Exploring the use of Congo red agar in improving the serotyping of non-serotypeable Shigella with In-silico evidence.

OBJECTIVES: To assess if congo red could make non-serotypeable Shigella strains serotypeable and to employ molecular docking to determine the basis of the same phenomenon. METHODS: We used 42 bacterial strains of non-serotypeable Shigella collected from 2012 to 2019 for this study. Each bacterial strain was freshly inoculated onto congo red agar and incubated at 37° C for 18-24 h. Bacterial colonies obtained were re-subjected to biochemical tests followed by serotyping and serogrouping. In-silico studies to investigate the interaction between MxiC protein of T3SS and O-antigen LPS with congo red were performed. RESULTS: Of the total 42 non-serotypeable Shigella studied, (26/42)62% were capable of being serotyped following the use of congo red agar, 65% were Shigella flexneri, 19% were Shigella dysenteriae, while 2 strains (7%) each of Shigella boydii and Shigella sonnei were detected. We observed no change in their biochemical properties. The in-silico molecular docking studies revealed high binding affinity between congo red and the B-Chain of Mxi C. Out of the 5 chains of the O-Antigen, congo red showed robust binding with the B-chain with the involvement of a cluster of hydrophobic interactions between them. This may have a crucial role in the conversion of non-serotypeable strains to serotypeable strains on exposure to congo red as observed in our study. CONCLUSION: Congo red agar as a medium converts a sizeable percentage of non-serotypeable Shigella strains to serotypeable Shigella strains.

Lack of Direct Correlation between Biofilm Formation and Antimicrobial Resistance in Clinical Staphylococcus epidermidis Isolates from an Italian Hospital.

Staphylococcus epidermidis is an opportunistic pathogen and a frequent cause of nosocomial infections. In this work, we show that, among 51 S. epidermidis isolates from an Italian hospital, only a minority displayed biofilm formation, regardless of their isolation source (peripheral blood, catheter, or skin wounds); however, among the biofilm-producing isolates, those from catheters were the most efficient in biofilm formation. Interestingly, most isolates including strong biofilm producers displayed production levels of PIA (polysaccharide intercellular adhesin), the main S. epidermidis extracellular polysaccharide, similar to reference S. epidermidis strains classified as non-biofilm formers, and much lower than those classified as intermediate or high biofilm formers, possibly suggesting that high levels of PIA production do not confer a particular advantage for clinical isolates. Finally, while for the reference S. epidermidis strains the biofilm production clearly correlated with the decreased sensitivity to antibiotics, in particular, protein synthesis inhibitors, in our clinical isolates, such positive correlation was limited to tetracycline. In contrast, we observed an inverse correlation between biofilm formation and the minimal inhibitory concentrations for levofloxacin and teicoplanin. In addition, in growth conditions favoring PIA production, the biofilm-forming isolates showed increased sensitivity to daptomycin, clindamycin, and erythromycin, with increased tolerance to the trimethoprim/sulfamethoxazole association. The lack of direct correlation between the biofilm production and increased tolerance to antibiotics in S. epidermidis isolates from a clinical setting would suggest, at least for some antimicrobials, the possible existence of a trade-off between the production of biofilm determinants and antibiotic resistance.

Anti-biofilm potential of Lactobacillus plantarum Y3 culture and its cell-free supernatant against multidrug-resistant uropathogen Escherichia coli U12.

Uropathogens develop biofilms on urinary catheters, resulting in persistent and chronic infections that are associated with resistance to antimicrobial therapy. Therefore, the current study was performed to control biofilm-associated urinary tract infections through assaying the anti-biofilm ability of lactic acid bacteria (LAB) against multidrug-resistant (MDR) uropathogens. Twenty LAB were obtained from pickles and fermented dairy products, and screened for their anti-biofilm and antimicrobial effects against MDR Escherichia coli U12 (ECU12). Lactobacillus plantarum Y3 (LPY3) (MT498405), showed the highest inhibitory effect and biofilm production. Pre-coating of a microtitre plate with LPY3 culture was more potent than co-incubation. Pre-coating with LPY3 culture generated a higher anti-biofilm effect with an adherence of 14.5% than cell free supernatant (CFS) (31.2%). Anti-biofilm effect of CFS was heat stable up to 100 °C with higher effect at pH 4-6. Pre-coating urinary catheter with LPY3 culture reduced the CFU/cm2 of ECU12 attached to the catheter for up to seven days. Meanwhile, CFS reduced the ECU12 CFU/cm2 for up to four days. Scanning electron microscope confirmed the reduction of ECU12 adherence to catheters after treatment with CFS. Therefore, Lactobacillus plantarum can be applied in medical devices as prophylactic agent and as a natural biointervention to treat urinary tract infections.

Incidence of biofilm formation among MRSA and MSSA clinical isolates from hospitalized patients in Israel.

AIM: To assess the biofilm-producing capacities of Staphylococcus aureus strains isolated from hospitalized patients in Israel. METHODS AND RESULTS: A total of 16 S. aureus (80 MRSA and 83 MSSA) from screening (nasal swab) and clinical samples (blood and wounds) were characterized. Biofilm-producing capacities were determined using two different biofilm detection assays: Congo Red agar (CRA) and microtiter plate (MtP). In addition, a real-time PCR analysis was performed to detect the presence of biofilm-associated genes (icaA and icaD) and mecA gene. The two assays showed similar biofilm production pattern (28.2% agreement). MRSA strains tended to be greater biofilm-producers than MSSA strains. The presence of mecA was associated with biofilm production (p = 0.030). Additionally, bacteria isolated from blood samples produced less biofilm compared to those from other sources. Finally, no association was found between icaA and icaD presence and biofilm production. CONCLUSION: This study supports earlier assumptions that biofilm formation depends strongly on environmental conditions. SIGNIFICANCE AND IMPACT OF STUDY: This study significantly improved our knowledge on the biofilm production capacity of S. aureus strains in Israel. Moreover, it revealed an association between the mecA gene and biofilm production. Finally, this study underscores the importance of further research to evaluate risk factors for biofilm production.

No Correlation between Biofilm-Forming Capacity and Antibiotic Resistance in Environmental Staphylococcus spp.: In Vitro Results.

The production of biofilms is a critical factor in facilitating the survival of Staphylococcus spp. in vivo and in protecting against various environmental noxa. The possible relationship between the antibiotic-resistant phenotype and biofilm-forming capacity has raised considerable interest. The purpose of the study was to assess the interdependence between biofilm-forming capacity and the antibiotic-resistant phenotype in 299 Staphylococcus spp. (S. aureus n = 143, non-aureus staphylococci [NAS] n = 156) of environmental origin. Antimicrobial susceptibility testing and detection of methicillin resistance (MR) was performed. The capacity of isolates to produce biofilms was assessed using Congo red agar (CRA) plates and a crystal violet microtiter-plate-based (CV-MTP) method. MR was identified in 46.9% of S. aureus and 53.8% of NAS isolates (p > 0.05), with resistance to most commonly used drugs being significantly higher in MR isolates compared to methicillin-susceptible isolates. Resistance rates were highest for clindamycin (57.9%), erythromycin (52.2%) and trimethoprim-sulfamethoxazole (51.1%), while susceptibility was retained for most last-resort drugs. Based on the CRA plates, biofilm was produced by 30.8% of S. aureus and 44.9% of NAS (p = 0.014), while based on the CV-MTP method, 51.7% of S. aureus and 62.8% of NAS were identified as strong biofilm producers, respectively (mean OD570 values: S. aureus: 0.779±0.471 vs. NAS: 1.053±0.551; p < 0.001). No significant differences in biofilm formation were observed based on MR (susceptible: 0.824 ± 0.325 vs. resistant: 0.896 ± 0.367; p = 0.101). However, pronounced differences in biofilm formation were identified based on rifampicin susceptibility (S: 0.784 ± 0.281 vs. R: 1.239 ± 0.286; p = 0.011). The mechanistic understanding of the mechanisms Staphylococcus spp. use to withstand harsh environmental and in vivo conditions is crucial to appropriately address the therapy and eradication of these pathogens.

Exopolysaccharides-Producing Biofilm Bacteria from Submerged Seawater Substrate for Bioremediation of Heavy Metal Contamination.

<b>Background and Objective:</b> The coastal environment is often polluted by various toxic compounds such as heavy metals. Exposure to these toxic compounds causes coastal bacteria to adapt so that they can be used as bioremediation agents for heavy metals. This study aims for finding and screening the ability of bacteria to produce exopolysaccharide biofilms and then determine the characteristics of bacterial isolates as agents candidates for heavy metal bioremediation in the coastal environment. <b>Materials and Methods:</b> Samples were collected on submerged seawater substrate from Bungus Coastal, Padang and West Sumatra, on the wet area that was exposed by seawater (on the rocks, on the wood and the ship, the lower out part on the ship that exposed to seawater). Bacterial isolation process using Marine Agar Medium. The isolate discovered then observed and purified. Furthermore, Congo Red Agar was used for bacteria screening for detecting EPS produced by biofilm bacteria. <b>Results:</b> The results of the isolation, found 9 bacterial isolates attached to the substrate submerged seawater. The screening results showed that isolates K4, K5 and K7 were positive as biofilm-forming bacteria as indicated by the colour change of the bacterial colonies to black on Congo Red Media after 24 hrs incubation. The characteristics of the three bacterial isolates were gram-negative, with cocci and bacilli cells form. <b>Conclusion:</b> Three isolates of positive exopolysaccharide biofilm bacteria that 1 isolate gram-negative coccus (K4) and the other 2 isolates (K5 and K7) were bacillus. Then, the 3 isolates can be used for remediation of metal contamination research in aquatic.

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.

INTRODUCTION: Staphylococcus 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. METHODS: A 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. RESULTS: There 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. DISCUSSION: Our study emphasizes the need for additional experiments to assess the role biofilms have in the pathogenesis of implant-associated and chronic S. aureus infections.

Staphylococcus aureus including MRSA nasal carriage among hospital exposed and unexposed medical students.

INTRODUCTION: Staphylococcus aureus is one of the most common human pathogen causing a wide range of infections. It is estimated that S.aureus colonizes the anterior nares in approximately 31% of the general population at any given time. The incidence of community acquired & hospital acquired S. aureus has been increasing over the past few decades, predominantly due to continuous upsurge in the drug resistant isolates. Moreover, globally the incidence of methicillin resistant S.aureus (MRSA) is progressively increasing. Hence, it would be imperative to screen all healthcare workers, interns and admitted patients for MRSA carriage and to treat all those who are found positive for the same. With the above background, the current study was undertaken to investigate the carrier rate of S. aureus (including MRSA) among hospital unexposed & exposed medical students. METHODS: A total of 181 medical students of Veer Chandra Singh Garhwali Government Institute of Medical Sciences & Research, Srinagar Garhwal, Uttarakhand. Study participants were broadly divided into two groups: hospital exposed group (n=107) and hospital unexposed group (n=74). Nasal swabs were obtained & cultured for the detection of S. aureus. Congo red agar and 0.1% Crystal Violet Assay were performed to observe the ability to form in vitro biofilm by S. aureus. RESULTS: Out of total 181 medical students 29.28% were found to be healthy carrier of S. aureus. Among the hospital exposed group 37.38% and among hospital unexposed group 17.57% were found to be healthy carrier of S. aureus. Only one student (hospital exposed group) was found to be positive for MRSA. Beta-lactamase production was noted in 90.57% strains of S. aureus while the significant rate of slime layer production was observed in 73.58% of strains. CONCLUSION: Prevalence of S. aureus nasal carriage increases with the duration of exposure to the hospital environment. The nasal carriage of S. aureus in medical students indicate the potential danger of dissemination of S. aureus including MRSA from them to the hospitalized patients which in turn complicates the treatment of same.

Evaluation of two fixation techniques for direct observation of biofilm formation of Bacillus subtilis in situ, on Congo red agar, using scanning electron microscopy.

BACKGROUND AND AIM: Direct observation, scanning electron microscopy (SEM) is a common method used for the observations of biofilms. N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide) (EDC) fixation method has proven to be a valuable fixation method in the observation of these biofilms. Still, it entails a method of biofilm fixation that can damage slim structures, leading to the impossible observation of biofilm development. In contrast, alcian blue and lysine (ABL) fixation technique appears more glycocalyx of biofilm, fully preserved samples, which may provide much insight into the development of B. subtilis biofilms. MATERIALS AND METHODS: Here, the evaluation of the fixation of ABL technique for the study of B. subtilis biofilms was carried out in situ, on Congo red agar. In doing so, the comparison to commonly use conventional EDC technique for sample fixation, and observation was carried out. Observations were based on SEM over 30 samples. RESULTS: Overall, ABL technique provided excellent observation of biofilms formed in situ, on Congo red agar, and revealed slime structures, which have not been observed, much in standard EDC fixation or earlier in other studies of these biofilms in B. subtilis. CONCLUSION: This study reported the appropriate use of ABL in the fixation technique for the preservation of biofilm of B. subtilis.

Study of biofilm formation in bacterial isolates from contact lens wearers.

Purpose: To detect biofilm forming capacity of bacterial isolates obtained from the conjunctiva, contact lens and accessories of contact lens wearers using phenotypic and genotypic methods. Methods: Bacterial strains were collected from the conjunctiva, contact lens and lens storage cases of contact lens wearers. The phenotypic detection of biofilm production was done using the tube method and congo red agar method. The biofilm-forming related genes, icaA, of Coagulase negative Staphylococcus (CONS) and Staphylococcus aureus, and pslA, of P. aeruginosa, were detected using PCR. Results: A total of 265 bacterial isolates which included S. aureus, CONS, Pseudomonas, Nil-fermenter Gram-negative bacilli (NFGNB), Bacillus spp, Diphtheroids, Micrococci, Klebsiella pneumonia, Klebsiella oxytoca, E. coli, Proteus mirabilis, Proteus vulgaris, Citrobacter koseri, Citrobacter freundii, Enterobacter cloacae, Moraxella were obtained. Of the 265 isolates, 53.5% were moderately positive, 33.2% strongly positive and 13.2% negative for biofilm production by tube method and 36.6% were moderately positive, 40% strongly positive and 23.3% negative for biofilm production by congo red agar method. Of the four S. aureus isolates, two (50%) showed the presence of icaA gene. Of the 23 CONS isolates, three (13%) showed the presence of icaA gene. All the Pseudomonas isolates were negative for presence pslA (1119 bp) gene though most of them were phenotypically positive for biofilm formation. Conclusion: Most of the bacterial isolates obtained from contact lens wearers had the potential to produce biofilms. Tube method and Congo red agar method exhibited significant statistical correlation (P-value = 0.006) and picked up a good number of biofilm-forming isolates, hence may be used for detection of biofilm production. The absence of biofilm-forming gene did not rule out the possibility for phenotypic biofilm production by bacteria.

Comparative evaluation of methods for the detection of biofilm formation in coagulase-negative staphylococci and correlation with antibiogram.

INTRODUCTION: Coagulase-negative staphylococci (CNS) are normal commensals of human skin and mucous membranes. The objective of the study was to determine the prevalence of CNS among clinical isolates, characterize them up to species level, compare the three conventional methods for detection of biofilm formation, and study their antimicrobial susceptibility pattern. MATERIALS AND METHODS: CNS were obtained from various clinical samples including blood, urine, central venous catheter tips, endotracheal tube aspirate, and pus during a 1-year period (July 1, 2014, to June 30, 2015). Characterization up to species level was done using biochemical tests, and biofilm formation was detected by tube adherence, Congo red agar, and tissue culture plate method. Antimicrobial susceptibility testing was performed following Clinical and Laboratory Standards Institute guidelines. RESULTS: A total of 71 CNS isolates, comprising of seven species were obtained. Staphylococcus epidermidis was the most common species followed by S. saprophyticus and S. haemolyticus. We detected biofilm formation in 71.8% of isolates. Considering the fact that tissue culture plate method is the gold standard, sensitivity of tube adherence method and Congo red agar method was found as 82% and 78%, respectively. The isolates exhibited high resistance toward penicillin (90%), azithromycin (60%), co-trimoxazole (60%), and ceftriaxone (40%), while all were susceptible to vancomycin and linezolid. Biofilm former isolates showed higher resistance than the non-formers. CONCLUSION: Among 71 CNS isolated, S. epidermidis was the most common isolate followed by S. saprophyticus and S. haemolyticus. Biofilm formation was detected in 71.8% of the isolates. All of the methods were effective in detecting biofilm-producing CNS strains. The antimicrobial resistance was significantly higher in biofilm formers than non-formers.

Comparison of biofilm formation by Staphylococcus aureus and Staphylococcus epidermidis strains isolated from sheep milk using three diagnostic methods.

In this study we investigated 24 strains of Staphylococcus aureus and 33 strains of Staphylococcus epidermidis isolated from milk of sheep with clinical mastitis, for their ability to form biofilms. Three methods for the determination of a biofilm were used. When evaluating the growth on Congo Red agar (CRA), 79.2% S. aureus strains and 72.7% S. epidermidis strains were positive for biofilm formation. The quantitative method of biofilm detection on a Microtitre Plate (MTP) revealed positive results for 75.0% of S. aureus samples and 75.8% for S. epidermidis samples. Using PCR method for determination of the presence of genes that affect formation of biofilms, the most frequently determined genes were eno in both S. aureus (18/24; 75.0%) and S. epidermidis strains (20/33; 60.6%). The genes icaAB and ebpS were detected in both S. aureus and S. epidermidis strains, and similarity between these strains was 12.5% - 15.1% and 4.2% - 6.0%, respectively. The bap was recorded only in S. epidermidis (3.0%). Statistical comparison of the level of biofilm formation was performed using Chi square test. There were no statistically significant differences in the amount of biofilm formation between two methods for detection of biofilm CRA and MTP (p>0.05). Comparison of all six monitored parameters showed no dependence of characteristics of the tested strains S. aureus and S. epidermidis at significance level α = 0.05. Biofilm formation by the bacteria isolated from 57 cases of clinical mastitis in sheep was confirmed. Sensitivity and specificity of the CRA method for S. aureus were 94.44% and 66.66%, respectively, and for S. epidermidis 92.0% and 87.5%, respectively. Both CRA and MTP methods can be recommended for the detection of biofilm production by S. aureus and S. epidermidis strains isolated from milk of sheep with clinical mastitis.

Evaluation of Different Phenotypic Techniques for the Detection of Slime Produced by Bacteria Isolated from Clinical Specimens.

UNLABELLED: INTRODUCTION : Microorganisms use various strategies for their survival in both the environment and in humans. Slime production by bacteria is one such mechanism by which microbes colonize on the indwelling prosthetic devices and form biofilms. Infections caused by such microorganisms are difficult to treat as the biofilm acts as a shield and protects microbes against antimicrobial agents. There are several methods for the detection of slime produced by bacteria, and they include both phenotypic and molecular methods. The present study evaluated the Congo red agar/broth method, Christensen's method, dye elution technique, and the latex agglutination method for the demonstration of slime production by different bacterial clinical isolates. MATERIALS & METHODS: We collected 151 bacterial clinical isolates (both gram-positive and gram-negative bacteria) from various specimens and tested them for the production of slime both by qualitative and quantitative tests. Congo red agar/broth method, Christensen's method, dye elution technique, and latex agglutination methods were used for detecting the slime or slime-like substance. RESULTS : We found that 103 (68.2%) strains were positive for slime production by Congo red agar/broth method. It was found that 18 (94.7%) strains of Klebsiella pneumoniae, 21 (84.0%) strains of S aureus and 25 (65.7%) strains of coagulase-negative Staphylococci were positive for slime or slime-like substances by Congo red agar/broth method. A total of 41.0% of the strains positive by Christensen's method and 15.2% of the strains by dye elution technique were found to be more adherent organisms and that have the potential to form biofilms. Only the gram-positive organisms showed nonspecific agglutination with latex suspension. CONCLUSION : Among the various phenotypic methods compared in this study the Congo red agar/broth method is a simple, economical, sensitive, and specific method that can be used by clinical microbiology laboratories for screening the strains for the presence of slime or slime-like substances.

Evaluation of Ica Gene in Comparison with Phenotypic Methods for Detection of Biofilm Production by Coagulase Negative Staphylococci in a Tertiary Care Hospital.

BACKGROUND: Biofilm producing bacteria are responsible for several chronic infections and are difficult to treat as they show much greater resistance to antibiotics. The major virulence factor determining the pathogenicity of CoNS has now well defined and found to be biofilm production. OBJECTIVE: The study was conducted to isolate and characterize Coagulase Negative Staphylococci (CoNS) and their ability to form biofilms was evaluated by phenotypic and genotypic methods. MATERIALS AND METHODS: A total of 96 clinical isolates of CoNS were characterized and subjected to biofilm detection by tissue culture plate method (TCP), tube method (TM), congo red agar method (CRA) and PCR. RESULTS: Staphylococcus epidermidis was the most commonly isolated species 76(79.17%). The ica gene was present in 35 (36.45%) of CoNS isolates which were detected as biofilm producers by TCP. Biofilm producing isolates showed higher antibiotic resistance(72.1%). Majority of biofilm producers had strong association with medical device related infections. CONCLUSION: To compare PCR based dectection method for presence of ica genes with TCP, the test share the specific identification rates. The sensitivity and specificity of TCP method in detection of biofilm was high in comparison with TM and CRA. TCP can be recommended as a general screening test for biofilm detection.

Evaluation of Multidrug Resistant Staphylococcus aureus and their Association with Biofilm Production in a Tertiary Care Hospital, Tripura, Northeast India.

BACKGROUND: High morbidity and mortality rates are associated with Methicillin-resistant Staphylococcus aureus (MRSA) because of development of multidrug resistance. Staphylococcus aureus (S. aureus) has the ability to colonize and form biofilms on biomaterials which is causing resistance towards antimicrobials and thus making them difficult to eradicate from the infected hosts. MATERIALS AND METHODS: Culture isolation, identification was done following standard protocol and antibiogram of the isolates were done. The detection of MRSA, Macrolide-Lincosamide-Streptogramin B resistance (MLSB), vancomycin resistance phenotypes were done by using cefoxitin disc diffusion test, D zone test and vancomycin E test. Biofilm was detected by Congo red agar method. RESULTS: A total of 100 (31.7%) S. aureus strains were isolated from 315 clinical specimens. The prevalence of MRSA was 47% (47/100) with 85.1% were homogeneous MRSA and 14.9% were heterogeneous. Out of 47 MRSA strains, 63.8% were Hospital acquired-MRSA (HA-MRSA) infections whereas rests 36.2% were caused by Community acquired-MRSA (CA-MRSA) strains. Maximum number of MRSA isolates belonged to group A biotype (34%). A 14.9% isolates were of nontypeable group. Out of 100 S. aureus isolates, the prevalence of Vancomycin resistant S. aureus (VRSA) was found to be 3%. The MLSB phenotypes showed that the rates of inducible MLSB (iMLSB), constitutive MLSB (cMLSB) and Macrolide-Streptogramin B (MSB) in case of MRSA to be 19.1%, 31.9% and 12.8%. Prevalence of low-level (MUP(L)) and high-level mupirocin resistance (MUP(H)) among MRSA was 19.1% and 6.4%. Biofilm production was found in 55% strains of S. aureus. Out of 47 MRSA strains 76.6%were producing biofilm in comparison to 38.8% in methicillin-sensitive S. aureus (MSSA). Higher degree of antibiotic resistance in biofilm producers was seen especially in case of ciprofloxacin, co-trimoxazole, rifampicin, kanamycin, erythromycin and clindamycin whereas gentamycin, tetracycline and penicillin resistance was more in non-biofilm producers. CONCLUSION: This study shows high rate of circulating MRSA with a majority of these isolates are multi-drug resistant of which mostly are biofilm producers in our hospital setup. Development of antimicrobial stewardship program based on the local epidemiological data and national guidelines is the need of the hour.

Investigation of biofilm production and icaA and icaD genes in Staphylococcus aureus isolated from heifers and cows with mastitis.

Biofilm formation and antimicrobial resistance of Staphylococcus aureus are important virulence factors in cases of mastitis in dairy cows. However, few studies have investigated mastitis strains isolated from heifers. Within this context, the objective of the present study was to investigate biofilm formation on Congo red agar, the presence of the icaA and icaD genes by polymerase chain reaction (PCR), and the percentage of in vitro antimicrobial resistance of 110 S. aureus isolates from mammary gland secretions of heifers and cows with mastitis. PCR detected the icaA and icaD genes in 98% and 100% of isolates, respectively. However, only 55.5% of all isolates produced a biofilm on Congo red agar. Antimicrobial susceptibility testing revealed that 47.0% of isolates from heifers and 70.4% of isolates from cows were resistant to at least one of the antimicrobial agents tested. Resistance to penicillin and/or ampicillin was the most frequent (44.5%). These results indicate the need to implement prophylactic and control measures of mastitis for heifers. Heifers and cows can carry resistant strains with the capacity of biofilm production, a fact representing a threat to public health and animal well-being and generating losses to dairy farmers.

Antiobiotic resistance pattern of biofilm-forming uropathogens isolated from catheterised patients in Pondicherry, India.

BACKGROUND: Microbial biofilms pose a public health problem for persons requiring indwelling medical devices, as micro-organisms in biofilms are difficult to treat with antimicrobial agents. Thus the present study includes biofilm formation and antibiotic resistance pattern of uropathogens in hospitalised patients with catheter associated urinary tract infections (UTI). METHOD: This prospective analysis included 100 urine samples from catheterised patients with symptoms of UTI over a period of six months. Following identification, all isolates were subjected to antibiotic sensitivity using modified Kirby- Bauer disc diffusion method. Detection of biofilms was done by tube adherence method and Congo red agar method. RESULTS: E.coli was found to be the most frequently isolated uropathogen 70%, followed by Klebsiella pneumoniae 16%, Pseudomonas aeruginosa 4%, Acinetobacter spp 2%, coagulase negative Staphylococci 6% and Enterococci Spp 2%. In the current study 60% of strains were in vitro positive for biofilm production. Biofilm positive isolates showed 93.3%, 83.3%, 73.3% and 80% resistance to nalidixic acid, ampicillin, cephotaxime and cotrimoxazole, respectively, compared to 70%, 60%, 35%, 60% resistance showed by biofilm non-producers for the respective antibiotics. Approximately 80% of the biofilm producing strains showed multidrug resistant phenotype CONCLUSION: To conclude E.coli was the most frequent isolate, of which 63% were biofilm producers. The antibiotic susceptibility pattern in the present study showed quinolones were the least active drug against uropathogens. The uropathogens showed the highest sensitivity to carbapenems. The next best alternatives were aminoglycosides. Significant correlation between biofilm production and multi-drug resistance was observed in our study.

Biofilm production by clinical Staphylococci strains from canine otitis

This study determined the species of 54 staphylococci isolates from canine otitis and their ability to produce biofilm through the Congo red agar method, confirmed by scanning electron microscopy. The most frequently identified species were S. intermedius and S. simulans. Results showed that 30% of the strains were biofilm producers.

In vitro anti-biofilm and anti-bacterial activity of Junceella juncea for its biomedical application.

OBJECTIVE: To investigate the anti-biofilm and anti-bacterial activity of Junceella juncea (J. juncea) against biofilm forming pathogenic strains. METHODS: Gorgonians were extracted with methanol and analysed with fourier transform infrared spectroscopy. Biofilm forming pathogens were identified by Congo red agar supplemented with sucrose. A quantitative spectrophotometric method was used to monitor in vitro biofilm reduction by microtitre plate assay. Anti-bacterial activity of methanolic gorgonian extract (MGE) was carried out by disc diffusion method followed by calculating the percentage of increase with crude methanol (CM). RESULTS: The presence of active functional group was exemplified by FT-IR spectroscopy. Dry, black, crystalline colonies confirm the production of extracellular polymeric substances responsible for biofilm formation in Congo red agar. MGE exhibited potential anti-biofilm activity against all tested bacterial strains. The anti-bacterial activity of methanolic extract was comparably higher in Salmonella typhii followed by Escherichia coli, Vibrio cholerae and Shigella flexneri. The overall percentage of increase was higher by 50.2% to CM. CONCLUSIONS: To conclude, anti-biofilm and anti-bacterial efficacy of J. juncea is impressive over biofilm producing pathogens and are good source for novel anti-bacterial compounds.

The Isolation and the Biofilm Formation of Uropathogens in the Patients with Catheter Associated Urinary Tract Infections (UTIs).

BACKGROUND: Urinary tract infections are the most commonly acquired bacterial infections and they account for an estimated 25-40% of the nosocomial infections. The microbial biofilms pose a public health problem for the persons who require indwelling medical devices, as the microorganisms in the biofilms are difficult to treat with antimicrobial agents. AIMS: The present study included the isolation and the biofilm formation of the uropathogens in patients with catheter associated urinary tract infections. METHODS AND MATERIALS: This prospective analysis which was carried out over a period of two months, included 50 urine samples from catheterized patients with symptoms of UTI. Following their isolation and identification, all the isolates were subjected to the biofilm detection by the tube adherence method and the Congo Red agar method. RESULTS: E.coli was found to be the most frequently isolated uropathogen 35(70%), followed by Klebsiella pneumoniae 8(16%), Pseudomona aeruginosa 2(4%), Acinetobacter spp 1(2%), coagulase negative Staphylococci 3(6%) and Enterococci spp 1(2%). In the current study, 30 (60%) strains were positive in vitro for the biofilm production. CONCLUSION: To conclude, there was significant bacteriuria in all the symptomatic catheterized patients and E.coli was the most frequent isolate. Diabetes (44%) was the most common factor which was associated with the UTIs in the catheterized patients.