Inhibition of the lectin pathway of complement activation reduces LPS-induced acute respiratory distress syndrome in mice.

Acute respiratory distress syndrome (ARDS) is a life-threatening disorder with a high rate of mortality. Complement activation in ARDS initiates a robust inflammatory reaction that can cause progressive endothelial injury in the lung. Here, we tested whether inhibition of the lectin pathway of complement could reduce the pathology and improve the outcomes in a murine model of LPS-induced lung injury that closely mimics ARDS in human. In vitro, LPS binds to murine and human collectin 11, human MBL and murine MBL-A, but not to C1q, the recognition subcomponent of the classical pathway. This binding initiates deposition of the complement activation products C3b, C4b and C5b-9 on LPS via the lectin pathway. HG-4, a monoclonal antibody that targets MASP-2, a key enzyme in the lectin pathway, inhibited lectin pathway functional activity in vitro, with an IC50 of circa 10nM. Administration of HG4 (5mg/kg) in mice led to almost complete inhibition of the lectin pathway activation for 48hrs, and 50% inhibition at 60hrs post administration. Inhibition of the lectin pathway in mice prior to LPS-induced lung injury improved all pathological markers tested. HG4 reduces the protein concentration in bronchoalveolar lavage fluid (p<0.0001) and levels of myeloid peroxide (p<0.0001), LDH (p<0.0001), TNFα and IL6 (both p<0.0001). Lung injury was significantly reduced (p<0.001) and the survival time of the mice increased (p<0.01). From the previous findings we concluded that inhibition of the lectin pathway has the potential to prevent ARDS pathology.

Characterization of Integrons and Quinolone Resistance in Clinical Escherichia coli Isolates in Mansoura City, Egypt.

Escherichia coli is a common pathogen in both humans and animals. Quinolones are used to treat infections caused by Gram-negative bacteria, but resistance genes emerged. Only scarce studies investigated the association between plasmid-mediated quinolone resistance (PMQR) genes and integrons in clinical isolates of E. coli. The current study investigated the prevalence of quinolone resistance and integrons among 134 clinical E. coli isolates. Eighty (59.70%) isolates were quinolone-resistant, and 60/134 (44.77%) isolates were integron positive with the predominance of class I integrons (98.33%). There was a significant association between quinolone resistance and the presence of integrons (P < 0.0001). Isolates from Urology and Nephrology Center and Gastroenterology Hospital were significantly quinolone-resistant and integron positive (P ≤ 0.0005). Detection of PMQR genes on plasmids of integron-positive isolates showed that the active efflux pump genes oqxAB and qepA had the highest prevalence (72.22%), followed by the aminoglycoside acetyltransferase gene (aac(6')-Ib-cr, 66.67%) and the quinolone resistance genes (qnr, 61.11%). Amplification and sequencing of integrons' variable regions illustrated that no quinolone resistance genes were detected, and the most predominant gene cassettes were for trimethoprim and aminoglycoside resistance including dfrA17, dfrB4, and dfrA17-aadA5. In conclusion, this study reported the high prevalence of PMQR genes and integrons among clinical E. coli isolates. Although PMQR genes are not cassette-born, they were associated with integrons' presence, which contributes to the widespread of quinolone resistance in Egypt.

Comparative Assessment of Different PCR-Based Typing Methods of Pseudomonas aeruginosa Isolates.

INTRODUCTION: Pseudomonas aeruginosa is one of the important causes of nosocomial infections. Analyzing the diversity of these isolates is important to control the diseases caused by them. Studies of molecular epidemiology depend on the application of typing methods. PURPOSE: This study aims to assess the performance of PCR- based typing techniques (RAPD, ribotyping, tDNA, and ERIC) in determining the genetic diversity of 44 P. aeruginosa urinary isolates. METHODS: Performance parameters were analyzed for each of the tested methods. The banding pattern was assessed by calculating polymorphism, genotypic gene diversity and the effective multiplex ratio. Moreover, strain diversity, typeability, and discriminatory power were used to measure the efficiency of typing methods. The congruence among typing methods was calculated by Rand's and Wallace coefficients. RESULTS: P-640 among RAPD primers and Ribo-2 among ribotyping primers were more informative as they gave high strain diversity, the highest number of clusters, and highest discriminatory power (ISD=70.45%, 29 clusters at 70% cutoff, DI=0.97 and ISD=75%, 25 clusters at 70% cutoff DI=0.969, respectively). Comparison of typing methods showed that RAPD-PCR gave the highest mean percent polymorphism per assay (76.85%) followed by ERIC-PCR. ERIC-PCR outperformed in most marker parameters; highest mean number of alleles, number of monomorphic bands per assay unit, mean genotypic gene diversity, effective multiplex ratio, and assay efficiency index. Calculated congruence revealed that individual methods demonstrate moderate to poor predictive power. Interestingly, this power increased by combining data obtained from another method. CONCLUSION: RAPD primer (P-640) had more discrimination power followed by ribo-2 and ERIC. The performance and predictive power of typing methods can be improved by combining data obtained from different methods as ERIC+OPA-02 and ERIC+P-640 combinations gave complete typeability and discrimination of isolates. ERIC, ERIC+OPA-02, and ERIC+P-640 combinations can provide finer discrimination and classification of P. aeruginosa strains than the other tested methods.

Effect of Sub-Minimum Inhibitory Concentrations of Tyrosol and EDTA on Quorum Sensing and Virulence of Pseudomonas aeruginosa.

INTRODUCTION: Pseudomonas aeruginosa is considered a dangerous pathogen, as it causes many human diseases, besides that it is resistant to almost all types of antibacterial agents. So, new strategies to overcome P. aeruginosa infection have evolved to attenuate its virulence factors and inhibit its quorum-sensing (QS) activity. PURPOSE: This study investigated the effect of tyrosol and EDTA as anti-quorum-sensing and antivirulence agents against P. aeruginosa PAO1. METHODS: Anti-quorum activity of sub-minimum inhibitory concentrations (sub-MICs) of tyrosol and EDTA was tested using Chromobacterium violaceum (CV 12,472) biosensor bioassay. Miller assay was used to assess the inhibition of QS signal molecules by ß-galactosidase activity determination. Also, their effects on the production of protease, lipase, lecithinase, and motility were tested. The inhibitory effects of these molecules on QS regulatory genes and exotoxins genes expression were evaluated by real-time PCR. RESULTS: Tyrosol and EDTA at sub-MICs inhibited the production of violacein pigment. Both compounds inhibited QS molecules production and their associated virulence factors (protease, lipase, lecithinase, and motility) (P≤ 0.05). Besides, the expression levels of QS regulatory genes (lasI, lasR, rhƖI, rhIR, pqsA, and pqsR) and exotoxins genes (exoS and exoY) were significantly reduced (P≤ 0.05). CONCLUSION: Both tyrosol and EDTA can be used to fight P. aeruginosa infection as anti-quorum-sensing and antivirulence agents at their sub-MICs.

Virulence characteristics and molecular relatedness of methicillin resistant Staphylococcus aureus harboring different staphylococcal cassette chromosome mec.

INTRODUCTION: Methicillin resistant Staphylococcus aureus (MRSA) is a versatile pathogen capable of causing multitude of human diseases. It is one of the most important nosocomial pathogen that implicated in community and healthcare associated infections. Therefore, this study aims to characterize different SCCmec elements found in MRSA isolates. Moreover, molecular typing was performed to investigate the genetic relatedness among MRSA isolates. METHODS: Phenotypic identification of MRSA was done by disc diffusion method. The MRSA isolates were typed based on the SCCmec, coa and agr genes. Phenotypic characterization included the detection of biofilm, lipase, protease, lecithinase, staphylokinase and hemagglutination. Also, hla, hlb, hlg, hld, tsst-1, psm-mec and mecI genes were detected genotypically. The correlation between the molecular types identified and the profile of virulence factors, clinical and geographical sources was determined for all isolates. RESULTS: Eighty five isolates were identified as MRSA. Eight types of SCCmec elements were detected among these isolates. Type V was the most observed type (56.47%). Regarding the correlation between SCCmec types and virulence factors, type V SCCmec exhibited a significant association with biofilm (p < 0.0001), staphylokinase (p = 0.0495) and tsst-1 (p = 0.0498). Molecular typing of coa gave an insight to the presence of specific types in specific hospital wards. Based on agr typing, agr I was the highest prevalent type in MRSA isolates (54.11%). CONCLUSION: There is an increase of MRSA infections particularly the community acquired with high variability in the distribution of virulence factors among different SCCmec types. The association between type III and V SCCmec with certain hospitals may be an evidence of nosocomial infection among these hospitals.

Emergence of class 1 to 3 integrons among members of Enterobacteriaceae in Egypt.

In spite of the role of integrons as the main contributor to multidrug resistance worldwide, their prevalence in Egypt is still underestimated. In this work, we announce the emergence of class 2 and 3 integrons among Enterobacteriacae isolates from Mansoura University Hospitals. Ninety-three clinical isolates were obtained from different clinical sources, among which 70% of E. coli, 94.8% of K. pneumoniae and 85.7% of Enterobacter spp. were assigned to be multidrug resistant (MDR). Subsequently, the occurrence of class 1-3 integrons was confirmed by multiplex PCR. Class 1 integron was the most predominant being harbored by 42.8%, 90% and 25% of MDR E. coli, K. pneumoniae and Enterobacter spp. isolates, respectively. This was followed by class 2 and 3 integrons which were, for the first time, reported in these hospitals. Also, coexistence of integrons 1and 2 was revealed in 36.9% of integron positive isolates. A significant association was noticed only between resistance to gentamicin and integron prevalence among MDR E. coli isolates (P = 0.02). In conclusion, this work represents the first report for detection of class 2 and 3 integrons, beside the previously detected class 1 integrons. This highlights the high incidence of integrons among MDR Enterobacteriacae isolates which indicates the selective pressure of antibiotics in these hospitals. Moreover, this study confirms the possibility of the use of integrons as markers for MDR identification.