Analysis of Salmonella enterica Serovar Typhi by Outer Membrane Protein (OMP) Profiling, Random Amplification of Polymorphic DNA (RAPD) and Pulsed Field Gel Electrophoresis (PFGE).

A number of countries, including developed countries, still have typhoid fever as a major problem resulting in frequent outbreaks. The importance of controlling spread of typhoid fever is well known and necessitates periodic studies to delineate epidemiological relationships. Although phage typing remains to be the preferred conventional method for characterisation of typhoid bacilli, it is of limited use due to prevalence of few predominant phage types in the country like India. Therefore, an effort has been made to assess three molecular methods [Outer Membrane Protein (OMP) Profiling, Random Amplification of Polymorphic DNA (RAPD) and Pulsed Field Gel Electrophoresis (PFGE)] for typing of Salmonella enterica serovar Typhi. 128 Salmonella enterica serovar Typhi isolates were identified using biotyping and serotyping followed by antimicrobial susceptibility testing. These isolates were further subjected to OMP analysis, RAPD and PFGE. PFGE (114 unique clusters) was found to be the most discriminatory method followed by RAPD (94 unique clusters) and OMP profiling (50 unique clusters). Multidrug resistant strains were well discriminated by all three methods used in the study. PFGE still remains the most preferred method for detailed epidemiological investigations. However, random amplification of polymorphic DNA and outer membrane protein profiling can also be considered for molecular discrimination of the isolates in the laboratories lacking high-end facilities.

Characterization of antimicrobial resistance markers & their stability in Salmonella enterica serovar Typhi.

BACKGROUND & OBJECTIVES: Typhoid fever is a major cause of morbidity and mortality in the developing countries including India. Resistance to multiple antimicrobial agents is an emerging global problem that has serious impact on the treatment of disease. There are many factors associated with the emergence of resistance. Most important of them is the acquisition and further transmission and spread of resistance markers among various bacterial species. Therefore, we conducted this study to characterize the resistance plasmids in terms of their transferability and stability among Salmonella enterica serovar Typhi. METHODS: Six multidrug-resistant S. Typhi isolates were evaluated for the stability and transfer of resistance markers. The resistance plasmids were also checked for the presence of RepHI1A replicon. RESULTS: All resistance markers were found to be transferred to the recipient through conjugation and transformation, except for nalidixic acid. None of the resistance plasmid was found to harbour RepHI1A replicon and therefore, did not belong to incompatibility group IncHI1. Resistance markers were found to be highly stable in all the isolates during serial passages and storage as stab cultures at different temperatures for different time periods. INTERPRETATION & CONCLUSIONS: Resistance markers for chloramphenicol, ampicillin, streptomycin and trimethoprim were transferred through conjugation as well as transformation whereas that for nalidixic acid was not transferred in any of the isolates. Markers for chloramphenicol and streptomycin resistance were found to be most stable during various storage conditions. Presence of small-sized non-IncHI1 resistance plasmids is a matter of concern due to their capability to exist inside the host, thereby increasing the possibility of their transmission and spread among S. Typhi and other bacterial species.

Antibiogram and characterization of resistance markers among Escherichia coli Isolates from urinary tract infections.

INTRODUCTION: Urinary tract infection is the most frequently diagnosed kidney and urologic disease, and Escherichia coli is by far its most common etiological agent. Uropathogenic E. coli are responsible for approximately 90% of urinary tract infections seen in individuals with ordinary anatomy therefore, it is essential to review the antibiogram of uropathogenic E. coli periodically to help clinicians decide on the appropriate therapy. METHODOLOGY: We evaluated E. coli isolated from urinary tract infections at the National Salmonella and Escherichia Centre for antibiogram, plasmid transferability and stability of resistance markers. RESULTS: In total, 90.9% of the isolates were found to be sensitive to nitrofurantoin while the highest proportion of the isolates was found to be resistant to nalidixic acid. Minimum inhibitory concentrations of all antimicrobials for different isolates were well within the limits specified by the Clinical and Laboratory Standards Institute.  Resistance against tetracycline was not transferred either by conjugation and transformation. Streptomycin resistance was found to be lost in the maximum number of tested isolates showing loss at the 10th, 15th and 20th passages. CONCLUSION: Changing trends in antibiotic resistance necessitates the periodic generation of antibiogram data to help health authorities revise treatment strategies for urinary tract infections caused by E. coli.

Antibiogram Profile of Salmonella enterica Serovar Typhi in India - A Two Year Study.

Typhoid fever continues to remain a major health problem in the developing world, and the emergence of multidrug-resistant (MDR) strains has further reduced therapeutic options for treatment of the disease. The National Salmonella and Escherichia Centre in Kasauli, India received 128 Salmonella Typhi isolates during 2008-2009. These were evaluated for antimicrobial resistance, prevalent resistotypes and the proportion of MDR strains, using standard methods for 11 antimicrobials. An abrupt decrease in the proportion of MDR strains was observed. Only 4.7% of the isolates were found to be MDR with resistotypes chloramphenicol-ampicillin-streptomycin-nalidixic acid-trimethoprim (C-AS-Na-Tr) and chloramphenicol-ampicillin-nalidixic acid-trimethoprim (C-A-Na-Tr), which is very low compared to other studies from India. Nalidixic acid resistance was found to be present in 93.8% of the isolates. Moreover, the difference in the mean minimum inhibitory concentration (MIC) of ciprofloxacin for nalidixic acid-resistant and nalidixic acid-sensitive strains was found to be statistically significant (p<0.001), which calls into question the further use of ciprofloxacin for the treatment of typhoid fever because of potential treatment failures. The low proportion of MDR strains increases the possibility of first-line drugs for the treatment of typhoid fever.

Re-emergence of susceptibility to conventionally used drugs among strains of Salmonella Typhi in central west India.

INTRODUCTION: Typhoid fever (enteric fever) is a global health problem causing high morbidity and mortality, especially in endemic areas such as India. The problem is exacerbated as the causative agent, Salmonella enterica subspecies enterica serovar Typhi (S. Typhi), rapidly develops resistance to drugs used in treatment. However, non-responsiveness of S. Typhi to quinolones has been reported simultaneously with the re-emergence of susceptibility to chloramphenicol. The present study investigates the re-emergence of sensitivity to conventionally used drugs among strains of S. Typhi in the central west part of India. METHODOLOGY: We evaluated 128 S. Typhi isolates received at the National Salmonella and Escherichia Centre for chloramphenicol, ampicillin and trimethoprim susceptibility using standard methods. Minimum inhibitory concentrations were also evaluated. RESULTS: The proportion of S. Typhi isolates showing susceptibility to chloramphenicol, ampicillin, and trimethoprim was 95.3%, 94.5%, and 94.5%, respectively. These findings may help the health authorities in reconsidering the addition of these antimicrobial drugs into the treatment regime of typhoid fever and therefore may help combat the problem of increasing resistance to quinolones and cephalosporins. CONCLUSION: The changing trends of S. Typhi resistance patterns necessitate reconsideration of conventionally used drugs in typhoid fever treatment in India. In the present study, S. Typhi strains exhibited increased susceptibility toward chloramphenicol, ampicillin and trimethoprim, therefore suggesting the possibility of their use for treatment of typhoid fever. 

High level of resistance to nalidixic acid in Salmonella enterica serovar Typhi in Central India.

BACKGROUND: Fluoroquinolones are the drugs of choice for the treatment of typhoid fever. But the recent increase in minimum inhibitory concentration (MIC) values of ciprofloxacin in Salmonella Typhi may result in delayed response and serious complications. Nalidixic acid resistance has been used as an indirect evidence of increased minimum inhibitory concentration for ciprofloxacin in Salmonella Typhi. METHODS: We evaluated the isolates received at the National Salmonella and Escherichia Centre for nalidixic acid and ciprofloxacin susceptibility using standard methods. Minimum inhibitory concentrations have also been evaluated. RESULTS: Ninety-six percent of the isolates were found to be nalidixic acid resistant while all isolates were found to be ciprofloxacin sensitive. The difference between minimum inhibitory concentration values of ciprofloxacin for nalidixic acid resistant and nalidixic acid sensitive isolates was found to be statistically significant. CONCLUSION: The study may be helpful in revising treatment strategies for the infections caused by nalidixic acid resistant Salmonella Typhi in the country.

Purification, potency and immunogenicity analysis of Vero cell culture-derived rabies vaccine: a comparative study of single-step column chromatography and zonal centrifuge purification.

Continuous Vero cell lines are more suitable for large-scale production of rabies vaccine. The purification of Vero cell-derived rabies vaccine is critical because of the residual cellular DNA and serum proteins. The perfection of techniques using column chromatography with different matrix material, gel filtration and zonal centrifugation is of paramount importance for the optimal purification of rabies vaccine, leaving minimal residual cellular DNA, below the permissible level of 100 pg per dose and serum protein content of 1 ppm. In this study the potency, immunogenicity and safety of Vero cell-derived rabies vaccines were compared following purification by densely or loosely packed DEAE-sepharose CL-6B columns with different bed heights or by zonal centrifugation. The optimal virus recovery and maximum removal of substrate DNA and serum proteins were achieved only when the sepharose CL-6B column bed height was maintained at a thickness of 2-2.5 cm. The rabies virus material was purified by layering over the matrix without applying pressure. DEAE-sepharose CL-6B column purification using a simplified, cost effective technique as described in this study enhances the antigen yield by 50% in comparison with zonal purification.

Process standardization for optimal virus recovery and removal of substrate DNA and bovine serum proteins in Vero cell-derived rabies vaccine.

Purification of a rabies vaccine by a single zonal centrifugation run was replaced by two runs with optimal standardization of the sucrose density gradient. As a result, significant reductions in the levels of substrate DNA and bovine serum protein in the Vero cell-derived human rabies vaccine were achieved. Following many trials, for the first run, loading of the 3.2-l capacity K-3 rotor with 1800 ml of 60% sucrose solution and 1400 ml of vaccine PBS buffer solution gave a satisfactory linear gradient. However, after the first run, the substrate DNA and bovine serum contents exceeded the required levels. After protamine sulphate and Tween-80 treatment of the concentrated inactivated material, a second run using the same procedure as in the first run was tried. However, these purification procedures resulted in low virus recovery. To achieve optimal virus recovery, and removal of substrate DNA and bovine serum protein, the peak fractions from the first run as indicated by the haemagglutination, sucrose concentration, and optical density values were pooled and the sucrose concentration of the pooled fractions was increased to 60%. A second (flotation) run was then carried out. Using this method, the virus recovery rate was more than 95% that of the first run, and the levels of cellular DNA and bovine serum protein were well within the acceptable limits of less than 100 pg/dose and one part per million, respectively. The substrate DNA was quantified by both radioactive labeling and non-radioactive biotin labeling methods. For the quantification of calf serum protein, a counter-immunoelectrophoresis method was developed and effectively applied. A potency assay was performed using the National Institutes of Health (NIH) and well-standardized in vitro single radial immuno diffusion (SRD) methods. Finally, an immunogenicity study was conducted with human volunteers and the results were confirmed by a rapid fluorescent focus inhibition test (RFFIT).