruginosa. The GA-AgNPs were characterized by UV-spectroscopy, X-ray diffraction, and high resolution-transmission electron microscopy analysis. The isolates were screened for their biofilm forming ability, using the Congo red agar, tube method and tissue culture plate assays. The biofilm forming ability was further validated and its inhibition by GA-AgNPs was demonstrated by performing the scanning electron microscopy (SEM) and confocal laser scanning microscopy. SEM analysis of GA-AgNPs treated bacteria revealed severely deformed and damaged cells. Double fluorescent staining with propidium iodide and concanavalin A-fluorescein isothiocyanate concurrently detected the bacterial cells and exopolysaccharides (EPS) matrix. The CLSM results exhibited the GA-AgNPs concentration dependent inhibition of bacterial growth and EPS matrix of the biofilm colonizers on the surface of plastic catheters. Treatment of catheters with GA-AgNPs at 50 µg ml(-1) has resulted in 95% inhibition of bacterial colonization. This study elucidated the significance of GA-AgNPs, as the next generation antimicrobials, in protection against the biofilm mediated infections caused by MDR P. aeruginosa. It is suggested that application of GA-AgNPs, as a surface coating material for dispensing antibacterial attributes to surgical implants and implements, could be a viable approach for controlling MDR pathogens after adequate validations in clinical settings.

BACTERIOLOGICAL STUDY ON STAPHYLOCOCCAL BOVINE CLINICAL MASTITIS WITH REFERENCE TO METHICILLIN-RESISTANT STAPH. AUREUS (MRSA)

This descriptive study was done on 101 milk samples obtained from clinically mastitic dairy cows in Assiut Governorate, Egypt. Staphylococcus aureus (S. aureus) was the main causative agent of clinical mastitis (34.65%) followed by S. saprophyticus (10.89%), S. intermedius and S. epidermidis (8.91%, for each). The other causative agents (non Staph. Spp .) were identified. Sensitivity test of S. aureus isolates was performed against 11 antimicrobial agents, where found that 21 S. aureus strains (60%) were methicillin resistant S. aureus (MRSA). Ten MRSA strains were subjected for: I- slime-producing factor on Congo Red Agar (CRA) plates phenotypically, as 6 isolates (60%) were positive for slime production. II- PCR which was optimized targeting mecA, icaA and icaD genes, where 5 isolates (50%) were positive for mecA gene. Six isolates (60%) and 8 isolates (80%) were positive for icaA and icaD genes, respectively. Five strains (50%) were positive for both icaA and icaD genes. Also 3 strains (30%) were positive for all mecA, icaA and icaD genes. Conclusion, it was concluded that bovine staphylococcal mastitis was the most predominant issue where S. aureus was the main cause. Detection of mecA gene in S. aureus isolates indicating that several cases suffering from S. aureus mastitis have an MRSA problem. Genotypic determination of mecA gene proved the most reliable method for detection of methicillin-resistant S. aureus. The present work paid an attention to the 3 MRSA strains (30%) were positive to all tested genes rather than slime production as the worst isolated strains all over this study (multidrug resistant, slime producing as well as carrying mecA, icaA and icaD genes). In vitro Enrofloxacin, Gentamicin and Doxycycline the most effective drugs for Staph. spp. clinical mastitis and should be recommended for treatment of such cases of bovine mastitis.

English

In view of compliance with increasingly stringent environmental legislation imposed by regional, national and supranational (for example, European Union) authorities, innovative environmental technologies for the treatment of dye-contaminated effluents are necessary in the color industry. In this study, azo (methyl orange (MO) and methyl red (MR)) and triphenylmethanes (malachite green (MG), fushin (F) and crystal violet (CV)) dyes were subjected to distinct treatment strains following an initial qualitative characterization. The effectiveness of their biodegradation using Staphylococcus epidermidis strain was compared with respect to parameters such as residual color, and toxicity on human cells. The Hep-2 cells were exposed to three azo and two triphenylmethane dyes at various concentrations. After 72 h exposure, the protein contents of the samples compared to the protein contents of non-exposed cells were measured. The level of protein content indicates the viability of the cells. The IC20 values show the limiting value of toxicity. The IC50 values before biodegradation show whether samples are clearly toxic. The IC50 value for the azo dyes (MO, MR) were 65 and 10 &mu;g/ml, the IC20 value were 10 and 5 &mu;g/ml. The IC50 for the triphenylmethane dyes (MG, F and CV) were 35, 25 and 30 &mu;g/ml, the IC20 value were 5, 5 and 10 &mu;g/ml. The viability of the cells was good, the protein content of the samples being over 80% compared to the non-exposed cells. The Hep-2 cell test indicated the toxicity of pure dyes before biodegradation; the dyes after decolorization had no adverse effect. The human keratinocyte Hep-2 cells seem to be a useful tool for the study of the purity/toxicity of dyes and other substances applied to textiles. In addition, the initial cytotoxicity of dyes was still present after the biodegradation, while it was completely removed through the bacterial treatment. Our results show that S. epidermidis was a slime-producer developing almost black colonies on Congo red agar plate. A significant increase in azoreductase, lignin peroxidase and laccase activities in the cells were obtained after complete decolorization. &nbsp; Key words: Azo dye, triphenylmethane dye, Staphylococcus epidermidis, cytotoxicity, slime production.

English

Staphylococcus spp. is a major cause of infections associated with urinary catheterization and other medical devices. Biofilm formation is an important step in the pathogenesis of these Staphylococci and depends on the expression of the icaADBC operon involved in the synthesis of a polysaccharide intercellular adhesion. In this study, forty-four (44) Staphylococcus spp. obtained from urinary catheters at the University Hospital of Tlemcen (North-West Algeria) were analyzed to investigate the presence or absence of the intercellular adhesion icaA and icaD genes, using the polymerase chain reaction (PCR). Phenotypic biofilm formation was examined by tissue culture plate (TCP) and Congo red agar (CRA) methods. Seventeen (17) of 44 isolates were shown to carry ica-specific DNA, 18 produced slime on CRA plates but only eight produced biofilm spontaneously on the polystyrene surfaces, under normal growth conditions. Upon induction by sugar, biofilm formation could be stimulated in seven (7) of nine (9)ica positive. Biofilm-negative isolates indicated that the icaADBC expression was down-regulated in these strains. Staphylococcus strains isolated from urinary catheters showed high levels of resistance to penicillin (98%) and gentamicine (75%). The data obtained shows the important role of ica-genes, the phenotypic variability of biofilm formation and the multi-resistance to antibiotics as virulence factors of staphylococcus spp. from urinary catheters.It confirms the complexity and diversity of regulation mechanisms implicated in biofilm formation. &nbsp; Key words: Staphylococcus spp., urinary catheter, Biofilm, ica operon, slime, tissue culture plate (TCP).