Culture Plate Method Research Articles

Correlation between Bio-Film Formation and Carbapenem Resistant Enterobacteriaceae Isolates Obtained from Various Clinical Specimens

Introduction: The ability to form bio-film is a universal trait of bacteria by attaching to the surfaces. Carbapenem resistance in Enterobacteriaceae might be endorsed by bio-film formation which augmented colonization of pathogens. Aim: To correlate between bio-film formation and carbapenem resistant Enterobacteriaceae isolates obtained from various clinical specimens and to compare the qualitative and quantitative assay for bio-film production. Method: Study was conducted on 150 strains of Enterobacteriaceae isolates. of these, carbapenem resistant Enterobacteriaceae isolates were subjected for bio-film formation by Modified Congo red agar method, tube adherence method and Tissue culture plate method. Results: Carbapenem resistant Enterobacteriaceae strains were found to be 60.66% and the rate of biofilm producers was 75.53% by any of the phenotypic method. Tissue culture plate method was found to be (67.6%) better than Modified Congo Red agar method (54.9%) and Tube adherence method (39.4%). The highest number of bio-film producers was isolated from urinary tract infections (36.61%). Conclusion: TCP method is most reliable, precise and sensitive method for detection of bio-film formation by Enterobacteriaceae isolates and is ideal to use as a general screening tool to detect bio-film production.

BIOFILM FORMATION AND ANTIBIOTIC SUSCEPTIBILITY OF UROPATHOGENS IN PATIENTS WITH CATHETER ASSOCIATED URINARY TRACT INFECTIONS IN IBB CITY -YEMEN

Biofilm formation by uropathogens on the surface of indwelling medical devices, can inflict obstinate or recurring infection, thought-provoking antimicrobial therapy. This study included 227 patients with indwelling urinary catheters and suffering from CAUTI. They were analyzed for biofilm formation and antibiogram susceptibility, 59.4% were males and 40.6% were females. Ensuing phenotypic identification of isolated bacteria, antibiotic sensitivity test was performed by modified Kirby–Bauer disc diffusion method following the Clinical and Laboratory Standards Institute (CLSI 2015) guidelines; Biofilm-forming uropathogens were detected by the tissue culture plate (TCA) method. The predominant uropathogen in catheter-associated UTIs (CAUTIs) was Escherichia coli 46.3%, followed by K. pneumonia 18.5%, P. aeruginosa 11.9%, 7%, S. coagulase negative 5.7%, S. aureus 4.8%, Enterobacter spp. 4.4%, E. faecalis 1.3%. The total rate of biofilm producer bacteria was 49.3% (21.1% high producers, 28.2% moderate producers). Prime biofilm producers were E. coli 60% with OR=8.6 (p=0.002), followed by K. pneumoniae 57.1% with OR=10.1 (p=0.002), and P. aeruginosa 37% with OR=6.6 (p=0.02). The biofilm producers bacteria were associated with >65year patients (OR=5.4, p>0.001), pre-UTI (OR=2.4,p<0.001), long duration of catheterization (OR=15.3, p<0.001), and diabetic mellitus (OR=3.5, p<0.001). Multidrug resistance associated with biofilm producers were greater than biofilm non-producers. Gram-negative biofilm producers found 100%, 100%, 88.6%, 82.9%, 81.9%, 80.9%, and 72.4%, 40%, 33% resistant to ampicillin , amoxyclave, cotrimoxazole, ceftraxone, nalidixic acid, ciprofloxacin, cefotaxime , nitrofurantoin and amikacin respectively. Gram-positive biofilm producers, however, were found 85.7%, 85.7%, 71.4%, 71.4%, 57.1% and 42.9% resistant to penicillin, erythromycin, cotrimoxazole, gentamycin, norfloxacin, and nitrofurantoin respectively. In conclusion, a high antimicrobial resistance was observed in biofilm producers than non-biofilm producers. Of recommended antimicrobial therapies for CAUTIs, ampicillin and amoxicillin-clavulanate were the least active antibiotics, whereas imipenem and amikacin were found as the most effectual for gram-negative biofilm producer. Likewise, penicillin and erythromycin were the least active antibiotics, whereas vancomycin, and rifampicin were found as the most effective antibiotic for Gram-positive biofilm producer.
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 Name: Dr. Müjde Eryılmaz 
 Affiliation: Ankara University, Turkey
 E-mail: meryilmaz@ankara.edu.tr
 
 Name: Dr. Tamer Mohamed Ali El Habibi
 Affiliation: Suez Canal University, Egypt.
 E-mail: tamer_hassan@pharm.suez.edu.eg
 
 Name: Dr. Ali Alyahawi
 Affiliation: Depatment of Pharmacy, Al-Razi University, Yemen.
 E-mail: alyahawipharm@yahoo.com
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Biological control of bacterial wilt of tomato caused by Ralstonia solanacearum using Pseudomonas species isolated from the rhizosphere of tomato plants

Tomato bacterial wilt, caused by Ralstonia solanacearum, is one of the devastating soil-borne diseases of tomato plants in Nigeria. We investigated the potentiality of indigenous Pseudomonas species in controlling bacterial wilt of tomato. Seven Pseudomonas species were isolated and identified from the rhizosphere of tomato plants. The isolates were screened for enzyme production, and in vitro antimicrobial activity against pathogenic R. solanacearum using dual plate culture method. The biocontrol and growth-promoting efficacy of the Pseudomonas species were studied in the screenhouse using a completely randomised design. P. aeruginosa, P. syringae and P. fluorescens produced lipase, protease and α-amylase enzymes. All the Pseudomonas isolates except P. aureofaciens inhibited the growth of R. solanacearum. Since the tested species reduced the incidence of bacterial wilt and promoted the growth of the tested tomato genotypes, the indigenous Pseudomonas strain could be used to develop active biocontrol agents of tomato bacterial wilt in Nigeria.

Interpreting the Results of the Conventional Plate Culture and Gene Detection Methods for Legionella Detection in Environmental Water Samples.

The conventional plate culture method is widely used as a method for detection of Legionella in environmental water samples, but to obtain results takes more than a week. Because it is much quicker, the gene detection method has become widespread as an alternative detection method. However, the results of gene detection and plate culture methods may differ even when the same sample is examined; the gene detection method shows a higher detection ratio than the plate culture method. The reason for this difference is that the plate culture method detects Legionella cells that have the ability to form colonies on an agar plate, whereas the gene detection method detects any Legionella genes present regardless of the state of the Legionella. In this paper, we consider the factors that cause differences between the results of the plate culture and gene detection methods, and how to interpret the results of each.

Evaluation of Legiolert for Quantification of Legionella pneumophila from Bath Water Samples.

Testing for Legionella spp. in public bath water samples is regulated in Japan. In this study, we used a total of 132 public bath water samples to compare the performance of Legiolert® and the conventional plate culture method for the enumeration of Legionella pneumophila. When Legiolert and plate culturing were performed at the same detection limit, L. pneumophila was detected in 26.5% of 132 samples by Legiolert, while 12.9% contained Legionella spp. (11.4% contained L. pneumophila) based on the plate culture method. Moreover, results of 83.3% of the total samples were consistent between the two methods, meaning that they were both positive or both negative. In this study, we demonstrated that Legiolert is a simpler and more effective method of monitoring for L. pneumophila in bath water samples.

Comparative evaluation of antibacterial efficacy of green coffee bean extract mouthwash and chlorhexidine mouthwash against Streptococcus mutans and Lactobacilli spp. – An in vitro study

INTRODUCTION: Dental caries can occur in an individual irrespective of age, which makes preventing the incidence of dental caries vital. The prevention of dental caries includes various types of oral hygiene measures. One such modality is the use of mouthwash. As chlorhexidine mouthwash causes several adverse effects, there is a need for herbal alternative. Green coffee bean extract is one such herbal alternative. AIM: The aim of this study is to compare and evaluate the antibacterial efficacy of green coffee bean extract mouthwash and chlorhexidine mouthwash against Streptococcus mutans and Lactobacilli spp. MATERIALS AND METHODS: The ethanolic extract of green coffee bean was obtained by treating it with cellulase and 30% ethanol. Minimum inhibitory concentration and minimum bactericidal concentration were obtained by the broth dilution method and culture plating method, respectively. Based on these values, 3% green coffee bean extract mouthwash was prepared, and the antibacterial efficacy against S. mutans and Lactobacilli spp. was tested using the direct contact test. The obtained data were analyzed using the Mann–Whitney U-test and Wilcoxon matched-pair test. RESULTS: Intergroup comparison using Mann–Whitney U-test showed that green coffee bean extract mouthwash is equally effective against S. mutans and Lactobacilli spp. as compared to chlorhexidine mouthwash. Wilcoxon matched-pairs test showed that the efficacy of green coffee bean extract mouthwash reduced over a period of 10, 30, and 60 min time intervals. CONCLUSION: Therefore, green coffee bean extract can be used as an alternative to chlorhexidine as mouthwash.

Investigating the prevalence of intestinal parasites with an emphasis on Strongyloides stercoralis infection in hospitalized patients: a regional report from Iran.

Intestinal parasitic infections such as strongyloidosis are more common among individuals with immune deficiency and sometimes accompanied by severe symptoms. The purpose of this cross-sectional study was to evaluate the prevalence of intestinal parasites with focus on strongyloidosis in hospitalized patients. A total number of 566 faecal samples were obtained from different wards and assessed by the use of direct smear, formalin-ether concentration, and agar plate culture procedures in order to find parasitic protozoa and helminthes. The findings revealed that 10.1% (n=57) of the examined samples were positive for intestinal parasites. The highest prevalence rate was related to Entamoeba coli (4.6%, n=26) and the lowest one was related to Strongyloides stercoralis (0.5%, n=3). In addition, Giardia lamblia prevalence rate was 3.2% (n=18) and the prevalence rate of Blastocystis hominis was 1.8% (n=10). The sensitivity of S. stercoralis diagnosis was equal for agar plate culture and formalin-ether concentration methods. This study demonstrated the significance of focus on intestinal parasites in hospitalized patients and highlighted the necessity of improving the insight in health care providers about the occurrence of parasitic infections especially strongyloidiasis in these patients.

Detection of Biofilm Production and Its Impact on Antibiotic Resistance Profile of Bacterial Isolates from Chronic Wound Infections.

Background:Microorganisms are known to be involved in the formation of biofilm. These biofilms are often seen in chronic wound infections, surgical site infections, implants etc., These are capable of causing recalcitrant infections and most of them are also known to possess high antibiotic resistance.Objectives:This study was conducted to detect the biofilm formation in bacterial isolates from chronic wound infections.Materials and Methods:In the present study, ninety two isolates from chronic wound infections were identified by MALDI-TOF-MS (bioMerieux) and VITEK-2-MS (bioMerieux). These isolates were further screened for biofilm formation by three methods i. e., Tissue Culture Plate method (TCP), Tube Method (TM) and Congo Red Agar (CRA) method. Impact of biofilm production was correlated with the antibiotic resistant pattern.Statistical Analysis:Statistical analysis was done for all three methods considering TCP as Gold Standard and parameters like senitivity and specificity of TM i.e. 47.2 and 100% respectively.Results:Out of 92 isolates, biofilm formation was seen in 72 isolates (78.2%) by TCP method. 64 isolates were strong biofilm producers, 8 isolates were moderate biofilm producers and 20 isolates were nonbiofilm producing. High prevalence of biofilm formation was seen in nonhealing ulcers infected with Staphylococcus aureus followed by Klebsiella pneumoniae.Conclusion:Among three screening methods used for detection of biofilm production, TCP method is considered to be a standard and most reliable for screening of biofilm formation in comparison to TM and CRA.

BIOFILM FORMATION AND ANTIBIOTIC SUSCEPTIBILITY OF STROKE PATIENT’S URINARY CATHETER ISOLATES AT BETHESDA HOSPITAL, YOGYAKARTA

Background: Antibiotic resistance currently challenges infectious disease management, specifically in combating biofilm formation. Biofilm might be developed on urinary catheter, in which stroke patients with urinary tract problem have higher risk of catheter associated urinary tract infection and problems related to antibiotic resistance. Aim: This study describes the microbe types from catheter isolates, measuring the antibiotic susceptibility and biofilm formation, especially in stroke patients at Bethesda Hospital, Yogyakarta. Method: This is a cross-sectional study, describing findings of specimens from stroke patients at Bethesda Hospital, since December 2018 to January 2019. The isolates were identified with standard method, the antibiotic susceptibility were tested with antibiotic disc on MHA medium, and the biofilm formation were assessed using tissue culture plate method. All procedure were done in Microbiology Laboratorium, FK UKDW. Result: Thirty three microbes were isolated from 30 specimens of urinary catheter (63,3% male, 36,3% female). There is 76% isolates incapable of developing biofilm, while 15% is moderate biofilm producer and 9% is strong biofilm producer. The susceptibility test showed 100% resistancy of ampicillin-sulbactam, tetracyclin, ketoconazole, itraconazole, and terbinafine. Around 50% of E. coli and 57% of K. pneumonias isolates is MDR, and 75% of A. baumanii is XDR. Conclusion: Isolated microbes from urinary catheter of stroke patients at Bethesda Hospital is dominated by E. coli, K. pneumoniae, and A. baumanii. Some of the microbes could form strong biofilm, and some of the antibiotics could not be used in the future due to their 100% resistancy on all specimens.

Effect of Psidium guajava L on Biofi lm Forming Multidrug Resistant Extended Spectrum Beta Lactamase (ESBL) Producing Pseudomonas aeruginosa

Objectives: In order to investigate the relative efficacy of Psidium guajava L (guava) leave, in vitro antibacterial effect of ethanolic extract and leaf tea of guava against pathogenic Pseudomonas aeruginosa was carried out.
 Methods: This study was carried out at Microbiology laboratory of KIST medical college and teaching hospital, Lalitpur. Pseudomonas aeruginosa was isolated from different clinical samples. Antibiotic susceptibility of the isolates was performed by Kirby-Bauer disk diffusion method. Biofilm formation was detected by microtitre culture plate method and ESBL production by combine disk methods. Antimicrobial activity of guava leaf tea and ethanol extract of leaf were determined by agar well diffusion method.
 Results: Ethanol extract of fresh guava leaves exhibited higher antibacterial activity than dry and fresh leaves tea, but significantly less than the standard antibiotics. In this study, 7% of the total bacterial isolates were P. aeruginosa with 65.30% of these isolates being MDR. Similary, significant number of these MDR strains ie. 83.67% of these P. aeruginosa isolates produced biofilm. While as 6.12% of the isolated were ESBL producer.
 Conclusion: The leaves extract of guava have shown effective result against P. aeruginosa and could serve as good source of antibacterial agents. Guava leaves extract can be an economic alternative to antibiotics. However, active compound of this extract need to be purified and pharmacologically tested before its application.

Isolation, identification, carbon utilization profile and control of Pythium graminicola, the causal agent of chilli damping‐off

AbstractPostemergence damping‐off of chilli caused by Pythium spp. is a common and serious problem in large chilli growing areas of India under the moist conditions that generally prevails during the sowing period. Therefore, in order to better understand this disease, an isolate belonging to the genus Pythium (Pythiales) was isolated from the infected chilli (Capsicum annuum L.) plant root parts collected from the fields of Chandauli district, Uttar Pradesh, India. Based on the congruence of cultural, morphological, cardinal growth rate and the sequence data analysis, the isolate was identified as Pythium graminicola. The molecular phylogenetic analysis based on ITS‐rDNA sequences clustered the isolate with representative sequences for P. graminicola from GenBank in the Pythium clade. The isolate carbon utilization profiles were characterized using Biolog FF MicroPlate method. The results revealed that the isolate used a wide range of carbon sources, mainly carbohydrates, but also amino acids, suggesting the use of metabolic routes that include glycolysis/gluconeogenesis. Moreover, an in vitro colony growth inhibition assay was performed to determine the influence of chemical (fungicides) and biological (bacteria and fungi) antagonists over the pathogen using the poison plate and dual culture method, respectively. Overall, the results revealed that the presence of aggressive broad range biocontrol agents can be used as an effective environmentally friendly approach for management and control of damping‐off in production systems. The antagonist can serve as a bio‐efficient and eco‐friendly alternative to synthetic fungicides for the development of an effective integrated pest management (IPM) system and obtaining higher yields.

Coagulase negative staphylococci in Anti-Cancer Center, Batna, Algeria: antibiotic resistance pattern, biofilm formation, and detection of mecA and icaAD genes

Background : Coagulase-negative staphylococci (CoNS) are normal microbial flora found on the skin and mucous membranes of mammals. Considered for a long time as avirulent commensals, these bacteria are now recognized as opportunistic pathogens by virtue of their high resistance to multiple antibiotics and capacity for biofilm formations, which made them important agents of nosocomial and community-acquired infections. The objectives of this study are to determine the antibiotic resistance pattern and biofilm formation, and to detect mecA and icaAD genes in clinical CoNS isolates from Batna’s Anti-Cancer Center (ACC) in Algeria. Methods : A total of 66 CoNS were isolated from different samples and identified by API Staph system. In vitro antibiotic susceptibility testing (AST) of each isolate to selected antibiotics was determined by the disk diffusion method, and minimum inhibitory concentrations (MICs) of oxacillin and vancomycin were determined by E-test. Biofilm formation was assessed by Tissue Culture Plate (TCP) and Congo Red Agar (CRA) methods. The polymerase chain reaction (PCR) was used to amplify mecA gene in 9 oxacillin-resistant and 1 oxacillinsensitive CoNS, and icaAD gene in 9 biofilm forming and 1 non-biofilm forming CoNS. Sequencing of the 16S rDNA of 1 mecA and 1 icaAD positive isolates was performed by the Sanger method. Results : Nine species of CoNS were identified, with Staphylococcus epidermidis (n=29, 44%) and Staphylococcus haemolyticus (n=15, 22.7%) constituting the largest proportion, and isolated mainly from the onco-haematology service unit of the center. The isolates were resistant to penicillin G (98.5%), cefoxitin (80.3%) and oxacillin (72.2%). The TCP method was more sensitive (89.4%) than CRA method (31.8%) in detecting biofilm formation. The mecA gene was detected in 66.7% (6/9) of oxacillin resistant CoNS and the icaAD gene in 55.6% (5/9) of TCP positive CoNS isolates Conclusion : Invitro resistance to methicillin (oxacillin) and biofilm formation were high among the CoNS isolates in this study, but the association of these with respective carriage of mecA and icaAD genes was low. Keywords : Coagulase negative staphylococci, identification, antibiotic resistance, biofilm, PCR

An in vitro experimental study of the effect of fosfomycin in combination with amikacin, ciprofloxacin or meropenem on biofilm formation by multidrug-resistant urinary isolates of Escherichia coli.

Background. This study was conducted to understand the effect of fosfomycin in combination with amikacin, ciprofloxacin or meropenem on biofilm formation by multidrug-resistant urinary isolates of Escherichia coli.Methods. Fifty urinary tract multidrug-resistant E. coli isolates that were known biofilm producers were studied. The MIC was determined using the agar dilution method for amikacin, ciprofloxacin, meropenem and fosfomycin. The fractional inhibitory concentration was determined for the combination of antibiotics followed by a time-kill assay. A tissue culture plate method was used to study the effect of the combination of antibiotics on biofilm formation.Results. The MICs of the isolates tested ranged from 0.25 to 32 µg ml-1 for fosfomycin, 1 to 1024 µg ml-1 for ciprofloxacin, 4 to 1024 µg ml-1 for amikacin, and 0.25 to 512 µg ml-1 for meropenem. The combination of fosfomycin with meropenem showed 68 % synergy, fosfomycin with amikacin 58 % synergy and fosfomycin with ciprofloxacin 6 % synergy. The combination also reduced the MIC of each antibiotic and none showed an antagonistic effect. Biofilm inhibition was best observed with the combination of fosfomycin with meropenem.Conclusion. The combination of fosfomycin with amikacin and fosfomycin with meropenem yielded a high percentage of synergy alongside an increased capacity to reduce biofilm formation when compared to combination with ciprofloxacin against multidrug-resistant E. coli. Fosfomycin in combination with other classes of antimicrobial agents has potential beneficial effects.

Biofilm Measurement, Site Specific Variation in Biofilm Production, Biofilm Typing: An Acinetobacter Trilogy

Acinetobacter baumannii is a member of the ESKAPE pathogens, notorious for causing multidrug resistant nosocomial infections worldwide. Biofilm has an important role in its persistence and spread in hospital environment, as well as its multidrug resistance potential. The present study was aimed at finding out whether the strength of Biofilm formation varies with infection of different organs / system. A total of 136 isolates of Acinetobacter baumannii were taken from a variety of samples. Strength of Biofilm formation, determined by Tissue culture plate method, was graded according to OD value in ELISA reader as nil, moderate and strong. It was seen that association between variations in biofilm forming capacity depending on different sites of infection was statistically significant. We suggest biofilm typing by this method for this pathogen, as a potentially feasible alternative in countries with cost restrained healthcare set ups. Moreover, biofilm typing report would be more meaningful to the clinicians as well.

Bacillus subtilis BS-2 and Peppermint Oil as Biocontrol Agents Against Botrytis cinerea

Abstract The purpose of this study was to assess the activity of Bacillus subtilis BS-2 and peppermint oil against Botrytis cinerea. In this study parameters such as the age and the density of the bacterial culture and the incubation temperature were taken into consideration. Furthermore, the cellulolytic activity of the bacterium was determined. The effect of peppermint oil was evaluated at a concentration range of 0.5-4.0 %. The research was conducted with a dual culture plate method. The influence of B. subtilis BS-2 and peppermint oil on the growth of B. cinerea was evaluated based on the growth rate index. It was noted that the bacterial culture occurred at an initial density of OD 560 = 1.0, cultivated at 30 °C for 48 hours demonstrated the strongest antagonistic effect (57.07 % inhibition). Furthermore, it was observed that the highest cellulolytic activity occurred on the bacteria incubated for 48 hours at 37 °C. The effect of mint oil, at the lowest concentration of 0.5-1.0 %, was much weaker on bacterial activity (1.1-12.1 % inhibition). The highest concentration (4.0 %) of mint oil caused the maximum inhibition (31.9 %) of the mycelial growth. B. subtilis BS-2 may be environmental-friendly alternatives for protecting plants against B. cinerea

Occurrence and biofilm forming ability of Pseudomonas aeruginosa in the water output of dental unit waterlines in a dental center in Alexandria, Egypt.

Dental unit waterlines (DUWLs) are notorious for being contaminated with different bacterial species including the opportunistic pathogen Pseudomonas aeruginosa which poses a risk to patients and professionals. This work aimed at studying the occurrence and biofilm-forming ability (BFA) of P. aeruginosa in the output of DUWLs in a dental center in Egypt. Water samples were collected from the outlets of the high-speed hand piece, the air/water syringe and the cup filler waterlines. Bacteriological analysis included heterotrophic plate count (HPC), isolation and identification of P. aeruginosa and determination of the antimicrobial susceptibility and the BFA of the isolates by tissue culture plate (TCP) method and tube method (TM). The average concentration of HPC bacteria in the output of the 3 DUWLs was 2.9×104 CFU/μL where 88.3% of the samples exceeded the Egyptian standards for drinking water (<50 CFU L). P. aeruginosa was isolated from nine cup filler samples (which had a water source different from the other waterlines). The isolates were sensitive to all tested antimicrobials. Of these nine isolates, 6, 5 and 4 were positive for BFA by TCP, modified TCP and TM, respectively. More stringent measures are required to ensure safer dental water; as the majority of studied samples exceeded the required HPC bacterial limit and P. aeruginosa isolates were detected. P. aeruginosa isolates from DUWLs may not be as resistant to antibiotics as what is reported in the literature about clinical isolates. Some P. aeruginosa isolates can colonize DUWLs despite their inability to form biofilms in experimental testing.

Colloidal gold immunochromatographic strip for rapid detection of Haemophilus influenzae

To establish a novel colloidal gold immunochromatography assay (GICA) for rapid, sensitive and accurate detection of Haemophilus influenzae infection by using the outer membrane protein P6 as detection target. First, the linear antigen epitope located in the extracellular domain of the P6 protein (GenBank accession number: AGH02799) was predicted by bioinformatics analysis. The region (62-75 aa of the protein) with strong antigen specificity was chosen and synthesized. Two rabbits were then immunized by the polypeptides (14 aa) for production of polyclonal antibodies. Then, the recombinant P6 proteins were also obtained to produce polyclonal antibodies. Finally, based on the two antibodies, a novel colloidal GICA for detection of Haemophilus influenzae infection was established and the specificity, sensitivity, repeatability and stability of this method were evaluated. At the same time, the method was tested in clinical simulation, and the plate culture method was used to verify its accuracy. The test strip for Haemophilus influenzae infection was successfully prepared. The detection limit of the test strip was as low as 1×105 CFU/mL and the whole process can be completed within 15 minutes. The strip specifically recognized Haemophilus influenzae and did not react with nine of other common respiratory pathogens such as Streptococcus pneumoniae, Moraxella catarrhalis, Mycoplasma pneumonia, and Legionella pneumophila. And the strips could be stored at 25 °C for at least 6 months without losing sensitivity or specificity. The coincidence rate between the results of 200 clinical samples and the plate culture method was 90.5%. Haemophilus influenzae protein P6, which possessed a high degree of surface antigen accessibility and antigencity, could be used as a marker for Haemophilus influenzae detection. The immunochromatographic colloidal gold test strip which bears the features of rapidity, convenience and sensitivity provides a unique tool for the on-site surveillance and diagnosis of Haemophilus influenzae infection in clinical test.

Development of a rapid viability polymerase chain reaction method for detection of Yersinia pestis

Due to the occurrence of natural plague outbreaks and its historical usage as a biological weapon, Yersinia pestis is considered one of the high-priority biological threat agents. It can remain viable in certain environments including water for >100 days. Because of its slow-growth characteristic, it usually takes three or more days to detect and confirm the identity of viable Y. pestis cells by PCR, serological, or biochemical assays when using the traditional microbiological plate-culture-based analysis, and that too, assuming faster growing microbes present in a water sample do not mask the Y. pestis colonies and interfere with analysis. Therefore, a rapid-viability Polymerase Chain Reaction (RV-PCR) method was developed for detection of Y. pestis. The RV-PCR method combines 24 h-incubation broth culture in a 48-well plate, and pre- and post-incubation differential PCR analyses, thereby allowing for rapid and high-throughput sample analysis compared with the current plate culture method. One chromosomal and two plasmid gene target-based real-time PCR assays were down-selected, showing ca. 10 genome equivalent detection; the chromosomal assay was then used for RV-PCR method development. A 101-cell level (10–99 cells) sensitivity of detection was demonstrated even with complex sample backgrounds including known PCR inhibitors (ferrous sulfate and humic acid), as well as metal oxides and microbes present in Arizona Test Dust (ATD). The method sensitivity was maintained in the presence of dead Y. pestis cells up to 104 cells per sample. While affording high-throughput and rapid sample analysis, the 48-well plate format used in this method for sample enrichment significantly reduced labor requirements and generation of BioSafety Level-3 (BSL-3) laboratory waste as compared to the usual microbiological plate-culture-based methods. This method may serve as a model for other vegetative bacterial pathogens.

Miniaturized fermentation in eppendorf tubes for the detection of antagonistic actinomycetes

Actinomycetes are recognized as a prolific source for bioactive metabolites. Screening adequate number of strains by appropriate high quality screening protocol determines success of drug discovery programs. To accomplish screening for antagonistic actinomycetes at higher rate, the fermentation stage of the initial screening needs to be miniaturized. The present study is attempted for miniaturized production of bioactive compounds from 40 actinomycetes isolated from less explored ecosystems. Actinomycetes were cultivated in eppendorf tubes (2 ml) using 0.5 ml of yeast extract malt extract agar and modified nutrient glucose agar medium for 10 days and extracted using 1 ml of methanol. The crude extracts were tested against S. aureus, B. subtilis and E. coli by disc diffusion method. All the isolates were also tested for antagonistic activity by conventional cross streak method and disc diffusion method using extracts prepared by plate culture method. Crude bioactive compounds produced from five selective isolates by shake flask method showed good activity. Antagonistic activity, requirements, time duration and cost effect of all the three methods were compared. Miniaturized production process in eppendorf tubes was found to be more advantageous and suitable to develop as high throughput screening (HTS) method for the detection of antagonistic actinomycetes particularly in resource poor laboratories settings.

Colorimetric Analysis of Alkaline Phosphatase Activity in S. aureus Biofilm.

Alkaline phosphatase (ALP) is a common enzyme expressed in both prokaryotic and eukaryotic cells. It catalyzes the hydrolysis of phosphate monoesters from many molecules at basic pH and plays an indispensable role in phosphate metabolism. In humans, eukaryotic ALP is one of the most frequently used enzymatic signals in diagnosing various diseases, such as cholestasis and rickets. In S. aureus, ALP is detected exclusively on the cell membrane; it is also expressed as a secretory form as well. Yet, little is known about its function in biofilm formation. The purpose of this manuscript is to develop a quick and reliable assay to measure ALP activity in S. aureus biofilm that does not require protein isolation. Using p-nitrophenyl phosphate (pNPP) as a substrate, we measured ALP activity in S. aureus biofilm formed in 96-well tissue culture plates. Activity was based on the formation of the soluble reaction product measured by 405 nm absorbance. The high throughput nature of the 96 well tissue culture plate method provides a sensitive and reproducible method for ALP activity assays. The same experimental set up can also be extended to measure other extracellular molecular markers related to biofilm formation.