Open Access Original Research Article

Molecular Detection of Mycobacterium tuberculosis Rifampicin Drug Resistance Using rpoB Gene Sequencing in Zimbabwe

Nyasha Chin’ombe, Wadzanai Manjeese, Ellen Munemo

Microbiology Research Journal International, Page 1-6
DOI: 10.9734/BMRJ/2016/28510

Aim: To detect Mtb rifampicin drug resistance mutations in Zimbabwe by rpoB gene sequencing.

Study Design:  This was a retrospective study.

Place of Study: The study was conducted in Zimbabwe in 2015 using archived Mtb isolates previously isolated throughout Zimbabwe during a national TB Survey.

Methodology: Archived Mtb isolates at the National Microbiology Reference Laboratory were retrieved. Genomic DNA from the isolates was extracted by the boiling method. The 81 bp region of rpoB gene that contains rifampicin drug resistance mutations was amplified by polymerase chain reaction (PCR). The PCR products were sequenced and sequences analyzed to determine mutations in the rpoB protein.

Results: RpoB mutations in 30 Mtb isolates were analyzed. Out of the 30 Mtb isolates, 19 (63.3%) had at least one codon mutation in the rpoB gene that resulted in a change of amino acid. The commonest mutation was on codons 531 (S>L), with a prevalence of 20%. Other mutations were detected at codons 511 (L>P) (3.3%), 516 (D>Y) (10.0%), 516 (D>V) (3.3%), 518 (N>H) (3.3%), 526 (H>D) (10.0%), 526 (H>L) (3.3%), 526 (H>C) (3.3%), 526 (H>Y) (3.3%) and 533 (L>P) (6.6%). The other 11 Mtb isolates (36.7%) did not have any mutations in the gene coding for drug resistance.

Conclusion: Genetic mutations that code for rifampicin drug resistance are prevalent in Mtb  isolates from Zimbabwe. Further studies need to be instituted to ascertain the scope and threat of the Mtb drug resistance.

Open Access Original Research Article

Detection, Survival, and Source of Inoculum of Pseudomonas syringae pv. syringae from Weeds and Plant Debris in Relation to Epidemiology of Bacterial Citrus Blast and Black Pit in Tunisia

Imen Mougou, Naima Boughalleb M'Hamdi

Microbiology Research Journal International, Page 1-10
DOI: 10.9734/BMRJ/2016/27954

Background: Citrus blast and black pit caused by Pseudomonas syringae pv. syringae occur in several citrus growing regions around the world. However, the sources of inoculums of Pseudomonas syringe responsible for this disease are not well defined.

Aims: To determine the survival of P. syringae pv. syringae in weeds and  plant debris, evaluate the pathogenicity of Pseudomonas syringae on citrus and to assess the potential for plant debris  andweeds serving as inoculum sources for this pathogen.

Settings and Design: This study was carried out in the Department of Biological Sciences and Plant Protection at the Institute of Science, Agriculture, Tunisia. For a period of six months.

Methodology: Strains of Pseudomonas syringae pv. syringae was   recovered  from plant  debris and symptomless weed species growing in citrus orchards. A total of 24 samples of weeds and four samples of plant debris were included in this study. The fluorescent strains cultivated on King’s medium were identified by LOPAT (Levan production, Oxidase, Pectolytic activity on potato slices, Arginine dehydrolase, HR on tobacco leaves) and GATTa tests (Gelatine hydrolysis, Aesculine hydrolysis, Tyrosine activity, Tarataric Acid usage). Pathogenicity test was carried out on orange (cv.Navel) fruits. PCR amplification for the detection of syrB gene was performed with the syrB primers.

Results: Forty six strains isolated from weeds and plants debris were gram negative by KOH test, levan-positive, oxidase-negative, pectolytic activity-negative, arginine dihydrolase-negative and tobacco hypersensitivity-positive. They showed the LOPAT characters of group Ia (+- - -+). Among all, the 46 strains were positive for gelatin liquefaction and aesculin hydrolysis but negative for tyrosinase activity and tartrate utilization (G+A+T-Ta-). Twenty eight of 43 (65.11%) strains were isolated from weeds, 18 of 22 (81.81%) strains isolated from plant debris were, pathogenic on mature orange (cv. Navel) fruits. The PCR amplification with the syrB primers yielded 752-bp fragments confirmed that the syrBgene was present in the 46 isolates. According to biochemical tests (LOPAT and GATTa), pathogenicity assay and PCR amplification with the syrB primers, the forty six isolated bacterial strains were identified as Pseudomonas syringae pv. syringae.

Open Access Original Research Article

Antimicrobial Susceptibility of Urinary Escherichia coli from Outpatients with Community Acquired Urinary Tract Infections, Report from Tertiary Health Care Center, Egypt

Ghada El-Saeed Mashaly

Microbiology Research Journal International, Page 1-6
DOI: 10.9734/BMRJ/2016/27150

Background: Urinary tract infection (UTI) is one of the most common infections at the community setting. Escherichia coli are the main agent of UTIs. Antibacterial resistance spreads rapidly among community acquired urinary E. coli.

Objectives: The aim of this prospective study was to describe antimicrobial susceptibility profile and prevalence of multidrug resistant (MDR) urinary E. coli isolated from outpatients with community acquired UTI.

Methods: Urine samples were collected from patients attending outpatient departments of Mansoura University Hospitals in Egypt presented with symptoms of UTI.  Samples were cultured and E. coli isolates were identified by colonial morphology, Gram stained film, and analytical profile index (API) 20E. Antibiotic sensitivity testing was performed by standard Kirby-Bauer disk diffusion method.

Results: One hundred and forty one E. coli uropathogen were isolated. The highest resistance was to beta lactam antibiotics. Amoxicillin susceptibility was 2.1%. Resistance to 3rd generation cephalosporins was (57.4%, 40.4%, 46.8% to cefotaxime, ceftazidime, and cefoperazone) respectively. The resistance to norfloxacin was (46.8%). While resistance to nitrofuantoin was (27.7%). The least resistance was to cefoperazone-sulbactam and amikacin (8.5% and 12.8% respectivelly). Most of the isolates were multidrug resistant (87.9%).

Conclusion: High level of antibiotic resistance among E. coli causing community acquired urinary tract infection.

Open Access Original Research Article

Biocontrol of Fusarium wilt of cucumber with Trichoderma longibrachiatum NGJ167 (Rifai)

T. Kehinde Kareem, O. Esther Ugoji, O. Olusimbo Aboaba

Microbiology Research Journal International, Page 1-11
DOI: 10.9734/BMRJ/2016/28208

Aim: This research investigated the use of Trichoderma longibrachiatum NGJ167 (Rifai) as a biocontrol agent of Fusarium wilt in cucumber varieties (Ashley and Marketmoor) both in the screenhouse and on the field.

Study Design: The screenhouse experiment was laid down in a Completely Randomized Design (CRD) while Randomized complete block design (RCBD) was used for the field experiment.

Place and Duration of Study: The study was conducted at the National Horticultural Research Institute, Ibadan between 2012 and 2013.

Methodology: Soils were inoculated with mycelial plugs of T. longibrachiatum NGJ167 before planting while the control soil was mock-inoculated with agar plugs of Potato dextrose agar (PDA). Two weeks after planting, F. oxysporum was inoculated into the soils in the screenhouse while natural infection was allowed to occur on the field. The biocontrol abilities of T. longibrachiatum NGJ167 on F. oxysporum was observed on disease incidence and severity and the fruit yield. The presence of T. longibrachiatum NGJ167 genes was detected in the treated cucumber fruits to ensure consumers’ safety.

Results: The control plants had higher incidence and severity of F. oxysporum than the T. longibrachiatum-treated plants. The T. longibrachiatum NGJ167-inoculated Marketmoor had higher fruit weight value of 200g in the screenhouse when compared with the control which had a fruit weight value of 133.33 g. On the field, T. longibrachiatum-treated Marketmoor produced the highest fruit weight of 220 g while the control had a mean weight of 120.6 g. Results also revealed that T. longibrachiatum DNAs were absent in the inoculated cucumber fruits.

Conclusion: The use of T. longibrachiatum NGJ167 as a biocontrol agent indicates its potentials in improving plant health in agriculture. The absence of T. longibrachiatum NGJ167 in the treated cucumber indicated that the consumption of such fruits will have no adverse effect on consumers’ health.

Open Access Original Research Article

Comparative Study of Blood Culture and Widal Agglutination Test from the Patients Suspected of Enteric Fever

Preety Chaudhary, Vijay Kumar Sharma, Anshu Kumari Chaudhary, Shashi Bhushan Chaturwedi, Anima Shrestha

Microbiology Research Journal International, Page 1-9
DOI: 10.9734/BMRJ/2016/26141

Aims: This study was performed to identify the enteric fever cases by both blood culture and Widal agglutination test and compare the results obtained from both methods.

Study Design: This research was carried out as hospital based descriptive cross-sectional study.

Methods: Blood samples collected aseptically from patients suspecting enteric fever were processed for identification of Salmonella species by blood culture and Widal agglutination test. The isolates were further subjected to antibiotic susceptibility testing according to CLSI guidelines. Total 1269 samples from the suspected patients were enrolled for this study and statistical analysis of the result was done by using 16.0 versions of SPSS.

Results: Among suspected patients studied, 70 (71%) and 29 (29%) cases were confirmed to be infected with S. typhi and S. paratyphi A respectively from blood culture. Out of total sera processed for Widal test, 263 samples gave agglutination with titre more than 1/80. The study showed sensitivity of 81.4% and specificity of 84.4%, positive predictive value of 31.5% and negative predictive value of 98.2% and the efficiency 84.4% of Widal test in compare to blood culture. S. typhi isolates sensitive to the classical first  line drugs- amoxycillin, chloramphenicol and cotrimoxazole were 94.3%, 97.1%  and  97.1% respectively while S. paratyphi A isolates sensitive were 68.9%, 96.5%, and 93.1% respectively. Fifty eight (82.9%) S. typhi isolates were nalidixic acid resistance while 25(86.2%) S. paratyphi A were nalidixic acid resistant. Also, 3(3.03%) multi-drug resistant isolates were confirmed to be nalidixic acid resistant.

Conclusion: The study showed blood culture remains the gold standard for enteric fever diagnosis. Widal test alone either positive or negative should not be considered  confirmatory  for enteric fever However  cut-off  titre  can  be  taken  in  the  diagnosis  and  Widal  test  can  be  helpful  in  making  a presumptive diagnosis of typhoid fever if interpreted with care. Azithromycin and Ceftriaxone were the most effective drugs for enteric fever cases.

Open Access Original Research Article

Screening of Antioxidant, Antibacterial and Antileishmanial Activities of Salvia officinalis L. Extracts from Morocco

Abdeslam Et- Touys, Hajiba Fellah, Meryem Mniouil, Abdelhakim Bouyahya, Nadia Dakka, El Hassane Abdennebi, Abderrahim Sadak, Youssef Bakri

Microbiology Research Journal International, Page 1-10
DOI: 10.9734/BMRJ/2016/28307

Aims: The aim of this study was to evaluate in vitro antioxidant and antimicrobial activities of organic extracts from Salvia officinalis L. (Lamiaceae) collected in the province of Ouezzane.

Study Design: Evaluation of in vitro antimicrobial and antioxidant activities of extracts and determination phenolic contents.

Place and Duration of Study: After plant collection from the Province of Ouezzane, further work was carried out in Parasitology Laboratory of the National Institute of health and Laboratory of Biochemistry-Immunology, Faculty of Science, Mohammed V University of Rabat, Morocco from Novembre 2015 to Mai 2016.

Methodology: The antioxidant activity was evaluated using DPPH scavanging assay. The antibacterial activity was tested against three reference strains (Staphylococcus aureus, Escherichia coli and Listeria monocytogenes serovar) by the diffusion method and the minimum inhibitory concentration (MIC) by microtitration assay. The antiparasitic activity was tested against Leishmania major using MTT (3- (4.5-dimethylthiazol- 2yl) -2.5-diphenyltetrazolium bromide) assay. The levels of polyphenols and flavonoids extracts were estimated by colorimetric assay.

Results: The methanol extract has shown a significant ability to trap the radical DPPH (IC50=65.655 µg/ml) compared to n-hexane and ethanol extracts. This value is higher than that of ascorbic acid (13.198 µg/ml) and Trolox (22.484 µg/ml) used as standards. The three extracts tests revealed the inhibitory power of three bacterial strains with a significant difference in the diameters of inhibition. The largest area was registered by the hexane and methanol extracts against S. aureus (22±8 mm), while the weakest area was 11±0.22 mm expressed by the ethanol extract against E. coli and the methanol extract against L. monocytogenes. The antileishmanial activity was moderate with a value of cytotoxicity (IC50) above 1 mg / ml. The extracts showed high concentrations of polyphenols and flavonoids, while biological activities were not very high when correlated with these levels.

Conclusion: These results will be completed by the determination of the active component and the extracts will be tested on other biological systems namely antifungal and antitumor activities.