Molecular characterization of staphylococcal cassette chromosome mec and virulence encoding genes in methicillin-resistant staphylococci at a medical center in Lebanon

Background: Methicillin-resistant staphylococci (MRS) are major human pathogens accounting for most hospital-acquired (HA) and community acquired (CA) infections worldwide. The recent increase in MRS in a medical center in Lebanon elicited the determination of SCCmec types, genotypes, and prevalence of Panton-Valentine leucociden (PVL) and toxic shock syndrome toxin-1 (TSST-1) among the MRS isolates.   Methods: Thirty-six MRS isolates collected between October 2010 and September 2011 at a medical center, Lebanon were typed using phenotypic and genotypic methods. Antimicrobial susceptibility was determined using the disk diffusion agar method. SCCmec typing was performed by multiplex PCR and sequence analysis. The prevalence of the genes encoding PVL and TSST-1 virulence factors and their transcription levels, were determined respectively by PCR and semi-quantitative real-time PCR. The genomic relatedness of the isolates was assessed by random amplified polymorphic DNA (RAPD) analysis.Results: Antimicrobial susceptibility revealed three distinct antibiotypes. The predominant SCCmec type found among the MRS isolates was type IVa (51%). Twenty-nine percent harbored SCCmec type III and 14% harbored SCCmec type II. One isolate harbored SCCmec type IVc, and another  harbored SCCmec type I. All methicillin-resistant Staphylococcus aureus (MRSA) isolates were negative for the gene encoding for PVL, and two were positive for the gene encoding for TSST-1. RAPD analysis demonstrated high genomic diversity among the MRS isolates.Conclusion: This study demonstrated the SCCmec types and the clonality of the MRS strains, allowing the differentiation between HA and CA-MRS strains. CA-MRS have  increased  in the hospital environment and rendered highly resistant to erythromycin and clindamycin

Comparative analysis of amplicon and metagenomic sequencing methods reveals key features in the evolution of animal metaorganisms

Background The interplay between hosts and their associated microbiome is now recognized as a fundamental basis of the ecology, evolution, and development of both players. These interdependencies inspired a new view of multicellular organisms as “metaorganisms.” The goal of the Collaborative Research Center “Origin and Function of Metaorganisms” is to understand why and how microbial communities form long-term associations with hosts from diverse taxonomic groups, ranging from sponges to humans in addition to plants. Methods In order to optimize the choice of analysis procedures, which may differ according to the host organism and question at hand, we systematically compared the two main technical approaches for profiling microbial communities, 16S rRNA gene amplicon and metagenomic shotgun sequencing across our panel of ten host taxa. This includes two commonly used 16S rRNA gene regions and two amplification procedures, thus totaling five different microbial profiles per host sample. Conclusion While 16S rRNA gene-based analyses are subject to much skepticism, we demonstrate that many aspects of bacterial community characterization are consistent across methods. The resulting insight facilitates the selection of appropriate methods across a wide range of host taxa. Overall, we recommend single- over multi-step amplification procedures, and although exceptions and trade-offs exist, the V3 V4 over the V1 V2 region of the 16S rRNA gene. Finally, by contrasting taxonomic and functional profiles and performing phylogenetic analysis, we provide important and novel insight into broad evolutionary patterns among metaorganisms, whereby the transition of animals from an aquatic to a terrestrial habitat marks a major event in the evolution of host-associated microbial composition

Exploring mechanisms of C. elegans microbiota-mediated protection

Animals and microorganisms live together, forming a network of associations between animal hosts and their resident microorganisms, the microbiota. One important beneficial microbiota function is protecting the host against pathogen infection, termed microbiota-mediated protection. Many studies describe microbiota-mediated protection in animals; however, the underlying mechanisms remain mostly elusive. In my Ph.D. thesis project, I investigated mechanisms of microbiota-mediated protection in the nematode model Caenorhabditis elegans. To understand microbiota-mediated protection mechanisms, I first identified C. elegans natural microbiota isolates that protect the host against infection with Bacillus thuringiensis. Then, I explored the underlying mechanisms of protection by these microbiota isolates both from the bacterial and the host side. On the bacterial side, I showed that: 1) Some microbiota isolates can protect the host by directly inhibiting pathogen growth via massetolide E production, a lipopeptide of the viscosin group, while others protect the host without producing massetolide E. 2) Microbiota isolates can produce biofilm in vitro, suggesting biofilm formation as a potential protection mechanism, which merits in vivo investigation. 3) Microbiota isolates cannot protect against B. thuringiensis toxins (Cry21Aa3 and Cry14Aa2) and are rendered pathogenic under certain pathogen exposure conditions, revealing the context-dependent dual nature of microbiota protective function. Moreover, in collaboration with colleagues, we characterized a more extensive selection of natural microbiota isolates of C. elegans and elucidated their metabolic contribution to the host using whole-genome sequencing, mathematical modeling, and experimentation. We showed that C. elegans microbiota could synthesize all the essential vitamins and amino acids needed by the nematode, reinforcing its beneficial role to the host. On the host side, I used a multi-omics approach to identify host genes and pathways required for microbiota-mediated protection. To this end, I obtained transcriptomic and proteomic datasets of the infected and uninfected worms treated or untreated with the protective microbiota isolates. I showed that: 1) Protective microbiota isolates influence the worm's innate immune response and cellular structural component functions. 2) Microbiota isolates induce changes in expression and abundances on both gene and protein levels, respectively, for galectins, C-type lectins, and lysozymes, suggesting their potential roles in microbiota-mediated host protection. Altogether, these findings indicate microbiota-mediated activation of innate immune response genes, suggesting that the microbiota isolates ‘prime’ the C. elegans immune response, increasing host preparedness for subsequent pathogen attack. Overall, the findings of this thesis provide valuable insights into mechanisms of microbiota-mediated protection on the bacterial and host sides, expanding our understanding of host-microbe interactions

Prevalence of carbapenem resistance encoding genes and corresponding MIC 90 in enterobacteriaceae at a tertiary care center in Lebanon

Background: The aim of this study was to correlate genes involved in carbapenem resistance to MIC levels among clinical ESBL and non-ESBL producing carbapenem resistant Enterobacteriaceae (CRE) isolates of Escherichia coli and Klebsiella pneumoniae. Materials and Methods: E. coli (n= 76) and K. pneumoniae (n=54), collected between July 2008 and July 2014, were analyzed. The MICs were determined against ertapenem (ERT), imipenem (IMP) and meropenem (MER). PCR was performed on all 130 isolates to amplify the resistance and outer membrane proteins (OMPs) encoding genes: bla OXA-48, blaNDM-1,blaTEM-1,blaCTX-M-15, ompCand ompF.Sequencing was performed on selected isolates.  Results: The prevalence of bla OXA-48, blaNDM-1,  blaTEM-1 and/or blaCTX-M-15 among E. coli isolates were 36%, 12%, 20% and 80%, respectively, while among K. pneumoniae they were 37%, 28%, 28% and 72%, respectively. K. pneumoniae isolates positive for any of these genes had an MIC90> 32µg/ml  against ERT, IMP and MER, while in E. coli isolates there was a variation in the MIC90 values. Porin impermeabilitieswere due to mutations in ompCand ompF genes in E. coli, and loss of ompCand ompF genes in K. pneumoniae,andincreased MIC90 values. The presence of more than one carbapenem resistance encoding gene and/ or ESBL encoding genedid not have an effect on the MIC90 value in K. pneumoniae isolates, while in E. coliit showed higher MIC90 values. Conclusion: Levels of MIC in CRE may largely depend on the type of resistance encoding genes, and porin impermeabilities. These resultsmay provide information for antibiotic regimen selection and epidemiological monitoring of resistance

Sites of colonization in hospitalized patients with infections caused by extended-spectrum beta-lactamase organisms: a prospective cohort study

Abstract Background The objective of this study was to determine whether patients infected with extended-spectrum beta-lactamase (ESBL)-producing organisms are colonized at multiple body sites. Methods This was a prospective cohort study at a tertiary care center in Beirut, Lebanon. Hospitalized patients with infections caused by ESBL-producing organisms were included. Cultures were obtained from the primary site of infection as well as from other sites (skin, nasopharynx, urine, rectum). Molecular analysis was performed on isolates to determine clonal relatedness. Results One hundred patients were included in the study. Only 22 patients had positive cultures from sites other than the primary site of infection. The most common ESBL gene was CTX-M-15 followed by TEM-1. In 11 of 22 patients, isolates collected from the same patient were 100% genetically related, while in the remaining patients, genomic relatedness ranged from 42.9% to 97.1%. Conclusions Colonization at sites other than the primary site of infection was not common among our patient population infected with ESBL-producing organisms. The dynamics of transmission of these bacterial strains should be studied in further prospective studies to determine the value of routine active surveillance and the need for expanded precautions in infected and colonized patients

Assessment of Combination Therapy in BALB/c Mice Injected With Carbapenem-Resistant Enterobacteriaceae Strains

Monotherapeutic options for carbapenem resistant infections are limited. Studies suggest that combination therapy may be associated with better outcomes than monotherapies. However, this is still controversial. This study assessed, the efficacy of combination therapy against carbapenem resistant Enterobacteriaceae harboring singly various ESBL or carbapenemase encoding genes. Thus, four isolates harboring either blaCTXM-15, blaCTXM-15 and blaOXA-48, blaNDM-1, or blaKPC-2 genes were selected for testing. Minimal Inhibitory Concentration (MIC) was determined by broth dilution method. Gene transcript levels on single and combined treatments were done in vitro and in vivo by q RT-PCR. Assessment of treatments was done in BALB/c mice according to a specific protocol. As such, the qRT-PCR revealed a significant decrease of transcript levels in all isolates upon using rifampicin or tigecycline, singly or in combination with colistin. However, variable levels were obtained using colistin singly or in combination with meropenem or fosfomycin. In vivo assessment showed that all combinations used were effective against isolates harboring blaCTXM-15, blaOXA-48, and blaNDM-1. Conversely, the most significant combination against the isolate harboring blaKPC-2 gene was colistin with carbapenem, fosfomycin, or kanamycin. As a conclusion, combination therapy selected based on the type of carbapenemase produced, appeared to be non-toxic and might be effective in BALB/c mice. Therefore, the use of a rationally optimized combination therapy might lead to better results than monotherapy, however, clinical trials are needed for human consumption