Phytic acid demonstrates rapid antibiofilm activity and inhibits biofilm formation when used as a surface conditioning agent

Root canal infections are associated with biofilms and are treated with chemical irrigants with a high success rate. However, treatment failure does arise, which is attributed primarily to resistance exhibited by biofilms. Currently used irrigants in root canal treatment have disadvantages, and there is therefore a need for more biocompatible alternatives with antibiofilm properties to reduce root canal treatment failure and complications. The aim of this study was to evaluate the in vitro antibiofilm properties of phytic acid (IP6), which is a potential alternative treatment agent. Single- and dual-species biofilms of Enterococcus faecalis and Candida albicans were developed on the well surfaces of 12-well plates and on hydroxyapatite (HA) coupons, and then exposed to IP6. In addition, selected HA coupons were preconditioned with IP6 before biofilm development. IP6 demonstrated bactericidal effects and altered the metabolic activity of biofilm cells. Confocal laser-scanning microscopy showed that IP6 caused significant and rapid reduction in live biofilm cells. At sublethal concentrations, IP6 did not alter the expression of tested virulence genes except for C. albicans hwp1, the expression of which was upregulated but not reflected by a change in hyphal transformation. IP6-preconditioned HA coupons led to extensive inhibition of dual-species biofilm formation. The results of this study highlight for the first time the antibiofilm inhibitory properties of IP6 and the potential for its exploitation in several clinical applications

The prevalence of novel periodontal pathogens and bacterial complexes in Stage II generalized periodontitis based on 16S rRNA next generation sequencing

Objective: To define the subgingival microbial profile associated with Stage II generalized periodontitis using next-generation sequencing and to determine the relative abundance of novel periodontal pathogens and bacterial complexes. Methodology: Subgingival biofilm samples were collected from 80 subjects diagnosed with Stage II generalized periodontitis. Bacterial DNA was extracted, and 16S rRNA-based bacterial profiling via next-generation sequencing was carried out. The bacterial composition and diversity of microbial communities based on the age and sex of the patients were analyzed. The bacterial species were organized into groups: bacterial complexes (red, orange, purple, yellow, and green), novel periodontal pathogens, periodontal health-related species, and unclassified periodontal species. The results were analyzed and statistically evaluated. Results: The highest number of bacteria belonged to the phylum Bacteroidetes and Firmicutes. In terms of relative abundance, the orange complex represented 18.99%, novel bacterial species (Fretibacterium spp. and Saccharibacteria spp.) comprised 17.34%, periodontal health-related species accounted for 16.75% and unclassified periodontal species represented (Leptotrichia spp. and Selenomonas spp.) 15.61%. Novel periodontal pathogens had outweighed the periodontal disease-related red complex (5.3%). The one-sample z-test performed was statistically significant at p<0.05. The Beta diversity based on the unweighted UniFrac distance at the species level demonstrated a total variance of 15.77% based on age and 39.19% on sex, which was not statistically significant. Conclusion: The bacterial species corresponding to the disease-related orange complex and novel periodontal pathogens are predominant in Stage II generalized periodontitis

Comparison of Molecular and Phenotypic Methods for the Detection and Characterization of Carbapenem Resistant Enterobacteriaceae

In recent years, there has been a rapid dissemination of carbapenem resistant Enterobacteriaceae (CRE). This study aimed to compare phenotypic and molecular methods for detection and characterization of CRE isolates at a large tertiary care hospital in Saudi Arabia. This study was carried out between January 2011 and November 2013 at the King Khalid University Hospital (KKUH) in Saudi Arabia. Determination of presence of extended-spectrum beta-lactamases (ESBL) and carbapenem resistance was in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines. Phenotypic classification was done by the MASTDISCSTM ID inhibitor combination disk method. Genotypic characterization of ESBL and carbapenemase genes was performed by the Check-MDR CT102. Diversilab rep-PCR was used for the determination of clonal relationship. Of the 883 ESBL-positive Enterobacteriaceae detected during the study period, 14 (1.6%) isolates were carbapenem resistant. Both the molecular genotypic characterization and phenotypic testing were in agreement in the detection of all 8 metalo-beta-lactamases (MBL) producing isolates. Of these 8 MBL-producers, 5 were positive for blaNDM gene and 3 were positive for blaVIM gene. Molecular method identified additional blaOXA gene isolates while MASTDISCSTM ID detected one AmpC producer isolate. Both methods agreed in identifying 2 carbapenem resistant isolates which were negative for carbapenemase genes. Diversilab rep-PCR analysis of the 9 Klebsiella pneumoniae isolates revealed polyclonal distribution into eight clusters. MASTDISCSTM ID is a reliable simple cheap phenotypic method for detection of majority of carbapenemase genes with the exception of the blaOXA gene. We recommend to use such method in the clinical laboratory

The Effect of Chlorhexidine on Bacterial Contamination of Hall Technique Elastomeric Orthodontic Separators and Gingival Health: A Pilot Study

Objective: To study the effect of chlorhexidine on elastomeric orthodontic separators (EOS) bacterial-colonisation and gingival-health in Hall technique (HT) patients. Material and Methods: Prospective in-vivo pilot clinical study of EOS bacterial colonisation and primary-molar gingival health assessment in 20 patients (mean age 5.45±1.27 years) requiring bilateral HT crowns (40 teeth). One side received 1-minute 0.12% chlorhexidine-soaked-EOSs (Chx-EOSs), and the other side dry-EOSs (NoChx-EOSs). The EOSs were removed five-days later and underwent a bacterial enumeration technique. Plaque (PI) and Gingival (GI) indices were assessed pre-, five-days and three-months post-treatment. Wilcoxon-Signed-Rank/McNemar-Chi-square statistics were used (p<0.05). Results: Baseline unused/packaged EOSs’ sterility check yielded zero colony-forming-units (CFU) per millilitre, but 100% of the used EOSs became colonised by oral-microorganisms. An overall trend of lower mean CFU count in Chx-EOSs (3.415± 0.78 x105 CFU/ml) compared to NoChx-EOSs (6.157±1.48 x105 CFU/ml) was observed (p=0.009). Both NoChx-EOSs and Chx-EOSs insertion sites showed evidence of gingivitis with no difference between PI and GI indices by site over time. Conclusion:There was a lower trend of bacterial colonization in chlorhexidine treated EOSs and an occurrence of gingivitis pre/post HT-treatment regardless of EOS type. The lack of difference in the gingival health may be inconclusive due to this pilot’s low power suggesting the need for robust large scale studies

Carbapenem resistant Enterobacterales in the United Arab Emirates: a retrospective analysis from 2010 to 2021

BackgroundCarbapenem-resistant Enterobacterales (CRE) are spreading in the United Arab Emirates (UAE) where their dissemination is facilitated by international travel, trade, and tourism. The objective of this study is to describe the longitudinal changes of CRE as reported by the national AMR surveillance system of the UAE.MethodsIn this study, we retrospectively describe CRE isolated from 317 surveillance sites, including 87 hospitals and 230 centers/clinics from 2010 to 2021. The associated clinical, demographic, and microbiological characteristics are presented by relying on the UAE national AMR surveillance program. Data was analyzed using WHONET microbiology laboratory database software (http://www.whonet.org).ResultsA total of 14,593 carbapenem resistant Enterobacterales were analyzed, of which 48.1% were carbapenem resistant Klebsiella pneumoniae (CRKp), 25.1% carbapenem resistant Escherichia coli (CREc), and 26.8% represented 72 other carbapenem resistant species. Carbapenem resistant strains were mostly associated with adults and isolated from urine samples (36.9% of CRKp and 66.6% of CREc) followed by respiratory samples (26.95% for CRKp) and soft tissue samples (19.5% for CRKp). Over the studied period carbapenem resistance rates remained high, especially in K. pneumoniae, and in 2021 were equivalent to 67.6% for imipenem, 76.2% for meropenem, and 91.6% for ertapenem. Nevertheless, there was a statistically significant decreasing trend for imipenem and meropenem resistance in Klebsiella species (p < 0.01) while the decrease in ertapenem resistance was non-significant. Concerning E. coli, there was a statistically significant decreasing trend for meropenem and imipenem resistance over the 12 years, while ertapenem resistance increased significantly with 83.8% of E. coli exhibiting ertapenem resistance in 2021. Resistance rates to ceftazidime and cefotaxime remained higher than 90% (in 2021) for CRKp and cefotaxime rates increased to 90.5% in 2021 for CREc. Starting 2014, resistance to colistin and tigecycline was observed in carbapenem resistant Enterobacterales. CRE were associated with a higher mortality (RR: 6.3), admission to ICU (RR 3.9), and increased length of stay (LOS; 10 excess inpatient days per CRE case).ConclusionThis study supports the need to monitor CRE in the UAE and draws attention to the significant increase of ertapenem resistance in E. coli. Future surveillance analysis should include a genetic description of carbapenem resistance to provide new strategies

Antimicrobial resistance in Streptococcus pneumoniae: a retrospective analysis of emerging trends in the United Arab Emirates from 2010 to 2021

IntroductionAlthough pneumococcal conjugate vaccines (PCV) have been effective in reducing the burden of Streptococcus pneumoniae infections, there is a paucity of data on the relationship with antimicrobial resistance (AMR) trends in the Arabian Gulf region. This study was carried out to assess S. pneumoniae resistance trends in the United Arab Emirates (UAE) where PCV-13 vaccination was introduced in 2011.MethodsRetrospective analysis of S. pneumoniae demographic and microbiological data collected as part of the national AMR surveillance program from 2010 to 2021 was carried out. A survey of reporting sites and hand searching of annual reports of local health authorities was carried out to identify data on S. pneumoniae serotypes as this is not included in the AMR surveillance database.ResultsFrom 2010 to 2021, 11,242 non-duplicate S. pneumoniae isolates were reported, increasing from 324 in 2010 to 1,115 in 2021. Factoring in annual increment in the number of surveillance sites, the number of isolates per site showed an upward trajectory from 2015 to 2018 and declined in 2020 with the onset of the pandemic. The majority of isolates (n/N = 5,751/11,242; 51.2%) were from respiratory tract specimens with 44.5% (n/N = 2,557/5,751) being nasal colonizers. Up to 11.9% (n/N = 1,337/11,242) were invasive pneumococcal disease (IPD) isolates obtained from sterile site specimens including blood (n = 1,262), cerebrospinal (n = 52), pleural (n = 19) and joint (n = 4) fluid; and were predominantly from pediatric patients. The downward trend for amoxicillin and for penicillin G at the non-meningitis and meningitis as well as oral penicillin breakpoints was statistically significant. In contrast, increasing trends of resistance were seen for levofloxacin, moxifloxacin, trimethoprim/sulfamethoxazole and erythromycin. IPD and non-IPD isolates showed similar demographic and AMR trends. None of the surveillance sites carried out S. pneumoniae serotyping and handsearching of annual reports did not yield this information.ConclusionThe increasing trend of pneumococcal disease and AMR with emergence of isolates with MDR phenotype despite is of concern. In the absence of S. pneumoniae serotyping the role of non-vaccine serotypes in driving this pattern remains unknown. There is an urgent need for serotype, genomic and AMR surveillance of S. pneumoniae isolates in the UAE

Lateral flow immunoassay for the Detection of Panton-Valentine leukocidin in Staphylococcus aureus from skin and soft tissue infections in the United Arab Emirates

Introduction: Panton Valentine leukocidin (PVL) is a virulence factor which is associated with methicillin sensitive and resistant Staphylococcus aureus (MSSA/MRSA) causing skin and soft tissue infections (SSTI). This study aimed to evaluate a novel lateral flow immunoassay (LFI) for PVL detection in S. aureus cultures and to describe their genotypic characterization. Methods: The study was carried out from January-August 2020 in Dubai, United Arab Emirates. S. aureus isolates associated with SSTI were tested for PVL detection using LFI. DNA microarray-based assays were used for molecular characterization including detection of pvl genes. Results: One-hundred thirty-five patients with a clinical diagnosis of SSTIs were recruited. Sixty-six patients received antibiotics, mostly beta lactams (n=36) and topical fusidic acid (n=15). One-hundred twenty-nine isolates (MRSA: n=43; MSSA: n=86) were tested by LFI and DNA microarrays. All 76 (58.9%) isolates which were unambiguously negative for the PVL in LFI were negative for pvl genes using the DNA microarray. All the LFI PVL positive isolates (n=53) had pvl genes detected. This translates into 100% each for sensitivity, specificity, positive and negative predictive values for the LFI. The LFI typically takes about 15 min inclusive of a 10 min incubation period. Predominant S. aureus clonal complexes (CC) were CC30 (n=18), CC22 (n=13), CC5 (n=12), CC1 (n=11), CC152 (n=8), CC15 (n=7); CC97 (n=7); CC8 and CC20 (n=6 each). Among MRSA, the proportion of pvl-positives (35/43; 81%) was higher than among MSSA (n/N=18/86; 21%). The fusidic acid resistance gene fusC was detected in 14 MRSA (33%) compared to 8 MSSA (9%). A co-carriage of fusC and pvl genes was present in 7 MRSA and in one MSSA. Conclusion: LFI shows excellent diagnostic accuracy indices for rapid identification of PVL in MSSA/MRSA in a setting with high prevalence of pvl+ve strains. The high occurrence of pvl and fusC genes in MRSA strains causing SSTI is of concern and needs constant surveillance

Emergence of novel methicillin resistant Staphylococcus aureus strains in a tertiary care facility in Tiyadh, Saudi Arabia

Purpose: There is a need for continuous surveillance of methicillin-resistant Staphylococcus aureus (MRSA) to identify emergence of new strains. We hypothesize that MRSA strains are evolving with ongoing acquisition of SCCmec elements. This study was carried out to evaluate the evolution of MRSA at a tertiary care facility in Saudi Arabia. Methods: MRSA isolates associated with invasive clinical infection, which were identified in 2017 at the microbiology laboratory, King Khalid University Hospital (KKUH) in Riyadh, Saudi Arabia, were studied. The molecular characterization of isolates was carried out using StaphyType DNA microarray (Alere Technologies GmbH/Abbott, Jena, Germany). Results: The 125 MRSA isolates studied belonged to 18 clonal complexes (CC) which were distributed into 32 strain assignments. The predominant CC were CC5 (n=30), CC6 (n=17), CC80 (n=13), CC22 (n=12), CC361 (n=12). The findings demonstrated the first identification of CC152, CC361 and CC1153 MRSA as well as ST5-MRSA-[I+fus], “Geraldine Clone”, CC6-MRSA-IV (PVL+) and CC88-MRSA-V (PVL+), WA MRSA-117 in Saudi Arabia. Four novel variants were identified: CC5-MRSA-[VI+fus+tirS], CC22-MRSA-[V/VT+fus](PVL+), CC152-MRSA-[V+fus](PVL+) and CC361-MRSA-[VT+fus]. Fifty-four isolates (n/N=54/125; 43.2%) including the novel strains carried the Q6GD50 SCCfusC gene while the Panton-Valentine leukocidin genes were present in 30.4% (n/N=38/125). Conclusion: The findings demonstrate an expanding MRSA repertoire in our setting including emergence of previously unreported clonal complexes and novel strains. The high carriage of fusC gene suggests a role for fusidic acid misuse in driving the evolution of the MRSA genome and underscores the need for increased monitoring of antibiotic use.Purpose: There is a need for continuous surveillance of methicillin-resistant Staphylococcus aureus (MRSA) to identify emergence of new strains. We hypothesize that MRSA strains are evolving with ongoing acquisition of SCCmec elements. This study was carried out to evaluate the evolution of MRSA at a tertiary care facility in Saudi Arabia. Methods: MRSA isolates associated with invasive clinical infection, which were identified in 2017 at the microbiology laboratory, King Khalid University Hospital (KKUH) in Riyadh, Saudi Arabia, were studied. The molecular characterization of isolates was carried out using StaphyType DNA microarray (Alere Technologies GmbH/Abbott, Jena, Germany). Results: The 125 MRSA isolates studied belonged to 18 clonal complexes (CC) which were distributed into 32 strain assignments. The predominant CC were CC5 (n=30), CC6 (n=17), CC80 (n=13), CC22 (n=12), CC361 (n=12). The findings demonstrated the first identification of CC152, CC361 and CC1153 MRSA as well as ST5-MRSA-[I+fus], “Geraldine Clone”, CC6-MRSA-IV (PVL+) and CC88-MRSA-V (PVL+), WA MRSA-117 in Saudi Arabia. Four novel variants were identified: CC5-MRSA-[VI+fus+tirS], CC22-MRSA-[V/VT+fus](PVL+), CC152-MRSA-[V+fus](PVL+) and CC361-MRSA-[VT+fus]. Fifty-four isolates (n/N=54/125; 43.2%) including the novel strains carried the Q6GD50 SCCfusC gene while the Panton-Valentine leukocidin genes were present in 30.4% (n/N=38/125). Conclusion: The findings demonstrate an expanding MRSA repertoire in our setting including emergence of previously unreported clonal complexes and novel strains. The high carriage of fusC gene suggests a role for fusidic acid misuse in driving the evolution of the MRSA genome and underscores the need for increased monitoring of antibiotic use

Microbial metabolic genes crucial for S. aureus biofilms: an insight from re-analysis of publicly available microarray datasets

Bacterial biofilms are microbial lifestyles found in all environments. Up to 80% of human infections and 60-70% of hospital-acquired infections have a biofilm origin, with Staphylococcus aureus one of the leading causes of these infections. Microorganisms in biofilms exhibit significant antimicrobial resistance which poses important treatment challenges, hence the urgent need to identify novel antibiofilm strategies. Microbes form biofilms in response to various factors, and once these 3-dimentional structures form they are highly recalcitrant to removal. The switch from planktonic lifestyle to the biofilm protected mode of growth results in a phenotypic shift in the behavior of the microorganisms in terms of growth rate and gene expression. Given these changes, investigation of microbial gene expression and their modulation at different stages of biofilm maturation is needed to provide vital insight into the behaviour of biofilm cells. In this study, we analysed publicly available transcriptomic dataset of S. aureus biofilm at different stages of maturation to identify consistently upregulated genes irrespective of the biofilm maturation stage. Our reanalysis identified a total of 6 differentially expressed genes upregulated in both 48 and 144-h old S. aureus biofilms. Functional analysis revealed that these genes encode for proteins which play a role in key microbial metabolic pathways. However, these genes, as yet, are unrelated or poorly studied in the context of biofilm. Moreover, the findings of this in silico work, suggest that these genes may represent potential novel targets for the development of more effective antibiofilm strategies against S. aureus biofilm-associated infections