Whole Genome Analyses Accurately Identify Neisseria spp. and Limit Taxonomic Ambiguity.

Genome sequencing facilitates the study of bacterial taxonomy and allows the re-evaluation of the taxonomic relationships between species. Here, we aimed to analyze the draft genomes of four commensal Neisseria clinical isolates from the semen of infertile Lebanese men. To determine the phylogenetic relationships among these strains and other Neisseria spp. and to confirm their identity at the genomic level, we compared the genomes of these four isolates with the complete genome sequences of Neisseria gonorrhoeae and Neisseria meningitidis and the draft genomes of Neisseria flavescens, Neisseria perflava, Neisseria mucosa, and Neisseria macacae that are available in the NCBI Genbank database. Our findings revealed that the WGS analysis accurately identified and corroborated the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) species identities of the Neisseria isolates. The combination of three well-established genome-based taxonomic tools (in silico DNA-DNA Hybridization, Ortho Average Nucleotide identity, and pangenomic studies) proved to be relatively the best identification approach. Notably, we also discovered that some Neisseria strains that are deposited in databases contain many taxonomical errors. The latter is very important and must be addressed to prevent misdiagnosis and missing emerging etiologies. We also highlight the need for robust cut-offs to delineate the species using genomic tools.

Challenges in identification of enteroinvasive Escherichia coli and Shigella spp. in Lebanon.

This study aimed to evaluate the routine identification tools available in Lebanon for differentiation of Escherichia coli and Shigella spp. The identification of 43 isolates defined as Shigella spp. by Api 20E was accessed using MALDI-TOF, serological testing, duplex PCR targeting ipaH (present in Shigella spp. and enteroinvasive E. coli "EIEC") and lacY (found in E. coli including EIEC but not Shigella spp.) as well as gyrB gene sequencing. Antibiotic susceptibility was investigated as well as Shiga-toxin production. All isolates were identified as E. coli by MALDI-TOF while the PCR showed a disparate group of 26 EIEC, 11 Shigella spp., 5 E. coli and 1 inactive E. coli. However, the sequencing of gyrB gene, which was recently described as a suitable marker for distinguishing E. coli and Shigella spp., identified all isolates as E. coli. Antibiotic resistance was noticeable against ß-lactams, rifampicin, trimethoprim-sulfamethoxazole, gentamicin, and ciprofloxacin. The most common variants of beta-lactamase genes were blaTEM-1, blaCTX-M-15, and blaCTX-M-3. A great discordance between the used methods in identification was revealed herein. An accurate identification technique able to distinguish E. coli from Shigella spp. in routine laboratories is a pressing need in order to select the appropriate treatment and assess the epidemiology of these bacteria.

Drug-Resistant Tuberculosis, Lebanon, 2016 - 2017.

In a 12-month nationwide study on the prevalence of drug-resistant tuberculosis (TB) in Lebanon, we identified 3 multidrug-resistant cases and 3 extensively drug-resistant TB cases in refugees, migrants, and 1 Lebanon resident. Enhanced diagnostics, particularly in major destinations for refugees, asylum seekers, and migrant workers, can inform treatment decisions and may help prevent the spread of drug-resistant TB.

Extrahuman epidemiology of Acinetobacter baumannii in Lebanon.

The presence of Acinetobacter baumannii outside hospitals is still a controversial issue. The objective of our study was to explore the extrahospital epidemiology of A. baumannii in Lebanon. From February 2012 to October 2013, a total of 73 water samples, 51 soil samples, 37 raw cow milk samples, 50 cow meat samples, 7 raw cheese samples, and 379 animal samples were analyzed by cultural methods for the presence of A. baumannii. Species identification was performed by rpoB gene sequencing. Antibiotic susceptibility was investigated, and the A. baumannii population was studied by two genotyping approaches: multilocus sequence typing (MLST) and blaOXA-51 sequence-based typing (SBT). A. baumannii was detected in 6.9% of water samples, 2.7% of milk samples, 8.0% of meat samples, 14.3% of cheese samples, and 7.7% of animal samples. All isolates showed a susceptible phenotype against most of the antibiotics tested and lacked carbapenemase-encoding genes, except one that harbored a blaOXA-143 gene. MLST analysis revealed the presence of 36 sequence types (STs), among which 24 were novel STs reported for the first time in this study. blaOXA-51 SBT showed the presence of 34 variants, among which 21 were novel and all were isolated from animal origins. Finally, 30 isolates had new partial rpoB sequences and were considered putative new Acinetobacter species. In conclusion, animals can be a potential reservoir for A. baumannii and the dissemination of new emerging carbapenemases. The roles of the novel animal clones identified in community-acquired infections should be investigated.

Molecular epidemiology of Acinetobacter baumannii in different hospitals in Tripoli, Lebanon using bla(OXA-51-like) sequence based typing.

BACKGROUND: A. baumannii has emerged as an important nosocomial pathogen with an outstanding ability to acquire multidrug resistant mechanisms. In this study, we investigate the molecular epidemiology and carbapenem resistance mechanisms of A. baumannii in Tripoli, Northern Lebanon. METHODS: One hundred sixteen non-duplicate isolates isolated between 2011 and 2013 in different hospitals in Tripoli, Lebanon from Lebanese patients and wounded Syrian patients during Syrian war were studied. Antibiotic susceptibility testing was determined by agar disc diffusion and Etest. Carbapenemase-encoding genes were investigated by PCR. All isolates were typed by bla OXA-51-like sequence based typing (SBT) and 57 isolates were also analysed by MLST using Pasteur's scheme followed by eBURST analysis. RESULTS: Of the 116 isolates, 70 (60 %) showed a carbapenem resistance phenotype. The bla OXA-23 with an upstream insertion of ISAba1 was the major carbapenem resistance mechanism and detected in 65 isolates. Five isolates, including four from wounded Syrian patients and one from a Lebanese patient, were positive for bla NDM-1. bla OXA-51-like SBT revealed the presence of 14 variants, where bla OXA-66 was the most common and present in 73 isolates, followed by bla OXA-69 in 20 isolates. MLST analysis identified 17 sequence types (ST) and showed a concordance with bla OXA-51-like SBT. Each clonal complex (CC) had a specific bla OXA-51-like sequence such as CC2, which harboured bla OXA-66 variant, and CC1 harbouring bla OXA-69 variant. NDM-1 producing isolates belonged to ST85 (4 Syrian isolates) and ST25 (1 Lebanese isolate). CONCLUSIONS: Our results showed a successful predominance of international clone 2 with a widespread occurrence of OXA-23 carbapenemase in Lebanese hospitals. These findings emphasise the urgent need of effective measures to control the spread of A. baumannii in this country.

Evaluation of SARS-CoV-2 anti-Spike antibody levels and breakthrough infection risk among vaccinated adults in North Lebanon.

Since March 2020, the COVID-19 pandemic has swiftly propagated, triggering a competitive race among medical firms to forge vaccines that thwart the infection. Lebanon initiated its vaccination campaign on February 14, 2021. Despite numerous studies conducted to elucidate the characteristics of immune responses elicited by vaccination, the topic remains unclear. Here, we aimed to track the progression of anti-spike SARS-CoV-2 antibody titers at two-time points (T1: shortly after the second vaccination dose, T2: six months later) within a cohort of 201 adults who received Pfizer-BioNTech (BNT162b2), AstraZeneca, or Sputnik V vaccines in North Lebanon. Blood specimens were obtained from participants, and antibody titers against SARS-CoV-2 were quantified through the Elecsys-Anti-SARS-CoV-2 S assay (Roche Diagnostics, Switzerland). We used univariate analysis and multivariable logistic regression models to predict determinants influencing the decline in immune response and the occurrence of breakthrough infections among vaccinated patients. Among the 201 participants, 141 exhibited unchanging levels of antibody titers between the two sample collections, 55 displayed waning antibody titers, and only five participants demonstrated heightened antibody levels. Notably, age emerged as the sole variable significantly linked to the waning immune response. Moreover, the BNT162b2 vaccine exhibited significantly higher efficacy concerning the occurrence of breakthrough infections when compared with the AstraZeneca vaccine. Overall, our study reflected the immune status of a sample of vaccinated adults in North Lebanon. Further studies on a larger scale are needed at the national level to follow the immune response after vaccination, especially after the addition of the third vaccination dose.

Genomic analysis of Shigella isolates from Lebanon reveals marked genetic diversity and antimicrobial resistance.

In this study, we characterized 54 clinical isolates of Shigella collected in North Lebanon between 2009 and 2017 through phenotypic and genomic analyses. The most prevalent serogroup was S. sonnei, accounting for 46.3 % (25/54) of the isolates, followed by S. flexneri (27.8 %, 15/54), S. boydii (18.5 %, 10/54) and S. dysenteriae (7.4 %, 4/54). Only three isolates were pan-susceptible, and 87 % (47/54) of the isolates had multidrug resistance phenotypes. Notably, 27.8 % (15/54) of the isolates were resistant to third-generation cephalosporins (3GCs) and 77.8 % (42/54) were resistant to nalidixic acid. 3GC resistance was mediated by the extended-spectrum beta-lactamase genes bla CTX-M-15 and bla CTX-M-3, which were present on various plasmids. Quinolone resistance was conferred by single point mutations in the gyrA DNA gyrase gene, leading to GyrA S83L, GyrA D87Y or GyrA S83A amino acid substitutions. This is the first study, to our knowledge, to provide genomic insights into the serotypes of Shigella circulating in Lebanon and the various antimicrobial resistance determinants carried by these strains.

Zoonotic Tuberculosis: A Neglected Disease in the Middle East and North Africa (MENA) Region.

Mycobacterium bovis is the etiologic agent of bovine tuberculosis (BTB), a serious infectious disease in both humans and animals. BTB is a zoonotic disease primarily affecting cattle and occasionally humans infected through close contact with infected hosts or the consumption of unpasteurized dairy products. Zoonotic tuberculosis is strongly associated with poverty and poor hygiene, and low- and middle-income countries bear the brunt of the disease. BTB has been increasingly recognized as a growing public health threat in developing countries. However, the lack of effective surveillance programs in many of these countries poses a barrier to accurately determining the true burden of this disease. Additionally, the control of BTB is threatened by the emergence of drug-resistant strains that affect the effectiveness of current treatment regimens. Here, we analyzed current trends in the epidemiology of the disease as well as the antimicrobial susceptibility patterns of M. bovis in the Middle East and North Africa (MENA) region, a region that includes several developing countries. Following PRISMA guidelines, a total of 90 studies conducted in the MENA region were selected. Our findings revealed that the prevalence of BTB among humans and cattle varied significantly according to the population size and country in the MENA region. Most of the available studies were based on culture and/or PCR strategies and were published without including data on antimicrobial resistance and molecular typing. Our findings highlighted the paramount need for the use of appropriate diagnostic tools and the implementation of sustainable control measures, especially at the human/animal interface, in the MENA region.

ShigaPass: an in silico tool predicting Shigella serotypes from whole-genome sequencing assemblies.

Shigella is one of the commonest causes of diarrhoea worldwide and a major public health problem. Shigella serotyping is based on a standardized scheme that splits Shigella strains into four serogroups and 60 serotypes on the basis of biochemical tests and O-antigen structures. This conventional serotyping method is laborious, time-consuming, impossible to automate, and requires a high level of expertise. Whole-genome sequencing (WGS) is becoming more affordable and is now used for routine surveillance, opening up possibilities for the development of much-needed accurate rapid typing methods. Here, we describe ShigaPass, a new in silico tool for predicting Shigella serotypes from WGS assemblies on the basis of rfb gene cluster DNA sequences, phage and plasmid-encoded O-antigen modification genes, seven housekeeping genes (EnteroBase's MLST scheme), fliC alleles and clustered regularly interspaced short palindromic repeats (CRISPR) spacers. Using 4879 genomes, including 4716 reference strains and clinical isolates of Shigella characterized with a panel of biochemical tests and serotyped by slide agglutination, we show here that ShigaPass outperforms all existing in silico tools, particularly for the identification of Shigella boydii and Shigella dysenteriae serotypes, with a correct serotype assignment rate of 98.5 % and a sensitivity rate (i.e. ability to make any prediction) of 100 %.

The Acinetobacter baumannii website (Ab-web): a multidisciplinary knowledge hub, communication platform, and workspace.

Acinetobacter baumannii is a Gram-negative bacterium increasingly implicated in hospital-acquired infections and outbreaks. Effective prevention and control of such infections are commonly challenged by the frequent emergence of multidrug-resistant strains. Here we introduce Ab-web (https://www.acinetobacterbaumannii.no), the first online platform for sharing expertise on A. baumannii. Ab-web is a species-centric knowledge hub, initially with 10 articles organized into two main sections, 'Overview' and 'Topics', and three themes, 'epidemiology', 'antibiotic resistance', and 'virulence'. The 'workspace' section provides a spot for colleagues to collaborate, build, and manage joint projects. Ab-web is a community-driven initiative amenable to constructive feedback and new ideas.

A global snapshot on the prevalent macrolide-resistant emm types of Group A Streptococcus worldwide, their phenotypes and their resistance marker genotypes during the last two decades: A systematic review.

Watchful epidemiological surveillance of macrolide-resistant Group A Streptococcus (MRGAS) clones is important owing to the evolutionary and epidemiological dynamic of GAS. Meanwhile, data on the global distribution of MRGAS emm types according to macrolide resistance phenotypes and genotypes are scant and need to be updated. For this, the present systematic review analyses a global set of extensively characterized MRGAS isolates from patients of diverse ages and clinical presentations over approximately two decades (2000 to 2020) and recaps the peculiar epidemiological features of the dominant MRGAS clones. Based on the inclusion and exclusion criteria, 53 articles (3593 macrolide-resistant and 15,951 susceptible isolates) distributed over 23 countries were dissected with a predominance of high-income countries over low-income ones. Although macrolide resistance in GAS is highly variable in different countries, its within-GAS distribution seems not to be random. emm pattern E, 13 major emm types (emm12, 4, 28, 77, 75, 11, 22, 92, 58, 60, 94, 63, 114) and 4 emm clusters (A-C4, E1, E6, and E2) were significantly associated with macrolide resistance. emm patterns A-C and D, 14 major emm types (emm89, 3, 6, 2, 44, 82, 87, 118, 5, 49, 81, 59, 227, 78) and 3 well-defined emm clusters (A-C5, E3, and D4) were significantly associated with macrolide susceptibility. Scrutinizing the tendency of each MRGAS emm type to be significantly associated with specific macrolide resistance phenotype or genotype, interesting vignettes are also unveiled. The 30-valent vaccine covers ~95% of MRGAS isolates. The presented data urge the importance of comprehensive nationwide sustained surveillance of MRGAS circulating clones particularly in Low and Middle income countries where sampling bias is high and GAS epidemiology is obfuscated and needs to be demystified.

Variants of Tn6924, a Novel Tn7 Family Transposon Carrying the blaNDM Metallo-ß-Lactamase and 14 Copies of the aphA6 Amikacin Resistance Genes Found in Acinetobacter baumannii.

Carbapenem resistance in Acinetobacter baumannii is primarily due to the global spread of two main clones that carry oxa23, oxa24, and oxa58. However, new carbapenem-resistant clones are emerging that are also resistant to a wide range of antibiotics. Strains belonging to ST85IP (Institut Pasteur) carry the blaNDM metallo-ß-lactamase carbapenem resistance gene. Here, we completed the genome sequence of an ST85IP strain, Cl300, recovered in 2015 in Lebanon, using a combination of Illumina MiSeq and Oxford Nanopore sequencing and a hybrid assembly approach. Cl300 is highly resistant to meropenem and amikacin, and consistent with this, a copy of the blaNDM carbapenem and 14 copies of the aphA6 amikacin resistance genes were found in the genome. Cl300 also contains the sul2 sulfonamide and the msr(E) macrolide resistance genes. All aphA6 copies and blaNDM are in a novel 76-kb Tn7 family transposon designated Tn6924. Like Tn7, Tn6924 is bounded by 29-bp inverted repeats with additional TnsB binding sites at each end. Several variants of Tn6924 were found in a set of diverse strains, including ST85IP strains as well as members of global clones 1 and 2. sul2 and msr(E) are in a 13.0-kb pseudocompound transposon (PCT) bounded by IS1008. ST85s represent a diverse group of strains, particularly in their antibiotic resistance gene content and the K and OC surface polysaccharide loci. Acquisition of Tn6924 by members of global clones indicates the significance of this transposon in spreading two clinically significant resistance genes, blaNDM and aphA6. IMPORTANCE To date, efforts to study the resistance mechanisms of carbapenem-resistant Acinetobacter baumannii have been largely focused on the two major globally distributed clones (GC1 and GC2). ST85 is an emerging sequence type, and unlike other clones, it is associated with the carriage of the blaNDM gene. Here, we completed the genome sequence of an ST85 strain and showed that blaNDM and 14 copies of the aphA6 amikacin resistance genes are in Tn6924, a novel Tn7 family transposon. Analysis of all publicly available ST85s predicted that all strains in the main lineage carry a variant of Tn6924. Variants of Tn6924 were also found in other clones, including GC1 and GC2. Tn6924 is an important mobile element given that it carries two clinically important resistance genes (blaNDM and aphA6) and has spread to other clones. Therefore, outbreaks caused by ST85s should be studied and tracked.

Emergence of a Neisseria flavescens clinical strain with a high level of third-generation cephalosporins resistance in Lebanon.

Third generation cephalosporins (3GC)-resistant Neisseria flavescens could cause difficult-to-treat infections. This study aimed to investigate the genetic determinants of 3GC resistance using whole genome sequence comparisons of three N. flavescens; one of which was 3GC-resistant, while two were susceptible. Mutations in penA, ponA, and porB might be associated with the 3GC-resistance.

Analysis of pCl107 a large plasmid carried by an ST25 Acinetobacter baumannii strain reveals a complex evolutionary history and links to multiple antibiotic resistance and metabolic pathways.

Acinetobacter baumannii has successfully spread during the last decades as one of the main critically important pathogens. However, many aspects including plasmids, are still under-investigated. Here, we report the complete sequence of an Acinetobacter baumannii strain, belonging to the ST25IP (Institut Pasteur) sequence type recovered in 2012 in Lebanon, using a combination of Illumina MiSeq and Oxford Nanopore sequencing and a hybrid assembly approach. This strain (Cl107) carries a 198 kb plasmid called pCl107 that encodes the MPFI conjugative transfer system. The plasmid carries the aacA1, aacC2, sul2, strAB, and tetA(B) antibiotic resistance genes. pCl107 region encompassing the sul2, strAB, tetA(B) is closely related to AbGRI1 chromosomal resistance islands, which are widespread in A. baumannii strains belonging to Global Clone 2. The resistance region found in pCl107 is one of the missing links in the evolutionary history of the AbGRI1 islands. pCl107 also contains a BREX Type 1 region and represents one of the two main evolution patterns observed in BREX clusters found in plasmids related to pCl107. pCl107 also harbours a ptx phosphonate metabolism module, which plays an ancestral structure compared to other large plasmids in ST25 strains. While the uric acid metabolic module found in pCl107 is incomplete, we identified possible ancestors from plasmids and chromosomes of Acinetobacter spp. Our analyses indicate a complex evolutionary history of plasmids related to pCl107 with many links to multiple antibiotic resistance and metabolic pathways.

Genome sequence of a multidrug-resistant Campylobacter coli strain isolated from a newborn with severe diarrhea in Lebanon.

A multidrug-resistant (MDR) Campylobacter coli (C. coli) strain was isolated from a 2-month-old newborn who suffered from severe diarrhea in Lebanon. Here, whole-genome sequencing (WGS) analysis was deployed to determine the genetic basis of antimicrobial resistance and virulence in the C. coli isolate and to identify its epidemiological background (sequence type). The identity of the isolate was confirmed using API® Campy, MALDI-TOF, and 16S rRNA gene sequencing analysis. The antimicrobial susceptibility phenotype was determined using the disk diffusion assay. Our analysis showed that resistance to macrolide and quinolone was potentially associated with the presence of multiple point mutations in antibiotic targets on the chromosomal DNA. Furthermore, tetracycline and aminoglycoside resistance were encoded by genes on a pTet plasmid. The blaOXA-61, which is associated with beta-lactam resistance, was also detected in the C. coli genome. A set of 30 genes associated with the virulence in C. coli was detected using WGS analysis. MLST analysis classified the isolate as belonging to a new sequence type (ST-9588), a member of ST-828 complex which is mainly associated with humans and chickens. Taking together, this study provides the first WGS analysis of Campylobacter isolated from Lebanon. The detection of a variety of AMR and virulence determinants strongly emphasizes the need for studying the burden of Campylobacter in Lebanon and the Middle East and North Africa (MENA) region, where information on campylobacteriosis is scant.

Trends in the epidemiology of dermatophytosis in the Middle East and North Africa region.

Dermatophytosis corresponds to a broad series of infections, mostly superficial, caused by a group of keratinophilic and keratinolytic filamentous fungi called dermatophytes. These mycoses are currently considered to be a major public health concern worldwide, particularly in developing countries such as those in the Middle East and North Africa (MENA) region. Here we compiled and discussed existing epidemiologic data on these infections in the MENA region. Most of the available studies were based on conventional diagnostic strategies and were published before the last taxonomic revision of dermatophytes. This has led to misidentifications, which might have resulted in the underestimation of the real burden of these infections in the MENA countries. Our analysis of the available literature highlights an urgent need for further studies based on reliable diagnostic tools and standard susceptibility testing methods for dermatophytosis, which represents a major challenge for these countries. This is crucial for guiding appropriate interventions and activating antifungal stewardship programs in the future.

Teleclinical Microbiology: An Innovative Approach to Providing Web-Enabled Diagnostic Laboratory Services in Syria.

OBJECTIVES: Telemedicine can compensate for the lack of health care specialists in response to protracted humanitarian crises. We sought to assess the usability of a teleclinical microbiology (TCM) program to provide diagnostic services in a hard-to-reach region of Syria. METHODS: A semimobile station was equipped with conventional micrograph and macrograph digital imaging systems. An electronic platform (Telemicrobiology in Humanitarian Crises, TmHC) was created to facilitate sharing, interpreting, and storing the results. A pilot study was conducted to identify the bacterial species and antimicrobial susceptibility pattern of 74 urinary clinical isolates. An experience survey was conducted to capture the feedback of 8 participants in the program. RESULTS: The TmHC platform (https://sdh.ngo/tmhc/) enabled systematic transmission of the laboratory records and co-interpretation of the results. The isolates were identified as Escherichia coli (n = 61), Klebsiella pneumoniae (n = 12), and Proteus mirabilis(n = 1). All the isolates were multidrug resistant. The performance of our TCM module was rated 4 (satisfying) and 5 (very satisfying) by 6 and 2 users, respectively. Data security of and cost-effectiveness were the main perceived concerns. CONCLUSIONS: Although we encountered several context-related obstacles, our TCM program managed to reach a highly vulnerable population of 4 million people confined in the northwest region of Syria.

Population structure analysis and laboratory monitoring of Shigella by core-genome multilocus sequence typing.

The laboratory surveillance of bacillary dysentery is based on a standardised Shigella typing scheme that classifies Shigella strains into four serogroups and more than 50 serotypes on the basis of biochemical tests and lipopolysaccharide O-antigen serotyping. Real-time genomic surveillance of Shigella infections has been implemented in several countries, but without the use of a standardised typing scheme. Here, we study over 4000 reference strains and clinical isolates of Shigella, covering all serotypes, with both the current serotyping scheme and the standardised EnteroBase core-genome multilocus sequence typing scheme (cgMLST). The Shigella genomes are grouped into eight phylogenetically distinct clusters, within the E. coli species. The cgMLST hierarchical clustering (HC) analysis at different levels of resolution (HC2000 to HC400) recognises the natural population structure of Shigella. By contrast, the serotyping scheme is affected by horizontal gene transfer, leading to a conflation of genetically unrelated Shigella strains and a separation of genetically related strains. The use of this cgMLST scheme will facilitate the transition from traditional phenotypic typing to routine whole-genome sequencing for the laboratory surveillance of Shigella infections.