Antifungal Activity of Lactobacillus plantarum ZZUA493 and Its Application to Extend the Shelf Life of Chinese Steamed Buns

Lactic acid bacteria (LAB) can produce many kinds of antifungal substances, which have been widely proven to have antifungal activity. In this study, 359 strains of LAB were screened for antifungal activity against Aspergillus niger (A. niger) using the 96-well microtiter plate method, and three showed strong activity. Of these, ZZUA493 showed a broad-spectrum antifungal ability against A. niger, Aspergillus oryzae, Trichoderma longibrachiatum, Aspergillus flavus and Fusarium graminearum. ZZUA493 was identified as Lactobacillus plantarum. Protease treatment, the removal of hydrogen peroxide with catalase and heat treatment had no effect on the antifungal activity of the cell-free supernatant (CFS) of ZZUA493; organic acids produced by ZZUA493 appeared to have an important role in fungal growth inhibition. The contents of lactic acid, acetic acid and phenyllactic acid in the CFS tended to be stable at 48 h, and amounted to 28.5, 15.5 and 0.075 mg/mL, respectively. In addition, adding ZZUA493, as an ingredient during their preparation, prolonged the shelf life of Chinese steamed buns. Overall, ZZUA493 appears to have good potential as a fungal inhibitor for food preservation.

In vitro activities of thiazolidione derivatives combined with daptomycin against clinical Enterococcus faecium strains

Background Previous reports have demonstrated two thiazolidione derivatives (H2-60 and H2-81) can robustly inhibit the planktonic growth and biofilm formation of S. epidermidis and S. aureus by targeting the histidine kinase YycG. Whereas the antibacterial and anti-biofilm activity of these two thiazolidione derivatives (H2-60 and H2-81) against Enterococcus faecium remains elusive. Here, the pET28a-YycG recombinant plasmid were in vitro expressed in E. coli competent cell BL21 (DE3) and induced to express YycG’ protein (conding HisKA and HATPase_c domain) by 0.5 mM IPTG and was purified by Ni – NTA agarose and then for the autophosphorylation test. Antimicrobial testing and time-killing assay were also be determined. Anti-biofilm activity of two derivatives with sub-MIC concentration towards positive biofilm producers of clinical E. faecium were detected using polystyrene microtiter plate and CLSM. Results The MICs of H2-60 and H2-81 in the clinical isolates of E. faecium were in the range from 3.125 mg/L to 25 mg/L. Moreover, either H2-60 or H2-81 showed the excellent bactericidal activity against E. faecium with monotherapy or its combination with daptomycin by time-killing assay. E. faecium planktonic cells can be decreased by H2-60 or H2-81 for more than 3 × log10 CFU/mL after 24 h treatment when combined with daptomycin. Furthermore, over 90% of E. faecium biofilm formation could markedly be inhibited by H2-60 and H2-81 at 1/4 × MIC value. In addition, the frequency of the eradicated viable cells embedded in mature biofilm were evaluated by the confocal laser microscopy, suggesting that of H2-60 combined with ampicillin or daptomycin was significantly high when compared with single treatment (78.17 and 74.48% vs. 41.59%, respectively, P < 0.01). Conclusion These two thiazolidione derivatives (H2-60 and H2-81) could directly impact the kinase phosphoration activity of YycG of E. faecium. H2-60 combined with daptomycin exhibit the excellent antibacterial and anti-biofilm activity against E. faecium by targeting YycG.

Isolation of Shewanella putrefaciens GRD 03 from Fish and Explication of Biofilm Adherence Potency on Different Substrates

Foodborne pathogens are the main threat and cause of food poisoning. The majority of food infections have been related to the biofilm formation of foodborne pathogens in the food industry. Shewanella putrefaciens (KX355803, GRD 03), a Gram-negative pathogen isolated from mackerel fish, was identified and recognized as a food spoilage bacterium and a strong biofilm producer. The adhesion or attachment ability of Shewanella putrefaciens was determined on steel, plastic, glass, PVC and wood. NB (Nutrient broth), LB (Luria-Bertani broth), TSB (Tryptic soy broth) and BHI (Brain heart infusion broth) were enriched with glucose and shows optimum for bacterial adhesion. In the microtiter plate method (MTP), the strong attachment was observed at 48 and 72 hours of incubation and significant differences were obtained at p < 0.05. As the incubation period increases, the OD value (Optical density) of samples also increase. Biofilm formation is the major cause cross-contamination, and shows resistance to certain disinfectants, which leads to environmental stress tolerance. This study suggested with optimum biofilm production of isolate from fish by using glucose enriched media on different substrates, also comparing different growth media provide a detailed idea about biofilm-forming ability at different incubation time intervals.

Correlation of biofilm production with antibiotic susceptibility pattern of Pseudomonas aeruginosa from various clinical specimens

Background: Pseudomonas aeruginosa is one of the most prevalent nosocomial pathogens that cause a life-threatening infection. One of the important characteristics of P. aeruginosa is biofilm formation which leads to antibiotic resistance. Aims and Objectives: The aim of the study was to study the antibiotic resistance pattern of P. aeruginosa isolates and correlation with their biofilm-production. Materials and Methods: A total of 87 P. aeruginosa isolates from different clinical specimens were processed and confirmed by conventional microbiological methods as per standard methodology. Antibiotic sensitivity testing was done for all isolates. Biofilm producing isolates were identified by the microtiter plate method (MTPM). Results: Of 87 P. aeruginosa isolates, majority were from pus 33 (38%), followed by urine 26 (30%), sputum 19 (22%), body fluids 7 (8%), and blood 2 (2%). Biofilm producing isolates showed more resistance in comparison to non-biofilm producers. The observed difference between biofilm formation for multidrug resistant and susceptible isolates was found to be statistically significant. Conclusion: MTPM method was an effective test for detection of biofilm formation and was also able to verify biofilm production by P. aeruginosa. This indicated a higher propensity among the clinical isolates of P. aeruginosa to form biofilm and revealed a positive correlation between biofilm formation and antibiotic resistance. This indicates the need for testing of even susceptible isolates for virulence factors such as biofilm production.

Antibacterial and antibiofilm effect of silver and gold nanoparticles in Uropathogenic Escherichia coli

Nosocomial infections of the urinary tract are the most common nosocomial infections. Infectious diseases currently pose a life threatening due to the increasing phenomenon of infectious agent resistance. One of the factors known to cause resistance to broad-spectrum antimicrobials agent is the susceptibility to biofilm formation. The use of silver nanoparticles (NPs) is now regarded as one of the most promising strategies to target infections associated with biofilms. This study aims to determine the application of nanotechnology in the form of metal nanoparticles in targeting the formation of biofilms on infectious agents. The antibacterial and antibiofilm activity test was performed on a 96-well microtiter plate on uropathogenic Escherichia coli (UPEC) at concentrations of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 ppm. The results showed the addition of silver and gold nanoparticles at different concentrations affected and inhibited the growth of UPEC and prevented the formation of biofilms, as indicated by a decrease in the OD value. In conclusion, silver and gold nanoparticles have antibacterial and antibiofilm effects on UPEC.

Different effects of sub-minimum inhibitory concentrations of gentamicin on the expression of genes involved in alginate production and biofilm formation of Pseudomonas aeruginosa

Background and Objectives: Antibiotics at sub-minimum inhibitory concentrations (sub-MIC) may alter bacterial viru- lence factors. The objective of this study was to investigate the effect of gentamicin at sub-MIC concentrations on the expres- sion of genes involved in alginate production and biofilm formation of Pseudomonas aeruginosa. Materials and Methods: The broth microdilution method was used to determine the MIC of gentamicin for three P. aeru- ginosa clinical isolates (P1-P3) and standard strains (PAO1 and 8821M). Alginate production and biofilm formation of the bacteria in the presence and absence of sub-MIC concentrations of gentamicin were measured using microtiter plate and carbazole assay, respectively. The real-time PCR method was used to determine the effect of gentamicin at sub-MIC con- centrations on the expression level of genes involved in biofilm formation (pelA and pslA) and alginate production (algD and algU). Results: Gentamicin at sub-MIC concentrations significantly reduced alginate production, biofilm formation, and the expres- sion of alginate and biofilm-encoding genes in clinical isolate P1. This inhibitory effect was also observed on the alginate production of 8821M strain and biofilm formation of PAO1strain. In clinical isolates, P2 and P3, alginate production, biofilm formation, and the expression of alginate and biofilm-encoding genes were significantly increased in exposure to sub-MIC concentrations of gentamicin. Conclusion: This study showed that different phenotypic changes in clinical isolates and standard strains of P. aeruginosa in exposure to sub-MIC concentrations of gentamicin are associated with changes in the expression of virulence genes. Further researches are required to understand the mechanisms involved in regulating the expression of virulence genes after exposure to sub-MIC concentrations of antibiotics.

ANTIBACTERIAL EFFECT OF NANNOCHLOROPSIS OCULATA AS ROOT CANAL STERILIZATION MATERIAL ON STREPTOCOCCUS MUTANS BIOFILM

ABSTRACTBackground: Streptococcus mutans in the most frequent microbiota that causes pulp necrosis because of caries. The microorganism that is colonized and embedded in the biofilm matrix is resistant to antimicrobials compared to planktonic cells. Root canal sterilization materials must have good biocompatibility with tissues. Nannochloropsis oculata is an algae that contains various compounds such as terpenoids, alkaloids, and flavonoids that have potential as antibacterial and antioxidant and can be used as alternative to root canal sterilization. Method: This research was true experimental laboratory research with post-test only control group design. The antibacterial potential of Nannochloropsis oculata was tested using the biofilm method, divided into 5 groups. The control group was: K- (aquadest), K+ (calcium hydroxide), and the treatment group was given Nannochloropsis oculata: P1 (0.625%), P2 (1.25%), and P3 (2.5 %). Congo Red method test was to determine the formation of biofilm that shows black strains on agar. While biofilm test with Microtiter Plate Assay to measure the value of biofilm that were inhibited in Optical Density (OD) value in the ELISA Reader. The lower the value, the more biofilm inhibited, with OD value, inhibition percentage could counted Result: The result of all treatment groups were increasing in percentage inhibition value shows inhibition in biofilm growth (p <0.05). Conclusion: Nannochloropsis oculata had an antibacterial effect on the biofilm of Streptococcus mutans

Development of a sensitive competitive enzyme-linked immunosorbent assay for serodiagnosis of Burkholderia mallei, a Tier 1 select agent

Glanders is a highly contagious and potentially serious disease caused by Burkholderia mallei, a Tier 1 select agent. In this study, we raised a monoclonal antibody (mAb) against the lipopolysaccharide (LPS) of B. mallei and developed a competitive enzyme-linked immunosorbent assay (cELISA) for B. mallei infection. Using the titrated optimal conditions of B. mallei-LPS (2 ng) for microtiter plate coating, sample serum dilution at 1:20 and 3.5 ng/μL anti-LPS mAb B5, the cutoff value of the cELISA was determined using serum samples from 136 glanders-free seronegative horses in Hong Kong. All calculated percentage inhibition (PI) values from these seronegative samples were below 39.6% inhibition (1.5 standard deviations above mean PI) and was used as the cutoff value. The diagnostic sensitivity of the developed LPS-based cELISA was first evaluated using sera from donkeys and mice inoculated with B. mallei. An increasing trend of PI values above the defined cELISA cutoff observed in the donkey and mouse sera suggested positive detection of anti-LPS antibodies. The sensitivity and specificity of the LPS-based cELISA was further evaluated using 31 serologically positive horse sera from glanders outbreaks in Bahrain and Kuwait, of which 30 were tested positive by the cELISA; and 21 seronegative horse sera and 20 seronegative donkey sera from Dubai, of which all were tested negative by the cELISA. A cELISA with high sensitivity (97.2%) and specificity (100%) for the detection of B. mallei antibodies in different animals was developed.

Barcoded Asaia bacteria enable mosquito in vivo screens and identify novel systemic insecticides and inhibitors of malaria transmission

This work addresses the need for new chemical matter in product development for control of pest insects and vector-borne diseases. We present a barcoding strategy that enables phenotypic screens of blood-feeding insects against small molecules in microtiter plate-based arrays and apply this to discovery of novel systemic insecticides and compounds that block malaria parasite development in the mosquito vector. Encoding of the blood meals was achieved through recombinant DNA-tagged Asaia bacteria that successfully colonised Aedes and Anopheles mosquitoes. An arrayed screen of a collection of pesticides showed that chemical classes of avermectins, phenylpyrazoles, and neonicotinoids were enriched for compounds with systemic adulticide activity against Anopheles. Using a luminescent Plasmodium falciparum reporter strain, barcoded screens identified 48 drug-like transmission-blocking compounds from a 400-compound antimicrobial library. The approach significantly increases the throughput in phenotypic screening campaigns using adult insects and identifies novel candidate small molecules for disease control.

GC-MS Analysis and Antimicrobial Assessment of Syzygium cumini (L.) Skeels Seed Ethanol Extract

People use plants to treat infections, and this has led to search of antimicrobials from medicinal plants. In this work, we evaluated the ethanol extract of Syzygium cumini seeds for their antibacterial and antifungal activities. Extraction was performed by maceration method using ethanol. The antimicrobial efficacy of the extract was assessed by agar well diffusion method against ten bacterial species, Bacillus cereus, Bacillus subtilis, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhi, Serratia marcescens, Staphylococcus aureus and Streptococcus mutans, and five fungal species, Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Candida albicans and Mucor sp. Minimum inhibitory concentrations (MICs) of the extract were determined by resazurin microtiter plate assay. Phytochemicals in the extract was identified by gas chromatography mass spectrometry (GC-MS) information. In agar well diffusion method, Gram-negative bacteria such as P. aeruginosa and S. marcescens, Gram-positive bacteria such as B. subtilis and E. faecalis and fungi A. fumigatus were more susceptible showing larger zones of inhibition. In resazurin method, low MICs were recorded for bacteria, B. cereus (<7.8 µg) and P. aeruginosa (15.6 µg) and fungi, A. fumigatus (31.2 µg). Fifteen compounds were identified by GC-MS profiling of the extract. The antimicrobial activity of the extract can be rightly related to the secondary metabolites in the ethanol extract of Syzygium cumini seeds.