Inducible Clindamycin Resistance and Biofilm Production among Staphylococci Isolated from Tertiary Care Hospitals in Nepal

Resistance to antibiotics, biofilm formation and the presence of virulence factors play important roles in increased mortality associated with infection by staphylococci. The macrolide lincosamide streptogramin B (MLSB) family of antibiotics is commonly used to treat infections by methicillin-resistant isolates. Clinical failure of clindamycin therapy has been reported due to multiple mechanisms that confer resistance to MLSB. This study aims to find the incidence of different phenotypes of MLSB resistance and biofilm production among staphylococci. A total of 375 staphylococci were isolated from different clinical samples, received from two tertiary care hospitals in Nepal. Methicillin resistance was detected by cefoxitin disc diffusion method and inducible clindamycin resistance by D test, according to CLSI guidelines. Biofilm formation was detected by the tissue culture plate method and PCR was used to detect ica genes. Of the total staphylococci isolates, 161 (42.9%) were Staphylococcus aureus, with 131 (81.4%) methicillin-resistant strains, and 214 (57.1%) isolates were coagulase-negative staphylococci, with 143 (66.8%) methicillin-resistant strains. The overall prevalence of constitutive MLSB (cMLSB) and inducible MLSB (iMLSB) phenotypes was 77 (20.5%) and 87 (23.2%), respectively. Both iMLSB and cMLSB phenotypes predominated in methicillin-resistant isolates. The tissue culture plate method detected biofilm formation in 174 (46.4%) isolates and ica genes in 86 (22.9%) isolates. Among biofilm producing isolates, cMLSB and iMLSB phenotypes were 35 (20.1%) and 27 (15.5%), respectively. The cMLSB and iMLSB were 11 (12.8%) and 19 (22.1%), respectively, in isolates possessing ica genes. Clindamycin resistance in the form of cMLSB and iMLSB, especially among MRSA, emphasizes the need for routine D tests to be performed in the lab.

Comparative study of antibiofilm activity of Lime juice and Lithium dioxide nanoparticles against E.coli isolated from local made cheese

Thirty specimens of fresh white cheese, in different markets at different cities of Iraq were analyzed microbiologically. Isolates of E.coli that have been collected from the samples of cheese, were investigated. Capacity for biofilm producing was demonstrated by two method, Tissue culture plate method (TCP) and agar (CRA). After that, antibiofilm activity of lime extract and LiO2NPs was studied as each one of them alone and then the synergistic effect was done by TCP method. The results showed that all E.coli isolates produce biofilm but in different degrees. The results also displayed that Lime extract and LiO2NPs had antibiofilm effect against E.coli when used alone and when the combination done between each one of these materials. In conclusion, it was observed that the specimens of fresh white cheese included in this study contained microbial contamination at a health-threatening level but elimination of this contamination can be done by using lime extract and LiO2NPs.

Screening and Characterization of Biofilm Formation by Staphylococcus Spp and Pseudomonas Spp Isolates from Diabetic Foot Ulcer

Diabetes mellitus is a very serious systemic disease worldwide. Around 416 million cases were estimated in 2015 worldwide, and are expected to reach 549 million in 2030 which is 8.6% higher. Diabetes mellitus is the leading cause of mortality and morbidity and is responsible for 3.8 million deaths annually. 25% of the diabetic patients are affected with foot infections out of which, 15% people are forced for limb amputation which affects the quality of life of the patients. Diabetic foot ulcer is a poly microbial infection mostly occurs due to Staphylococcus spp and Pseudomonas spp and pose serious complications. Bacteria are the cause for much type of diseases and generate resistance to wide range of biofilm forming. Biofilms constitute reservoirs of pathogens and are associated with resistance to antimicrobial agents and chronic infection. The study included 156 patients (59% male and 41% female) suffering diabetic foot ulcer whose pus culture was isolated. The identification of isolates for both gram-negative and gram-positive organisms was done as per the procedures mentioned in Bergey’s manual of Determinative Bacteriology. Further, MALDI- TOF (Matrix-Assisted Laser Desorption/Ionization) was used to confirm the identification of the isolates using classical methods. Staphylococcus spp (65%) and Pseudomonas spp (35%) biofilm producing isolates were identified for Congo red method assay and Tissue culture plate method. Results of biofilm production in positive, intermediate, negative differentiation on the Congo red plate assay and Tissue culture plate method assays were analyzed.

Biofilm formation by bacteria isolated from intensive care units of a tertiary care hospital, with special relevance to its risk factors

Background: The purpose of this study was to detect biofilm formation by bacterial isolates from patients with device associated infection admitted in intensive care units (ICUs), to compare the three methods used for detection of bioiflm, to compare the antimicrobial susceptibility pattern of the biofilm producers with the non-producers and to study the risk factors associated with biofilm formation.Methods: A total of 115 bacterial isolates from patients with device associated infection admitted in different ICU for a period of one year was included in the study. These clinical isolates were detected for biofilm formation by tissue culture plate method, tube method and Congo red agar method. Kirby-Bauer disc diffusion method of antibiotic susceptibility was performed on all isolates.Results: Out of the 115 bacterial isolates, 71 were biofilm producers. Tissue culture plate method detected the maximum number of biofilm producers (61.7%). The maximum number of biofilm producers were isolated from tracheal aspirate and endotracheal tubes (52.1%) followed by blood (17%) and urine (12.6%) respectively. The predominant biofilm producing isolates were Klebsiella pneumoniae (39.4%), Staphylococcus aureus (19.7%) and Pseudomonas aeruginosa (16.9%). Multi drug resistance among the biofilm producers was significantly higher than the non-biofilm producers (p value=0.0125). The risk of biofilm formation was seen to increase with the increase in duration of hospital stay (p value=0.0092, statistically very significant).Conclusions: From this study it was found that a high degree of biofilm producers were isolated from patients on indwelling devices. Tissue culture plate was found to be the most accurate method. The degree of multidrug resistance among the bioiflm producers was significantly higher than the non-producers.

Phenotypic and genotypic characterization of biofilm producing clinical coagulase negative staphylococci from Nepal and their antibiotic susceptibility pattern

Background Coagulase-negative staphylococci (CNS) survive as commensals of skin, anterior nares and external canals of human and were regarded as non-infectious pathogens. However, they are emerging as a major cause of nosocomial infectious due to their ability to form biofilms and high resistance to several classes of antibiotics. This study examines the biofilm forming abilities of 214 clinical CNS isolates using phenotypic and genotypic methods, and determines their antibiotic susceptibility patterns. Methods A total of 214 clinical isolates collected from different clinical samples were identified as CNS and their antibiotic susceptibility determined by CLSI guidelines. The biofilm forming ability of all isolates was determined by three phenotypic methods; Congo red agar (CRA) method, tube adherence method (TM) and tissue culture plate (TCP) method and by genotypic method for the detection of icaAD genes. Results Among all the isolates, S. epidermidis (57.5%) was found the most frequently, followed by S. saprophyticus (18.7%), S. haemolyticus (11.2%), S. hominis (7%), and S. capitis (5.6%). Antibiotic susceptibility pattern demonstrated 91.6% isolates were resistant to penicillin and 66.8% to cefoxitin while 91.1% isolates were susceptible to chloramphenicol. Constitutive and inducible clindamycin resistant phenotype as measured by D-test was seen among 28% and 14.5% of isolates respectively. Tissue culture plate method detected biofilm production in 42.1% isolate followed by 31.8% through tube method while 20.1% isolates were found to produce slime in Congo red agar method. The genotypic assay revealed presence of icaA and icaD genes in 19.2% isolates. Conclusion The study shows a high prevalence of biofilm formation and inducible clindamycin resistance in CNS isolates, indicating the importance of in-vitro biofilm production test and D-test in routine laboratory diagnostics. Implementation of efficient diagnostic techniques for detection of biofilm production in clinical samples can help manage staphylococcal infections and minimize risks of treatment failures in hospitals.

The Role of Biofilm Formation in Antibiotic Resistance of Bacteria Isolated from Saliva of Patients with Dental Caries

Objectives: This study aim to determine the bacterial diversity, biofilm forming ability and the antimicrobial resistance of bacteria isolated from saliva of patients with dental caries conditions with the using of 16S rRNA gene sequencing technique for identification of the most virulent isolates. Methods: Isolation and identification of microorganisms were done employing standard bacteriologic techniques, followed by biofilm detection using tissue culture plate method. The strong biofilm forming isolates were selected for antibiotic susceptibility test against selected antibiotics using disk diffusion technique. In order to identify the selected isolates. The genomic DNA obtained following the extraction process were used for the amplification of the bacterial 16S rRNA gene. Results: A total of 137 bacterial isolates were obtained and identified as belonging to 21 genera. Tissue culture plate (TCP) method were employed for screening the isolates according to its biofilm forming ability, its showed that 55 (40.1%) of the total isolates were strong, 57 (41.6%) were moderate and 25 (18.3%) were weak biofilm producers. The antimicrobial susceptibility test showed the multi antibiotics resistance of the strong biofilm former isolates to the conventional antibiotics. Enterococcus faecalis isolates showed the highest biofilm formation and antibiotic resistance. The 16S rRNA gene for two of these isolates have been amplified using PCR and the product sequenced, analyzed and registered in the National Center for Biotechnology Information (NCBI) as UKMS1 and UKMS2 and the accession numbers KX960104.1 and KX960105.1 respectively. Conclusion: The study has revealed that antimicrobial resistance of bacteria isolates from saliva of patients with dental caries conditions is associated with biofilm formation. Other uncommon pathogenic bacteria were also isolated in this study as a result of the use of non-selective enrichment medium for culturing. Enterococcus faecalis isolates indicated the highest biofilm formation and antibiotic resistance.

Correlation between antimicrobial resistance and biofilm formation capability among Klebsiella pneumoniae strains isolated from hospitalized patients in Iran

Background Klebsiella pneumoniae is a common cause of nosocomial infections. Antibiotic resistance and ability to form biofilm, as two key virulence factors of K. pneumoniae, are involved in the persistence of infections. The purpose of this study was to investigate the correlation between antimicrobial resistance and biofilm formation capability among K. pneumoniae strains isolated from hospitalized patients in Iran. Methods Over a 10-month period, a total of 100 non-duplicate K. pneumoniae strains were collected. Antibiotic susceptibility was determined by Kirby–Bauer disk diffusion method according to CLSI. Biofilm production was assessed by tissue culture plate method. Finally, polymerase chain reaction was conducted to detect four families of carbapenemase: blaIMP, blaVIM, blaNDM, blaOXA−48; biofilm formation associated genes: treC, wza, luxS; and K. pneumoniae confirming gene: rpoB. Results Most of the isolates were resistant to trimethoprim-sulfamethoxazole (52 %), cefotaxime (51 %), cefepime (43 %), and ceftriaxone (43 %). Among all the 100 isolates, 67 were multidrug-resistant (MDR), and 11 were extensively drug-resistant (XDR). The prevalence of the blaVIM, blaIMP, blaNDM, and blaOXA−48 genes were 7 , 11 , 5 , and 28 %, respectively. The results of biofilm formation in the tissue culture plate assay indicated that 75 (75 %) strains could produce biofilm and only 25 (25 %) isolates were not able to form biofilm. Among these isolates, 25 % formed fully established biofilms, 19 % were categorized as moderately biofilm-producing, 31 % formed weak biofilms, and 25 % were non-biofilm-producers. The antimicrobial resistance among biofilm former strains was found to be significantly higher than that of non-biofilm former strains (p < 0.05). Molecular distribution of biofilm formation genes revealed that 98 , 96 , and 34 % of the isolates carried luxS, treC, and wza genes, respectively. Conclusions The rise of antibiotic resistance among biofilm-producer strains demonstrates a serious concern about limited treatment options in the hospital settings. All of the data suggest that fundamental actions and introduction of novel strategies for controlling of K. pneumoniae biofilm-related infections is essential.

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

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

Effect of Glucose Induction on Biofilm Density in Clinical Isolate Acinetobacter baumannii Patients in Intensive Care Unit of Dr. Soetomo Hospital, Surabaya

This study aimed to analyze the effect of glucose induction on the clinical isolate biofilm density of Acinetobacter baumannii. Thirteen clinical isolates of A. baumannii non biofilm forming were collected from non-DM patients who were treated at the ICU of Dr. Soetomo Hospital, Surabaya, was treated with the addition of 0.08% glucose, 0.15% glucose, 0.2% glucose, and 0.4% glucose in TSB growth media, followed by biofilm density examination with Tissue Culture Plate Method (TCPM) using 96 wells flatbottomed polyesterene tissue culture plate and read by autoreader ELISA with a wavelength of 630 nm (OD630). Biofilm density obtained was analyzed using ANOVA statistical analysis. The results of OD630 showed that the biofilm density increased significantly at the addition of 0.2% and 0.4% glucose. There was a significant increase in biofilm density at the addition of 0.2% and 0.4% glucose so that the management of blood sugar levels in ICU patients was needed before and when medical devices were installed.

Biofilm-Formation in Clonally Unrelated Multidrug-Resistant Acinetobacter baumannii Isolates

This study analyzed the genotype, antibiotic resistance, and biofilm formation of Acinetobacter baumannii strains and assessed the correlation between biofilm formation, antibiotic resistance, and biofilm-related risk factors. A total of 207 non-replicate multi-drug-resistant A. baumannii strains were prospectively isolated. Phenotypic identification and antimicrobial susceptibility testing were carried out. Isolate biofilm formation ability was evaluated using the tissue culture plate (TCP), Congo red agar, and tube methods. Clonal relatedness between the strains was assessed by enterobacterial repetitive intergenic consensus-PCR genotyping. Of the 207 isolates, 52.5% originated from an intensive care unit setting, and pan resistance was observed against ceftazidime and cefepime, with elevated resistance (99–94%) to piperacillin/tazobactam, imipenem, levofloxacin, and ciprofloxacin. alongside high susceptibility to tigecycline (97.8%). The Tissue culture plate, Tube method, and Congo red agar methods revealed that 53.6%, 20.8%, and 2.7% of the strains were strong biofilm producers, respectively, while a significant correlation was observed between biofilm formation and device-originating respiratory isolates (p = 0.0009) and between biofilm formation in colonized vs. true infection isolates (p = 0.0001). No correlation was detected between antibiotic resistance and biofilm formation capacity, and the majority of isolates were clonally unrelated. These findings highlight the urgent need for implementing strict infection control measures in clinical settings.