Inhibitory Effect of Terminalia chebula Retz. on MDR Shigella flexneri Invasion of HeLa Cells.

Shigella spp. (Shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei) cause bacillary dysentery (shigellosis), which is characterized by bloody mucous diarrhoea. Although a variety of antibiotics have been effective for treatment of shigellosis, options are becoming limited due to globally emerging drug resistance. In the present study, in vitro antibacterial activity of methyl gallate (MG) isolated from Terminalia chebula was determined by performing MIC, minimal bactericidal concentration (MBC) and time-kill kinetic studies. Bacterial membrane-damaging activity of MG was determined by membrane perturbation and transmission electron microscopy (TEM). Cellular drug accumulation, cell infection and assessment of intracellular activities of MG and reference antibiotics were performed using HeLa cell cultures. The bactericidal activity of MG against multidrug-resistant (MDR) Shigella spp. in comparison with other commonly used drugs including fluoroquinolone was demonstrated here. TEM findings in the present study revealed that MG caused the total disintegration of inner and outer membranes, and leakage of the cytoplasmic contents of S. dysenteriae. The level of accumulation of MG and tetracycline in HeLa cells incubated for 24 h was relatively higher than that of ciprofloxacin and nalidixic acid (ratio of intracellular concentration/extracellular concentration of antibiotic for MG and tetracycline>ciprofloxacin and nalidixic acid). The viable number of intracellular S. dysenteriae was decreased in a time-dependent manner in the presence of MG (4 × MBC) and reduced to zero within 20 h. The significant intracellular activities of MG suggested that it could potentially be used as an effective antibacterial agent for the treatment of severe infections caused by MDR Shigella spp.

Background: Shigella is one of the most serious pathogens associated with bloody diarrhea in children. The empiric antibiotic therapy of enteric illness with blood streaked stool leads to emergence of multi drug resistant (MDR) Shigella. The condition gets exacerbated by presence of integrons that facilitate the horizontal spread. Virulence genes associated with MDR Shigella modulate the patient outcome, particularly in children. Objectives: The present study was aiming at isolation of MDR Shigella from children with diarrheal sickness and characterization of those isolates as regarding presence of class 1 integrase and other virulence genes. Methods: Four hundred and ninety patients under the age of five suffering from diarrheal illness were examined for presence of Shigella in their stool specimens. MDR Shigella was determined using the antibiotic susceptibility testing by disc diffusion method; those isolates were tested for presence of class 1 integrase by PCR. Multiplex PCR assay was used to determine the presence of virulence genes, virA, ial, sen, set1A, set1B, sat, ipaBCD, ipaH and stx in the MDR Shigella isolates. Results: The isolation rate of Shigella from pediatric patients was 5.3%. Most of the isolated Shigella (57.7%) were from infants between 12 and 23 month. 73.1% of the identified Shigella were MDR. intI1 gene was present in 78.9% of MDR isolates. Muliplex PCR revealed that ipaH and ipaBCD, virA, sat, ial, set1A and set1B, sen were detected in 94.7%, 78.9%, 73.7%, 68.4%, 42.1%, 36.8% of the MDR Shigella isolates respectively. Conclusion: The MDR isolates represented a considerable percentage of Shigella detected in pediatric patients. Presence of intI1 gene in most of MDR Shigella reflects the higher possibility of resistant strains spread. Existence of a variety of virulence genes in those isolates is an important indicator of serious disease outcome.

Methicillin-resistant Staphylococcus aureus (MRSA) infection is a global health problem that needs to be answered by developing a synthetic or natural antibacterial to overcome the problem. The honey pineapple peel contains potentially antibacterial compounds such as bromelain and flavonoid. This study aims to determine the antibacterial activity of honey pineapple peel extract (Ananas comosus [L] Merr.) against MRSA. The design of this study was a true experimental study with a post-test control group design. This study examined the antibacterial activity of honey pineapple peel extract at concentrations of 100%, 50%, 25%, 12.5%, 6.25%, 3.125%, and 1.56% against MRSA bacteria by measuring minimum inhibitory concentration (MIC) with dilution method and minimum bactericidal concentration (MBC). The results of this study show that MIC was obtained at concentrations of 50 % and MBC at extract with a concentration of 100 %. There is an antibacterial activity of honey pineapple peel extract (Ananas comosus [L] Merr.) against MRSA.
 Keywords: Antibacterial Activity, Honey Pineapple Peel, MRSA

Background and Objectives: Chemical preservatives are commonly used to prevent food from quick spoiling and oxidizing. Despite their effect in increasing shelf life of foods, these compounds can have side effects. Residues of chemical compounds in foods increase microbial resistance. This study aims to investigate the antioxidant and antimicrobial properties of lemon verbena (Lippia citriodora L.) ethanolic extract against some gram-positive and gram-negative bacteria. Methods: In this experimental study, The soaking method and ethanol solvent was used for extraction. Antioxidant activities of different concentrations of extract were assessed using DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity test and compared with butylated hydroxyanisole (BHA) as a synthetic antioxidant. Antimicrobial effect of extract on Staphylococcus aureus, Listeria innocua, Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa and Salmonella typhimurium was determined by disk diffusion and well diffusion methods and measuring minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Experimental data were analyzed in SPSS software, version 20 using analysis of variance and the mean values were compared using Duncan’s multiple range test. Results: The amounts of phenols and flavonoids in the ethanolic extract of were 96.47±0.35 mg of gallic acid/g of extract and 31.07±0.42 mg of quercetin/g of extract, respectively. The half-maximal inhibitory concentration of lemon verbena extract was 11.79±0.76 μg/mL, while it was 10.16±0.29 μg/mL for BHA. In both disk diffusion and well diffusion methods, with the increase of the concentration of extract, the inhibition zone diameter increased. The ethanolic extract of lemon verbena at the concentration of 12.5 mg/mL had no inhibitory effect on gram-negative bacteria growth. In both methods, the extract had the greatest effect on S. aureus. The MIC of ethanolic extract ranged from 8 to 128 mg/mL, depending on the type of bacteria (gram-positive or gram-negative). Conclusion: The ethanolic extract of lemon verbena has higher antimicrobial effects on gram-positive bacteria. Therefore, it can be used in food and pharmaceutical industries as a natural antimicrobial agent. Considering the increasing resistance of bacteria to chemical antibiotics, more studies on lemon verbena and its antibacterial compounds are needed for treating infections.

Alkaloids extracted from different parts (leaf, stem, stem bark, and fruits) of Terminalia chebula were screened for antimicrobial activity against nine bacteria (Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Staphylococcus aureus, Bacillus subtilis, Raoultella planticola, Enterobacter aerogens, Agrobacterium tumefaciens, and Klebsiella pneumoniae) and two fungi (Aspergillus flavus and Aspergillus niger) and one yeast (Candida albicans). Minimum inhibitory concentration, Minimum bactericidal/fungicidal concentration, and Total activity of the extracts, against each sensitive test pathogen, were also evaluated. Alkaloids from all plant parts showed good antimicrobial activity against almost all the test microorganisms except A. niger, against which, none of the tested extracts showed activity. The largest zone of inhibition (IZ 20.75 mm) was observed against P. aeruginosa. The total activity of the leaf alkaloid was found to be the same and the highest (256.41ml/g) was against E. aerogens and A. tumefaciens. Key words: Alkaloids, T. chebula, antimicrobial activity, minimum inhibitory concentration, minimum bactericidal concentration, minimum fungicidal concentration, E. aerogens

Anticancer activity of an ehnomedicinal plant Croton caudatus Geiseler, Kam sabut in cultured HeLa cells

Piper betle L. has traditionally been used in alternative medicine in different countries for various therapeutic purposes, including as an anti-infective agent. However, studies reported in the literature are mainly on its activities on drug susceptible bacterial strains. This study determined the antimicrobial activities of its ethanol, methanol, and supercritical CO2 extracts on clinical isolates of multiple drug resistant bacteria which have been identified by the Infectious Disease Society of America as among the currently more challenging strains in clinical management. Assay methods included the standard disc diffusion method and the broth microdilution method for the determination of the minimum inhibitory concentration (MIC) and the minimum bactericidal concentrations (MBC) of the extracts for the test microorganisms. This study revealed the bactericidal activities of all the P. betle leaf crude extracts on methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), extended spectrum β-lactamase-producing Enterobacteriaceae, carbapenem-resistant Enterobacteriaceae, and metallo-β-lactamase-producing Pseudomonas aeruginosa and Acinetobacter baumannii, with minimum bactericidal concentrations that ranged from 19μg/ml to 1250 μg/ml. The extracts proved to be more potent against the Gram positive MRSA and VRE than for the Gram negative test bacteria. VRE isolates were more susceptible to all the extracts than the MRSA isolates. Generally, the ethanol extracts proved to be more potent than the methanol extracts and supercritical CO2 extracts as shown by their lower MICs for both the Gram positive and Gram negative MDRs. MTT cytotoxicity assay showed that the highest concentration (100 μg/ml) of P. betle ethanol extract tested was not toxic to normal human dermal fibroblasts (HDFn). Data from the study firmly established P. betle as an alternative source of anti-infectives against multiple drug resistant bacteria.

Lipoic acid increases glutathione production and enhances the effect of mercury in human cell lines

Background:Cassia fistula, is a flowering plant and a member of Fabaceae family. Its leaves are compound of 4 - 8 pairs of opposite leaflets. There are many Cassia species around the world which are used in herbal medicine.Objectives:This study was designed to examine in vitro anti-bacterial activity of methanolic and ethanolic extracts of C. fistula native to Khuzestan, Iran.Materials and Methods:The microbial inhibitory effect of methanolic and ethanolic extracts of C. fistula was tested on 3 Gram positive: Bacillus cereus, Staphylococcus aureus and S. epidermidis and 5 Gram negative: Salmonella Typhi, Kelebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and Proteus mirabilis bacterial species using disc diffusion method at various concentrations. The minimum inhibitory and bactericidal concentrations (MIC and MBC) were measured by the tube dilution assay.Results:The extract of C. fistula was effective against B. cereus, S. aureus, S. epidermidis, E. coli and K. pneumoniae. The most susceptible microorganisms to ethanolic and methanolic extracts were E. coli and K. pneumoniae, respectively. Also B. cereus and S. aureus showed the least sensitivity to ethanolic and methanolic extracts, respectively. The MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) of ethanolic extracts against S. aureus, E. coli, S. epidermidis and K. pneumoniae were also determined.Conclusions:With respect to the obtained results and regarding to the daily increase of the resistant microbial strains to the commercial antibiotics, it can be concluded that these extracts can be proper candidates of antibacterial substance against pathogenic bacterial species especially S. aureus, E. coli, K. pneumoniae and S. epidermidis.

The evidence and prevalence of multidrug-resistant (MDR) Shigella spp. poses a serious global threat to public health and the economy. Food- or water-borne MDR Shigella spp. demands an alternate strategy to counteract this threat. In this regard, phage therapy has garnered great interest from medical practitioners and researchers as a potential way to combat MDR pathogens. In this observation, we isolated Shigella phages from environmental water samples and tested against various clinically isolated MDR Shigella spp. In this study, we have defined the isolation and detailed physical and genomic characterizations of two phages Sfin-2 and Sfin-6 from environmental water samples. The phages exhibited potent lytic activity against Shigella flexneri, Shigella dysenteriae, and Shigella sonnei. They showed absorption within 5–10 min, a burst size ranging from ~74 to 265 PFU/cell, and a latent period of 5–20 min. The phages were stable at a broad pH range and survived an hour at 50°C. The purified phages Sfin-2 and Sfin-6 belong to the Siphoviridae family with an isometric head (64.90 ± 2.04 nm and 62.42 ± 4.04 nm, respectively) and a non-contractile tail (145 ± 8.5 nm and 148.47 ± 14.5 nm, respectively). The in silico analysis concluded that the size of the genomic DNA of the Sfin-2 phage is 50,390 bp with a GC content of 44.90%, while the genome size of the Sfin-6 phage is 50,523 bp with a GC content of 48.30%. A total of 85 and 83 putative open reading frames (ORFs) were predicted in the Sfin-2 and Sfin-6 phages, respectively. Furthermore, a comparative genomic and phylogenetic analysis revealed that both phages represented different isolates and novel members of the T1-like phages. Sfin-2 and Sfin-6 phages, either individually or in a cocktail form, showed a significant reduction in the viable Shigella count on raw chicken samples after 72 h of incubation. Therefore, these results indicate that these phages might have a potential role in therapeutic approaches designed for shigellosis patients as well as in the biological control of MDR Shigella spp. in the poultry or food industry during the course of meat storage.

This study was conducted to carryout preliminary phytochemical analysis and in vitro antimicrobial activities of aqueous and ethanolic root and stem bark extracts of Ficus sycomorus. Qualitative phytochemical analysis for tannins, saponin, terpenoids, flavonoids, alkaloids, glycosides, steroids, phenols, and reducing sugar was done using standard methods. The antimicrobial activities of the extracts were tested against four micro- organisms; Escherichia coli, Staphylococcus aureus, Shigella dysentrae, and Salmonella typhi. Agar well diffusion method was used for the antimicrobial studies. Phytochemical screening of both root and stem bark aqueous extracts showed the presence of tannin, saponin, terpenoid, flavonoid, alkaloids, glycoside, steroid, reducing sugar, and phenol. Glycoside was not detected in both the aqueous and ethanolic extracts of the root bark. The result of the antimicrobial studies showed that the aqueous root extract have higher antimicrobial activity ranging from (2-12 mm) on the tested microorganisms than aqueous stem bark extract (3-9 mm), while for ethanol extract both stem and root bark extract has almost the same effect or antimicrobial activity on the tested pathogens ranging from (2-15 mm) which is having higher activity compared to the aqueous extracts. The Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) of both the extracts were found to be 50 mg/mL and 100 mg/mL respectively. From this study, it can, therefore, be concluded that the root and stem bark extract is a potential antimicrobial agent which support the claim of the traditional users of this plant in herbal medicine for the treatment of diseases that are of microbial origin.

PurposeThe last few decades have witnessed a rapid and global increase in multidrug-resistant bacteria (MDR) emergence.MethodsThe aim of the current study is to isolate the most common MDR bacteria from dairy farms and beef slaughterhouses followed by evaluation of their antimicrobial resistance pattern and assessment of the antibacterial activity of AgNPs-H2O2 as an alternative to conventional antibiotics. In this regard, 200 samples were collected from two dairy farms and one beef slaughterhouse located in Dakhliya Governorate, Egypt.ResultsInterestingly, out of 120 collected samples from dairy farms, the prevalence of the isolated strains was 26.7, 23.3, 21.7, 16.7, and 11.7% for S. typhimurium, E. coli O157:H7, L. monocytogenes, K. pneumoniae and P. aeruginosa, respectively. Meanwhile, the overall prevalence was 30, 25, 22.5, 17.5, and 5% for E. coli O157:H7, L. monocytogenes, S. typhimurium, P. aeruginosa, and K. pneumoniae, respectively, for the 80 samples collected from a beef slaughterhouse. The antimicrobial susceptibility pattern elucidated that all isolated strains exhibited resistance to at least four of the tested antimicrobials, with multiple-antibiotic resistance index values (MAR) ranging between 0.44 and 0.88. Furthermore, the commercial AgNPs-H2O2 product was characterized by transmission electron microscopy (TEM) and zeta potential that showed spherical particles with a surface charge of −0.192 mV. The antimicrobial activity of synergized nano-silver (AgNP) with H2O2 product toward MDR strains was assessed via measuring minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and time-kill curve.ConclusionThe present data report high prevalence rates of MDR pathogens in dairy farms and abattoirs. More importantly, AgNPs-H2O2 exerted broad-spectrum bactericidal activity toward MDR bacterial strains, suggesting their promising usage as safe, ecofriendly, cost-effective antibacterial agents. To our knowledge, this study is a pioneer in investigating the potential alternative antimicrobial role of silver nanoparticles for control of multiple drug-resistant pathogens in Egypt.

Shigella and Salmonella infections represent a major health problem, particularly in the developing countries. 165 million cases of Shigella infection occur annually worldwide with 1.1 million deaths with most of the causality in children under 5 years of age. Plasmid mediated fluoroquinolone resistance is on the rise among enteric pathogens. This study was aimed to investigate the horizontal transfer of fluoroquinolone resistant gene harbouring plasmid from multi-drug resistant (MDR) Shigella spp. to susceptible Salmonella spp ., which are closely related among diarrheal infections. A total of 67 MDR Shigella spp. were isolated from faeces samples from January to December 2014. Isolates were identified by standard biochemical tests and further serotyped by slide agglutination test with commercial antisera. Antimicrobial susceptibility testing was performed as per Kirby-Bauer disc diffusion method. 20 MDR Shigella spp were selected in random for further analysis. Quinolone resistance genes ( qnr A, qnr B and qnr S) were identified by colony PCR for all isolates. Plasmids were isolated from 20 isolates for confirmation of the presence of qnr genes. MDR Shigella isolates ( n = 2) , harbouring plasmid with qnr S gene was conjugated with completely susceptible Salmonella spp. to observe horizontal gene transfer. Selection of conjugates were done using antibiotic medium. Next generation sequencing (NGS) was performed to confirm the identity of the conjugated plasmid. 6 out of 20 MDR Shigella isolates had qnr S genes in addition to one qnr B gene in one isolate. The qnr S containing plasmid was observed to successfully transfer from MDR Shigella isolates ( n = 2) to susceptible Salmonella spp. through conjugation. PCR of the transferred plasmid confirmed the presence of qnr S gene. NGS confirmed that the same qnr S harbouring plasmid was transferred from Shigella spp . to Salmonella spp . This study clearly demonstrates that the plasmid donated by Shigella spp. can be naturally acquired by Salmonella spp ., which poses a greater threat for rapid spread of fluoroquinolone resistance among enteric pathogens. Continuous surveillance of plasmids containing antimicrobial resistance genes is crucial for control of further spread of fluoroquinolone resistance.

Shigella and Salmonella infections represent a major health problem, particularly in the developing countries. 165 million cases of Shigella infection occur annually worldwide with 1.1 million deaths with most of the causality in children under 5 years of age. Plasmid mediated fluoroquinolone resistance is on the rise among enteric pathogens. This study was aimed to investigate the horizontal transfer of fluoroquinolone resistant gene harbouring plasmid from multi-drug resistant (MDR) Shigella spp. to susceptible Salmonella spp ., which are closely related among diarrheal infections. A total of 67 MDR Shigella spp. were isolated from faeces samples from January to December 2014. Isolates were identified by standard biochemical tests and further serotyped by slide agglutination test with commercial antisera. Antimicrobial susceptibility testing was performed as per Kirby-Bauer disc diffusion method. 20 MDR Shigella spp were selected in random for further analysis. Quinolone resistance genes ( qnr A, qnr B and qnr S) were identified by colony PCR for all isolates. Plasmids were isolated from 20 isolates for confirmation of the presence of qnr genes. MDR Shigella isolates ( n = 2) , harbouring plasmid with qnr S gene was conjugated with completely susceptible Salmonella spp. to observe horizontal gene transfer. Selection of conjugates were done using antibiotic medium. Next generation sequencing (NGS) was performed to confirm the identity of the conjugated plasmid. 6 out of 20 MDR Shigella isolates had qnr S genes in addition to one qnr B gene in one isolate. The qnr S containing plasmid was observed to successfully transfer from MDR Shigella isolates ( n = 2) to susceptible Salmonella spp. through conjugation. PCR of the transferred plasmid confirmed the presence of qnr S gene. NGS confirmed that the same qnr S harbouring plasmid was transferred from Shigella spp . to Salmonella spp . This study clearly demonstrates that the plasmid donated by Shigella spp. can be naturally acquired by Salmonella spp ., which poses a greater threat for rapid spread of fluoroquinolone resistance among enteric pathogens. Continuous surveillance of plasmids containing antimicrobial resistance genes is crucial for control of further spread of fluoroquinolone resistance.

Summary Rabbits were immunized by repeated intravenous injections of culture fluid in which HeLa cells had been grown and ruptured. No anaphylactic reactions were observed with the schedule followed. The serum of the rabbits before immunization showed no untoward effect on HeLa cells in stationary tube culture. After immunization the rabbits’ serum developed the capacity to agglutinate and lyse HeLa cells in culture. In contrast, serum of rabbits immunized with maintenance solution without cellular antigens did not affect HeLa cells in culture. The anti-HeLa effect could be demonstrated inmedium containing either horse serum or human serum. The anti-HeLa property of antiserum could be removed by adsorption with HeLa cell suspension. The reaction on six other tissues of human origin cultured on glass have been tested, but the specificity of the reaction needs further study. The “antibody” responsible was resistant to exposure to 56°C for one-half hour, or for two weeks at 4°C. The anti-HeLa effect was not found to be enhanced by the presence of fresh normal guinea pig serum at 1:15 or 1:20 final dilution. However, in the presence of 1:4 dilution of normal guinea pig serum a predominantly lytic effect occurred when the serum was fresh, in contrast to the agglutination reaction observed in inactivated serum. The possibility of such an anti-tissue reaction should be considered when attempting to produce antisera to agents growing in tissue culture. The reaction may prove useful for releasing parasitic organisms from the intracellular position in tissue culture. It may also serve in studies of host-parasite relationships by employing antibodies to antigens of cellular as well as of parasitic origin.