Nezar Noor Al-hebshi

Khat (Catha edulis)—an updated review

The habit of chewing fresh leaves and twigs of khat (Catha edulis) for their stimulating amphetamine‐like effects is highly prevalent in East Africa and southwest on the Arabic peninsula. There is an extensive literature on khat providing information about its history, botany, production, geographical distribution, chemistry and pharmacology, and exploring the social, economic, medical, psychological and oral aspects related to its use. Some of this literature dates as early as the 11th century; however, most of it appeared after the first scientific description of khat by Peter Forskal in 1775. This review provides a panorama of khat and the various aspects of its use. A non‐technical description of the plant chemistry and pharmacology is included. The medical, psychological and oral aspects are emphasized, and the current knowledge about the microbiological effects of khat is also presented.

In vitro effects of crude khat extracts on the growth, colonization, and glucosyltransferases of Streptococcus mutans

Millions of Yemenites, East Africans, and immigrants to Western countries chew khat daily for its amphetamine-like effects. There is little information in the literature concerning the possible effects of the habit on oral microbiota. Our objective was to study in vitro crude khat extract effects on Streptococcus mutans growth and sucrose-dependent colonization, and on its glucosyltransferase (GTF) activity and production. Three khat cultivars were used. Lyophilized crude aqueous khat extracts were applied to the different assays at concentrations of 0–1% (w/v). Sucrose-dependent colonization was assessed as the ability of Streptococcus mutans UA159 to form adherent biofilms in glass culture tubes. Colony forming units (CFUs) in the planktonic phase served as a measure of bacterial growth, while CFUs in the biofilm phase were used to quantify viability in the biofilms. GTFs activity was tested by incubating a crude GTFs preparation with sucrose and determining the amount of water-soluble and water-insoluble glucans formed. GTFs production was assayed by comparing intensities of GTF bands in Western blots of extracts from control and khat-containing cultures. The khat extracts effectively inhibited biofilm formation. The minimum biofilm inhibitory concentration (MBIC) varied among the cultivars (0.25–1%). The extracts also inhibited synthesis of both glucan types, particularly insoluble glucans (average 85% inhibition at 1%), with significant differences among the cultivars. However, khat increased bacterial growth and at sub-MBIC also viability within biofilms; there were no inter-cultivar differences. It is shown that khat leaves contain water-soluble constituents that inhibit some cariogenic properties of S. mutans in vitro.

Effect of khat chewing on periodontal pathogens in subgingival biofilm from chronic periodontitis patients

AIMS Existing in vitro and in vivo data suggest that khat may have a favorable effect on periodontal microbiota. The purpose of this study was to assess the effect of khat chewing on major periodontal pathogens in subgingival plaque samples from subjects with chronic periodontitis. MATERIALS AND METHODS 40 subgingival plaque samples were obtained from periodontitis and healthy sites of 10 khat chewers (40 y median age) and 10 khat non-chewers (37.5 y median age) with chronic periodontitis. Absolute and relative counts of 6 periodontal pathogens were determined in each sample using highly sensitive and specific Taqman real-time PCR assays. Data were analyzed using an ordinal regression model. RESULTS Significantly more total bacteria were detected in samples from the periodontitis sites of the khat chewers (OR=20). Treponema denticola was present at significantly higher absolute counts at the healthy as well as periodontitis sites of the khat chewers (OR=3.13 and 13, respectively). However, the khat chewers harbored significantly lower absolute counts of Porphyromonas gingivalis at the healthy sites (OR=0.07). Furthermore, khat chewing was significantly associated with lower relative counts of Porphyromonas gingivalis, fusobacterium ssp., prevotella ssp. and Parvimonas micra-like species in subgingival plaque samples from both healthy and periodontitis sites (OR=0.11-0.33). Only Treponema denticola was found in higher relative counts at the healthy sites of the khat chewers (OR=2.98). CONCLUSIONS Overall, there was a lower burden of pathogens in the khat chewers. Findings from the current study are suggestive of a potential prebiotic effect for khat on periodontal microbiota.

Robust species taxonomy assignment algorithm for 16S rRNA NGS reads: application to oral carcinoma samples

Background Usefulness of next-generation sequencing (NGS) in assessing bacteria associated with oral squamous cell carcinoma (OSCC) has been undermined by inability to classify reads to the species level. Objective The purpose of this study was to develop a robust algorithm for species-level classification of NGS reads from oral samples and to pilot test it for profiling bacteria within OSCC tissues. Methods Bacterial 16S V1-V3 libraries were prepared from three OSCC DNA samples and sequenced using 454s FLX chemistry. High-quality, well-aligned, and non-chimeric reads ≥350 bp were classified using a novel, multi-stage algorithm that involves matching reads to reference sequences in revised versions of the Human Oral Microbiome Database (HOMD), HOMD extended (HOMDEXT), and Greengene Gold (GGG) at alignment coverage and percentage identity ≥98%, followed by assignment to species level based on top hit reference sequences. Priority was given to hits in HOMD, then HOMDEXT and finally GGG. Unmatched reads were subject to operational taxonomic unit analysis. Results Nearly, 92.8% of the reads were matched to updated-HOMD 13.2, 1.83% to trusted-HOMDEXT, and 1.36% to modified-GGG. Of all matched reads, 99.6% were classified to species level. A total of 228 species-level taxa were identified, representing 11 phyla; the most abundant were Proteobacteria, Bacteroidetes, Firmicutes, Fusobacteria, and Actinobacteria. Thirty-five species-level taxa were detected in all samples. On average, Prevotella oris, Neisseria flava, Neisseria flavescens/subflava, Fusobacterium nucleatum ss polymorphum, Aggregatibacter segnis, Streptococcus mitis, and Fusobacterium periodontium were the most abundant. Bacteroides fragilis, a species rarely isolated from the oral cavity, was detected in two samples. Conclusion This multi-stage algorithm maximizes the fraction of reads classified to the species level while ensuring reliable classification by giving priority to the human, oral reference set. Applying the algorithm to OSCC samples revealed high diversity. In addition to oral taxa, a number of human, non-oral taxa were also identified, some of which are rarely detected in the oral cavity.

Emerging role of bacteria in oral carcinogenesis: a review with special reference to perio-pathogenic bacteria

Oral cancer, primarily oral squamous cell carcinoma (OSCC), continues to be a major global health problem with high incidence and low survival rates. While the major risk factors for this malignancy, mostly lifestyle related, have been identified, around 15% of oral cancer cases remain unexplained. In light of evidence implicating bacteria in the aetiology of some cancer types, several epidemiological studies have been conducted in the last decade, employing methodologies ranging from traditional culture techniques to 16S rRNA metagenomics, to assess the possible role of bacteria in OSCC. While these studies have demonstrated differences in microbial composition between cancerous and healthy tissues, they have failed to agree on specific bacteria or patterns of oral microbial dysbiosis to implicate in OSCC. On the contrary, some oral taxa, particularly Porphyromonas gingivalis and Fusobacterium nucleatum, show strong oral carcinogenic potential in vitro and in animal studies. Bacteria are thought to contribute to oral carcinogenesis via inhibition of apoptosis, activation of cell proliferation, promotion of cellular invasion, induction of chronic inflammation, and production of carcinogens. This narrative review provides a critical analysis of and an update on the association between bacteria and oral carcinogenesis and the possible mechanisms underlying it.

Nonsyndromic cleft lip with or without cleft palate in arab populations: Genetic analysis of 15 risk loci in a novel case–control sample recruited in Yemen

BACKGROUND Nonsyndromic orofacial clefting (nsOFC) is among the most common of all congenital disorders and has a genetically complex etiology. Based on embryological and epidemiological data, the phenotype can be differentiated into nonsyndromic cleft lip with or without cleft palate (nsCL/P) and nonsyndromic cleft palate only, with nsCL/P being the most frequent form. Recent genetic research, predominantly performed in populations from Europe and Asia, has identified numerous genetic susceptibility loci for nsCL/P. As only few data are available concerning genetic susceptibility to nsCL/P in Arab populations, we investigated a newly recruited nsOFC sample from Yemen. METHODS For each of the 15 currently known nsCL/P risk loci, the top single-nucleotide polymorphism (plus nine back-up variants) were genotyped in 242 nsCL/P cases and 420 healthy controls. RESULTS Single-marker association analysis revealed significant associations for four loci (8q24, 9q22, 10q25, 13q31). The strongest association was for the European high risk locus at 8q24 (Pcorrected  = 5.09 × 10(-4) ; heterozygous odds ratio = 1.74 (1.22-2.47), homozygous odds ratio = 2.47 (1.55-3.93). Five additional loci (1q32.2, 3q12, 8q21, 17q22, 20q12) showed nominal significance that did not withstand correction for multiple testing. Although the six remaining loci (1p22, 1p36, 2p21, 3p11, 15q22, 17p13) failed to reach nominal significance, the risk alleles were in the same direction as in the discovery studies. CONCLUSION The results suggest that four of the 15 analyzed nsCL/P risk loci which were identified in European and Asian ethnicities significantly confer risk for nsCL/P in Arab populations.

Quantitative analysis of classical and new putative periodontal pathogens in subgingival biofilm: a case–control study

BACKGROUND AND OBJECTIVES A number of species/phylotypes have been newly implicated as putative periopathogens. The objective of this study was to explore associations among classical and new pathogens in subgingival biofilm and to assess their relative importance to chronic periodontitis. MATERIAL AND METHODS Pooled subgingival biofilm samples were obtained from 40 patients with chronic periodontitis and 40 healthy controls. Taqman q-PCR assays were used to determine the absolute and relative counts of Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Parvimonas micra, Filifactor alocis, oral Synergistetes and oral TM7s. Microbial associations were assessed using cluster analysis. Different statistical models were used to explore associations between microbial parameters and periodontitis. RESULTS The median log and relative counts were lowest for TM7s (4.4 and 0.0016%, respectively) and highest for oral Synergistetes (7.2 and 1.4%, respectively). Oral Synergistetes clustered strongly with the red complex, particularly T. forsythia (100% rescaled similarity). All species/phylotypes except TM7s were significantly associated with periodontitis (Mann-Whitney test; p ≤ 0.005). However, P. gingivalis and F. alocis lost association after adjusting for confounders (ordinal regression). In receiving operator characteristic curve analysis, the log counts of oral Synergistetes were the best markers of periodontitis (82.5% sensitivity and specificity), followed by those of T. forsythia, P. micra and T. denticola. In prediction analysis, however, P. micra was the only microbial predictor of periodontal parameters. CONCLUSIONS Oral Synergistetes are presented here as new members of the red complex, with relative importance to periodontitis exceeding that of the classical members. P. micra is shown as an important periodontal pathogen warranting more attention.

Viral infection and oral habits as risk factors for oral squamous cell carcinoma in Yemen: a case-control study

OBJECTIVE The role of qat chewing, tobacco (shammah) dipping, smoking, alcohol drinking, and oral viral infection as risk factors for oral squamous cell carcinoma (OSCC) in Yemen was assessed. STUDY DESIGN A total of 60 cases of OSCC and 120 age- and gender-matched controls were analyzed with respect to demographic data, history of oral habits, and the presence of human papillomavirus (HPV)-16, HPV-18, or Epstein-Barr virus (EBV) as determined by Taqman quantitative polymerase chain reaction. Logistic regression analysis was used to identify independent predictors of the disease. RESULTS Shammah use was the only risk factor for OSCC, with an odds ratio of 12.6 (CI, 3.3-48.2) and 39 (CI, 14-105) for the ex-users and current users, respectively. The association of shammah use alone with OSCC exceeded that of shammah use in combination with qat chewing, smoking, or both. EBV infection, smoking, and qat chewing showed no association with OSCC, while neither HPV-16 nor HPV-18 were detected in any sample. CONCLUSIONS Shammah use is a major risk factor for oral cancer in Yemen.

Inflammatory Bacteriome Featuring Fusobacterium nucleatum and Pseudomonas aeruginosa Identified in Association with Oral Squamous Cell Carcinoma

Studies on the possible association between bacteria and oral squamous cell carcinoma (OSCC) remain inconclusive, largely due to methodological variations/limitations. The objective of this study was to characterize the species composition as well as functional potential of the bacteriome associated with OSCC. DNA obtained from 20 fresh OSCC biopsies (cases) and 20 deep-epithelium swabs (matched control subjects) was sequenced for the V1-V3 region using Illumina’s 2 × 300 bp chemistry. High quality, non-chimeric merged reads were classified to species level using a prioritized BLASTN-algorithm. Downstream analyses were performed using QIIME, PICRUSt, and LEfSe. Fusobacterium nucleatum subsp. polymorphum was the most significantly overrepresented species in the tumors followed by Pseudomonas aeruginosa and Campylobacter sp. Oral taxon 44, while Streptococcus mitis, Rothia mucilaginosa and Haemophilus parainfluenzae were the most significantly abundant in the controls. Functional prediction showed that genes involved in bacterial mobility, flagellar assembly, bacterial chemotaxis and LPS synthesis were enriched in the tumors while those responsible for DNA repair and combination, purine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, ribosome biogenesis and glycolysis/gluconeogenesis were significantly associated with the controls. This is the first epidemiological evidence for association of F. nucleatum and P. aeruginosa with OSCC. Functionally, an “inflammatory bacteriome” is enriched in OSSC.

Subgingival periodontal pathogens associated with chronic periodontitis in Yemenis

BackgroundSubgingival microbial profile associated with periodontitis has been reported to significantly differ by geographical location. The purpose of this study was to assess the association between a panel of putative periodontal bacterial pathogens and chronic periodontitis among Yemenis.MethodsSubgingival DNA samples were obtained from diseased and healthy sites of 20 non-smoking, moderate to severe chronic periodontitis subjects. Absolute counts (bacterial DNA copies per sample) and relative counts (% total bacteria) of seven periopathogenic species/genera representative of the red and orange complexes were determined using Taqman q-PCR assays.ResultsThe q-PCR assays showed excellent linearity (R2 > 0.99) and a sensitivity of 100 copies/sample. The detection rate was 100% for all tested species/genera except for P. gingivalis and A. actinomycetemcomitans that were detected at 97.5% and 67.5%, respectively. The median log absolute counts were in the range of 2.41-6.53 copies per sample while median relative counts were in the range of 0.001-0.77%, both being highest for fusobacteria and lowest for A. actinomycetemcomitans. Significant interspecies correlations were observed. Adjusting for multiple comparisons (P≤0.0063), only T. forsythia, T. denticola and P. micra maintained significant association with periodontal destruction. The latter species, however, showed the strongest association and was found in higher proportions at the periodontitis sites across all subjects (3.39 median fold increase). No significant differences were observed for P. gingivalis.ConclusionsP. micra rather than P. gingivalis appears as a keystone pathogen in this Yemeni Sample. However, these findings need to be validated in a larger-scale study before they can be claimed to represent ethnic variations in pathogens’ association with periodontitis.