Open Access Short communication

Multi-locus Genotyping Reveals High Occurrence of Mixed Assemblages in Giardia duodenalis within a Limited Geographical Boundary

Avik Kumar Mukherjee, Sumallya Karmakar, Dibyendu Raj, Sandipan Ganguly

Microbiology Research Journal International, Page 190-197
DOI: 10.9734/BMRJ/2013/2812

Aim: To determine the common genotypes of Giardia duodenalis causing diarrhea in the study region and to assess the extent of genetic polymorphism among them.
Study Design: Stool samples were collected from the patients attending IDBG Hospital, Kolkata with diarrheal complaints through a systemic sampling technique and were screened for Giardia duodenalis. The G. duodenalis positive samples were subjected to molecular genotyping through ‘PCR - Direct DNA sequencing’ procedure. All the sequence data obtained were incorporated into MEGA 4 software for multiple alignment and validation followed by phylogenetic analysis. The genotyping data obtained are stored in Excel spreadsheets and incorporated into EpiInfo 3.1 for analyzing possible association of genotype outcome with common physical factors such as age, sex etc.
Place and Duration of Study: Department of parasitology, National Institute of Cholera and Enteric Diseases, Kolkata, India from July 2009 to November 2011.
Methodology: A total of 68 Giardia duodenalis positive stool samples were identified from the diarrhea patients attending IDBG hospital in the city and were subjected to multi-locus genotyping. Fragments of ß-giardin, Glutamate-dehydrogenase and Triosephosphate-isomerase genes of Giardia were amplified from those samples with specific primers and sequenced. All the sequences were analyzed using MEGA 4 software for obtaining the genotyping results.
Results: Multi-locus genotyping identified 13 isolates as assemblage A and 41 as assemblage B, whereas 14 of them could not be assigned in a particular group. Detailed phylogenetic analysis revealed that multiple genotypes were observed in those 14 isolates depending upon the marker loci.
Conclusion: The study could produce a preliminary idea about the G. duodenalis genotypes found in Kolkata city. High percentage of mixed assemblages in the study population also revealed the presence of genetic diversity among a small population of diarrheal patient within a limited geographical boundary. It has also hypothesized the possibility of inter-assemblage genetic exchange among Giardia.

Open Access Original Research Article

Detecting Latent Tuberculosis Infection Prior to Kidney Transplantation in a Tertiary Hospital in Saudi Arabia: Comparison of the T-SPOT.TB Test and Tuberculin Test

Hoda Abdel Hadi Hassan, Mahmoud Shorman, Abdel Rahman E. l. Housawi, Mohamed Y. Elsammak

Microbiology Research Journal International, Page 116-127
DOI: 10.9734/BMRJ/2013/2926

Background: Tuberculosis (TB) remains a major worldwide public health problem with over 8.8 million newly diagnosed cases in 2010. Patients with end-stage renal disease (ESRD) who are on hemodialysis (HD) have a significantly higher incidence of Mycobacterium tuberculosis infection or disease than healthy individuals. Most cases of active tuberculosis (TB) in patients with ESRD are due to the reactivation of a latent infection, and this patient group is at roughly 10- to 25-fold higher risk for reactivating TB infection than the general population. Candidates for solid organ transplantation are routinely screened for latent tuberculosis infection (LTBI). In this study we aimed to compare Tuberculin Skin Test (TST) with T-SPOT.TB, for the detection of LTBI in candidates for kidney transplantation.
Methods: Prospective study of 133 HD Patients who did not have a diagnosis of active TB diseases or LTBI previously referred, through a 5-month period, to our institutions. Forty four kidney donors without evidence of renal insufficiency or immunocompromising conditions by medical history served as control group. All patients were tested with tuberculin (TST), and T-Spot.TB and the results were compared.
Results: In donors, the concordance between the T-SPOT and the TST was moderate (90.9 %, к=0.46). Forty of 44 donors (90.9%) had concordance results between the T-Spot TB and TST.In hemodialysis patients, the concordance between the T-SPOT.TB and the TST was poor (60.15 %, к=0.07). Fifty three of 133 patients (40%) had discordant results between the T-SPOT.TB and TST. Of these, 13 patients had a positive TST but negative T-SPOT.TB and 40 had a positive T-SPOT.TB but a negative TST.
Conclusion: Our data strongly argue against the use of TST in screening of LTBI in HD patients. T-SPOT.TB test in dialysis patients correlated better than TST with the risk of TB infection (e.g. increased age and low body mass index). It is a more reliable and powerful diagnostic tool than TST. However, further studies should be carried out to determine the tests with higher sensitivity and most permitted specificity.

Open Access Original Research Article

Biodiversity and Enterotoxigenic Potential of Staphylococci Isolated from Raw and Spontaneously Fermented Camel Milk

Patrick Murigu Kamau Njage, Stefania Dolci, Christoph Jans, John Wangoh, Christophe Lacroix, Leo Meile

Microbiology Research Journal International, Page 128-138
DOI: 10.9734/BMRJ/2013/2896

Aims: To characterise the diversity, genotypic and phenotypic properties of coagulase negative and coagulase positive staphylococci from camel milk.
Place and Duration of Study: Laboratory of Food Biotechnology, Department of Health Sciences and Technology (D-HEST), Swiss Federal Institute of Technology, Zurich, Switzerland, between July 2009 and June 2011.
Methodology: Staphylococci isolated from 59 raw and spontaneously fermented camel milk (suusac) samples from Kenya and Somalia were identified, pheno- and genotypically characterized. Preliminary screening of colonies was done by catalase test, Gram staining reactions, clumping factor/protein A and microscopy. Further identification was done by 23S rDNA species PCR, thermostable nuclease gene (nuc) PCR and rep-PCR followed by staphylococcal genus ID32 Staph system and coagulase negative species specific PCR. PCR amplification of the genes encoding capsular polysaccharides cap5 and cap8, and staphylococcal enterotoxins SEA to SEE and SEG to SEJ was also carried out.
Results: From a total of 235 BP medium isolates, staphylococci were 146 (62 %) of which, 66 (45 %) were Staphylococcus aureus. S. epidermidis accounted for 43 % of the coagulase negative staphylococci (CNS). The rest of the CNS were 25 % S. simulans, 16.3 % S. saprophyticus, 2.5 % S. haemolyticus, 2.5% S. hyicus, 2.5 % S. xylosus, 2.5 % S. lentus, 1.3 % S. carnosus and 1.3 % S. microti. Capsular polysaccharide gene cap5 was present in 15 % and cap8 in 23 % of the S. aureus isolates. Enterotoxin genes were detected in 47 % of the staphylococci with sej in 33 %, seb in 6 %, sed in 5 % and seg in 3 % of the isolates. Within the species enterotoxin genes were detected in 100 %, 64.7 %, 38.5 % and 22.7 % of the S. simulans, S. epidermidis, S. sapropyticus and S. aureus respectively.
Conclusion: The diversity of CNS is remarkable and the prevalence of enterotoxin genes amongst CNS and CPS further informs generalizations for other milk and hygienic situations in similar production environment.

Open Access Original Research Article

Prevalence of seg, seh and sei Genes among Clinical and Nasal Staphylococcus aureus Isolates in Palestine

Ghaleb Adwan, Kamel Adwan, Naser Jarrar, Yousef Salama, Ali Barakat

Microbiology Research Journal International, Page 139-149
DOI: 10.9734/BMRJ/2013/2913

Aims: To investigate the presence of the staphylococcal enterotoxin genes seg, seh and sei among clinical and nasal isolates.
Place and Duration of Study: Department of Biology and Biotechnology, An-Najah N. University, Palestine, in 2011.
Methodology: A total 124 S. aureus isolates were collected, forty three were nasal and 81 were clinical isolates. PCR technique was used to detect enterotoxin genes seg, seh and sei, mecA gene and analysis of SCCmec types. Enterotoxigenic strains were also typed using coagulase typing kit.
Results: Fifty two (41.9%) isolates were positive for one or more of these enterotoxin genes. The prevalence of toxin genes among S. aureus isolated from nasal swabs 25/43 (58.1%) was higher than those isolated from clinical samples 27/81 (33.3%). Combination of the toxin genes was noted only in MSSA isolate from both nasal swabs and clinical samples. Distribution of toxin genes in MSSA isolates was higher (49.5%) than those in MRSA isolates (21.2%). SCCmec typing showed that the MRSA enterotoxigenic strain were belonged to types II, III and IVa. MRSA strains were found to belong to coagulase serotypes II, III and VII, while MSSA strains were belonged to serotypes II-VII. In nasal samples, 16/25 (64.0%) of enterotoxigenic strains showed the genotype seg+/sei+, while in clinical samples 1/27 (3.7%), 1/27 (3.7%) and 3/27 (11.1%) of enterotoxigenic strains showed the genotypes seg+/seh+, seg+/sei+ and seg+/seh+/sei+, respectively. This study showed that the majority of the isolates 42/124 (33.9%) were seg+, while none of nasal strains harbored seh gene.
Conclusion: The prevalence of seg, seh and sei genes in the S. aureus isolated from nasal swabs differed significantly from those obtained from clinical samples, as well as the prevalence of the same genes in MSSA differed significantly from those in MRSA. In addition, S. aureus isolates from clinical and nasal swabs could serve as a possible reservoir of newly described seg, seh and sei genes.

Open Access Original Research Article

Farnesol Sensitivity of Serum Induced Yeast to Hyphae Morphogenesis: A Study on Fifty Clinical Isolates of Candida albicans

Vimal S. Rathod, Jayant S. Raut, Sharmila S. Raut, Sanjay R. More, Sankunny Mohan Karuppayil

Microbiology Research Journal International, Page 150-157
DOI: 10.9734/BMRJ/2013/2939

Aim: Objective of this study was to examine farnesol sensitivity of yeast to hyphae dimorphism in clinical isolates of Candida albicans.
Study Design: Variations in virulence attributes contribute to variations in pathogenicity of C. albicans. Ability to switch from yeast to hyphae morphology is an important virulence factor. Farnesol, a quorum sensing molecule is known to play an important role in the regulation of C. albicans morphogenesis. Analysis of farnesol susceptibility of yeast to hyphae conversion may reveal a factor responsible for variation in pathogenicity among clinical isolates of C. albicans.
Place and Duration of Study: SCG Medical College & SGGS Memorial Hospital, and School of Life Sciences, SRTM University, Nanded, India. Duration of this study was, December 2008 to December 2010.
Methodology: Fifty clinical isolates of C. albicans were recovered from body fluids (such as, sputum, blood, urine, vaginal swab, tracheal swab, throat swab, feces, pus and cerebrospinal fluid, etc.) of patients with different clinical manifestations, in the tertiary care center hospital. Presumptive identification of C. albicans was done on HiCHROM agar-Candida, while confirmation was done by Germ tube formation assay, Carbohydrate assimilation and Corn meal agar test. Serum induced yeast to hyphae morphogenesis in C. albicans was performed in 96 well plates. Recent methodology of micro broth dilution was used for farnesol susceptibility testing in fifty clinical isolates.
Results: Farnesol prevented hyphae formation in a concentration dependent manner, in the range 25 to 400 µM. Inhibition of ≥ 50% hyphae was considered as significant reduction in morphogenesis. MIC70 for farnesol mediated inhibition of morphogenesis in C. albicans was at 200 µM. Mean values for percentage inhibition of morphogenesis in fifty strains was compared by analysis of variance (ANOVA). P = 0.05 was considered significant.
Conclusion: Susceptibility of yeast to hyphae morphogenesis to the quorum sensing molecule farnesol, varied significantly among clinical isolates of C. albicans. We hypothesize that variation in farnesol sensitivity may be a factor responsible for variable dissemination and infection ability of C. albicans.

Open Access Original Research Article

Persistent Bactericidal Action by a Silver Disinfectant on Surfaces of Hospital Furniture

Sonia De Lorenzi, Italo Barrai, Gianfranco Finzi, Paola Cugini, Germano Salvatorelli

Microbiology Research Journal International, Page 158-164
DOI: 10.9734/BMRJ/2013/2761

Aims: In hospitals, surfaces are often colonized by potentially pathogenic micro organisms which can remain alive for long periods of time, thus playing a major role in hospital-acquired infections. One way to overcome this drawback could be to use disinfectants with long-term action. Recent studies have shown that not only do disinfectants containing silver present an immediate effect, reducing the surface bacterial load, but that this action also appears persistent in time. This work assesses the bactericidal activity of a long-lasting disinfectant complex composed of silicon oxide, silver ions and a cationic surfactant (BACTERCLINE ENERGY BLAST) applied on the surfaces of two surgery ward rooms and left in place for different amounts of time (15 min and 72 hours).
Study Design: An experimental study.
Place and Duration of Study: The investigation was carried out in two rooms of a surgical ward at the Villa Erbosa health care facility in Bologna (Italy) over a period of seven weeks.
Methodology: The samples were taken using contact plates (diameter: 24 cm2) containing an adequate agar culture medium (Tryptic Soy Agar). After incubation at the temperature of 36±1ºC for 24 and 48h, the number of colonies was counted and the statistical analysis of results was performed.
Results: The product was able to achieve a high degree of decontamination (around 90%) immediately after application and that, after 72 hours, decontamination remained at about 55%. The results are statistically significant.
Conclusion: It may be concluded that the tested product could be profitably used to decontaminate surfaces in hospital wards.

Open Access Original Research Article

Lactobacillus rhamnosus GG Protects Cells from Clostridium difficile Toxins

Eden Ephraim, Ronald D. Schultz, Nasia Safdar

Microbiology Research Journal International, Page 165-175
DOI: 10.9734/BMRJ/2013/3068

Aims: To determine the anti-cytotoxic effects of Lactobacillus rhamnosus GG (LGG) against extracellular and intracellular Clostridium difficile toxins.
Study Design: Co-culture system.
Place and Duration of Study: Division of Infectious Diseases, Department of Medicine, School of Medicine and Public Health and Department of Pathobiological Sciences, School of Veterinary Medicine, Madison, Wisconsin, between April 2010 and August 2011.
Methodology: In this study, we investigated the effects of a probiotic LGG (Culturelle®) against a toxigenic C. difficile strain (ATCC 9689) and a non-toxigenic C. difficile strain (ATCC 700057) in a co-culture system. Co-cultures were prepared with 3 ml of 1:10, 1:100 or 1:1000 dilution of an overnight culture of LGG and 2 ml of 1:100 dilution of either the toxigenic or the non-toxigenic strain. Cytotoxic effects of cell-free culture supernatants (CFS) and cell lysates of the toxigenic strain on Vero cells were evaluated after co-culturing. The relative abundance of toxin A (TcdA) and Toxin B (TcdB) genes in 72 h co-cultures were determined using real time PCR.
Results: In co-cultures with 1:10 or 1:100 dilution of LGG, counts of the toxigenic C. difficile strain were about one log unit lower than control pure cultures after incubation for 48 h. In all co-cultures, counts of the non-toxigenic strain were two log units lower than those of controls. Accordingly, LGG resulted in a significant decrease (p < 0.05) in the relative abundance of TcdA and TcdB in target DNA prepared from co-cultures containing the 1:10 or the 1:100 dilution of the probiotic. Co-culturing the toxigenic strain with the probiotic (1:10 and 1:100) decreased (P < 0.05) the cytotoxic effect of both extracellular and intracellular clostridial toxins resulting in up to 30% increase in cell viability.
Conclusion: LGG inhibits the growth of C. difficile in a dose-dependent manner and protects cells from C. difficile induced cytotoxicity.

Open Access Original Research Article

Assessment of Microbiological Quality of Yogurt Sold By Street Vendors in Onitsha Metropolis, Anambra State, Nigeria

V. O. Ifeanyi, E. O. Ihesiaba, O. M. Muomaife, C. Ikenga

Microbiology Research Journal International, Page 198-205
DOI: 10.9734/BMRJ/2013/1801

Four brands of yogurt sold by street vendors in Onitsha Metropolis, Anambra State, Eastern Nigeriia were sampled, the pH was determined and microbiological assessments were conducted in order to ascertain the total heterotrophic bacteria, coliforms and yeast in the samples (A – D) during a seven day period. The results revealed that values of pH monitored varied from 3.69 – 4.50 while a total of five bacteria species belonging to Escherichia coli, Staphylococcus aureus, Streptococcus, Lactobacillus and Bacillus species, and three fungi species belonging to Aspergillus, Rhizopus and Saccharomyces were isolated from the samples. Sample B had the highest mean heterotrophic bacteria count with a value of 6.1 x 105 cfu/ml. Statistical analysis of heterotrophic bacteria count among the 4 sample groups had p-value =0.0000374. There is a significant difference in the heterotrophic bacteria count among the groups. Low titre values of starter cultures were recorded in the control samples. Escherichia coli, an indicator of coliform was detected in all the samples and the value of 4.4 x 105 cfu/ml was observed in sample B. Coliforms, S. aureus, Bacillus species and fungi were not detected from control samples purchased directly from the producing companies. Statistical analysis of coliform count in the four groups had p-value=0.529296. There was no significant difference in the coliform count among the 4 sample groups at α =0.05 and p-value =0.529296. The findings suggest that the yogurt traded by street vendors in Onitsha Metropolis has poor microbiological quality control. This poses danger to public health. Therefore, attention of the appropriate government agencies and manufacturers is needed to ensure that sale of yogurt by vendors is done in most appropriate condition and in a mobile refrigerator to maintain adequate temperature, thereby, reduce contamination.

Open Access Review Article

Genetic Instability and Herpes Simplex Virus

H. S. Zakaryan, Z. A. Karalyan

Microbiology Research Journal International, Page 176-189
DOI: 10.9734/BMRJ/2013/2736

Although inhibitory mechanisms that safeguard cells against DNA damages occur in all cells, genetic instability is widely present throughout living world. Some well known viruses cause alterations in host genome, leading to the formation of malignant transformations. Throughout this review we discuss some forms of instabilities caused by herpes simplex virus. Herpes simplex virus may induce chromosomal instabilities by various mechanisms, such as by formation of syncytium or ICP0-induced degradation of constitutive centromere proteins as well as may induce accumulation of point mutations by means of oxidative stress in neurons. We believe that further investigation of the ability of herpes simplex virus to cause genetic instability may help us to increase our understanding about the nature of this phenomenon.