Antimicrobial Activity of Five Calcium Silicate Based Root Canal Sealers against a Multispecies Engineered Biofilm: An In Vitro Study.

AIM: The present study's objective was to compare the impact of CerasealR, total fill BC SealerR, Bio-C SealerR, AH Plus BioceramicR, and K-BiocerR on the elimination of a multispecies' endodontic biofilm at 3, 7 and 14 days. MATERIALS AND METHODS: A total of 20 freshly extracted, caries-free premolars were prepared for the study to create dentinal disks. For the multispecies biofilm formation, Enterococcus faecalis, Proteus mirabilis, Pseudomonas aeruginosa, and Candida albicans were cultured and used to inoculate hydroxyapatite discs. After incubation, the biofilms were placed on blotting papers in petri dishes with an orthodontic bend. Different root canal sealers, including CeraSeal, total Fill BC Sealer, Bio-C Sealer, AH Plus Bioceramic, K-Biocer, and Sealite, were injected into the bend, facilitating contact with the biofilms. The samples were divided into seven groups, including a negative control. At specific intervals, 3, 7, and 14 days, 3 biofilm samples from each group were collected, diluted, and plated on Agar media for colony counting and analysis. RESULTS: In all tested groups, the total bacterial count significantly decreased between day 3 and 14 (p < 0.05) with no statistically significant differences among the different sealers' groups at all-time points for the total bacterial count, E. faecalis count, and P. mirabilis count. However, Sealite demonstrated the most consistent effectiveness in reducing bacterial counts across multiple categories. The sealite group was capable of decreasing the C. albicans count significantly between day 3 and day 14 (p < 0.05) in comparison with the bioceramic groups. CONCLUSION: All sealers had antibacterial activity against the multispecies biofilm between day 3 and day 14. The ascending order of sealers in terms of their effectiveness in killing bacteria, based on the provided results, is as follows: Sealite, Bio-C Sealer, AH Plus, CeraSeal, TotalFill, and K-Biocer. However, there were no statistically significant differences in the bacterial counts among the different sealer groups at any time point. CLINICAL SIGNIFICANCE: The role of sealers in combating biofilm-associated infections highlights their potential clinical utility in preserving root canal health. Understanding the antimicrobial properties of these sealers is vital for informed decision-making in selecting the most effective materials for improved treatment outcomes and long-term success in endodontic procedures.

In vitro evaluation of three engineered multispecies endodontic biofilms on a dentinal disk substrate.

The aim of this study was the development of a complex multispecies endodontic biofilm using Candida albicans, Proteus mirabilis and Pseudomonas aeruginosa on a biofilm of Enterococcus faecalis in a dentinal substrate design. The endodontic pathology is a biofilm-mediated infection, and the aim of root canal therapy is to reduce, as much as possible, the bacterial population. Thus, it is important to develop a laboratory endodontic biofilm to test the effect of new irrigation and obturation techniques on reduction of bacterial count. The culture of Enterococcus faecalis from ATCC 29212 began with aerobic cultivation on blood agar, followed by transfer to Brain Heart Infusion (BHI) broth with 5% sucrose. Incubation occurred in a shaker at 37 °C for 24 h, followed by an additional 24-h static phase. After 10 d, Proteus mirabilis, Pseudomonas aeruginosa, and Candida albicans were introduced sequentially in three distinct groups. Group 1: the order of addition was Candida albicans, Proteus mirabilis, and Pseudomonas aeruginosa; Group 2: the order was Pseudomonas aeruginosa, Candida albicans, and Proteus mirabilis; and Group 3: Proteus mirabilis, Pseudomonas aeruginosa, and Candida albicans. After 16 days, the biofilm was carefully extracted, transferred to sterile BHI, and dissected using a sterile needle technique. Subsequently, an optical density test, bacterial counts, and colony enumeration were performed on various agar plates. Group 2 in which Pseudomonas aeruginosa was added directly after Enterococcus faecalis followed by Candida albicans and Proteus mirabilis showed significantly greater total bacterial count than the other two groups.

Resistome Diversity and Dissemination of WHO Priority Antibiotic Resistant Pathogens in Lebanese Estuaries.

Anthropogenic pressure is known to be a key driver of antimicrobial resistance (AMR) dissemination in the environment. Especially in lower income countries, with poor infrastructure, the level of AMR dissemination is high. Therefore, we assessed the levels and diversity of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in Lebanese rivers at estuaries' sites (n = 72) of the Mediterranean Sea in spring 2017 and winter 2018. METHODS: A combined approach using culture techniques and high throughput qPCR were applied to identify ARB and ARGs in rivers along the Lebanese coast. RESULTS: Multidrug-resistant Gram-negative (Enterobacterales and Pseudomonas spp.) and Gram-positive bacterial pathogens were isolated. Levels of ARGs were highest in the winter campaign and areas with high anthropogenic activities and population growth with an influx of refugees. CONCLUSION: Qualitative analysis of ARB and the analysis of the Lebanese estuaries' resistome revealed critical levels of contamination with pathogenic bacteria and provided significant information about the spread of ARGs in anthropogenically impacted estuaries.

Bactericidal effects and stability of LL-37 and CAMA in the presence of human lung epithelial cells.

Cationic antimicrobial peptides (CAMPs) are important actors in host innate immunity and represent a promising alternative to combat antibiotic resistance. Here, the bactericidal activity of two CAMPs (LL-37 and CAMA) was evaluated against Pseudomonas aeruginosa (PA) in the presence of IB3-1 cells, a cell line derived from patients with cystic fibrosis. The two CAMPs exerted different effects on PA survival depending on the timing of their administration. We observed a greater bactericidal effect when IB3-1 cells were pretreated with sub-minimum bactericidal concentrations (Sub-MBCs) of the CAMPs prior to infection. These findings suggest that CAMPs induce the production of factors by IB3-1 cells that improve their bactericidal action. However, we observed no bactericidal effect when supra-minimum bactericidal concentrations (Supra-MBCs) of the CAMPs were added to IB3-1 cells at the same time or after infection. Western-blot analysis showed a large decrease in LL-37 levels in supernatants of infected IB3-1 cells and an increase in LL-37 binding to these cells after LL-37 administration. LL-37 induced a weak inflammatory response in the cells without being toxic. In conclusion, our findings suggest a potential prophylactic action of CAMPs. The bactericidal effects were low when the CAMPs were added after cell infection, likely due to degradation of CAMPs by bacterial or epithelial cell proteases and/or due to adherence of CAMPs to cells becoming less available for direct bacterial killing.

Occurrence of the Colistin Resistance Gene mcr-1 and Additional Antibiotic Resistance Genes in ESBL/AmpC-Producing Escherichia coli from Poultry in Lebanon: A Nationwide Survey.

The objective of the present study was to determine genomic characteristics of expanded-spectrum cephalosporin (ESC)-resistant Escherichia coli spreading in healthy broilers in Lebanon in 2018. Rectal swabs (n = 280) from 56 farms were screened for the presence of ESC-resistant E. coli isolates. Antimicrobial susceptibility and extended-spectrum ß-lactamase (ESBL)/AmpC production were determined by the disk diffusion method. Whole-genome sequencing (WGS) of 102 representative isolates of E. coli was performed to determine their phylogenetic diversity, serotypes, sequence types (ST), acquired resistance genes, and virulence-associated genes. Fifty-two out of 56 farms housed broilers carrying ESC-resistant E. coli isolates. These farms had large and recurrent antimicrobial practices, using, for some of them, critically important antibiotics for prophylactic and therapeutic purposes. Among the 102 sequenced multidrug-resistant (MDR) E. coli isolates, the proportion of ESBL, plasmid-mediated AmpC ß-lactamase (pAmpC) producers, and ESBL/pAmpC coproducers was 60%, 27.6%, and 12.4%, respectively. The most prevalent ESBL/pAmpC genes were blaCMY-2, blaCTX-M-3, blaCTX-M-15, blaCTX-M-27, and blaCTX-M-14b (n = 42, n = 31, n =15, n = 9, and n = 7, respectively). These ESBL/pAmpC producers were distributed in different STs, most being well-known avian-associated and sometimes pathogenic STs (ST-10, ST-48, ST-93, ST-115, ST-117, and ST-457). Phylogenetic single nucleotide polymorphism (SNP) analysis confirmed their genetic diversity and wide dispersion across the Lebanese territory. Most isolates were also resistant to ciprofloxacin (101/102 with 3 QRDR mutations), and 19/102 isolates from 11 unrelated STs also carried the mobile resistance gene mcr-1. This survey illustrates the alarming prevalence of MDR E. coli resistant to medically important antibiotics in broilers in Lebanon. This advocates the need for surveillance programs of antimicrobial resistance in Lebanon and the reduction of excessive use of antibiotics to limit the spread of MDR E. coli in food-producing animals. IMPORTANCE Poultry production is a main contributor of the global trend of antimicrobial resistance arising from food-producing animals worldwide. In Lebanon, inappropriate use of antibiotics is frequent in chickens for prophylactic reasons and to improve productivity, resulting in an alarming prevalence of extended-spectrum ß-lactamase (ESBL)/AmpC-producing Escherichia coli, also resistant to other medically important antibiotics (i.e., colistin and ciprofloxacin). Their complex genomic epidemiology highlighted by an important genetic diversity suggests that these resistance determinants are largely spreading in enteric bacteria in Lebanese poultry. Further molecular surveillance is needed to understand the country-specific epidemiology of ESBL/AmpC and mcr-1 genes in Lebanese poultry production. In addition, decisive interventions are urgently needed in order to ban the use of critically important antibiotics for human medicine in food-producing animals and limit the spread of antibiotic resistance in Lebanon.

Assessment of the performance of CHROMagar KPC and Xpert Carba-R assay for the detection of carbapenem-resistant bacteria in rectal swabs: First comparative study from Abu Dhabi, United Arab Emirates.

OBJECTIVES: The objective of this study was to evaluate the performance of CHROMagar™ KPC compared with Xpert® Carba-R assay for the detection of carbapenem-resistant bacterial isolates from rectal swabs. METHODS: Rectal swabs were obtained from patients admitted to Cleveland Clinic Abu Dhabi (United Arab Emirates) over a period of 7 months and were screened for carbapenem resistance by either culture on CHROMagar KPC or carbapenemase production using the Xpert Carba-R molecular method. Further testing for carbapenem susceptibility of isolates recovered from CHROMagar KPC was performed using VITEK®2. RESULTS: A total of 1813 rectal swabs were screened, of which 61 (3.4%) were positive for carbapenem resistance by either one or both methods. Both methods were equally efficient in detecting carbapenem resistance in 37/61 swabs (60.7%), mostly positive for Klebsiella pneumoniae (22 isolates), of which 40.9% (9/22) carried blaOXA-48-like and blaNDM. Xpert Carba-R assay detected 12 additional swabs with negative CHROMagar KPC culture and revealed additional carbapenemase-producing organisms carrying blaOXA-48-like and/or blaNDM. CHROMagar KPC recovered organisms in nine swabs not detected by the genotypic method, 44.4% of which were K. pneumoniae. Three swabs yielded false-positive results (carbapenem-susceptible organisms) by both methods. Sensitivity and specificity were, respectively, 75.4% and 99.8% for CHROMagar KPC and 80% and 99.8% for Xpert Carba-R. CONCLUSION: This comparative study of CHROMagar KPC versus Xpert Carba-R in rectal swabs showed a slightly higher sensitivity for the PCR-based method. Whilst CHROMagar KPC provides a less expensive screening method, Xpert Carba-R may be more accurate and faster.

Phenotypic and genotypic detection of ß-lactamases in Acinetobacter spp. isolates recovered from Lebanese patients over a 1-year period.

OBJECTIVES: The aim of this study was to determine the prevalence of ß-lactamases in Acinetobacter spp. recovered from Lebanese patients over a 1-year period using phenotypic and molecular methods. METHODS: A total of 100 non-duplicate consecutive Acinetobacter spp. isolates were collected from various clinical specimens. Antimicrobial susceptibility testing was performed by the disk diffusion method. Susceptibility to colistin, imipenem and meropenem was determined by broth microdilution. The ß-lactamase inhibitors phenylboronic acid, cloxacillin and ethylene diamine tetra-acetic acid (EDTA) were used for presumptive detection of KPC-type ß-lactamase, AmpC ß-lactamase and metallo-ß-lactamase (MBL), respectively. Simplex PCR was conducted for molecular detection of ß-lactamases. Trilocus PCR typing was performed to determine the clonality of the isolates. RESULTS: Among the 100 Acinetobacter spp. isolates, 78% were resistant to imipenem and 84% to meropenem. Only one isolate was resistant to colistin by the microdilution method. Phenotypically, 23% of the isolates were presumptively diagnosed as producing extended-spectrum ß-lactamase (ESBL), 15% as producing KPC and 4% MBL, whilst 5% were diagnosed as overproducing AmpC ß-lactamase. The blaOXA-51-like gene was detected in 99% of isolates, blaADC in 93%, blaOXA-23-like in 77% and blaOXA-24/40-like in 3%. Trilocus PCR identified 86% (82/95) of the Acinetobacter baumannii isolates as international clone II (IC II). CONCLUSIONS: A high rate of carbapenem resistance, with a predominance of OXA-23-like and IC II, was shown in this study. Moreover, the inhibitor-based method was shown not to be accurate for the prediction of carbapenemases in A. baumannii.

Heterogeneity of Carbapenem Resistance Mechanisms Among Gram-Negative Pathogens in Lebanon: Results of the First Cross-Sectional Countrywide Study.

Carbapenem-resistant Gram-negative pathogens have progressively disseminated to different countries worldwide, presenting a serious public health concern. The aims of this study were to determine the prevalence of carbapenem resistance in Gram-negative bacteria in Lebanon, to elucidate molecular mechanisms, and to identify genetic relatedness of incriminated strains. Carbapenem nonsusceptible Enterobacteriaceae, Acinetobacter baumannii, and Pseudomonas were collected from 11 Lebanese hospitals in 2012. Antimicrobial susceptibility was assessed with phenotypic tests, genes encoding carbapenemases were screened via PCR-sequencing, and genetic relatedness was examined by PGFE and ERIC-PCR. A total of 398 nonrepetitive carbapenem nonsusceptible isolates were studied, of which 44 were Enterobacteriaceae, 142 were A. baumannii, and 212 were Pseudomonas. Among Enterobacteriaceae, 70.4% carried blaOXA-48-like gene on IncL/M-type plasmids, while acquired AmpC cephalosporinases, extended-spectrum-ß-lactamases, and efflux-pump were additional contributors to carbapenem resistance. Among A. baumannii, 90% produced OXA-23 and GES-11 and carried insertion sequence ISAba1 upstream and adjacent to blaOXA-23 and blaAcinetobacter-derived cephalosporinases. Among Pseudomonas, 16% harbored VIM-2, 4.2% IMP-2, and 1.4% IMP-1 metallo-ß-lactamases. Fingerprint analysis indicated that the spread of OXA-48-like carbapenemases was mostly mediated by horizontal transfer, while OXA-23 and GES-11 diffusion in A. baumannii and VIM-2 diffusion in P. aeruginosa were primarily due to clonal dissemination. This study is the first nationwide investigation of carbapenem resistance in Lebanon, showing low level of resistance in Enterobacteriaceae, and higher levels in A. baumannii and Pseudomonas. With current changes in the region, continuous surveillance of carbapenem resistance is crucial.

SURVEILLANCE of CARBAPENEM NON-SUSCEPTIBLE GRAM NEGATIVE STRAINS and CHARACTERIZATION of CARBAPENEMASES of CLASSES A, B and D in a LEBANESE HOSPITAL.

The production of carbapenem-hydrolyzing enzymes has been recognized as one of the most currently relevant resistance mechanisms in gram negative bacterial isolates, and is being detected in various countries. In Lebanon, carbapenem resistance was studied among gram negative pathogens collected from a university hospital from January to June of years 2011 and 2012. All isolates were subjected to phenotypic tests including antibiotic susceptibility, cloxacillin effect, modified Hodge test, and Etest for metallo-ß-lactamase detection. They were also subjected to genotyping by PCR sequencing to characterize ß-lactamases. Between January and June 2011, 48 carbapenem non-susceptible strains were collected. Of these, one Klebsiella pneumoniae harbored OXA-48 and insertion sequence IS 1999; four Acinetobacter baumanni harbored simultaneously OXA-23 and GES-11, and three Pseudomonas harbored VIM-2 carbapenemase. Between January and June 2012, 100 carbapenem non-susceptible strains were collected. Of these, one K. pneumoniae harbored simultaneously OXA-48, IS 1999, and an acquired AmpC of the ACC group; four Serratia marcescens harbored OXA-48, while among eight A. baumannii, one strain co-harbored OXA-23 and GES-11, six harbored OXA-23 and one OXA-24. Fifteen P, aeruginosa and two Pseudomonas species harbored VIM-2; two P. aeruginosa strains produced IMP-1 and two others IMP-2. This epidemiological survey demonstrates the presence of carbapenemases of Ambler classes A, B, and D in a Lebanese hospital and indicates increase in the number and variety of such enzymes.

Susceptibility trends and molecular characterization of Gram-negative bacilli associated with urinary tract and intra-abdominal infections in Jordan and Lebanon: SMART 2011-2013.

OBJECTIVES: To investigate phenotypic and genotypic patterns of antimicrobial resistance among Gram-negative bacilli associated with urinary tract infection (UTI) and intra-abdominal infection (IAI) in medical centres of Jordan and Lebanon. METHODS: Gram-negative bacilli from the SMART study, collected between the years 2011 and 2013, were first identified at local laboratories. These isolates were shipped to a central laboratory where re-identification, susceptibility testing, and molecular characterization were performed using standard methods. RESULTS: Among the 523 UTI-associated isolates, Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis were the most frequent (70%, 14%, and 5%, respectively). E. coli, K. pneumoniae, and Pseudomonas aeruginosa were the most frequent species among the 527 IAI-associated isolates (46%, 14%, and 12%, respectively). Incidence rates of extended-spectrum beta-lactamase (ESBL) producers among UTI-associated E. coli, K. pneumoniae, and P. mirabilis were 43%, 54%, and 4%, respectively. Corresponding rates among IAI-associated isolates were 49%, 56%, and 12%, respectively. Acinetobacter baumannii and P. aeruginosa isolates showed very disturbing low susceptibility patterns. CTX-M-15 was the most prevalent ESBL produced. Seventeen isolates were non-susceptible to carbapenems (estimated prevalence of 1.6%). CONCLUSIONS: The alarmingly high rates of ESBL production and emergence of carbapenemases emphasize the urgent need to develop antimicrobial stewardship initiatives and to maintain antimicrobial resistance surveillance systems.

Brain abscesses in HIV-positive patients due to Pneumocystis jiroveci.

Cerebral toxoplasmosis is common in AIDS patients; however, pneumocystosis of the brain is rarely documented. We report a patient with AIDS hospitalized for brain abscesses. Stereotactic brain biopsy with immunofluorescence staining was positive for Pneumocystis jiroveci. The patient received high doses of cotrimoxazole and had a favorable clinical course.

Countrywide spread of community- and hospital-acquired extended-spectrum beta-lactamase (CTX-M-15)-producing Enterobacteriaceae in Lebanon.

A prospective study was carried out to assess the extent of carriage of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae at both hospital and community levels in Lebanon. A total of 1,442 fecal samples were collected from hospital-based patients and 58 from health care workers of six Lebanese tertiary care general hospitals located in different areas of Lebanon between January and March 2003. A total of 382 fecal samples were also collected from healthy subjects between April and June 2003. The samples analysis led to the identification of 118 strains as ESBL producers based on the synergistic effects between clavulanate and selected beta-lactams (ceftazidime and cefotaxime). These strains were isolated from 72 subjects: 61 patients, 2 health care workers, and 9 healthy subjects. One representative strain per subject was selected, and a total of 72 nonduplicate ESBL producers, including a high majority of Escherichia coli (n = 56), Klebsiella pneumoniae (n = 9), Enterobacter cloacae (n = 6), and Citrobacter freundii (n = 1), were characterized. The molecular analysis revealed that the majority of the strains (83%) express CTX-M-15 ESBL (pI 8.6). SHV-5a ESBL (pI 8.2) was produced by 18% of the strains. DNA macrorestriction analysis of ESBL-producing E. coli presented 38 different genotypes, revealing the absence of clonal link among these strains. In addition to the fact that the present study highlights the emergence and the countrywide dissemination of CTX-M-15-producing E. coli in Lebanon, it represents the first report of an SHV-5a-producing C. freundii.