Acyl Homoserine Lactone Sensitised Streptococcus Pyogenes Differentially Regulates the Transcriptional Expression of Early Growth Response 1 (EGR1) in Epithelial and Macrophage Cells.

The host transcriptional activator Early growth response 1 (EGR1) plays a vital role in cell cycle and differentiation, cell proliferation, and regulation of cytokines and several growth factors. It is an immediate-early gene that is expressed as an initial response to various environmental stimuli. Bacterial infection is one such factor that can trigger the expression of EGR1 in host. Therefore, it is imperative to understand expression of EGR1 during early stages of host-pathogen interaction. Streptococcus pyogenes is an opportunistic bacteria causing skin and respiratory tract infections in humans. The quorum-sensing molecule, N-(3-oxododecanoyl)-l-homoserine lactone (Oxo-C12), not synthesised by S. pyogenes, can be sensed by S. pyogenes leading to molecular changes in the pathogen. In this study, we investigated the role of Oxo-C12 on EGR1 regulation in lung epithelial and murine macrophage cell line upon S. pyogenes infection. We report that Oxo-C12 sensitised S. pyogenes upregulates the transcriptional expression of EGR1 through ERK1/2 pathway. It was observed that EGR1 was not involved in the intial attachment of S. pyogenes to A549 cells. However, inhibition of EGR1 in macrophage cell line, J774A.1, through the ERK1/2 pathway resulted in decreased adhesion of S. pyogenes. The EGR1 upregulation by Oxo-C12 sensitised S. pyogenes plays a vital role in enhancing the survival of S. pyogenes in murine macrophages, leading to persistent infection. Thus, understanding the molecular modulation in the host during bacterial infection will further help develop therapeutics to target specific sites.

Effect of respiratory tract co-colonizers on initial attachment of Neisseria meningitidis.

Respiratory tract is a complex system comprising of unique microbiota inhabitants. Neisseria meningitidis, Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa and Klebsiella pneumoniae are few prevalent bacteria in the community composition during lung infections. Although, N. meningitidis resides asymptomatically in nasopharynx of the human host, it can cause fatal infections like meningitis. However, factors affecting transit from carriage to symptomatic infection are not well understood. Various host metabolites and environmental conditions affect the virulence of bacteria. Here, we report that presence of co-colonizers significantly reduces the initial attachment of N. meningitidis to A549 nasopharyngeal epithelial cells. Further, significant decrease in invasion to A549 nasopharyngeal epithelial cells was observed. Moreover, survival in J774A.1 murine macrophage also increases significantly when conditioned media (CM) from S. pyogenes and L. rhamnosus is used for culturing N. meningitidis. The increase in survival could be attributed to increased capsule synthesis. The gene expression studies revealed increased expression of siaC and ctrB in CM prepared from the growth S. pyogenes and L. rhamnosus. Overall, the results suggest change in the virulence of N. meningitidis is assisted by lung microbiota.

Polyamine as a microenvironment factor in resistance to antibiotics.

One of the main issues in modern medicine is the decrease in the efficacy of antibiotic therapy against resistant microorganisms. The advent of antimicrobial resistance has added significantly to the impact of infectious diseases, in number of infections, as well as added healthcare costs. The development of antibiotic tolerance and resistance is influenced by a variety of environmental variables, and it is important to identify these environmental factors as part of any strategy for combating antibiotic resistance. The review aims to emphasize that biogenic polyamines are one of such environmental cues that impacts the antibiotic resistance in bacteria. The biogenic polyamines can help bacteria acquire resistance to antibiotics either by regulating the level of number of porin channels in the outer membrane, by modifying the outer membrane liposaccharides or by protecting macromolecule from antibiotic stress. Thus, understanding the way polyamines function in bacteria can thus be beneficial while designing the drugs to combat diseases.

Antibiotics modulates the virulence of Neisseria meningitidis by regulating capsule synthesis.

The opportunistic pathogens residing are frequently exposed to range of antimicrobials which affects virulence attributes. Neisseria meningitidis, is a host-restricted commensal of human upper respiratory tract which is subjected to a variety of stresses within the host, including antibiotic exposure. One of the most important virulence factors for pathogenesis is the meningococcal lipo-oligosaccharide capsule. Role of capsules in antimicrobial resistance and persistence is not yet established. In this study, different virulence factors of N. meningitidis were examined in presence of sub-MIC of four antibiotics: penicillin, ciprofloxacin, erythromycin and chloramphenicol. We observed increased production of the capsule by N. meningitidis when grown in the presence of penicillin, erythromycin, and chloramphenicol at sub-inhibitory concentration. Capsular production increase concurrently with increased resistance to inducing antibiotic which also confers increased survival in human serum. Finally, we show that increased capsule production in response to antibiotic exposure is aided by siaC, ctrB, lipA gene expression. These findings show that capsule synthesis, a major pathogenicity determinant, is regulated in response to antibiotic stress. Our findings support a model in which gene expression changes caused by ineffective antibiotic treatment cause N. meningitidis transition between states of low and high virulence potential, contributing to pathogen's opportunistic nature.

CRISPR in Modulating Antibiotic Resistance of ESKAPE Pathogens.

The ESKAPE (Enterococcus spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) isolates both from the clinical settings and food products are demonstrated to gain resistance to multiple antimicrobials. Therefore, the ESKAPE pathogens pose a serious threat to public health, which warrants specific attention to developing alternative novel therapeutics. The clustered regularly interspaced short palindromic repeats associated (CRISPR-Cas) system is one of the novel methods for managing antibiotic-resistant strains. Specific Cas nucleases can be programmed against bacterial genomic sequences to decrease bacterial resistance to antibiotics. Moreover, a few CRISPR-Cas nucleases have the ability to the sequence-specific killing of bacterial strains. However, some pathogens acquire antibiotic resistance due to the presence of the CRISPR-Cas system. In brief, there is a wide range of functional diversity of CRISPR-Cas systems in bacterial pathogens. Hence, to be an effective and safe infection treatment strategy, a comprehensive understanding of the role of CRISPR-Cas systems in modulating antibiotic resistance in ESKAPE pathogens is essential. The present review summarizes all the mechanisms by which CRISPR confers and prevents antibiotic resistance in ESKAPE. The review also emphasizes the relationship between CRISPR-Cas systems, biofilm formation, and antibiotic resistance in ESKAPE.

Lactic acid bacteria: prominent player in the fight against human pathogens.

INTRODUCTION: The human microbiome is a unique repository of diverse bacteria. Over 1000 microbial species reside in the human gut, which predominantly influences the host's internal environment and plays a significant role in host health. Lactic acid bacteria have long been employed for multiple purposes, ranging from food to medicines. Lactobacilli, which are often used in commercial food fermentation, have improved to the point that they might be helpful in medical applications. AREAS COVERED: This review summarises various clinical and experimental evidence on efficacy of lactobacilli in treating a wide range of infections. Both laboratory based and clinical studies have been discussed. EXPERT OPINION: Lactobacilli are widely accepted as safe biological treatments and host immune modulators (GRAS- Generally regarded as safe) by the US Food and Drug Administration and Qualified Presumption of Safety. Understanding the molecular mechanisms of lactobacilli in the treatment and pathogenicity of bacterial infections can help with the prediction and development of innovative therapeutics aimed at pathogens which have gained resistance to antimicrobials. To formulate effective lactobacilli based therapy significant research on the effectiveness of different lactobacilli strains and its association with demographic distribution is required. Also, the side effects of such therapy needs to be evaluated.

Spermidine promotes lysozyme tolerance and acid stress resistance in Streptococcus pyogenes M3.

Streptococcus pyogenes are Gram-positive opportunistic pathogens residing in the human nasopharynx and skin. Changes in environmental conditions, such as pH, temperature and availability of essential ions, can stimulate the expression of S. pyogenes virulence factors. One such factor could be the availability of an extracellular pool of polyamines. Polyamines are synthesized from amino acids, and are universally present in the environment. Polyamines have been implicated in the ecology of pathogenesis by modulating quorum sensing, host adaptation and virulence. Polyamines mediate pathogenesis and help the pathogen resist environmental stress. In this study, we investigated the ability of the polyamine, spermidine, to promote acid stress survival of S. pyogenes. S. pyogenes does not synthesize spermidine, but the extracellular pool of spermidine constituted by the host and microbiome could be utilized as a signalling molecule. We report that spermidine promotes acid stress resistance in S. pyogenes. Moreover, spermidine affects the morphology of S. pyogenes by decreasing the cell size and increasing the dltA gene expression. Along with dltA, spermidine upregulated the gene expression of cell wall-modifying genes such as mur, pgdA, pepO and srtA, which might help the bacteria to resist acidic stress.

The source of carbon and nitrogen differentially affects the survival of Neisseria meningitidis in macrophages and epithelial cells.

Neisseria meningitidis is a commensal of human nasopharynx which under certain unidentified conditions could lead to fulminant meningitis or sepsis. Availability of nutrients is essential for bacterial growth and virulence. The metabolic adaptations allow N. meningitidis to utilize host resources, colonize and cause virulence functions which are a crucial for the invasive infection. During colonization meningococci encounters a range of microenvironments involving fluctuations in the availability of carbon and nitrogen source. Therefore, the characterization of virulence factors of N. meningitidis under different microenvironmental conditions is a prime requisite to understand pathogenesis; however, the role of nutrients is not well understood. Here, we explore the expression of virulence phenotype leading to symptomatic behaviour as affected by available carbon and nitrogen sources. We evaluate the effect of carbon or nitrogen source on growth, adhesion to epithelial cells, macrophage infectivity, capsule formation and virulence gene expression of N. meningitidis. It was found that lactate, pyruvate, and acetate facilitate survival of N. meningitidis in macrophages. While in epithelial cells, the survival of N. meningitidis is negatively affected by the presence of lactate and pyruvate.

Availability of polyamines affects virulence and survival of Neisseria meningitidis.

Neisseria meningitidis is a Gram-negative human-restricted pathogen that asymptomatically resides in the human respiratory tract. Meningococcal meningitis and sepsis both are caused by N. meningitidis. The bacterium must adhere to host epithelial cells in order to colonize effectively. The factors that determine the initial attachment to the host and dispersal, are not well understood. Metabolites released by the host may aid in meningococcal colonization and dissemination. Polyamines are aliphatic polycations that assist in cell survival and proliferation. The virulence properties of N. meningitidis after exposure to polyamines were investigated. Adhesion to nasopharyngeal epithelial cells increased in the presence of spermine. Also, the relative expression of adhesin, pilE increased in the presence of spermine. Further, relative expression of ctrA, ctrB and lipB was upregulated in the presence of spermidine, indicating increased capsule formation. Upregulated capsule synthesis of N. meningitidis in the presence of spermidine allows it to survive in murine macrophages. The study suggests the importance of the extracellular pool of polyamines in promoting virulence in N. meningitidis.

Spermidine enhances the survival of Streptococcus pyogenes M3 under oxidative stress.

Streptococcus pyogenes, a host-restricted gram-positive pathogen during infection, initially adheres to the epithelia of the nasopharynx and respiratory tract of the human host, followed by disseminating to other organs and evading the host immune system. Upon phagocytosis, S. pyogenes encounters oxidative stress inside the macrophages. The role of polyamines in regulating various physiological functions including stress resistance in bacteria has been reported widely. Since S. pyogenes lacks the machinery for the biosynthesis of polyamines, the study aimed to understand the role of extracellular polyamines in the survival of S. pyogenes under oxidative stress environments. S. pyogenes being a catalase-negative organism, we report that its survival within the macrophages and H2 O2 is enhanced by the presence of spermidine. The increased survival can be attributed to the upregulation of oxidative stress response genes such as sodM, npx, and mtsABC. In addition, spermidine influences the upregulation of virulence factors such as sagA, slo, and hasA. Also, spermidine leads to a decrease in hydrophobicity of the cell membrane and an increase in hyaluronic acid. This study suggests a role for extracellular spermidine in the survival of S. pyogenes under oxidative stress environments. Recognizing the factors that modulate S. pyogenes survival and virulence under stress will assist in understanding its interactions with the host.

Exposure to Acyl Homoserine Lactone Enhances Survival of Streptococcus pyogenes in Murine Macrophages.

Streptococcus pyogenes is an opportunistic pathogen causing infections of the skin and upper respiratory tract of the human host. Due to the polymicrobial community present in the human host, S. pyogenes comes across several interspecies signalling molecules. Among these molecules, N-(3-oxododecanoyl)-L-homoserine lactone (Oxo-C12) modulates the morphology, thereby enhancing virulence characteristics of S. pyogenes. After the initial attachment of the bacteria to the host cell, the pathogen needs to invade the host immune system for a successful infection to occur. The host immune system is activated upon infection, where macrophages engulf the pathogen, thereby killing the bacteria. However, S. pyogenes have evolved various strategies to evade the host immune response. In this study, we investigate the role of Oxo-C12 in enhancing the survival of S. pyogenes M3 in murine macrophages. The observed Oxo-C12-mediated increased survival in murine macrophages was through increased lysozyme and acid stress resistance. Moreover, Oxo-C12 increased the survival of S. pyogenes in normal human serum. Thus, understanding the role of interspecies signalling in enhancing the survival strategies of S. pyogenes in the host will further help fill the gap for therapeutics development.

CRISPR/Cas 9-Based Editing in the Production of Bioactive Molecules.

Plants, fungi, and bacteria synthesize a wide range of secondary metabolites that exhibit diverse biological activities. These bioactives, due to their potential benefits in research and therapeutics, have gained immense industrial importance. There is a need to synthesize these bioactives at significantly higher concentrations using cost-effective measures to be economically viable. However, the broader study of industrially important secondary metabolites has been hindered, thus, far due to a shortage of reliable, comparatively easy, and highly effective gene manipulation techniques. With the advent of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas), there is a revolution in the field of genetic engineering. CRISPR/Cas system, due to its simplicity and ease of use. This has widened its application in plant breeding, strain improvement, and engineering the metabolic pathways involved in the biochemical synthesis of industrially valuable bioactive. This review briefly introduces the CRISPR/Cas9 system and summarizes the applications of CRISPR/Cas9-mediated editing tools for the production of plant and fungal-derived bioactives.

Interspecies signaling affects virulence related morphological characteristics of Streptococcus pyogenes M3.

Streptococcus pyogenes is a Gram-positive human-specific pathogen that asymptomatically colonizes the human respiratory tract. The factors affecting the colonization to the host is not clearly understood. Adherence of the pathogen to host epithelial cell is the initial step for a successful colonization process. In the host, bacteria live in a polymicrobial community; thus, the signaling mediated between the bacteria plays a significant role in the colonization of the pathogen to the host. Thus, the effect of acyl-homoserine lactone, secreted by Gram-negative bacteria on the adhesion properties of S. pyogenes M3 strain was examined. N-(3-Oxododecanoyl)-L-homoserine lactone (Oxo-C12) increased the cell size as well as hydrophobicity of S. pyogenes. qPCR data revealed that the expression of sagA and hasA was negatively affected by Oxo-C12. Moreover, Oxo-C12 leads to changes in the morphological characteristic of S. pyogenes, further promoting adherence to host epithelia and biofilm formation on abiotic surface. The study demonstrates the role of Oxo-C12 as a factor that can promote virulence in S. pyogenes M3.

Benchmarking of 16S rRNA gene databases using known strain sequences.

16S rRNA gene analysis is the most convenient and robust method for microbiome studies. Inaccurate taxonomic assignment of bacterial strains could have deleterious effects as all downstream analyses rely heavily on the accurate assessment of microbial taxonomy. The use of mock communities to check the reliability of the results has been suggested. However, often the mock communities used in most of the studies represent only a small fraction of taxa and are used mostly as validation of sequencing run to estimate sequencing artifacts. Moreover, a large number of databases and tools available for classification and taxonomic assignment of the 16S rRNA gene make it challenging to select the best-suited method for a particular dataset. In the present study, we used authentic and validly published 16S rRNA gene type strain sequences (full length, V3-V4 region) and analyzed them using a widely used QIIME pipeline along with different parameters of OTU clustering and QIIME compatible databases. Data Analysis Measures (DAM) revealed a high discrepancy in ratifying the taxonomy at different taxonomic hierarchies. Beta diversity analysis showed clear segregation of different DAMs. Limited differences were observed in reference data set analysis using partial (V3-V4) and full-length 16S rRNA gene sequences, which signify the reliability of partial 16S rRNA gene sequences in microbiome studies. Our analysis also highlights common discrepancies observed at various taxonomic levels using various methods and databases.

Foodborne ESKAPE Biofilms and Antimicrobial Resistance: lessons Learned from Clinical Isolates.

The ESKAPE pathogens (Enterococcus spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) are identified to be multidrug-resistant (MDR), extensively drug-resistant (XDR), and pan drug-resistant (PDR); thereby, imposing severe challenges in the treatment of associated infections. ESKAPE pathogens colonize on various biotic and abiotic surfaces; biofilms formed by these pathogens are a potential source for food contamination. Moreover, biofilms play a pivotal role in the development of antimicrobial-resistant (AMR) strains. Hence, the frequent isolation of antimicrobial-resistant ESKAPE pathogens from food products across the globe imposes a threat to public health. A comprehensive understanding of the adhesion signaling involved in the polymicrobial and single-species biofilm will assist in developing alternative preservation techniques and novel therapeutic strategies to combat ESKAPE pathogens. The review provides a comprehensive overview of the signaling mechanisms that prevail in the ESKAPE pathogens for adhesion to abiotic and biotic surfaces and molecular mechanisms associated with poly-microbial biofilm-assisted AMR in ESKAPE.

Pore-forming toxins of foodborne pathogens.

Pore-forming toxins (PFTs) are water-soluble molecules that have been identified as the most crucial virulence factors during bacterial pathogenesis. PFTs disrupt the host cell membrane to internalize or to deliver other bacterial or virulence factors for establishing infections. Disruption of the host cell membrane by PFTs can lead to uncontrollable exchanges between the extracellular and the intracellular matrix, thereby disturbing the cellular homeostasis. Recent studies have provided insights into the molecular mechanism of PFTs during pathogenesis. Evidence also suggests the activation of several signal transduction pathways in the host cell on recognition of PFTs. Additionally, numerous distinctive host defense mechanisms as well as membrane repair mechanisms have been reported; however, studies reveal that PFTs aid in host immune evasion of the bacteria through numerous pathways. PFTs have been primarily associated with foodborne pathogens. Infection and death from diseases by consuming contaminated food are a constant threat to public health worldwide, affecting socioeconomic development. Moreover, the emergence of new foodborne pathogens has led to the rise of bacterial antimicrobial resistance affecting the population. Hence, this review focuses on the role of PFTs secreted by foodborne pathogens. The review highlights the molecular mechanism of foodborne bacterial PFTs, assisting bacterial survival from the host immune responses and understanding the downstream mechanism in the activation of various signaling pathways in the host upon PFT recognition. PFT research is a remarkable and an important field for exploring novel and broad applications of antimicrobial compounds as therapeutics.

Early growth response 1 (EGR1) activation in initial stages of host-pathogen interactions.

The factors that determine the outcomes of host-pathogen interactions, such as host specificity, tissue specificity, and transition from asymptomatic to symptomatic behavior of a pathogen, are yet to be deciphered. The initial interaction of a pathogen with host and host-associated factors play a crucial role in deciding such outcomes. One of the several host-factors that contribute to bacterial adhesion and the outcome of an infection is the activation of early growth response 1 (EGR1). EGR1 is an initial response transcriptional regulator that plays a vital role in regulating cell growth, differentiation, and survival. EGR1 expression is seen in most of the mammalian tissues. Multiple post-translational modifications occur, which modulate the EGR1 transcriptional activity. Upon activation, EGR1 can transactivate several genes with diverse cellular functions, including transcriptional regulatory proteins and cell proliferation. EGR1 has also been identified as a potential mediator of inflammatory gene expression. Recent studies have highlighted the role of EGR1 as a potent signaling molecule that facilitates bacterial adhesion to host epithelial cells, thus modulating colonization pathways. The pathways for the regulation of EGR1 during host-pathogen interaction remain yet unidentified. The review focuses on the role and regulation of EGR1 during host-pathogen interaction.

Polyamines in the virulence of bacterial pathogens of respiratory tract.

Polyamines are positively charged hydrocarbons that are essential for the growth and cellular maintenance in prokaryotes and eukaryotes. Polyamines have been demonstrated to play a role in bacterial pathogenicity and biofilm formation. However, the role of extracellular polyamines as a signaling molecule in the regulation of virulence is not investigated in detail. The bacterial pathogens residing in the respiratory tract remain asymptomatic for an extended period; however, the factors that lead to symptomatic behavior are poorly understood. Further investigation to understand the relation between the host-secreted factors and virulence of pathogenic bacteria in the respiratory tract may provide insights into the pathogenesis of respiratory tract infections. Polyamines produced within the bacterial cell are generally sequestered. Therefore, the pool of extracellular polyamines formed by secretion of the commensals and the host may be one of the signaling molecules that might contribute toward the alterations in the expression of virulence factors in bacterial pathogens. Besides, convergent mechanisms of polyamine biosynthesis do exist across the border of species and genus level. Also, several novel polyamine transporters in the host and bacteria remain yet to be identified. The review focuses on the role of polyamines in the expression of virulence phenotypes and biofilm formation of the respiratory tract pathogens.

Isolation and characterization of Escherichia coli serotype O157:H7 and other verotoxin-producing E. coli in healthy Indian cattle.

BACKGROUND AND AIM: Cattle are the main reservoir of Escherichia coli O157:H7 and other verotoxigenic E. coli (VTEC); therefore, there is an increased risk of infection to humans by either direct or indirect mode of transmissions. However, the prevalence of E. coli O157:H7 in the healthy cattle population of India is yet to be ascertained. This study aimed to screen the dairy cattle in and around Pune, Maharashtra, India, for verotoxin-producing E. coli O157:H7. MATERIALS AND METHODS: A total of 257 rectal swabs were collected from 15 different organized and unorganized dairy farms of Pune during the period, January-March 2015. The screening involved enrichment in EC broth followed by differential identification on MacConkey sorbitol agar. The presumptive positive isolates were further confirmed by multiplex polymerase chain reaction (PCR) using primers specific to rfbE (O157), fliC (H7), VT1 (MK1), and VT2 (MK2). Vero-toxicity and antibiotic sensitivity were examined in PCR confirmed isolates. RESULTS: Out of the 257 samples analyzed, 1.9% (2/105) were positive for O157:H7 and 39% (41/105) were positive for VTEC. Two PCR confirmed positive O157:H7 strains and two randomly selected PCR-positive VT strains exhibited in vitro cytopathic effect on Vero cells on day-7 post-inoculation. Antibiotic sensitivity profiling of O157:H7 strains exhibited resistance against penicillin G, kanamycin, ampicillin, tetracycline, gentamycin, cefotaxime, streptomycin, and piperacillin. CONCLUSION: These findings reveal the presence of pathogenic E. coli O157:H7 in the healthy cattle of Pune; in a situation, wherein regular surveillance for O157:H7 is not a norm. Therefore, the findings presented herein warrant routine surveillance and public awareness to prevent the transfer of such pathogens and manage health risks to the public.

Role of interspecies bacterial communication in the virulence of pathogenic bacteria.

Bacteria live in a polymicrobial community where it interacts with biotic and abiotic factors using specific signalling molecules. Acyl homoserine lactones, autoinducing peptides, bacteriocins and polyamines are a few signals documented for interspecies signalling. The signalling system could be used for a coordinated behaviour categorised as Quorum sensing (QS). QS is a term used to define a cell - cell communication process amongst bacteria that helps to gather cell density information and regulate gene expression accordingly. QS had been demonstrated to play a pivotal role in bacterial pathogenesis by regulating the expression of different virulence factors affecting adhesion, invasion and survival within a tissue. In the current review, we discuss the role of interspecies bacterial communication in pathogenicity. The molecules involved in the interspecies bacterial communication affecting virulence factors required for the establishment of infection have been discussed in detail to gain an insight for development of strategies that can be proposed to combat bacterial infections by attenuating their communication systems. The knowledge on the role of interspecies bacterial communication on virulence will assist in understanding the factors affecting symptomatic and asymptomatic infections.