NanoNose Detector Turns Epidemic Intelligence into a Predictive Science
CHAPEL HILL, N.C.--(BUSINESS WIRE)--QualSec (OTCBB: QLSCB):
In 1918, a strain of influenza, popularly known as the Spanish flu, ravaged the globe, killing an estimated 2.5% of the world's population. Now, concerns about new pandemics have led the global public health community to seek early warning systems to identify disease "hot spots" and enable rapid responses to prevent epidemics. The technological breakthrough that epidemiologists are looking for exists with QualSec’s NanoNose.
As environmental changes worldwide bring people in greater contact with animals, zoonoses - diseases that can transfer between humans and animals – are increasingly emerging as threats, including diseases like avian influenza, hantavirus, Ebola and West Nile viruses. QualSec’s NanoNose uses patented breakthroughs in nanotechnology for rapid data collection, allowing health officials better disease surveillance and vulnerability mapping.
This electronic nose technology developed by QualSec will be able to rapidly identify "hot spots" that could become epidemic nodes. Rapid detection is crucial for effective epidemic intelligence.
QualSec’s NanoNose is highly portable and rugged, and can be easily deployed to remote areas and used by operators with minimal training to detect epidemics before they spread. Though the sensor is designed to process data individually, it can be linked into a wireless network and transmit findings of disease to a central response center, where global health organizations can react to disease outbreaks.
For further details on the company and its products please visit the company's website at www.qualsensors.com.
QualSec believes that this press release contains forward-looking statements as that term is defined by the Private Securities Litigation Reform Act of 1995. Such forward-looking statements are subject to risks and uncertainties. Such statements are based on management's current expectations and are subject to facts that could cause results to differ materially from the forward-looking statements. For further information you are encouraged to view QualSec's filings with the Securities and Exchange Commission, including its annual report on Form 10K for the period ending December 31, 2007. The Company assumes no obligation to update the information contained in this press release.
Saturday, September 27, 2008
Tuesday, August 26, 2008
Changing Pattern of Clostridium Difficile Associated Diarrhoea in a Tertiary Care Hospital: A 5 Year Retrospective Study
Changing Pattern of Clostridium Difficile Associated Diarrhoea in a Tertiary Care Hospital: A 5 Year Retrospective Study
Posted on: Tuesday, 26 August 2008, 03:00 CDT
By Chaudhry, Rama Joshy, Lovely; Kumar, Lalit; Dhawan, Benu
Background & objectives: Frequent use of broad spectrum antibiotics in hospitalized patients has increased the incidence of Clostridium difficile diarrhoea in recent years. In our tertiary care hospital in north India, C. difficile was responsible for 15 per cent of cases of nosocomial diarrhoea in 1999. A retrospective study was carried out to determine the frequency of C. difficile associated diarrhoea (CdAD) in our hospital, and to assess the effect of awareness among the hospital personnel and control measures taken to present C. difficile infection following the previous report. Methods: A retrospective chart review of all suspected cases of CdAD diagnosed at the hospital from January 2001 to December 2005 was done. Clinical specimens comprised 524 stool samples. All the samples were analyzed for C. difficile using culture and ELISA for toxin A and B. Attempts were made to type isolates using antibiogram, SDS-PAGE, gas liquid chromatography (GLC), PCR for toxin A and B gene fragments and restriction fragment length polymorphism (RFLP).
Results: A total of 37 (7.1%) specimens were positive for C. difficile toxin (11.2% in 2001, 9.4% in 2002, 8.6% in 2003, 5% in 2004 and 4% in 2005). The highest number of C. difficile toxin positive cases were from stool samples of patients hospitalized in the haematology/oncology ward (67.5% of all positive cases) followed by gastrointestinal surgery, neurology and nephrology wards. Of the C. difficile toxin positive samples, 15 (41%) were also positive for C. difficile culture. The isolates were grouped in to one, 3 and 5 groups using antibiogram, SDS-PAGE and PCR RFLP respectively. We observed an increase in the number of stool specimens tested for C. difficile infection but a decrease in C. difficile positives.
Interpretation & conclusions: A decrease in the number of C. difficile positive cases were noted during the 5 year study period though number of samples tested was increased. This may be due to stringent surveillance and an improved antibiotic policy followed in the hospital.
Diarrhoea is one of the most frequent side effects of antibiotic treatment. The symptoms may vary from slight abdominal discomfort to severe diarrhoea to colitis1. The aetiology of antibiotic associated diarrhoea (AAD) varies. The disruption of normal enteric flora caused by antibiotics may lead to overgrowth of pathogens and functional disturbances of the intestinal carbohydrate and bile acid metabolism, resulting in osmotic diarrhoea1. Allergic, toxic and pharmacological effects of antibiotics may also affect the intestinal mucosa and motility2. Cytotoxin producing Clostridium difficile has been reported to be the causative agent of approximately 20 per cent of AAD and of nearly all cases of pseudomembraneous colitis, the most severe manifestation of AAD1. Because of the frequent use of broad spectrum antibiotics, the incidence of C. difficile diarrhoea has risen dramatically in recent years3,4. Established guidelines should be followed to minimize exposure to the pathogen which include judicious use of antibiotics, rapid detection of C. difficile by immunoassays for toxin A and B, isolation of patients who had C. difficile associated diarrhoea (CdAD), proper disinfection of objects and education of staff members5. In our hospital which is a tertiary care hospital in north India, C. difficile was responsible for 15 per cent of cases of nosocomial diarrhoea in 1999(6). Standard control measures were implemented in our hospital to minimize the spread of this nosocomial pathogen after this report. This retrospective analysis was carried out in continuation of our earlier study6 to determine the effect of awareness and control measures taken to contain C. difficile infection in our hospital during the subsequent years.
Material & Methods
The study comprised retrospective analysis of faecal specimens from 524 patients suspected on clinical grounds to have CdAD2. The patients were hospitalized in All India Institute of Medical Sciences, New Delhi, India, over a period of 5 yr (January 1, 2001 - December 31, 2005). These included 80 patients in 2001, 96 in 2002, 92 in 2003, 106 in 2004 and 150 patients in 2005 respectively. Of these, 53 per cent were males and 82.4 per cent were in all age group > 12-60 yr (Table I).
Clinical information about the cause of diarrhoea underlying disease and antimicrobial therapy was obtained by reviewing the patient charts. A patient was considered to have CdAD if AAD was present and a stool specimen was positive in a toxin dependent C. difficile assay.
Sample collection and isolation of C. difficile: All the stool specimens were processed immediately for culture of C. difficile and stool aliquots were stored at -20[degrees]C for <72 h till they were tested for C. difficile toxin A and B. Spore selection was performed using 95 per cent ethanol and culture for C. difficile was done on cycloserine cefoxitin fructose agar (CCFA) and brain heart infusion agar (BHIA) as described elsewhere6. Concurrently, a loopful of stool specimen was inoculated into Robertsons cooked meat broth and incubated at 37[degrees]C for 48 h.
The plates were incubated anaerobically at 37[degrees]C in an anaerobic jar for 48 h. After incubation, the plates were examined and colonies which resembled C. difficile were Gram stained and identified by biochemical reactions using standard methods7.
When culture plate were negative for C. difficile, subcultures were made from cooked meat broth onto CCFA and BHIA and incubated anaerobically at 37[degrees]C up to 5 days before being discarded as negative.
Enzyme immunoassay for toxin A and B: Detection of enterotoxin and cytotoxin (toxin A and toxin B) of C. difficile was performed on the stool specimens by a double sandwich enzyme-linked immunosorbent assay technique using a commercial kit (Premier toxins A & B; Meridian Diagnostics, Inc., Cincinnati, Ohio, USA). The assay was performed according to the manufacturer's instructions.
Characterization of C. difficile isolates: All the C. difficile isolates were characterized phenotypically using antibiogram, SDS- PAGE6, gas liquid chromatography (GLC)7, and genotypically using PCR for toxin A gene and RFLP8,9.
Antibiogram typing - Antibiogram patterns were determined by disc diffusion method10. The antibiotics tested were chloramphenicol (30 [mu]g), penicillin G (10 units), clindamycin (2 [mu]g), vancomycin (5 [mu]g); metronidazole (5 [mu]g), tetracycline (30 [mu]g) and erythromycin (10 [mu]g). The results were expressed as susceptible or resistant.
Analysis of volatile fatty acids by gas liquid chromatography (GLC): All isolates were inoculated to cooked meat broth and incubated anaerobically for 48 h or more for GLC analysis to detect volatile fatty acids produced as metabolic end products. 1 ml of RCM broth was acidified with 0.2 ml of 50 per cent sulphuric acid and extracted with 1 ml of diethyl ether. The mixture was shaken vigorously and centrifuged at 176 g for 3 min; 1.5 [mu]l of the extracted ether layer was injected to the injection port of preconditioned GLC column with a 10 [mu]l Hamilton syringe. Chromatography was performed on a Nucon Series 5700, fitted with a flame ionization detector (FID)7. Operating conditions were as follows: carrier gas (oxygen free nitrogen): 60 ml/min oven temperature: detector 240[degrees]C, column 175[degrees]C, injector 240[degrees]C, attenuation 4X, sensitivity of the detector was set at 1000X. Fatty acids were identified by comparing the retention times of peaks in the test samples with those of known standard solutions which were examined each day7.
PCR assay for toxin gene fragments: The presence of toxin A gene in all isolates of C. difficile was determined by specific PCR using published primers8. PCR to detect the toxin B gene was performed in the C. difficile isolates using primers that had been developed and validated by Gumerlock et at9 to yield a 399-bp fragment for toxin B gene. PCR was performed in a 25 [mu]l reaction volume. Each reaction tube contained 1 X buffer (10 mm Tris HCl, pH 8.3, 50 mm KCl, 2.5 mm MgCl^sub 2^, 0.001% gelatin), each deoxynuclotide at a concentration of 100 mm (MBI, Fermentas, USA), each primer at a concentration of 20 pmol, 1.25 U Taq polymerase (MBI, Fermantas, USA) and 10 [mu]l of DNA. PCR was performed for 2 min at 95[degrees]C followed by 30 cycles of 1 min of denaturation at 95[degrees]C, 1 min of annealing at 52[degrees]C and 1 min of extension at 72[degrees]C. After the 30th cycle, extension was continued for an additional 10 min. 10 [mu]l of the amplified product was analyzed in 1 per cent agarose gel stained with ethidium bromide.
PCR-restriction fragment length polymorphism (RFLP) analysis: The amplified toxin A gene fragment was then digested with Alu I(10 units) restriction enzyme, under conditions recommended by the supplier (MBI-Fermentas). These digests were then subjected to electrophoresis on 2.5 per cent agarose gel at 60 V, along side a PCR size marker (100 bps, Sigma, USA).
Comparisons of patterns were performed visually. Strains with patterns differing alteast by one band were assigned to different types.
Results
A total of 524 stool specimens were analyzed for C. difficile from suspected cases of CdAD. The maximum number of C. difficile suspected cases were from oncology ward (378 cases, 72%), followed by other wards such as gastrointestinal surgery, neurology, nephrology and other medical wards. A total of 95 per cent of the analyzed group were on multiple antibiotics which included, 65 per cent on cephalosporins, 35 per cent on quinolones, 43 per cent on aminoglycosides, 12 per cent on macrolides, 69 per cent in vancomycin and metronidazole. Of the analyzed group, 37 (7.1%) patients were positive for C. difficile infection by the toxin dependent assay. Of these, 9 samples (11.2%) were positive in 2001, 9 (9.4%) in 2002, 8 (8.6%) in 2003, 5 (5%) in 2004 and 6 patients (4%) in 2005 (Fig. 1). Fifteen (41%) of the 37 toxin positive stool samples were also positive for C. difficile by culture. Eight of the 37 toxin positive cases expired, the cause of death was not directly related to C. difficile diarrhoea, although this might have been a contributory factor. Other pathogenic clostridia isolated from the patient group included C. perfringens (2.5%).
The highest number of C. difficile toxin positive cases were from stool samples of patients hospitalized in the haematology/oncology ward (25 samples, 67.5% of all positive cases), followed by gastrointestinal surgery, neurology and nephrology wards. Recovery rates of C. difficile in patient populations surveyed and summarized in Table II.
Of the 37 positive cases, 19 (51%) were males; 32 patients (86%) experienced diarrhoea during antibiotic treatment or within 15 days after the start of antibiotic treatment. The median time of occurrence of symptoms was 7 days (ranges 0-16 days) after start of antibiotic treatment and 8 days after admittance to hospital. All the patients were on multiple drugs and 50 per cent of the positive cases were on 3rd generation cephalosporins. None of the positive cases was on clindamycin. C. difficile positive cases were treated with metronidazole or vancomycin.
Antibiogram grouped all 15 isolates together as all were sensitive to erythromycin, chloramphenicol, penicillin, tetracyclin, clindamycin, vancomycin and metronidazole.
The identical fatty acid producers were grouped into 2 groups based on the production of isocaproic acid. Except one, all the isolates were producing isocaproic acid.
Based on the protein profiles observed on SDSPAGE, the isolates were placed into 3 groups; 12 isolates in group A, 2 in group B and 1 in group C.
PCR and RFLP analysis: All the ...lates were positive for toxin A (1.2 kb fragment) ... (399 bp) gene by PCR. Five different restriction profiles were obtained using Alu I endonucleases. The isolates were classified into five RFLP groups. The most frequent RFLP type was group I (6 isolates) group II, III, IV and V had 5, 2, 1 and 1 isolates respectively.
Discussion
C. difficile is considered as the most frequent aetiological agent of nosocomial diarrhoea occurring in hospitalized patients, spreading easily to the environment, the hands of health care workers and subsequently to other patients, particularly in large hospitals12. A trend of increasing prevalence of C. difficile has been reported in Europe and USA during the past 10 years13.
In our hospital C. difficile was found to be responsible for 15 per cent of the cases of nosocomial diarrhoea in our earlier study6. In this study, C. difficile was isolated mainly from patients in the haematology/ oncology wards. This points to the high risk areas for nosocomial spread of C. difficile isolates14. However, the percentage of infection showed a gradual decrease during the recent years.
Standard laboratory methods for diagnosing these infections include stool culture and identification of bacterial isolate, faecal toxin detection and C. difficile antigen detection. The culture lacks specificity due to the possible faecal carriage of non- toxigenic isolates, therefore many laboratories rely on toxin detection rather than culure for diagnosis of C. difficile infection15. A European survey of diagnostic methods for C. difficile, showed that culture of the organism is performed only in few countries. Mostly C. difficile toxin EIAs were used for diagnosis of CdAD16. In this study we used ELISA for toxin A, B and culture for diagnosing C. difficile infection. However, to the previous study6 we used C. difficile toxin A dependent ELISA for the analysis.
There was a gradual decrease in the percentage of ... infection during 2001 and 2005. The fact ... the 22 culture negative cases were on ... or vancomycin at the time of sample ... might be responsible for the decrease in isolation of organism as compared with the ELISA.
Older age, female gender and a prolonged hospital stay have been identified as risk factors in hospitalized CdAD patients17. In the current study, there was no gender prevalence among the positive cases and the median age of positive cases were 39 yr. However, highest percentage of culture positives was seen among patients >60 yr of age. Prolonged courses of antibiotic treatment have been related to an increased risk of AAD18,19. The median time for occurrence of symptoms was 7 days after the start of treatment in the present study, which was in accordance with other studies1,20. This suggests that disturbance of the normal colonic flora, eventually resulting in diarrhoea, takes place within about one week of antibiotic treatment. Prolonged duration of hospital stay has also been reported to be associated with AAD and CdAD19,21. In the present study, the median time of hospital stay was 8 days.
AAD was found to be frequently associated with cephalosporins, clindamycin and broad spectrum penicillins and quinolones22-25. In this study, about 50 per cent of our CdAD cases were on cephalosporins. However, since all the patients were on multiple antibiotics, the association with a particular group was not identified.
Discontinuation of antibiotic therapy withdraws the offending agents but is often not appropriate if the indication for such therapy was correct. An alternative is to change to antibiotics that do not belong to the high risk groups for induction of CdAD, such as quinolones, sulphonamides, parenteral aminoglycosides, cotrimoxazole, etc26. Metronidazole is suggested as the first line drug for the treatment of C. difficile infection2, and therefore the policy of the use of metronidazole in the treatment of suspected CdAD in our hospital is justified.
No nosocomial outbreak of C. difficile was reported during the study period. In this study we found antibiogram was least discriminatory of the typing strategies evaluated. The detection of short chain fatty acids by GLC is commonly utilized in bacteriological laboratories to identify anaerobes27. As all the isolates were positive for toxin A gene, we looked forward to analyze the variability of toxin A gene among C. difficile isolates by RFLP analysis. As the sequence analysis of the amplified 1.2 kbp toxin A gene fragment does not show any restriction sites for the previously reported restriction enzymes like Hinc II, Hind III, Ace I, EcoR I28, we decided to examine the amplified gene structure using restriction enzyme AIu I (5' AG[arrow down]CT 3', 3'TC[arrow up]GA 5'), which showed multiple restriction sites (8 sites) in the amplicon.
Although PCR-RFLP types 1 and II clustered some patient isolates, there was no epidemiological association between them. The locations where these patients were housed were different, and were admitted at different time periods. Better discriminatory methods such as pulsed field gel electrophoresis (PFGE) or ribotyping may be used to analyze the epidemiology of the pathogen.
In our recent prospective study, all the C. perfringens isolates were analyzed for the presence of enterotoxin by reverse passive later agglutination (RPLA), ELISA and by PCR assay for the presence of enterotoxin gene29. Of these, two were positive by PCR, RPLA and ELISA for C. perfringens enterotoxin. None of these samples had a co- infection with C. difficile.
Prevention of C. difficile infection is challenging. A change in antibiotic policy and implementation of standard infection control measures reduce the incidence of C. difficile symptomatic infections30,31. Combined approach, involving effective control measures, the use of rapid and sensitive techniques for laboratory diagnosis as well as prudent use of antibiotics, is necessary to reduce morbidity and mortality due to C. difficile associated infections in hospitalized patients.
In conclusion, we observed a decrease in the number of C. difficile toxin positive cases during the 5 yr of the study though there was an increase in the number of stool specimens tested per year for C. difficile. This possibly could be a result of stringent surveillance and antibiotic policy followed in our hospital especially in high risk areas such as haematology/oncology wards. Secondly, the use of clindamycin has been minimized in the hospital. Thirdly, antibiotics effective against C. difficile such as metronidazole have been included as the first line drugs in suspected CdAD cases. Isolation of the patients having C. difficile infection and regular awareness programmes conducted in the hospital might also have contributed.
Acknowledgment
Authors thank Ms. Sonam, Ms. Poornima and Shri Madho Prasad for technical assistance and acknowledge the Indian Council of Medical Research (ICMR), New Delhi, for financial suppport.
References
1. Wistrom J, Norrby SR, Myhre EB, Eriksson S, Granstron G, Legergrea, et al. Frequency of antibiotic-associated diarrhoea in 2462 antibiotic-treated hospitalized patients: a prospective study. J Antimicrob Chemother 2001; 47 : 43-50.
2. Hogenauer C, Hammer HF, Krejs GJ, Reisinger EC. Mechanisms and management of antibiotic-associated diarrhea. Clin Infect Dis 1998; 27 : 702-10.
3. Dallal RM, Harbrecht BG, Boujoukas AJ, Sirio CA, Farkas LM, Lee KK, et al. Fulminant Clostridium difficile: an underappreciated and increasing cause of death and complications. Ann Surg 2002; 235 : 363-72. 4. Kyne L, Hamel MB, Polavaram R, Kelly CP. Health care costs and mortality associated with nosocomial diarrhea due to Clostridium difficile. Clin Infect Dis 2002; 34 : 346-53.
5. Schroeder MS. Clostridium difficile-associated diarrhea. Am Fam Physician 2005; 71 : 921-8.
6. Dhawan B, Chaudhry R, Sharma N Incidence of Clostridium difficile infection: a prospective study in an Indian hospital. J Hosp Infect 1999; 43 : 275-80.
7. Sutter VL, Citron DM, Edelstein MAC, Finegold SM. Wadsworth Anaerobic Manual. Belmont, California: Star Publishers, 1985. p. 71- 5.
8. Kato N, Ou CY, Kato H, Bartley SL, Brown VK, Dowell VR Jr, et al. Identification of toxigenic Clostridium difficile by the polymerase chain reaction. J Clin Microbiol 1991; 29 : 33-7.
9. Gumerlock PH, Tang YJ, Weiss JB, Silva J Jr. Specific detection of toxigenic strains of Clostridium difficile in stool specimens. J Clin Microbiol 1993; 3 : 507-11.
10. Lambe DW Jr., Laslie WW. A standard disc diffusion method for antibiotic susceptability testing for anaerobes; four years antibiotic data. In: Lambe DW Jr, Gena RJ, Carson KJM, editors. Anaerobic bacteria-selected topics. New York: Plenum Press, 1980.
11. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage. Nature 1970; 227 : 680-5.
12. Wroblewska MM, Swoboda-Kopec E, Rokosz A, Nurzynska G, Bednarska A, Luczak M. Detection of Clostridium difficile and its toxin A (TcdA) in stool specimens from hospitalised patients. Pol J Microbiol 2005; 54 : 111-5.
13. Wongwanich S, Pongpech P, Dhiraputra C, Huttayananont S, Sawanpanyalert P. Characteristics of Clostridium difficile strains isolated from asymptomatic individuals and from diarrheal patients. Clin Microbiol Infect 2001; 7: 438-41.
14. Blot E, Escande MC, Besson D, Barbut F, Granpeix C, Asselain B, et al. Outbreak of C. difficile related diarrhea in an adult oncology unit: risk factors and microbiological characteristics. J Hosp Infect 1993; 53: 187-92.
15. Wilcox MH, Fawley WN, Settle CD, Davidson A. Recurrence of symptoms in Clostridium difficile infection-relapse or reinfection? J Hosp Infect 1998; 38 : 93-100.
16. Barbut F, Delmee M, Brazier JS, Petit JC, Poxton IR, Rupnik M, et al. ESCMID Study Group on Clostridium difficile (ESGCD). A European survey of diagnostic methods and testing protocols for Clostridium difficile. Clin Microbiol Infect 2003; 9 : 989-96.
17. Al-Eidan FA, McElnay JC, Scott MG, Kearney MP. Clostridium difficile-associated diarrhoea in hospitalised patients. J Clin Pharm Ther 2000; 25 : 101-9.
18. Spencer R. The role of antimicrobial agents in the aetiology of Clostridium difficile-associated disease. J Antimicrob Chemother 1998; 41 : 21-7.
19. Bignardi GE. Risk factors for Clostridium difficile infection. J Hosp Infect 1998; 40 : 1-15.
20. Thamilikitkul V, Danpakdi K, Chokloikaew S. Incidence of diarrhea and Clostridium difficile toxin in stools from hospitalized patients receiving Clindamycin, beta lactams, and nonantibiotic medications. J Clin Gastroenterol 1996; 22 : 161-3.
21. Clabots CR, Johnson S, Olson MM, Peterson LR, Gerding DN. Acquisition of Clostridium difficile by hospitalized patients: evidence for colonized new admissions as a source of infection. J Infect Dis 1992; 166 : 561-7.
22. Impallomeni M, Galletly NP, Wort SU, Starr JM, Rogers TR. Increased risk of diarrhea caused by Clostridium difficile in elderly patients receiving cefotaxime. BMJ 1995; 311 : 1345-6.
23. Pear SM, Williamson TH, Bettin KM, Gerding DN, Galgiani JN. Decrease in nosocomial Clostridium difficile-associated diarrhea by restricting clindamycin use. Ann Intern Med 1994; 720 : 272-7.
24. Pepin J, Saheb N, Coulombe MA, Alary ME, Carrivean MP, Authier S, et al. Emergence of fluoroquinolones as the predominant risk factor for Clostridium difficile-associated diarrhea: a cohort study during an epidemic in Quebec. Clin Infect Dis 2005; 41 : 1254- 60.
25. Muto CA, Pokrywka M, Shutt K, Mendelsohn AS, Nouri K, Posey K, et al. A large outbreak of Clostridium difficile-associated disease with an unexpected proportion of deaths and colectomies at a teaching hospital following increased fluoroquinolone use. Infect Control Hosp Epidemiol 2005; 26 : 273-80
26. Bartlett JG. Antibiotic associated diarrhea. Clin Infect Dis 1992; 15 : 573-81.
27. Pepersack F, Labbe M, Nonhoff C, Schoutens E. Use of gas- liquid chromatography as a screening test for toxigenic Clostridium difficile in diarrhoeal stools. J Clin Pathol 1983; 36 : 1233-6.
28. McFarland LV, Surawicz CM, Rubin M, Fekety R, Elmer GW, Greenberg RN. Recurrent Clostridium difficile disease: Epidemiology and clinical characteristics. Infect Control Hosp Epidemiol 1999; 20 : 43-50.
29. Joshy L, Chaudhry R, Dhawan B, Kumar L, Das BK. Incidence and characterization of Clostridium perfringens isolated from antibiotic- associated diarrhoeal patients: a prospective study in an Indian hospital. J Hosp Infect 2006; 63 : 323-9.
30. Khan R, Cheesbrough J. Impact of changes in antibiotic policy on Clostridium difficile-associated diarrhoea (CDAD) over a five- year period in a district general hospital. J Hosp infect 2003; 54 : 104-8.
31. Riley TV. Nosocomial diarrhoea due to Clostridium difficile. Curr Opin Infect Dis 2004; 17 : 323-7.
Rama Chaudhry, Lovely Joshy, Lalit Kumar* & Benu Dhawan
Departments of Microbiology, * Medical Oncology, Institute- Rotary Cancer Hospital
All India Institute of Medical Sciences, New Delhi, India
Received December 7, 2006
Reprint requests: Dr Rama Chaudhry, Professor, Department of Microbiology, All India Institute of Medical Sciences
New Delhi 110029, India
e-mail: drramach@rediffmail.com, ramach003@yahoo.com
Copyright Indian Council of Medical Research Apr 2008
(c) 2008 Indian Journal of Medical Research. Provided by ProQuest LLC. All rights Reserved.
Source: Indian Journal of Medical Research
Posted on: Tuesday, 26 August 2008, 03:00 CDT
By Chaudhry, Rama Joshy, Lovely; Kumar, Lalit; Dhawan, Benu
Background & objectives: Frequent use of broad spectrum antibiotics in hospitalized patients has increased the incidence of Clostridium difficile diarrhoea in recent years. In our tertiary care hospital in north India, C. difficile was responsible for 15 per cent of cases of nosocomial diarrhoea in 1999. A retrospective study was carried out to determine the frequency of C. difficile associated diarrhoea (CdAD) in our hospital, and to assess the effect of awareness among the hospital personnel and control measures taken to present C. difficile infection following the previous report. Methods: A retrospective chart review of all suspected cases of CdAD diagnosed at the hospital from January 2001 to December 2005 was done. Clinical specimens comprised 524 stool samples. All the samples were analyzed for C. difficile using culture and ELISA for toxin A and B. Attempts were made to type isolates using antibiogram, SDS-PAGE, gas liquid chromatography (GLC), PCR for toxin A and B gene fragments and restriction fragment length polymorphism (RFLP).
Results: A total of 37 (7.1%) specimens were positive for C. difficile toxin (11.2% in 2001, 9.4% in 2002, 8.6% in 2003, 5% in 2004 and 4% in 2005). The highest number of C. difficile toxin positive cases were from stool samples of patients hospitalized in the haematology/oncology ward (67.5% of all positive cases) followed by gastrointestinal surgery, neurology and nephrology wards. Of the C. difficile toxin positive samples, 15 (41%) were also positive for C. difficile culture. The isolates were grouped in to one, 3 and 5 groups using antibiogram, SDS-PAGE and PCR RFLP respectively. We observed an increase in the number of stool specimens tested for C. difficile infection but a decrease in C. difficile positives.
Interpretation & conclusions: A decrease in the number of C. difficile positive cases were noted during the 5 year study period though number of samples tested was increased. This may be due to stringent surveillance and an improved antibiotic policy followed in the hospital.
Diarrhoea is one of the most frequent side effects of antibiotic treatment. The symptoms may vary from slight abdominal discomfort to severe diarrhoea to colitis1. The aetiology of antibiotic associated diarrhoea (AAD) varies. The disruption of normal enteric flora caused by antibiotics may lead to overgrowth of pathogens and functional disturbances of the intestinal carbohydrate and bile acid metabolism, resulting in osmotic diarrhoea1. Allergic, toxic and pharmacological effects of antibiotics may also affect the intestinal mucosa and motility2. Cytotoxin producing Clostridium difficile has been reported to be the causative agent of approximately 20 per cent of AAD and of nearly all cases of pseudomembraneous colitis, the most severe manifestation of AAD1. Because of the frequent use of broad spectrum antibiotics, the incidence of C. difficile diarrhoea has risen dramatically in recent years3,4. Established guidelines should be followed to minimize exposure to the pathogen which include judicious use of antibiotics, rapid detection of C. difficile by immunoassays for toxin A and B, isolation of patients who had C. difficile associated diarrhoea (CdAD), proper disinfection of objects and education of staff members5. In our hospital which is a tertiary care hospital in north India, C. difficile was responsible for 15 per cent of cases of nosocomial diarrhoea in 1999(6). Standard control measures were implemented in our hospital to minimize the spread of this nosocomial pathogen after this report. This retrospective analysis was carried out in continuation of our earlier study6 to determine the effect of awareness and control measures taken to contain C. difficile infection in our hospital during the subsequent years.
Material & Methods
The study comprised retrospective analysis of faecal specimens from 524 patients suspected on clinical grounds to have CdAD2. The patients were hospitalized in All India Institute of Medical Sciences, New Delhi, India, over a period of 5 yr (January 1, 2001 - December 31, 2005). These included 80 patients in 2001, 96 in 2002, 92 in 2003, 106 in 2004 and 150 patients in 2005 respectively. Of these, 53 per cent were males and 82.4 per cent were in all age group > 12-60 yr (Table I).
Clinical information about the cause of diarrhoea underlying disease and antimicrobial therapy was obtained by reviewing the patient charts. A patient was considered to have CdAD if AAD was present and a stool specimen was positive in a toxin dependent C. difficile assay.
Sample collection and isolation of C. difficile: All the stool specimens were processed immediately for culture of C. difficile and stool aliquots were stored at -20[degrees]C for <72 h till they were tested for C. difficile toxin A and B. Spore selection was performed using 95 per cent ethanol and culture for C. difficile was done on cycloserine cefoxitin fructose agar (CCFA) and brain heart infusion agar (BHIA) as described elsewhere6. Concurrently, a loopful of stool specimen was inoculated into Robertsons cooked meat broth and incubated at 37[degrees]C for 48 h.
The plates were incubated anaerobically at 37[degrees]C in an anaerobic jar for 48 h. After incubation, the plates were examined and colonies which resembled C. difficile were Gram stained and identified by biochemical reactions using standard methods7.
When culture plate were negative for C. difficile, subcultures were made from cooked meat broth onto CCFA and BHIA and incubated anaerobically at 37[degrees]C up to 5 days before being discarded as negative.
Enzyme immunoassay for toxin A and B: Detection of enterotoxin and cytotoxin (toxin A and toxin B) of C. difficile was performed on the stool specimens by a double sandwich enzyme-linked immunosorbent assay technique using a commercial kit (Premier toxins A & B; Meridian Diagnostics, Inc., Cincinnati, Ohio, USA). The assay was performed according to the manufacturer's instructions.
Characterization of C. difficile isolates: All the C. difficile isolates were characterized phenotypically using antibiogram, SDS- PAGE6, gas liquid chromatography (GLC)7, and genotypically using PCR for toxin A gene and RFLP8,9.
Antibiogram typing - Antibiogram patterns were determined by disc diffusion method10. The antibiotics tested were chloramphenicol (30 [mu]g), penicillin G (10 units), clindamycin (2 [mu]g), vancomycin (5 [mu]g); metronidazole (5 [mu]g), tetracycline (30 [mu]g) and erythromycin (10 [mu]g). The results were expressed as susceptible or resistant.
Analysis of volatile fatty acids by gas liquid chromatography (GLC): All isolates were inoculated to cooked meat broth and incubated anaerobically for 48 h or more for GLC analysis to detect volatile fatty acids produced as metabolic end products. 1 ml of RCM broth was acidified with 0.2 ml of 50 per cent sulphuric acid and extracted with 1 ml of diethyl ether. The mixture was shaken vigorously and centrifuged at 176 g for 3 min; 1.5 [mu]l of the extracted ether layer was injected to the injection port of preconditioned GLC column with a 10 [mu]l Hamilton syringe. Chromatography was performed on a Nucon Series 5700, fitted with a flame ionization detector (FID)7. Operating conditions were as follows: carrier gas (oxygen free nitrogen): 60 ml/min oven temperature: detector 240[degrees]C, column 175[degrees]C, injector 240[degrees]C, attenuation 4X, sensitivity of the detector was set at 1000X. Fatty acids were identified by comparing the retention times of peaks in the test samples with those of known standard solutions which were examined each day7.
PCR assay for toxin gene fragments: The presence of toxin A gene in all isolates of C. difficile was determined by specific PCR using published primers8. PCR to detect the toxin B gene was performed in the C. difficile isolates using primers that had been developed and validated by Gumerlock et at9 to yield a 399-bp fragment for toxin B gene. PCR was performed in a 25 [mu]l reaction volume. Each reaction tube contained 1 X buffer (10 mm Tris HCl, pH 8.3, 50 mm KCl, 2.5 mm MgCl^sub 2^, 0.001% gelatin), each deoxynuclotide at a concentration of 100 mm (MBI, Fermentas, USA), each primer at a concentration of 20 pmol, 1.25 U Taq polymerase (MBI, Fermantas, USA) and 10 [mu]l of DNA. PCR was performed for 2 min at 95[degrees]C followed by 30 cycles of 1 min of denaturation at 95[degrees]C, 1 min of annealing at 52[degrees]C and 1 min of extension at 72[degrees]C. After the 30th cycle, extension was continued for an additional 10 min. 10 [mu]l of the amplified product was analyzed in 1 per cent agarose gel stained with ethidium bromide.
PCR-restriction fragment length polymorphism (RFLP) analysis: The amplified toxin A gene fragment was then digested with Alu I(10 units) restriction enzyme, under conditions recommended by the supplier (MBI-Fermentas). These digests were then subjected to electrophoresis on 2.5 per cent agarose gel at 60 V, along side a PCR size marker (100 bps, Sigma, USA).
Comparisons of patterns were performed visually. Strains with patterns differing alteast by one band were assigned to different types.
Results
A total of 524 stool specimens were analyzed for C. difficile from suspected cases of CdAD. The maximum number of C. difficile suspected cases were from oncology ward (378 cases, 72%), followed by other wards such as gastrointestinal surgery, neurology, nephrology and other medical wards. A total of 95 per cent of the analyzed group were on multiple antibiotics which included, 65 per cent on cephalosporins, 35 per cent on quinolones, 43 per cent on aminoglycosides, 12 per cent on macrolides, 69 per cent in vancomycin and metronidazole. Of the analyzed group, 37 (7.1%) patients were positive for C. difficile infection by the toxin dependent assay. Of these, 9 samples (11.2%) were positive in 2001, 9 (9.4%) in 2002, 8 (8.6%) in 2003, 5 (5%) in 2004 and 6 patients (4%) in 2005 (Fig. 1). Fifteen (41%) of the 37 toxin positive stool samples were also positive for C. difficile by culture. Eight of the 37 toxin positive cases expired, the cause of death was not directly related to C. difficile diarrhoea, although this might have been a contributory factor. Other pathogenic clostridia isolated from the patient group included C. perfringens (2.5%).
The highest number of C. difficile toxin positive cases were from stool samples of patients hospitalized in the haematology/oncology ward (25 samples, 67.5% of all positive cases), followed by gastrointestinal surgery, neurology and nephrology wards. Recovery rates of C. difficile in patient populations surveyed and summarized in Table II.
Of the 37 positive cases, 19 (51%) were males; 32 patients (86%) experienced diarrhoea during antibiotic treatment or within 15 days after the start of antibiotic treatment. The median time of occurrence of symptoms was 7 days (ranges 0-16 days) after start of antibiotic treatment and 8 days after admittance to hospital. All the patients were on multiple drugs and 50 per cent of the positive cases were on 3rd generation cephalosporins. None of the positive cases was on clindamycin. C. difficile positive cases were treated with metronidazole or vancomycin.
Antibiogram grouped all 15 isolates together as all were sensitive to erythromycin, chloramphenicol, penicillin, tetracyclin, clindamycin, vancomycin and metronidazole.
The identical fatty acid producers were grouped into 2 groups based on the production of isocaproic acid. Except one, all the isolates were producing isocaproic acid.
Based on the protein profiles observed on SDSPAGE, the isolates were placed into 3 groups; 12 isolates in group A, 2 in group B and 1 in group C.
PCR and RFLP analysis: All the ...lates were positive for toxin A (1.2 kb fragment) ... (399 bp) gene by PCR. Five different restriction profiles were obtained using Alu I endonucleases. The isolates were classified into five RFLP groups. The most frequent RFLP type was group I (6 isolates) group II, III, IV and V had 5, 2, 1 and 1 isolates respectively.
Discussion
C. difficile is considered as the most frequent aetiological agent of nosocomial diarrhoea occurring in hospitalized patients, spreading easily to the environment, the hands of health care workers and subsequently to other patients, particularly in large hospitals12. A trend of increasing prevalence of C. difficile has been reported in Europe and USA during the past 10 years13.
In our hospital C. difficile was found to be responsible for 15 per cent of the cases of nosocomial diarrhoea in our earlier study6. In this study, C. difficile was isolated mainly from patients in the haematology/ oncology wards. This points to the high risk areas for nosocomial spread of C. difficile isolates14. However, the percentage of infection showed a gradual decrease during the recent years.
Standard laboratory methods for diagnosing these infections include stool culture and identification of bacterial isolate, faecal toxin detection and C. difficile antigen detection. The culture lacks specificity due to the possible faecal carriage of non- toxigenic isolates, therefore many laboratories rely on toxin detection rather than culure for diagnosis of C. difficile infection15. A European survey of diagnostic methods for C. difficile, showed that culture of the organism is performed only in few countries. Mostly C. difficile toxin EIAs were used for diagnosis of CdAD16. In this study we used ELISA for toxin A, B and culture for diagnosing C. difficile infection. However, to the previous study6 we used C. difficile toxin A dependent ELISA for the analysis.
There was a gradual decrease in the percentage of ... infection during 2001 and 2005. The fact ... the 22 culture negative cases were on ... or vancomycin at the time of sample ... might be responsible for the decrease in isolation of organism as compared with the ELISA.
Older age, female gender and a prolonged hospital stay have been identified as risk factors in hospitalized CdAD patients17. In the current study, there was no gender prevalence among the positive cases and the median age of positive cases were 39 yr. However, highest percentage of culture positives was seen among patients >60 yr of age. Prolonged courses of antibiotic treatment have been related to an increased risk of AAD18,19. The median time for occurrence of symptoms was 7 days after the start of treatment in the present study, which was in accordance with other studies1,20. This suggests that disturbance of the normal colonic flora, eventually resulting in diarrhoea, takes place within about one week of antibiotic treatment. Prolonged duration of hospital stay has also been reported to be associated with AAD and CdAD19,21. In the present study, the median time of hospital stay was 8 days.
AAD was found to be frequently associated with cephalosporins, clindamycin and broad spectrum penicillins and quinolones22-25. In this study, about 50 per cent of our CdAD cases were on cephalosporins. However, since all the patients were on multiple antibiotics, the association with a particular group was not identified.
Discontinuation of antibiotic therapy withdraws the offending agents but is often not appropriate if the indication for such therapy was correct. An alternative is to change to antibiotics that do not belong to the high risk groups for induction of CdAD, such as quinolones, sulphonamides, parenteral aminoglycosides, cotrimoxazole, etc26. Metronidazole is suggested as the first line drug for the treatment of C. difficile infection2, and therefore the policy of the use of metronidazole in the treatment of suspected CdAD in our hospital is justified.
No nosocomial outbreak of C. difficile was reported during the study period. In this study we found antibiogram was least discriminatory of the typing strategies evaluated. The detection of short chain fatty acids by GLC is commonly utilized in bacteriological laboratories to identify anaerobes27. As all the isolates were positive for toxin A gene, we looked forward to analyze the variability of toxin A gene among C. difficile isolates by RFLP analysis. As the sequence analysis of the amplified 1.2 kbp toxin A gene fragment does not show any restriction sites for the previously reported restriction enzymes like Hinc II, Hind III, Ace I, EcoR I28, we decided to examine the amplified gene structure using restriction enzyme AIu I (5' AG[arrow down]CT 3', 3'TC[arrow up]GA 5'), which showed multiple restriction sites (8 sites) in the amplicon.
Although PCR-RFLP types 1 and II clustered some patient isolates, there was no epidemiological association between them. The locations where these patients were housed were different, and were admitted at different time periods. Better discriminatory methods such as pulsed field gel electrophoresis (PFGE) or ribotyping may be used to analyze the epidemiology of the pathogen.
In our recent prospective study, all the C. perfringens isolates were analyzed for the presence of enterotoxin by reverse passive later agglutination (RPLA), ELISA and by PCR assay for the presence of enterotoxin gene29. Of these, two were positive by PCR, RPLA and ELISA for C. perfringens enterotoxin. None of these samples had a co- infection with C. difficile.
Prevention of C. difficile infection is challenging. A change in antibiotic policy and implementation of standard infection control measures reduce the incidence of C. difficile symptomatic infections30,31. Combined approach, involving effective control measures, the use of rapid and sensitive techniques for laboratory diagnosis as well as prudent use of antibiotics, is necessary to reduce morbidity and mortality due to C. difficile associated infections in hospitalized patients.
In conclusion, we observed a decrease in the number of C. difficile toxin positive cases during the 5 yr of the study though there was an increase in the number of stool specimens tested per year for C. difficile. This possibly could be a result of stringent surveillance and antibiotic policy followed in our hospital especially in high risk areas such as haematology/oncology wards. Secondly, the use of clindamycin has been minimized in the hospital. Thirdly, antibiotics effective against C. difficile such as metronidazole have been included as the first line drugs in suspected CdAD cases. Isolation of the patients having C. difficile infection and regular awareness programmes conducted in the hospital might also have contributed.
Acknowledgment
Authors thank Ms. Sonam, Ms. Poornima and Shri Madho Prasad for technical assistance and acknowledge the Indian Council of Medical Research (ICMR), New Delhi, for financial suppport.
References
1. Wistrom J, Norrby SR, Myhre EB, Eriksson S, Granstron G, Legergrea, et al. Frequency of antibiotic-associated diarrhoea in 2462 antibiotic-treated hospitalized patients: a prospective study. J Antimicrob Chemother 2001; 47 : 43-50.
2. Hogenauer C, Hammer HF, Krejs GJ, Reisinger EC. Mechanisms and management of antibiotic-associated diarrhea. Clin Infect Dis 1998; 27 : 702-10.
3. Dallal RM, Harbrecht BG, Boujoukas AJ, Sirio CA, Farkas LM, Lee KK, et al. Fulminant Clostridium difficile: an underappreciated and increasing cause of death and complications. Ann Surg 2002; 235 : 363-72. 4. Kyne L, Hamel MB, Polavaram R, Kelly CP. Health care costs and mortality associated with nosocomial diarrhea due to Clostridium difficile. Clin Infect Dis 2002; 34 : 346-53.
5. Schroeder MS. Clostridium difficile-associated diarrhea. Am Fam Physician 2005; 71 : 921-8.
6. Dhawan B, Chaudhry R, Sharma N Incidence of Clostridium difficile infection: a prospective study in an Indian hospital. J Hosp Infect 1999; 43 : 275-80.
7. Sutter VL, Citron DM, Edelstein MAC, Finegold SM. Wadsworth Anaerobic Manual. Belmont, California: Star Publishers, 1985. p. 71- 5.
8. Kato N, Ou CY, Kato H, Bartley SL, Brown VK, Dowell VR Jr, et al. Identification of toxigenic Clostridium difficile by the polymerase chain reaction. J Clin Microbiol 1991; 29 : 33-7.
9. Gumerlock PH, Tang YJ, Weiss JB, Silva J Jr. Specific detection of toxigenic strains of Clostridium difficile in stool specimens. J Clin Microbiol 1993; 3 : 507-11.
10. Lambe DW Jr., Laslie WW. A standard disc diffusion method for antibiotic susceptability testing for anaerobes; four years antibiotic data. In: Lambe DW Jr, Gena RJ, Carson KJM, editors. Anaerobic bacteria-selected topics. New York: Plenum Press, 1980.
11. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage. Nature 1970; 227 : 680-5.
12. Wroblewska MM, Swoboda-Kopec E, Rokosz A, Nurzynska G, Bednarska A, Luczak M. Detection of Clostridium difficile and its toxin A (TcdA) in stool specimens from hospitalised patients. Pol J Microbiol 2005; 54 : 111-5.
13. Wongwanich S, Pongpech P, Dhiraputra C, Huttayananont S, Sawanpanyalert P. Characteristics of Clostridium difficile strains isolated from asymptomatic individuals and from diarrheal patients. Clin Microbiol Infect 2001; 7: 438-41.
14. Blot E, Escande MC, Besson D, Barbut F, Granpeix C, Asselain B, et al. Outbreak of C. difficile related diarrhea in an adult oncology unit: risk factors and microbiological characteristics. J Hosp Infect 1993; 53: 187-92.
15. Wilcox MH, Fawley WN, Settle CD, Davidson A. Recurrence of symptoms in Clostridium difficile infection-relapse or reinfection? J Hosp Infect 1998; 38 : 93-100.
16. Barbut F, Delmee M, Brazier JS, Petit JC, Poxton IR, Rupnik M, et al. ESCMID Study Group on Clostridium difficile (ESGCD). A European survey of diagnostic methods and testing protocols for Clostridium difficile. Clin Microbiol Infect 2003; 9 : 989-96.
17. Al-Eidan FA, McElnay JC, Scott MG, Kearney MP. Clostridium difficile-associated diarrhoea in hospitalised patients. J Clin Pharm Ther 2000; 25 : 101-9.
18. Spencer R. The role of antimicrobial agents in the aetiology of Clostridium difficile-associated disease. J Antimicrob Chemother 1998; 41 : 21-7.
19. Bignardi GE. Risk factors for Clostridium difficile infection. J Hosp Infect 1998; 40 : 1-15.
20. Thamilikitkul V, Danpakdi K, Chokloikaew S. Incidence of diarrhea and Clostridium difficile toxin in stools from hospitalized patients receiving Clindamycin, beta lactams, and nonantibiotic medications. J Clin Gastroenterol 1996; 22 : 161-3.
21. Clabots CR, Johnson S, Olson MM, Peterson LR, Gerding DN. Acquisition of Clostridium difficile by hospitalized patients: evidence for colonized new admissions as a source of infection. J Infect Dis 1992; 166 : 561-7.
22. Impallomeni M, Galletly NP, Wort SU, Starr JM, Rogers TR. Increased risk of diarrhea caused by Clostridium difficile in elderly patients receiving cefotaxime. BMJ 1995; 311 : 1345-6.
23. Pear SM, Williamson TH, Bettin KM, Gerding DN, Galgiani JN. Decrease in nosocomial Clostridium difficile-associated diarrhea by restricting clindamycin use. Ann Intern Med 1994; 720 : 272-7.
24. Pepin J, Saheb N, Coulombe MA, Alary ME, Carrivean MP, Authier S, et al. Emergence of fluoroquinolones as the predominant risk factor for Clostridium difficile-associated diarrhea: a cohort study during an epidemic in Quebec. Clin Infect Dis 2005; 41 : 1254- 60.
25. Muto CA, Pokrywka M, Shutt K, Mendelsohn AS, Nouri K, Posey K, et al. A large outbreak of Clostridium difficile-associated disease with an unexpected proportion of deaths and colectomies at a teaching hospital following increased fluoroquinolone use. Infect Control Hosp Epidemiol 2005; 26 : 273-80
26. Bartlett JG. Antibiotic associated diarrhea. Clin Infect Dis 1992; 15 : 573-81.
27. Pepersack F, Labbe M, Nonhoff C, Schoutens E. Use of gas- liquid chromatography as a screening test for toxigenic Clostridium difficile in diarrhoeal stools. J Clin Pathol 1983; 36 : 1233-6.
28. McFarland LV, Surawicz CM, Rubin M, Fekety R, Elmer GW, Greenberg RN. Recurrent Clostridium difficile disease: Epidemiology and clinical characteristics. Infect Control Hosp Epidemiol 1999; 20 : 43-50.
29. Joshy L, Chaudhry R, Dhawan B, Kumar L, Das BK. Incidence and characterization of Clostridium perfringens isolated from antibiotic- associated diarrhoeal patients: a prospective study in an Indian hospital. J Hosp Infect 2006; 63 : 323-9.
30. Khan R, Cheesbrough J. Impact of changes in antibiotic policy on Clostridium difficile-associated diarrhoea (CDAD) over a five- year period in a district general hospital. J Hosp infect 2003; 54 : 104-8.
31. Riley TV. Nosocomial diarrhoea due to Clostridium difficile. Curr Opin Infect Dis 2004; 17 : 323-7.
Rama Chaudhry, Lovely Joshy, Lalit Kumar* & Benu Dhawan
Departments of Microbiology, * Medical Oncology, Institute- Rotary Cancer Hospital
All India Institute of Medical Sciences, New Delhi, India
Received December 7, 2006
Reprint requests: Dr Rama Chaudhry, Professor, Department of Microbiology, All India Institute of Medical Sciences
New Delhi 110029, India
e-mail: drramach@rediffmail.com, ramach003@yahoo.com
Copyright Indian Council of Medical Research Apr 2008
(c) 2008 Indian Journal of Medical Research. Provided by ProQuest LLC. All rights Reserved.
Source: Indian Journal of Medical Research
Sunday, August 24, 2008
Nanotubes could help study retrovirus transmission between human cells
Recent findings by medical researchers indicate that naturally occurring nanotubes may serve as tunnels that protect retroviruses and bacteria in transit from diseased to healthy cells — a fact that may explain why vaccines fare poorly against some invaders.
To better study the missions of these intercellular nanotubes, scientists have sought the means to form them quickly and easily in test tubes.
Sandia National Laboratories researchers have now learned serendipitously to form nanotubes with surprising ease.
"Our work is the first to show that the formation of nanotubes is not complicated, but can be a general effect of protein-membrane interactions alone," says Darryl Sasaki of Sandia's Bioscience and Energy Center.
Sandia is a National Nuclear Security Administration laboratory.
The tunnel-like structures have been recognized only recently as tiny but important bodily channels for the good, the bad, and the informational.
In addition to providing protected transport to certain diseases, the nanotubes also seem to help trundle bacteria to their doom in the tentacles of microphages. Lastly, the nanotubes may provide avenues to send and receive information (in the form of chemical molecules) from cell to cell far faster than their random dispersal into the bloodstream would permit.
Given the discovery of this radically different transportation system operating within human tissues, it was natural for researchers to attempt to duplicate the formation of the nanotubes. In their labs, they experimented with giant lipid vesicles that appeared to mimic key aspects of the cellular membrane.
Giant lipid vesicles resemble micron-sized spherical soap bubbles that exist in water. They are composed of a lipid bilayer membrane only five nanometers thick.
The object for experimenters was to create conditions in which the spheres would morph into cylinders of nanometer radii.
But researchers had difficulties, says Sasaki, perhaps because they used a composite lipid called egg PC that requires unnecessarily high energies to bend into a tubular shape.
Egg PC is inexpensive, readily available, and offers good, stable membrane properties. It is the usual lipid of choice in forming nanocylinders via mechanical stretching techniques.
But Sandia postdoctoral researcher Haiqing Lui instead used POPC — a single pure lipid requiring half the bending energy of egg PC.
She was trying to generate nanotubes by a completely different approach that involved the use of motor proteins to stretch naturally occurring membranes into tubes.
Working with Sandia researcher George Bachand, she serendipitously found that interaction of the POPC membrane with a high affinity protein called streptavidin alone was enough to form the nanotubes.
"Perhaps this information — linking membrane bending energy with nanotube formation — may provide some clue about the membrane structure and the cell's ability to form such intercellular connections," Sasaki says.
The formation was confirmed by Sandia researcher Carl Hayden, who characterized the nanotube formation through a confocal imaging microscope. The custom instrument allows pixel-by-pixel examination of the protein interaction with the membranes comprising the nanotubes by detecting the spectrum and lifetimes of fluorescent labels on the proteins.
Nanotube formation had been noticed previously by cell biologists, but they had dismissed the tiny outgrowths as "junk — an aberration of cells growing in culture," says Sasaki. "The reason they were only noticed recently as trafficking routes is because of labeling studies that marked organelles and proteins. This allowed a focused look at what these nanostructures might be used for."
It became clear, says Sasaki, that the organelles were being transported with "specific directionality" on the backs of motor proteins within the tubes, rather than randomly.
Three-dimensional networks of nanotubes also are found to be created by macrophages — part of the police force of the body — grown in culture, says George. The tubes in appearance and function resemble a kind of spider web, capturing bacterium and transporting them to the macrophages, which eat them.
Source : DOE/Sandia National Laboratories
To better study the missions of these intercellular nanotubes, scientists have sought the means to form them quickly and easily in test tubes.
Sandia National Laboratories researchers have now learned serendipitously to form nanotubes with surprising ease.
"Our work is the first to show that the formation of nanotubes is not complicated, but can be a general effect of protein-membrane interactions alone," says Darryl Sasaki of Sandia's Bioscience and Energy Center.
Sandia is a National Nuclear Security Administration laboratory.
The tunnel-like structures have been recognized only recently as tiny but important bodily channels for the good, the bad, and the informational.
In addition to providing protected transport to certain diseases, the nanotubes also seem to help trundle bacteria to their doom in the tentacles of microphages. Lastly, the nanotubes may provide avenues to send and receive information (in the form of chemical molecules) from cell to cell far faster than their random dispersal into the bloodstream would permit.
Given the discovery of this radically different transportation system operating within human tissues, it was natural for researchers to attempt to duplicate the formation of the nanotubes. In their labs, they experimented with giant lipid vesicles that appeared to mimic key aspects of the cellular membrane.
Giant lipid vesicles resemble micron-sized spherical soap bubbles that exist in water. They are composed of a lipid bilayer membrane only five nanometers thick.
The object for experimenters was to create conditions in which the spheres would morph into cylinders of nanometer radii.
But researchers had difficulties, says Sasaki, perhaps because they used a composite lipid called egg PC that requires unnecessarily high energies to bend into a tubular shape.
Egg PC is inexpensive, readily available, and offers good, stable membrane properties. It is the usual lipid of choice in forming nanocylinders via mechanical stretching techniques.
But Sandia postdoctoral researcher Haiqing Lui instead used POPC — a single pure lipid requiring half the bending energy of egg PC.
She was trying to generate nanotubes by a completely different approach that involved the use of motor proteins to stretch naturally occurring membranes into tubes.
Working with Sandia researcher George Bachand, she serendipitously found that interaction of the POPC membrane with a high affinity protein called streptavidin alone was enough to form the nanotubes.
"Perhaps this information — linking membrane bending energy with nanotube formation — may provide some clue about the membrane structure and the cell's ability to form such intercellular connections," Sasaki says.
The formation was confirmed by Sandia researcher Carl Hayden, who characterized the nanotube formation through a confocal imaging microscope. The custom instrument allows pixel-by-pixel examination of the protein interaction with the membranes comprising the nanotubes by detecting the spectrum and lifetimes of fluorescent labels on the proteins.
Nanotube formation had been noticed previously by cell biologists, but they had dismissed the tiny outgrowths as "junk — an aberration of cells growing in culture," says Sasaki. "The reason they were only noticed recently as trafficking routes is because of labeling studies that marked organelles and proteins. This allowed a focused look at what these nanostructures might be used for."
It became clear, says Sasaki, that the organelles were being transported with "specific directionality" on the backs of motor proteins within the tubes, rather than randomly.
Three-dimensional networks of nanotubes also are found to be created by macrophages — part of the police force of the body — grown in culture, says George. The tubes in appearance and function resemble a kind of spider web, capturing bacterium and transporting them to the macrophages, which eat them.
Source : DOE/Sandia National Laboratories
Worms do calculus to find meals or avoid unpleasantness
Thanks to salt and hot chili peppers, researchers have found a calculus-computing center that tells a roundworm to go forward toward dinner or turn to broaden the search. It's a computational mechanism, they say, that is similar to what drives hungry college students to a pizza.
These behavior-driving calculations, according to a paper published in the July 3 issue of the journal Nature, are done "in a tiny, specialized computer inside a primitive roundworm," says principal investigator Shawn Lockery, a University of Oregon biologist and member of the UO Institute of Neuroscience.
In their paper, the researchers documented how two related, closely located chemosensory neurons, acting in tandem, regulate behavior. The left neuron controls an on switch, while the opposing right one an off switch. These sister neurons are situated much like the two nostrils or two eyes of mammals. Together these neurons are known as ASE for antagonistic sensory cues.
It's possible, Lockery said, that the discovery someday could help research aimed at treating at least some of the 200,000 people in the United States who annually seek medical treatment, according to records of the National Institutes of Health, for problems involving taste and smell.
"This computer does some nice calculus, differentiating the rate of change of the strength of various tastes," Lockery said. "The worm uses this information to find food and to avoid poisons."
Lockery and colleagues predicted the existence of a derivative-crunching mechanism in the Journal of Neuroscience in 1999 based on findings that nematodes change directions based on taste and smell.
"In effect, they have two nostrils or two tongues but they are so close together that it is really like having one nostril or one tongue, and yet they find their way around quite effectively," Lockery said. "We knew from behavioral experiments that nematodes were doing the same thing that humans were doing, but only from the view of behavioral responses. We didn't know what was going on in the brain."
To get there, Lockery and colleagues used new imaging and molecular tools, along with some genetic engineering of their worms.
In one experiment, these chemosensory neurons carried a fluorescent protein that changed color based on neuronal activity. In another experiment, the neurons carried receptor proteins that recognize capsaicin, the active component in chili peppers.
Researchers found that when concentrations of salt were high, fluorescent proteins change from blue to yellow, showing that the left neuron (ASEL) was active as the worms continued forward movement. When salt levels were reduced, the right neuron (ASER) activated but generated a different behavior; the worms began a turning, or searching, motion.
"At this point, we wanted to know if these neurons really are controlling behavior. If ASEL really signals that things are getting better, then, if you could artificially activate ASEL the animals ought to go straight like a human going directly toward the pizza," Lockery said. "Conversely, if you activate the ASER the animals ought to turn to find their goal."
Such was the case, according to the capsaicin-receptor experiment. When the pepper ingredient was spread on turning worms with receptor proteins in the left neuron, they straightened their motion. Likewise, capsaicin applied to worms with the receptors in their right neurons caused them to change from turning motion to forward crawling.
"We have discovered a tiny, specialized computer inside a primitive round worm," Lockery said. "The computer calculates the rate of change of the strengths, or concentrations, of various tastes. The worm uses this information to find food and to avoid poisons."
Evidence for such on and off switching cells in other chemosensory networks of mammals, he added, "There are strong indications that a similar device exists in the human nervous system."
Source : University of Oregon
These behavior-driving calculations, according to a paper published in the July 3 issue of the journal Nature, are done "in a tiny, specialized computer inside a primitive roundworm," says principal investigator Shawn Lockery, a University of Oregon biologist and member of the UO Institute of Neuroscience.
In their paper, the researchers documented how two related, closely located chemosensory neurons, acting in tandem, regulate behavior. The left neuron controls an on switch, while the opposing right one an off switch. These sister neurons are situated much like the two nostrils or two eyes of mammals. Together these neurons are known as ASE for antagonistic sensory cues.
It's possible, Lockery said, that the discovery someday could help research aimed at treating at least some of the 200,000 people in the United States who annually seek medical treatment, according to records of the National Institutes of Health, for problems involving taste and smell.
"This computer does some nice calculus, differentiating the rate of change of the strength of various tastes," Lockery said. "The worm uses this information to find food and to avoid poisons."
Lockery and colleagues predicted the existence of a derivative-crunching mechanism in the Journal of Neuroscience in 1999 based on findings that nematodes change directions based on taste and smell.
"In effect, they have two nostrils or two tongues but they are so close together that it is really like having one nostril or one tongue, and yet they find their way around quite effectively," Lockery said. "We knew from behavioral experiments that nematodes were doing the same thing that humans were doing, but only from the view of behavioral responses. We didn't know what was going on in the brain."
To get there, Lockery and colleagues used new imaging and molecular tools, along with some genetic engineering of their worms.
In one experiment, these chemosensory neurons carried a fluorescent protein that changed color based on neuronal activity. In another experiment, the neurons carried receptor proteins that recognize capsaicin, the active component in chili peppers.
Researchers found that when concentrations of salt were high, fluorescent proteins change from blue to yellow, showing that the left neuron (ASEL) was active as the worms continued forward movement. When salt levels were reduced, the right neuron (ASER) activated but generated a different behavior; the worms began a turning, or searching, motion.
"At this point, we wanted to know if these neurons really are controlling behavior. If ASEL really signals that things are getting better, then, if you could artificially activate ASEL the animals ought to go straight like a human going directly toward the pizza," Lockery said. "Conversely, if you activate the ASER the animals ought to turn to find their goal."
Such was the case, according to the capsaicin-receptor experiment. When the pepper ingredient was spread on turning worms with receptor proteins in the left neuron, they straightened their motion. Likewise, capsaicin applied to worms with the receptors in their right neurons caused them to change from turning motion to forward crawling.
"We have discovered a tiny, specialized computer inside a primitive round worm," Lockery said. "The computer calculates the rate of change of the strengths, or concentrations, of various tastes. The worm uses this information to find food and to avoid poisons."
Evidence for such on and off switching cells in other chemosensory networks of mammals, he added, "There are strong indications that a similar device exists in the human nervous system."
Source : University of Oregon
New antibiotic beats superbugs at their own game
The problem with antibiotics is that, eventually, bacteria outsmart them and become resistant. But by targeting the gene that confers such resistance, a new drug may be able to finally outwit them. Rockefeller University scientists tested the new drug, called Ceftobiprole, against some of the deadliest strains of multidrug-resistant Staphylococcus aureus (MRSA) bacteria, which are responsible for the great majority of staphylococcal infections worldwide, both in hospitals and in the community.
The research, to be published in the August 2008 issue of the journal Antimicrobial Agents and Chemotherapy and available online now, looked at how well Ceftobiprole worked against bacterial clones that had already developed resistance to other drugs. In every case, Ceftobiprole won. "It just knocked out the cells 100 percent," says the study's lead investigator, Alexander Tomasz, head of the Laboratory of Microbiology at Rockefeller.
Previous research had already shown that -- in general -- Ceftobiprole was highly effective against most clinical isolates of S. aureus. "Instead, we looked more carefully at the highly resistant cells that already occur in such clinical isolates at very low frequency -- maybe in one bacterium in every 1,000," says Tomasz. Ceftobiprole was able to kill these resistant cells.
Never before has an antibiotic been tested this way. "In the history of antibiotic development, an antibiotic arrives on the scene, and sooner or later resistant bacteria emerge," Tomasz says. "We sought to test in advance which would win this particular chess game: the new drug, or the bacteria that now cause human deaths."
In an ominous new "move" in this chess game, S. aureus strains with resistance to vancomycin (VRSA), a different class of antibiotics, also began to appear in hospitals in the United States. Ceftobiprole was also able to kill these new resistant VRSA strains.
The drug is effective because the chemists who developed Ceftobiprole managed to outwit the bacteria at their own game, Tomasz says. The broad-spectrum antibiotic was discovered by Basilea Pharmaceuticals, based in Basel, Switzerland, and is being developed in the U.S. and worldwide by Johnson & Johnson. The research was supported by Johnson & Johnson along with a grant from the U.S. Public Health Service.
Source : Rockefeller University
The research, to be published in the August 2008 issue of the journal Antimicrobial Agents and Chemotherapy and available online now, looked at how well Ceftobiprole worked against bacterial clones that had already developed resistance to other drugs. In every case, Ceftobiprole won. "It just knocked out the cells 100 percent," says the study's lead investigator, Alexander Tomasz, head of the Laboratory of Microbiology at Rockefeller.
Previous research had already shown that -- in general -- Ceftobiprole was highly effective against most clinical isolates of S. aureus. "Instead, we looked more carefully at the highly resistant cells that already occur in such clinical isolates at very low frequency -- maybe in one bacterium in every 1,000," says Tomasz. Ceftobiprole was able to kill these resistant cells.
Never before has an antibiotic been tested this way. "In the history of antibiotic development, an antibiotic arrives on the scene, and sooner or later resistant bacteria emerge," Tomasz says. "We sought to test in advance which would win this particular chess game: the new drug, or the bacteria that now cause human deaths."
In an ominous new "move" in this chess game, S. aureus strains with resistance to vancomycin (VRSA), a different class of antibiotics, also began to appear in hospitals in the United States. Ceftobiprole was also able to kill these new resistant VRSA strains.
The drug is effective because the chemists who developed Ceftobiprole managed to outwit the bacteria at their own game, Tomasz says. The broad-spectrum antibiotic was discovered by Basilea Pharmaceuticals, based in Basel, Switzerland, and is being developed in the U.S. and worldwide by Johnson & Johnson. The research was supported by Johnson & Johnson along with a grant from the U.S. Public Health Service.
Source : Rockefeller University
Pandemic mutations in bird flu revealed
Scientists have discovered how bird flu adapts in patients, offering a new way to monitor the disease and prevent a pandemic, according to research published in the August issue of the Journal of General Virology. Highly pathogenic H5N1 avian influenza virus has spread through at least 45 countries in 3 continents. Despite its ability to spread, it cannot be transmitted efficiently from human to human. This indicates it is not fully adapted to its new host species, the human. However, this new research reveals mutations in the virus that may result in a pandemic.
"The mutations needed for the emergence of a potential pandemic virus are likely to originate and be selected within infected human tissues," said Professor Dr Prasert Auewarakul from Mahidol University, Thailand. "We analyzed specific molecules called haemagglutinin on viruses derived from fatal human cases. Our results suggest new candidate mutations that may allow bird flu to adapt to humans."
Viruses with a high mutation rate such as influenza virus usually exist as a swarm of variants, each slightly different from the others. These are called H5N1 bird flu quasispecies. Professor Dr Auewarakul and his colleagues found that some mutations in the quasispecies were more frequent than others, which indicates they may be adaptive changes that make the virus more efficient at infecting humans. Most of these mutations were found in the area required for the virus to bind to the host cell.
"This study shows that the H5N1 virus is adapting each time it infects a human," said Professor Dr Auewarakul. "Such adaptations may lead to the emergence of a virus that can cause a pandemic. Our research highlights the need to control infection and transmission to humans to prevent further adaptations."
The research has provided genetic markers to help scientists monitor bird flu viruses with pandemic potential. This means they will be able to detect potentially dangerous strains and prevent a pandemic. The research also gives new insights into the mechanism of the genesis of a pandemic strain.
"Our approach could be used to screen for mutations with significant functional impact," said Professor Dr Auewarakul. "It is a new method of searching for changes in H5N1 viruses that are required for the emergence of a pandemic virus. We hope it will help us to prevent a pandemic in the future."
Source : Society for General Microbiology
"The mutations needed for the emergence of a potential pandemic virus are likely to originate and be selected within infected human tissues," said Professor Dr Prasert Auewarakul from Mahidol University, Thailand. "We analyzed specific molecules called haemagglutinin on viruses derived from fatal human cases. Our results suggest new candidate mutations that may allow bird flu to adapt to humans."
Viruses with a high mutation rate such as influenza virus usually exist as a swarm of variants, each slightly different from the others. These are called H5N1 bird flu quasispecies. Professor Dr Auewarakul and his colleagues found that some mutations in the quasispecies were more frequent than others, which indicates they may be adaptive changes that make the virus more efficient at infecting humans. Most of these mutations were found in the area required for the virus to bind to the host cell.
"This study shows that the H5N1 virus is adapting each time it infects a human," said Professor Dr Auewarakul. "Such adaptations may lead to the emergence of a virus that can cause a pandemic. Our research highlights the need to control infection and transmission to humans to prevent further adaptations."
The research has provided genetic markers to help scientists monitor bird flu viruses with pandemic potential. This means they will be able to detect potentially dangerous strains and prevent a pandemic. The research also gives new insights into the mechanism of the genesis of a pandemic strain.
"Our approach could be used to screen for mutations with significant functional impact," said Professor Dr Auewarakul. "It is a new method of searching for changes in H5N1 viruses that are required for the emergence of a pandemic virus. We hope it will help us to prevent a pandemic in the future."
Source : Society for General Microbiology
New study sheds light on how intracellular pathogens trigger the immune system
Disease-causing microbes like the food-borne bacterium Listeria monocytogenes specialize in invading and replicating inside their animal hosts' own cells, making them particularly tricky to defeat. Now, a new study led by biologists at the University of California, Berkeley, has identified a molecular alarm system in which the intracellular pathogen sends out signals that kick the immune response into gear.
The findings, to be reported the week of July 14 in the journal Proceedings of the National Academy of Sciences, shed light on how the cells recognize and destroy the pathogenic bugs living within their walls, and may even provide new targets for the research and development of new vaccines and drugs.
The pathogens' signals come from multidrug resistance transporters (MDRs), membrane proteins used by a wide variety of organisms to pump out a broad range of molecules from their systems. Similar transporters have been linked in other studies to the development of resistance to multiple drugs that are toxic to the pathogen. This study is the first to connect multidrug resistance transporters directly to stimulation of the immune system, although the nature of the molecules that the bacteria are spitting out remains unclear.
"For the MDRs to work, the pathogen needs to be alive, so this study actually shows how the immune system can tell the difference between a living, harmful microbe and one that is dead," said the study's principal investigator, Daniel Portnoy, a UC Berkeley professor with joint appointments in the Department of Molecular and Cell Biology and the School of Public Health, and associate director of the Berkeley Center for Emerging and Neglected Diseases. "This is important because you don't want the immune system to overreact to non-threats, which is what happens in autoimmune disorders such as inflammatory bowel disease, asthma and multiple sclerosis."
The Listeria bacterium makes headlines when it contaminates deli meats, raw cheeses, cole slaw and other foods. According to the Centers for Disease Control and Prevention, Listeria causes some 2,500 infections and 500 deaths each year, and at greatest risk are people who have weakened immune systems or are pregnant.
The bacteria first trick immune cells into swallowing them, where they become encased in bubbles called vacuoles. The bacteria become dangerous when they break out of these bubbles into the cells' internal fluid, or cytosol, to multiply and spread the infection. The role of MDRs is not clearly known, but the results of this study plainly show that one particular MDR transporter is necessary for the host to respond to the infection, the authors said. In addition, overexpression of this or other related MDRs leads to an enhanced host immune response.
"The only way the bug molecule enters into the cytosol is if the bacterium is virulent," said Portnoy, who is also a member of UC Berkeley's Health Sciences Initiative. "We know that there are different immune system receptors in different compartments of a cell, but until this paper, it was not understood exactly how the cytosolic surveillance system was triggered. Our findings suggest that the molecules pumped out by the pathogen while it's in the cellular fluid help the immune system gauge whether a bacterium is a threat based upon its location inside the cell."
The researchers isolated the role of multidrug resistance transporters by manipulating specific genes in the bacteria that controlled their expression and then measuring how increased or decreased activity by the transporter proteins impacted levels of interferon beta, a protein produced by the immune system that rally more disease-fighting cells when infections are detected.
They found that greater MDR expression led to greater stimulation of the immune system, as measured by interferon beta levels.
Strains of Listeria with higher levels of MDR expression increased interferon beta levels up to 20-fold compared with unmodified, wild-type Listeria in cell cultures, the study found. Tests in mice infected with those same mutant strains of Listeria had bacterial loads that were 20 times lower in their livers, although the researchers could not attribute the decreased levels solely to the higher levels of interferon beta.
"This paper raises the classic issue of the tug-of-war in the evolution of the host and the pathogen; it's a never-ending arms race," said Gregory Crimmins, UC Berkeley graduate student in molecular and cell biology who, along with former UC Berkeley post-doctoral researcher Anat Herskovits, was the study's co-lead author.
The study results could provide clues to the actions of other intracellular pathogens, such as the bacteria responsible for tuberculosis and Legionnaires' disease, since they also activate similar immune mechanisms, the researchers said.
Crimmins noted that better understanding of how the class of interferons in this study is triggered could have implications for a variety of diseases. "Type I interferons have wide-ranging effects on the immune system, and are used to treat multiple sclerosis, hepatitis C and some types of cancer," he said. "The strains generated in this study may provide novel insight into the role of Type I interferons in coordinating the host immune response."
"By understanding the pathways of innate immunity, we can better understand acquired immunity, and that is important for vaccine development," added Portnoy. "The concept of making safe but fully effective vaccines is still a challenge, especially for intracellular pathogens."
Portnoy pointed out that weakened Listeria is already being used to develop cancer vaccines by Anza Pharmaceuticals, a Concord-based biopharmaceutical company with which he consults.
Source : University of California - Berkeley
The findings, to be reported the week of July 14 in the journal Proceedings of the National Academy of Sciences, shed light on how the cells recognize and destroy the pathogenic bugs living within their walls, and may even provide new targets for the research and development of new vaccines and drugs.
The pathogens' signals come from multidrug resistance transporters (MDRs), membrane proteins used by a wide variety of organisms to pump out a broad range of molecules from their systems. Similar transporters have been linked in other studies to the development of resistance to multiple drugs that are toxic to the pathogen. This study is the first to connect multidrug resistance transporters directly to stimulation of the immune system, although the nature of the molecules that the bacteria are spitting out remains unclear.
"For the MDRs to work, the pathogen needs to be alive, so this study actually shows how the immune system can tell the difference between a living, harmful microbe and one that is dead," said the study's principal investigator, Daniel Portnoy, a UC Berkeley professor with joint appointments in the Department of Molecular and Cell Biology and the School of Public Health, and associate director of the Berkeley Center for Emerging and Neglected Diseases. "This is important because you don't want the immune system to overreact to non-threats, which is what happens in autoimmune disorders such as inflammatory bowel disease, asthma and multiple sclerosis."
The Listeria bacterium makes headlines when it contaminates deli meats, raw cheeses, cole slaw and other foods. According to the Centers for Disease Control and Prevention, Listeria causes some 2,500 infections and 500 deaths each year, and at greatest risk are people who have weakened immune systems or are pregnant.
The bacteria first trick immune cells into swallowing them, where they become encased in bubbles called vacuoles. The bacteria become dangerous when they break out of these bubbles into the cells' internal fluid, or cytosol, to multiply and spread the infection. The role of MDRs is not clearly known, but the results of this study plainly show that one particular MDR transporter is necessary for the host to respond to the infection, the authors said. In addition, overexpression of this or other related MDRs leads to an enhanced host immune response.
"The only way the bug molecule enters into the cytosol is if the bacterium is virulent," said Portnoy, who is also a member of UC Berkeley's Health Sciences Initiative. "We know that there are different immune system receptors in different compartments of a cell, but until this paper, it was not understood exactly how the cytosolic surveillance system was triggered. Our findings suggest that the molecules pumped out by the pathogen while it's in the cellular fluid help the immune system gauge whether a bacterium is a threat based upon its location inside the cell."
The researchers isolated the role of multidrug resistance transporters by manipulating specific genes in the bacteria that controlled their expression and then measuring how increased or decreased activity by the transporter proteins impacted levels of interferon beta, a protein produced by the immune system that rally more disease-fighting cells when infections are detected.
They found that greater MDR expression led to greater stimulation of the immune system, as measured by interferon beta levels.
Strains of Listeria with higher levels of MDR expression increased interferon beta levels up to 20-fold compared with unmodified, wild-type Listeria in cell cultures, the study found. Tests in mice infected with those same mutant strains of Listeria had bacterial loads that were 20 times lower in their livers, although the researchers could not attribute the decreased levels solely to the higher levels of interferon beta.
"This paper raises the classic issue of the tug-of-war in the evolution of the host and the pathogen; it's a never-ending arms race," said Gregory Crimmins, UC Berkeley graduate student in molecular and cell biology who, along with former UC Berkeley post-doctoral researcher Anat Herskovits, was the study's co-lead author.
The study results could provide clues to the actions of other intracellular pathogens, such as the bacteria responsible for tuberculosis and Legionnaires' disease, since they also activate similar immune mechanisms, the researchers said.
Crimmins noted that better understanding of how the class of interferons in this study is triggered could have implications for a variety of diseases. "Type I interferons have wide-ranging effects on the immune system, and are used to treat multiple sclerosis, hepatitis C and some types of cancer," he said. "The strains generated in this study may provide novel insight into the role of Type I interferons in coordinating the host immune response."
"By understanding the pathways of innate immunity, we can better understand acquired immunity, and that is important for vaccine development," added Portnoy. "The concept of making safe but fully effective vaccines is still a challenge, especially for intracellular pathogens."
Portnoy pointed out that weakened Listeria is already being used to develop cancer vaccines by Anza Pharmaceuticals, a Concord-based biopharmaceutical company with which he consults.
Source : University of California - Berkeley
Subscribe to:
Posts (Atom)
