Tamara Salloum

Draft Genome Sequences of Acinetobacter baumannii Strains Harboring the blaNDM-1 Gene Isolated in Lebanon from Civilians Wounded during the Syrian Civil War.

ABSTRACT We present here the draft genome sequences of multidrug-resistant blaNDM-1-positive Acinetobacter baumannii strains ACMH-6200 and ACMH-6201, isolated in north Lebanon from civilians wounded during the Syrian civil war. The draft genomes were contained in 217 contigs for ACMH-6200 and 83 contigs for ACMH-6201, including a combined 3,997,237 bases for ACMH-6200 and 3,983,110 bases for ACMH-6201, with 39% and 38.9% G+C content, respectively.

Detection, molecular typing and phylogenetic analysis of Leishmania isolated from cases of leishmaniasis among Syrian refugees in Lebanon

Leishmania is a parasitic protozoan with more than two-dozen species causing the disease leishmaniasis. It is transmitted to humans through the bite of an infected female phlebotomine sand-fly vector. In the past two years the incidence of leishmaniasis has been drastically increasing in Lebanon. This was in parallel with the deterioration of the security in Syria forcing thousands to flee and seek shelter in poorly maintained refugee camps and collective shelters. Cutaneous leishmaniasis (CL) is now considered a public health problem, but its epidemiology has not been fully elucidated. To our knowledge, this is the first study comparing two different molecular methods for the detection and identification of Leishmania tropica in Lebanon. Two molecular typing methods of 39 FFPE Leishmania isolates were used: the ITS1-PCR RFLP and the nested ITS1-5.8S rDNA gene amplification followed by sequencing and phylogenetic analysis. The efficiency of these two techniques in Leishmania identification was compared and the phylogenetic relationships among these isolates were illustrated based on the neighbor-joining (NJ) method. The results were statistically correlated with the parasitic index (PI). The DNA storage in formalin-fixed paraffin embedded (FFPE) tissues was assessed as well. The parasites identified were all L. tropica as determined by both techniques. ITS1-5.8S rDNA gene based typing proved to be more sensitive in the detection of parasites (positive in 69.2% of the isolates) as opposed to the ITS1-PCR RFLP method that was successful in identifying L. tropica in only 43.6% of the isolates. Sequencing and phylogenetic analysis revealed high levels of heterogeneity. A statistically significant correlation was observed between PI and the results of the nested ITS1-5.8S rDNA gene PCR. Genotyping at the species level is essential for monitoring the relative frequency of CL in the Mediterranean area that is correlated to three different Leishmania species (Leishmania infantum, Leishmania major and L. tropica), each characterized by distinct epidemiological features. The obtained results highlight the need to find a universally accepted diagnostic tool for Leishmania typing.

16S–23S rRNA Gene Intergenic Spacer Region Variability Helps Resolve Closely Related Sphingomonads

Sphingomonads comprise a physiologically versatile group many of which appear to be adapted to oligotrophic environments, but several also had features in their genomes indicative of host associations. In this study, the extent variability of the 16S–23S rDNA intergenic spacer (ITS) sequences of 14 ATCC reference sphingomonad strains and 23 isolates recovered from drinking water was investigated through PCR amplification and sequencing. Sequencing analysis of the 16S–23S rRNA gene ITS region revealed that the ITS sizes for all studied isolates varied between 415 and 849 bp, while their G+C content was 42.2–57.9 mol%. Five distinct ITS types were identified: ITSnone (without tRNA genes), ITSAla(TGC), ITSAla(TGC)+Ile(GAT), ITSIle(GAT)+Ala(TGC), and ITS Ile(GAT)+Pseudo. All of the identified tRNAAla(TGC) molecules consisted of 73 bases, and all of the tRNAIle(GAT) molecules consisted of 74 bases. We also detected striking variability in the size of the ITS region among the various examined isolates. Highest variability was detected within the ITS-2. The importance of this study is that this is the first comparison of the 16S–23S rDNA ITS sequence similarities and tRNA genes from sphingomonads. Collectively the data obtained in this study revealed the heterogeneity and extent of variability within the ITS region compared to the 16S rRNA gene within closely related isolates. Sequence and length polymorphisms within the ITS region along with the ITS types (tRNA-containing or lacking and the type of tRNA) and ITS-2 size and sequence similarities allowed us to overcome the limitation we previously encountered in resolving closely related isolates based on the 16S rRNA gene sequence.

Molecular characterization of Carbapenem resistant Escherichia coli recovered from a tertiary hospital in Lebanon

The emergence of carbapenem resistant Escherichia coli represents a serious public health concern. This study investigated the resistome, virulence, plasmids content and clonality of 27 carbapenem resistant E. coli isolated from 27 hospitalized patients at the American University of Beirut Medical Center (AUBMC) in Lebanon between 2012 and 2016. Whole-genome sequencing (WGS) data were used to identify resistance determinants. Multilocus sequence typing (MLST), pulsed field gel electrophoresis (PFGE), phylogenetic grouping and PCR-based replicon typing (PBRT) were also performed. The 27 isolates were distributed into 15 STs, of which ST405 (14.8%; n = 4) was the most prevalent. All of the 27 isolates were carbapenem resistant and 20 (74%) were extended-spectrum β-lactamase (ESBL) gene carriers. The predominant detected carbapenemases were blaOXA-48 (48.1%; n = 13) and blaOXA-181 (7.4%; n = 2), for the ESBLs it was blaCTX-M-15 (55.6%; n = 15) and blaCTX-M-24 (18.5%; n = 5), and for the AmpC-type β–lactamases, blaCMY-42 (40.7%; n = 11) and blaCMY-2 (3.7%; n = 1). Thirteen replicons were identified among the 27 E. coli isolates including: IncL/M, IncFIA, IncFIB, IncFII, IncI1, and IncX3. PFGE revealed a high genetic diversity with the 27 isolates being grouped in 21 different pulsotypes. SNPs analysis and PFGE showed a possible clonal dissemination of ST405, ST1284, ST354 and ST410 and the dominance of certain STs, monitoring of which could help in elucidating routes of transmission. This study represents the first WGS-based in depth analysis of the resistomes and mobilomes of carbapenem resistant E. coli in Lebanon.

Insights into the genome diversity and virulence of two clinical isolates of Burkholderia cenocepacia

Purpose. Burkholderia cenocepacia is among the most common members of the Burkholderia cepacia complex (Bcc) isolated from patients with cystic fibrosis (CF). The factors triggering the high rates of morbidity and mortality in CF patients are not well elucidated. In this study, we aim to highlight the genome diversity of two clinical isolates of B. cenocepacia through comparative genome analysis. Methodology. The repertoire of virulence factors and resistance genes compared to reference strains J2315 and K56–2 was elucidated. The isolates were screened for the presence of phages and insertion sequences. Two methods were combined to obtain an accurate prediction of genomic islands (GIs): the cumulative GC profile and the IslandViewer web tool. To study evolutionary relatedness, whole genome‐based single‐nucleotide polymorphism (wgSNP) analysis was also performed with 43 publically available strains of the Bcc of various sequence types. Results/Key findings. Genome‐based species identification of the two isolates BC‐AUH and BC‐BMEH confirmed the species as B. cenocepacia. Both belonged to ST‐602, a double‐locus variant of ST‐32 (CC31), genomovar IIIA, and carried a large number of antibiotic resistance genes. Eighteen GIs were predicted in BC‐AUH and BC‐BMEH, occupying 9.3 and 6.1 % of the respective genomes. Comparison to J2315 revealed 89 and 85 genes unique to BC‐BMEH and BC‐AUH, respectively. Additionally, 1823 intergenic SNPs were detected between BC‐BMEH and BC‐AUH. Conclusion. This study mapped existing genetic variations in B. cenocepacia associated with notorious outcomes in CF patients, and the data obtained provide comprehensive, genome‐inferred insights and multifactorial examination of an important human pathogen.

Genomic Mapping of ST85 blaNDM-1 and blaOXA-94 producing Acinetobacter baumannii Isolates from Syrian Civil War Victims

OBJECTIVES The rapid emergence of carbapenem-resistant Acinetobacter baumannii is a global health concern. A comparative genomic analysis was performed on two ST85 A. baumannii strains harboring blaNDM-1 and blaOXA-94 collected in Lebanon from Syrian Civil War victims. METHODS Genome sequencing data of ACMH-6200 and ACMH-6201 were used for in silico extraction of multilocus sequence types (MLST), resistance genes, and virulence factors. Plasmids were genetically mapped in silico and using PCR-based replicon typing (PBRT). The genetic environment of blaNDM-1 and blaOXA-94 was determined, and whole-genome single nucleotide polymorphism (wgSNP) analysis in comparison with 41 publicly available A. baumannii genomes was performed. RESULTS Tn125 carrying blaNDM-1 was truncated by the insertion of ISAba14 downstream of dct, generating ΔTn125. blaOXA-94 was upstream of ISAba13 and ISAba17. Resistance to ceftazidime could be attributed to AmpC cephalosporinase encoded by blaADC-25, and to blaNDM-1 on plasmids. GyrA (S83L) and ParC (S80L) substitutions conferred resistance to fluoroquinolones. wgSNP analysis separated the isolates based on their sequence types. CONCLUSIONS The role of refugees in the transmission of antimicrobial resistance in developing countries is understudied. As such, this study sheds light on the correlation between population mobility and the importation of drug-resistant pathogens. It also highlights the manifold mechanisms underlying antibiotic resistance in A. baumannii.

Genomic Characterization of MDR Escherichia coli Harboring blaOXA-48 on the IncL/M-type Plasmid Isolated from Blood Stream Infection

Escherichia coli is responsible for a wide variety of community and hospital acquired extraintestinal infections, and the emergence of ESBL resistant isolates is a major clinical concern. In this study, we characterized the genomic attributes of an OXA-48 and CTX-M-3 producing E. coli EC-IMP153. Whole-genome initial assembly produced 146 contigs with a combined 5,504,170 bp in size and a G+C content of 50.5%. wgSNPs-based phylogenetic comparison with 36 publically available genomes was also performed. Comprehensive genomic analysis showed that EC-IMP153 belonged to sequence type ST-405 and harbored several resistance determinants including the β-lactam resistance genes blaOXA-48, blaCTX-M-3, blaTEM-1B, blaOXA-1, and blaCMY-70, aminoglycoside fyuA and aac(3)IId, tetracycline tet(A) and tet(R), and fluoroquinolone gyrA, parC, and mfd resistance determinants. Plasmids with the following incompatibility groups were detected in silico and confirmed using PBRT: IncI1-α, IncL, IncW, Col (BS512), and IncF. To our knowledge this is the first in-depth genomic analysis of an OXA-48 producing E. coli ST-405 isolated from a patient in Lebanon and linked to a blood stream infection. Continuous monitoring is necessary to better understand the continued diffusion of such pathogens, especially in view of the population movements triggered by unrest in the Middle East.

Genome sequencing and comparative analysis of an NDM-1-producing Klebsiella pneumoniae ST15 isolated from a refugee patient

Abstract The escalating problem of antibiotic resistance, specifically cabarpenemase and extended-spectrum β-lacatamase (ESBL) producing K. pneumoniae strains, is directly correlated with increased patient morbidity and mortality and prolonged hospitalization and costs. In this study, a comprehensive genomic analysis encompassing the resistomics, virulence repertoire and mobile genetic elements of an NDM-1 positive ESBL-producing K. pneumoniae EA-MEH ST15 isolated from a urine sample collected from a Syrian refugee was conducted. Illumina paired-end libraries were prepared and sequenced resulting in 892,300 high-quality reads. The initial assembly produced 329 contigs with a combined 5,954,825 bp and a 56.5% G+C content. Resistome analysis revealed the presence of several β-lactamases including NDM-1, SHV-28, CTX-M-15 and OXA-1 in addition to 18 other genes encoding for resistance, among which are aph(3′)-Ia, aac(6′)Ib-cr, armA, strB, strA and aadA2 genes. Additionally, five plasmids IncFIB(Mar), IncHI1B, IncFIB(pKPHS1), IncFIB(K) and IncFII(K) and four integrated phages were detected. In silico MLST analysis revealed that the isolate was of sequence type ST15. To our knowledge this is the first in-depth genomic analysis of a NDM-1 positive K. pneumoniae ST15 in Lebanon associated with the recent population migration. The potential dissemination of such MDR strains is an important public health concern.

Typing and comparative genome analysis of Brucella melitensis isolated from Lebanon

Brucella melitensis is the main causative agent of the zoonotic disease brucellosis. This study aimed at typing and characterizing genetic variation in 33 Brucella isolates recovered from patients in Lebanon. Bruce-ladder multiplex PCR and PCR-RFLP of omp31, omp2a and omp2b were performed. Sixteen representative isolates were chosen for draft-genome sequencing and analyzed to determine variations in virulence, resistance, genomic islands, prophages and insertion sequences. Comparative whole-genome single nucleotide polymorphism analysis was also performed. The isolates were confirmed to be B. melitensis. Genome analysis revealed multiple virulence determinants and efflux pumps. Genome comparisons and single nucleotide polymorphisms divided the isolates based on geographical distribution but revealed high levels of similarity between the strains. Sequence divergence in B. melitensis was mainly due to lateral gene transfer of mobile elements. This is the first report of an in-depth genomic characterization of B. melitensis in Lebanon.