Open Access Original Research Article

The Evaluation of the Combination of Caspofungin with Voriconazole and Amphotericin B against Clinical Candida krusei Isolates by Etest and Disk Diffusion Methods

Yasemin Oz, Abdurrahman Kiremitci, Filiz Aksit, Nuri Kiraz

Microbiology Research Journal International, Page 497-508
DOI: 10.9734/BMRJ/2014/6950

Background: C. krusei is an opportunistic fungal pathogen that known of its intrinsic fluconazole resistance and its frequency is increasing especially among hematology patients. The increase in the frequency of high mortality fungal infections have accelerated the efforts of new drug development with broad spectrum, low toxicity and the studies including their combination. However, there is no standardized method to evaluate the activity of drug combinations.

Aims: To evaluate the activity of caspofungin (CAS) with voriconazole (VOR) and amphotericin B (AMB) alone and in combination and the utility of Etest and disk diffusion methods for antifungal combinations.

Methodology: The minimum inhibitory concentrations of VOR, CAS and AMB against 30 clinical C. krusei isolates were determined by using Etest, disk diffusion and reference broth microdilution methods. Combinations of CAS with VOR and CAS with AMB were evaluated using disk diffusion (three different ways) and Etest (two different ways) methods.

Results: All isolates tested were susceptible to VOR and CAS in vitro by all three methods. Categorical agreements of Etest and disk diffusion methods with reference microdilutiontest were 100% for CAS and VOR (for each method), 86.7% and 50% for AMB, respectively. In the all ways of both combination methods, we did not observe distinctly antagonistic or synergic interaction. 

Conclusion: Etest and disk diffusion could be easy, convenient, and nontime-consuming alternative methods to evaluate the antifungal combinations. The combinations of CAS with VOR and AMB exhibited promising results because of an apparent antagonistic interaction was not detected in this study

Open Access Original Research Article

Enzymatic Activity of Lactobacillus Grown in a Sweet Potato Base Medium

Saeed A. Hayek, Aboghasem Shahbazi, Mulumebet Worku, Salam A. Ibrahim

Microbiology Research Journal International, Page 509-522
DOI: 10.9734/BMRJ/2014/5921

Aim: To study the impact of sweet potatoes on α-glucosidase, β-glucosidase, acid phosphatase, and phytase activity of Lactobacillus.

Study Design: Enzymatic activity of seven strains of Lactobacillus grown in a sweet potato medium (SPM) was determined and compared to the standard lactobacilli MRS. Strains having the highest enzymatic activity were further enhanced by metal ions.

Place and Duration of Study: Food Microbiology and Biotechnology Laboratory, North Carolina A&T State University, Greensboro, NC, USA, between September 2012 and May 2013.

Methodology: Lactobacillus strains were grown in SPM and MRS at 37ºC for 16 h. At the end of incubation, bacterial population (log CFU/mL) was determined by plating and enzymatic activity was determined spectrophotometrically using the corresponding substrate.

Results: Lactobacillus strains continue to grow in SPM and MRS and reached averages of 10.98±0.49 and 10.92±0.55 log CFU/mL respectively. Growth of Lactobacillus strains in SPM led to higher β-glucosidase, acid phosphatase, and phytase activity than MRS. Strains of L. reuteri (CF2-7F and SD2112) grown in SPM showed the highest acid phosphatase (15.84±1.05 and 20.56±1.49 Ph U/mL), and phytase (0.66±0.14 and 0.65±0.11 Ph U/mL) respectively. The highest β-glucosidase (36.04±3.16 Glu U/mL) activity was obtained from L. delbrueckii subsp. bulgaricus SR35 grown in SPM. In addition, acid phosphatase and phytase produced by L. reuteri CF2-7F growing in SPM were further increased by the addition of Mn2+ (70.1 and 41.8%) or Mg2+ (94.7 and 20.9%) respectively. β-glucosidase activity of L. reuteri was increased in a range of 4.1 to 130.6% due to the addition of metal ions.

Conclusion: Components in sweet potatoes could increase enzymatic activity of Lactobacillus and the addition of metal ions could further produce an enhanced level of these enzymes

Open Access Original Research Article

In-vitro Effect of Iron on the Growth Density of Mycobacterium tuberculosis and Drug Susceptibility Testing

Rasheeda H. Abdalla, Ali M. S. Eleragi, Salaheddin O. Hassan, Nuha Y. Ibrahim, Mohamed S. El Sanousi

Microbiology Research Journal International, Page 523-529
DOI: 10.9734/BMRJ/2014/7049

This work was carried out to study the effect of iron on the growth density of Mycobacterium tuberculosis as well as drug susceptibility testing. Fifty two smear-positive acid fast bacilli out of 100 sputum specimens were obtained from patients who were referred to National Health Laboratory, Federal Ministry of Health, Sudan. The smear positive specimens were cultured onto control LJ medium and other three sets of LJ medium containing ferrous sulfate (iron) with different concentration; 100 mg/l, 200 mg/l and 400 mg/l. The growth was graded as negative (0 = no growth), +1 (1-19 colonies), +2 (20-100 colonies) +3 (100-200 colonies), +4 (200-500 colonies) and +5 (more than 500 colonies). At the same time, proportion method was applied to test susceptibility of mycobacterial isolates (20 isolates) to anti-tuberculosis drugs; isoniazid (INH), rifampicin (RIF), ethambutol (EMB) and streptomycin (STM) in the presence and absence of ferrous sulfate. Chi squire test was used to analyze categorical variables and the significance level was set at P = .05. Collectively, the results showed that the growth density of M. tuberculosis increased significantly when ferrous sulfate was added to LJ medium. Resistance to the four first line anti-tuberculosis drugs (INH, RIF, EMB, STM), INH and EMB were also respectively increased from 10% to 15℅, 20% to 40% and 10% to 15% in the presence of ferrous sulfate. Contrary to other findings, it was observed that addition of ferrous sulfate to LJ medium enhanced the activity of STM while no effect was seen on RIF and multi-drug resistance (INH and RIF).

The study concluded that the growth density of M. tuberculosis can be enhanced by supplementing LJ medium with 100-200 mg/l iron. This would be useful in recovery of the organisms from specimens with a low bacterial load particularly in laboratories in low income setting. Supplementation of LJ medium with iron must be avoided if drug susceptibility testing is required.

Open Access Original Research Article

Direct and Rapid Diagnosis of Extra-Pulmonary Tuberculosis in Archival Tissue by Nested and Touchdown PCR

L. F. Nimri, Z. Al-joulani, I. Matalka

Microbiology Research Journal International, Page 530-539
DOI: 10.9734/BMRJ/2014/8427

Aims: To identify and differentiate mycobacterium spp. in archival formalin-fixed, paraffin-embedded tissue by PCR to supply additional differential diagnostic method for Mycobacterium infections, where tuberculosis had been tested for by histopathological methods but without culturing. 

Place and Duration of Study: Department of Medical Laboratory Sciences, Department of Pathology and Microbiology (Faculty of Medicine) and King Abdulla University Hospital, between January 2004 and July 2010.

Methodology: Fifty-six extra-pulmonary specimens of formalin-fixed, paraffin-embedded tissue obtained from patients showing granulomatus inflammation and/or other histopathologic features. Specimens were analyzed by a hemi-nested PCR assay targeting the gene encoding for 16S ribosomal RNA, which is common to all mycobacteria spp. The PCR positive specimens were amplified by a touch-down PCR targeting a fragment in the insertion sequence IS6110 specific to Mycobacterium tuberculosis complex. Results were compared to acid fast bacilli stain and with histopathology of each specimen.

Results: M. tuberculosis complex DNA was detected in 27 (48.2%) specimens, and non- tuberculous mycobacteria in four specimens, compared to 10 (18%) specimens that were positive by acid fast staining. The positive cases were observed more in the lymph nodes, and pleural specimens.

Conclusions: This study is among few studies to use the touch-down PCR assay as a promising auxiliary tool for the diagnosis of extra-pulmonary tuberculosis in archival tissue specimens. It could be used in conjunction with routine laboratory tests (e.g., cultures, acid fast staining) and clinical criteria of the patient to increase the accurate diagnosis of such cases. It is also recommended that culture be routinely done for all tuberculosis suspected cases.

Open Access Original Research Article

High Pressure Processing of Chicken Meat: Change in Total Aerobic Counts after Pressure Treatment and During Chilled Storage

Sami Bulut, Nicolas Chapleau, Marie de Lamballerie, Alain Le-Bail

Microbiology Research Journal International, Page 540-549
DOI: 10.9734/BMRJ/2014/8214

Aims: To investigate the effect of pressure, pressurization time, pressurization temperature and their interaction on inactivation and recovery of Listeria innocua inoculated in minced chicken meat.

Study Design: Effect of the parameters of high pressure processing (HPP) on the inactivation of L. innocua were studied by response surface methodology using Box-Behnken design.

Place and Duration of Study: Study conducted during an 11 months postdoctoral study at Agriculture, Agronomy and Food Sciences Department at LUNAM Université, Oniris, Nantes, France.

Methodology: Minced chicken meat inoculated with  Listeria innocua strain ATCC 33090 to give a total aerobic count (TAC) of 108 cfu/g and samples were subjected to high pressures of 200, 300, 400 MPa, temperatures of 0ºC, 20ºC, 40ºC and holding times of 5, 10 and 15 minutes. Survival of L. innocua was determined by TAC immediately after pressure treatment and during 35 days of storage at 3ºC.

Results: Survival of L. innocua decreased with increasing pressure and pressurization time. Effect of pressurization temperature on survival of bacteria was not linear, giving higher reduction at 0ºC and 40ºC compared to treatments at 20ºC. Analysis of variance (ANOVA) showed that pressure (P<.001), time (P<.001), temperature (P=.05) and interaction of pressure and temperature (P=.05) were significant parameters. After a 10 min treatment at 400 MPa and 0ºC, no survival of microorganisms was detected immediately after pressure treatment. However, TAC increased during storage and reached to about the initial level of microbial load (108cfu/g) in all samples after 35 days of storage at 3ºC.

Conclusion: Undetected survival of microorganisms in a nutrient rich food immediately after a pressure treatment does not necessarily mean total inactivation of the microorganisms. Injured microbial cells could recover during the refrigerated storage and compromise the safety of pressure treated foods. Therefore, care must be taken to ensure the safety of high pressure processed foods.

Open Access Original Research Article

Phytochemical Analysis and Chromatographic Studies of Pergularia tomentosa L. and Mitracarpus scaber Zucc

S. A. Shinkafi

Microbiology Research Journal International, Page 550-559
DOI: 10.9734/BMRJ/2014/3534

Qualitative phytochemical analysis was carried out on Pergularia tomentosa and Mitracarpus scaber (leaves), the results revealed the presence of Tannins, Flavonoids, Alkaloids, Saponins, Glycosides, Saponin Glycosides, Cardiacglycosides, Anthraquinone and Steroids.The quantitative phytochemical analysis of the plants indicated high contents of total saponins and flavonoids compounds (4.44g% and 4.32g% saponins and 4.28g% and 4.51g% for flavonoids of P. tomentosa and M. scaber respectively). Results obtained from the analysis of variance indicated significant difference between the values of different weights of phytochemical components obtained. (P≤0.5) at 5% confidence level. Post experimental analysis using the least significant difference test (LSD = 0.69) showed that saponins and flavonoids compounds stand out different from other compounds. Extaction of the plants was carried out using organic solvents, (chloroform and N- Hexane). Column chromatographic fractionation of the active extracts of the plants revealed five fractions in each of the extracts, (CHL1 to CHL5 andHX1 to HX5).The phytochemical analysis of the active fractions indicated Saponins and Flavonoids compounds in large amount. Thin layer chromatography (TLC) of Saponins and Flavonoids compounds revealed the retension factor values (Rf)   as 0.869 and 0.92 when compared with the standards gymnemic acid and quercetin as 0.862 and 0.92 respectively

Open Access Original Research Article

Multiple Antimicrobial Resistance in Vibrio spp Isolated from River and Aquaculture Water Sources in Imo State, Nigeria

C. I. Chikwendu, S. N. Ibe, G. C. Okpokwasili

Microbiology Research Journal International, Page 560-569
DOI: 10.9734/BMRJ/2014/4896

Aim: To study multiple antimicrobial resistances in Vibrio spp. isolated from river and aquaculture water sources in Imo State Nigeria.

Methodology: A total of 157 Vibrio isolates from river and aquaculture water sources were analysed for multiple antimicrobial resistance during a 6 month period. Antimicrobial resistance profile was determined by the Kirby-Bauer technique, while the phenotypic expression of β-lactamase production was performed by the double disk diffusion method. PCR was used to screen isolates for the presence of β-lactamase resistance genes.

Results: The isolates from river water expressed high resistance rates (81.3 to 97.8%) to the following antimicrobials: mezlocillin, doxycycline, tetracycline, carbenicillin and ampicillin, while resistance rate to kanamycin was moderate at 40.9%. Resistance rates for the aquaculture water Isolates were also high for the same antibiotics as the river water isolates, while resistance rate to kanamycin was low to moderate at 32.8%. Phenotypic screening of isolates for ESβL production showed the isolates were resistant to β-lactam antimicrobials and the β-lactamase inhibitor of amoxicillin/clavulnic acid combination. Gel electrophoresis of PCR products showed amplification for blaTEM of size 964bp.

Conclusion: Results showed the presence of highly resistant Vibrio isolates from the sampled environmental sources. The presence of resistance markers among the isolates in this study infers that they could be agents of transfer of resistance to other bacterial pathogens found in river and aquaculture water

Open Access Original Research Article

Protection against Helicobacter pylori Infection in BALB/c Mice by Oral or Intramuscular Administration of Multicomponent Vaccine of rCagA +LPS +CpG

D. Esmaeili, A. Mobarez, A. H. Salmanian, A. Zavaran

Microbiology Research Journal International, Page 570-582
DOI: 10.9734/BMRJ/2014/5160

Aims: In this research, the protective effects of this multicomponent vaccine were investigated using the BALB/c mice model.

Place and Duration of Study: This study was performed in Laboratory of Bacteriology, University of Tarbiat Modares, Tehran, IRAN, 2011 and 2012.

Methodology:  BALB/c mice were immunized with different formulations three times orally followed by two times intramuscularly (i.m.) at 10-day intervals. The protective effects of two component vaccines plus CpG Adjutants were assessed after H. pylori ss1 challenge in different studies. The specific IgG antibodies titers in sera were studied by using ELISA, and Antigen specific IL-4, IL-10, IL-12, TGFβ and IFN-γ responses were measured in spleen of immunized mice after challenge using ELISA test. Clearance of H. pylori carried out according to standard protocol.

Results: In this study the IgG1/IgG2a ratio in the mice immunized with rCagA and rCagA plus CpG was <1. Analysis of lymphocyte proliferation of mice showed that one microgram rCagA increases lymphocytes proliferation excellent compared to control group. CpG oligodeoxy nucleotides are known for their ability to induce entirely Th1-biased immune responses. Immunization of mice with H. pylori rcagA+LPS+CpG induced a strong local and systemic Th1 immune response. Only mice immunized with rCagA+LPS+CpG and LPS+CpG secreted significantly more IFNγ than others (P<0.05). Protection were correlated with an increase in H. pylori-specific interleukin-12 and IFN-γ and both immunoglobulin G1 (IgG1) and IgG2a serum titers following challenge.

Conclusion:  Because of strong Th1 response, mice were protected from infection with H. pylori 6-fold reduction in the number of H. pylori in the gastric mucosa compared to no immunized mice. This study illustrates the crucial role of the immunization route and immunogenic candidate.


Open Access Original Research Article

Enteropathogens Associated with Childhood Diarrheal Cases seen at a Tertiary Hospital in Nguru, Yobe State of Nigeria

K. O. Okon, M. G.Nguru, M. Y.Bularafa, H. U. Mohammed, Z. A. Baba, U. Hamza, R. T. Akuhwa, C. U. Aguoru

Microbiology Research Journal International, Page 583-591
DOI: 10.9734/BMRJ/2014/4737

Aim: Childhood diarrheal diseases are common clinical episodes seen among children under 5 years old in the developing countries of sub-Saharan Africa and Asia. Epidemiological information of enteropathogens associated with childhood diarrhea will  provides clinical information to alliterate and enhance effective therapy management in our hospital.

Study Design: Retrospective analysis of enteropathogens associated with childhood diarrheal cases.

Place and Duration: The study was carried out in Federal Medical Centre, Nguru, over one year period from January to December, 2010.

Methodology: Fecal specimens were collected from patients presented with childhood diarrheal symptoms seen at the tertiary hospital at Nguru, Nigeria over the study period. Standard microbiological methods were employed in the enteropathogens detection. A total of 144 diarrheic fecal specimens were examined for existence of enteropathogens. The breakdowns of associated clinical diagnosis are as follows, gastroenteritis, 14 (9.7%), diarrhea, 80 (55.6%), dysentery, 31 (21.5%) and mucoid/bloody stool, 19 (13.2%).

Results: Of the 144 specimens analysed, enteropathogens were found in 89 (61.8%), 41 (46.1%) parasites and 48 (53.9%) bacterial cases respectively. Only two bacterial groups were identified, 43 (29.9%) were Escherichia coli and 5 (3.5%) belonged to Shigella spp. Among the parasites, Enteamoeba histolytica was the most prevalent (31 isolates, 21.5%), followed by Ascaris lumbricoides with 7 isolates (4.9%), Taenia saginata with 2 isolates (1.4%) and Hookworm with only 1 isolate (0.4%). Statistical significant difference was observed when the isolation frequency of enteropathogens was compared with the age-group and associated clinical diagnosis of the patients (p<0.02). Co-infections were observed in 16 (12.2%) cases, including 10 (62.5%) cases of E. coli / E. histolytica and one case (6.3%) of A. lumbricoides and Shigella spp.

Conclusion: The frequency of enteropathogens detected in this study was similar with those reported in other studies. In addition, it provides the epidemiological information on enteropathogens associated with childhood diarrhea in the studied region and serves as a guide to pediatricians towards empirical therapy

Open Access Review Article

A Review of Fusobacterium necrophorum Infections in Humans

Emelda E. Chukwu, Francisca O. Nwaokorie, Akitoye O. Coker

Microbiology Research Journal International, Page 480-496
DOI: 10.9734/BMRJ/2014/6634

The incidence of human infections caused by Fusobacterium necrophorum is recently on the increase and this is attributed largely to alteration in antibiotic usage pattern, malnutrition and poor oral hygiene. These infections are usually acquired exogenously from animals such as dogs, livestock or humans and ranges from mild sore throat to severe infections like Lemierre’s syndrome and Cancrum oris (NOMA). Fusobacterium necrophorum species produce characteristic toxins and virulent factors which are responsible for the severity of infections. Confirming the presence of these species is recommended during suspected infections.  It would help in providing information on the antimicrobial sensitivity pattern so as to guide treatment and control of these severe infections as well as for epidemiological purposes. This review summarizes human infections associated with F. necrophorum providing information on their epidemiology, risk factors, pathogenicity, diagnosis and treatment