Author Correction: A CRISPR/Cas system mediates bacterial innate immune evasion and virulence.

Change history: We could not replicate the results in Fig. 2a and g of this Letter, and new information has revealed a flaw in the interpretation of Fig. 2h. As a result, we do not have evidence to support RNA degradation as the mechanism that underlies Cas9-mediated regulation of FTN_1103 mRNA expression; see accompanying Amendment. This has not been corrected online.

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.

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.

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.

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.

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.

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.

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.

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.

Host cell-derived lactate functions as an effector molecule in Neisseria meningitidis microcolony dispersal.

The development of meningococcal disease, caused by the human pathogen Neisseria meningitidis, is preceded by the colonization of the epithelial layer in the nasopharynx. After initial adhesion to host cells meningococci form aggregates, through pilus-pilus interactions, termed microcolonies from which the bacteria later detach. Dispersal from microcolonies enables access to new colonization sites and facilitates the crossing of the cell barrier; however, this process is poorly understood. In this study, we used live-cell imaging to investigate the process of N. meningitidis microcolony dispersal. We show that direct contact with host cells is not required for microcolony dispersal, instead accumulation of a host-derived effector molecule induces microcolony dispersal. By using a host-cell free approach, we demonstrated that lactate, secreted from host cells, initiate rapid dispersal of microcolonies. Interestingly, metabolic utilization of lactate by the bacteria was not required for induction of dispersal, suggesting that lactate plays a role as a signaling molecule. Furthermore, Neisseria gonorrhoeae microcolony dispersal could also be induced by lactate. These findings reveal a role of host-secreted lactate in microcolony dispersal and virulence of pathogenic Neisseria.

A CRISPR/Cas system mediates bacterial innate immune evasion and virulence.

CRISPR/Cas (clustered regularly interspaced palindromic repeats/CRISPR-associated) systems are a bacterial defence against invading foreign nucleic acids derived from bacteriophages or exogenous plasmids. These systems use an array of small CRISPR RNAs (crRNAs) consisting of repetitive sequences flanking unique spacers to recognize their targets, and conserved Cas proteins to mediate target degradation. Recent studies have suggested that these systems may have broader functions in bacterial physiology, and it is unknown if they regulate expression of endogenous genes. Here we demonstrate that the Cas protein Cas9 of Francisella novicida uses a unique, small, CRISPR/Cas-associated RNA (scaRNA) to repress an endogenous transcript encoding a bacterial lipoprotein. As bacterial lipoproteins trigger a proinflammatory innate immune response aimed at combating pathogens, CRISPR/Cas-mediated repression of bacterial lipoprotein expression is critical for F. novicida to dampen this host response and promote virulence. Because Cas9 proteins are highly enriched in pathogenic and commensal bacteria, our work indicates that CRISPR/Cas-mediated gene regulation may broadly contribute to the regulation of endogenous bacterial genes, particularly during the interaction of such bacteria with eukaryotic hosts.

Lactobacilli Reduce Helicobacter pylori Attachment to Host Gastric Epithelial Cells by Inhibiting Adhesion Gene Expression.

The human gastrointestinal tract, including the harsh environment of the stomach, harbors a large variety of bacteria, of which Lactobacillus species are prominent members. The molecular mechanisms by which species of lactobacilli interfere with pathogen colonization are not fully characterized. In this study, we aimed to study the effect of lactobacillus strains upon the initial attachment of Helicobacter pylori to host cells. Here we report a novel mechanism by which lactobacilli inhibit adherence of the gastric pathogen H. pylori In a screen with Lactobacillus isolates, we found that only a few could reduce adherence of H. pylori to gastric epithelial cells. Decreased attachment was not due to competition for space or to lactobacillus-mediated killing of the pathogen. Instead, we show that lactobacilli act on H. pylori directly by an effector molecule that is released into the medium. This effector molecule acts on H. pylori by inhibiting expression of the adhesin-encoding gene sabA Finally, we verified that inhibitory lactobacilli reduced H. pylori colonization in an in vivo model. In conclusion, certain Lactobacillus strains affect pathogen adherence by inhibiting sabA expression and thereby reducing H. pylori binding capacity.

Properties of putrescine uptake by PotFGHI and PuuP and their physiological significance in Escherichia coli.

Properties of putrescine uptake by PotFGHI and PuuP and their physiological significance were studied using a polyamine biosynthesis and uptake deficient Escherichia coli KK3131 transformed with pACYC184 containing potFGHI or puuP. Putrescine uptake activity of E. coli KK3131 transformed with pACYC184-PotFGHI was higher than that of E. coli 3131 transformed with pACYC-PuuP when cells were cultured in the absence of putrescine. Putrescine uptake by PotFGHI was both ATP and membrane potential dependent, while that by PuuP was membrane potential dependent. Feedback inhibition by polyamines occurred at the PotFGHI uptake system but not at the PuuP uptake system. Expression of PuuP was reduced in the presence of PuuR, a negative regulator for PuuP, and expression of PuuR was positively regulated by glucose, which reduces the level of cAMP. The complex of cAMP and CRP (cAMP receptor protein) inhibited the expression of PuuR in the absence of glucose. Thus, the growth rate of E. coli KK3131 in the presence of both 0.4% (22.2 mM) glucose and 10 mM putrescine was in the order of cells transformed with pACYC-PotFGHI > pACYC-PuuP > pACYC-PuuP + PuuR, which was parallel with the polyamine content in cells. The results indicate that PotFGHI is necessary for rapid cell growth in the presence of glucose as an energy source. When glucose in medium was depleted, however, PuuP was absolutely necessary for cell growth in the presence of putrescine, because accumulation of putrescine to a high level by PuuP was necessary for utilization of putrescine as an energy source.

Bacteriophages Mediating Effective Elimination of Multidrug-Resistant Avian Pathogenic Escherichia coli.

Background: Avian pathogenic Escherichia coli (APEC) causes colibacillosis and septicemia; in certain cases, mortality leads to economic losses and elicits potential foodborne zoonotic risk. The study aimed to determine the prevalence of APEC pathotypes and serotypes in poultry, followed by characterization for virulence markers and antibiotic sensitivity and analysis of lytic efficacy of bacteriophages in the eradication of APEC. Methods: We successfully isolated and characterized 34 E. coli isolates from poultry farms. The lytic efficacy of seven bacteriophages, as well as a phage cocktail, was evaluated for biological control of multiple drug resistance (MDR) APEC. Results: A total of 67.65% of isolated E. coli were APEC. A total of 94.11% of the isolates were multidrug-resistant bacteria harboring virulence genes. The lytic ability of seven bacteriophages ranged from 0.98% to 36.76%, with a cocktail of EscoΦA-06 and ΦA-07 exhibiting lysis of 48.04% isolates. Conclusion: As serological variability in APEC limits the application and development of vaccines, the findings support the employment of bacteriophages against elimination of MDR APEC in poultry settings.

Streptomycin inhibits quorum sensing in Acinetobacter baumannii.

Streptomycin at subinhibitory concentrations was found to inhibit quorum sensing in Acinetobacter baumannii. Conditioned medium prepared by growth of A. baumannii in the presence of subinhibitory concentrations of streptomycin exhibited reduced activation of two quorum-sensing-regulated genes, abaI, encoding an autoinducer synthase, and A1S_0112. The reduced expression of AbaI resulted in greatly decreased levels of 3-OH-C(12)-HSL as confirmed by direct analysis using thin-layer chromatography. The effect on acyl-homoserine lactone (AHL) signal production was specific to streptomycin, as gentamicin and myomycin had no significant effect at subinhibitory levels.

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.

Novel mechanism for fluoroquinolone resistance in Acinetobacter baumannii.

An EZ::TNTnp transposon insertion in an open reading frame of unknown function (ncr) in Acinetobacter baumannii resulted in an 8-fold increase in ciprofloxacin resistance (Cip(r)). Transposon insertions in an ncr mutant that reduced Cip(r) back to wild type mapped to three genes encoding subunits of the RecCBD exonuclease. The ncr mutation increased transcription of the recCBD genes, and overexpression of the recCBD genes in a wild-type background resulted in a 4-fold increase in Cip(r).

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.