Multiple Virulence Research Articles

Regulation of the Sae Two-Component System by Branched-Chain Fatty Acids in Staphylococcus aureus.

ABSTRACTStaphylococcus aureus is a ubiquitous Gram-positive bacterium and an opportunistic human pathogen. S. aureus pathogenesis relies on a complex network of regulatory factors that adjust gene expression. Two important factors in this network are CodY, a repressor protein responsive to nutrient availability, and the SaeRS two-component system (TCS), which responds to neutrophil-produced factors. Our previous work revealed that CodY regulates the secretion of many toxins indirectly via Sae through an unknown mechanism. We report that disruption of codY results in increased levels of phosphorylated SaeR (SaeR~P) and that codY mutant cell membranes contain a higher percentage of branched-chain fatty acids (BCFAs) than do wild-type membranes, prompting us to hypothesize that changes to membrane composition modulate the activity of the SaeS sensor kinase. Disrupting the lpdA gene encoding dihydrolipoyl dehydrogenase, which is critical for BCFA synthesis, significantly reduced the abundance of SaeR, phosphorylated SaeR, and BCFAs in the membrane, resulting in reduced toxin production and attenuated virulence. Lower SaeR levels could be explained in part by reduced stability. Sae activity in the lpdA mutant could be complemented genetically and chemically with exogenous short- or full-length BCFAs. Intriguingly, lack of lpdA also alters the activity of other TCSs, suggesting a specific BCFA requirement managing the basal activity of multiple TCSs. These results reveal a novel method of posttranscriptional virulence regulation via BCFA synthesis, potentially linking CodY activity to multiple virulence regulators in S. aureus.

An Unprecedented Tolerance to Deletion of the Periplasmic Chaperones SurA, Skp, and DegP in the Nosocomial Pathogen Acinetobacter baumannii.

ABSTRACTThe outer membrane (OM) of Gram-negative bacteria efficiently protects from harmful environmental stresses such as antibiotics, disinfectants, or dryness. The main constituents of the OM are integral OM β-barrel proteins (OMPs). In Gram-negative bacteria such as Escherichia coli, Yersinia enterocolitica, and Pseudomonas aeruginosa, the insertion of OMPs depends on a sophisticated biogenesis pathway. This comprises the SecYEG translocon, which enables inner membrane (IM) passage; the chaperones SurA, Skp, and DegP, which facilitate the passage of β-barrel OMPs through the periplasm; and the β-barrel assembly machinery (BAM), which facilitates insertion into the OM. In E. coli, Y. enterocolitica, and P. aeruginosa, the deletion of SurA is particularly detrimental and leads to a loss of OM integrity, sensitization to antibiotic treatment, and reduced virulence. In search of targets that could be exploited to develop compounds that interfere with OM integrity in Acinetobacter baumannii, we employed the multidrug-resistant strain AB5075 to generate single gene knockout strains lacking individual periplasmic chaperones. In contrast to E. coli, Y. enterocolitica, and P. aeruginosa, AB5075 tolerates the lack of SurA, Skp, or DegP with only weak mutant phenotypes. While the double knockout strains ΔsurAΔskp and ΔsurAΔdegP are conditionally lethal in E. coli, all double deletions were well tolerated by AB5075. Strikingly, even a triple-knockout strain of AB5075, lacking surA, skp, and degP, was viable.IMPORTANCE Acinetobacter baumannii is a major threat to human health due to its ability to persist in the hospital environment, resistance to antibiotic treatment, and ability to deploy multiple and redundant virulence factors. In a rising number of cases, infections with multidrug-resistant A. baumannii end up fatally, because all antibiotic treatment options fail. Thus, novel targets have to be identified and alternative therapeutics have to be developed. The knockout of periplasmic chaperones has previously proven to significantly reduce virulence and even break antibiotic resistance in other Gram-negative pathogens. Our study in A. baumannii demonstrates how variable the importance of the periplasmic chaperones SurA, Skp, and DegP can be and suggests the existence of mechanisms allowing A. baumannii to cope with the lack of the three periplasmic chaperones.

Virulence Characteristics and Emerging Therapies for Biofilm-Forming Acinetobacter baumannii: A Review.

Simple SummaryAcinetobacter baumannii (A. baumannii) is one of the ESKAPE organisms and has the competency to build biofilms. These biofilms account for the most nosocomial infections all over the world. This review reflects on the various physicochemical and environmental factors such as adhesion, pili expression, growth surfaces, drug-resistant genes, and virulence factors that profoundly affect its resistant forte. Emerging drug-resistant issues and limitations to newer drugs are other factors affecting the hospital environment. Here, we discuss newer and alternative methods that can significantly enhance the susceptibility to Acinetobacter spp. Many new antibiotics are under trials, such as GSK-3342830, The Cefiderocol (S-649266), Fimsbactin, and similar. On the other hand, we can also see the impact of traditional medicine and the secondary metabolites of these natural products’ application in searching for new treatments. The field of nanoparticles has demonstrated effective antimicrobial actions and has exhibited encouraging results in the field of nanomedicine. The use of various phages such as vWUPSU and phage ISTD as an alternative treatment for its specificity and effectiveness is being investigated. Cathelicidins obtained synthetically or from natural sources can effectively produce antimicrobial activity in the micromolar range. Radioimmunotherapy and photodynamic therapy have boundless prospects if explored as a therapeutic antimicrobial strategy.Acinetobacter species is one of the most prevailing nosocomial pathogens with a potent ability to develop antimicrobial resistance. It commonly causes infections where there is a prolonged utilization of medical devices such as CSF shunts, catheters, endotracheal tubes, and similar. There are several strains of Acinetobacter (A) species (spp), among which the majority are pathogenic to humans, but A. baumannii are entirely resistant to several clinically available antibiotics. The crucial mechanism that renders them a multidrug-resistant strain is their potent ability to synthesize biofilms. Biofilms provide ample opportunity for the microorganisms to withstand the harsh environment and further cause chronic infections. Several studies have enumerated multiple physiological and virulence factors responsible for the production and maintenance of biofilms. To further enhance our understanding of this pathogen, in this review, we discuss its taxonomy, pathogenesis, current treatment options, global resistance rates, mechanisms of its resistance against various groups of antimicrobials, and future therapeutics.

Hcp Proteins of the Type VI Secretion System Promote Avian Pathogenic E. coli DE205B (O2:K1) to Induce Meningitis in Rats.

Avian pathogenic Escherichia coli (APEC) is an important extra-intestinal pathogenic E. coli (ExPEC), which often causes systemic infection in poultry and causes great economic loss to the breeding industry. In addition, as a major source of human ExPEC infection, the potential zoonotic risk of APEC has been an ongoing concern. Previous studies have pointed out that APEC is a potential zoonotic pathogen, which has high homology with human pathogenic E. coli such as uro-pathogenic E. coli (UPEC) and neonatal meningitis E. coli (NMEC), shares multiple virulence factors and can cause mammalian diseases. Previous studies have reported that O18 and O78 could cause different degrees of meningitis in neonatal rats, and different serotypes had different degrees of zoonotic risk. Here, we compared APEC DE205B (O2:K1) with NMEC RS218 (O18:K1:H7) by phylogenetic analysis and virulence gene identification to analyze the potential risk of DE205B in zoonotic diseases. We found that DE205B possessed a variety of virulence factors associated with meningitis and, through phylogenetic analysis, had high homology with RS218. DE205B could colonize the cerebrospinal fluid (CSF) of rats, and cause meningitis and nerve damage. Symptoms and pathological changes in the brain were similar to RS218. In addition, we found that DE205B had a complete T6SS, of which Hcp protein was its important structural protein. Hcp1 induced cytoskeleton rearrangement in human brain microvascular endothelial cells (HBMECs), and Hcp2 was mainly involved in the invasion of DE205B in vitro. In the meningitis model of rats, deletion of hcp2 gene reduced survival in the blood and the brain invasiveness of DE205B. Compared with WT group, Δhcp2 group induced lower inflammation and neutrophils infiltration in brain tissue, alleviating the process of meningitis. Together, these results suggested that APEC DE205B had close genetic similarities to NMEC RS218, and a similar mechanism in causing meningitis and being a risk for zoonosis. This APEC serotype provided a basis for zoonotic research.

Tackling recalcitrant Pseudomonas aeruginosa infections in critical illness via anti-virulence monotherapy

Intestinal barrier derangement allows intestinal bacteria and their products to translocate to the systemic circulation. Pseudomonas aeruginosa (PA) superimposed infection in critically ill patients increases gut permeability and leads to gut-driven sepsis. PA infections are challenging due to multi-drug resistance (MDR), biofilms, and/or antibiotic tolerance. Inhibition of the quorum-sensing transcriptional regulator MvfR(PqsR) is a desirable anti-PA anti-virulence strategy as MvfR controls multiple acute and chronic virulence functions. Here we show that MvfR promotes intestinal permeability and report potent anti-MvfR compounds, the N-Aryl Malonamides (NAMs), resulting from extensive structure-activity-relationship studies and thorough assessment of the inhibition of MvfR-controlled virulence functions. This class of anti-virulence non-native ligand-based agents has a half-maximal inhibitory concentration in the nanomolar range and strong target engagement. Using a NAM lead in monotherapy protects murine intestinal barrier function, abolishes MvfR-regulated small molecules, ameliorates bacterial dissemination, and lowers inflammatory cytokines. This study demonstrates the importance of MvfR in PA-driven intestinal permeability. It underscores the utility of anti-MvfR agents in maintaining gut mucosal integrity, which should be part of any successful strategy to prevent/treat PA infections and associated gut-derived sepsis in critical illness settings. NAMs provide for the development of crucial preventive/therapeutic monotherapy options against untreatable MDR PA infections.

Protein expression profiling, in silico classification and pathway analysis of cariogenic bacteria Streptococcus mutans under bacitracin stress conditions.

Introduction. Streptococcus mutans is a cariogenic bacterium that causes dental caries as well as being implicated in other dental pathologies and infective endocarditis. Bacitracin is a bactericidal antibiotic that induces cell wall stress in Gram-positive bacteria.Gap Statement. S. mutans is among the most characterized Gram-positive bacteria. However, the transcriptome and proteome of S. mutans have received less attention, and they are actually key in understanding the pathogenesis of any bacteria. In this study, we extracted the whole proteome of S. mutans grown under bacitracin stress. Such a proteome is anticipated to offer deep insights related to physiological dynamic fluctuations and, consequently, it may provide 'proteomic signatures' to be identified as potential targets.Aim. The aim of the study is to explore the general stress response that S. mutans exhibits at the proteome level when cell wall stress is imposed on it.Methodology. A sub-MIC concentration of bacitracin was added to the growth media of S. mutans followed by whole-cell protein extraction. The proteome was then subjected to high-throughput proteomics analysis, i.e. liquid chromatography tandem mass spectrometry (LC-MS/MS). Differentially expressed proteins obtained through LC-MS/MS underwent analyses such as gene ontology, KEGG (Kyoto Encyclopaedia of Genes and Genomes) and DAVID (Database for Annotation, Visualization and Integrated Discovery) analysis, and STRING for functional annotation, pathway enrichment and protein-protein interaction (PPI) networks, respectively. These proteins were also categorized into functional classes using the PANTHER (Protein Annotation Through Evolutionary Relationship) classification system.Result. LC-MS/MS produced data from 321 identified proteins. From these, 41 and 30 were found to be significantly over- (≥2 fold change) and underexpressed (≤0.4 fold change), respectively. In the upregulated proteins we mostly observed sortases and proteins involved in the EPS biosynthesis pathway, whereas among the downregulated proteins the majority related to glycolysis.Conclusion. The sortase family of proteins appear to be potential targets because they regulate various virulence factors and therefore can be targeted to inhibit multiple virulence pathways simultaneously. This study offers an understanding of proteomic fluctuations in response to cell wall stress and can thus help in identifying key players mediating virulence.

Presence of Extended Spectrum Beta Lactamase, Virulence Genes and Resistance Determinants in Biofilm Forming Klebsiella pneumoniae Isolated from Food Sources: A Potent Risk to the Consumers

Foodborne diseases and infection caused by associated pathogens is a public health concern. Majority of the investigations focus on common foodborne pathogens like Vibrio parahaemolyticus, Escherichia coli, Listeria monocytogenes, Shigella, Salmonella and Staphylococcus aureus. Limited knowledge has been accounted on Klebsiella pneumoniae. Presence of multidrug-resistant K. pneumoniae in the food supply is disturbing. Hence, this study assessed the presence of K. pneumoniae isolates from food samples (fresh vegetables and chicken), ascertained the presence of drug-resistant phenotypes, extended spectrum beta lactamase production, antibiotic resistance determinants, genes associated with virulence and their ability to form biofilm. Resistance towards ceftazidime and tetracycline was noted among all the isolates in the study, while they exhibited sensitivity to chloramphenicol and co-trimoxazole. All the isolates were potent ESBL producers carrying at least one ESBL encoding genes. Plasmid mediated quinolone resistance gene was detected in one isolate each from onion and chicken respectively. The isolates marked the absence of tetracycline and chloramphenicol resistance genes. Multiple virulence genes (ureA, khe, fimH, mrkD, wabG, uge and elt) were possessed by each of the isolates. K. pneumoniae from chicken and cucumber were moderate biofilm formers and those from tomato exhibited weak biofilm formation. Increased expression of the mrkA gene and reduction in the expression of the biofilm forming gene fimH gene was observed among the biofilm formers. One of the moderate and non-biofilm formers exhibited increased mrkD gene expression. The results from our study stipulate, that raw vegetables and meat serve as dormant source of drug-resistant and virulent K. pneumoniae.

7,8-Dihydroxyflavone attenuates the virulence of Staphylococcus aureus by inhibiting alpha-hemolysin.

Staphylococcus aureus (S. aureus), a Gram-positive bacteria, is an incurable cause of hospital and community-acquired infections. Inhibition bacterial virulence is a viable strategy against S. aureus infections based on the multiple virulence factors secreted by S. aureus. Alpha-hemolysin (Hla) plays a crucial role in bacteria virulence without affecting bacterial viability. Here, we identified that 7,8-Dihydroxyflavone (7,8-DHF), a natural compound, was able to decrease the expression of and did not affect the in vitro growth of S. aureus USA300 at a concentration of 32μg/mL. It was verified by western blot and RT-qPCR that the natural compound could inhibit the transcription and translation of Hla. Further mechanism studies revealed that 7,8-DHF has a negative effect on transcriptional regulator agrA and RNAIII, preventing the upregulation of virulence gene. Cytotoxicity assays showed that 7,8-DHF did not produce significant cytotoxicity to A549 cells. Animal experiments showed that the combination of 7,8-DHF and vancomycin had a more significant therapeutic effect on S. aureus infection, reflecting the synergistic effect of 7,8-DHF with antibiotics. In conclusion, 7,8-DHF was able to target Hla to protect host cells from hemolysis while limiting the development of bacterial resistance.

Loss of rpoE Encoding the δ-Factor of RNA Polymerase Impacts Pathophysiology of the Streptococcus pyogenes M1T1 Strain 5448.

Streptococcus pyogenes, also known as the Group A Streptococcus (GAS), is a Gram-positive bacterial pathogen of major clinical significance. Despite remaining relatively susceptible to conventional antimicrobial therapeutics, GAS still causes millions of infections and hundreds of thousands of deaths each year worldwide. Thus, a need for prophylactic and therapeutic interventions for GAS is in great demand. In this study, we investigated the importance of the gene encoding the delta (δ) subunit of the GAS RNA polymerase, rpoE, for its impact on virulence during skin and soft-tissue infection. A defined 5448 mutant with an insertionally-inactivated rpoE gene was defective for survival in whole human blood and was attenuated for both disseminated lethality and lesion size upon mono-culture infection in mouse soft tissue. Furthermore, the mutant had reduced competitive fitness when co-infected with wild type (WT) 5448 in the mouse model. We were unable to attribute this attenuation to any observable growth defect, although colony size and the ability to grow at higher temperatures were both affected when grown with nutrient-rich THY media. RNA-seq of GAS grown in THY to late log phase found that mutation of rpoE significantly impacted (>2-fold) the expression of 429 total genes (205 upregulated, 224 downregulated), including multiple virulence and “housekeeping” genes. The arc operon encoding the arginine deiminase (ADI) pathway was the most upregulated in the rpoE mutant and this could be confirmed phenotypically. Taken together, these findings demonstrate that the delta (δ) subunit of RNA polymerase is vital in GAS gene expression and virulence.

Quorum Sensing Regulation of a Major Facilitator Superfamily Transporter Affects Multiple Streptococcal Virulence Factors.

Cell-cell signaling mediated by Rgg-family transcription factors and their cognate pheromones is conserved in Firmicutes, including all streptococci. In Streptococcus pyogenes, or group A strep (GAS), one of these systems, the Rgg2/3 quorum sensing (QS) system, has been shown to regulate phenotypes, including cellular aggregation and biofilm formation, lysozyme resistance, and macrophage immunosuppression. Here, we show the abundance of several secreted virulence factors (streptolysin O, SpyCEP, and M protein) decreases upon induction of QS. The main mechanism underlying the changes in protein levels appears to be transcriptional, occurs downstream of the QS circuit, and is dysregulated by the deletion of an Rgg2/3 QS-regulated major facilitator superfamily (MFS) transporter. Additionally, we identify this MFS transporter as the factor responsible for a previously observed increase in aminoglycoside sensitivity in QS-induced cells. IMPORTANCE The production of virulence factors is a tightly regulated process in bacterial pathogens. Efforts to elucidate the mechanisms by which genes are regulated may advance the understanding of factors influencing pathogen behavior or cellular physiology. This work finds expression of a major facilitator superfamily (MFS) transporter, which is governed by a quorum sensing (QS) system, impacts the expression of multiple virulence factors and accounts for QS-dependent antibiotic susceptibility. Although the mechanism underlying this effect is not clear, MFS orthologs with high sequence similarity from S. pneumoniae and S. porcinus were unable to substitute indicating substrate specificity of the GAS MFS gene. These findings demonstrate novel associations between expression of a transmembrane transporter and virulence factor expression and aminoglycoside transport.

Genetic characterization of hypervirulent Klebsiella pneumoniae responsible for acute death in captive marmosets.

Klebsiella pneumoniae is a Gram-negative bacterium implicated as the causative pathogen in several medical health issues with different strains causing different pathologies including pneumonia, bloodstream infections, meningitis and infections from wounds or surgery. In this study, four captive African marmosets housed in Thailand were found dead. Necropsy and histology revealed congestion of hearts, kidneys and adrenal glands. Twenty-four bacterial isolates were obtained from these four animals with all isolates yielding identical phenotypes indicative of K. pneumoniae based on classical identification schema. All the isolates show the susceptibility to amikacin, cephalexin, doxycycline, gentamicin, and enrofloxacin with intermediate susceptibility to amoxycillin/clavulanic acid. One isolate (20P167W) was chosen for genome analysis and determined to belong to sequence type 65 (ST65). The genome of 20P167W possessed multiple virulence genes including mrk gene cluster and iro and iuc gene cluster (salmochelin and aerobactin, respectively) as well as multiple antibiotic resistance genes including blaSHV−67, blaSHV−11, oqxA, oqxB, and fosA genes resembling those found in human isolates; this isolate has a close genetic relationship with isolates from humans in Ireland, but not from Thailand and California sea lions. Phylogenetic studies using SNP show that there was no relation between genetic and geographic distributions of all known strains typing ST65, suggesting that ST65 strains may spread worldwide through multiple international transmission events rather than by local expansions in humans and/or animals. We also predict that K. pneumoniae ST65 has an ability to acquire genetic mobile element from other bacteria, which would allow Klebsiella to become an even greater public health concern.

Protein interactome mapping of Porphyromonas gingivalis provides insights into the formation of the PorQ-Z complex of the type IX secretion system.

Porphyromonas gingivalis is an anaerobic Gram-negative human oral pathogen highly associated with the more severe forms of periodontal disease. Porphyromonas gingivalis utilises the type IX secretion system (T9SS) to transport ∼30 cargo proteins, including multiple virulence factors, to the cell surface. The T9SS is a multiprotein system consisting of at least 20 proteins, and recently, we characterised the protein interactome of these components. Similar to the T9SS, almost all biological processes are mediated through protein-protein interactions (PPIs). Therefore, mapping PPIs is important to understand the biological functions of many proteins in P. gingivalis. Herein, we provide native migration profiles of over 1000 P. gingivalis proteins. Using the T9SS, we demonstrate that our dataset is a useful resource for identifying novel protein interactions. Using this dataset and further analysis of T9SS P. gingivalis mutants, we discover new mechanistic insights into the formation of the PorQ-Z complex of the T9SS. This dataset is a valuable resource for studies of P. gingivalis.

Genetic Characterization of Enterobacter hormaechei Co-Harboring bla NDM-1 and mcr-9 Causing Upper Respiratory Tract Infection.

PurposeWith the spread of multiple drug-resistant bacteria, blaNDM-1 and mcr-9 have been detected in various bacteria worldwide. However, the simultaneous detection of blaNDM-1 and mcr-9 in Enterobacter hormaechei has been rarely reported. This study identified an E. hormaechei strain carrying both blaNDM-1 and mcr-9. We investigated the genetic characteristics of these two resistance genes in detail, elucidating various potential mechanisms by which they may be transmitted.MethodsBacterial genomic features and possible origins were assessed by whole-genome sequencing (WGS) with Illumina and PacBio platforms and phylogenetic analysis. Subsequent investigations were performed, including antimicrobial susceptibility testing and multilocus sequence typing (MLST).ResultsWe isolated an E. hormaechei strain DY1901 carrying both blaNDM-1 and mcr-9 from the sputum sample. Susceptibility testing showed that the isolate was multidrug-resistant. Multiple antibiotic resistance genes and virulence genes are widely distributed in DY1901. S1-PFGE, Southern blotting, and plasmid replicon typing showed that DY1901 carried four plasmids. The plasmid carrying mcr-9 was 259Kb in size and belonged to IncHI2, while the plasmid carrying blaNDM-1 was 45Kb in length and belonged to IncX3.ConclusionThe E. hormaechei strain isolated in this study has a broad antibiotic resistance spectrum, posing a challenge to clinical treatment. Plasmids carrying mcr-9 are fusion plasmids, and those taking NDM are widely disseminated in China, suggesting that we should conduct routine genomic surveillance on such plasmids to curb the spread of drug-resistant bacteria in the region.

Pathological and mycological characterization of pulmonary Aspergillus fumigatus infection producing gliotoxin in a captive African grey parrot (Psittacus erithacus)

Aspergillosis, the main causative agent of which is Aspergillus fumigatus, causes mortality in all types of birds. Gliotoxin (GT), one of the multiple virulence factors of A. fumigatus, has a variety of immunosuppressive effects. The corpse of an African grey parrot (Psittacus erithacus) was sent for necropsy and diagnostic rule-out. The lungs were enlarged, firm, and had dark-red coloration, on the parietal faces of both lungs, some semi-circular caseous necrosis areas were observed. The caudal thoracic and abdominal air sacs were thickened and contained a fibrin-heterophilic exudate. Histopathologically, a necrotic and granulomatous bronchopneumonia was observed with intralesional hyphae with characteristics compatible with Aspergillus sp. that were positive with Grocott´s staining. Fibrinous and heterophilic airsacculitis was found in the air sacs. A. fumigatus was isolated from lungs, characterized using serial microcultures, and confirmed using polymerase chain reaction. In addition, GT production was detected in vitro from the culture filtrate in which the isolate was grown; the organic extract was analysed via thin-layer chromatography. This is the first detection of GT in a case of pulmonary aspergillosis in a parrot, which could help to understand the pathogenesis of the disease in psittacines.

Actinomycin D: a novel Pseudomonas aeruginosa quorum sensing inhibitor from the endophyte Streptomyces cyaneochromogenes RC1.

The infections caused by Pseudomonas aeruginosa are difficult to treat due to its multidrug resistance. A promising strategy for controlling P. aeruginosa infection is targeting the quorum sensing (QS) system. Actinomycin D isolated from the metabolite of endophyte Streptomyces cyaneochromogenes RC1 exhibited good anti-QS activity against P. aeruginosa PAO1. Actinomycin D (50, 100, and 200μg/mL) significantly inhibited the motility as well as reduced the production of multiple virulence factors including pyocyanin, protease, rhamnolipid, and siderophores. The images of confocal laser scanning microscopy and scanning electron microscopy revealed that the treatment of actinomycin D resulted in a looser and flatter biofilm structure. Real-time quantitative PCR analysis showed that the expression of QS-related genes lasI, rhlI, rhlR, pqsR, pslA, and pilA were downregulated dramatically. The production of QS signaling molecules N-(3-oxododecanoyl)-L-homoserine lactone and N-butanoyl-L-homoserine lactone were also decreased by actinomycin D. These findings suggest that actinomycin D, a potent in vitro anti-virulence agent, is a promising candidate to treat P. aeruginosa infection by interfering with the QS systems.

Molecular Determination of Virulence Factor Genes of Acinetobacter baumannii Isolates from Clinical Specimens

Acinetobacter baumannii is a gram negative coccobacilli present abundantly in nature, in soil and water. Acinetobacter baumannii is considered as a major cause of nosocomial infections affecting mainly ICU patients and other hospitalised patients. Both intrinsic and acquired antibiotic resistance of A. baumannii account for a significant cause of outbreaks. Significant levels of morbidity and mortality have been reported with outbreaks and common infections include ventilator associated pneumonia and bacteraemia. A.baumannii is also a common cause of bloodstream infections in the intensive care setting. Multiple virulence factors are required for the pathogenesis of infections by Gram negative bacteria including A. baumannii. Possession of specialized virulence genes enables pathogens to infect hosts efficiently. Virulence factors of A. baumannii were less identified compared to other Gram negative bacteria. Hence this study was done to identify the major virulence factor genes from the clinical isolates of multidrug resistant A. baumannii from a tertiary care hospital. A preliminary study was done to determine the prevalence of Acinetobacter infections in the region and then the isolates were subjected to determine the antibiotic sensitivity and to various molecular typing. Various clinical specimens like blood, urine, abscess, vaginal swab were analyzed and 15% of the isolates was confirmed and identified as to be resistant to carbapenems. A molecular typing was done to identify the genes conferring virulence factors. Presence of different genes like Bap, Omp A, EpsA, ptk, AdeG were screened from the isolates. : , , , ,

The integration host factor regulates multiple virulence pathways in bacterial pathogen Dickeya zeae MS2.

Dickeya zeae is an aggressive bacterial phytopathogen that infects a wide range of host plants. It has been reported that integration host factor (IHF), a nucleoid‐associated protein consisting of IHFα and IHFβ subunits, regulates gene expression by influencing nucleoid structure and DNA bending. To define the role of IHF in the pathogenesis of D. zeae MS2, we deleted either and both of the IHF subunit encoding genes ihfA and ihfB, which significantly reduced the production of cell wall‐degrading enzymes (CWDEs), an unknown novel phytotoxin and the virulence factor‐modulating (VFM) quorum‐sensing (QS) signal, cell motility, biofilm formation, and thereafter the infection ability towards both potato slices and banana seedlings. To characterize the regulatory pathways of IHF protein associated with virulence, IHF binding sites (consensus sequence 5′‐WATCAANNNNTTR‐3′) were predicted and 272 binding sites were found throughout the genome. The expression of 110 tested genes was affected by IHF. Electrophoretic mobility shift assay (EMSA) showed direct interaction of IhfA protein with the promoters of vfmE, speA, pipR, fis, slyA, prtD, hrpL, hecB, hcp, indA, hdaA, flhD, pilT, gcpJ, arcA, arcB, and lysR. This study clarified the contribution of IHF in the pathogenic process of D. zeae by controlling the production of VFM and putrescine QS signals, phytotoxin, and indigoidine, the luxR‐solo system, Fis, SlyA, and FlhD transcriptional regulators, and secretion systems from type I to type VI. Characterization of the regulatory networks of IHF in D. zeae provides a target for prevention and control of plant soft rot disease.

Phylogenetic Classification and Functional Review of Autotransporters.

Autotransporters are the core component of a molecular nano-machine that delivers cargo proteins across the outer membrane of Gram-negative bacteria. Part of the type V secretion system, this large family of proteins play a central role in controlling bacterial interactions with their environment by promoting adhesion to surfaces, biofilm formation, host colonization and invasion as well as cytotoxicity and immunomodulation. As such, autotransporters are key facilitators of fitness and pathogenesis and enable co-operation or competition with other bacteria. Recent years have witnessed a dramatic increase in the number of autotransporter sequences reported and a steady rise in functional studies, which further link these proteins to multiple virulence phenotypes. In this review we provide an overview of our current knowledge on classical autotransporter proteins, the archetype of this protein superfamily. We also carry out a phylogenetic analysis of their functional domains and present a new classification system for this exquisitely diverse group of bacterial proteins. The sixteen phylogenetic divisions identified establish sensible relationships between well characterized autotransporters and inform structural and functional predictions of uncharacterized proteins, which may guide future research aimed at addressing multiple unanswered aspects in this group of therapeutically important bacterial factors.

Distribution of Leukocidins, Exfoliative Toxins, and Selected Resistance Genes Among Methicillin-resistant and Methicillin-sensitive Staphylococcus aureus Clinical Strains in Egypt

Background: Infection with Staphylococcus aureus (S.aureus) is an increasing health problem worldwide. This pathogen has multiple virulence factors that contribute to its pathogenesis in a wide range of diseases. The present study aimed to investigate the prevalence of leukocidins, exfoliative toxins, and common antimicrobial resistance genes among Methicillin-Resistant Staphylococcus aureus (MRSA) and Methicillin-Sensitive Staphylococcus aureus (MSSA) strains collected from various clinical sources in Egypt. Methods: Isolates were identified as S.aureus by the standard microbiological methods. Methicillin resistance was detected phenotypically by cefoxitin disc diffusion method and genotypically by PCR for detection of mecA gene. PCR was also used to detect the presence of leukocidin genes (LukD, LukE, LukF-PV, and LukS-PV), exfoliative toxin genes (eta and etb), and antibiotic resistance genes (tetK, tetM, ermA, ermC, msrA, and aacA-aphD). Results: About 50.5% of tested isolates were methicillin resistant by cefoxitin disc assay, while mecA gene was amplified in 64.6% of isolates. The highest prevalent toxin gene was lukE (93%) and the least prevalent one was eta (1%). The resistance genes tetK and tetM were detected in nearly 50% of the tested strains but lower prevalence rates were recorded for aacA-aphD, msrA, ermA, and ermC genes. Conclusion: Methicillin resistance was highly prevalent among tested S.aureus strains. Regarding the studied virulence and resistance genes, no significant difference was detected between MRSA and MSSA strains, except for ermA gene p<0.05 which was highly prevalent in MRSA strains. So, the variation between MRSA and MSSA strains in the response to treatment may be attributed to the resistance of MRSA strains to all β-lactams in addition to other possible acquired resistance mechanisms. Accordingly, fewer options of antimicrobial medications are available to treat MRSA infections.

Subinhibitory Concentrations of Antibiotics Exacerbate Staphylococcal Infection by Inducing Bacterial Virulence.

ABSTRACTAntibiotics are widely used for the treatment of bacterial infections. However, injudicious use of antibiotics based on an empirical method may lead to the emergence of resistant strains. Despite appropriate administration of antibiotics, their concentrations may remain subinhibitory in the body, due to individual variations in tissue distribution and metabolism rates. This may promote bacterial virulence and complicate the treatment strategies. To investigate whether the administration of certain classes of antibiotics will induce bacterial virulence and worsen the infection under in vivo conditions. Different classes of antibiotics were tested in vitro for their ability to induce virulence in a methicillin-resistant S. aureus strain Mu3 and clinical isolates. Antibiotic-induced pathogenicity was assessed in vivo using mouse peritonitis and bacteremia models. In vitro, β-lactam antibiotics and tetracyclines induced the expression of multiple surface-associated virulence factors as well as the secretion of toxins. In peritonitis and bacteremia models, mice infected with MRSA and treated with ampicillin, ceftazidime, or tetracycline showed enhanced bacterial pathogenicity. The release of induced virulence factors in vivo was confirmed in a histological examination. Subinhibitory concentrations of antibiotics belonging to β-lactam and tetracycline aggravated infection by inducing staphylococcal virulence in vivo. Thus, when antibiotics are required, it is preferable to employ combination therapy and to initiate the appropriate treatment plan, following diagnosis. Our findings emphasize the risks associated with antibiotic-based therapy and underline the need for alternative therapeutic options.IMPORTANCE Antibiotics are widely applied to treat infectious diseases. Empirically treatment with incorrect antibiotics, or even correct antibiotics always falls into subinhibitory concentrations, due to dosing, distribution, or secretion. In this study, we have systematically evaluated in vitro virulence induction effect of antibiotics and in vivo exacerbated infection. The major highlight of this work is to prove the β-lactam and tetracyclines antibiotics exacerbated disease is due to their induction effect on staphylococcal virulence. This phenomenon is common and suggests that if β-lactam antibiotics remain the first line of defense during empirical therapy, we either need to increase patient reliability or the treatment approach may improve in the future when paired with anti-virulence drugs.