The Moraxella catarrhalis AdhC-FghA system is important for formaldehyde detoxification and protection against pulmonary clearance.

Multidrug-resistant clinical isolates of Moraxella catarrhalis have emerged, increasing the demand for the identification of new treatment and prevention strategies. A thorough understanding of how M. catarrhalis can establish an infection and respond to different stressors encountered in the host is crucial for new drug-target identification. Formaldehyde is a highly cytotoxic compound that can be produced endogenously as a by-product of metabolism and exogenously from environmental sources. Pathways responsible for formaldehyde detoxification are thus essential and are found in all domains of life. The current work investigated the role of the system consisting of the S-hydroxymethyl alcohol dehydrogenase (AdhC), a Zn-dependent class III alcohol dehydrogenase, and the S-formyl glutathione hydrolase (FghA) in the formaldehyde detoxification process in M. catarrhalis. Bioinformatics showed that the components of the system are conserved across the species and are highly similar to those of Streptococcus pneumoniae, which share the same biological niche. Isogenic mutants were constructed to study the function of the system in M. catarrhalis. A single fghA knockout mutant did not confer sensitivity to formaldehyde, while the adhC-fghA double mutant is formaldehyde-sensitive. In addition, both mutants were significantly cleared in a murine pulmonary model of infection as compared to the wild type, demonstrating the system's importance for this pathogen's virulence. The respective phenotypes were reversed upon the genetic complementation of the mutants. To date, this is the first study investigating the role of the AdhC-FghA system in formaldehyde detoxification and pathogenesis of M. catarrhalis.

Dihydrophenazine: a multifunctional new weapon that kills multidrug-resistant Acinetobacter baumannii and restores carbapenem and oxidative stress susceptibilities.

AIMS: The current work aims to fully characterize a new antimicrobial agent against Acinetobacter baumannii, which continues to represent a growing threat to healthcare settings worldwide. With minimal treatment options due to the extensive spread of resistance to almost all the available antimicrobials, the hunt for new antimicrobial agents is a high priority. METHODS AND RESULTS: An Egyptian soil-derived bacterium strain NHM-077B proved to be a promising source for a new antimicrobial agent. Bio-guided fractionation of the culture supernatants of NHM-077B followed by chemical structure elucidation identified the active antimicrobial agent as 1-hydroxy phenazine. Chemical synthesis yielded more derivatives, including dihydrophenazine (DHP), which proved to be the most potent against A. baumannii, yet it exhibited a marginally safe cytotoxicity profile against human skin fibroblasts. Proteomics analysis of the cells treated with DHP revealed multiple proteins with altered expression that could be correlated to the observed phenotypes and potential mechanism of the antimicrobial action of DHP. DHP is a multipronged agent that affects membrane integrity, increases susceptibility to oxidative stress, interferes with amino acids/protein synthesis, and modulates virulence-related proteins. Interestingly, DHP in subinhibitory concentrations re-sensitizes the highly virulent carbapenem-resistant A. baumannii strain AB5075 to carbapenems providing great hope in regaining some of the benefits of this important class of antibiotics. CONCLUSIONS: This work underscores the potential of DHP as a promising new agent with multifunctional roles as both a classical and nonconventional antimicrobial agent that is urgently needed.

γ-Glutamyltransferase as a Novel Virulence Factor of Acinetobacter baumannii Inducing Alveolar Wall Destruction and Renal Damage in Systemic Disease.

A thorough understanding of Acinetobacter baumannii pathogenicity is the key to identifying novel drug targets. In the current study, we characterize the γ-glutamyltransferase enzyme (GGT) as a novel virulence factor. A GGT assay showed that the enzyme is secreted via the type II secretion system and results in higher extracellular activity for the hypervirulent AB5075 than the laboratory-adapted strain American Type Culture Collection 17978. Enzyme-linked immunosorbent assay revealed that the former secretes larger amounts of GGT, and a rifampicin messenger RNA stability study showed that one reason for this could be the longer AB5075 ggt transcript half-life. Infection models confirmed that GGT is required for the virulence of A. baumannii. Finally, we show that clinical isolates with significantly higher extracellular GGT activity resulted in more severe infections, and assay of immune response and tissue damage markers confirm this correlation. The current findings establish for the first time the role of the GGT in the pathogenicity of A. baumannii.

Comparative proteomics analyses of Acinetobacter baumannii strains ATCC 17978 and AB5075 reveal the differential role of type II secretion system secretomes in lung colonization and ciprofloxacin resistance.

Acinetobacter baumannii is an emerging nosocomial pathogen with alarming antibiotic resistance profiles. A better understanding of the virulence and resistance mechanisms of this pathogen is necessary for identifying new methods to combat its infections in a more efficient way. In this regard, the type II secretion system (T2SS) of A. baumannii is an attractive target majorly secreting lipid-metabolizing enzymes and contributes significantly to its virulence. No attempts have been made to study the differential role, and the nature of T2SS secreted proteins among different strains of A. baumannii. In this study, we compare T2SS substrates and functions between A. baumannii strains ATCC 17978, and the MDR highly virulent strain AB5075. The functional categories of the T2-secreted proteins were analyzed, and the virulence potential of the tested strains was compared in vivo using a murine pneumonia model. Biofilm formation was compared using crystal violet assay in micro-titer plates. The contribution to antibiotic resistance was measured by determining the minimum inhibitory concentration (MIC) of different classes of antibiotic. Results indicate that the T2SS secretome gives a colonization advantage to AB5075 over ATCC 1797 but is more important for biofilm formation by the latter. Transposon insertional inactivation of the general secretory pathway protein D (gspD), which is a key component in the structure of the T2SS, significantly increased the MIC of AB5075 to ciprofloxacin. Our report is the first to describe the strain-dependent evolution of the T2SS secretome in relation to the virulence and antibiotic resistance attributes of Gram-negative species.

Complexity of Complement Resistance Factors Expressed by Acinetobacter baumannii Needed for Survival in Human Serum.

Acinetobacter baumannii is a bacterial pathogen with increasing impact in healthcare settings, due in part to this organism's resistance to many antimicrobial agents, with pneumonia and bacteremia as the most common manifestations of disease. A significant proportion of clinically relevant A. baumannii strains are resistant to killing by normal human serum (NHS), an observation supported in this study by showing that 12 out of 15 genetically diverse strains of A. baumannii are resistant to NHS killing. To expand our understanding of the genetic basis of A. baumannii serum resistance, a transposon (Tn) sequencing (Tn-seq) approach was used to identify genes contributing to this trait. An ordered Tn library in strain AB5075 with insertions in every nonessential gene was subjected to selection in NHS. We identified 50 genes essential for the survival of A. baumannii in NHS, including already known serum resistance factors, and many novel genes not previously associated with serum resistance. This latter group included the maintenance of lipid asymmetry genetic pathway as a key determinant in protecting A. baumannii from the bactericidal activity of NHS via the alternative complement pathway. Follow-up studies validated the role of eight additional genes identified by Tn-seq in A. baumannii resistance to killing by NHS but not by normal mouse serum, highlighting the human species specificity of A. baumannii serum resistance. The identification of a large number of genes essential for serum resistance in A. baumannii indicates the degree of complexity needed for this phenotype, which might reflect a general pattern that pathogens rely on to cause serious infections.

Adaptation to Potassium-Limitation Is Essential for Acinetobacter baumannii Pneumonia Pathogenesis.

BACKGROUND: Acinetobacter baumannii is challenging the healthcare community as the cause of a wide range of untreatable infections. New targets need to be explored for the development of therapeutics. METHODS: The potassium-dependent protein (Kdp) system was investigated via bioinformatics and genetic tools. An isogenic mutant was constructed in kdpE and complemented in trans Gene expression and the ability to grow under potassium-limited conditions were investigated. Finally, the role of KdpE in virulence was examined in the murine pneumonia model. RESULTS: The A. baumannii Kdp system has a distinct arrangement and is well conserved among A. baumannii strains. The genes encoding the 5 members of the system are transcriptionally linked. kdpE is upregulated >70-fold under potassium-limited conditions. The ΔkdpE mutant showed a significant growth defect under potassium-limited conditions and in the colonization of mice lungs. These defects could be restored upon introducing kdpE on a multiple-copy plasmid. Proteomic analyses indicated that KdpE could be regulating several proteins with potential involvement in pathogenesis. CONCLUSIONS: For the first time, A. baumannii KdpE is shown to be crucial to pneumonia onset, and targeting this system can be a viable approach to treating these fatal infections.

The secretome of Acinetobacter baumannii ATCC 17978 type II secretion system reveals a novel plasmid encoded phospholipase that could be implicated in lung colonization.

Acinetobacter baumannii infections are compounded with a striking lack of treatment options. In many Gram-negative bacteria, secreted proteins play an important early role in avoiding host defences. Typically, these proteins are targeted to the external environment or into host cells using dedicated transport systems. Despite the fact that medically relevant species of Acinetobacter possess a type II secretion system (T2SS), only recently, its significance as an important pathway for delivering virulence factors has gained attention. Using in silico analysis to characterize the genetic determinants of the T2SS, which are found clustered in other organisms, in Acinetobacter species, they appear to have a unique genetic organization and are distributed throughout the genome. When compared to other T2SS orthologs, individual components of the T2SS apparatus showed the highest similarity to those of Pseudomonas aeruginosa. A mutant of Acinetobacter baumannii strain ATCC 17978 lacking the secretin component of the T2SS (ΔgspD), together with a trans-complemented mutant, were tested in a series of in vitro and in vivo assays to determine the role of T2SS in pathogenicity. The ΔgspD mutant displayed decreased lipolytic activity, associated with attenuated colonization ability in a murine pneumonia model. These phenotypes are linked to LipAN, a novel plasmid-encoded phospholipase, identified through mass spectroscopy as a T2SS substrate. Recombinant LipAN showed specific phospholipase activity in vitro. Proteomics on the T2-dependent secretome of ATCC 17978 strain revealed its potential dedication to the secretion of a number of lipolytic enzymes, among others which could contribute to its virulence. This study highlights the role of T2SS as an active contributor to the virulence of A. baumannii potentially through secretion of a newly identified phospholipase.

Association Between Combined Presence of Hepatitis C Virus and Polymorphisms in Different Genes With Toxicities of Methotrexate and 6-Mercaptopurine in Children With Acute Lymphoblastic Leukemia.

BACKGROUND: The aim of the present study is to determine the correlation of hepatitis C virus (HCV) infection and polymorphisms in different genes with toxicity of either methotrexate (MTX) or 6-mercaptopurine (6-MP) administered to children with acute lymphoblastic leukemia (ALL). PROCEDURE: One hundred children with low-risk ALL, who were treated according to the St. Jude Total therapy XV, were recruited. The recruited children were receiving MTX and 6-MP during maintenance phase. Patients were excluded from the study if they had other types of leukemia. Genotyping analyses for the thiopurine methyltransferase (TPMT), methylenetetrahydrofolate reductase (MTHFR), and glutathione S-transferase (GST) genes were performed using a combination of polymerase chain reaction (PCR) and PCR-RFLP (where RFLP is restriction fragment length polymorphism) protocols. Relevant clinical data on adverse drug reactions were collected objectively (blinded to genotypes) from the patient medical records. RESULTS: There was a significant correlation between the combined presence of HCV and TPMT*3B G460A gene polymorphisms and grades 2-4 hepatotoxicity as aspartate aminotransferase (AST) elevation (P < 0.04). The same observation was seen when comparing either the presence of HCV alone or the presence of the gene polymorphism alone. A significant association between the combined presence of HCV and MTHFR C677T polymorphism and grades 2-4 hepatotoxicity as alanine aminotransferase (ALT), AST, and alkaline phosphatase (ALP) elevation was observed (P values <0.001, 0.02, and 0.001, respectively). The presence of HCV infection had a significant negative effect on hepatic transaminases. CONCLUSIONS: The present data support a role for combining analysis of genetic variation in drug-metabolizing enzymes and the presence of HCV in the assessment of specific drugs toxicities in multiagent chemotherapeutic treatment regimens.

Acinetobacter baumannii universal stress protein A plays a pivotal role in stress response and is essential for pneumonia and sepsis pathogenesis.

Acinetobacter baumannii is one of the most significant threats to global public health. This threat is compounded by the fact that A. baumannii is rapidly becoming resistant to all relevant antimicrobials. Identifying key microbial factors through which A. baumannii resists hostile host environment is paramount to the development of novel antimicrobials targeting infections caused by this emerging pathogen. An attractive target could be a molecule that plays a role in the pathogenesis and stress response of A. baumannii. Accordingly, the universal stress protein A (UspA) was chosen to be fully investigated in this study. A platform of A. baumannii constructs, expressing various levels of the uspA gene ranging from zero to thirteen folds of wild-type level, and a recombinant E. coli strain, were employed to investigate the role of UspA in vitro stress and in vivo pathogenesis. The UspA protein plays a significant role in protecting A. baumannii from H(2)O(2), low pH, and the respiratory toxin 2,4-DNP. A. baumannii UspA protein plays an essential role in two of the deadliest types of infection caused by A. baumannii; pneumonia and sepsis. This distinguishes A. baumannii UspA from its closely related homolog, the Staphylococcus aureus Usp2, as well as from the less similar Burkholderia glumae Usps. Heterologous and overexpression experiments suggest that UspA mediates its role via an indirect mechanism. Our study highlights the role of UspA as an important contributor to the A. baumannii stress and virulence machineries, and polishes it as a plausible target for new therapeutics.

Development of an Immunochromatographic Strip Using Conjugated Gold Nanoparticles for the Rapid Detection of Klebsiella pneumoniae Causing Neonatal Sepsis.

Neonatal sepsis is a leading cause of death among newborns and infants, especially in the developing world. The problem is compounded by the delays in pinpointing the causative agent of the infection. This is reflected in increasing mortality associated with these cases and the spread of multi-drug-resistant bacteria. In this work, we deployed bioinformatics and proteomics analyses to determine a promising target that could be used for the identification of a major neonatal sepsis causative agent, Klebsiella pneumoniae. A 19 amino acid peptide from a hypothetical outer membrane was found to be very specific to the species, well conserved among its strains, surface exposed, and expressed in conditions simulating infection. Antibodies against the selected peptide were conjugated to gold nanoparticles and incorporated into an immunochromatographic strip. The developed strip was able to detect as low as 105 CFU/mL of K. pneumoniae. Regarding specificity, it showed negative results with both Escherichia coli and Enterobacter cloacae. More importantly, in a pilot study using neonatal sepsis cases blood specimens, the developed strip selectively gave positive results within 20 min with those infected with K. pneumoniae without prior sample processing. However, it gave negative results in cases infected with other bacterial species.

A Novel Surface-Exposed Polypeptide Is Successfully Employed as a Target for Developing a Prototype One-Step Immunochromatographic Strip for Specific and Sensitive Direct Detection of Staphylococcus aureus Causing Neonatal Sepsis.

Neonatal sepsis is a life-threatening condition and Staphylococcus aureus is one of its major causes. However, to date, no rapid and sensitive diagnostic tool has been developed for its direct detection. Bioinformatics analyses identified a surface-exposed 112-amino acid polypeptide of the cell wall protein NWMN_1649, a surface protein involved in cell aggregation and biofilm formation, as being a species-specific and highly conserved moiety. The polypeptide was cloned, purified, and used to immunize mice to raise specific immunoglobulins. The purified antibodies were conjugated to gold nano-particles and used to assemble an immunochromatographic strip (ICS). The developed prototype ICS detected as low as 5 µg purified polypeptide and 102 CFU/mL S. aureus within 15 min. The strip showed superior ability to directly detect S. aureus in neonatal sepsis blood specimens without prior sample processing. Moreover, it showed no cross-reaction in specimens infected with two other major causes of neonatal sepsis; coagulase-negative staphylococci and Klebsiella pneumoniae. The selected NWMN_1649-derived polypeptide demonstrates success as a promising biomolecule upon which a prototype ICS has been developed. This ICS provides a rapid, direct, sensitive, and specific option for the detection of S. aureus causing neonatal sepsis. Such a tool is urgently needed especially in resources-limited countries.

A Rapid Lysostaphin Production Approach and a Convenient Novel Lysostaphin Loaded Nano-emulgel; As a Sustainable Low-Cost Methicillin-Resistant Staphylococcus aureus Combating Platform.

Staphylococcus aureus is a Gram-positive pathogen that is capable of infecting almost every organ in the human body. Alarmingly, the rapid emergence of methicillin-resistant S.aureus strains (MRSA) jeopardizes the available treatment options. Herein, we propose sustainable, low-cost production of recombinant lysostaphin (rLST), which is a native bacteriocin destroying the staphylococcal cell wall through its endopeptidase activity. We combined the use of E. coli BL21(DE3)/pET15b, factorial design, and simple Ni-NTA affinity chromatography to optimize rLST production. The enzyme yield was up to 50 mg/L culture, surpassing reported systems. Our rLST demonstrated superlative biofilm combating ability by inhibiting staphylococcal biofilms formation and detachment of already formed biofilms, compared to vancomycin and linezolid. Furthermore, we aimed at developing a novel rLST topical formula targeting staphylococcal skin infections. The phase inversion composition (PIC) method fulfilled this aim with its simple preparatory steps and affordable components. LST nano-emulgel (LNEG) was able to extend active LST release up to 8 h and cure skin infections in a murine skin model. We are introducing a rapid, convenient rLST production platform with an outcome of pure, active rLST incorporated into an effective LNEG formula with scaling-up potential to satisfy the needs of both research and therapeutic purposes.

Development of Biphenylthiazoles Exhibiting Improved Pharmacokinetics and Potent Activity Against Intracellular Staphylococcus aureus.

Exploring the structure-activity relationship (SAR) at the cationic part of arylthiazole antibiotics revealed hydrazine as an active moiety. The main objective of the study is to overcome the inherited toxicity associated with the free hydrazine. A series of hydrocarbon bridges was inserted in between the groups, to separate the two amino groups. Hence, the aminomethylpiperidine-containing analog 16 was identified as a new promising antibacterial agent with efficient antibacterial and pharmacokinetic profiles. Briefly, compound 16 outperformed vancomycin in terms of the antibacterial spectrum against vancomycin-resistant staphylococcal and enterococcal strains with minimum inhibitory concentrations (MICs) ranging from 2 to 4 µg/mL, which is a faster bactericidal mode of action, completely eradicating the high staphylococcal burden within 6-8 h, and it has a unique ability to completely clear intracellular staphylococci. In addition, the initial pharmacokinetic assessment confirmed the high metabolic stability of compound 16 (biological half-life >4 h); it had a good extravascular distribution and maintained a plasma concentration higher than the average MIC value for over 12 h. Moreover, compound 16 significantly reduced MRSA burden in an in vivo MRSA skin infection mouse experiment. These attributes collectively suggest that compound 16 is a good therapeutic candidate for invasive staphylococcal and enterococcal infections. From a mechanistic point of view, compound 16 inhibited undecaprenyl diphosphate phosphatase (UppP) with an IC50 value of 29 µM.

Acinetobacter: an emerging pathogen with a versatile secretome.

Acinetobacter baumannii is a notorious pathogen that has emerged as a healthcare nightmare in recent years because it causes serious infections that are associated with high morbidity and mortality rates. Due to its exceptional ability to acquire resistance to almost all available antibiotics, A. baumannii is currently ranked as the first pathogen on the World Health Organization's priority list for the development of new antibiotics. The versatile range of effectors secreted by A. baumannii represents a large proportion of the virulence arsenal identified in this bacterium to date. Thus, these factors, together with the secretory machinery responsible for their extrusion into the extracellular milieu, are key targets for novel therapeutics that are greatly needed to combat this deadly pathogen. In this review, we provide a comprehensive, up-to-date overview of the organization and regulatory aspects of the Acinetobacter secretion systems, with a special emphasis on their versatile substrates that could be targeted to fight the deadly infections caused by this elusive pathogen.

Development and validation of LC-MS/MS assay for the simultaneous determination of methotrexate, 6-mercaptopurine and its active metabolite 6-thioguanine in plasma of children with acute lymphoblastic leukemia: Correlation with genetic polymorphism.

Individualized therapy is a recent approach aiming to specify dosage regimen for each patient according to its genetic state. Cancer chemotherapy requires continuous monitoring of the plasma concentration levels of active forms of cytotoxic drugs and subsequent dose adjustment. In order to attain optimum therapeutic efficacy, correlation to pharmacogenetics data is crucial. In this study, a specific, accurate and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) has been developed for determination of methotrexate (MTX), 6-mercaptopurine (MP) and its metabolite 6-thioguanine nucleotide (TG) in human plasma. Based on the basic character of the studied compounds, solid phase extraction using a strong cation exchanger was found the optimum approach to achieve good extraction recovery. Chromatographic separation was carried out using RP-HPLC and isocratic elution by acetonitrile: 0.1% aqueous formic acid (85:15v/v) with a flow rate of 0.8mL/min at 40°C. The detection was performed by tandem mass spectrometry in MRM mode via electrospray ionization source in positive ionization mode. Analysis was carried out within 1.0min over a concentration range of 6.25-200.00ng/mL for the studied analytes. Validation was carried out according to FDA guidelines for bioanalytical method validation and satisfactory results were obtained. The applicability of the assay for the monitoring of the MTX, MP and TG and subsequent application to personalized therapy was demonstrated in a clinical study on children with acute lymphoblastic leukemia (ALL). Results confirmed the need for implementation of reliable analysis tools for therapeutic dose adjustment.

The effect of α1-antitrypsin deficiency combined with increased bacterial loads on chronic obstructive pulmonary disease pharmacotherapy: A prospective, parallel, controlled pilot study.

Chronic obstructive pulmonary disease (COPD) is caused by α1-antitrypsin deficiency (AATD) genetic susceptibility and exacerbated by infection. The current pilot study aimed at studying the combined effect of AATD and bacterial loads on the efficacy of COPD conventional pharmacotherapy. Fifty-nine subjects (29 controls and 30 COPD patients) were tested for genetic AATD and respiratory function. The bacterial loads were determined to the patients' group who were then given a long acting beta-agonist and corticosteroid inhaler for 6 months. Nineteen percent of the studied group were Pi∗MZ (heterozygote deficiency variant), Pi∗S (5%) (milder deficiency variant), Pi∗ZZ (10%) (the most common deficiency variant), and Pi∗Mmalton (2%) (very rare deficiency variant). The patients' sputum contained from 0 to 8 × 108 CFU/mL pathogenic bacteria. The forced vital capacity (FVC6) values of the AAT non-deficient group significantly improved after 3 and 6 months. Patients lacking AATD and pathogenic bacteria showed significant improvement in forced expiratory volume (FEV1), FEV1/FVC6, FVC6, and 6 min walk distance (6MWD) after 6 months. However, patients with AATD and pathogenic bacteria showed only significant improvement in FEV1 and FEV1/FVC6. The findings of this pilot study highlight for the first time the role of the combined AATD and pathogenic bacterial loads on the efficacy of COPD treatment.