Double-layer Agar Technique Research Articles

Applications of bacteriophage in combination with nisin for controlling multidrug-resistant Bacillus cereus in broth and various food matrices

This study focused on the isolation and characterization of bacteriophages with specific activity against toxin-producing and multidrug-resistant strains of Bacillus cereus sensu stricto (B. cereus s. s.). Ten different samples yielded six bacteriophages by utilizing the double-layer agar technique. The most promising phage, vB_BceS-M2, was selected based on its broad host range and robust lytic activity against various B. cereus s. s. strains. The phage vB_BceS-M2 had a circular double-stranded DNA genome of 56,482 bp. This phage exhibited stability over a wide range of temperatures and pH values, which is crucial for its potential application in food matrices. The combined effect of phage vB_BceS-M2 and nisin, a widely used antimicrobial peptide, was investigated to enhance antimicrobial efficacy against B. cereus in food. The results suggested that nisin showed synergy and combined effect with the phage, potentially overcoming the growth of phage-resistant bacteria in the broth. Furthermore, practical applications were conducted in various liquid and solid food matrices, including whole and skimmed milk, boiled rice, cheese, and frozen meatballs, both at 4 and 25 °C. Phage vB_BceS-M2, either alone or in combination with nisin, reduced the growth rate of B. cereus in foods other than whole milk. The combination of bacteriophage and nisin showed promise for the development of effective antimicrobial interventions to counteract toxigenic and antibiotic-resistant B. cereus in food.

Isolation of Escherichia coli bacteriophages from Domestic Wastewater in Tangerang Selatan

In recent years, there has been a continuous increase in the prevalence of antibiotic resistance among bacteria responsible for infectious diseases. Several studies have shown that the occurrence of the COVID-19 pandemic is a major factor contributing to this rapid increase. To overcome this challenge, one promising approach is the use of bacteriophages as a treatment option. Therefore, this study aims to isolate E. coli lytic bacteriophages as an alternative antibiotic for treating secondary bacterial infections of COVID-19. Isolation of bacteriophages was carried out using the plaque assay method with double layer agar technique. The isolate obtained was purified and tested for effectiveness in lysing E. coli ATCC 25922 bacterial cells. The results showed that E. coli had resistance to antibiotics, such as Amoxicillin, Clindamycin, Ciprofloxacin, Azithromycin, and Trimethoprim/sulfamethoxazole. BEB1 isolate obtained from domestic waste had activity in inhibiting the growth of the test bacteria with an effectiveness of 72% at the 25th hour. Based on these findings, it had the potential to be developed as an alternative antibiotic agent to overcome infectious diseases caused by E. coli ATCC 25922.

The potency of bacteriophages isolated from chicken intestine and beef tribe to control biofilm-forming bacteria, Bacillus subtilis

Biofilm becomes one of the crucial food safety problems in the food industry as the formation of biofilm can be a source of contamination. To deal with the problem, an industry generally employs physical and chemical methods including sanitizers, disinfectants, and antimicrobials to remove biofilm. However, the use of these methods may bring about new problems, which are bacterial resistance in the biofilm and the risk for product contamination. New strategies to deal with bacterial biofilms are needed. Bacteriophages (phages), as a green alternative to chemical, have re-emerged as a promising approach to treat bacterial biofilm. In the present study, the potential of lytic phages which have antibiofilm activity on biofilm-forming bacteria (Bacillus subtilis), were isolated from chicken intestines and beef tripe obtained from Indonesian traditional markets using host cells obtained isolated from these samples. Phages isolation was conducted by using double layer agar technique. A lytic test of phages was administered on biofilm-forming bacteria. The difference of turbidity level between control (which were not infected by phages) and the test tubes containing host bacteria infected by phages was investigated. The infection time for the production of phages was determined based on the level of clarity of the media in the test tube with a longer lysate addition time. Three phages were isolated namely: ϕBS6, ϕBS8, and ϕUA7. It showed the ability to inhibit B. subtilis as biofilm-forming spoilage bacteria. The best inhibition results were obtained from ϕBS6. Infection with ϕBS6 in B. subtilis lead to 0.5 log cycle decreased in bacterial cells. This study showed that isolated phages might be used as a potential approach for handling the problem of biofilm formation by B. subtilis.

Inactivation of phage phiX174 by UV254 and free chlorine: Structure impairment and function loss

Disinfection is widely applied in water and wastewater treatment to inactivate viruses. However, the inactivation mechanism associated with viral structural alteration during disinfection is still not clear. In this work, inactivation of bacteriophage phiX174 by ultraviolet radiation (UV254) and free chlorine (FC), two most commonly used disinfection processes, was studied at the molecular level to investigate the relationship between phiX174 genome impairment and virus inactivation, and the correlation between protein impairment and function loss. Double-layer agar technique, quantitative real-time polymerase chain reaction (qPCR), real-time reverse transcription-polymerase chain reaction (RT-qPCR), and liquid chromatography-tandem mass spectrometry techniques (LC-MS/MS), together with structure impairment and function experiments were implemented to quantitatively analyze the inactivation and damage to genome and proteins of phiX174. Results showed that UV254 and FC could effectively inactivate phiX174 at the practical doses (UV254 dose of 30 mJ/cm2, and FC of 1–3 mg/L) used in water treatment plants, accompanied with the damage to viral genome and proteins. Specifically, a UV254 irradiation dose of 9.6 mJ/cm2, and FC at an initial concentration of 1 mg/L at 4 min could lead to a 4-log10 inactivation. Nevertheless, the combination of these two methods at selected doses played no significant synergistic disinfection effect. During UV254 disinfection, the proportion of phiX174 with damaged genome was similar with that of the inactivated phiX174. In addition, UV254 and FC could disrupt proteins of phiX174 such as H protein, thereby hindering the physiological function associated with these proteins. With these findings, it is suggested that UV254 and FC disinfection could hinder the injection of the viral genome into host cells, thus resulting in the inactivation of phiX174. This work provides a comprehensive study of the inactivation mechanism of phiX174, which can enhance the applicability of UV254 and FC in water treatment plants, and facilitate the design and optimization of disinfection technologies for virus control in drinking water and wastewater worldwide to ensure the biosafety.

Predatory efficacy of Bdellovibrio stolpii isolated from the wastewater sources against the multidrug-resistant clinical isolates

Abstract The present study investigated the predatory efficacy of Bdellovibrio isolated from untreated hospital wastewater sources against human pathogens. Bdellovibrio is a Gram-negative, motile, uniflagellate predatory bacteria present in the environment, which directly predates on other bacteria, including human pathogens. In the present study, 30 hospital effluent samples were collected and screened for Bdellovibrio. A total of 11 Bdellovibrio isolates were obtained by the double-layer agar technique. All the isolates were identified by amplification of the 16S rDNA coding region using polymerase chain reaction (PCR) and confirmed as Bdellovibrio stolpii. The lytic activity of confirmed isolates was investigated against four Gram-negative bacteria Pseudomonas aeruginosa ADW44, Pseudomonas aeruginosa 27853(ATCC Strain), V. cholerae and Salmonella typhimurium of clinical origin obtained from the institutional repository. Among the 11 isolates, three Bdellovibrio isolates NBd1, YBd1 and RBd1 demonstrated the ability to prey upon the tested clinical isolates. To the best of our knowledge, this is the first report on the isolation of B. stolpii from hospital wastewater settings in India with broad and high bacteriolytic activity.

Killing Efficiency and Full Genome Sequencing of Bacteriophage BAG1 Targeting Endodontic Clinical Strain of Enterococcus faecalis

Objectives: Bacterial biofilms can increase the survival of bacteria through providing resistance to conventional disinfectants as well as antibiotics, thus biofilm considered a serious risk to human health with hug loss. Consequently, novel approaches to deal with bacterial biofilms are required. Viruses that may eradicate bacteria are known as bacteriophages, they are also known as “bacteria eaters.” Due to their activity on bacteria, bacteriophages are distinct from other organisms and do not pose a threat to human. Consequently, they are regarded as secure substitutes for antibiotics in the management of bacterial infections. Aim of the Study: In this study, we report the full genome sequencing of bacteriophage, namely BAG1 that targeting an endodontic clinical strain of Enterococcus faecalis (K3). Furthermore, we evaluate it killing efficiency to eliminate E. faecalis in both planktonic media and dentine slabs. Materials and Methods: We used the previously isolated endodontic E. faecalis clinical strain, namely K3 and enterococcus bacteriophage BAG1 in this study by spotting on the double layer agar technique. DNA extracted proceeded with NORGEN phage DNA isolation kit. Phage full genome sequencing was performed using Illumina nova Seq 600, 150 bp PE. In addition, the killing capacity of BAG1 phage against E. faecalis K3 was measured by infecting 5 × 106 E. faecalis with BAG1 phage at multiplicity of infection of 0.1. Furthermore, the biofilm fraction and the number of E. faecalis colony-forming unit in planktonic media were measured after infecting 24 dentine slabs with K3 bacteria and BAG1 phage. SYTO® 9 green LIVE/DEAD® BacLight Bacterial Viability Kits were used to visualize K3 biofilm on dentine slabs using fluorescent microscope. Results: Full Genome Organization of BAG1 phage was circular composed of 68 open reading frame with GC percentage of 34.56%. An efficient killer reflecting by completely eliminating K3 strain within 180 min in killing test. In addition, the result revealed that BAG1 highly significant to eliminate K3 bacteria in both planktonic broth and biofilm fraction obtained from dentine slabs infection with the endodontic K3 clinical strain. Conclusion: Our results support that anti E. faecalis bacteriophage BAG1 significantly eliminate biofilm of E. faecalis K3 on dentine slabs with a lytic properties due to the absence of lysogenic genes which make it a suitable substitute to adjunctive anti E. faecalis therapy.

Effect of Antibiotics and Glycerol on Improving Bacteriophage Detection and Enumeration

Antibiotic-resistant bacteria are a growing issue in aquaculture, and phage therapy offers an alternative approach to the use of antibiotics. Phages for phage therapy can be isolated, identified and enumerated using a Double-Layer Agar (DLA) technique. While some phages form large and well-defined plaques that can be enumerated when plated with the DLA technique, some produce small and turbid plaques that are difficult to detect and count. A phage, Aeromonas phages-T65, was isolated from Aeromonas hydrophila, which formed small and turbid plaques making it hard to count. The use of antibiotics and chemicals have been suggested as a way to increase plaque size for easy detection and enumeration of phage plaques. To increase the plaque size of Aeromonas phages-T65, antibiotics and glycerol were added to the culture media, and tested at different concentrations. Application of penicillin-streptomycin and/or glycerol showed an increase in plaque size and the biggest improvement in plaque size were observed using either 40 U/μl penicillin-streptomycin or a 5 % glycerol in the top agar layer of the DLA technique. These findings suggest that such modifications could be implemented into the standard DLA overlay method to investigate new Aeromonas phages for the development of phage therapy against Aeromonas spp. in aquaculture.

Characterization of a lytic EBP bacteriophage with large size genome against Enterobacter cloacae.

Enterobacter cloacae (E.cloacae) is an emerging nosocomial pathogen that had acquired antibiotic resistance against multiple classes of antibiotics. The current study was aimed to isolate and characterize lytic bacteriophage against E.cloacae. The bacteriophage EBP was isolated from a sewage water sample using E.cloacae as a host strain by double-layer agar technique. EBP was found stabile at a wide range of temperatures (25, 37, 60, and 80°C) and pH (5, 6, 7, 8, and 9) with antibacterial activity up to 24h of infection. The latent period of EBP was 20min with a burst size of 252 phages per cell. It showed a narrow host range and infected 12/21 (57%) isolates of E.cloacae tested. It has helical symmetry with a head size of 105 and 120nm long tail with contractile sheath. The EBP has 179.1kb long double-stranded DNA genome with 44.8% GC content. Majority of identified ORFs (187/281) were encoding putative proteins with unknown function. Necessary replication enzymes, structural proteins, and lytic enzymes were detected in the genome of EBP. Phylogenetic analysis revealed that EBP closely resembles with Coronobacter phage vB_CsaM_IeN, vB_CsaM_IeE, vB_CsaM_IeB, and Citrobacter phage Margaery. Based on electron microscopy and molecular characterization, EBP was classified as a Myoviridae phage.

Characterization and Lytic Activity of Isolated Escherichia coli Bacteriophages against Escherichia coli in vitro.

Background: Escherichia coli (E. coli) is the most common cause of urinary tract infection (UTI) and typically treated with antibiotics. Unrestricted use of antibiotics may lead to the emergence of antibiotic-resistant bacteria. The present study aimed to isolate and characterize phages against E. coli from infected urine samples and to determine the lytic activity of phages against E. coli in vitro.Methods: The present experimental study was conducted in the Laboratory of Bouali Sina Hospital (Sari, Iran) in May 2018. E. coli was identified from nine urine samples of patients with UTI using conventional microbiological methods. Bacteriophages were isolated from the infected urine specimens, and their lytic activity was determined using the spot test. The titer of the bacteriophages was measured using the double-layer agar technique. The morphology of the bacteriophages was revealed using transmission electron microscopy, and the latent time period and burst size were determined. Data were analyzed using the SPSS software package.Results: E. coli was isolated from nine infected urine samples. The lytic activity of bacteriophages against E. coli was determined using the spot test by observing the formation of inhibition zones. Transmission electron microscopy showed E. coli phages belonging to the Myoviridae family. The latent time period was 20 minutes with a burst size of 1,200 plaque-forming unit (PFU) per infected cell. The results of the double-layer agar assay showed that the titer of bacteriophages was 20×108 PFU/mL.Conclusion: The E. coli bacteriophage was isolated from infected urine samples and characterized, and their lytic activity against E. coli was determined in vitro.

Isolation of Lytic Acinetobacter baumannii Phage vB_Acib_C_A10 from Iraq pond waters and Comparing Its Antibacterial Effect with Cefotaxime Antibiotic

Bacteriophages are viruses that attack bacteria and lead to their lysis in an efficient and highly specific manner. These phages could be an ideal option for microbial control. These natural enemies of bacteria were used as therapeutic agents before the advent of antibiotics. Currently, with the rapid spread of multidrug resistant bacteria, phage therapy can be an effective alternative treatment for antibiotic resistant bacteria. This study evaluated the effectiveness of bacteriophages in removing Cefotaxime-resistant clinical Acinetobacter baumannii strains (CTX_RAB) in vitro. Our A. baumannii strains were isolated and identified by standard and genetic methods. The antibiogram resistant was ascertained using phenotypic and genotypic method for cefotaxime antibiotics. The bacteriophages were isolated from environmental water samples. They were exposed to the host bacteria by the double-layer agar technique (DLA) to observe plaques. Cross reaction of the phages on test A.baumannii strains was performed to determine broader-spectrum phages. We successfully isolated Bacteriophage vB_Acib_C_A10 (ф Acib_A10) active against clinical strains of CTX_RAB by enrichichment from activated pond water samlies using representatives of those strains. Purified bacteriophage suspensions obtained were tested on a range of clinical isolates that included representatives of multiple strains of each of the international clonal lineages, as well as minor and sporadic strains. An effective bacteriophage was isolated for each strain. Examination by transmission electron microscopy revealed bacteriophage of the Corticoviridae family. The crossreaction showed phages which affect more than six A.baumannii strains. They can be a good choice for clinical therapeutic use. Conclusions: According to the results, six strains were resistant to all concentration of cefotaxime antibiotics. However, for each of these resistant bacteria one bacteriophage was isolated from environmental samples, which showed the effectiveness of Effective bacteriophages to remove clinically resistant A. baumannii in vitro.

Optimized Conditions for Pharmaceuticals and Personal Care Products Removal by Ozonation Using Response Surface Methodology

Bacteriophages are viruses that attack bacteria and lead to their lysis in an efficient and highly specific manner. These phages could be an ideal option for microbial control. These natural enemies of bacteria were used as therapeutic agents before the advent of antibiotics. Currently, with the rapid spread of multidrug resistant bacteria, phage therapy can be an effective alternative treatment for antibiotic resistant bacteria. This study evaluated the effectiveness of bacteriophages in removing Cefotaxime-resistant clinical Acinetobacter baumannii strains (CTX_RAB) in vitro. Our A. baumannii strains were isolated and identified by standard and genetic methods. The antibiogram resistant was ascertained using phenotypic and genotypic method for cefotaxime antibiotics. The bacteriophages were isolated from environmental water samples. They were exposed to the host bacteria by the double-layer agar technique (DLA) to observe plaques. Cross reaction of the phages on test A.baumannii strains was performed to determine broader-spectrum phages. We successfully isolated Bacteriophage vB_Acib_C_A10 (ф Acib_A10) active against clinical strains of CTX_RAB by enrichichment from activated pond water samlies using representatives of those strains. Purified bacteriophage suspensions obtained were tested on a range of clinical isolates that included representatives of multiple strains of each of the international clonal lineages, as well as minor and sporadic strains. An effective bacteriophage was isolated for each strain. Examination by transmission electron microscopy revealed bacteriophage of the Corticoviridae family. The crossreaction showed phages which affect more than six A.baumannii strains. They can be a good choice for clinical therapeutic use. Conclusions: According to the results, six strains were resistant to all concentration of cefotaxime antibiotics. However, for each of these resistant bacteria one bacteriophage was isolated from environmental samples, which showed the effectiveness of Effective bacteriophages to remove clinically resistant A. baumannii in vitro.

Biomimetic hydroxyapatite nanocrystals are an active carrier for Salmonella bacteriophages.

PurposeThe use of bacteriophages represents a valid alternative to conventional antimicrobial treatments, overcoming the widespread bacterial antibiotic resistance phenomenon. In this work, we evaluated whether biomimetic hydroxyapatite (HA) nanocrystals are able to enhance some properties of bacteriophages. The final goal of this study was to demonstrate that biomimetic HA nanocrystals can be used for bacteriophage delivery in the context of bacterial infections, and contribute – at the same time – to enhance some of the biological properties of the same bacteriophages such as stability, preservation, antimicrobial activity, and so on.Materials and methodsPhage isolation and characterization were carried out by using Mitomycin C and following double-layer agar technique. The biomimetic HA water suspension was synthesized in order to obtain nanocrystals with plate-like morphology and nanometric dimensions. The interaction of phages with the HA was investigated by dynamic light scattering and Zeta potential analyses. The cytotoxicity and intracellular killing activities of the phage–HA complex were evaluated in human hepatocellular carcinoma HepG2 cells. The bacterial inhibition capacity of the complex was assessed on chicken minced meat samples infected with Salmonella Rissen.ResultsOur data highlighted that the biomimetic HA nanocrystal–bacteriophage complex was more stable and more effective than phages alone in all tested experimental conditions.ConclusionOur results evidenced the important contribution of biomimetic HA nanocrystals: they act as an excellent carrier for bacteriophage delivery and enhance its biological characteristics. This study confirmed the significant role of the mineral HA when it is complexed with biological entities like bacteriophages, as it has been shown for molecules such as lactoferrin.

The application of bacteriophages as novel indicators of viral pathogens in wastewater treatment systems

Many wastewater treatment technologies have been shown to remove bacterial pathogens more effectively than viral pathogens and, in aquatic environments, levels of traditional faecal indicator bacteria (FIB) do not appear to correlate consistently with levels of human viral pathogens. There is, therefore, a need for novel viral indicators of faecal pollution and surrogates of viral pathogens, especially given the increasing importance of indirect and direct wastewater reuse. Potential candidates include bacteriophages (phages) and the study described here sought to elucidate the relationship between three groups of phages (somatic coliphages (SOMPH), F-RNA coliphages (F-RNAPH) and human-specific phages infecting B. fragilis (Bf124PH) – enumeration using double layer agar technique) and viral pathogens (human adenovirus (HuAdV) and norovirus (NoV) – enumeration using molecular methods) through full-scale municipal wastewater treatment processes. FIB (faecal coliforms (FC) and intestinal enterococci (ENT) – enumeration using membrane filtration) were also monitored. Samples were collected every fortnight, during a twelve-month period, at each stage of four full-scale wastewater treatment plants (WWTP) in southern England (two activated sludge (AS) and two trickling filter (TF) plants) (n = 360 samples). FIB and SOMPH were consistently found in all samples tested, whereas F-RNAPH, Bf124PH and HuAdV were less frequently detected, especially following AS treatment. The detection rate of NoV was low and consequently discussion of this group of viruses is limited. Concentrations of SOMPH and FIB were statistically higher (p value < 0.05) than concentrations of F-RNAPH, Bf124PH and HuAdV in raw wastewater. FIB were more effectively removed than phages in both systems. Removal rates of HuAdV were similar to those of phages at the secondary treatment stage of both systems. In TF systems, HuAdV were removed at the same rate as F-RNAPH, but at lower rates than SOMPH and Bf124PH. The findings suggest that phages (in particular SOMPH) are better indicators of the fate of viral pathogens in WWTP than existing FIB and that these organisms may have a useful role to play in future sanitation safety planning.

English

Bacteriophage virolysins or lytic enzymes are bacterial peptidoglycan hydrolases responsible for lysing bacterial cells. Consequently, they are used as enzybiotics alongside with bacteriophage therapy to remedy multi-drug resistant Salmonella typhi. The objective of this study was to evaluate the potentiality of lytic bacteriophages and their virolysins in curing multi-drug resistant S. typhi. S. typhi was isolated and identified according to WHO and ISO guidelines. Antibiotics susceptibilities were tested using CLSI recommendations. Correspondingly, bacteriophage-lysing efficiency was assayed by plaques formation using the double-layer agar technique. Virolysins were extracted using ultracentrifugation and purified by dialysis after buffering in ammonium sulfate. Virolysins activity was determined by measuring the reaction mixtures spectrophotometrically (bacteria incubated as substrate in 37°C for 4 h). The phages and virolysins kinetics exponential rates were calculated using specific differential equations. Susceptibility data plotted based on antibiogram criteria confirmed that 33% of S. typhi isolates were multi-drug resistant. For bacteriophage replication and multiplicity of infection, phages were amplified to produce the maximum particles of titers. The phage titration data fit on sonogram revealed exponential decay of S. typhi incubated for 12 h. Meanwhile, the enzyme kinetics exponential decay on double reciprocal plot showed irretrievable relationship of host decay in 4 h. Since phages depend on their lytic cycle in lysing bacterial host, their enzymes have more capability in decaying the host; besides they are safe and time-saving when used in the treatment of antibiotics resistant S. typhi. Key words: Antibiotics, bacteriophage, enzybiotics, Salmonella typhi, virolysins.

Endophytic fungi harbored in Panax notoginseng: diversity and potential as biological control agents against host plant pathogens of root-rot disease

BackgroundEndophytic fungi play an important role in balancing the ecosystem and boosting host growth. In the present study, we investigated the endophytic fungal diversity of healthy Panax notoginseng and evaluated its potential antimicrobial activity against five major phytopathogens causing root-rot of P. notoginseng. MethodsA culture-dependent technique, combining morphological and molecular methods, was used to analyze endophytic fungal diversity. A double-layer agar technique was used to challenge the phytopathogens of P. notoginseng. ResultsA total of 89 fungi were obtained from the roots, stems, leaves, and seeds of P. notoginseng, and 41 isolates representing different morphotypes were selected for taxonomic characterization. The fungal isolates belonged to Ascomycota (96.6%) and Zygomycota (3.4%). All isolates were classified to 23 genera and an unknown taxon belonging to Sordariomycetes. The number of isolates obtained from different tissues ranged from 12 to 42 for leaves and roots, respectively. The selected endophytic fungal isolates were challenged by the root-rot pathogens Alternaria panax, Fusarium oxysporum, Fusarium solani, Phoma herbarum, and Mycocentrospora acerina. Twenty-six of the 41 isolates (63.4%) exhibited activity against at least one of the pathogens tested. ConclusionOur results suggested that P. notoginseng harbors diversified endophytic fungi that would provide a basis for the identification of new bioactive compounds, and for effective biocontrol of notoginseng root rot.

Characterization of a new virulent phage infecting the lactic acid bacterium Oenococcus oeni

ΦOE33PA is a new virulent siphophage infecting Oenococcus oeni that was isolated from a red wine collected in Pauillac (France). Although the phage could not lysogenize its host, a conserved sequence within the integrase genes harbored by oenophages could be detected, and corresponded to a B-type integrase. The phage host range encompassed ten out of the 38 O. oeni strains tested. One-step growth kinetics revealed latent and burst periods of 4 and 5 h, respectively, with a burst size of about 45 plaque-forming units per infected cell. The phage had a distinctive restriction profile when compared with Φ10 MC, another B-type oenophage previously isolated in our laboratory. Incubation in wine could inactivate high-titer suspensions of ΦOE33PA in a short time at room temperature. However, encapsidated phage DNA could still be detected by real time PCR, and these non-infectious viruses dominated the wine samples showing that direct enumeration of phages in wine samples using the double-layer agar technique only informs about the quantity of residual infectious phages. Kinetics studies throughout the fermentation using both qPCR as well as plating should now provide reliable understanding of the phage dynamics during wine making.

Removal of Rotavirus and Bacteriophages by Membrane Bioreactor Technology from Sewage.

Human enteric viruses constitute a public health concern due to their low infectious dose and their resistance to environmental factors and to inactivation processes. We aimed at assessing the performance of a laboratory scale Submerged membrane bioreactor (SMBR) treating abattoir wastewaters for Rotavirus (RV) and total coliphages removal. We also aimed at evaluating removal efficiency of enteric viruses through conventional activated sludge treatment by measuring concentrations of total coliphages, considered as fecal and viral contamination indicators, with double-layer agar technique. The Log10 reduction values of bacteriophages ranged from 1.06 to 1.47. Effluents were analyzed to investigate and quantify RV, hepatitis A virus (HAV), Hepatitis E virus (HEV), Noroviruses genogroup I (NoV GI) and genogroup II (NoVGII), and Enterovirus (EV) by real-time PCR, using standardized detection kits (ceeramTools detection kits(®)). All effluent samples were positive for RV; concentrations ranged from 5.2 × 10(5) to 1.3 × 10(7) genome copies/L. These results highlight the inefficiency of conventional biological process for viral removal. A complete removal of RV during Membrane Bioreactor treatment was obtained. To the best of our knowledge, this is the first study providing an evidence of removal of RV simultaneously with total coliphages by SMBR.

Bacteriophages as indicators of human and animal faecal contamination in raw and treated wastewaters from Tunisia

We aimed at quantifying bacteriophages in raw and treated wastewaters of human and animal origin in Tunisia to assess their usefulness for tracking the origin of faecal pollution and in the follow-up of effectiveness of water treatments process. The concentrations of bacteriophages in wastewater samples were determined by double layer agar technique. Somatic coliphages and F-specific RNA bacteriophages were present in all types of samples in high concentrations. The values of Escherichia coli were variable depending on geographical location. On the other hand, bacteriophages infecting strain GA17 were detected preferably when human faecal contamination was occurred. Bacteriophages appear as a feasible and widely applicable manner to detect faecal contamination in Tunisia. On the other hand, phages infecting GA17 could be good markers for tracking the origin of faecal pollution in the area studied. The reuse of treated wastewaters can be a solution to meet the needs of water in the geographical area of study. Bacteriophages seem to predict differently the presence of faecal contamination in water than bacterial indicators. Consequently, they can be a valuable additional tool to improve water resources management for minimizing health risks.

Effective Phages as Green Antimicrobial Agents Against Antibiotic-Resistant Hospital Escherichia coli.

Background:Bacteriophages are viruses that attack bacteria and lead to their lysis in an efficient and highly specific manner. These natural enemies of bacteria were used as therapeutic agents before the advent of antibiotics. Currently, with the rapid spread of multi-drug resistant bacteria, phage therapy can be an effective alternative treatment for antibiotic resistant bacteria.Objectives:This study evaluated the effectiveness of bacteriophages in removing antibiotic-resistant clinical Escherichia coli strains in vitro and in vivo.Patients and Methods:Different samples were taken from bed sore and foot ulcers of patients with diabetes. E. coli strains were isolated and identified by standard methods. The antibiogram was ascertained using the Kirby Bauer disc diffusion method for ten antibiotics. The bacteriophages were isolated from environmental water samples. They were exposed to the host bacteria by the double-layer agar technique (DLA) to observe plaques. Cross reaction of the phages on test E. coli strains was performed to determine broader-spectrum phages. Phage TPR7 was selected for animal trials. Five groups of mice including a control group, bacterial group, phage group, antibiotic therapy group and phage therapy group, were examined.Results:Ten E. coli strains were isolated from hospital samples. They showed high resistance to the used antibiotics. An effective bacteriophage was isolated for each strain. The cross-reaction showed phages which affect more than six E. coli strains. They can be a good choice for clinical therapeutic use. In animal trials the group challenged with phages after being infected showed similar results as the group treated with gentamicin after being infected. In both groups infection was removed after 48 hours.Conclusions:According to the results, six strains were resistant to six or seven antibiotics and all strains were at least resistant to two antibiotics. However, for each of these resistant bacteria one bacteriophage was isolated from environmental samples, which showed the effectiveness of bacteriophages to remove clinically resistant E. coli strains. Effective phages in vitro showed effective results in vivo as well.

Occurrence of bacteriophages infecting Aeromonas, Enterobacter, and Klebsiella in water and association with contamination sources in Thailand

The co-residence of bacteriophages and their bacterial hosts in humans, animals, and environmental sources directed the use of bacteriophages to track the origins of the pathogenic bacteria that can be found in contaminated water. The objective of this study was to enumerate bacteriophages of Aeromonas caviae (AecaKS148), Enterobacter sp. (EnspKS513), and Klebsiella pneumoniae (KlpnKS648) in water and evaluate their association with contamination sources (human vs. animals). Bacterial host strains were isolated from untreated wastewater in Bangkok, Thailand. A double-layer agar technique was used to detect bacteriophages. All three bacteriophages were detected in polluted canal samples, with likely contamination from human wastewater, whereas none was found in non-polluted river samples. AecaKS148 was found to be associated with human fecal sources, while EnspKS513 and KlpnKS648 seemed to be equally prevalent in both human and animal fecal sources. Both bacteriophages were also present in polluted canals that could receive contamination from other fecal sources or the environment. In conclusion, all three bacteriophages were successfully monitored in Bangkok, Thailand. This study provided an example of bacteriophages for potential use as source identifiers of pathogen contamination. The results from this study will assist in controlling sources of pathogen contamination, especially in developing countries.