Efficacy of Indolicidin,Cecropin A (1-7)-Melittin (CAMA) and Their Combination Against Biofilm-Forming Multidrug-Resistant Enteroaggregative Escherichia coli

The present study examined the anti-biofilm efficacy of two short-chain antimicrobial peptides (AMPs), namely, indolicidin and cecropin A (1-7)-melittin (CAMA) against biofilm-forming multidrug-resistant enteroaggregative Escherichia coli (MDR-EAEC) isolates. The typical EAEC isolates re-validated by PCR and confirmed using HEp-2 cell adherence assay was subjected to antibiotic susceptibility testing to confirm its MDR status. The biofilm-forming ability of MDR-EAEC isolates was assessed by Congo red binding, microtitre plate assays and hydrophobicity index; broth microdilution technique was employed to determine minimum inhibitory concentrations (MICs) and minimum biofilm eradication concentrations (MBECs). The obtained MIC and MBEC values for both AMPs were evaluated alone and in combination against MDR-EAEC biofilms using crystal violet (CV) staining and confocal microscopy-based live/dead cell quantification methods. All the three MDR-EAEC strains revealed weak to strong biofilm-forming ability and were found to be electron-donating and weakly electron-accepting (hydrophobicity index). Also, highly significant (P < 0.001) time-dependent hydrodynamic growth of the three MDR-EAEC strains was observed at 48 h of incubation in Dulbecco’s modified Eagle’s medium (DMEM) containing 0.45% D-glucose. AMPs and their combination were able to inhibit the initial biofilm formation at 24 h and 48 h as evidenced by CV staining and confocal quantification. Further, the application of AMPs (individually and combination) against the preformed MDR-EAEC biofilms resulted in highly significant eradication (P < 0.001) at 24 h post treatment. However, significant differences were not observed between AMP treatments (individually or in combination). The AMPs seem to be an effective candidates for further investigations such as safety, stability and appropriate biofilm-forming MDR-EAEC animal models.

     

Ecology of Listeria monocytogenes and Listeria species in India: the occurrence,resistance to biocides,genomic landscape and biocontrol

Listeria monocytogenes, the causative agent of listeriosis, has been implicated in increasing foodborne outbreaks worldwide. The disease is manifested in various forms ranging from severe sepsis in immune-compromised individuals, febrile gastroenteritis, still birth, abortions and meningoencephalitis. In India, data from studies on the detection and molecular epidemiological analysis of L. monocytogenes are only recently emerging. The presence of Listeria in different ecological niches has been recorded from India, including foods, soil, vegetables, mangrove swamps, seafood, freshwater fishes, clinical cases, and also insects. The organism has also been isolated from women with spontaneous abortions, miscarriage or recurrent obstetric history, aborted foetuses, animal clinical cases and wildlife samples. A novel species of Listeria has also been characterized. Listeria monocytogenes strains isolated from clinical, environmental, and foods showed biofilm-forming abilities. Listeria monocytogenes serotype 4b isolates of ST328, a predominant and unique ST observed in India, was repeatedly isolated from different sources, times, and geographical locations. Here, we reviewed the occurrence of Listeria in different sources in India, its resistance to biocides, and provide epidemiological analysis on its genomic landscape.     

Listeria monocytogenes in spontaneous abortions in humans and its detection by multiplex PCR

AIM: To assess the extent of Listeria monocytogenes in causation of human spontaneous abortions by isolation methods and PCR analysis for the presence of virulence-associated genes. METHODS AND RESULTS: A total of 305 samples comprising blood, urine, placental bits, faecal and vaginal swabs were collected from 61 patients with spontaneous abortions. Listeria spp. were isolated from 10 samples collected from nine (14.8%) patients. Confirmation of these isolates was based on biochemical tests, haemolysis on blood agar, CAMP test, phosphatidylinositol-specific phospholipase C (PI-PLC) assay followed by in vivo pathogenicity tests and multiplex PCR to detect virulence-associated genes (prfA, plcA, hlyA, actA and iap). Three isolates were confirmed as L. monocytogenes. Of these, two isolates turned out to be pathogenic and found to posses all five genes. However, the remaining two haemolytic L. monocytogenes isolates lacking the plcA gene and activity in the PI-PLC assay were found to be nonpathogenic by in vivo tests. CONCLUSIONS: The occurrence of pathogenic L. monocytogenes in cases of spontaneous abortions was 3.3%. It seems that the plcA gene and its expression have an important role as essential virulence determinants in pathogenic Listeria spp. SIGNIFICANCE AND IMPACT OF THE STUDY: The recovery of pathogenic L. monocytogenes isolates from cases of spontaneous abortion indicates the significance of listeric infection in pregnant women.     

Isolation and Confirmation of <Emphasis Type="Italic">Listeria</Emphasis> Species from Seafood off Goa Region by Polymerase Chain Reaction

Several food borne outbreaks have highlighted the importance of Listeria monocytogenes to the public health and have been recognized as an emerging, important food borne pathogen, and a causative agent of listerioses. A number of genes are involved in the manifestation of Listeria virulence, hlyA is one among them. In the present study, 111 marine fish samples including prawns, finfishes and bivalves were screened for the presence of Listeria species. The isolates were characterized biochemically and further L. monocytogenes were confirmed by polymerase chain reaction (PCR) technique using the hlyA gene as a tool to differentiate between L. monocytogenes and other non-pathogenic Listeria species. Out of 111 samples 5 (4.5%) samples were positive for L. monocytogenes. Among the three different types of samples bivalves were found to have maximum percent (12.5) of L. monocytogenes followed by prawns (3.84) and finfishes (2.9). Among all the 111 samples, 15 (13.51%) samples were positive for other Listeria species. It was observed that Listeria occurrence is more in shellfishes than in fin fishes. All the isolates were sensitive towards five different antibiotics in sequence ciprofloxacin > sulphafurazole > norfloxacin > ampicillin and gentamicin.     

Biotechnological applications of Listeria's sophisticated infection strategies

Listeria monocytogenes is a Gram‐positive bacterium that is able to survive both in the environment and to invade and multiply within eukaryotic cells. Currently L. monocytogenes represents one of the most well‐studied and characterized microorganisms in bacterial pathogenesis. A hallmark of L. monocytogenes virulence is its ability to breach bodily barriers such as the intestinal epithelium, the blood–brain barrier as well as the placental barrier to cause severe systemic disease. Curiously, this theme is repeated at the level of the interaction between the individual cell and the bacterium where its virulence factors contribute to the ability of the bacteria to breach cellular barriers. L. monocytogenes is a model to study metabolic requirements of bacteria growing in an intracellular environment, modulation of signalling pathways in the infected cell and interactions with cellular defences involving innate and adaptive immunity. Technical advances such as the creation of LISTERIA‐susceptible mouse strains, had added interest in the study of the natural pathogenesis of the disease via oral infection. The use of attenuated strains of L. monocytogenes as vaccines has gained considerable interest because they can be used to express heterologous antigens as well as to somatically deliver recombinant DNA to eukaryotic cells. A novel vaccine concept, the use of non‐viable but metabolically active bacteria to induced immunoprotective responses, has been developed with L. monocytogenes. In this mini‐review, we review the strategies used by L. monocytogenes to subvert the cellular functions at different stages of the infection cycle in the host and examine how these properties are being exploited in biotechnological and clinical applications.     

Antibacterial efficacy of in-house designed cell-penetrating peptide against multi-drug resistant strains of Salmonella Enteritidis and Salmonella Typhimurium

The in vitro antibacterial efficacy of an in-house designed cell-penetrating peptide (CPP) variant of Cecropin A (1–7)-Melittin (CAMA) (CAMA-CPP) against the characterized multi-drug resistant (MDR) field strains of Salmonella Enteritidis and Salmonella Typhimurium were evaluated and compared with two identified CPPs namely, P7 and APP, keeping CAMA as control. Initially, the minimum inhibitory concentration (MIC) (μg ml⁻¹) of in-house designed CAMA-CPP, APP and CAMA was determined to be 3.91, whereas that of P7 was 7.81; however, the minimum bactericidal concentration (MBC) of all the peptides were twice the MIC. CAMA-CPP and CAMA were found to be stable under different conditions (high-end temperatures, proteinase-K, cationic salts, pH and serum) when compared to the other CPPs. Moreover, CAMA-CPP exhibited negligible cytotoxicity in HEp-2 and RAW 264.7 cell lines as well as haemolysis in the sheep and human erythrocytes with no adverse effects against the commensal gut lactobacilli. In vitro time-kill assay revealed that the MBC levels of CAMA-CPP and APP could eliminate the intracellular MDR-Salmonella infections from mammalian cell lines; however, CAMA and P7 peptides were ineffective. CAMA-CPP appears to be a promising antimicrobial candidate and opens up further avenues for its in vivo clinical translation.     

Multiplex PCR assay for species identification of bovine mastitis pathogens

Aim: To develop and evaluate a multiplex PCR (mPCR) assay for simultaneous detection of 10 bacterial species causing bovine mastitis namely, Staphylococcus aureus, Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus sciuri, Staphylococcus haemolyticus, Staphylococcus simulans, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis and Escherichia coli in milk. Methods and Results: A two‐tube mPCR assay was developed. The accuracy of the mPCR was evaluated using 56 standard reference strains and 705 strains comprising of E. coli (n = 99), staphylococci (n = 522) and streptococci (n = 84). The threshold of detection of the mPCR assay was 10 fg of genomic DNA and <10³ CFU ml^?1. A comparative evaluation of mPCR with culture method using 115 milk samples from subclinical mastitis showed mPCR to be more efficacious. Subsequently, the mPCR showed successful detection of target bacteria, when applied directly for the assessment of 36 bulk milk samples. Conclusion: The developed mPCR assay was found to be simple, rapid, reliable and specific in species identification of 10 bacteria at a time. Significance and Impact of the Study: The assay will be useful for the detection of mastitis, testing bacteriological safety of milk and for species level differentiation. The assay will be of value in the dairy sector for diagnosis and research. The early and accurate identification of pathogens will enable timely interventions for the treatment and control of bovine mastitis.     

Rapid Identification and Typing of Listeria Species by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

Listeria monocytogenes is a food-borne pathogen that is the causative agent of human listeriosis, an opportunistic infection that primarily infects pregnant women and immunologically compromised individuals. Rapid, accurate discrimination between Listeria strains is essential for appropriate therapeutic management and timely intervention for infection control. A rapid method involving matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) that shows promise for identification of Listeria species and typing and even allows for differentiation at the level of clonal lineages among pathogenic strains of L. monocytogenes is presented. A total of 146 strains of different Listeria species and serotypes as well as clinical isolates were analyzed. The method was compared with the pulsed-field gel electrophoresis analysis of 48 Listeria strains comprising L. monocytogenes strains isolated from food-borne epidemics and sporadic cases, isolates representing different serotypes, and a number of Listeria strains whose genomes have been completely sequenced. Following a short inactivation/extraction procedure, cell material from a bacterial colony was deposited on a sample target, dried, overlaid with a matrix necessary for the MALDI process, and analyzed by MALDI-TOF MS. This technique examines the chemistry of major proteins, yielding profile spectra consisting of a series of peaks, a characteristic “fingerprint” mainly derived from ribosomal proteins. Specimens can be prepared in a few minutes from plate or liquid cultures, and a spectrum can be obtained within 1 minute. Mass spectra derived from Listeria isolates showed characteristic peaks, conserved at both the species and lineage levels. MALDI-TOF MS fingerprinting may have potential for Listeria identification and subtyping and may improve infection control measures.     

Green Synthesized Silver Nanoparticles Using Lactobacillus Acidophilus as an Antioxidant,Antimicrobial, and Antibiofilm Agent Against Multi-drug Resistant Enteroaggregative Escherichia Coli

Probiotics and Antimicrobial Proteins - The present study was envisaged to employ the green synthesis and characterization of silver nanoparticles (AgNPs) using the potential probiotic strain...