Viable Pathogens Research Articles

Soil Solarization: an Eco-friendly Management Technique of Damping-off of Tomato

The present study was conducted at experimental farm and laboratory of Dept. of Plant Pathology of Dr. YSPUHF, Nauni, Solan Himachal Pradesh in May 2017–November 2018. The objective of the study was to evaluate the effect of thermal water bathas well as soil solarization on soil temperature and viability of soil borne pathogens of tomato. The disease occurs in two phases i.e. pre-emergence and post emergence that leads to severe infection and crop loss. These pathogens are more likely to influence the development of damping-off disease and their eradication remains a difficult task. Among various eco-friendly methods of disease management, soil solarization is a recourse technique and has been attempted in the present study in mulched and un-mulched beds at three depths i.e., 5, 10 and 20 cm to check the effect of soil solarization on soil temperature. Nursery beds covered with polythene mulch noticed rise in soil temperature and data was recorded for two consecutive crop seasons i.e., 2018 and 2019, where average/ mean weekly temperature was recorded in range of 35.99º to 43.87ºC and 29.25º to 41.68ºC, respectively. The highest temperature was recorded for the nursery beds with 5 cm depth and vice-versa. Overall, conclusive evidence of the survival and killing of pathogens F. oxysporum and R. solani was observed for the corresponding 10 to 20 days and 30 to 40 days schedule of the soil solarization that represents the existence of pathogens.

Pairing Colicins B and E5 with Bdellovibrio bacteriovorus To Eradicate Carbapenem- and Colistin-Resistant Strains of Escherichia coli.

While diverse antibacterials are available in nature, each possesses their own strengths and limitations. One such antibacterial is colicins, proteinaceous toxins that are produced by strains of E. coli to subvert the growth or viability of other E. coli strains. Similarly, predatory bacteria, of which Bdellovibrio bacteriovorus is well-known, are microbes that actively predate on and consume other Gram-negative bacterial strains. While they are all quite active as antibacterials, they also present some limitations: rapid resistance development to colicins while predation does not completely kill their prey. Within this study, therefore, we evaluated the impact of two different colicins (colicin B [ColB] and colicin E5 [ColE5]) and B. bacteriovorus HD100 either individually or together against four clinical isolates of E. coli that are resistant to either colistin or carbapenem. While the ColB and ColE5 were quickly active when used alone, causing a significant loss in viability (>3-log) in susceptible populations after only 3 h, the pathogens always grew afterwards and had final cell densities that were similar with their respective controls. Predation with B. bacteriovorus HD100, in contrast, was most pronounced after 24 h (>3-log reduction in each pathogen viability but never complete). When combined, better killing efficiencies were observed with several of the pathogens, with complete eradication realized for two (<100 viable pathogens per mL). Given the diversity of colicins in nature and the broad-spectrum activities of B. bacteriovorus strains, the results presented here suggest there is a massive potential to control pathogens when they are used together. IMPORTANCE The coupled impact of drug resistance with reduced antibiotic development has placed humankind at a postantibiotic crossroads where antibiotic alternatives are desperately needed. Consequently, we discuss here the combined effectiveness of two vastly different classes of antibacterials, namely, colicins and a predatory bacterium (i.e.,Bdellovibrio bacteriovorus HD100), against two priority pathogenic groups, colistin- and carbapenem-resistant strains of E. coli. While each is effective in its own manner, these antibacterials also display limitations, i.e., the rapid appearance of mutations that confer resistance to the colicins while predatory bacteria do not completely kill their prey. Here, we show these limitations can be overcome using combined treatments of these antibacterials, with two pathogenic E. coli populations completely eradicated within 24 h. Given the diversity of colicins and the broad-spectrum activities of B. bacteriovorus strains, the results presented here suggests there is a massive potential to control pathogens when they are used together.

Sensitive detection of viable salmonella bacteria based on tertiary cascade signal amplification via splintR ligase ligation-PCR amplification-CRISPR/Cas12a cleavage

Several viable Salmonella bacteria are capable of causing severe human diseases and huge economic losses. In this regard, viable Salmonella bacteria detection techniques that can identify small numbers of microbial cells are highly valuable. Here, we present a detection method (referred to as SPC) based on the amplification of tertiary signals using splintR ligase ligation, PCR amplification and CRISPR/Cas12a cleavage. The detection limit of the SPC assay was 6 copies (HilA RNA) and 10CFU (cell). Based on Intracellular HilA RNA detection, this assay can be used to distinguish between viable and dead Salmonella. In addition, it is able to detect multiple serotypes of Salmonella and has been successfully used to detect Salmonella in milk or isolated from farms. Overall, this assay is a promising test for viable pathogens detection and biosafety control.

Highly efficient photonic PCR system based on plasmonic heating of gold nanofilms

The polymerase chain reaction (PCR) is a standard molecular method that has the potential to solve the need for accurate, viable, and immediate infectious pathogen detection at point-of-care (POC) centers in different fields such as pathogen identification, and forensics. In this work, we present a photonic PCR thermocycler that achieves strong optical absorption, more efficient gene amplification, and further improves the effect of temperature distribution through the PCR sample using two gold nanofilms (AuNFs) and a high-power light-emitting diode chip (LED). The photonic device achieved higher heating and cooling rates of 13.20 null°C/s and 7.92 null°C/s, respectively on average, with uniform and reliable temperature distribution throughout the sample with negligible deviations. During amplification cycles, maximum temperatures attained had variations less than 1 °C at 90 °C, less than 0.8 °C at 55 °C, and less than 0.5 °C at 72 °C, showing uniform heating which yielded more accurate and reliable results. Using the fabricated device, PCR amplification for a bacteria genomic DNA was performed in 7.5 minutes for 30 thermal cycles with a sample volume of 20 μL. Due to the device's simple configuration and increased heat transfer rates, this photonic platform will be a highly efficient choice for rapid POC applications in developing countries.

Potential Application of the WST-8-mPMS Assay for Rapid Viable Microorganism Detection.

To ensure clean drinking water, viable pathogens in water must be rapidly and efficiently screened. The traditional culture or spread-plate process-the conventional standard for bacterial detection-is laborious, time-consuming, and unsuitable for rapid detection. Therefore, we developed a colorimetric assay for rapid microorganism detection using a metabolism-based approach. The reaction between a viable microorganism and the combination of 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium sodium salt (WST-8) and 1-methoxy-5-methylphenazinium methyl sulfate (mPMS) results in a color change. In combination with a microplate reader, WST-8-mPMS reactivity was leveraged to develop a colorimetric assay for the rapid detection of various bacteria. The detection limit of the WST-8-mPMS assay for both gram-negative and gram-positive bacteria was evaluated. This WST-8-mPMS assay can be used to perform colorimetrical semi-quantitative detection of various bacterial strains in buffers or culture media within 1 h without incubation before the reaction. The easy-to-use, robust, rapid, and sensitive nature of this novel assay demonstrates its potential for practical and medical use for microorganism detection.

A novel bacteriocin against multiple foodborne pathogens from Lacticaseibacillus rhamnosus isolated from juice ferments: ATF perfusion-based preparation of viable cells, characterization, antibacterial and antibiofilm activity

Foodborne pathogens and their biofilms pose a risk to human health through food chain. However, the bacteriocin resources combating this threat are still limited. Here, Lacticaseibacillus rhamnosus, one of the most used probiotics in food industry, was prepared on a large scale using alternating tangential flow (ATF) perfusion-based technology. Compared to the conventional fed-batch approach, ATF perfusion remarkably increased the viable cells of L. rhamnosus CLK 101 to 11.93 ± 0.14 log CFU/mL. Based on obtained viable cells, we purified and characterized a novel bacteriocin CLK_01 with a broad spectrum of activity against both Gram-positive and Gram-negative foodborne pathogens. LC-MS/MS analysis revealed that CLK_01 has a molecular mass of 701.49 Da and a hydrophobic amino acid composition of I–K–K–V-T-I. As a novel bacteriocin, CLK_01 showed high thermal stability and acid-base tolerance over 25–121 °C and pH 2–10. It significantly reduced cell viability of bacterial pathogens (p < 0.001), and strongly inhibited their biofilm formation. Scanning electron microscopy demonstrated deformation of pathogenic cells caused by CLK_01, leading to cytoplasmic content leakage and bacterial death. Summarily, we employed ATF perfusion to obtain viable L. rhamnosus, and presented that bacteriocin CLK_01 could serve as a promising biopreservative for controlling foodborne pathogenic bacteria and their biofilms.

Progress of viable but non-culturable (VBNC) state foodborne pathogens in milk and dairy products

Progress of viable but non-culturable (VBNC) state foodborne pathogens in milk and dairy products

Mechanism and Efficacy of Cu2O-Treated Fabric.

Pathogenic bacteria can remain viable on fabrics for several days and therefore are a source of infection. Antimicrobial fabrics are a potential method of reducing such infections, and advances in antimicrobial fabrics can be enhanced by knowledge of how the fabric kills bacteria. Metal oxides have been considered and used as antimicrobial ingredients in self-sanitizing surfaces, including in clinical settings. In this work, we examine how the addition of cuprous oxide (Cu2O) particles to polypropylene fibers kills bacteria. First, we show that the addition of the Cu2O particles reduces the viability of common hospital pathogens, Staphylococcus aureus, Pseudomonas aeruginosa, and Streptococcus pneumoniae, by 99.9% after 30 min of contact with the treated polypropylene. Then, we demonstrate that the main killing effect is due to the drying of the bacteria onto the cuprous oxide particles. There is also a weaker effect due to free Cu+ ions that dissolve into the liquid. Other dissolved species were unimportant. Chelation of these Cu+ ions in soluble form or precipitation removes their antimicrobial activity.

Dual-mode visual detection strategies of viable pathogens for point-of-care testing

Here, we introduce a power-free foldable poly(methyl methacrylate) (PMMA) microdevice fully integrating DNA extraction, amplification, and visual detection, realized in novel dual modes – colorimetric and aggregate formation – using 4-Aminoantipyrine (4-AP) for monitoring pathogens. The microdevice contains two parts: reaction and detection zones. A sealing film was utilized to connect the two zones and make the device foldable. The FTA card was deposited in the reaction zone for DNA extraction, followed by loop-mediated isothermal amplification (LAMP) at 65 °C for 45 min. When the detection zone is folded toward the reaction zone, paper discs modified with 4-AP placed in the detection zone are delivered to the reaction zone. Specifically, in the presence of LAMP amplicons, 4-AP is oxidized into antipyrine red or generates aggregates by interacting with copper sulfate, forming copper hybrid nanostructure (Cu-hNs). In the absence of LAMP amplicons, 4-AP is not oxidized and maintains yellow color or fails to form aggregates. Furthermore, we introduced the ethidium homodimer-1 (EthD-1) to identify viable bacteria. EthD-1 penetrated the compromised membranes of nonviable cells and prevented further DNA amplification by intercalating with the DNA. In this way, only samples containing viable cells displayed color change or formed aggregates upon reaction with 4-AP. Using this method, SARS-CoV-2 RNA and Enterococcus faecium were identified by naked eye, with the limit of detection of 103 copies/μL and 102 CFU/mL, respectively, within 60 min. The introduced microdevice can be used for rapidly monitoring viable pathogens and controlling outbreaks of infectious disease in resource-limited settings.

Assessment of Infectious Diseases Risks From Dental Aerosols in Real-World Settings.

Infectious diseases physicians are leaders in assessing the health risks in a variety of community settings. An understudied area with substantial controversy is the safety of dental aerosols. Previous studies have used in vitro experimental designs and/or indirect measures to evaluate bacteria and viruses from dental surfaces. However, these findings may overestimate the occupational risks of dental aerosols. The purpose of this study was to directly measure dental aerosol composition to assess the health risks for dental healthcare personnel and patients. We used a variety of aerosol instruments to capture and measure the bacterial, viral, and inorganic composition of aerosols during a variety of common dental procedures and in a variety of dental office layouts. Equipment was placed in close proximity to dentists during each procedure to best approximate the health risk hazards from the perspective of dental healthcare personnel. Devices used to capture aerosols were set at physiologic respiration rates. Oral suction devices were per the discretion of the dentist. We detected very few bacteria and no viruses in dental aerosols-regardless of office layout. The bacteria identified were most consistent with either environmental or oral microbiota, suggesting a low risk of transmission of viable pathogens from patients to dental healthcare personnel. When analyzing restorative procedures involving amalgam removal, we detected inorganic elements consistent with amalgam fillings. Aerosols generating from dental procedures pose a low health risk for bacterial and likely viral pathogens when common aerosol mitigation interventions, such as suction devices, are employed.

Impact of a Purified Blueberry Extract on In Vitro Probiotic Mucin-Adhesion and Its Effect on Probiotic/Intestinal Pathogen Systems.

Several arguments have been made to substantiate the need for natural antimicrobials for the food industry. With blueberry extracts, the most compelling are both their healthy connotation and the possibility of obtaining a multipurpose solution that can be an antioxidant, colorant, and antimicrobial. From an antimicrobial perspective, as blueberry/anthocyanin-rich extracts have been associated with a capacity to inhibit harmful bacteria while causing little to no inhibition on potential probiotic microorganisms, the study of potential benefits that come from synergies between the extract and probiotics may be of particular interest. Therefore, the present work aimed to evaluate the effect of an anthocyanin-rich extract on the adhesion of five different probiotics as well as their effect on the probiotics’ capacity to compete with or block pathogen adhesion to a mucin/BSA-treated surface. The results showed that, despite some loss of probiotic adhesion, the combined presence of extract and probiotic is more effective in reducing the overall amount of adhered viable pathogen cells than the PROBIOTIC alone, regardless of the probiotic/pathogen system considered. Furthermore, in some instances, the combination of the extract with Bifidobacterium animalis Bo allowed for almost complete inhibition of pathogen adhesion.

Nondestructive and multiplex differentiation of pathogenic microorganisms from spoilage microflora on seafood using paper chromogenic array and neural network

Non-destructive detection of human foodborne pathogens is critical to ensuring food safety and public health. Here, we report a new method using a paper chromogenic array coupled with a machine learning neural network (PCA-NN) to detect viable pathogens in the presence of background microflora and spoilage microbe in seafood via volatile organic compounds sensing. Morganella morganii and Shewanella putrefaciens were used as the model pathogen and spoilage bacteria. The study evaluated microbial detection in monoculture and cocktail multiplex detection. The accuracy of PCA-NN detection was first assessed on standard media and later validated on cod and salmon as real seafood models with pathogenic and spoilage bacteria, as well as background microflora. In this study PCA-NN method successfully identified pathogenic microorganisms from microflora with or without the prevalent spoilage microbe, Shewanella putrefaciens in seafood, with accuracies ranging from 90% to 99%. This approach has the potential to advance smart packaging by achieving nondestructive pathogen surveillance on food without enrichment, incubation, or other sample preparation.

Nitrate enrichment does not affect enteropathogenic Escherichia coli in aquatic microcosms but may affect other strains present in aquatic habitats.

Eutrophication of the planet’s aquatic systems is increasing at an unprecedented rate. In freshwater systems, nitrate—one of the nutrients responsible for eutrophication—is linked to biodiversity losses and ecosystem degradation. One of the main sources of freshwater nitrate pollution in New Zealand is agriculture. New Zealand’s pastoral farming system relies heavily on the application of chemical fertilisers. These fertilisers in combination with animal urine, also high in nitrogen, result in high rates of nitrogen leaching into adjacent aquatic systems. In addition to nitrogen, livestock waste commonly carries human and animal enteropathogenic bacteria, many of which can survive in freshwater environments. Two strains of enteropathogenic bacteria found in New Zealand cattle, are K99 and Shiga-toxin producing Escherichia coli (STEC). To better understand the effects of ambient nitrate concentrations in the water column on environmental enteropathogenic bacteria survival, a microcosm experiment with three nitrate-nitrogen concentrations (0, 1, and 3 mg NO3-N /L), two enteropathogenic bacterial strains (STEC O26—human, and K99—animal), and two water types (sterile and containing natural microbiota) was run. Both STEC O26 and K99 reached 500 CFU/10 ml in both water types at all three nitrate concentrations within 24 hours and remained at those levels for the full 91 days of the experiment. Although enteropathogenic strains showed no response to water column nitrate concentrations, the survival of background Escherichia coli, imported as part of the in-stream microbiota did, surviving longer in 1 and 3 mg NO3-N/Lconcentrations (P < 0.001). While further work is needed to fully understand how nitrate enrichment and in-stream microbiota may affect the viability of human and animal pathogens in freshwater systems, it is clear that these two New Zealand strains of STEC O26 and K99 can persist in river water for extended periods alongside some natural microbiota.

Characterization of Bacillus anthracis replication and persistence on environmental substrates associated with wildlife anthrax outbreaks.

Anthrax is a zoonosis caused by the environmentally maintained, spore-forming bacterium Bacillus anthracis, affecting humans, livestock, and wildlife nearly worldwide. Bacterial spores are ingested, inhaled, and may be mechanically transmitted by biting insects or injection as occurs during heroin-associated human cases. Herbivorous hoofstock are very susceptible to anthrax. When these hosts die of anthrax, a localized infectious zone (LIZ) forms in the area surrounding the carcass as it is scavenged and decomposes, where viable populations of vegetative B. anthracis and spores contaminate the environment. In many settings, necrophagous flies contaminate the outer carcass, surrounding soils, and vegetation with viable pathogen while scavenging. Field observations in Texas have confirmed this process and identified primary browse species (e.g., persimmon) are contaminated. However, there are limited data available on B. anthracis survival on environmental substrates immediately following host death at a LIZ. Toward this, we simulated fly contamination by inoculating live-attenuated, fully virulent laboratory-adapted, and fully virulent wild B. anthracis strains on untreated leaves and rocks for 2, 5, and 7 days. At each time point after inoculation, the number of vegetative cells and spores were determined. Sporulation rates were extracted from these different time points to enable comparison of sporulation speeds between B. anthracis strains with different natural histories. We found all B. anthracis strains used in this study could multiply for 2 or more days post inoculation and persist on leaves and rocks for at least seven days with variation by strain. We found differences in sporulation rates between laboratory-adapted strains and wild isolates, with the live-attenuated strain sporulating fastest, followed by the wild isolates, then laboratory-adapted virulent strains. Extrapolating our wild strain lab results to potential contamination, a single blow fly may contaminate leaves with up to 8.62 x 105 spores per day and a single carcass may host thousands of flies. Replication outside of the carcass and rapid sporulation confirms the LIZ extends beyond the carcass for several days after formation and supports the necrophagous fly transmission pathway for amplifying cases during an outbreak. We note caution must be taken when extrapolating replication and sporulation rates from live-attenuated and laboratory-adapted strains of B. anthracis.

Clinically relevant pathogens on surfaces display differences in survival and transcriptomic response in relation to probiotic and traditional cleaning strategies

Indoor surfaces are paradoxically presumed to be both colonized by pathogens, necessitating disinfection, and “microbial wastelands.” In these resource-poor, dry environments, competition and decay are thought to be important drivers of microbial community composition. However, the relative contributions of these two processes have not been specifically evaluated. To bridge this knowledge gap, we used microcosms to evaluate whether interspecies interactions occur on surfaces. We combined transcriptomics and traditional microbiology techniques to investigate whether competition occurred between two clinically important pathogens, Acinetobacter baumannii and Klebsiella pneumoniae, and a probiotic cleaner containing a consortium of Bacillus species. Probiotic cleaning seeks to take advantage of ecological principles such as competitive exclusion, thus using benign microorganisms to inhibit viable pathogens, but there is limited evidence that competitive exclusion in fact occurs in environments of interest (i.e., indoor surfaces). Our results indicate that competition in this setting has a negligible impact on community composition but may influence the functions expressed by active organisms. Although Bacillus spp. remained viable on surfaces for an extended period of time after application, viable colony forming units (CFUs) of A. baumannii recovered following exposure to a chemical-based detergent with and without Bacillus spp. showed no statistical difference. Similarly, for K. pneumoniae, there were small statistical differences in CFUs between cleaning scenarios with or without Bacillus spp. in the chemical-based detergent. The transcriptome of A. baumannii with and without Bacillus spp. exposure shared a high degree of similarity in overall gene expression, but the transcriptome of K. pneumoniae differed in overall gene expression, including reduced response in genes related to antimicrobial resistance. Together, these results highlight the need to fully understand the underlying biological and ecological mechanisms for community assembly and function on indoor surfaces, as well as having practical implications for cleaning and disinfection strategies for infection prevention.

PCR Mediated Nucleic Acid Molecular Recognition Technology for Detection of Viable and Dead Foodborne Pathogens.

Living foodborne pathogens pose a serious threat to public and population health. To ensure food safety, it is necessary to complete the detection of viable bacteria in a short time (several hours to 1 day). However, the traditional methods by bacterial culture, as the gold standard, are cumbersome and time-consuming. To break through the resultant research bottleneck, PCR mediated nucleic acid molecular recognition technologies, including RNA-based reverse transcriptase PCR (RT-PCR) and DNA-based viability PCR (vPCR) have been developed in recent years. They not only sensitively amplify detection signals and quickly report detection results, but also distinguish viable and dead bacteria. Therefore, this review introduces these PCR-mediated techniques independent of culture for viable and dead foodborne pathogen detection from the nucleic acid molecular recognition principal level and describes their whole-process applications in food quality supervision, which provides a useful reference for the development of detection of foodborne pathogens in the future.

Application of natamycin and farnesol as bioprotection agents to inhibit biofilm formation of yeasts and foodborne bacterial pathogens in apple juice processing lines

Biofilms serve as a reservoir for pathogenic and spoilage microorganisms, and their removal from different surfaces is a recurring problem in the beverage industry. This study aimed to investigate the effect of a combination of natamycin (NAT, 0.01 mmol/l) and farnesol (FAR, 0.6 mmol/l) against biofilms on ultrafiltration (UF) membranes and stainless steel (SS) surfaces using apple juice as food matrix. The co-adhesion of Rhodotorula mucilaginosa, Candida tropicalis, C. krusei and C. kefyr (mixed-yeast) with Listeria monocytogenes, Salmonella enterica or Escherichia coli O157:H7 (multi-species) in presence of NAT + FAR was evaluated for 2, 24, 48 h. In biofilms treated with NAT + FAR were observed by cell quantification and microscopy, inhibition of the filamentous yeast forms, disruption of the tri-dimensional structure and a high detachment of yeast cells. NAT + FAR affected the biofilms independently of the surfaces used and the presence (or not) of bacteria. L. monocytogenes was the most susceptible (p < 0.001) in multi-species biofilms, followed by E. coli O157:H7 on both surfaces (p < 0.001), whereas the growth of S. enterica was reduced (p < 0.05) in SS but not in UF-membranes (p > 0.05). Since the combination NAT + FAR affected the structure and viability of yeast species and foodborne pathogens in multi-species biofilms developed on UF-membranes and SS surfaces, the combination proposed could be considered a promising control agent to prevent biofilms in apple juice processing lines.

Epidemiological and immunological gains from solar water disinfection: Fact or wishful thinking?

There is still no consensus on the impact of using solar disinfection (SODIS) to reduce the prevalence of waterborne gastrointestinal diseases. The reported reduction in diarrhoea prevalence among SODIS users has been attributed to the consumption of water free of viable pathogens. However, it has also been suggested that ingestion of SODIS-inactivated pathogens may induce protective immunological changes that may also contribute to a reduction in the frequency of diarrhoea. The present study aimed to critically review the epidemiological and immunological gains of using SODIS. We critically reviewed 22 articles published in English, selected from 2118 records systematically retrieved from the databases. All trials (except one) reported a significant reduction in diarrhoea prevalence among children using SODIS, but some of the data from trials report contrary findings. All in vitro and in vivo assays indicate that SODIS-inactivated pathogenic bacteria have the potential to induce immunological alterations that may result in protective immunological effects. Studies with a low risk of bias are still awaited to confirm the ability to use SODIS to reduce the prevalence of diarrhoea. Reducing the prevalence of diarrhoea depends on the success of SODIS delivery strategies in inducing behavioural changes in communities that result in the production of SODIS-compliant outcomes. The results of trials reporting a reduction in the prevalence of diarrhoea due to the use of SODIS seem to support the hypothesis of the contribution of the protective immunological effect against diarrhoea in SODIS users.

Antibacterial activity of Bacillus licheniformis B6 against viability and biofilm formation of foodborne pathogens of health importance.

We studied a strain of Bacillus isolated from an artisanal tannery in Salta, Argentina. It was identified as Bacillus licheniformis B6 by 16S phylogenetic analysis and MALDI TOF (GenBank accession code No. KP776730). The synthesis of lipopeptides by B6 and their antibacterial activity against clinical pathogenic strains was analyzed both in the cell-free supernatant (CFS) and in the crude fraction of lipopeptides (LF). Overall, the CFS did not significantly reduce the viability of the studied strains (Staphylococcus aureus 269 and ATCC 43,300, Escherichia coli 4591 and 25,922, Klebsiella sp. 1087 and 1101). However, LF at 9mg/mL reduced the viability of those pathogenic strains by 2 and 3 log orders compared to those of the control. When the effects of LF and ampicillin were compared, they showed different sensitivity against pathogenic strains. For example, E. coli 4591 was the strain most resistant to ampicillin, requiring 250mg/mL of antibiotic to achieve the same inhibitory effect as 9mg/mL of B6 LF. SEM observations of the effect of LF on biofilm formation by E. coli 4591 and Klebsiella sp. 1087 clearly showed that biofilm structures were destabilized, these strains turning into weak biofilm formers. Signals in the CFS and LF corresponding to kurstakin and iturin were identified by MALDI TOF. Interestingly, surfactin was detected, rather than lichenysin, the expected lipopeptide in B. licheniformis species. Signals of bacitracin and fengycins were also found, the latter with a higher number of homologues and relative intensity in the LF than the other lipopeptides. These results show that the lipopeptides synthesized by B. licheniformis B6 have both potential antibacterial and anti-biofilm activity against pathogenic bacteria of health importance.

Potential Bioactive Compounds from Marine Streptomyces sp. and Their In Vitro Antibiofilm and Antibacterial Activities Against Antimicrobial-Resistant Clinical Pathogens.

Antimicrobial resistance issues have risen dramatically in recent years, posing a severe concern to humans worldwide. The urgent need to find novel compounds for pharmaceutical applications prompts the research of under-explored environments such as marine ecosystems. The present study was designed to discover novel secondary metabolites, and we have isolated about 30 actinomycetes from the marine soil samples collected in Thondi (Ramanathapuram, Tamil Nadu, India), where most isolates are associated with the genus Streptomyces. Out of 30, one potentially active strain (Streptomyces sp. SRMA3) was identified using primary and secondary screening methods against the drug-resistant clinical pathogens. The active metabolites extracted from the selected active isolate were subjected to partial purification and characterization using Fourier transform infrared spectrophotometer (FTIR) and gas chromatography-mass spectroscopy (GC-MS) analysis. The minimum inhibitory concentration (MIC) value was determined for the active metabolite. Further, the partially purified active fraction was revealed for its antibacterial and antibiofilm activity against drug-resistant clinical pathogens. Light and fluorescence microscopy detected the viability and adhesion of the biofilm-forming drug-resistant pathogens. Growth curve analysis showed that the active metabolite has the potential to inhibit drug-resistant pathogens. The synergistic effect of active metabolite with commercial antibiotics also revealed that it could enhance the activity of antibiotics in antimicrobial resistance pathogens. This study shows that the isolated Streptomyces sp. SRMA3 is a potentially active strain, and the metabolite derived from this strain has a good antibacterial and antibiofilm activity against antimicrobially resistant clinical pathogens and could be used for various biotechnological applications.