Isolation, characterization, and molecular identification of soil bacteria showing antibacterial activity against human pathogenic bacteria

Background The present study dealt with the screening of soil bacteria with antibacterial activity from different locations in Bangalore, India. Antibiotics play the role of self-defense mechanism for the bacteria and are produced as secondary metabolites to protect themselves from other competitive microorganisms. The need for new antibiotics arose as the pathogenic bacteria acquire resistance to various antibiotics meant for treating human diseases. Given the importance of antibiotics of bacterial origin, standard techniques have been used to isolate and characterize the soil bacteria which showed antibacterial activity. Results The isolated bacteria were tested against human pathogenic bacteria like Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae by primary and secondary screening methods. The isolates PR1, PR2, and PR3 were confirmed to have antibacterial activity against S. aureus, E. coli, P. aeruginosa, and K. pneumoniae by both methods. Studies on the effect of filter sterilization, autoclaving, and proteinase K treatment on culture filtrates showed filter sterilization as the best method. The effect of different carbon and nitrogen sources on the antibacterial activity showed that preference by each isolate differed for carbon and nitrogen requirements. The isolates PR1, PR2, and PR3 were identified as Bacillus aryabhattai strain PR-D07, Arthrobacter humicola strain PR-F07, and Neomicrococcus lactis strain PR-F11 through 16S rRNA sequencing. Conclusion Findings from this research work are encouraging and could proceed further to applied aspects. Only 3 bacterial isolates out of 263 isolates from soil samples displayed antibacterial activity against human pathogens S. aureus, E. coli, P. aeruginosa, and K. pneumoniae. They were identified as B. aryabhattai, A. humicola, and N. lactis by 16S rRNA studies and all of them are Gram-positive. Each isolate preferred different carbon and nitrogen sources for their enhanced antibacterial activity. Efficacy of the culture filtrates of these isolates was tested by filter sterilization, autoclaving, and proteinase K treatment. Filter-sterilized culture filtrates showed higher antibacterial activity than other treatments. A comparison of the antibacterial activity of culture filtrates and antibiotic streptomycin produced an inhibition zone of 18.5 mm and 15.5 mm respectively. This is the first report on the antibacterial activity of all the 3 bacterial strains (B. aryabhattai strain PR-D07, A. humicola strain PR-F07, and N. lactis strain PR-F11), against all the human pathogens, mentioned earlier. It is also found that the antibiotic factor is proteinaceous as proteinase K considerably reduced the antibacterial activity of the culture filtrates. With the above significant results, these 3 bacteria are considered to be promising candidates for the isolation of new antibacterial agents.

Validation of saliva sampling as an alternative to oro-nasopharyngeal swab for detection of SARS-CoV-2 using unextracted rRT-PCR with the Allplex 2019-nCoV assay

Introduction. The current severe acute respiratory syndrome-associated coronavirus-2 (SARS-CoV-2) pandemic has stressed the global supply chain for specialized equipment, including flocked swabs. Hypothesis. Saliva could be a potential alternative specimen source for diagnosis of SARS-CoV-2 infection by reverse-transcriptase PCR (RT-PCR). Aim. To compare the detection efficiency of SARS-CoV-2 RNA in saliva and oro-nasopharyngeal swab (ONPS) specimens. Methodology. Patients recruited from hospital provided paired saliva and ONPS specimens. We performed manual or automated RT-PCR with prior proteinase K treatment without RNA extraction using the Seegene Allplex 2019 nCoV assay. Results. Of the 773 specimen pairs, 165 (21.3 %) had at least one positive sample. Additionally, 138 specimens tested positive by both sampling methods. Fifteen and 12 cases were detected only by nasopharyngeal swab and saliva, respectively. The sensitivity of ONPS (153/165; 92.7 %; 95 % CI: 88.8–96.7) was similar to that of saliva (150/165; 90.9 %; 95 % CI: 86.5–95.3; P=0.5). In patients with symptoms for ≤ 10 days, the sensitivity of ONPS (118/126; 93.7 %; 95 % CI: 89.4–97.9) was similar to that of saliva (122/126; 96.8 %; 95 % CI: 93.8–99.9 %; P=0.9). However, the sensitivity of ONPS (20/22; 95.2 %; 95 % CI: 86.1–100) was higher than that of saliva (16/22; 71.4 %; 95 % CI: 52.1–90.8) in patients with symptoms for more than 10 days. Conclusions. Saliva sampling is an acceptable alternative to ONPS for diagnosing SARS-CoV-2 infection in symptomatic individuals displaying symptoms for ≤ 10 days. These results reinforce the need to expand the use of saliva samples, which are self-collected and do not require swabs.

Generation of Infectious Mimivirus Virions Through Inoculation of Viral DNA Within Acanthamoeba castellanii Shows Involvement of Five Proteins, Essentially Uncharacterized

One of the most curious findings associated with the discovery of Acanthamoeba polyphaga mimivirus (APMV) was the presence of many proteins and RNAs within the virion. Although some hypotheses on their role in Acanthamoeba infection have been put forward, none have been validated. In this study, we directly transfected mimivirus DNA with or without additional proteinase K treatment to extracted DNA into Acanthamoeba castellanii. In this way, it was possible to generate infectious APMV virions, but only without extra proteinase K treatment of extracted DNA. The virus genomes before and after transfection were identical. We searched for the remaining DNA-associated proteins that were digested by proteinase K and could visualize at least five putative proteins. Matrix-assisted laser desorption/ionization time-of-flight and liquid chromatography–mass spectrometry comparison with protein databases allowed the identification of four hypothetical proteins—L442, L724, L829, and R387—and putative GMC-type oxidoreductase R135. We believe that L442 plays a major role in this protein–DNA interaction. In the future, expression in vectors and then diffraction of X-rays by protein crystals could help reveal the exact structure of this protein and its precise role.

A faster and less costly alternative for RNA extraction of SARS-CoV-2 using proteinase k treatment followed by thermal shock

One of the biggest challenges during the pandemic has been obtaining and maintaining critical material to conduct the increasing demand for molecular tests. Sometimes, the lack of suppliers and the global shortage of these reagents, a consequence of the high demand, make it difficult to detect and diagnose patients with suspected SARS-CoV-2 infection, negatively impacting the control of virus spread. Many alternatives have enabled the continuous processing of samples and have presented a decrease in time and cost. These measures thus allow broad testing of the population and should be ideal for controlling the disease. In this sense, we compared the SARS-CoV-2 molecular detection effectiveness by Real time RT-PCR using two different protocols for RNA extraction. The experiments were conducted in the National Institute of Health (INS) from Peru. We compared Ct values average (experimental triplicate) results from two different targets, a viral and internal control. All samples were extracted in parallel using a commercial kit and our alternative protocol–samples submitted to proteinase K treatment (3 μg/μL, 56°C for 10 minutes) followed by thermal shock (98°C for 5 minutes followed by 4°C for 2 minutes); the agreement between results was 100% in the samples tested. In addition, we compared the COVID-19 positivity between six epidemiological weeks: the initial two in that the Real time RT-PCR reactions were conducted using RNA extracted by commercial kit, followed by two other using RNA obtained by our kit-free method, and the last two using kit once again; they did not differ significantly. We concluded that our in-house method is an easy, fast, and cost-effective alternative method for extracting RNA and conducing molecular diagnosis of COVID-19.

Production of Membrane Vesicles in Listeria monocytogenes Cultured with or without Sub-Inhibitory Concentrations of Antibiotics and Their Innate Immune Responses In Vitro

Listeriosis is a food-borne illness caused by Listeria monocytogenes. Ampicillin (AMP) alone or in combination with gentamicin (GEN) is the first-line treatment option. Membrane vesicle (MV) production in L. monocytogenes under antibiotic stress conditions and pathologic roles of these MVs in hosts have not been reported yet. Thus, the aim of this study was to investigate the production of MVs in L. monocytogenes cultured with sub-minimum inhibitory concentrations (MICs) of AMP, GEN, or trimethoprim/sulfamethoxazole (SXT) and determine pathologic effects of these MVs in colon epithelial Caco-2 cells. L. monocytogenes cultured in tryptic soy broth with 1/2 MIC of AMP, GEN, or SXT produced 6.0, 2.9, or 1.5 times more MV particles, respectively, than bacteria cultured without antibiotics. MVs from L. monocytogenes cultured with AMP (MVAMP), GEN (MVGEN), or SXT (MVSXT) were more cytotoxic to Caco-2 cell than MVs obtained from cultivation without antibiotics (MVTSB). MVAMP induced more expression of tumor necrosis factor (TNF)-α gene than MVTSB, MVGEN and MVSXT, whereas MVTSB induced more expression of interleukin (IL)-1β and IL-8 genes than other MVs. Expression of pro-inflammatory cytokine genes by L. monocytogenes MVs was significantly inhibited by proteinase K treatment of MVs. In conclusion, antibiotic stress can trigger the biogenesis of MVs in L. monocytogenes and MVs produced by L. monocytogenes exposed to sub-MIC of AMP can induce strong pro-inflammatory responses by expressing TNF-α gene in host cells, which may contribute to the pathology of listeriosis.

Antagonistic Potential of Dairy Origin Enterococcus faecium Against Multidrug-Resistant Foodborne Pathogens

Probiotic potential of Enterococcus spp. is widely investigated around the globe. The biochemically and molecular characterized E. faecium strains isolated from Dahi (continental yogurt) were evaluated to tolerate simulated gastric environment, bile, sodium chloride, temperature, and pH. The safety was assessed by disc diffusion, broth microdilution, antibiotic resistance genes screening, and hemolytic ability. Enterococci survived simulated gastrointestinal conditions and depicted growth at temperature (15 to ≥42°C), pH (≤2.5 to ≥9.5), 0.3% bile salt and 3% NaCl. All strains were sensitive to ampicillin, vancomycin, kanamycin, gentamicin, streptomycin, tetracycline and ciprofloxacin and harbored vanR, vanX, qnrB2, qnrS, tetK, and tetW resistance genes. E. faecium strains inhibited the E. coli (85%) and S. Typhi (50%) whereas the 10% cell-free culture supernatant (CFCS) of E. faecium halted the growth of E. coli while 15% CFCS completely suppressed S. Typhi. The cell-free culture supernatant retained antibacterial nature after pH and proteinase K treatment, however, it lost activity after heat treatment (≥95°C). The genetic screening revealed that all isolates are capable to produce putrescine biogenic amine. Further assessment of strains for lack of infectivity, cytotoxicity in animals, adhesion to Caco-2 cells and characterization of enterocins is essential to conclude the probiotic potential of these strains.

1194. Identification and Characterization of Extracellular Inducers of Persistence in Staphylococcus epidermidis and Staphylococcus aureus

Background This study describes the identification and partial characterization of persistence inducing factors (PIF) from Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis). Persistence is an epigenetic process that results in tolerance of bacterial cells to antibiotic treatment, which can result in chronic human infections. Methods Others have demonstrated a significant increase in persister numbers during mid-log phase. Inducers of this mid-log increase have yet to be identified in staphylococci. Optical density at 600 nm (OD600) was used instead of time to determine when persister numbers increased during logarithmic growth. Concentrated culture filtrates (CCF) from S. epidermidis RP62A and S. aureus SH1000 were obtained at various OD600’s and following incubation at 16 h. The CCF’s were used to develop a persistence inducing factor (PIF) assay. The PIF assay was used to partially characterize PIF from S. epidermidis RP62A and S. aureus SH1000 for relative molecular weight, temperature and protease sensitivity and inter-species communications. Results Optimal OD600’s for the S. epidermidis RP62A and S. aureus SH1000 PIF assays were 2.0 and 0.5, respectively. The highest PIF activity for both species was from CCF following incubation overnight (16 h). S. epidermidis RP62A’s PIF activity was decreased by storage at 4o C (2 weeks or longer) but not following incubation at 20o C (16 h), 37o C (1 h) or 100o C (15 min). S. aureus SH1000’s PIF activity was decreased following storage at 4o C (2 week or longer) and after boiling at 100oC for 5 min but not after incubation at 37o C (1 h). PIF activity from both species was less than 3,000 Mrr. Proteinase-K treatment of S. aureus SH1000 PIF decreased activity but did not decrease PIF activity of S. epidermidis RP62A. PIF from S. epidermidis RP62A did not increase persister numbers when used to treat S. aureus SH1000 cells nor did PIF from S. aureus SH1000 increase persister numbers in S. epidermidis RP62A cells. Conclusion Previous attempts to discover PIF’s for staphylococcal species were unsuccessful due to the time-based means used to identify mid-log. Both staphylococcal species appear to produce unique, extracellular, low-molecular-weight inducers of persistence (PIF) when assayed using an OD600-based PIF assay. Disclosures All Authors: No reported disclosures

Potential of Cell-Free Supernatant from Lactobacillus plantarum NIBR97, Including Novel Bacteriocins, as a Natural Alternative to Chemical Disinfectants

The recent pandemic of coronavirus disease 2019 (COVID-19) has increased demand for chemical disinfectants, which can be potentially hazardous to users. Here, we suggest that the cell-free supernatant from Lactobacillus plantarum NIBR97, including novel bacteriocins, has potential as a natural alternative to chemical disinfectants. It exhibits significant antibacterial activities against a broad range of pathogens, and was observed by scanning electron microscopy (SEM) to cause cellular lysis through pore formation in bacterial membranes, implying that its antibacterial activity may be mediated by peptides or proteins and supported by proteinase K treatment. It also showed significant antiviral activities against HIV-based lentivirus and influenza A/H3N2, causing lentiviral lysis through envelope collapse. Furthermore, whole-genome sequencing revealed that NIBR97 has diverse antimicrobial peptides, and among them are five novel bacteriocins, designated as plantaricin 1 to 5. Plantaricin 3 and 5 in particular showed both antibacterial and antiviral activities. SEM revealed that plantaricin 3 causes direct damage to both bacterial membranes and viral envelopes, while plantaricin 5 damaged only bacterial membranes, implying different antiviral mechanisms. Our data suggest that the cell-free supernatant from L. plantarum NIBR97, including novel bacteriocins, is potentially useful as a natural alternative to chemical disinfectants.

Helicobacter pylori Biofilm Confers Antibiotic Tolerance in Part via A Protein-Dependent Mechanism

Helicobacter pylori, a WHO class I carcinogen, is one of the most successful human pathogens colonizing the stomach of over 4.4 billion of the world’s population. Antibiotic therapy represents the best solution but poor response rates have hampered the elimination of H. pylori. A growing body of evidence suggests that H. pylori forms biofilms, but the role of this growth mode in infection remains elusive. Here, we demonstrate that H. pylori cells within a biofilm are tolerant to multiple antibiotics in a manner that depends partially on extracellular proteins. Biofilm-forming cells were tolerant to multiple antibiotics that target distinct pathways, including amoxicillin, clarithromycin, and tetracycline. Furthermore, this tolerance was significantly dampened following proteinase K treatment. These data suggest that H. pylori adapts its phenotype during biofilm growth resulting in decreased antibiotic susceptibility but this tolerance can be partially ameliorated by extracellular protease treatment.