Agar Plate Dilution Method Research Articles

Evaluation of Antifungal and anti-aflatoxin B1 efficacy of some crude extracts of Chamaerops humilis L. against Aspergillus flavus isolated from peanuts (Arachis hypogea L.)

The present study strengthens the food preservative potential of Chamaerops humilis extracts viz. hexane extract, chloroform extract, ethanol extract, chloroform extract, and methanol extract based on their antifungal, antiaflatoxin, and antioxidant efficacy. The isolation of molds from peanuts were carried out by the suspension-dilution technique and inoculation on agar medium. Antifungal determinations were conducted using the agar plate and liquid dilution methods. The effect of C. humilis L. extracts on the production of AFB1 was determined in a SMKY broth. The molds isolated were of the genera Aspergillus, Penicillium sp., Fusarium sp., and Alternaria sp. with A. flavus (BBH-6) which is identified as the highest AFB1 producer. The minimum inhibitory concentration of extracts against the toxigenic strain of A. flavus ranged between 3.00 mg/ml and 3.50 mg/ml. The extracts were found more efficacious as they inhibited the dry mycelium weight and aflatoxin production of the aflatoxigenic strain A. flavus (BBH-6) at lower concentrations. The extracts showed fungitoxic spectrum against four molds. The IC50 value of C. humilis L. ranged between 140.4 and 189.46 μg/mg, oxidation of linoleic acid was moderately inhibited by the extracts ranges between 53.54 and 69.33%, while their total phenolic content ranged between 47.80 and 115.71 μg/ml. Based on the findings of present investigation, C. humilis L. can be introduced as a proposed solution for formulating plant-based preservative food additives.

The Quinone-Derived Small Molecule M5N32 Is an Effective Anti-Helicobacter pylori Agent Both In Vivo and In Vitro.

Helicobacter pylori has become increasingly resistant to all commonly used clinical antibiotics. Therefore, new anti-H. pylori drugs need to be identified. Recently, quinones were found to inhibit growth of H. pylori with quinone-derived small-molecule compounds identified as having antitumor effects. The minimum inhibitory concentrations of the compounds against H. pylori were measured by agar plate dilution method. The inhibition of biofilm formation by the compounds was assessed by SYTO9-PI double staining. The reactive oxygen species induced by the compounds were detected by DCFH-DA stain. The clearance effects of the compounds for H. pylori in mouse were evaluated by counting colony-forming units and hematoxylin and eosin staining. Our results revealed strong inhibition of M5N32 in vitro against H. pylori in both the planktonic and biofilm-forming states. Resistance to M5N32 was not developed in successive generations of the bacteria. In vivo, the combination of M5N32 and omeprazole showed enhanced effects in comparison to the standard triple therapy. M5N32 was nontoxic to normal tissues. M5N32 is effective in the treatment of H. pylori infections, providing potential development of anti-H. pylori medicines in the treatment of H. pylori infections.

Revealing the novel effect of Jinghua Weikang capsule against the antibiotic resistance of Helicobacter pylori.

BackgroundHelicobacter pylori (H. pylori) infects half of the human population globally. Eradication rates with triple or quadruple therapy have decreased owing to the increasing rate of antibiotic resistance. Jinghua Weikang capsule (JWC) is the first and most popular Chinese patent medicine approved by the state for the treatment of gastritis and peptic ulcers caused by H. pylori infection in China. Previous studies have found that JWC has a certain bactericidal effect on drug-resistant H. pylori and its major component, Chenopodium ambrosioides L. inhibits biofilm formation, but the mechanism remains unclear. This study focused on drug-resistant H. pylori and explored whether JWC could reverse drug resistance and its related mechanisms.MethodThe agar plate dilution method, E-test method, and killing kinetics assay were used to evaluate the bactericidal effect of JWC on antibiotic-resistant H. pylori and its effect on antibiotic resistance. Sanger sequencing was used to detect mutations in drug resistance genes. The crystal violet method, scanning electron microscopy, and confocal laser scanning microscopy were used to evaluate the effects of JWC on biofilms. qPCR was performed to evaluate the effect of JWC on the expression of efflux pump-related genes. qPCR and immunofluorescence were used to evaluate the effects of JWC on H. pylori adhesion.ResultsJWC showed considerable antibacterial activity against drug-resistant H. pylori strains, with minimum inhibitory concentration (MIC) values ranging from 64 to 1,024 μg/ml. The MIC of metronidazole (MTZ) against H. pylori 26,695–16R decreased from 64 to 6 μg/ml after treatment with 1/2 MIC of JWC. The resistance of H. pylori 26,695–16R to MTZ was reversed by JWC, and its effect was better than that of PaβN and CCCP. H. pylori 26,695–16R is a moderate biofilm-forming strain, and JWC (16–64 μg/ml) can inhibit the formation of biofilms in H. pylori 26,695–16R. JWC reduced the expression of HP0605-HP0607 (hefABC), HP0971-HP0969 (hefDEF), HP1327-HP1329 (hefGHI), and HP1489-HP1487. JWC reduced the adhesion of H. pylori to GES-1 cells and the expression of adhesives NapA, SabA, and BabA.ConclusionThe reversal of MTZ resistance by JWC may be achieved through the adhesin/efflux pump-biofilm pathway.

Isolation, genomic characterization, and mushroom growth-promoting effect of the first fungus-derived Rhizobium.

Polyporus umbellatus is a well-known edible and medicinal mushroom, and some bacteria isolated from mushroom sclerotia may have beneficial effects on their host. These mushroom growth-promoting bacteria (MGPBs) are of great significance in the mushroom production. In this work, we aimed to isolate and identify MGPBs from P. umbellatus sclerotia. Using the agar plate dilution method, strain CACMS001 was isolated from P. umbellatus sclerotia. The genome of CACMS001 was sequenced using PacBio platform, and the phylogenomic analysis indicated that CACMS001 could not be assigned to known Rhizobium species. In co-culture experiments, CACMS001 increased the mycelial growth of P. umbellatus and Armillaria gallica and increased xylanase activity in A. gallica. Comparative genomic analysis showed that CACMS001 lost almost all nitrogen fixation genes but specially acquired one redox cofactor cluster with pqqE, pqqD, pqqC, and pqqB involved in the synthesis of pyrroloquinoline quinone, a peptide-derived redox participating in phosphate solubilization activity. Strain CACMS001 has the capacity to solubilize phosphate using Pikovskaya medium, and phnA and phoU involved in this process in CACMS001 were revealed by quantitative real-time PCR. CACMS001 is a new potential Rhizobium species and is the first identified MGPB belonging to Rhizobium. This novel bacterium would play a vital part in P. umbellatus, A. gallica, and other mushroom cultivation.

In Vitro Pharmacodynamics and Bactericidal Mechanism of Fungal Defensin-Derived Peptides NZX and P2 against Streptococcus agalactiae

(1) Background: Based on the hazard of Streptococcus agalactiae to human and animal health and the increasing drug resistance, it is urgent to develop new antimicrobial agents with high bactericidal activity and low drug resistance against S. agalactiae. This study aims to investigate in vitro pharmacodynamics and bactericidal mechanism of fungal defensin-derived peptides NZX and P2 against S. agalactiae. (2) Methods: Minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC) were determined by broth dilution method and AGAR plate dilution method. Cell membrane integrity was determined by flow cytometer. Cell morphological changes were observed by scanning electron microscope (SEM) and transmission electron microscope (TEM). (3) Results: MIC values (NZX: 0.11 μM, P2: 0.91 μM) and MPC (NZX: 1.82 μM) showed their higher antibacterial activity and stronger inhibition ability of drug resistance mutation. The bactericidal mechanism was elucidated that P2 caused S. agalactiae ACCC 61733 cells to deform, bound to the cell wall, and perturbed cell membrane, resulting in K+ leakage, membrane hyperpolarization, ATP release, and reduced cell contents. Compared with P2, NZX focuses on the cell wall, and it bound to the cell wall causing cells boundary disappearance. (4) Conclusion: NZX and P2 are promising antimicrobial agents for streptococcicosis treatment.

IN VITRO ANTIBACTERIAL ACTIVITY OF GREEN TEA EXTRACT AGAINST MULTIDRUG-RESISTANT BACTERIA

Objectives: This study aimed to evaluate the antimicrobial activity of ethanolic extract of green tea (Camellia sinensis) against multidrug-resistant strains of the pathogenic bacteria: Extended-spectrum-β-lactamase (ESBL) producing Enterobacteriaceae, Carbapenem-resistant Enterobacteriaceae (CRE), and Metallo-β-lactamase (MBL) producing Pseudomonas aeruginosa strains. Methods: In this cross-sectional study, the antibacterial activity of ethanolic extract of commercial green tea against the 23 multidrug-resistant test strains was evaluated by the Agar well diffusion method, and the minimum inhibitory concentration of the extract for the test strains was determined by Agar plate dilution method. Results: Ethanolic extract of green tea was found to exhibit a remarkably significant antimicrobial activity against the ATCC (American type culture collection) control strains: E. coli ATCC 25922 and P. aeruginosa ATCC 27853 with slightly higher activity against later as compared to the former. The extract exhibited a significant antibacterial activity against multidrug-resistant bacterial strains. The highest activity was shown against ESBL producing strains, followed by CRE strains and the least activity against MBL producers. Conclusion: This study strongly depicts that the ethanolic extract of green tea exhibits significant antibacterial activity even against multidrug-resistant strains. Hence, such plant extracts could be a potential source of bioactive lead compounds that could be utilized in developing herbal antimicrobials as an alternative strategy for tackling the problem of antimicrobial resistance.

Study on the value of antibiotic-resistant gene detection in Helicobacter pylori in China.

The aim of the present study was to explore the value of detecting antibiotic-resistant genes in Helicobacter pylori (H. pylori) and the association between genotype and antibiotic resistance. Two gastric mucosa samples from each H. pylori-positive patient were collected. Each patient's H. pylori sample was cultured in vitro, and the agar plate dilution method was conducted. In addition, all patient samples were analyzed for the detection of antibiotic resistance-related mutant genes and VacA gene genotypes. The association between VacA genotypes and antibiotic resistance was also determined and the value of mutant gene detection in predicting H. pylori resistance to antibiotics was evaluated. In total, 133 H. pylori-positive patients were enrolled. A total of 22 strains of H. pylori failed to grow in in vitro culture and 25 strains were negative in a H. pylori gene test. Among 108 strains detected by PCR, a total of 39 VacA s1m1 strains, 69 VacA s1m2 strains and no VacA s2 strain were identified. There was no significant association between VacA genotypes and antibiotic resistance. The mutation rates of G616A in the rdxA gene, T87A, G91A, A91G and G91T in the gyrA gene and A2143G and A2142G in the 23S rRNA gene were 32.1, 32.3, 22.6, 12.9, 6.5, 81.8 and 0.0%, respectively. Among these mutant sites, the mutation coincidence rates were as follows, according to the agar plate dilution method: rdxA G616A (81.8%), gyrA G91T (66.7%), gyrA G91A (54.5%), 23 S rRNA A2143G (49.1%), gyrA T87A (45.5%), gyrA A91G (33.3%), penicillin-binding protein 1 (PBP1) C556G (0.0%), PBP1 A562T (0.0%), PBP1 A562G (0.0%) and 16 S rRNA 926-927 (AT-GT) (0.0%). VacA m subtypes were not associated with H. pylori antibiotic resistance. In conclusion, the present findings suggested that the detection of related mutant genes had a clinical application value in predicting the antibiotic resistance of H. pylori, particularly resistance to clarithromycin and levofloxacin.

Dog/cat-origin quinolone-resistant Streptococcus agalactiae isolates with point mutations in quinolone resistance-determining regions: Relatedness with clonal complex 10

ObjectiveWe aimed to investigate dog/cat-origin quinolone-resistant Streptococcus agalactiae isolates with point mutations in quinolone resistance-determining regions (QRDRs) and to define the relatedness between quinolone-resistant isolates and their microbiological features of capsular genotype, sequence type (ST)/clonal complex (CC), and antimicrobial resistance (AMR) gene. MethodsWith dog/cat-origin 22 isolates, type strain, and human-origin 6 isolates, we performed antimicrobial susceptibility testing by agar plate dilution method using levofloxacin, ciprofloxacin, and moxifloxacin. We also determined amino acid sequences in QRDRs of gyrA/gyrB/parC/parE genes and their point mutations. We conducted capsular genotyping, multilocus sequence typing, and AMR genotyping in our previous investigations. Correlations between quinolone-resistant population and their microbiological features were examined. ResultsWe found dog/cat-origin seven (31.8%) quinolone-resistant isolates harboring minimum inhibitory concentrations (MICs) of levofloxacin 16–32 μg/mL, ciprofloxacin 32 μg/mL, and moxifloxacin 2–4 μg/mL: human three isolates indicated MICs of levofloxacin 16–64 μg/mL, ciprofloxacin 32 μg/mL, and moxifloxacin 2–16 μg/mL. Point mutations Ser81Leu in gyrA and Ser79Phe/Ser79Tyr/Asp83Asn/Gly128Asp in parC were observed among these resistant isolates: mutations Leu495Ile/Val503Ile in parE was found among quinolone-nonresistant isolates. There was a significant correlation between dog/cat-origin quinolone-resistant population and ST10 (p = 0.023)/CC10 (p = 0.021). ConclusionTo our best knowledge, this is the first report assessing dog/cat-origin quinolone-resistant S. agalactiae. Our observations could be applied in future, by veterinarians while treating dogs and cats with clinical symptoms/signs suggestive of streptococcal infections.

Inhibition of penicillin-binding protein 2a (PBP2a) in methicillin resistant Staphylococcus aureus (MRSA) by combination of oxacillin and a bioactive compound from Ramalinaroesleri

Lichens are known to be useful and important in ethanopharmacology since ages and still possess substantial interest in alternative medical practices around the world. The intent of this investigation was to evaluate and to understand the antibacterial potential of usnic acid which was isolated from Himalyan fruticose lichen Ramalina roesleri. Usnic acid is predicted for its pharmaceutical properties through in -silico studies. Binding efficiency of usnic acid with Penicillin binding protein-PBP2a, a protein which is responsible for conferring resistance in Staphylococcus aureus was accessed using in-silico interaction assays comparing with oxacillin and ceftaroline. Further, the validation of in-silico modelling was checked by determining the antibacterial potential of usnic acid against methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates. In total, 28 clinical isolates collected from hospitals/medical students were included in the study and the anti-Staphylococcal activity was determined using agar plate dilution method followed by time-kill kinetics and synergistic studies. The scanning electron microscopic (SEM) pictures were obtained to show the cell wall disruption of MRSA by usnic acid.Docking results clearly indicated the enhanced binding potential of usnic acid (Glide XP G Score: 10.968; Glide energy −64.869) with PBP2a which is better than the energy range of reference compound, oxacillin (Glide XP G Score: 6.596; Glide energy −53.285) and roughly comparable to the co-crystallized ligand ceftaroline (Glide XP G Score: 12.20; Glide energy −70.322). Cefteroline is known to be more active against MRSA compared to oxacillin. The minimum inhibitory concentrations (MICs) of usnic acid against the clinical isolates of MRSA and reference strain (NCTC-6571) were in the range of 32–128 μg/ml. The high affinity of usnic acid to bind with PBP2a which is demonstrated via in-silico studies is further confirmed by the impressive inhibitory activity of usnic acid on MRSA clinical isolates.

In vitro screening for the antifungal activity of some Egyptian plant extracts against the fish pathogen Saprolegnia parasitica

Saprolegniasis which is caused mainly by Saprolegnia sp. is the main cause of the winter mass kills in northern freshwater fish farms in Egypt. Various treatment protocols were used to control it, but they are not favoured due to toxicity and cost of treatment; thus, the search for safer economic alternative is ongoing. In this study, 50/59 plant/plant parts methanolic extracts were screened for the possible anti-fungal activity against a locally isolated Saprolegnia parasitica strain by agar plate dilution method. The screening revealed a significant inhibition of S. parasitica produced by 8 plant extracts, in which the highest inhibition rate was produced by Cyperus esculentus rhizomes, and the least efficacy amongst them belonged to Juniperus phoenicea fruits. Afterwards, the minimum inhibitory concentration (MIC) by the macrodilution broth assay was performed for them, which revealed that C. esculentus and Carthamus tinctorius extracts were the most effective with concentration of 0.1 ppm while the least activity among the effective extracts belonged to Chenopodium murale, Ipomoea batatas and Luffa aegyptiaca (fruit rend) with concentration of 10 ppm. In the present study, it was concluded that the methanolic extracts of C. esculentus rhizomes, C. murale whole plant, C. tinctorius flowers, I. batatas leaves, J. phoenicea fruits, L. aegyptiaca leaves and fruit rend, and Ricinus communis leaves, showed high inhibitory action against Saprolegnia in low concentrations, which may lead to their future usage in the aquaculture application.

A Newly Discovered Drug Resistance Gene rfaF In Helicobacter pylori.

BackgroundThe purpose of this study was to understand the function of rfaF gene in Helicobacter pylori antibiotic resistance.MethodsThe gene homologous recombination method was used for knockout and complementation of H. pylori rfaF gene. Various constructed strains were analysed for drug sensitivity to amoxicillin (AMO), tetracycline (TET), clarithromycin (CLA), metronidazole (MET), levofloxacin (LEV), and chloramphenicol (CHL) by agar plate dilution method. Drug sensitivity was further confirmed using a growth inhibition curve. Ethidium bromide (EB) accumulation experiments were performed to assess cell membrane permeability. PCR and sequence analysis were used to detect the rfaF gene.ResultsThe minimum inhibitory concentrations (MIC) of TET, CHL, AMO, and CLA in 11,637 rfaF knockout strain (ΔrfaF strain) were 4, 4, 2, and 2 times higher than those in 11,637 wild type (WT) strain, respectively. A multidrug-resistant (MDR) ΔrfaF strain also displayed the same trend; however, the degrees of increase were relatively small. Growth inhibition experiments indicated that the growth of the 11,637 ΔrfaF strain was higher with antibiotics at the MIC of the 11,637 WT strain than that of 11,637 rfaF-complemented strain (ΔrfaF/rfaF strain), whereas the 11,637 WT strain did not exhibit any growth. The 11,637 ΔrfaF strain was significantly reduced compared with the cumulative EB fluorescence intensity of the 11,637 WT and of 11,637ΔrfaF/rfaF strain, and the same trend appeared in the MDR strain. Among the 10 clinical strains, 9 clinical strains were found to have mutations in the conserved sequence of rfaF amino acids.ConclusionWe found a new drug resistance gene, rfaF, in H. pylori, which changes the permeability of cell membrane to confer cross-resistance to AMO, TET, CLA, and CHL and is involved in clinical strain drug resistance. It can be used as a drug target.

A multicenter study of hospital infection with carbapenem-resistant Acinetobacter baumannii

Objective To investigate the epidemiologic features of interhospital, intrahospital, interdepartmental and intradepartmental infections of carbapenem-resistant Acinetobacter baumannii (CRAB) isolated from clinical specimens. Methods From September 2015 to September 2016, 540 non-repetitive strains of Acinetobacter baumannii were clinically isolated from 38 tertiary hospitals in Anhui Province, of which 460 strains were confirmed by antimicrobial resistance surveillance in Anhui Province. Agar plate dilution method was used to determine the MIC of 16 antibiotics and to select 312 CRAB isolates for drug sensitivity test. Pulsed field gel electrophoresis (PFGE) was used to analyze homology of 145 strains. PCR and clonal sequencing were used to determine the corresponding genotypes. Results All the 312 CRAB strains were multiple antibiotic resistant strains. The drug resistance rates to imipenem and meropenem were 67.9% and 70.6%, respectively. All the 145 strains were divided into 80 different types (PT1-PT80) by PFGE. Only one strain was found in the 60 types and two or more strains were found in the other 20 types. Genotype blaOXA-51 was detected in 145 strains (100%), genotype blaOXA-23 was detected in 134 of strains (92.41%), genotype blaOXA-24 was detected in 1 strain (0.69%), and subtype of blaIMP-4 was detected in 4 strains (2.76%). Genotype blaOXA-58, blaVIM, blaSIM, blaNDM and blaKPC were undetectable. Conclusions Homologous analysis indicates that all 145 strains of CRAB exhibit the interhospital, intrahospital, interdepartmental and intradepartmental transmission. Genotype blaOXA-51 is the inherent drug resistant associated gene in the chromosome of AB and genotype blaOXA-23 is the most common carbapenemase gene in Anhui Province and the main genotypes of resistance transmission. Key words: Acinetobacter baumannii; Drug resistance; Cross infection; Carbapenemase; PFGE

Evidence of antimicrobial and disinfectant resistance in a remote, isolated wild pig population

The spread of antimicrobial resistant E. coli within the environment is a global concern. Wildlife such as feral pigs have been identified as a possible reservoir of antimicrobial resistant bacteria. A cross-sectional survey of free-ranging, feral pigs within the Kimberley region of northwestern Australia was conducted to estimate the prevalence of antimicrobial and disinfectant resistant E. coli in this population. Of the 493 faecal samples collected, 115 E. coli isolates were randomly selected and their identity confirmed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). Isolates were screened for susceptibility to 14 veterinary antimicrobials (including antimicrobials not permitted for use in Australia) using MIC broth microdilution using Sensititre™ (BOPO6F) and breakpoints according to CLSI and EUCAST guidelines. Isolates also underwent disinfectant susceptibility testing to six disinfectants at their recommended concentration for use as well as at a twofold dilution, based on methods adapted from the CLSI agar plate dilution method. A moderate prevalence of resistance was observed to sulfadimethoxine (50.4%; 58/115) and florfenicol (27.0%; 31/115). A low prevalence of resistance was estimated to chlortetracycline (5.2%; 6/115) and multi-drug resistance was only identified in 1.7% (2/115) of E.coli isolates tested. Isolates were susceptible to five of the six disinfectants screened. Feral pigs could potentially act as a reservoir of antimicrobial resistance in the environment with possible implications for domestic livestock. The role that feral pigs might play in transmission of antimicrobial resistance requires further investigation, and the occurrence of resistance in such isolated populations needs to be considered when attempting to infer source attribution of antimicrobial resistance in livestock and human populations.

Antibiotic resistance patterns of methicillin-resistant Staphylococcus aureus (MRSA) isolated from livestock and associated farmers in Anuradhapura, Sri Lanka.

The animal husbandry comes to play an important role according to new economic reforms of the rural economy in South Asia including Sri Lanka, and the rural farming community has a poor knowledge about hygienic issues of animal husbandry, which can lead to spread of pathogenic bacterial strains from animals to humans. Our study was conducted to evaluate methicillin susceptible Staphylococcus aureus (MRSA) colonization and its antimicrobial resistance pattern among livestock (n=188) and related farmers (n=94) in Anuradhapura District, Sri Lanka. S. aureus isolates were identified using mannitol salt agar, coagulase test and DNAase test. The agar plate dilution method was conducted to determine the minimum inhibitory concentration (MIC) of oxacillin against S. aureus. Antimicrobial susceptibility testing for other antibiotics was performed against MRSA isolates using antibiotic containing discs. To assess the MRSA transmission from livestock to humans, we have grouped MRSA strains according to antimicrobial susceptibility patterns against the tested antibiotics. Among MRSA isolates, 14 different groups with similar MIC and antibiotic susceptibility patterns were identified. Of those, 2 groups amongst pigs and pig farmers showed a significant relationship (p=0.031). The other groups did not show any significant relationship between animals and the farmers. The percentages of MRSA prevalence in pigs and pig farmers were 26.6% each, in poultry and poultry farmers 8.3% and 13.3% respectively, in cattle and cattle farmers 8.3% and 3%. Compared to human MRSA isolates, animal isolates were significantly more resistant to ciprofloxacin (p=0.031), gentamicin (p=0.010) and clindamycin (p=0.011). Similarly, animal methicillin susceptible Staphylococcus aureus (MSSA) isolates were significantly more resistant to ciprofloxacin (p=0.022) and doxycycline (p=0.012). Pig farming showed a higher prevalence and 2.4 times higher risk (OR=2.4, CI95%: 1.2-4.8) of likely transmission of MRSA between animals and humans than cattle and poultry farming. Overall, 65% of MRSA and MSSA animal isolates were multidrug resistant.

Increased incidences of multidrug-resistant gonorrhea in Taiwanese men: Experiences from a single institute

ObjectivesOur objective was to examine the detection rate of routine urine cultures and genital discharge swab cultures for the detection of gonococcal urethritis in male patients. In addition, we evaluated the results of urinalysis for gonococcal infections and investigated the drug sensitivity of Neisseria gonorrhoeae strains isolated from these patients. Materials and methodsFrom August 2009 to October 2015, 202 men diagnosed with gonococcal infection, based on the results of a urine culture or genital discharge swab culture, were enrolled in this study. Initial clinical symptoms and urinalysis results were collected. The susceptibility of N. gonorrhoeae to penicillin, cefmetazole, cefotaxime, ceftazidime, cefuroxime, ceftriaxone, and ofloxacin were determined using agar plate dilution method. ResultsThe mean age of the patients was 29.67 ± 10.39 years, and 157 of the 202 male patients (87.6%) were aged between 15 years and 34 years. We determined that the diagnostic value of genital discharge swab culture was significantly higher than that of the urine culture (90.9% vs. 67.4%, respectively). Genital or urethral discharge is the major symptom in these patients (88.89%). The susceptibility of N. gonorrhoeae to cefuroxime, cefmetazole, cefotaxime, ceftazidime, penicillin, and ciproxin revealed an increasing prevalence of resistant strains in recent years. ConclusionGenital discharge swab culture is a more effective method than urine culture to detect gonorrhea in patients with dysuria and urethral discharge. The increasing numbers of antibiotic-resistant N. gonorrhoeae strains is a major problem in treating gonococcal urethritis.

The prevalence and epidemiology of plasmid-mediated penicillin and tetracycline resistance among Neisseria gonorrhoeae isolates in Guangzhou, China, 2002-2012.

BackgroundGonococcal antimicrobial resistance is a global problem. Different resistance plasmids have emerged and spread among the isolates of Neisseria gonorrhoeae worldwide and in China. We conducted this study to monitor the plasmid-mediated penicillin and tetracycline resistance among N. gonorrhoeae isolates in Guangzhou from 2002 to 2012.MethodsConsecutive isolates of N. gonorrhoeae were collected from outpatients with gonorrhea attending the STD clinic in Guangdong Provincial Centre for Skin Diseases and STIs Control and Prevention. Penicillinase-producing N. gonorrhoeae (PPNG) isolates were analyzed by the paper acidometric method. Plasmid-mediated resistance to tetracycline in N. gonorrhoeae (TRNG) isolates was screened by the agar plate dilution method. Plasmid types were determined for TRNG and PPNG isolates using polymerase chain reaction (PCR). Minimum inhibitory concentrations (MICs) to penicillin and tetracycline were detected by the agar plate dilution.ResultsOf 1378 consecutive N. gonorrhoeae isolates, 429 PPNG and 639 TRNG isolates were identified. The prevalence of PPNG, TRNG, and PPNG/TRNG increased from 18.3 to 47.1 % (χ2 = 31.57, p < 0.001), from 29.4 to 52.1 % (χ2 = 16.28, p < 0.001) and from 10.0 to 26.2 % (χ2 = 10.46, p < 0.001) between 2002 and 2012, respectively. Genotyping of plasmids among PPNGs showed that the majority (93.7 %) of the isolates were the Asian type plasmids, while the African type plasmid emerged in 2008 and rapidly increased to 14.0 % in 2012 (χ2 = 25.03, p < 0.001). For TRNGs, all 639 isolates carried the Dutch type plasmid. MICs of penicillin G and tetracycline persisted at high levels and the MIC90s were 32-fold higher than the resistant cutoff point over 11 years. The prevalence rates of penicillin- and tetracycline-resistant N. gonorrhoeae varied from 90.9 to 91.1 % and from 88.3 to 89.3 % during 2002 to 2012, respectively.ConclusionsResistance to penicillin and tetracycline among N. gonorrhoeae isolates remained at high levels in Guangzhou. The Asian type PPNG continued to spread and Dutch type TRNG was still the dominant strain. The African type PPNG has emerged and is spreading rapidly.

Taxonomy and Antimicrobial Activity of an Actinomycete Strain Isolated from Swallow Bird Mud Nest

An actinobacterial strain designated as BN-6 was isolated from swallow bird mud nest by agar plate dilution method. It showed the morphological, physiological and biochemical characteristics of the genus Streptomyces. On the basis of 16S rRNA gene sequence analysis and physiological tests, this isolate was found to be most closely related with Streptomyces vinaceusdrappus NRRL2363. The strain BN-6 produced a broad spectrum active metabolite in Glucose Soybean meal (GS) medium and was extracted with HP Dianion resin. The resin extracted product showed activity against Gram-positive, Gram-negative bacteria, acid fast bacilli and fugal cultures, suggesting it broad spectrum characteristics. The bioautography on silica gel plates using Micrococcus luteus MTCC106 as test organism indicated the presence of a single metabolite having a Rf value of 3.2 and 9.1 in two different solvent systems. The characteristic peak obtained in UV–VIS spectrophotometer at 214 nm and chemical analysis indicates that it may be peptide class of metabolite.

Detection and epidemiology of drug resistance related blaOXA-23 and armA genes of Acinetobacter baumannii

Objective To investigate the drug resistant genes against carbapenems, aminoglycosides and quinolones and the molecular epidemiology of clinical isolates of Acinetobacter baumannii. Methods Forty non-duplicate strains of Acinetobacter baumannii were collected from clinical specimens in First Affiliated Hospital of Wenzhou Medical University. The identification of strains was conducted by Vitek 2 Compact system. The susceptibilities to antimicrobials commonly used were determined by agar plate dilution method and broth microdilution method. The presence of class B metalloenzyme-encoding genes (blaIMP, blaVIM,blaNDM,blaSIM,blaGIM), class D cabapenemase-encoding genes (blaOXA-23,blaOXA-48,blaOXA-58), 16S rRNA methylase genes (armA, rmtB) and quinolone resistance-determining regions (QRDR) in gyrA and parC were detected by polymerase chain reaction (PCR) and sequenced. Chromosomal or plasmid location of blaOXA-23 and armA genes were assessed by Southern blot. Multiple loci sequence classification (MLST) was performed to analyze the molecular epidemiology of these strains. Results All of the 40 isolates were multi-drug resistant Acinetobacter baumannii (MDR-AB) and showed high level resistance to all of the tested antimicrobial agents excluding colistin and tigecycline. The positive rates of blaOXA-23 and armA were 90% and 95%, respectively. All of the 40 isolates carried QRDR mutations in gyrA and parC genes, leading to the Ser83→Leu and the Ser80→Leu amino-acid substitutions, respectively. Southern blot showed the chromosomal location of blaOXA-23 and armA genes. Six different ST (ST191, ST381, ST373, ST426, ST208 and ST207) were assigned for these isolates by MLST and the most dominant clones were ST191 (23/40) and ST381 (10/40). Conclusions The predominant cabapenemase-encoding gene and 16S rRNA methylase gene of Acinetobacter baumannii isolates in First Affiliated Hospital of Wenzhou Medical University are blaOXA-23 and armA, respectively, which may be located on the chromosome and vertically transmit the drug resistance. ST191 MDR-AB with blaOXA-23 and armA gene clonally spread in this hospital. Key words: Acinetobacter baumannii; Drug resistance, multiple; Carbapenemase; 16S rRNA methylase; MLST

Antimicrobial activity of fruits, leaves, seeds and stems extracts of Ziziphus spina -Christi

Petroleum ether , chloroform , methanol and aqueous extracts of the fruits, leaves, seeds and stems of Ziziphus spina -Christi belonging to the family Rhamnaceae were screened for their antimicrobial activity against six standard bacteria: two Gram positive ( Bacillus subtilis ,Staphylococcus aureus), four Gram negative ( Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris and Pseudomonas aeruginosa ) and two fungi ( Aspergillus niger and Candida albicans ) using the cup plate agar diffusion method. The methanol extracts of all parts showed the highest activity against the bacteria tested followed by chloroform then petroleum ether. The aqueous extracts of all parts were inactive against all bacterial organisms. All four parts of the plant extracts showed no antifungal activity against the two fungi tested. The minimum inhibitory concentrations of the most active methanol extracts of fruits, leaves and stems were determine against standard bacteria using the agar plate dilution method. The antibacterial activity of five reference drugs and the antifungal activity of two reference drugs were determined against six bacteria and two fungi and their activities were compared with the activity of the plant extracts.

Antimicrobial activity of the leaves essential oil of Eucalyptus camaldulensis Dehnh

Essential oil from the leaves of Eucalyptus camaldulensis Dehnh. belonging to the family Myrtaceae was tested for its antimicrobial activity against six standard bacteria, two Gram positive bacteria ( Bacillus subtilis, Staphylococcus aureus ), four Gram negative ( Escherichia coli , Klebsiella pneumoniae , Proteus vulgaris, Pseudomonas aeruginosa ) and two standard fungi ( Aspergillus niger, Candida albicans ) using the cup plate agar diffusion method. The essential oil of E. camaldulensis showed high activity (22mm) against S.aureus, (20mm) against E.coli and C. albicans . It also possessed moderate activity (17mm) against B.subtilis, Kl. Pneumoniae and Pr. vulgaris ,(18mm) against Ps. aeruginosa and (16mm) against A.niger . The minimum inhibitory concentrations (MICs) of E. camaldulensis essential oil against standard bacteria were determined using the agar plate dilution method and the MICs were12.5 µg/ml. The essential oil of E. camaldulensis was further tested against fifty clinical isolates, collected randomly from patients of Khartoum Teaching Hospital laboratory and Soba Hospital. The antibacterial activity of two reference drugs and the antifungal activity of two reference drugs were determined against six bacteria and two fungi and their activities were compared with the activity of essential oil of E. camaldulensis leaves. The essential oil of E. camaldulensis leaves can be of potential use as antimicrobial agents.