Isolation and partial characterization of phosphate solubilizing bacteria isolated from soil and marine samples.

In the present study the potential of indigenous bacterial isolates from soil rhizosphere and marine environment to promote plant growth was determined. Eight bacterial strains isolated from soil and marine samples were characterized for the phosphate solubilizing activity. Qualitative and quantitative estimation of phosphate solubilization is done. MIC of antibiotic and heavy metals were checked for these strains. Strains show a diverse pattern of antibiotic and heavy metals resistance.

Drug resistance profile and biofilm forming potential of Pseudomonas aeruginosa isolated from contact lenses in Karachi-Pakistan.

BACKGROUND: The contaminated contact lens provides Pseudomonas aeruginosa an ideal site for attachment and biofilm production. Continuous contact of the eye to the biofilm-infested lens can lead to serious ocular diseases, such as keratitis (corneal ulcers). The biofilms also prevent effective penetration of the antibiotics, which increase the chances of antibiotic resistance. METHODS: For this study, 22 Pseudomonas aeruginosa isolates were obtained from 36 contact lenses and 14 contact lens protective fluid samples. These isolates were tested against eight commonly used antibiotics using Kirby-Bauer disk diffusion method. The biofilm forming potential of these isolates was also evaluated using various qualitative and quantitative techniques. Finally, a relationship between biofilm formation and antibiotic resistance was also examined. RESULTS: The isolates of Pseudomonas aeruginosa tested were found resistant to most of the antibiotics tested. Qualitative and quantitative biofilm analysis revealed that most of the isolates exhibited strong biofilm production. The biofilm production was significantly higher in isolates that were multi-drug resistant (p < 0.0001). CONCLUSION: Our study indicates that multi-drug resistant, biofilm forming Pseudomonas aeruginosa isolates are mainly involved in contact lens associated infections. This appears to be the first report from Pakistan, which analyzes both antibiotic resistance profile and biofilm forming potential of Pseudomonas aeruginosa isolates from contact lens of the patients with contact lens associated infections.

In vitro antituberculosis activities of the constituents isolated from Haloxylon salicornicum.

In vitro antituberculosis activities of fractions and pure compounds (1-20) including seven triterpenes, two alkaloids, two cycloheximide derivatives, two coumarins six sterol derivatives and a long chain alcohol, respectively, isolated from Haloxylon salicornicum were determined against Mycobacterium tuberculosis H37Rv. Actively growing cultures were tested by rapid colorimetric method while the stationary phase cultures were tested by drug exposure methods for bactericidal activity. The MIC values were found to be 50 microg/ml for compounds 15, 19 and 20 where as rest of the compounds invariably showed MIC value of 100 microg/ml against the logarithmic phase culture. These were compare to Isoniazid as a control drug. The compounds exhibited no activity against the stationary phase culture of M. tuberculosis H37Rv up to 200 microg/ml. Further studies are required to investigate the in vivo efficacies and activities of the compounds in combination with antimicrobials that are already being used for TB therapy.

Evidence for a "Founder Effect" among HIV-infected injection drug users (IDUs) in Pakistan.

BACKGROUND: We have previously reported a HIV-1 subtype A infection in a community of injection drug users (IDUs) in Karachi, Pakistan. We now show that this infection among the IDUs may have originated from a single source. METHODS: Phylogenetic analysis was performed of partial gag sequences, generated using PCR, from 26 HIV-positive IDU samples. RESULTS: Our results showed formation of a tight monophyletic group with an intra-sequence identity of < 98% indicating a "founder effect". Our data indicate that the HIV-1 epidemic in this community of IDUs may have been transmitted by an HIV positive overseas contract worker who admitted to having contact with commercial sex workers during stay abroad. CONCLUSION: Specific measures need to implemented to control transmission of HIV infection in Pakistan through infected migrant workers.

Investigation of a community outbreak of typhoid fever associated with drinking water.

BACKGROUND: This report is about the investigation of an outbreak of typhoid fever claimed three human lives and left more than 300 people suffered within one week. The aim of this report is to draw the attention of global health community towards the areas that are still far from basic human essentialities. METHODS: A total of 250 suspected cases of typhoid fever were interviewed, out of which 100 were selected for sample collection on the basis of criteria included temperature > 38 degrees C since the onset of outbreak, abdominal discomfort, diarrhea, vomiting and weakness. Food and water samples were also collected and analyzed microbiologically. RESULTS: Inhabitants of village lived in poor and unhygienic conditions with no proper water supply or sewage disposal facilities and other basic necessities of life. They consumed water from a nearby well which was the only available source of drinking water. Epidemiological evidences revealed the gross contamination of well with dead and decaying animal bodies, their fecal material and garbage. Microbiological analysis of household and well water samples revealed the presence of heavy bacterial load with an average total aerobic count 106-109 CFU/ml. A number of Gram positive and Gram negative bacteria including Escherichia coli, Klebsiella, Bacillus species, Staphylococcus species, Enterobacter species, and Pseudomonas aeruginosa were isolated. Lab investigations confirmed the presence of multidrug resistant strain of Salmonella enterica serovar Typhi in 100% well water, 65% household water samples and 2% food items. 22% of clinical stool samples were tested positive with Salmonella enterica serover Typhi CONCLUSIONS: This study indicated the possible involvement of well water in outbreaks. In order to avoid such outbreaks in future, we contacted the local health authorities and urged them to immediately make arrangements for safe drinking water supply.

Prevalence of HCV and HIV infections in 2005-Earthquake-affected areas of Pakistan.

BACKGROUND: On October 8, 2005, an earthquake of magnitude 7.6 hit the Northern parts of Pakistan. In the post-earthquake scenario, overcrowding, improper sewage disposal, contamination of food and drinking water, hasty surgical procedures, and unscreened blood transfusions to earthquake victims most likely promotes the spread of infections already prevalent in the area. OBJECTIVE: The objective of the study reported here was to determine the prevalence of Human Immunodeficiency and Hepatitis C viruses (respectively, HIV and HCV) in the earthquake-affected communities of Pakistan. The samples were analyzed 2 months and then again 11 months after the earthquake to estimate the burden of HIV and HCV in these areas, and to determine any rise in the prevalence of these viral infections as a result of the earthquake. METHODS: Blood samples were initially collected during December, 2005 to March 2006, from 245 inhabitants of the earthquake-affected areas. These samples were screened for HCV and HIV, using immunochromatography and Enzyme-Linked Immuno-Sorbent Assay (ELISA). RESULTS: Out of 245 samples tested, 8 (3.26%) were found positive for HCV, and 0 (0.0%) for HIV, indicating the existence of HCV infection in the earthquake-stricken areas. The same methods were used to analyze the samples collected in the second round of screening in the same area, in September, 2006 - 11 months after the earthquake. This time 290 blood samples were collected, out of which 16 (5.51%) samples were positive for HCV, and 0 for HIV. CONCLUSION: A slightly higher prevalence of HCV was recorded 11 months after the earthquake; this increase, however, was not statistically significant. None of the study participants was found HIV-infected.

Association of oral Helicobacter pylori with gastric complications.

AIM: This study was aimed to identify the presence of Helicobacter pylori (H. pylori) genes in oral mucosa and find out their relationship between oral H. pylori infection and gastric complications. METHODS: This study is a case control study consists of 567 subjects with periodontal infection (278 gastric complication cases and 289 controls normal gastric intestinal mucosa) with age range of 20-80 years. Oral health status was recorded by calculating oral hygiene index (OHI), probing depths (PD) and clinical attachment loss (CAL). Each participant provided gastric biopsy and plaque samples which were subjected to H. pylori detection. Polymerase chain reaction (PCR) with different primers specifically ß globulin, 16SrRNA, babA, cagA, ureA, ureC and vacA gene was performed which were then analyzed using gel electrophoresis. RESULTS: No significant differences (χ2 = 11.873, p value > 0.05) were observed between oral H. pylori and gastric infections/complications. However, H. pylori increase the risk of developing gastro-esophageal reflux grade II (OR = 1.458, 95%CI = 0.659-3.226), normal upper GIT mucosa with lax esophageal sphincters (OR = 1.215, 95%CI = 0.285-5.181) and duodenal ulcer/duodenitis (OR = 2.187, 95%CI = 0.225-21.278). This study also showed a significant increased risk of gastritis with babA gene. CONCLUSION: Oral pathogenic H. pylori genes may enhance the severity of the gastric infection.

Prognostic markers in HIV mono-and co-infected individuals: A study from Karachi-Pakistan.

BACKGROUND: Multiple infections are the most common problem among HIV infected individuals. The prognostic impact of these co infections in HIV-population in resource-limited countries like Pakistan has not been fully elucidated. The aim of this study was to assess CD4 and hemoglobin (Hb) levels in patients with HIV mono infection and HIV co-infection with Hepatitis C (HCV), Hepatitis B (HBV) and Mycobacterium tuberculosis (MTB). METHODS: A total of 207 HIV positive patients were assessed for CD4 cells count and hemoglobin levels after confirmation of HIV by rapid tests as well as PCR. CD4 counts were performed via flow cytometry whereas hemoglobin levels were performed by Sysmex K-4500 auto-analyzer. RESULTS: Out of 207 patients, 22 patients were found to be HIV mono-infected, while 185 patients were HIV positive along with co-infections of MTB or HCV or HBV. We found significant positive correlation between HB levels and CD4 count across the studied group (r=0.30 in HIV mono-infected group, r=0.23 in HIV co-infected group, p<0.05) at baseline. However, majority of the low hemoglobin levels (<8g/dl) and low CD4 count (<200cells/ul) cases were observed particularly in HIV/TB co-infections. CONCLUSION: This study documents the prognostic value of hemoglobin assessment in HIV patients. The results indicate that decreasing Hb levels correlate with decreasing CD4 counts. It is emphasizing that Hb measurement may be used as an inexpensive surrogate marker as compared to CD4 analysis for disease progression in HIV patients. In addition, low Hb levels may also indicate presence of under lying co-infections, particularly, with M. tuberculosis (MTB).

Synergistic antimicrobial activity of Camellia sinensis and Juglans regia against multidrug-resistant bacteria.

Synergistic combinations of antimicrobial agents with different mechanisms of action have been introduced as more successful strategies to combat infections involving multidrug resistant (MDR) bacteria. In this study, we investigated synergistic antimicrobial activity of Camellia sinensis and Juglans regia which are commonly used plants with different antimicrobial agents. Antimicrobial susceptibility of 350 Gram-positive and Gram-negative strains belonging to 10 different bacterial species, was tested against Camellia sinensis and Juglans regia extracts. Minimum inhibitory concentrations (MICs) were determined by agar dilution and microbroth dilution assays. Plant extracts were tested for synergistic antimicrobial activity with different antimicrobial agents by checkerboard titration, Etest/agar incorporation assays, and time kill kinetics. Extract treated and untreated bacteria were subjected to transmission electron microscopy to see the effect on bacterial cell morphology. Camellia sinensis extract showed higher antibacterial activity against MDR S. Typhi, alone and in combination with nalidixic acid, than to susceptible isolates." We further explore anti-staphylococcal activity of Juglans regia that lead to the changes in bacterial cell morphology indicating the cell wall of Gram-positive bacteria as possible target of action. The synergistic combination of Juglans regia and oxacillin reverted oxacillin resistance of methicillin resistant Staphylococcus aureus (MRSA) strains in vitro. This study provides novel information about antimicrobial and synergistic activity of Camellia sinensis and Juglans regia against MDR pathogens.

Detection of genes for superantigen toxins in methicillin-resistant Staphylococcus aureus clinical isolates in Karachi.

OBJECTIVE: To detect genes for enterotoxins, exfoliative and toxic shock syndrome toxins in Staphylococcus aureus (S. aureus) strains isolated from clinical specimens. STUDY DESIGN: Cross-sectional observational study. PLACE AND DURATION OF STUDY: Department of Molecular Genetics, Dr. Ziauddin Hospital, Karachi, from January to December 2010. METHODOLOGY: Two hundred and ninety eight S. aureus clinical isolates were obtained from various clinical samples received at Dr. Ziauddin Hospital, Karachi. Out of these, 115 were detected as methicillin resistant (MRSA) by cefoxitin disk diffusion test showing a prevalence rate of 38.6%. Detection of individual toxin genes was performed by Polymerase Chain Reaction (PCR) by using only one primer pair for each tube. Uniplex primers were preferred as multiplex primers are longer in base pairs and have the potential for cross reaction due to non-specific binding and increase in optimization time. RESULTS: The possession of a single gene or more than a single gene in MRSA isolates was found in 61.73% of clinical samples; the highest number was found in pus swab, followed by sputum, blood, urethral swab, and urine. The prevalence of toxin genes was higher in MRSA as compared to methicillin sensitive (MSSA) isolates (19.12%). CONCLUSION: PCR detects strains possessing toxin genes independent of their expression. The possession of genes for super-antigens seems to be a frequent and habitual trait of S. aureus more so in MRSA.

Prevalence, diversity and disease association of Helicobacter pylori in dyspeptic patients from Pakistan.

INTRODUCTION: The etiological association of Helicobacter pylori with gastric ulcer (GU), gastric cancer (GC), and duodenal ulcer (DU) is well-known. Understanding the epidemiology of H. pylori facilitates the estimation of disease burden in a certain population. This study presents the diversity of H. pylori genotypes and their association with different clinical outcomes among dyspeptic patients in Pakistan over a period of four years. METHODOLOGY: Gastric biopsy samples from a total of 450 dyspeptic individualswere subjected to PCR, genotypingand histology. RESULTS: A total of 201 (45%) cases were found positive for H. pylori. The detection rate was high in GU (91%), DU (86%) and GC (83%) cases compared with those cases who had intact gastric mucosa (18%). Histology revealed the presence of infection in 68% of cases of mild/chronic nonspecific gastritis with others belonging to the GU sequel. cagA gene carriage was observed in 104 (51%) cases or mostly from DU, GU and GC groups, of which 97 were Western type strains while 3 were East-Asian type strains that are rarely observed in South Asia. vacA allelic variant s1am1 was most commonly observed, followed by s1am2, and s1bm1, with direct correlation in diseased cases (gastritis, GU, DU and GC). Prevalent genotypic combinations were s1am1/cagA- in gastritis and s1am1/cagA+ in DU, GU, and GC. CONCLUSIONS: Our study indicates the predominant circulation of Western type cagA and vacAs1am1 type H. pylori strains in Pakistan.

Fluoroquinolones inhibit HCV by targeting its helicase.

BACKGROUND: HCV has infected >170 million individuals worldwide. Effective therapy against HCV is still lacking and there is a need to develop potent drugs against the virus. In the present study, we have employed two culture models to test the activity of fluoroquinolone drugs against HCV: a subgenomic replicon that is able to replicate independently in the cell line Huh-8 and the Huh-7 cell culture model that employs cells transfected with synthetic HCV RNA to produce the infectious HCV particles. Fluoroquinolones have also been shown to have inhibitory activity against certain viruses, possibly by targeting the viral helicase. To tease out the mechanism of the antiviral activity of fluoroquinolones, their effect on HCV NS3 helicase protein was also tested. METHODS: Huh-7 cells producing the HCV virion as well as Huh-8 cells were grown in the presence or absence of 12 different fluoroquinolones. Afterwards, Huh-7 and Huh-8 cells were lysed and viral RNA was extracted. The extracted RNA was reverse transcribed and quantified by real-time quantitative PCR. Fluoroquinolones were also tested on purified NS3 protein in a molecular-beacon-based in vitro helicase assay. RESULTS: To varying degrees, all of the tested fluoroquinolones effectively inhibited HCV replication in both Huh-7 and Huh-8 culture models. The inhibition of HCV NS3 helicase activity was also observed with all 12 of the fluoroquinolones. CONCLUSIONS: Fluoroquinolones inhibit HCV replication possibly by targeting the HCV NS3 helicase. These drugs hold promise for the treatment of HCV infection.

Prevalence of Hepatitis B virus genotype D in females in Karachi, Pakistan.

BACKGROUND: Hepatitis B virus (HBV) is an etiological agent of acute and chronic liver disease existing throughout the world. The high genetic variability of HBV genome is reflected by eight genotypes (A to H), and each genotype has characteristic geographical distribution, which is important epidemiologically. Previous studies from the province of Sindh, Pakistan, have reported that genotypes A and D as prevalent HBV genotypes. The aim of the study was to investigate the prevalence of HBV genotypes in physically healthy females at two universities in Karachi, Sindh, Pakistan. METHODOLOGY: Blood was collected from a total of 4,000 healthy female volunteer students and serum samples obtained were screened for Hepatitis B surface antigen (HBsAg), and anti-HBs antibodies by immunochromatography and ELISA. Genotyping was conducted for 6 HBV genotypes (A through F). Both genotyping and sequencing data of HBV positive females are described. RESULTS: Out of 4,000 volunteers, 180 (4.5%) tested positive for HBsAg and 20 (0.5%) were positive for HBs antibodies. All 180 serum samples were genotyped by PCR and sequencing analyses was conducted for 21 samples. Out of 180 HBsAg positive samples, 150 showed a single HBV D genotype infection; 29 showed co-infection of genotypes B and D; and 1 exhibited co-infection of genotypes C and D. Twenty-one representative samples were selected randomly from genotypes B, D, and C for sequencing and each isolate clustered with respective reference genotype sequence, thus validating the genotyping strategy. CONCLUSION: Genotype D appears to be the dominant genotype prevalent in Karachi's otherwise healthy female population.