Genetic and Structural Basis of Colistin Resistance in Klebsiella pneumoniae: Unraveling the Molecular Mechanisms.

BACKGROUND: Antimicrobial Antimicrobial resistance (AMR), together with multi-drug resistant (MDR), mainly among Gram-negative bacteria, has been on the rise. Colistin (polymyxin E) remains one of the primary available last resorts to treat infections by MDR bacteria with the rapid emergence of global resistance. OBJECTIVES: Since the exact mechanism of bacterial resistance to colistin remains unfolded, this study warranted elucidating the underlying mechanism of colistin resistance and heteroresistance among carbapenem-resistant (CR) Klebsiella pneumoniae isolates. METHODS: Molecular analysis was carried out on the resistant isolates using a genome-wide characterization approach, and MALDI-TOF MS for lipid A. RESULTS: Among the 32 CR K. pneumoniae isolates, several isolates showed resistance and intermediate resistance, to colistin. The seven isolates with intermediate resistance exhibited the "skip-well" phenomenon, attributed to the presence of resistant subpopulations. The three isolates with full resistance to colistin showed ions using MALDI-TOF MS at m/z 1840 and 1824 representing bisphosphorylated and hexaacylated lipid A with or without hydroxylation, at position C'-2 of the fatty acyl chain, respectively. Studying the genetic environment of mgrB locus revealed the presence of insertion sequences that disrupted the mgrB locus in the three colistin resistant isolates: IS1R and IS903B. CONCLUSIONS: Our findings showed that colistin resistance/heteroresistance was inducible with mutations in chromosomal regulatory networks controlling lipid A moiety and IS sequences disrupting the mgrB gene, leading to elevated MIC values and treatment failure. Different treatment strategies should be employed to avoid colistin heteroresistance-linked treatment failures, mainly through combination therapy using colistin with carbapenems, aminoglycosides, or tigecycline.

A dynamic epibiont community associated with the bone-eating polychaete genus Osedax.

Osedax, the deep-sea annelid found at sunken whalefalls, is known to host Oceanospirillales bacterial endosymbionts intracellularly in specialized roots, which help it feed exclusively on vertebrate bones. Past studies, however, have also made mention of external bacteria on their trunks. During a 14-yr study, we reveal a dynamic, yet persistent, shift of Campylobacterales integrated into the epidermis of Osedax, which change over time as the whale carcass degrades on the sea floor. The Campylobacterales associated with seven species of Osedax, which comprise 67% of the bacterial community on the trunk, appear initially dominated by the genus Arcobacter (at early time points <24 mo), the Sulfurospirillum at intermediate stages (~50 mo), and the Sulfurimonas at later stages (>140 mo) of whale carcass decomposition. Metagenome analysis of the epibiont metabolic capabilities suggests potential for a transition from heterotrophy to autotrophy and differences in their capacity to metabolize oxygen, carbon, nitrogen, and sulfur. Compared to free-living relatives, the Osedax epibiont genomes were enriched in transposable elements, implicating genetic exchange on the host surface, and contained numerous secretions systems with eukaryotic-like protein (ELP) domains, suggesting a long evolutionary history with these enigmatic, yet widely distributed deep-sea worms. IMPORTANCE Symbiotic associations are widespread in nature and we can expect to find them in every type of ecological niche. In the last twenty years, the myriad of functions, interactions and species comprising microbe-host associations has fueled a surge of interest and appreciation for symbiosis. During this 14-year study, we reveal a dynamic population of bacterial epibionts, integrated into the epidermis of 7 species of a deep-sea worm group that feeds exclusively on the remains of marine mammals. The bacterial genomes provide clues of a long evolutionary history with these enigmatic worms. On the host surface, they exchange genes and appear to undergo ecological succession, as the whale carcass habitat degrades over time, similar to what is observed for some free-living communities. These, and other annelid worms are important keystone species for diverse deep-sea environments, yet the role of attached external bacteria in supporting host health has received relatively little attention.

Distinct genomic routes underlie transitions to specialised symbiotic lifestyles in deep-sea annelid worms.

Bacterial symbioses allow annelids to colonise extreme ecological niches, such as hydrothermal vents and whale falls. Yet, the genetic principles sustaining these symbioses remain unclear. Here, we show that different genomic adaptations underpin the symbioses of phylogenetically related annelids with distinct nutritional strategies. Genome compaction and extensive gene losses distinguish the heterotrophic symbiosis of the bone-eating worm Osedax frankpressi from the chemoautotrophic symbiosis of deep-sea Vestimentifera. Osedax's endosymbionts complement many of the host's metabolic deficiencies, including the loss of pathways to recycle nitrogen and synthesise some amino acids. Osedax's endosymbionts possess the glyoxylate cycle, which could allow more efficient catabolism of bone-derived nutrients and the production of carbohydrates from fatty acids. Unlike in most Vestimentifera, innate immunity genes are reduced in O. frankpressi, which, however, has an expansion of matrix metalloproteases to digest collagen. Our study supports that distinct nutritional interactions influence host genome evolution differently in highly specialised symbioses.

First report of plasmid-mediated colistin resistance mcr-8.1 gene from a clinical Klebsiella pneumoniae isolate from Lebanon.

Colistin is considered as a last resort treatment for infections caused by multidrug-resistant Enterobacteriaceae. Plasmid-mediated mobile colistin resistance (mcr) genes contributed to the global spread of colistin resistance. This is the first report of plasmid-mediated colistin resistance mcr-8 gene from a clinical Klebsiella pneumoniae K9 isolate recovered from Lebanon. The isolate was characterized phenotypically and genotypically through both short and long read whole-genome sequencing, plasmid typing and conjugation assays. k9 belonged to sequence type 15 and harbored 31 antimicrobial resistance genes. The mcr-8.1 variant was carried on a novel ~ 300 kb multireplicon plasmid having IncFIA, IncR and IncHI1B. The plasmid was conjugative and carried a plethora of antimicrobial resistance determinants. The introduction of novel mcr variants in Lebanon poses an alarming health concern. Surveillance and screening for colistin resistant Enterobacteriaceae and mcr in livestock, animal farms, imported meat and poultry is highly recommended along with monitoring antibiotic use.

SARS-CoV-2 and ORF3a: Nonsynonymous Mutations, Functional Domains, and Viral Pathogenesis.

The effect of the rapid accumulation of nonsynonymous mutations on the pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is not yet known. The 3a protein is unique to SARS-CoV and is essential for disease pathogenesis. Our study aimed at determining the nonsynonymous mutations in the 3a protein in SARS-CoV-2 and determining and characterizing the protein's structure and spatial orientation in comparison to those of 3a in SARS-CoV. A total of 51 different nonsynonymous amino acid substitutions were detected in the 3a proteins among 2,782 SARS-CoV-2 strains. We observed microclonality within the ORF3a gene tree defined by nonsynonymous mutations separating the isolates into distinct subpopulations. We detected and identified six functional domains (I to VI) in the SARS-CoV-2 3a protein. The functional domains were linked to virulence, infectivity, ion channel formation, and virus release. Our study showed the importance of conserved functional domains across the species barrier and revealed the possible role of the 3a protein in the viral life cycle. Observations reported in this study merit experimental confirmation.IMPORTANCE At the surge of the coronavirus disease 2019 (COVID-19) pandemic, we detected and identified six functional domains (I to VI) in the SARS-CoV-2 3a protein. Our analysis showed that the functional domains were linked to virulence, infectivity, ion channel formation, and virus release in SARS-CoV-2 3a. Our study also revealed the functional importance of conserved domains across the species barrier. Observations reported in this study merit experimental confirmation.

Detailed characterization of an IncFII plasmid carrying blaOXA-48 from Lebanon.

BACKGROUND: The spread of carbapenem-resistant Enterobacteriaceae is an important challenge and an increasing healthcare problem. OXA-48 is a class D carbapenemase that is usually localized on a conjugative plasmid belonging to the IncL incompatibility group. METHODS: In this study, we used a combination of short- and long-read WGS approaches and molecular typing techniques to characterize the genetic environment of the smallest reported 27 029 bp IncFII plasmid carrying blaOXA-48 (pLAU-OXA48). RESULTS: The plasmid recovered from a clinical Escherichia coli isolate was positive for blaOXA-48, which was located within the Tn6237 composite transposon. Primers targeting junctions between the IncF fragment and Tn6237 for the rapid identification of pLAU-OXA48-like plasmids were designed. CONCLUSIONS: To our knowledge, this is the first report showing the complete sequence of an IncFII plasmid carrying blaOXA-48 within Tn6237 using hybrid assembly of long- and short-read sequencing.

Whole-Genome-Sequence-Based Characterization of Extensively Drug-Resistant Acinetobacter baumannii Hospital Outbreak.

Carbapenem-resistant Acinetobacter baumannii (CRAB) is an important opportunistic pathogen linked to a variety of nosocomial infections and hospital outbreaks worldwide. This study aimed at investigating and characterizing a CRAB outbreak at a large tertiary hospital in Lebanon. A total of 41 isolates were collected and analyzed using pulsed-field gel electrophoresis (PFGE). Whole-genome sequencing (WGS) was performed on all the isolates, and long-read PacBio sequencing was used to generate reference genomes. The multilocus sequence types (MLST), repertoire of resistance genes, and virulence factors were determined from the sequencing data. The plasmid content was analyzed both in silico and using the A. baumannii PCR-based replicon typing (AB-PBRT) method. Genome analysis initially revealed two clones, one carrying blaOXA-23 on Tn2006 (ST-1305, ST-195, and ST-218) and another carrying blaOXA-72 on pMAL-1 (ST-502 and ST-2059, a new ST), with the latter having two subclones, as revealed using the Bayesian transmission network. All isolates were extensively drug resistant (XDR). WGS analysis revealed the transmission pathways and demonstrated the diversity of CRAB isolates and mobile genetic elements in this health care setting. Outbreak detection using WGS and immediate implementation of infection control measures contribute to restraining the spread and decreasing mortality.IMPORTANCE Carbapenem-resistant Acinetobacter baumannii (CRAB) has been implicated in hospital outbreaks worldwide. Here, we present a whole-genome-based investigation of an extensively drug-resistant CRAB outbreak rapidly spreading and causing high incidences of mortality at numerous wards of a large tertiary hospital in Lebanon. This is the first study of its kind in the region. Two circulating clones were identified using a combination of molecular typing approaches, short- and long-read sequencing and Bayesian transmission network analysis. One clone carried blaOXA-23 on Tn2006 (ST-1305, ST-195, and ST-218), and another carried blaOXA-72 on a pMAL-1 plasmid (ST-502 and ST-2059, a new ST). A pMAL-2 plasmid was circulating between the two clones. The approaches implemented in this study and the obtained findings facilitate the tracking of outbreak scenarios in Lebanon and the region at large.

Genomic Features of Vibrio parahaemolyticus from Lebanon and Comparison to Globally Diverse Strains by Whole-Genome Sequencing.

Gastroenteritis is a disease that can be caused by virulent strains of Vibrio parahaemolyticus in humans upon the consumption of contaminated seafood. In summer 2017, a sudden increase in the number of patients suffering from gastroenteritis due to a V. parahaemolyticus infection was observed at the Middle East Institute of Health University Hospital in Lebanon. The aim of this study was to analyze the isolates recovered from stool specimens, and to compare them using different phenotypic assays, genomic profiling techniques, and whole-genome sequencing, to achieve a better understanding of the current V. parahaemolyticus strains available in Lebanon. Virulence potential was analyzed based on the detection of the hemolysins: thermostable direct hemolysin (tdh), thermostable direct hemolysin-related hemolysin (trh), and thermolabile hemolysin (tlh). Resistance was determined by testing antibiotic susceptibility and performing PCR assays for ß-lactamases and quinolone resistance determinants. Genetic relatedness was verified by multilocus sequence typing, pulsed-field gel electrophoresis, and whole genome-based single nucleotide polymorphism analysis. All of the isolates had the tdh+, trh-, group-specific PCR+ genotype, which is a characteristic of the O3:K6 pandemic clone. The isolates were resistant to ampicillin (100%), ceftazidime (86%), ticarcillin (14%), and amikacin (14%), belonged to the sequence type ST3, and had very similar phylogenetic fingerprints. The isolates undertaken in this study exhibited almost identical resistance, virulence, and phylogenetic patterns, confirming an outbreak linked to the spread of the pandemic O3:K6 serotype in the country.

Genome Mining and Comparative Analysis of Streptococcus intermedius Causing Brain Abscess in a Child.

Streptococcus intermedius (SI) is associated with prolonged hospitalization and low survival rates. The genetic mechanisms involved in brain abscess development and genome evolution in comparison to other members of the Streptococcus anginosus group are understudied. We performed a whole-genome comparative analysis of an SI isolate, LAU_SINT, associated with brain abscess following sinusitis with all SI genomes in addition to S. constellatus and S. anginosus. Selective pressure on virulence factors, phages, pan-genome evolution and single-nucleotide polymorphism analysis were assessed. The structural details of the type seven secretion system (T7SS) was elucidated and compared with different organisms. ily and nanA were both abundant and conserved. Nisin resistance determinants were found in 47% of the isolates. Pan-genome and SNPs-based analysis didn't reveal significant geo-patterns. Our results showed that two SC isolates were misidentified as SI. We propose the presence of four T7SS modules (I⁻IV) located on various genomic islands. We detected a variety of factors linked to metal ions binding on the GIs carrying T7SS. This is the first detailed report characterizing the T7SS and its link to nisin resistance and metal ions binding in SI. These and yet uncharacterized T7SS transmembrane proteins merit further studies and could represent potential therapeutic targets.

Molecular Characterization of Carbapenem Resistant Klebsiella pneumoniae and Klebsiella quasipneumoniae Isolated from Lebanon.

Klebsiella pneumoniae is a Gram-negative organism and a major public health threat. In this study, we used whole-genome sequences to characterize 32 carbapenem-resistant K. pneumoniae (CRKP) and two carbapenem-resistant K. quasipneumoniae (CRKQ). Antimicrobial resistance was assessed using disk diffusion and E-test, while virulence was assessed in silico. The capsule type was determined by sequencing the wzi gene. The plasmid diversity was assessed by PCR-based replicon typing to detect the plasmid incompatibility (Inc) groups. The genetic relatedness was determined by multilocus sequence typing, pan-genome, and recombination analysis. All of the isolates were resistant to ertapenem together with imipenem and/or meropenem. Phenotypic resistance was due to blaOXA-48, blaNDM-1, blaNDM-7, or the coupling of ESBLs and outer membrane porin modifications. This is the first comprehensive study reporting on the WGS of CRKP and the first detection of CRKQ in the region. The presence and dissemination of CRKP and CRKQ, with some additionally having characteristics of hypervirulent clones such as the hypermucoviscous phenotype and the capsular type K2, are particularly concerning. Additionally, mining the completely sequenced K. pneumoniae genomes revealed the key roles of mobile genetic elements in the spread of antibiotic resistance and in understanding the epidemiology of these clinically significant pathogens.

First insights on the genetic diversity of MDR Mycobacterium tuberculosis in Lebanon.

BACKGROUND: Lebanon hosts a heterogeneous population coming from underdeveloped and developing countries, resulting in increasing incidences of tuberculosis over the past years. The genetic heterogeneity and lineages associated with tuberculosis, along with their resistance determinants have not been studied at the genomic level previously in the region. METHODS: Isolates were recovered from the American University of Beirut Medical Center (AUBMC). Antimicrobial susceptibility profiles were determined using the MGIT automated system for the first-line drugs at AUBMC, while second-line drug susceptibility was tested at Mayo Clinic Laboratories. Whole Genome Sequencing (WGS) was performed to classify mycobacterial lineages and highlight single nucleotide mutations causing resistance to both 1st line and 2nd line antimicrobials. wgSNP analysis provided insights on the phylogeny of the isolates along with spoligotyping and core genomic SNVs, IS6110 insertion sites, and variable number tandem repeats (VNTR). RESULTS: The analyzed isolates carry distinct resistance determinants to isoniazid, rifampicin, ethambutol, quinolones, and streptomycin. The isolates belonged to different lineages including the Euro/American lineage (Lineage 4) (53.8%), M. bovis (15.4%) and Delhi/Central Asia (Lineage 1) (15.4%), Beijing/East Asia (Lineage 2) (7.7%), and East Africa/Indian Ocean lineage (Lineage 3) (7.7%) showing great phylogenetic differences at the genomic level. CONCLUSIONS: The population diversity in Lebanon holds an equally diverse and uncharacterized population of drug resistant mycobacteria. To achieve the WHO "END-TB" milestones of 2025 and 2035, Lebanon must decrease TB incidences by 95% in the next decade. This can only be done through WGS-based patient centered diagnosis with higher throughput and genomic resolution to improve treatment outcomes and to monitor transmission patterns.