Novel silver nanoparticle-antibiotic combinations as promising antibacterial and anti-biofilm candidates against multiple-antibiotic resistant ESKAPE microorganisms.

HYPOTHESIS: The emergence of Multiple Antibiotic Resistance (MAR) in ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) pathogens is a global challenge to public health. The inherent antimicrobial nature of silver nanoparticles (AgNPs) makes them promising antimicrobial candidates against antibiotic-resistant pathogens. This study explores the combination of AgNPs with antibiotics (SACs) to create new antimicrobial agents effective against MAR ESKAPE microorganisms. METHODS: AgNPs were synthesized using Streptococcus pneumoniae ATCC 49619 and characterized for structure and surface properties. The SACs were tested against ESKAPE microorganisms using growth kinetics and time-kill curve methods. The effect of SACs on bacterial biofilms and the disruption of cell membranes was determined. The in-vitro cytotoxicity effect of the AgNPs was also studied. FINDINGS: The synthesized AgNPs (spherical, 7.37±4.55 nm diameter) were antimicrobial against MAR ESKAPE microorganisms. The SACs showed synergy with multiple conventional antibiotics, reducing their antibacterial concentrations up to 32-fold. Growth kinetics and time-kill studies confirmed the growth retardation effect and bactericidal activity of SACs. Mechanistic studies suggested that these biofilm-eradicating SACs probably resulted in the loss of bacterial cell membrane integrity, leading to leakage of the cytoplasmic content. The AgNPs were highly cytotoxic against skin melanoma cells but non-cytotoxic to normal Vero cells.

Isolation and Identification of Waterborne Antibiotic-Resistant Bacteria and Molecular Characterization of their Antibiotic Resistance Genes.

The development and spread of antibiotic resistance (AR) through microbiota associated with freshwater bodies is a major global health concern. In the present study, freshwater samples were collected and analyzed with respect to the total bacterial diversity and AR genes (ARGs) using both conventional culture-based techniques and a high-throughput culture-independent metagenomic approach. This paper presents a systematic protocol for the enumeration of the total and antibiotic-resistant culturable bacteria from freshwater samples and the determination of phenotypic and genotypic resistance in the culturable isolates. Further, we report the use of whole metagenomic analysis of the total metagenomic DNA extracted from the freshwater sample for the identification of the overall bacterial diversity, including non-culturable bacteria, and the identification of the total pool of different ARGs (resistome) in the water body. Following these detailed protocols, we observed a high antibiotic-resistant bacteria load in the range of 9.6 × 105-1.2 × 109 CFU/mL. Most isolates were resistant to the multiple tested antibiotics, including cefotaxime, ampicillin, levofloxacin, chloramphenicol, ceftriaxone, gentamicin, neomycin, trimethoprim, and ciprofloxacin, with multiple antibiotic resistance (MAR) indexes of ≥0.2, indicating high levels of resistance in the isolates. The 16S rRNA sequencing identified potential human pathogens, such as Klebsiella pneumoniae, and opportunistic bacteria, such as Comamonas spp., Micrococcus spp., Arthrobacter spp., and Aeromonas spp. The molecular characterization of the isolates showed the presence of various ARGs, such as blaTEM, blaCTX-M (ß-lactams), aadA, aac (6')-Ib (aminoglycosides), and dfr1 (trimethoprims), which was also confirmed by the whole metagenomic DNA analysis. A high prevalence of other ARGs encoding for antibiotic efflux pumps-mtrA, macB, mdtA, acrD, ß-lactamases-SMB-1, VIM-20, ccrA, ampC, blaZ, the chloramphenicol acetyltransferase gene catB10, and the rifampicin resistance gene rphB-was also detected in the metagenomic DNA. With the help of the protocols discussed in this study, we confirmed the presence of waterborne MAR bacteria with diverse AR phenotypic and genotypic traits. Thus, whole metagenomic DNA analysis can be used as a complementary technique to conventional culture-based techniques to determine the overall AR status of a water body.

FEAMR: A Database for Surveillance of Food and Environment-Associated Antimicrobial Resistance.

The rapid dissemination of antimicrobial resistance (AMR) has emerged as a serious health problem on an unprecedented global scale. AMR is predicted to kill more than 10 million people annually by 2050 leading to huge economic losses worldwide. Therefore, urgent action is required at the national as well as international levels to avert this looming crisis. Effective surveillance can play an important role in the containment of AMR spread by providing data to help determine AMR hotspots, predict an outbreak, maintain proper stewardship and propose immediate and future plans of action in this respect. Although many existing databases provide genetic and molecular information on AMR in microorganisms, there is no dedicated database of AMR from non-clinical samples. The FEAMR database is a one-of-its-kind database to provide manually collated and curated information on the prevalence of AMR in food and the environment. For designing the FEAMR webpage, Microsoft Visual Studio with HTML, CSS, ASP.NET, Bootstrap for the front-end and C# for the back-end were used. The FEAMR database is a free access resource ( https://feamrudbt-amrlab.mu.ac.in/ ), accepting verified food- and environment-related AMR submissions from across the globe. To the best of our knowledge, it is probably the first database providing AMR-related surveillance data from non-clinical samples. It is designed from the 'One Health Approach' perspective to help in the containment of global AMR spread. Flowsheet of steps for making FEAMR database 1. Research articles relating to Antimicrobial Resistance (AMR) were searched on the internet. 2. Data relating to AMR were retrieved from these articles and stored in an MS-Excel sheet. 3. The web pages of the FEAMR database (DB) were created using Microsoft Visual Studio (MVS) and its various tools. HTML, CSS, ASP.NET and Bootstrap were used for the front end and C# used for the back-end of the website. 4. The DB of FEAMR was created using MS SQL Server which was controlled by SQL Server Management Studio (SSMS). 5. The data from the MS-Excel sheet in step 2 was stored in the SQL server and displayed on the web page using GridView tool of MVS and C#. The database created was then uploaded on the University of Mumbai (UoM) website, where it can be accessed by all users having the link to the DB ( https://feamrudbt-amrlab.mu.ac.in/ ).

DNA repair pathways important for the survival of Escherichia coli to hydrogen peroxide mediated killing.

Escherichia coli exposed to 1-3 mM hydrogen peroxide undergo killing which is designated as the mode-one killing which is a result of oxidative DNA damage. Oxidative stress mediated DNA damage can be repaired by various DNA repair pathways like base excision repair, nucleotide excision repair and homologous recombination repair. In this study we have investigated the role of multiple DNA repair pathways in survival to oxidative killing and assessed their relative importance. Results show that both nucleotide excision repair pathway as well as the RecF pathway of recombination repair are important for repair of the DNA damage caused by exposure to hydrogen peroxide. The study also provides the evidence that RecG helicase which is known for the resolution of Holliday junction intermediates plays a critical role in the survival of mode-one killing by peroxide. There is a severe impact on the survival of repair mutants when parameters like aeration and growth medium are changed. Low aeration and growth in minimal medium provide significant protection from the mode-one killing suggesting that under natural conditions Escherichia coli cells are likely to be protected from the oxidative stress mediated DNA damage.

Characterization of multiple antibiotic resistance of culturable microorganisms and metagenomic analysis of total microbial diversity of marine fish sold in retail shops in Mumbai, India.

Marine fish species were analyzed for culturable and total metagenomic microbial diversity, antibiotic resistance (AR) pattern, and horizontal gene transfer in culturable microorganisms. We observed a high AR microbial load of 3 to 4 log CFU g-1. Many fish pathogens like Providencia, Staphylococcus, Klebsiella pneumoniae, Enterobacter, Vagococcus, and Aeromonas veronii were isolated. Photobacterium and Vibrio were two major fish and human pathogens which were identified in the fish metagenome. Other pathogens that were identified were Shewanella, Acinetobacter, Psychrobacter, and Flavobacterium. Most of these pathogens were resistant to multiple antibiotics such as erythromycin, kanamycin, neomycin, streptomycin, penicillin, cefotaxime, bacitracin, rifampicin, trimethoprim, ciprofloxacin, and doxycycline with a high multiple antibiotic resistance index of 0.54-0.77. The fish microflora showed high prevalence of AR genes like bla TEM, Class I integron, tetA, aph(3')-IIIa, ermB, aadA, and sul1. Nineteen of 26 AR isolates harbored Class I integrons showing high co-resistance to trimethoprim, kanamycin, doxycycline, and cefotaxime. Mobile R-plasmids from 6 of the 12 AR pathogens were transferred to recipient E. coli after conjugation. The transconjugants harbored the same R-plasmid carrying bla CTX-M, dfr1, tetA, bla TEM, and cat genes. This study confirms that fish is a potential carrier of AR pathogens which can enter the human gut via food chain. To the best of our knowledge, this is the first study in the Indian subcontinent reporting a direct evidence of spread of AR pathogens to humans from specific marine fish consumption.

The CRISPR-Cas immune system: biology, mechanisms and applications.

Viruses are a common threat to cellular life, not the least to bacteria and archaea who constitute the majority of life on Earth. Consequently, a variety of mechanisms to resist virus infection has evolved. A recent discovery is the adaptive immune system in prokaryotes, a type of system previously thought to be present only in vertebrates. The system, called CRISPR-Cas, provide sequence-specific adaptive immunity and fundamentally affect our understanding of virus-host interaction. CRISPR-based immunity acts by integrating short virus sequences in the cell's CRISPR locus, allowing the cell to remember, recognize and clear infections. There has been rapid advancement in our understanding of this immune system and its applications, but there are many aspects that await elucidation making the field an exciting area of research. This review provides an overview of the field and highlights unresolved issues.

Feasibility and challenges in the development of immunocontraceptive vaccine based on zona pellucida glycoproteins.

The zona pellucida (ZP) glycoproteins play a crucial role during fertilization and thus are considered as important target antigens for the development of immunocontraceptive vaccines aiming to inhibit fertility at a pre-fertilization stage. In order to evaluate the immunocontraceptive potential of ZP glycoproteins, bonnet monkey (Macaca radiata) ZP2, ZP3 and ZP4 have been cloned and expressed using either E. coli or baculovirus expression systems. Active immunization studies with the recombinant ZP glycoproteins in female baboons (Papio anubis) and bonnet monkeys revealed curtailment of fertility. In order to minimize the ovarian pathology, synthetic peptides corresponding to B cell epitopes that are devoid of 'oophoritogenic' T cell epitopes were designed and their in vitro immunocontraceptive potential explored. There are several issues that need to be addressed before ZP glycoproteins based immunocontraceptive vaccines become feasible for use in humans. Nonetheless, the utility of such a vaccine is imminent for controlling wild life population. In this direction, active immunization of female non-descript dogs with recombinant canine ZP3 conjugated to diphtheria toxoid led to curtailment of fertility. Further, canine ZP3 has also been expressed in insect cells as a fusion protein with rabies virus glycoprotein G (RV-G), an antigen that is involved in providing protection against rabies. The immunogenicity of such a recombinant protein and its potential to curtail fertility was explored both in female mice and dogs. Simultaneously, DNA vaccine encoding canine ZP3 and RV-G have been made and evaluated for their immunogenicity. The results obtained so far, current shortcomings and the possible ways to circumvent these have been discussed in the present manuscript.

DNA vaccine for rabies: relevance of the trans-membrane domain of the glycoprotein in generating an antibody response.

Various studies have demonstrated the potential of immunization with DNA vaccines encoding the rabies virus glycoprotein (RV-G) to elicit humoral responses. In the present study, we have designed four constructs using a VR1020 vector, wherein the RV-G ectodomain has been cloned without the signal sequence (SS) and the trans-membrane domain (TD) (rGVR), without the SS but with the TD (rGVRt), with the SS but without the TD (rGVRs) and with the SS and the TD (rGVRst), under the control of a cytomegalovirus (CMV) promoter, and downstream of the tissue plasminogen activator (TPA) signal sequence. In addition, RV-G has been expressed as a His6 tag fusion protein, both in Escherichia coli as well as in baculovirus expression systems. Using a prime-boost strategy, BALB/cJ mice administered with the rGVRt construct either in saline (intramuscularly) or adsorbed onto gold microcarriers (delivered intradermally by gene gun) generated the highest rabies virus neutralizing antibody (RVNA) titers. Inclusion of the SS, in addition to the TD (rGVRst), led to a significant decrease in RVNA titers, compared to the rGVRt construct. The DNA vaccine construct lacking both the SS and the TD domain and the vaccine having only the SS generated lower antibody responses, compared to the rGVRt construct. After priming with DNA vaccine, boosting with both E. coli- as well as baculovirus-expressed rRV-G led to an increase in the RVNA titers. The present results demonstrate that a DNA vaccine encoding the full-length sequence of the ectodomain plus TD of the mature native RV-G is capable of expressing an 'ideal' immunogen to produce RVNA titers.

Update on zona pellucida glycoproteins based contraceptive vaccine.

Zona pellucida (ZP) glycoproteins, due to their critical role in mammalian fertilization, have been proposed as candidate immunogens for development of a contraceptive vaccine. Active immunization studies in a variety of animal species, employing either native or recombinant zona proteins, has established their contraceptive potential. Hence, ZP glycoprotein-based contraceptive vaccines have a very good potential for controlling wild life population. To make it a realistic proposition, additional research inputs are required to develop new potent adjuvants and novel practical strategies for vaccine delivery. The observed ovarian dysfunction, often associated with immunization by ZP glycoproteins, is one of the major obstacles for their application in the control of human population. Ongoing studies to delineate epitopes of ZP glycoproteins that will generate an immune response capable of inhibiting fertility without any untoward effects on ovarian functions will help in determining their feasibility for human use.

Characterization of immune response in mice to plasmid DNA encoding dog zona pellucida glycoprotein-3.

To investigate the immunogenicity of plasmid DNA encoding dog zona pellucida glycoprotein-3 (dZP3), the cDNA corresponding to dZP3, was cloned in mammalian expression vector VR1020 downstream of tissue plasminogen activator signal sequence under cytomegalovirus promoter (VRdZP3). In vitro transfection of COS-1 mammalian cells with VRdZP3 plasmid DNA led to its cytosolic expression. The expressed dZP3 has an apparent molecular weight of 45kDa as compared to calculated molecular weight of 38.4 kDa, suggesting possible glycosylation. Immunization of male BALB/cJ mice with VRdZP3 plasmid DNA in saline, by electroporation or adsorbed onto gold microcarriers (delivered by gene gun) generated antibody response against Escherichia coli expressed recombinant dZP3 (r-dZP3). Administration of r-dZP3 in saline following immunization with plasmid DNA led to boosting of the antibody response. Although mice immunized with gene gun exhibited highest antibody titres, the differences in the antibody titres seen by the three modes of plasmid DNA delivery were not statistically significant (P>0.05). Interestingly, female mice immunized with VRdZP3 plasmid DNA using gene gun also generated antibodies against r-dZP3. A dominant IgG1 isotype response was observed in mice immunized with VRdZP3 plasmid DNA using gene gun as compared to a mixed IgG1-IgG2a isotype response when delivered in saline or by electroporation. Immunization with VRdZP3 plasmid DNA also generated cell mediated immune response. The antibodies generated by VRdZP3 plasmid DNA recognized dog native zona pellucida. These studies for the first time, demonstrate the feasibility of generating an immune response to ZP3 by DNA vaccine and that the antibodies thus generated recognize native zona pellucida.

Antibodies generated in response to plasmid DNA encoding zona pellucida glycoprotein-B inhibit in vitro human sperm-egg binding.

To investigate the immunogenicity of plasmid DNA encoding bonnet monkey (Macaca radiata) zona pellucida (ZP) glycoprotein-B (bmZPB), the cDNA corresponding to bmZPB, excluding the N-terminal signal sequence and C-terminus transmembrane-like domain, was cloned in mammalian expression vector VR1020 downstream of tissue plasminogen activator signal sequence under cytomegalovirus promoter (VRbmZPB). In vitro transfection of COS-1, COS-7, CHO, HEK-293, and UM-449 mammalian cells with VRbmZPB plasmid DNA led to the expression of bmZPB. Expression of bmZPB in transfected cells was cytosolic. Flow cytometry analysis of COS-1 cells transfected with VRbmZPB revealed that approximately 15% cells expressed bmZPB. The expressed bmZPB has an apparent molecular weight of 57 kDa. Immunization of male BALB/cJ mice with VRbmZPB plasmid DNA in saline as compared to VR1020 immunized group, elicited significant antibodies against E. coli expressed recombinant bmZPB as evaluated in ELISA. The antibodies generated by VRbmZPB plasmid DNA recognized bonnet monkey as well as human ZP. The immune sera obtained from mice immunized with VRbmZPB plasmid DNA also inhibited, in vitro, the binding of spermatozoa to the ZP in the hemizona assay. These studies, for the first time, demonstrate the feasibility of DNA vaccine to generate antibodies against ZP that recognize native protein and inhibit human sperm-oocyte binding.