If You're Not Confused, You're Not Paying Attention: Ochrobactrum Is Not Brucella.

Bacteria of the genus Brucella are facultative intracellular parasites that cause brucellosis, a severe animal and human disease. Recently, a group of taxonomists merged the brucellae with the primarily free-living, phylogenetically related Ochrobactrum spp. in the genus Brucella. This change, founded only on global genomic analysis and the fortuitous isolation of some opportunistic Ochrobactrum spp. from medically compromised patients, has been automatically included in culture collections and databases. We argue that clinical and environmental microbiologists should not accept this nomenclature, and we advise against its use because (i) it was presented without in-depth phylogenetic analyses and did not consider alternative taxonomic solutions; (ii) it was launched without the input of experts in brucellosis or Ochrobactrum; (iii) it applies a non-consensus genus concept that disregards taxonomically relevant differences in structure, physiology, population structure, core-pangenome assemblies, genome structure, genomic traits, clinical features, treatment, prevention, diagnosis, genus description rules, and, above all, pathogenicity; and (iv) placing these two bacterial groups in the same genus creates risks for veterinarians, medical doctors, clinical laboratories, health authorities, and legislators who deal with brucellosis, a disease that is particularly relevant in low- and middle-income countries. Based on all this information, we urge microbiologists, bacterial collections, genomic databases, journals, and public health boards to keep the Brucella and Ochrobactrum genera separate to avoid further bewilderment and harm.

The burden of laboratory-confirmed influenza infection in Lebanon between 2008 and 2016: a single tertiary care center experience.

BACKGROUND: Influenza is a major cause of morbidity and mortality worldwide. Following the 2009 pandemic, there was widened interest in studying influenza burden in all regions. However, since data from the World Health Organization (WHO) Middle East and North Africa (MENA) region remain limited, we aimed to contribute to the understanding of influenza burden in Lebanon. METHODS: A retrospective chart review extending over a period of 8 seasons from Jan 1st, 2008 till June 30th, 2016 at a tertiary care center in Beirut was performed. All cases confirmed to have influenza based on rapid antigen detection or/and polymerase chain reaction on a respiratory sample were included for analysis. Data on epidemiology, clinical presentation, complications, antiviral use and mortality were collected for analysis. RESULTS: A total of 1829 cases of laboratory-confirmed influenza were identified. Average annual positivity rate was 14% (positive tests over total requested). Both influenza A and B co-circulated in each season with predominance of influenza A. Influenza virus started circulating in December and peaked in January and February. The age group of 19-50 years accounted for the largest proportion of cases (22.5%) followed by the age group of 5-19 years (18%). Pneumonia was the most common complication reported in 33% of cases. Mortality reached 3.8%. The two extremes of age (< 2 years and ≥ 65 years) were associated with a more severe course of disease, hospitalization, intensive care unit (ICU) admission, complications, and mortality rate. Of all the identified cases, 26% were hospitalized. Moderate-to-severe disease was more likely in influenza B cases but no difference in mortality was reported between the two types. Antivirals were prescribed in 68.8% and antibiotics in 41% of cases. There seemed to be an increasing trend in the number of diagnosed and hospitalized cases over the years of the study. CONCLUSION: Patients with laboratory-confirmed influenza at our center had a high rate of hospitalization and mortality. A population based prospective surveillance study is needed to better estimate the burden of Influenza in Lebanon that would help formulate a policy on influenza control.

Molecular characterization, toxin detection and resistance testing of human clinical Clostridium difficile isolates from Lebanon.

Clostridium (Clostridioides) difficile is the main cause for nosocomial diarrhoea in industrialised nations. Epidemiologic data on the pathogen's occurrence in other world regions are still scarce. In this context we characterized with phenotypic and molecular genetic methods C. difficile isolates stemming from hospitalised patients with diarrhoea in Lebanon. From 129 stool samples of symptomatic patients at a tertiary care University hospital in Lebanon, a total of 107 C. difficile strains were cultivated and underwent ribotyping, toxin gene detection and antibiotic resistance testing. Ribotype 014 (RT014, 16.8%) predominated, followed by RT002 (9.3%), RT106 (8.4%) and RT070 (6.5%). Binary toxin gene-positive isolates (RT023, RT078 and RT126) were rarely detected and RT027 was absent. Interestingly, within one isolate only the toxin A gene (tcdA) was detected. Multiple-locus variable-number tandem repeat analysis (MLVA) revealed strong strain diversity in most RTs. The isolates were sensitive to metronidazole and vancomycin, and only a small proportion of strains displayed resistance against moxifloxacin, rifampicin, and clarithromycin (5.6%, 1.9%, and 2.8%), respectively. The data indicate that the genetic strain composition of Lebanese strains differs markedly from the situation seen in Europe and North America. Especially the epidemic RTs seen in the latter regions were almost absent in Lebanon. Interestingly, most strains showed almost no resistance to commonly used antibiotics that are suspected to play a major role in the development of C. difficile infection, despite frequent use of these antibiotics in Lebanon. Thus, the role of antimicrobial resistance as a major driving force for infection development remains uncertain in this area.

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.

TUBERCULOSIS BURDEN IN LEBANON: EVOLUTION AND CURRENT STATUS.

BACKGROUND: In the absence of surveillance data and consolidated information about tuberculosis (TB) and its drug resistance burden in Lebanon, this retrospective study was conducted to shed light on TB and its other relevant aspects over the last 15 years in this country. METHODS: To generate the TB data for this retrospective study, two main sources were used: 1) the records of patients in the National Tuberculosis Program (NTP); 2) the results of the Clinical Microbiology Laboratory (CML) at the American University of Beirut Medical Center (AUBMC). The TB data review pertained to its epidemiological aspect, implementation of the directly observed therapy strategy (DOTS) all over, the gender distribution, the impact of high risk groups (non-national population, Syrian refugees, patients with multi-drug resistance-TB [MDR-TB] and the inmate population) on the trend of TB in Lebanon between 1999 and 2013. Reviewed also are TB in children, extrapulmonary tuberculosis and the mycobacterium other than tuberculosis (MOTT). RESULTS: During the last 15 years, 7548 TB cases were diagnosed and evaluated at the NTP. After the decreasing of TB incidence from 13/100 000 population in 2001 to 9/100 000 in 2006, the incidence started to increase in 2007, reaching 20/100 000 in 2013, mostly due to increased cases among non-national population. Fluctuations in TB rates over the years were seen among children, inmates, MDR-TB, and HIV patients. MOTT isolates recovery rates also fluctuated during the study period, M. simiae being the most common. CONCLUSION: After the decreasing trends of TB incidence between 1999 and 2006, Lebanon has been experiencing an increasing incidence in tuberculosis population since 2007. This is mainly attributed to the dramatic increase of TB patients among non-nationals and the influx of Syrian refugees. The ongoing collaboration between the public and private sectors, improvements of the surveillance system and TB control are important factors for successful elimination of TB in this country.

Hydatid disease: the Lebanese contribution.

Hydatid disease (HD) or cystic echinococcosis (CE) has been an important zoonotic disease causing medical, economic and public health problems in many parts of the world, including South America, North Africa, Turkey, and Middle Eastern countries. Humans as well as animals, primarily sheep and cattle, are infected by the ingestion of food, usually leafy vegetables, contaminated with the eggs (oncospheres) of the dog tapeworm Echinococcus granulosus. Hydatid cysts, which are the larval stage of the parasite, are located mostly in the liver and lungs of the infected host. Because of its chronic endemicity in Lebanon and neighboring countries, this disease has constituted an integral part of research studies conducted by medical doctors and researchers in Lebanon, mostly spearheaded by those at the American University of Beirut (AUB) and its medical center (AUBMC) since the early turn of the last century (1920s). Over 130 wide ranging studies were published; some were innovative e.g. the introduction of the once famous Indirect Haemagglutination (IHA) test for serodiagnosis, and the use of dilute cetrimide as a protoscolicidal agent during surgery. Although the incidence of HD is decreasing in our country, it has acquired increasing public health concern and is considered as an emerging or re-emerging disease in many parts of the world. In this review, we shed light on the numerous studies/publications done in Lebanon as a tribute to those researchers who have impacted the literature of HD in many aspects. The latter include epidemiology and ways of transmission, clinical features and radiological tools for diagnosis, serodiagnosis and immunology, and investigation of different therapeutic modalities for different aspects of the disease. Moreover, consolidating these studies in this review would hopefully represent the historic foundation for interested researchers and investigators, especially in this country, to pursue and build on such studies. The advances in technology, and the availability and utilization of new methodologies will hopefully help find more reliable and efficient ways for the diagnosis, and management of this disease.

Molecular characterization of carbapenem and ceftazidime-avibactam-resistant Enterobacterales and horizontal spread of bla NDM-5 gene at a Lebanese medical center.

Introduction: In the battle against multidrug-resistant bacterial infections, ceftazidime- avibactam (CZA) stands as a pivotal defense, particularly against carbapenemresistant (CR) Gram-negative pathogens. However, the rise in resistance against this drug poses a significant threat to its effectiveness, highlighting the critical need for in-depth studies about its resistance mechanisms. Methods: This research focuses on the genomic characterization of CR- and CZA-resistant Escherichia coli (n=26) and Klebsiella pneumoniae (n=34) strains, harboring the blaNDM and/or blaOXA-48-like genes, at a major Lebanese tertiary care medical center, using whole genome sequencing (WGS). Results: Our findings revealed a notable prevalence of blaNDM in all K. pneumoniae strains isolates, with 27 of these also harboring blaOXA-48. On the other hand, E. coli strains predominantly carried the blaNDM-5 gene. Whole genome sequencing (WGS) identified a predominance of ST383 among K. pneumoniae strains, which possessed a multi-replicon IncFIB-IncHI1B plasmid harboring the blaNDM-5. Additionally, various Inc group plasmids in K. pneumoniae across multiple sequence types were found to carry the blaNDM. Similarly, diverse STs of E. coli were observed to carry blaNDM-5 on different plasmids. Discussion: The study underscores NDM carbapenemases as a paramount resistance mechanism in Lebanon,jeopardizing critical last-resort treatments. It also illuminates the role of varied sequence types and mobile genetic elements in the spread of NDM resistance,stressing the urgent need for strategies to mitigate this threat, especially in nosocomial infections.

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.

Molecular Characteristics of Colistin Resistance in Acinetobacter baumannii and the Activity of Antimicrobial Combination Therapy in a Tertiary Care Medical Center in Lebanon.

(1) Background: Infections with pan-drug-resistant (PDR) bacteria, such as A. baumannii, are becoming increasingly common, especially in healthcare facilities. In this study, we selected 15 colistin-resistant clinical A. baumannii isolates from a hospital in Beirut, Lebanon, to test combination therapies and determine their sequence types (STs) and the mechanism of colistin resistance using whole-genome sequencing (WGS). (2) Methods: Antimicrobial susceptibility testing via broth microdilution against 12 antimicrobials from different classes and growth rate assays were performed. A checkerboard assay was conducted on PDR isolates using six different antimicrobials, each in combination with colistin. Genomic DNA was extracted from all isolates and subjected to WGS. (3) Results: All isolates were resistant to all tested antimicrobials with the one exception that was susceptible to gentamicin. Combining colistin with either meropenem, ceftolozane-tazobactam, or teicoplanin showed synergistic activity. Sequencing data revealed that 67% of the isolates belonged to Pasteur ST2 and 33% to ST187. Furthermore, these isolates harbored a number of resistance genes, including blaOXA-23. Mutations in the pmrC gene were behind colistin resistance. (4) Conclusions: With the rise in antimicrobial resistance and the absence of novel antimicrobial production, alternative treatments must be found. The combination therapy results from this study suggest treatment options for PDR ST2 A. baumannii-infected patients.

Prevalence of intestinal parasites during pre- and post-COVID-19 pandemic at a tertiary care center in Lebanon.

INTRODUCTION: Intestinal parasitic infections (IPIs) are a major medical and public health problem, especially in developing countries. This study aimed at comparing the prevalence and types of IPI during pre- and post-COVID-19 pandemics, and with data reported in Lebanon a decade ago. METHODOLOGY: Stool specimen results from a total of 4,451 and 4,158 patients were examined using the concentration method during the pre-covid (2017-2018) and post-covid (2020-2021) pandemic periods, respectively. Demographic information related to patient's age and gender was recorded. RESULTS: The overall positive detected parasites among the total tested in these two periods were 589 (13.2%) and 310 (7.5%), respectively. The protozoa accounted for most parasites (e.g., Blastocystis hominis, Entamoeba coli (E. coli), Entamoeba histolytica, and Giardia lamblia). Only B. hominis and E. coli showed significant differences; B. hominis was more prevalent in the post-covid period (33.5%) whereas E. coli in the pre-covid phase (44.5%). Among gender, E. histolytica was higher in males during the post-covid period (13.3% vs. 6.3%). Regarding age, adults (between 26 and 55 years) had the highest prevalence, with a noticeable decrease among the elderly in the post-covid time. Compared to the previous decade, the prevalence of B. hominis and E. coli remained higher, and that of E. histolytica and G. lamblia was almost the same. CONCLUSIONS: These findings indicate an overall reduction in the prevalence of IPI during the post-covid period, though IPIs persistence remains high. This highlights the need for enhancing public health awareness efforts to improve hygiene and sanitation to reduce parasitic prevalence in Lebanon.

Whole Genome-Based Characterization of Multidrug Resistant Enterobacter and Klebsiella aerogenes Isolates from Lebanon.

Enterobacter spp. and Klebsiella aerogenes are rod-shaped Gram-negative opportunistic pathogens. This study aimed at the molecular and genomic characterization of multidrug resistant Enterobacter spp. and K. aerogenes isolates recovered from hospitalized patients in a tertiary care hospital in Lebanon. A total of 59 Enterobacter spp. clinical isolates consisting of 41 carbapenem-resistant and 18 susceptible by Etest were included in this study. Genotypic identification through whole-genome sequencing (WGS) was performed and confirmed in silico. Resistance and plasmid profiles were studied using ResFinder4.0 and Plasmid-Finder2.1. Multilocus sequence typing (MLST) was used to determine the isolates' clonality. Using the average nucleotide identity (ANI) we identified and confirmed that 47 (80%) isolates were E. hormaechei, 11 (18%) were Klebsiella aerogenes and 1 (2%) was an E. cloacae. Carbapenem-resistance was detected among 41 isolates all showing an MIC90 of ≥ 32 µg/mL for ertapenem, imipenem, and meropenem. blaNDM-1 (58.5%), blaACT-16 (54%), and blaOXA-1 (54%) were the most common detected ß-lactamases, while blaCTX-M-15 (68%) was the main detected extended-spectrum ß-lactamase (ESBL) encoding gene. Chromosomal ampC, carbapenemase encoding genes, and porin modifications were among the detected carbapenem resistance determinants. The carbapenemase encoding genes were linked to three well-defined plasmid Inc groups, IncFII/IncFIB, IncX3, and IncL. MLST typing revealed the diversity within the studied isolates, with ST114 being the most common among the studied E. hormaechei.: The spread of carbapenem-resistant isolates in clinical settings in Lebanon is a serious challenge. Screening and continuous monitoring through WGS analysis could effectively limit the dissemination of drug-resistant isolates in hospitalized patients. IMPORTANCE Drug resistance is an increasing global public health threat that involves most disease-causing organisms and antimicrobial drugs. Drug-resistant organisms spread in health care settings, and resistance to multiple drugs is common. Our study demonstrated the mechanisms leading to resistance against the last resort antimicrobial agents among members of the Enterobacteriaceae family. The spread of carbapenem-resistant bacteria in clinical settings is a serious challenge. Screening and continuous monitoring could effectively limit the dissemination of drug-resistant isolates in hospitalized patients.

Genomic surveillance of SARS-CoV-2 in COVID-19 vaccinated healthcare workers in Lebanon.

BACKGROUND: The emergence of SARS-CoV-2 variants including the Delta and Omicron along with waning of vaccine-induced immunity over time contributed to increased rates of breakthrough infection specifically among healthcare workers (HCWs). SARS-CoV-2 genomic surveillance is an important tool for timely detection and characterization of circulating variants as well as monitoring the emergence of new strains. Our study is the first national SARS-CoV-2 genomic surveillance among HCWs in Lebanon. METHODS: We collected 250 nasopharyngeal swabs from HCWs across Lebanon between December 2021 and January 2022. Data on the date of positive PCR, vaccination status, specific occupation, and hospitalization status of participants were collected. Extracted viral RNA from nasopharyngeal swabs was converted to cDNA, library prepped using the coronaHIT method, followed by whole genome sequencing on the Illumina NextSeq 500 platform. RESULTS: A total of 133 (57.1%) samples belonging to the Omicron (BA.1.1) sub-lineage were identified, as well as 44 (18.9%) samples belonging to the BA.1 sub-lineage, 28 (12%) belonging to the BA.2 sub-lineage, and only 15 (6.6%) samples belonging to the Delta variant sub-lineage B.1.617.2. These results show that Lebanon followed the global trend in terms of circulating SARS-CoV-2 variants with Delta rapidly replaced by the Omicron variant. CONCLUSION: This study underscores the importance of continuous genomic surveillance programs in Lebanon for the timely detection and characterization of circulating variants. The latter is critical to guide public health policy making and to timely implement public health interventions.

The under investigated facet of the COVID-19 pandemic: Molecular analysis of secondary bacterial infections at a COVID dedicated intensive care unit within a tertiary care center in Lebanon.

Background: The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide. Secondary bacterial infections are associated with unfavorable outcomes in respiratory viral infections. This study aimed at determining the prevalence of secondary bacterial infections in COVID-19 patients admitted at a tertiary medical center in Lebanon. Methodology: From May till November, 2020, a total of 26 Gram-negative isolates were recovered from 16 patients during the course of their COVID-19 infection with Escherichia coli being the most prevalent. The isolates were assessed for their antimicrobial susceptibility by broth microdilution against 19 antimicrobial agents from different classes. Whole genome sequencing of 13 isolates allowed the mining of antimicrobial resistance (AMR) determinants as well as mobile genetic elements and sequence types (ST). Finally, broth microdilution with three different efflux pump inhibitors [theobromine, conessine and PheArg-ß-naphthylamide (PAßN)] was done. Results: Antimicrobial susceptibility testing showed that out of the 26 Gram-negative isolates, 1 (4%) was extensively drug resistant and 14 (54%) were multi-drug resistant (MDR). Whole genome sequencing results revealed a plethora of AMR determinants among the 13 sequenced isolates. Moreover, the 9 Enterobacterales and 4 Pseudomonas aeruginosa sequenced isolates belonged to 9 and 2 different ST, respectively. Using a variety of efflux pump inhibitors we demonstrated that only PAßN had a significant effect when combined with levofloxacin, and the latter regained its activity against two P. aeruginosa isolates. Conclusion: The identification of carbapenem and colistin resistant Gram-negative bacilli causing secondary bacterial infections in critical patients diagnosed with COVID-19 should be of high concern. Additionally, it is crucial to monitor and track AMR, post-COVID pandemic, in order to better understand the effect of this disease on AMR exacerbation.

Molecular characterization and differential effects of levofloxacin and ciprofloxacin on the potential for developing quinolone resistance among clinical Pseudomonas aeruginosa isolates.

Background: Fluoroquinolones are some of the most used antimicrobial agents for the treatment of Pseudomonas aeruginosa. This study aimed at exploring the differential activity of ciprofloxacin and levofloxacin on the selection of resistance among P. aeruginosa isolates at our medical center. Methods: 233 P. aeruginosa clinical isolates were included in this study. Antimicrobial susceptibility testing (AST) was done using disk diffusion and broth microdilution assays. Random Amplification of Polymorphic DNA (RAPD) was done to determine the genetic relatedness between the isolates. Induction of resistance against ciprofloxacin and levofloxacin was done on 19 isolates. Fitness cost assay was done on the 38 induced mutants and their parental isolates. Finally, whole genome sequencing was done on 16 induced mutants and their 8 parental isolates. Results: AST results showed that aztreonam had the highest non-susceptibility. RAPD results identified 18 clusters. The 19 P. aeruginosa isolates that were induced against ciprofloxacin and levofloxacin yielded MICs ranging between 16 and 256 µg/mL. Levofloxacin required fewer passages in 10 isolates and the same number of passages in 9 isolates as compared to ciprofloxacin to reach their breakpoints. Fitness cost results showed that 12 and 10 induced mutants against ciprofloxacin and levofloxacin, respectively, had higher fitness cost when compared to their parental isolates. Whole genome sequencing results showed that resistance to ciprofloxacin and levofloxacin in sequenced mutants were mainly associated with alterations in gyrA, gyrB and parC genes. Conclusion: Understanding resistance patterns and risk factors associated with infections is crucial to decrease the emerging threat of antimicrobial resistance.

Genotypes and serotype distribution of macrolide resistant invasive and non-invasive Streptococcus pneumoniae isolates from Lebanon.

BACKGROUND: This study determined macrolide resistance genotypes in clinical isolates of Streptococcus pneumoniae from multiple medical centers in Lebanon and assessed the serotype distribution in relation to these mechanism(s) of resistance and the source of isolate recovery. METHODS: Forty four macrolide resistant and 21 macrolide susceptible S. pneumoniae clinical isolates were tested for antimicrobial susceptibility according to CLSI guidelines (2008) and underwent molecular characterization. Serotyping of these isolates was performed by Multiplex PCR-based serotype deduction using CDC protocols. PCR amplification of macrolide resistant erm (encoding methylase) and mef (encoding macrolide efflux pump protein) genes was carried out. RESULTS: Among 44 isolates resistant to erythromycin, 35 were resistant to penicillin and 18 to ceftriaxone. Examination of 44 macrolide resistant isolates by PCR showed that 16 isolates harbored the erm(B) gene, 8 isolates harbored the mef gene, and 14 isolates harbored both the erm(B) and mef genes. There was no amplification by PCR of the erm(B) or mef genes in 6 isolates. Seven different capsular serotypes 2, 9V/9A,12F, 14,19A, 19F, and 23, were detected by multiplex PCR serotype deduction in 35 of 44 macrolide resistant isolates, with 19F being the most prevalent serotype. With the exception of serotype 2, all serotypes were invasive. Isolates belonging to the invasive serotypes 14 and 19F harbored both erm(B) and mef genes. Nine of the 44 macrolide resistant isolates were non-serotypable by our protocols. CONCLUSION: Macrolide resistance in S. pneumoniae in Lebanon is mainly through target site modification but is also mediated through efflux pumps, with serotype 19F having dual resistance and being the most prevalent and invasive.

In vitro activity of fosfomycin and other antimicrobials against uropathogenic Escherichia coli and Klebsiella pneumoniae at a tertiary care center in Lebanon.

BACKGROUND: The incidence of urinary tract infections (UTIs) caused by extended-spectrum-beta-lactamase (ESBL) producing Escherichia coli (ESBL-EC) and Klebsiella pneumoniae (ESBL-KP) has been on the rise limiting oral treatment options. Fosfomycin was reported to be highly efficacious against these organisms, however, data is lacking in Lebanon and the Middle East. OBJECTIVE: To determine the in vitro activity of fosfomycin against ESBL and non-ESBL-producing E. coli and K. pneumoniae uropathogens in Lebanon. METHODS: A total of 542 consecutive non-duplicate isolates of ESBL-producing E. coli (n = 374) and K. pneumoniae (n = 168), and 291 isolates of non-ESBL-producing E. coli (n = 236) and K. pneumoniae (n = 55) were recovered from urine specimens of patients tested at the American University of Beirut Medical Center (AUBMC) during 2010. Each isolate was tested against a battery of 13 antimicrobials by disk diffusion according to the guidelines of CLSI testing and result interpretation criteria. RESULTS: The fosfomycin susceptibility for ESBL-producing vs. non-ESBL-producing isolates was 86% vs. 97% for E. coli and 62% vs. 78% for K. pneumoniae. This activity of fosfomycin among ESBL-EC and ESBL-KP was generally higher than cefepime (26% & 30%), ciprofloxacin (24% & 41%), Trimeth/sulfa (26% & 19%), Pip/taz (75% and 45%), gentamicin (45% & 42%), and tobramycin (32% & 26%). On the other hand, higher activity against both species of ESBL-producing bacteria was shown by amikacin (96% & 79%) and imipenem (99.7% & 98.8%). Nitroflurantoin was highly active against ESBL-EC (95%) but not against ESBL-KP (29%). CONCLUSION: Fosfomycin shows good activity, being higher against ESBL-producing E. coli than K. pneumoniae uropathogens in Lebanon.

A reflection on bacterial resistance to antimicrobial agents at a major tertiary care center in Lebanon over a decade.

BACKGROUND: Antimicrobial resistance has been inflecting deleterious health and economic consequences locally and globally. This study addresses the patterns and trends of bacterial resistance to antimicrobial agents over a decade, at a major tertiary care center in Beirut. METHODS: Data on bacterial susceptibility patterns at the CAP accredited Clinical Microbiology Laboratory is analyzed from January 2000 to November 2011, along with related different studies conducted during this period. RESULTS: Increasing rates of ESBL-producing isolates were noted for Escherichia coli, Klebsiella pneumoniae, Salmonella spp. and Shigella spp. Resistance to carbapenems remains problematic in Acinetobacter spp, and Pseudomonas aeruginosa, and started emerging in E. coli and K. pneumoniae. Tigecycline and colistin maintained excellent activity against most ESBL and carbapenem resistant bacteria relevant to the treatment by these agents. Resistance to quinolones is being encountered in Streptococcus pneumoniae, Haemophilus influenzae, Salmonella spp. and Shigella spp. Methicillin resistant Staphylococcus aureus (MRSA), though remaining relatively high, showed decreasing trends of resistance, while vancomycin maintain uniform activity. Rare and sporadic vancomycin resistant strains in enterococci are encountered. Macrolide and clindamycin increasing rates of resistance is noted in S. pneumoniae, group A streptococci, S. aureus, viridans streptococci and some others. CONCLUSION: Physicians should be aware of the local epidemiology of antimicrobial resistance to properly guide the initial therapy. These resistance problems can be attributed to uncontrolled use of antimicrobial agents, thus, highlighting the need for antimicrobial stewardship to curb this threat.

Genomic Characterization of Mutli-Drug Resistant Pseudomonas aeruginosa Clinical Isolates: Evaluation and Determination of Ceftolozane/Tazobactam Activity and Resistance Mechanisms.

Resistance to ceftolozane/tazobactam (C/T) in Pseudomonas aeruginosa is a health concern. In this study, we conducted a whole-genome-based molecular characterization to correlate resistance patterns and ß-lactamases with C/T resistance among multi-drug resistant P. aeruginosa clinical isolates. Resistance profiles for 25 P. aeruginosa clinical isolates were examined using disk diffusion assay. Minimal inhibitory concentrations (MIC) for C/T were determined by broth microdilution. Whole-genome sequencing was used to check for antimicrobial resistance determinants and reveal their genetic context. The clonal relatedness was evaluated using MLST, PFGE, and serotyping. All the isolates were resistant to C/T. At least two ß-lactamases were detected in each with the blaOXA-4, blaOXA-10, blaOXA-50, and blaOXA-395 being the most common. blaIMP-15, blaNDM-1, or blaVIM-2, metallo-ß-lactamases, were associated with C/T MIC >256 µg/mL. Eight AmpC variants were identified, and PDC-3 was the most common. We also determined the clonal relatedness of the isolates and showed that they grouped into 11 sequence types (STs) some corresponding to widespread clonal complexes (ST111, ST233, and ST357). C/T resistance was likely driven by the acquired OXA ß-lactamases such as OXA-10, and OXA-50, ESBLs GES-1, GES-15, and VEB-1, and metallo- ß-lactamases IMP-15, NDM-1, and VIM-2. Collectively, our results revealed C/T resistance determinants and patterns in multi-drug resistant P. aeruginosa clinical isolates. Surveillance programs should be implemented and maintained to better track and define resistance mechanisms and how they accumulate and interact.

The impact of vaccination on the burden of invasive pneumococcal disease from a nationwide surveillance program in Lebanon: an unexpected increase in mortality driven by non-vaccine serotypes.

BACKGROUND: The impact of pneumococcal conjugate vaccines (PCVs) on the burden of invasive pneumococcal disease (IPD) and serotype distribution was examined across age groups from data collected by the Lebanese Inter-Hospital Pneumococcal Surveillance Program. METHODS: Between 2005 and 2020, 593 invasive Streptococcus pneumoniae isolates were collected from 79 hospitals throughout Lebanon. Serotypes and antimicrobial resistance (AMR) profiles were identified, and trends compared over 3 eras: PCV7, post-PCV7/ pre-PCV13, and PCV13 eras. RESULTS: The prevalence of PCV7 serotypes decreased significantly from 43.6% in the PCV7 era to 17.8% during the PCV13 era (p<0.001). PCV13-only serotypes remained stable in the PCV13 compared to the post-PCV7 eras, especially serotypes 1 and 3, whereas non-vaccine types (NVT) increased throughout the study period, especially 24 and 16F. The mortality rate increased substantially from 12.5% (PCV7 era) to 24.8% (PCV13 era). A significant decrease in AMR was observed across the three study eras. CONCLUSION: PCVs substantially impacted IPD and AMR in vaccinated and unvaccinated populations despite an increase in mortality driven by NVT. Broadening the recommendation of vaccination to include older age-groups, using higher valency vaccines, and implementing stringent antimicrobial stewardship are likely to further impact the burden of IPD.

Nationwide proficiency assessment of bacterial identification and antimicrobial susceptibility testing among 110 laboratories in Lebanon.

INTRODUCTION: Proficiency testing (PT) is one of the most valuable and important activities for the Clinical Microbiology Laboratories (CML) to enroll in to ensure the accuracy and reliability of results. This first time conducted nationwide study was warranted to assess the PT performance activity among CML in Lebanon. METHODOLOGY: Four training and PT activities were organized for 110 nationwide laboratories involved in providing clinical microbiology services. In each PT activity, five different bacterial species were distributed to each laboratory to provide identification (ID) and antimicrobial susceptibility testing (AMST) according to prior discussions and guidelines. RESULTS: The percentages of labs that correctly identified the bacterial species and performed the relevant AMST to it, respectively, were as follows: S. aureus, (100% and 67.8%); Enterococcus faecalis (71% and 82%); Listeria monocytogenes (75% and 61%); Streptococcus agalactiae (86% and 71%); Corynebacterium amycolatum (7% and 33 %); Pseudomonas aeruginosa, (93 % and 53.4%); Klebsiella pneumoniae, (97% and 67.7%); Salmonella typhi ESBL (87 % and 66%); Enterobacter aerogenes (89% and 59%) and Stenotrophomonas maltophilia (84 % and 65%). The resistant types for the species were specified by labs as carbapenem resistant (CR) K. pneumoniae in 78 %, CR E. aerogenes in 34 %, MRSA in 83 %, and VRE in 80.5%. CONCLUSIONS: The wide variation as well as the overall humble scoring of accurate results reflects the dire need for the MOPH to establish and maintain a PT activity program, and entrust the reference laboratory to provide continuing education and training sessions.