Culture Plate Method Research Articles

Biofilm Formation and its Association with Gram Negative Sepsis Pathogenicity

Background and objectives: Biofilm formation is an important virulence factor that protects an organism from antimicrobial agents as well as host immune effectors, thus allowing organisms to invade, survive, and cause persistent-reoccurring infection in host cells. The aim of this study was to investigate the ability of sepsis-causing gram-negative bacteria to form biofilms, evaluate the association between antibiotic resistance pattern and biofilm formation, determine the role and influence of biofilm formation on pathogenicity and clinical outcome of sepsis. Methods: A prospective study conducted from October 2020 to August 2021, non-replicated gram-negative bacteria isolates were recovered from blood samples of patients with suspected bacteremia, sepsis, and sepsis shock and identified using biochemical procedures. Antimicrobial susceptibility patterns of GNB isolates were determined using the Kirby-Bauer disc diffusion method and interpreted using CLSI guidelines. The ability of GNB isolates to form biofilm was assessed using Congo red agar and the tissue culture plate method. Results: Of the 160 Gram-negative bacteria tested, biofilm formation was seen in 73 (45.63%) isolates. Isolates are Klebsiella pneumoniae (39.73%), Acinetobacter spp. (34.25%), Escherichia coli (23.29%), Pseudomonas aeruginosa (1.37%), and other non-fermenters (1.37%). Isolates were highly resistant to cephalosporins, fluoroquinolones, and the penicillin group of antibiotics. No statistical relationship was found between resistance pattern, clinical outcome, and biofilm formation. Conclusion: In the current study, we found that 45.63% of gram-negative bacteria causing sepsis were biofilm producers. Klebsiella pneumonia isolates exhibited the highest levels of biofilm formation and antimicrobial resistance. Based on the strength of biofilm formation, most isolates were weak biofilm producers, and there was no statistical correlation between the formation of biofilms and antimicrobial resistance, indicating that the formation of biofilms was not a determining factor for resistance.

Label-free and culture-free quantification of lactic acid bacteria using ring-shaped interdigitated electrodes

Quantification of lactic acid bacteria has great significance in the probiotic industry. However, conventional bacteria quantification methods usually take more than 24 hrs to perform, which is not ideal for manufacturing since production proceeds to different stages at a fast pace. To address this problem, a rapid bacteria quantification platform was developed, which utilized ring-shaped interdigitated electrodes to concentrate bacteria particles via electrokinetic forces. The application of an AC signal with 500 Hz and 12 VPP resulted in the collection of bacteria cells suspended in a medium of low conductivity (<15 µS/cm), which was then analyzed with an image analysis software. This was used for samples with bacteria concentrations ranging from 1 × 104 to 1 × 106 CFU/mL. The analysis is inexpensive (<5 USD per test), requires only a small sample volume (160 µL/test), and had a fast total analysis time (within 10 min per sample), which is much faster compared with plate culture methods. The accuracy was verified with blind tests using 61 production line samples and obtained a 100 % predictive accuracy. It is suitable for situations that require rapid bacteria detection and has a high potential for commercial use.

Comparative Evaluation ofBiofilm Detection Methods among Uropathogenic Gram-Positive and Gram Negative Bacteria Isolated from Catheterized Urine Sample and its antibiogram

Background: Biofilm formation is a potential virulence characteristic exhibited by bacteria resulting in the severity of many infections. It will give rise to multidrug resistant strains disturbing the effective management of many chronic infections. Objectives: The present study aims to focus on the biofilm detection of uropathogenic bacteria by three distinct techniques. The sensitivity and specificity for all the three methods is evaluated. The present study also demonstrates the antibiogram of biofilm producing bacteria.Materials and Method: The study includes 89bacterial isolates from catheterized patients with urinary tract infections admitted in ICUs, dialysis units and various wards. Formation of biofilm was detected by: tube method (TM), the tissue culture plate (TCP) method and Congo Red Agar (CRA) method. According to CLSI guidelines the antimicrobial susceptibility test was performed among the biofilm forming bacteria.Results: Out of 89 bacterial isolates, 33 were gram positive cocci and 56 were gram negative bacilli. Among the gram positive cocci, 28 (84.8%) andgram negative bacilli, 50(89.2%) formed biofilm. Sensitivity of TCM, TM, and CRM methods were 98.3%, 71.4% and 18.6% respectively. For biofilm forming gram positive bacteria, the maximum antibiotic resistance was achieved towards Penicillin (89%) followed by Amoxyclav (78%). In the case of gram negative bacteria gentamicin showed maximum resistance in 88% followed by Ciprofloxacin in 80% of the isolates. Conclusion: Biofilm and multi-drug resistance plays a vital role in the pathogenesis of catheterized urinary tract infection (CAUTI). Evaluation of Biofilm among uropathogenic bacteria helps to manage clinically resulting in better prognosis. Tissue culture plate techniqueis very effectivein detecting biofilm that can be suggested in diagnostic laboratories. Keywords: Biofilm, virulence factor, multidrug resistance,CAUTI.

Investigating the Role of Antibiotics on Induction, Inhibition and Eradication of Biofilms of Poultry Associated Escherichia coli Isolated from Retail Chicken Meat.

Widespread use of antibiotics as growth promoters and prophylactic agents has dramatic consequences for the development of antibiotic resistance. In this study, we investigated effects of selected antibiotics on bacterial biofilms and performed extensive antibiotic and VF profiling of poultry-meat associated E. coli strains. Antibiotic susceptibility was performed by a disc diffusion method, followed by molecular screening of resistance and virulence determinants. Further biofilm formation assays, MIC-p, MIC-b, MBIC and MBEC, were performed using standard tissue culture plate method. In total, 83 (75%) samples were confirmed as E. coli from poultry sources, 26 different antibiotics were tested, and maximum numbers of the isolates were resistant to lincomycin (100%), while the least resistance was seen against cefotaxime (1%) and polymyxin B (1%). Overall, 48% of the isolates were ESBL producers and 40% showed carbapenemase activity; important virulence genes were detected in following percentages: fimH32 (39%), papC21 (25%), iutA34 (41%), kpsMT-II23 (28%), papEF9 (11%), papGII22 (27%) and fyuA13 (16%). Colistin showed remarkable anti-biofilm activity, while at sub-MIC levels, gentamicin, ceftriaxone and enrofloxin significantly (p &lt; 0.01) inhibited the biofilms. A strong induction of bacterial biofilm, after exposure to sub-minimal levels of colistin clearly indicates risk of bacterial overgrowth in a farm environment, while use of colistin aggravates the risk of emergence of colistin resistant Enterobacteriaceae, a highly undesirable public health scenario.

Characterization of biofilm formation by Mycobacterium smegmatis during different environmental stress conditions: An in-vitro study

Aim: In-vitro characterisation of biofilm produced by Mycobacterium smegmatis (M. smegmatis), a surrogate model for biofilm production by Mycobacteria, and to evaluate the impact of different environmental stress on mycobacterial growth and biofilm formation. Methodology: M. smegmatis biofilms were studied using tissue culture plate and tube adherence methods. Confocal Laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) were used to study the 3D structure and surface morphology, respectively. Additionally, the effect of different environmental stress, such as the absence of essential ions, exposure to harsh environmental conditions, such as acidic environment or oxidative stress, on mycobacterial biofilm formation and mycobacterial growth was assessed. Results: All the exposures, except for carbon supplemented media had a detrimental effect on the number of viable counts and on biofilm formation by mycobacteria (p&lt;0.001). Growth in low pH and oxidative stress was found to be maximum showing reduction by 98% when compared with control. Interpretation: Our findings present various environmental conditions that profoundly affect biofilm formation and thus, may find practical implications in future as effective mycobacterial control strategies having attributes of mycobacterial growth as well as biofilm inhibition. Key words: Biofilm, Environmental stress, Multi-drug resistance, Mycobacterium

Detection of Biofilm Formation by Escherichia coli and Staphylococcus aureus Associated with Canine Pyometra

Background: Antibiotic resistance is one of the major problems encountered in the therapy of canine pyometra. The ability of bacteria to form biofilm is implicated as one of the factors responsible for this. Escherichia coli and Staphylococcus aureus are the predominant bacteria associated with pyometra in canines and are known for their biofilm formation. Keeping in this view, a preliminary study was conducted to detect the biofilm forming strains of E. coli and S. aureus, if any, associated with canine pyometra. Methods: A total of 25 samples were collected, which included uterine discharges from cases of closed pyometra and anterior vaginal swabs from open pyometra. The isolates of E. coli and S. aureus were identified based on the cultural, morphological and biochemical characteristics. These isolates were subjected to antibiotic sensitivity test employing disc diffusion method. For biofilm detection, the isolates were screened by Congo red agar method, tube method and tissue culture plate method. Result: From 25 samples, two Streptococcus spp., thirteen Staphylococcus spp., seven E.coli, five Klebsiella spp. and two Pseudomonas spp. were isolated. All the isolates were found to be multi-drug resistant on antibiogram. The tissue culture plate and Congo red agar method was found more sensitive to detect the biofilm formation by S. aureus and E.coli isolates, respectively. The biofilm forming strains showed higher degree of antibiotic resistance in comparison with non-formers, indicating it as one of the major reasons for failure of antibiotic therapy in canine pyometra.

Comparative qualitative and quantitative analysis of lactic acid bacteria by molecular methods in different Greek cheeses

In the present research communication, we report on identification and quantification of four main lactic acid bacteria (LAB) genera (Lactococcus, Lactobacillus, Streptococcus and Leuconostoc), most common in Greek cheeses, by a novel culture-independent method. More specifically, new primers were designed to be used in both multiplex PCR for simultaneous identification and in real-time PCR for quantification of the LAB. The method was validated by applying it in parallel to culture-dependent method in a variety of cheeses from different Greek geographical locations, of different animal milk origins and of different production methods. While the standard plate culture method showed absence of Leuconostoc sp. in all cheeses, the culture-independent methods detected all four LAB genera studied. Furthermore, the relative presence of the four genera detected by the culture-independent method showed a pattern present in almost all cheese samples tested, indicating Lactococcus genus as the dominant one.

Detection and Analysis of Resistance Genes in Escherichia coli Bacteria Isolated from Children in Baghdad

Urinary tract infections are common diseases and Escherichia coli is the most common cause of this disease, especially in infants and children. The 200 samples were collected for the period from July to December 2020 from the Central Children’s Hospital in Baghdad from infants and children and cultured on MacConkey and blood agar for primary isolation of E. coli bacteria. The VITEK 2 system was used for biochemical tests and final identification. Antibiotic susceptibility for 16 antibiotics was determined using the VITEK 2 system, as well as the ability for biofilm formation using the tissue culture plate method, while DNA was isolated for molecular antibiotic resistance genes tetA, tetB, CITM, sul1, sul2, and sul3 using the standard polymerase chain reaction. Results showed that 40/62 isolates (64%) belonged to E. coli bacteria (32 females and 8 males), and isolates were of varying resistance to antibiotics, with the highest resistance to ampicillin and sulfamethoxazole and the lowest resistance to tigecycline and most MDR. The percentage of biofilm formation ability was 62.5%. The ratio of resistance genes CITM, tetA, tetB, sul1, and sul2 for E. coli bacteria was 5%, 45%, 12.5%, 35%, and 40%, respectively, while the sul3 gene did not appear in isolation from any clinical local isolation. So, E. coli isolates isolated from children showed a high extent of antibiotic resistance as the genetic sequencing of resistance genes showed that there were many mutations with a type of transmission or silence or transition. In conclusion, E. coli isolated from children was highly resistant to antibiotics and had the ability to form biofilms as well as the presence of a resistance gene that could be transmitted among bacteria and spread antibiotic resistance.

To study drug resistance &amp; biofilm production in gram negative isolates from clinical samples

Biofilms are groups of microorganism encased in a matrix of extracellular polysaccharide (slime), called polysaccharide intercellular adhesion (PIA). Bacteria commonly involved include , , , and . The present study was performed to identify antibiotic resistance pattern &amp; their ability to form biofilm in gram negative clinical isolates. All clinical samples received in laboratory for microbial culture during study period of 12 months (2017 to 2018) were included in this study Antibiotic susceptibility testing, ESBL &amp; MBL detection was done for clinical isolates. Biofilm productions were determined by Congo red agar method, Christenson’s Test Tube method and Tissue culture plate method. 327 gram negative isolates were detected. Maximum were (32.72%) followed by (28.44%), (16.51%), (16.51%), Citrobacter species (3.97%). Maximum isolates showed resistance to ampicillin (93.27%) followed by amoxiclave (87.46%), ceftazidime (74%). Out of 327 GNB isolates, biofilm produced by 64 (19.57%) isolates by Tissue culture plate (TCP) method, 38(11.62%) by Congo red agar (CRA) method and 23 (7.03%) by Tube method. Maximum biofilm were detected in (24.29%). There is increase prevalence of multidrug resistant&amp; biofilm forming bacteria. The routine monitoring of multidrug resistance pattern &amp; biofilm detection can be recommended in clinical laboratories to guide proper antibiotic treatment.

Detection and quantification of Sclerotinia sclerotiorum infestation of canola petals using a clearing and staining technique

Petal testing can be used to determine the presence of Sclerotinia sclerotiorum inoculum during canola flowering as an indicator of disease risk and the potential need for fungicide application. Traditionally, the culture plate method has been used to test petals, but this takes up to 7 days to obtain results, which is a drawback in terms of making timely fungicide use decisions. Therefore, the availability of a simple, faster detection technique for S. sclerotiorum infected canola petals would be an advantage. We have documented and compared a canola petal clearing and staining technique with the standard culture plate method to study sclerotinia ascospore infestation. The staining technique was found to be a rapid, simple and reliable method compared to the traditional culture plate system.

Biofilm and Antimicrobial Resistance of Clinical and Environmental Isolates of Pseudomonas aeruginosa

Objectives: To evaluate the clinical and environmental ability of P. aeruginosa isolates to make biofilms and to determine their antimicrobial susceptibility patterns. Methods: Environmental swabs (144), air (28) and water samples (12) were collected from various wards of Gaza strip hospitals. Additionally, 158 clinical P. aeruginosa isolates were obtained from the microbiology laboratories of the same hospitals between July 21, 2019 and January 21, 2020. Samples were cultured and bacterial identification was performed using standard microbiological procedures. PCR was used to confirm the identity of P. aeruginosa. P. aeruginosa isolates were tested for their antimicrobial susceptibility patterns by the agar disk diffusion method. Both qualitative and quantitative methods assessed the biofilm formation by crystal violet and safranin stains. Results: Among the P. aeruginosa isolates (N=150), 90.0% were resistant for ceftazidime, (36.7%) aztreonam, (29.3%) gentamicin, (27.3%) levofloxacin, (22.0%) meropenem, (14.0%) piperacillin, (10.0%) amikacin and (9.3%) imipenem. The results for biofilm formation by tube method showed that 78.0% and 71.3% of the isolates were biofilm producer by crystal violet and safranin methods, respectively. Microtiter plate method demonstrated that 94.0% and 96.0% were biofilm producers by crystal violet and safranin, respectively. In addition, there was a statistical significance between the meropenem resistance and biofilm-forming ability of the isolates. Conclusions: High resistance rates were detected among P. aeruginosa isolates. The lowest rate of resistance was to imipenem and amikacin. As for the biofilm assessment, the tissue culture plate method showed higher detection rates than the tube method.

Novel PCR detection of CRISPR/Cas systems in Pseudomonas aeruginosa and its correlation with antibiotic resistance

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated proteins) systems are considered as acquired immune mechanisms in Gram-positive and Gram-negative bacteria and also in archaea. They provide resistance/immunity to attacking bacteriophages or mobile genetic elements as integrative conjugative elements (ICE) as well as plasmid transformation. As an opportunistic pathogen, Pseudomonas aeruginosa has been held responsible for serious infections especially in hospitalized and immunocompromised patients. Three subtypes of type I CRISPR system (I-C, I-E, & I-F1) have been detected in P. aeruginosa genomes. In this work, P. aeruginosa isolates were collected from different clinical sources, and the three CRISPR/Cas subtypes (I-C, I-E, & I-F1) were detected via singleplex and multiplex PCR techniques using novel universal primers that were designed specifically in this study. CRISPR subtypes I-C, I-E, and I-F1 were detected in 10, 9, and 13 isolates, respectively. Furthermore, antimicrobial susceptibility of CRISPR/Cas-positive and negative isolates to different antibiotics and the capacity of biofilm formation were detected using disc diffusion method and tissue culture plate method, respectively. There was a significant correlation between the presence/absence of CRISPR/Cas system and both antimicrobial susceptibility to some antibiotics and biofilm-forming capacity among P. aeruginosa clinical isolates.Key points• A novel multiplex–PCR for detection of CRISPR/Cas-positive strains of P. aeruginosa.• Understand the correlation between CRISPR/Cas systems and other characters of P. aeruginosa.• Correlation between antimicrobial susceptibility and CRISPR systems in P. aeruginosa.

Biofilm formation by ESBL-producing uropathogenic E. coli

Introduction: E. coli is considered to be the leading cause of urinary tract infections. (UTIs) and is responsible for both community acquired and hospital acquired UTIs. The ability of this bacterium to form biofilm on various devices like catheters plays an important role in progression of the disease. Biofilm production in extended spectrum beta lactamases (ESBLs) producing organisms provides a favourable environment for the exchange of resistance genes and may also facilitate widespread dissemination.&#x0D; Methods: The urine samples were collected from the suspected cases of urinary tract infections and were cultured on cystine lactose electrolyte deficient (CLED) agar plates to isolate the desired UPEC strains. All the isolated E. coli strains were subjected to ESBL detection by double disk approximation method and to biofilm formation by tissue culture plate method of biofilm detection. All the results were statistically analysed using k2 test with p value at &lt;0.05 level of significance.&#x0D; Results: Out of all the gram negative bacilli isolated from urine samples, E. coli alone accounted for 90% of the isolates. Of all the UPEC isolates subjected to ESBL detection 50% of the strains turned out to be the ESBL producers and 35% of the strains showed biofilm formation.&#x0D; Conclusion:&#x0D; &#x0D; The majority of the work in literature sites the data samples collected from indoor patients, where these isolates coli strains which produced ESBLs were mainly a strong biofilm former as compared to non-ESBL producer strains.&#x0D; In this study in spite of all the coli strains being outpatient isolates still the correlation between ESBL production and biofilm formation holds true.&#x0D; We found a significant correlation between biofilm formation and ESBL production. The detection of ESBL production and biofilm formation pattern will help in development of newer and more effective treatment.&#x0D;

Determination of Enterococcal Virulence Factors Expression and Impact of Biofilm Formation on Antimicrobial Susceptibility Pattern

Introduction: Enterococcus spp. has become recognized as a significant cause of hospital-acquired infections. Two main virulence factors namely gelatinase and hemolysin of Enterococci have been proved to cause severe infections. In addition, biofilm formation is causing infection by enhancing the persistence of Enterococci in medical indwelling devices. Therefore, the study aimed to evaluate the presence of hemolysin, gelatinase, and biofilm formation in Enterococcus spp. and the impact of biofilm on antimicrobial susceptibility patterns.&#x0D; Materials &amp; Methods: Total 104 Enterococcal isolates obtained from different clinical samples were included in the study for expression of virulence factors. All isolates were evaluated for biofilm formation by the tissue culture plate method. Hemolysin production was checked by using 5% sheep blood agar and gelatinase production by peptone yeast extract agar containing 3% gelatin. Antimicrobial susceptibility testing was done by the Vitek2 compact automated system.&#x0D; Results: Out of 104 isolates, 1(1%) were strong biofilm producers while 4(3.85% %) and 54(52%) were moderate and weak biofilm producers respectively. Hemolysin production was observed in 19(18%) isolates and gelatinase production was universal.&#x0D; Conclusion: Biofilm-producing strains showed higher resistance to beta-lactam drugs and high-level aminoglycosides. Hence, amongst three virulence factors, studying biofilm formation can be an important tool to develop a hospital’s antibiotic policy and other virulence factors can be helpful to understand the pathogenesis of infection caused by Enterococcus spp. as well as for antimicrobial usage strategies.

Investigation of Biofilm Formation Properties of Coagulase Negative Staphylococci Isolated from Catheter-Related Bloodstream Infections

In view of the significant negative impact of biofilm-mediated infection on patient health and the necessity of a reliable phenotypic method to detect biofilm producers, this study aimed to demonstrate phenotypic and molecular biofilm formation in coagulase-negative staphylococci (CoNS) isolated from catheter related infections and to compare the methods used with each other. The study was also aimed to determine the biofilm eradication effect of vancomycin in order to guide for the treatment. For the detection of biofilm formation, a total of 154 CoNS clinical isolates of which 30 being causative agents of catheter related bloodstream infection (CRBSI) (isolated from both the catheter tip and blood cultures of 15 patients), 89 being isolated from peripheral blood cultures of patients without a central venous catheter (CVC) (13 of them were bloodstream infection agents, 76 of them were contaminant), and 35 being isolated as catheter colonizer, were screened by tissue culture plate (TCP), Congo red agar (CRA) method and polymerase chain reaction (icaA, icaD and IS256). Vancomycin minimum inhibitory concentration (MIC) and minimum biofilm eradicating concentration (MBEC) values were determined. The pulsed field gel electrophoresis (PFGE) method was used to show the clonal relationship between CoNS isolated from the catheter tips and peripheral blood of patients with CRBSI. Of the 154 CoNS isolates included in the study, 38.9% were Staphylococcus epidermidis (n= 60), 34.4% were Staphylococcus haemolyticus (n= 53), 20.7% were Staphylococcus hominis (n= 32), and 3.8% were detected as Staphylococcus capitis (n= 6). In our study, biofilm formation was shown in 31.8% with the CRA method and in 68.1% with the TCP method. By TCP method, 22% (n= 34) were determined as weak, 31.2% (n= 48) medium and 14.9% (n= 23) strong biofilm producers. While the sensitivity of the CRA method was found to be low for isolates that were determined as weak positive in the microplate method, the high sensitivity of the CRA method for isolates with medium and strong positivity was found remarkable. The positivity rates of icaA, icaD and IS256 genes in a total of 154 CoNS isolates were found to be 40 (25.9%), 57 (37%) and 77 (50%), respectively. In total, at least one gene positivity was detected in 107 (69.5%) isolates. Single gene positivity was detected in 55 (35.7%), two gene positivity in 35 (22.7%) and three gene positivity was detected in 17 (11%) of the included CoNS. Biofilm formation (four weak, four medium, two strong) was detected by microplate method in 10 of 47 CoNS isolates (five S.epidermidis, three S.hominis, one S.haemolyticus and one S.capitis) in which no genes were detected. Vancomycin MBEC/ MIC values were found to be high and it was observed that as the biofilm forming power of the isolates increased, the MBEC/MIC ratio also increased. The CoNS isolated from the catheter samples and blood of patients diagnosed with CRBSI had a 100% similar profile with PFGE except for one unevaluable isolate. The tissue culture plate (TCP) method was found to be most sensitive, accurate and reproducible screening method for detection of biofilm formation by staphylococci and has the advantage of being a quantitative model to study the adherence of staphylococci. The presence of the icaAD and IS256 gene is not always associated with in vitro biofilm formation. For this reason, it is more appropriate to use more than one method together for the evaluation of biofilm formation. It was thought that the use of reliable methods to specifically detect biofilms could be helpful in diseases that are difficult to treat. Considering the high rates of biofilm and antimicrobial resistance of biofilm-forming isolates in biomedical device associated infections, it was determined that it would not be sufficient to evaluate only the MIC results for susceptibility results.

Promoting Resuscitation of the VBNC Bacteria and Enrichment of Naphthalene-degrading Bacteria from Oil-contaminated Soils with the Rpf4 from Rhodococcus erythropolis KB1

Nowadays, more than 90% of bacteria in nature are in a viable but not culturable (VBNC) state, and they cannot be separated by traditional plate culture methods. However, resuscitation-promoting factors (Rpf) participate in recovery of dormant cells and promoting bacterial growth. The Rpf4 of Rhodococcus erythropolis KB1 is a Rpf like protein of Micrococcus luteus and contained the Rpf domain and LysM domain. In this study, an expression vector for the rpf4 gene was constructed and expressed in Escherichia coli. The recombinant protein Rpf4 was purified by Ni2+-affinity chromatography. The recombinant protein showed a muralytic activity of 549 ± 28.77 U/mg. The purified Rpf4 could increase R. erythropolis KB1 cell proliferation and improve the resuscitation of R. erythropolis cells that were VBNC. The Rpf4 altered the diversity of naphthalene-degrading bacteria enriched from oil-contaminated soils. Based on the high-throughput sequencing, we found that Proteobacteria and Actinobacteria phyla increased from 76.10% and 0.029% to 98.23% and 0.401% after the Rpf4 treatment, while the Bacteroidetes phylum decreased from 22.70% to 0.76%. The genera Enterobacter, Citrobacter, and Acinetobacter increased from 0.0054%, 0.001%, and 0.745% to 19.02%, 6.34% and 2.49%. Some new genera such as Bifidobacterium, Limnobacter, Alloprevotella, Hydrogenophaga, and Nevskia were founded in the treatment group. Meanwhile, 23 naphthalene-degrading bacteria were isolated by adding the Rpf4 protein to the oil-contaminated soil samples. Study on the naphthalene-degrading bacteria will provide a basis for the microbial degradation of naphthalene and other polycyclic aromatic hydrocarbons and discover the wider application value of Rpf.

Evaluation of a Most Probable Number Method for Detection and Quantification of Legionella pneumophila.

The detection and enumeration of Legionella pneumophila (L. pneumophila) in water is crucial for water quality management, human health and has been a research hotspot worldwide. Due to the time-consuming and complicated operation of the plate culture method, it is necessary to adopt a fast and effective method for application. The present study aimed to comprehensively evaluate the performance and applicability of the MPN method by comparing its qualitative and quantitative results with the GB/T 18204.5-2013 and ISO methods, respectively. The qualitative results showed that 372 samples (53%) were negative for both methods; 315 samples (45%) were positively determined by the MPN method, compared with 211 samples (30%) using GB/T 18204.5-2013. The difference in the detection rate between the two methods was statistically significant. In addition, the quantitative results showed that the concentration of L. pneumophila by the MPN method was greater than ISO 11731 and the difference was statistically significant. However, the two methods were different but highly correlated (r = 0.965, p < 0.001). The specificity and sensitivity of the MPN method were 89.85% and 95.73%, respectively. Overall, the results demonstrated that the MPN method has higher sensitivity, a simple operation process and good application prospects in the routine monitoring of L. pneumophila from water samples.

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.&#x0D;

Phenotypic and genotypic study associated with biofilm formation for E. coli isolated from UPEC

UTI is a serious health problem with respect to antibiotic resistance and biofilms formation being the prime cause for the antibiotic resistance. In current study sixty E.coli isolated collected from patients with urinary tract infection. In this study, biofilm formation were detect by phenotypic and genotypic methods. The detection of biofilm formation by phenotypic method was carried out by Congo Red Agar (CRA) method and tissue culture plate (TCP) method and the genotypic method performed by detection present some gene associated with biofilm formation which include fliC ,csgA ,fimH and papC.. The results showed that 54(90%) from E. coli produced strong and moderate slime layer. The present study showed that 57(95%) from E. coli produced strong and moderate biofilm. Genotypic study of E. coli biofilm-associated genes were found in various frequencies in all isolates that included fliC (63.3%), csgA (73.3%), fimH (90%) and papC (76.6%).

Antagonistic Potential Marine Bacteria Against Luminescent Vibriosis Pathogen Vibrio Harveyi Obtained from Shrimp Farm, Marakkanam, Tamil Nadu, India

Aquaculture farming practices in India and other countries are facing a lot of economic loss due to luminescent vibriosis.Ecological and cost-effective practices may have been warranted to combat this tricky disease. A total of 57 morphologicallydifferent marine bacterial isolates were isolated from samples collected at different aquaculture ponds in Marakkanam, Tamil Nadu,India and screened for their antibacterial potential against luminescent vibriosis causing bacteria Vibrio harveyi. All marine bacterialisolates were screened for antibacterial activity by cross streak method against two different isolates V. harveyi GNC01 and V.harveyi GNC03. Secondary screening of antagonistic isolates by dual culture plate method and agar well diffusion method leads tothe identification of antagonistic isolates. The antagonistic isolates were characterized by molecular taxonomy (16S rRNA genesequence) and phylogeny and identified as Neobacillus niacini (GNC04), Bacillus halotolerans (GNC06), Bacillus amyloliquefaciens(GNC16), Lacticaseibacillus paracasei (GNC23) and Lactiplantibacillus plantarum (GNC24). The minimum inhibitory concentrationof all these selected bacterial crude secondary metabolites against both Vibrio harveyi isolates GNC01 and GNC03 ranged between0.062 to 0.50 mg/mL. The GC-MS profiling of the ethyl acetate extract revealed the presence of antibacterial compounds such asOxime-,methoxy-phenyl (26.63%), 2,3-Dihydroxybutane (9.52%), Phosphite, menthyldimethyl- (2.83%), Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)- (2.07%). The results of this study suggest that Lactiplantibacillus plantarum (GNC24) is apotential source for antagonistic secondary metabolites against luminescent vibriosis causing bacteria Vibrio harveyi. Further,biological control formulation with these isolates and their metabolites will be a reward for the integrated pest management inthe aquaculture sector.