Nanopore sequencing for identification and characterization of antimicrobial-resistant Escherichia coli and Salmonella spp. from tilapia and shrimp sold at wet markets in Dhaka, Bangladesh.

Wet markets in low-and middle-income countries are often reported to have inadequate sanitation resulting in fecal contamination of sold produce. Consumption of contaminated wet market-sourced foods has been linked to individual illness and disease outbreaks. This pilot study, conducted in two major wet markets in Dhaka city, Bangladesh during a 4-month period in 2021 aimed to assess the occurrence and characteristics of Escherichia coli and non-typhoidal Salmonella spp. (NTS) from tilapia (Oreochromis niloticus) and shrimp (Penaeus monodon). Fifty-four individuals of each species were collected. The identity of the bacterial isolates was confirmed by PCR and their susceptibility toward 15 antimicrobials was tested by disk diffusion. The whole genome of 15 E. coli and nine Salmonella spp. were sequenced using Oxford Nanopore Technology. E. coli was present in 60-74% of tilapia muscle tissue and 41-44% of shrimp muscle tissue. Salmonella spp. was found in skin (29%) and gills (26%) of tilapia, and occasionally in muscle and intestinal samples of shrimp. The E. coli had several Multilocus sequence typing and serotypes and limited antimicrobial resistance (AMR) determinants, such as point mutations on glpT and pmrB. One E. coli (BD17) from tilapia carried resistance genes for beta-lactams, quinolones, and tetracycline. All the E. coli belonged to commensal phylogroups B1 and A and showed no Shiga-toxin and other virulence genes, confirming their commensal non-pathogenic status. Among the Salmonella isolates, five belonged to Kentucky serovar and had similar AMR genes and phenotypic resistance patterns. Three strains of this serovar were ST198, often associated with human disease, carried the same resistance genes, and were genetically related to strains from the region. The two undetermined sequence types of S. Kentucky were distantly related and positioned in a separate phylogenetic clade. Two Brunei serovar isolates, one Augustenborg isolate, and one Hartford isolate showed different resistance profiles. This study revealed high fecal contamination levels in tilapia and shrimp sold at two main wet markets in Dhaka. Together with the occurrence of Salmonella spp., including S. Kentucky ST198, a well-known human pathogen, these results stress the need to improve hygienic practices and sanitation standards at markets to improve food safety and protect consumer health.

High prevalence of vancomycin non-susceptible and multi-drug resistant enterococci in farmed animals and fresh retail meats in Bangladesh.

The emergence of antimicrobial resistant Enterococcus spp., a main cause of untreatable nosocomial infection, in food animals and dissemination to humans is a public health risk. The study was performed to determine the prevalence and antimicrobial resistance, and virulence characteristics of Enterococcus faecalis and Enterococcus faecium in food animals and meats in Bangladesh. Enterococcus spp., were confirmed using sodA gene specific PCR, and antimicrobial resistance and virulence properties were characterized by PCR. Enterococcus spp. were recovered from 57% of the collected samples (n = 201/352). Farm samples yielded significantly higher (p ≤ 0.05) prevalence (62%) than that of retail meat samples (41%). E. faecalis (52%) is most frequently isolated species. Greater proportions of isolates exhibited resistance to tetracycline (74%), erythromycin (65%) and ciprofloxacin (34%). Fifty-one isolates are vancomycin non-susceptible enterococci (VNSE), of which forty-seven are MDR and twenty are linezolid resistant, a last line drug for VNSE. Virulence factors such as gelatinase (gelE), aggregation factor (asa1) and sex pheromone (cpd) are detected along with vancomycin resistance gene (vanA, vanB and vanC2/C3) in VNSE isolates. The high prevalence of MDR enterococci in food animals and retail meats may cause consumers infections with concomitant reduction of available therapeutic options.

Virulence and antimicrobial resistance profile of non-typhoidal Salmonella enterica serovars recovered from poultry processing environments at wet markets in Dhaka, Bangladesh.

The rapid emergence of virulent and multidrug-resistant (MDR) non-typhoidal Salmonella (NTS) enterica serovars is a growing public health concern globally. The present study focused on the assessment of the pathogenicity and antimicrobial resistance (AMR) profiling of NTS enterica serovars isolated from the chicken processing environments at wet markets in Dhaka, Bangladesh. A total of 870 samples consisting of carcass dressing water (CDW), chopping board swabs (CBS), and knife swabs (KS) were collected from 29 wet markets. The prevalence of Salmonella was found to be 20% in CDW, 19.31% in CBS, and 17.58% in KS, respectively. Meanwhile, the MDR Salmonella was found to be 72.41%, 73.21%, and 68.62% in CDW, CBS, and KS, respectively. All isolates were screened by polymerase chain reaction (PCR) for eight virulence genes, namely invA, agfA, IpfA, hilA, sivH, sefA, sopE, and spvC. The S. Enteritidis and untyped Salmonella isolates harbored all virulence genes while S. Typhimurium isolates carried six virulence genes, except sefA and spvC. Phenotypic resistance revealed decreased susceptibility to ciprofloxacin, streptomycin, ampicillin, tetracycline, gentamicin, sulfamethoxazole-trimethoprim, amoxicillin-clavulanic acid, and azithromycin. Genotypic resistance showed a higher prevalence of plasmid-mediated blaTEM followed by tetA, sul1, sul2, sul3, and strA/B genes. The phenotypic and genotypic resistance profiles of the isolates showed a harmonic and symmetrical trend. According to the findings, MDR and virulent NTS enterica serovars predominate in wet market conditions and can easily enter the human food chain. The chi-square analysis showed significantly higher associations among the phenotypic resistance, genotypic resistance and virulence genes in CDW, CBS, and KS respectively (p < 0.05).

Molecular Detection of Colistin Resistance mcr-1 Gene in Multidrug-Resistant Escherichia coli Isolated from Chicken.

Zoonotic and antimicrobial-resistant Escherichia coli (hereafter, E. coli) is a global public health threat which can lead to detrimental effects on human health. Here, we aim to investigate the antimicrobial resistance and the presence of mcr-1 gene in E. coli isolated from chicken feces. Ninety-four E. coli isolates were obtained from samples collected from different locations in Bangladesh, and the isolates were identified using conventional microbiological tests. Phenotypic disk diffusion tests using 20 antimicrobial agents were performed according to CLSI-EUCAST guidelines, and minimum inhibitory concentrations (MICs) were determined for a subset of samples. E. coli isolates showed high resistance to colistin (88.30%), ciprofloxacin (77.66%), trimethoprim/sulfamethoxazole (76.60%), tigecycline (75.53%), and enrofloxacin (71.28%). Additionally, the pathotype eaeA gene was confirmed in ten randomly selected E. coli isolates using primer-specific polymerase chain reaction (PCR). The presence of mcr-1 gene was confirmed using PCR and sequencing analysis in six out of ten E. coli isolates. Furthermore, sequencing and phylogenetic analyses revealed a similarity between the catalytic domain of Neisseria meningitidis lipooligosaccharide phosphoethanolamine transferase A (LptA) and MCR proteins, indicating that the six tested isolates were colistin resistant. Finally, the findings of the present study showed that E. coli isolated from chicken harbored mcr-1 gene, and multidrug and colistin resistance. These findings accentuate the need to implement strict measures to limit the imprudent use of antibiotics, particularly colistin, in agriculture and poultry farms.

Erratum: Genetic Evolution and Transmission Dynamics of Clade 2.3.2.1a Highly Pathogenic Avian Influenza A/H5N1 Viruses in Bangladesh.

[This corrects the article DOI: 10.1093/ve/veaa046.].

Knowledge, Attitudes and Practices Regarding Antibiotic Use and Resistance among Veterinary Students in Bangladesh.

The use of antibiotics in animals for both therapeutic and non-therapeutic purposes is a major driver of the emergence and spread of antimicrobial resistance (AMR). While several studies have investigated prescription and consumption patterns in humans, little attention has been paid to the veterinary sector. A cross-sectional study was conducted in 3002 veterinary students (VS) and non-medical students (NMS) from 12 universities in Bangladesh to explore their knowledge, attitudes and practices (KAP) about antibiotics and AMR using a self-administered questionnaire, and assess the influence of the veterinary curriculum. KAP regarding antibiotic use and AMR was significantly higher in veterinary than non-medical students, and in first-year than final-year students. However, gaps in knowledge and practices were highlighted, suggesting deficiencies in training. Moreover, final-year veterinary students were found to be more likely than first-year students to use antibiotics without instructions, which could indicate deficiencies in their curriculum. Although the study suggested a positive impact of the veterinary curriculum on KAP about antibiotics and AMR in Bangladesh, critical gaps remain that are likely to contribute to inadequate use in their future practice. Therefore, there is scope for improving educational programs on AMR in professional curricula.

Efficacy of two vaccines against recent emergent antigenic variants of clade 2.3.2.1a highly pathogenic avian influenza viruses in Bangladesh.

H5N1 highly pathogenic avian influenza viruses (HPAIVs) have caused outbreaks in poultry in Bangladesh since 2007. While clade 2.2.2 and 2.3.4.2 HPAIVs have not been detected since 2012, clade 2.3.2.1a viruses have caused continuous outbreaks since 2012 despite the use of vaccines. In this study, we evaluated the efficacy of two H5 vaccines licensed in Bangladesh, RE-6 inactivated vaccine, and a recombinant herpesvirus of turkeys vaccine with an H5 insert (rHVT-H5), for protection against recent field viruses in chickens. We selected three viruses for efficacy tests (A/chicken/Bangladesh/NRL-AI-3237/2017, A/crow/Bangladesh/NRL-AI-8471/2017 and A/chicken/Bangladesh/NRL-AI-8323/2017) from 36 H5 viruses isolated from Bangladesh between 2016 and 2018 by comparing the amino acid sequences at five antigenic sites (A-E) and analyzing hemagglutination inhibition (HI) titers with reference antisera. The RE-6 and rHVT-H5 vaccines both conferred 80-100% clinical protection (i.e. reduced morbidity and mortality) against the three challenge viruses with no significant differences in protection. In addition, both vaccines significantly decreased viral shedding from infected chickens as compared to challenge control chickens. Based on these metrics, the current licensed H5 vaccines protected chickens against the recent field viruses. However, the A/crow/Bangladesh/NRL-AI-8471/2017 virus exhibited antigenic divergence including: several unique amino acid changes in antigenic epitope sites A and B and was a serological outlier in cross HI tests as visualized on the antigenic map. The continuing emergence of such antigenic variants which could alter the dominant antigenicity of field viruses should be continuously monitored and vaccines should be updated if field efficacy declines.

Virulence and Antimicrobial Resistance Profiles of Salmonella enterica Serovars Isolated from Chicken at Wet Markets in Dhaka, Bangladesh.

Virulent and multi drug resistant (MDR) Salmonellaenterica is a foremost cause of foodborne diseases and had serious public health concern globally. The present study was undertaken to identify the pathogenicity and antimicrobial resistance (AMR) profiles of Salmonellaenterica serovars recovered from chicken at wet markets in Dhaka, Bangladesh. A total of 870 cecal contents of broiler, sonali, and native chickens were collected from 29 wet markets. The overall prevalence of S. Typhimurium, S. Enteritidis, and untyped Salmonella spp., were found to be 3.67%, 0.57%, and 1.95% respectively. All isolates were screened by polymerase chain reaction (PCR) for eight virulence genes, namely invA, agfA, IpfA, hilA, sivH, sefA, sopE, and spvC. S. Enteritidis isolates carried all virulence genes whilst S. Typhimurium isolates carried six virulence genes except sefA and spvC. A diverse phenotypic and genotypic AMR pattern was found. Harmonic descending trends of resistance patterns were observed among the broiler, sonali, and native chickens. Interestingly, virulent and MDR Salmonella enterica serovars were found in native chicken, although antimicrobials were not used in their production cycle. The research findings anticipate that virulent and MDR Salmonella enterica are roaming in the wet markets which can easily anchor to the vendor, consumers, and in the food chain.

Behavioral preventive measures and the use of medicines and herbal products among the public in response to Covid-19 in Bangladesh: A cross-sectional study.

The present study was conducted to assess the behavioral preventive measures and the use of medicines and herbal foods/products among the public in response to Covid-19. A cross-sectional survey comprised of 1222 participants was conducted from 27 June to 20 July 2020. Kruskal-Wallis test was used to identify the differences in behavioral preventive practices across different demographic categories. To identify the factors associated with the use of preventive medicines and herbal foods/products, multivariable logistic regression was performed. Most participants adopted the recommended preventive practices such as washing hands more frequently (87.5%), staying home more often (85.5%), avoiding crowds (86%), and wearing masks (91.6%). About half of the smokers reported a decreased rate of smoking during the pandemic. Also, 14.8% took medicines, 57.6% took herbal foods/products, and 11.2% took both medicines and herbal foods/products as preventive measure against Covid-19. Arsenicum album, vitamin supplements, and zinc supplements were the most commonly used preventive medicines. Gender, age, and fear of Covid-19 were significantly associated with the use of both preventive medicines and herbal foods/products. For the management of Covid-19 related symptoms, paracetamol, antihistamines, antibiotics, and mineral (zinc and calcium) supplements were used most often. Most participants sought information from non-medical sources while using medicines and herbal products. Moreover, potentially inappropriate and unnecessary use of certain drugs was identified.

Comparison of pathogenicity of subtype H9 avian influenza wild-type viruses from a wide geographic origin expressing mono-, di-, or tri-basic hemagglutinin cleavage sites.

An intravenous pathogenicity index (IVPI) of > 1.2 in chickens or, in case of subtypes H5 and H7, expression of a polybasic hemagglutinin cleavage site (HACS), signals high pathogenicity (HP). Viruses of the H9N2-G1 lineage, which spread across Asia and Africa, are classified to be of low pathogenicity although, in the field, they became associated with severe clinical signs and epizootics in chickens. Here we report on a pre-eminent trait of recent H9N2-G1 isolates from Bangladesh and India, which express a tribasic HACS (motif PAKSKR-GLF; reminiscent of an HPAIV-like polybasic HACS) and compare their features to H9Nx viruses with di- and monobasic HACS from other phylogenetic and geographic origins. In an in vitro assay, the tribasic HACS of H9N2 was processed by furin-like proteases similar to bona fide H5 HPAIV while some dibasic sites showed increased cleavability but monobasic HACS none. Yet, all viruses remained trypsin-dependent in cell culture. In ovo, only tribasic H9N2 viruses were found to replicate in a grossly extended spectrum of embryonic organs. In contrast to all subtype H5/H7 HPAI viruses, tribasic H9N2 viruses did not replicate in endothelial cells either in the chorio-allantoic membrane or in other embryonic tissues. By IVPI, all H9Nx isolates proved to be of low pathogenicity. Pathogenicity assessment of tribasic H9N2-G1 viruses remains problematic. It cannot be excluded that the formation of a third basic amino acid in the HACS forms an intermediate step towards a gain in pathogenicity. Continued observation of the evolution of these viruses in the field is recommended.

Evaluation of potential risk of transmission of avian influenza A viruses at live bird markets in response to unusual crow die-offs in Bangladesh.

In response to unusual crow die-offs from avian influenza A(H5N1) virus infection during January-February 2017 in Dhaka, Bangladesh, a One Health team assessed potential infection risks in live bird markets (LBMs). Evidence of aerosolized avian influenza A viruses was detected in LBMs and in the respiratory tracts of market workers, indicating exposure and potential for infection. This study highlighted the importance of surveillance platforms with a coordinated One Health strategy to investigate and mitigate zoonotic risk.

Genetic evolution and transmission dynamics of clade 2.3.2.1a highly pathogenic avian influenza A/H5N1 viruses in Bangladesh.

Asian lineage A/H5N1 highly pathogenic avian influenza viruses (HPAIVs) have been responsible for continuous outbreaks in Bangladesh since 2007. Although clades 2.2.2 and 2.3.4.2 HPAIVs have disappeared since poultry vaccination was introduced in 2012, clade 2.3.2.1a viruses have continued to be detected in Bangladesh. In this study, we identified A/H9N2 (n = 15), A/H5N1 (n = 19), and A/H5N1-A/H9N2 (n = 18) mixed viruses from live bird markets, chicken farms, and wild house crows (Corvus splendens) in Bangladesh from 2016 to 2018. We analyzed the genetic sequences of the H5 HPAIVs, to better understand the evolutionary history of clade 2.3.2.1a viruses in Bangladesh. Although seven HA genetic subgroups (B1-B7) and six genotypes (G1, G1.1, G1.2, G2, G2.1, and G2.2) have been identified in Bangladesh, only subgroup B7 and genotypes G2, G2.1, and G2.2 were detected after 2016. The replacement of G1 genotype by G2 in Bangladesh was possibly due to vaccination and viral competition in duck populations. Initially, genetic diversity decreased after introduction of vaccination in 2012, but in 2015, genetic diversity increased and was associated with the emergence of genotype G2. Our phylodynamic analysis suggests that domestic Anseriformes, including ducks and geese, may have played a major role in persistence, spread, evolution, and genotype replacement of clade 2.3.2.1a HPAIVs in Bangladesh. Thus, improvements in biosecurity and monitoring of domestic Anseriformes are needed for more effective control of HPAI in Bangladesh.

Prevalence of Avian Influenza A(H5) and A(H9) Viruses in Live Bird Markets, Bangladesh.

We conducted a cross-sectional study in live bird markets (LBMs) in Dhaka and Chittagong, Bangladesh, to estimate the prevalence of avian influenza A(H5) and A(H9) viruses in different types of poultry and environmental areas by using Bayesian hierarchical logistic regression models. We detected these viruses in nearly all LBMs. Prevalence of A(H5) virus was higher in waterfowl than in chickens, whereas prevalence of A(H9) virus was higher in chickens than in waterfowl and, among chicken types, in industrial broilers than in cross-breeds and indigenous breeds. LBMs with >1 wholesaler were more frequently contaminated by A(H5) virus than retail-only LBMs. Prevalence of A(H9) virus in poultry and level of environmental contamination were also higher in LBMs with >1 wholesaler. We found a high level of circulation of both avian influenza viruses in surveyed LBMs. Prevalence was influenced by type of poultry, environmental site, and trading.

Identification of Novel Non-secosteroidal Vitamin D Receptor Agonists with Potent Cardioprotective Effects and devoid of Hypercalcemia.

Vitamin D regulates many biological processes, but its clinical utility is limited by its hypercalcemic effect. Using a virtual screening platform to search novel chemical probes that activate the vitamin D signaling, we report discovery of novel non-steroidal small-molecule compounds that activate the vitamin D receptor (VDR), but are devoid of hypercalcemia. A lead compound (known as VDR 4-1) demonstrated potent transcriptional activities in a VDR reporter gene assay, and significantly ameliorated cardiac hypertrophy in cell culture studies and in animal models. VDR 4-1 also effectively suppressed secondary hyperparathyroidism in 1α-hydroxylase knockout mice. In contrast to 1α,25-dihydroxyvitamin D3 (1,25-D3 or calcitriol), a naturally occurring VDR agonist, VDR 4-1 therapy even at high doses did not induce hypercalcemia. These findings were accompanied by a lack of upregulation of calcium transport genes in kidney and in the gut providing a mechanism for the lack of hypercalcemia. Furthermore, VDR 4-1 therapy significantly suppressed cardiac hypertrophy and progression to heart failure in both vitamin D deficient and normal mice without inducing significant hypercalcemia. In conclusion, we have identified a unique VDR agonist compound with beneficial effects in mouse models of hyperparathyroidism and heart failure without inducing significant hypercalcemia.

Hydrogen peroxide-activatable antioxidant prodrug as a targeted therapeutic agent for ischemia-reperfusion injury.

Overproduction of hydrogen peroxide (H2O2) causes oxidative stress and is the main culprit in the pathogenesis of ischemia/reperfusion (I/R) injury. Suppression of oxidative stress is therefore critical in the treatment of I/R injury. Here, we report H2O2-activatable antioxidant prodrug (BRAP) that is capable of specifically targeting the site of oxidative stress and exerting anti-inflammatory and anti-apoptotic activities. BRAP with a self-immolative boronic ester protecting group was designed to scavenge H2O2 and release HBA (p-hydroxybenzyl alcohol) with antioxidant and anti-inflammatory activities. BRAP exerted potent antioxidant and anti-inflammatory activity in lipopolysaccharide (LPS)- and H2O2-stimulated cells by suppressing the generation of ROS and pro-inflammatory cytokines. In mouse models of hepatic I/R and cardiac I/R, BRAP exerted potent antioxidant, anti-inflammatory and anti-apoptotic activities due to the synergistic effects of H2O2-scavenging boronic esters and therapeutic HBA. In addition, administration of high doses of BRAP daily for 7 days showed no renal or hepatic function abnormalities. Therefore BRAP has tremendous therapeutic potential as H2O2-activatable antioxidant prodrug for the treatment of I/R injuries.

Exaggerated hypertensive response to combretastatin A-4 phosphate in hypertensive rats: Effective pharmacological inhibition by diltiazem.

Combretastatin A-4 phosphate (CA4P), a tubulin depolymerizing agent, shows promise in anti-cancer therapy and is associated with dose-dependent transient hypertension. The cardiac consequence of this hypertensive effect is unknown. This study was conducted to examine the cardiotoxic effect of CA4P on a rat model of hypertension. Hypertensive rats were created by feeding a 6% high salt (HS) diet to Dahl salt sensitive (DSS) rats for 2.5weeks. Cardiac toxicity was measured using serum troponin I levels 24h after CA4P administration. In rats fed HS diet, there was a significant increase in mean arterial blood pressure (MAP) from baseline, which was further increased by 80% following CA4P administration with peak systolic blood pressure (BP) of 247mmHg. Treatment with the calcium channel blockers, diltiazem and nicardipine, completely inhibited the hypertensive effects of CA4P. Nitroglycerin or enalapril, however, failed to completely block the hypertensive effects of CA4P. CA4P injection also significantly increased the cardiac troponin I level in hypertensive rats though pretreatment with diltiazem effectively blocked troponin I increase after CA4P administration. Based on these findings, an exaggerated hypertensive response to CA4P is associated with myocardial damage in hypertensive rats. Calcium channel blockers effectively blocked both CA4P induced hypertension and cardiac damage.

Endothelin A receptor antagonist, atrasentan, attenuates renal and cardiac dysfunction in Dahl salt-hypertensive rats in a blood pressure independent manner.

Proteinuria is a hallmark of chronic kidney disease (CKD) and cardiovascular disease (CVD), and a good predictor of clinical outcome. Selective endothelin A (ETA) receptor antagonist used with renin-angiotensin system (RAS) inhibitors prevents development of proteinuria in CKD. However, whether the improvement in proteinuria would have beneficial effects on CVD, independent of RAS inhibition, is not well understood. In this study, we investigated whether atrasentan, an ETA receptor antagonist, has renal and cardiovascular effects independent of RAS inhibition. Male Dahl salt sensitive (DSS) rats, at six weeks of age, received water with or without different doses of atrasentan and/or enalapril under high salt (HS) diet or normal diet (ND) for 6 weeks. At the end of 12th week, atrasentan at a moderate dose significantly attenuated proteinuria and serum creatinine without reducing mean arterial pressure (MAP), thereby preventing cardiac hypertrophy and improving cardiac function. ACE inhibitor enalapril at a dose that did not significantly lowered BP, attenuated cardiac hypertrophy while moderately improving cardiac function without reducing proteinuria and serum creatinine level. Nonetheless, combined therapy of atrasentan and enalapril that does not altering BP exerted additional cardioprotective effect. Based on these findings, we conclude that BP independent monotherapy of ETA receptor antagonist attenuates the progression of CKD and significantly mitigates CVD independent of RAS inhibition.

Comparative anti-inflammatory effects of anti-arthritic herbal medicines and ibuprofen.

Non-steroidal anti-inflammatory drugs (NSAIDS), such as ibuprofen, are widely used over-the-counter drugs to treat arthritis, but they are often associated with side effects. Herbal medicines have been used to treat various diseases such as arthritis, but the scientific profiles are not well understood. In this study, we examined, in comparison with ibuprofen, the inhibitory effects on various inflammatory markers of the most commonly used herbal medicines to treat arthritis, boswellia (Boswellia sapindales), licorice (Glycyrrhiza glabra), guggul (Commiphora wightii), and neem (Azadirachta indica). To elicit inflammatory response, we exposed mouse myoblast C2C12 cells to lipopolysaccharide (LPS). Tumor necrosis factor-alpha (TNF-α) and monocyte chemotactic protein-1 (MCP-1), which are cytokines activated during an inflammatory response, were determined. The optimal non-toxic concentration was determined by exposing different concentrations of drugs (from 0.01 to 10 mg/mL). Cell death measurement revealed that the drug concentrations lower than 0.05 mg/mL were non-toxic concentrations for each drug, and these doses were used for the main experiments. We found that neem and licorice showed robust anti-inflammatory responses compared with ibuprofen. However, boswellia and guggul did not demonstrate significant anti-inflammatory responses. We concluded that neem and licorice are more effective than ibuprofen in suppressing LPS-induced inflammation in C2C12 cells.