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Indian Journal of Medical Microbiology

vol. 26, No. 1

INDIAN JOURNAL OF MEDICAL MICROBIOLOGY (Publication of Indian Association of Medical Microbiologists)

ISSN 0255-0857

Volume 26

Number 1

January-March, 2008


Guest Editorial Novel HIV Prevention Strategies: The Case for Andhra Pradesh JA Schneider


Review Article Chikungunya Fever: A Re-emerging Viral Infection M Chhabra, V Mittal, D Bhattacharya, UVS Rana, S Lal


Special Article Fabrication and Evaluation of a Sequence-specific Oligonucleotide Miniarray for Molecular Genotyping J Iqbal, F Hänel, A Ruryk, GV Limmon, A Tretiakov, M Dürst, HP Saluz .......13

Original Articles A Comparison of PCR Detection of Meca with Oxacillin Disk Susceptibility Testing in Different Media and Sceptor Automated System for both Staphylococcus aureus and Coagulase-negative Staphylococci Isolates S Ercis, B Sancak, G Hasçelik


Effect of Exposure to Hydrogen Peroxide on the Virulence of Escherichia coli A Hegde, GK Bhat, S Mallya


A Low Molecular Weight Es-20 Protein Released In Vivo and In Vitro with Diagnostic Potential in Lymph Node Tuberculosis N Shende, V Upadhye, S Kumar, BC Harinath .......29 Community-based Study on Seroprevalence of Herpes Simplex Virus Type 2 Infection in New Delhi R Chawla, P Bhalla, K Bhalla, M Meghachandra Singh, S Garg .......34 Changing Patterns of Vibrio cholerae in Sevagram Between 1990 and 2005 P Narang, DK Mendiratta, VS Deotale, R Narang


Rapid Serodiagnosis of Leptospirosis by Latex Agglutination Test and Flow-through Assay TMA Senthilkumar, M Subathra, M Phil, P Ramadass, V Ramaswamy


High Level Ciprofloxacin Resistance in Salmonella enterica Isolated from Blood R Raveendran, C Wattal, A Sharma, JK Oberoi, KJ Prasad, S Datta


Role of Enteric Fever in Ileal Perforations: An Overstated Problem in Tropics? MR Capoor, D Nair, MS Chintamani, J Khanna, P Aggarwal, D Bhatnagar


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January-March 2008

Brief Communications Evaluation of a Modified Double-disc Synergy Test for Detection of Extended Spectrum β-lactamases in Ampc β-lactamase-producing Proteus mirabilis MKR Khan, SS Thukral, R Gaind .......58 Antimicrobial Susceptibility Profile of Neisseria gonorrhoeae at STI Clinic C Shilpee, VG Ramachandran, S Das, SN Bhattacharya


Detection of Extra-cellular Enzymes of Anaerobic Gram-negative Bacteria from Clinically Diseased and Healthy Sites JM Nagmoti, CS Patil, MB Nagmoti, MB Mutnal


Haemagglutination and Siderophore Production as the Urovirulence Markers of Uropathogenic Escherichia coli MA Vagarali, SG Karadesai, CS Patil, SC Metgud, MB Mutnal


The use of Dried Blood Spots on Filter Paper for the Diagnosis of HIV-1 in Infants Born to HIV Seropositive Women S Mini Jacob, D Anitha, R Vishwanath, S Parameshwari, NM Samuel


Evaluation of the Usefulness of Phage Amplification Technology in the Diagnosis of Patients with Paucibacillary Tuberculosis D Biswas, A Deb, P Gupta, R Prasad, KS Negi .......75

Case Reports Cytomegalovirus Oesophagitis in a Patient with Non-hodgkin’s Lymphoma SS Hingmire, G Biswas, A Bakshi, S Desai, S Dighe, R Nair, S Gupta, PM Parikh


Hydatid Cyst of Mediastinum S Sehgal, B Mishra, A Thakur, V Dogra, PS Loomba, A Banerjee


Ochrobactrum anthropi Septicaemia U Arora, S Kaur, P Devi


Intestinal Myiasis Caused by Muscina stabulans S Shivekar, K Senthil, R Srinivasan, L Sureshbabu, P Chand, J Shanmugam, R Gopal


Pyopericardium Due To Group D Streptococcus K Karthikeyan, KR Rajesh, H Poornima, R Bharathidasan, KN Brahmadathan, R Indra Priyadharsini


Pleural Effusion: A Rare Complication of Hepatitis A A Bukulmez, R Koken, H Melek, O Dogru, F Ovali


Correspondence Prevalence of Inducible AmpC β-lactamase-Producing Pseudomonas aeruginosa in a Tertiary Care Hospital in Northern India A Bhattacharjee, S Anupurba, A Gaur, MR Sen .......89 Parental History of Ulcer and the Prevalence of Helicobacter pylori Infection in their Offspring KS Ahmed, AA Khan, JD Ahi, CM Habibullah


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Indian Journal of Medical Microbiology

vol. 26, No. 1

Ciprofloxacin Breakpoints in Enteric Fever - Time to Revise our Susceptibility Criteria C Rodrigues, N Jai Kumar, J Lalwani, A Mehta


West Nile Virus in the Blood Donors in UAE M Alfaresi, A Elkoush


Estimation of Antibodies To HBsAg in Vaccinated Health Care Workers TV Rao, IJ Suseela, KA Sathiavathy


Seroprevalence of Rubella Among Urban and Rural Bangladeshi Women Emphasises the Need for Rubella Vaccination of Pre-pubertal Girls A Nessa, MN Islam, S Tabassum, SU Munshi, M Ahmed, R Karim .......94 Novel Digestion Patterns with Hepatitis B Virus Strains from the Indian Subcontinent Detected using Restriction Fragment Length Polymorphism P Vivekanandan, HDJ Daniel, S Raghuraman, D Daniel, RV Shaji, G Sridharan, G Chandy, P Abraham .......96 Acute Urticaria Associated with Dicrocoelium dendriticum Infestation A Sing, K Tybus, I Fackler


Book Reviews


Guidelines to Authors


The copies of the journal to members of the association are sent by ordinary post. The editorial board, association or publisher will not be responsible for non-receipt of copies. If any of the members wish to receive the copies by registered post or courier, kindly contact the journal’s / publisher’s office. If a copy returns due to incomplete, incorrect or changed address of a member on two consecutive occasions, the names of such members will be deleted from the mailing list of the journal. Providing complete, correct and up-to-date address is the responsibility of the members. Copies are sent to subscribers and members directly from the publisher’s address; it is illegal to acquire copies from any other source. If a copy is received for personal use as a member of the association/society, one cannot resale or giveaway the copy for commercial or library use.

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Indian Journal of Medical Microbiology, (2008) 26(1): 25-8 January-March 2008

Original Article


Abstract Purpose: To eliminate pathogenic bacteria, the host presents conditions that are stressful for bacteria. Oxidative stress arises when the concentration of pro-oxidants like hydrogen peroxide (H2O2) and superoxide anion increases to a level over the basal defence capacity of the cell. In the present study, we studied the effect of oxidative stress on the production of certain virulence factors by Escherichia coli. Methods: E. coli was exposed to oxidative stress by growing in the presence of different concentrations of H2O2. The effect of oxidative stress on the expression of surface hydrophobicity, adherence, haemolysin production, serum resistance and phagocytosis was studied. Results: Oxidative stress caused a signiÞcant decrease in the expression of all the virulence factors of E. coli. Conclusions: Synthesis of virulence factors can be signiÞcantly altered by oxidative stress and such changes may affect the pathogenicity of E. coli. Key words: Escherichia coli, oxidative stress, virulence

Stress is deÞned as any deviation from the optimal growth conditions that result in a reduced growth rate. A cell confronted with an abrupt change in its immediate surrounding environment suffers stress.[1] The causes of stress are limitation of essential nutrients such as iron, suboptimal physical conditions such as acidic pH, oxidative stress such as those caused by increased levels of superoxide anion and H2O2 due to oxidative bursts, osmotic stress caused by changes in external osmolarity, that could result in swelling and bursting of the cell in hypotonic environments or in plasmolysis and dehydration in hypertonic conditions. The virulence factors of Escherichia coli are multiple and unusually complex, affecting pathogenicity in combination with one another. The pathogenic processes that operate in a given infection always involve more than one virulence factor. These factors usually interact in so complicated a manner that the precise mechanisms still remain to be deÞned, but attempts to do so have been made.[2-5] The common virulence factors include surface hydrophobicity, colonization factor, capsule, serum resistance, resistance to phagocytosis, haemolysins, enterotoxins and siderophores. In the present study, we determined the effect of oxidative stress on the adherence, surface hydrophobicity, hemolysin production, serum resistance and phagocytosis of E. coli. Materials and Methods Bacterial strains Two standard strains of E. coli Microbial Type Culture *Corresponding author (email: ) Department of Microbiology, Kasturba Medical College, Mangalore 575 001, Karnataka, India Received: 14-06-06 Accepted: 26-04-07

Collection (MTCC, India) 723, E. coli MTCC 729 procured from MTCC and two clinical isolates of E. coli, E. coli A 030 (isolated from blood of a septicaemic patient) and E. coli A 105 (isolated from urine of a patient with symptomatic urinary tract infection) were used in the present study. The clinical isolates were characterized. Preparation of the bacterial inoculum Each bacterial strain from nutrient agar slope was subcultured twice in chemically deÞned medium (CDM) of Snyder and Koch,[3] each time at 37 °C for 24 hours. After the Þnal subculture, the cells were washed three times in sterile physiological saline (0.85% w/v NaCl) and Þnally suspended in saline to get a solution of OD600 0.1 (corresponds to approximately 1 × 108 cells/mL, conÞrmed by surface plating on nutrient agar). Effect of oxidative stress CDM containing three different molar concentrations of H2O2 (0.1, 0.2 and 0.3 mM) was prepared.[4] These media were dispensed aseptically in 10 mL quantities in three 100 mL Erlenmeyer ßasks. Bacterial suspension prepared as described above was used for inoculating the culture media, to achieve an initial concentration of approximately 1 × 106 cells/mL in each ßask. The ßasks were incubated at 37 °C in a rotary water bath at a speed of 160 rpm for 24 hours. The cultures were centrifuged at 3000×g for 10 minutes. The supernatant was discarded and the packed cells were used for the determination of surface hydrophobicity, adherence, haemolysin production, serum resistance and phagocytosis. Effect of stress on the surface hydrophobicity of E. coli The effect of oxidative stress on the surface hydrophobicity of E. coli was studied by quantitative surface


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Indian Journal of Medical Microbiology

hydrophobicity assay,[6] Samples of cultures prepared as described above were washed with sterile phosphate buffered saline (PBS) and then suspended in PBS to get a density of 0.3 at OD600 (OD Initial). Three millilitres of bacterial suspension was mixed with 0.3 mL of p-xylene and vortexed for 1 minute and left for 30 minutes at room temperature. The hydrophobicity index was calculated by applying the following formula: OD I – OD F HI = ___________ × 100 OD I Effect of stress on bacterial adherence to epithelial cells The effect of oxidative stress on adherence properties of E. coli was studied by bacterial adherence assay.[7] Bacterial inoculum was prepared as described above. One millilitre of bacterial culture containing about 1 × 106 cells/mL was mixed with 1 mL of vaginal epithelial cell suspension containing about 1 × 105 cells/mL and incubated at 37 °C for 30 min in a shaker water bath. The cells were centrifuged and the supernatant was Þltered to remove free bacteria. The cells were air dried, alcohol Þxed and stained for 2 minute with dilute carbol fuchsin. The cell preparation was then examined with oil immersion objective. The number of bacteria adherent to 50 cells were counted and mean was calculated. Effect of stress on the production of haemolysin Effect of oxidative stress on the production of haemolysin by E. coli was studied by the quantitative α-haemolysin assay.[8] Samples of cultures prepared as described above were centrifuged at 3000×g for 10 minutes. The supernatant was diluted in calcium saline and 1 mL of it was mixed with 1% (v/v) sheep erythrocyte suspension and incubated at 37 °C for 1 hour. At the end of the incubation period, 2 mL of saline was added to each tube exhibiting partial haemolysis. The tubes were centrifuged at 1500 rpm for 10 minutes to pellet the unlysed erythrocytes. The supernatant ßuid was separated and the OD540 was determined using the control tube as blank. The 50% haemolysis standard was prepared by mixing 1 mL of 1 % (v/v) sheep erythrocyte suspension and 3 mL of diluent without sodium chloride. Inverse of the dilution which caused 50% lysis was recorded as HU 50 (50% haemolytic units). Effect of stress on serum resistance of E. coli Effect of oxidative stress on the serum resistance of bacteria was evaluated by quantitative serum bactericidal assay.[9] Serum was obtained from healthy donors on the day of each test. Bacterial suspension (0.5 mL) was mixed with 1.5 mL of fresh undiluted serum and incubated at 37 °C. The viable count was determined at the beginning of incubation (0 h) and after 1, 2, 3 h of incubation by surface plating. Each bacterium was tested at least three times. The number of bacteria killed was calculated by applying

vol. 26, No. 1

the following formula: Initial count – Final count %Death = _______________________ × 100 Initial count Effect of stress on the phagocytosis of E. coli The effect of oxidative stress on the phagocytosis of E. coli was studied using polymorphonuclear leukocytes (PMNL).[10] One millilitre of mixture containing 425 µL of E. coli (1 × 107 cells/mL), 425 µL PMNLs (5 × 106 cells/mL) and 150 µL of healthy human serum (Þnal concentration of 15%v/v) was made in polystyrene tube and rotated slowly at 37 °C for 1 hour. From this mixture, 0.1 mL was added to 9.9 mL of distilled water to lyse the PMNLs and the viable count was determined by surface plating on nutrient agar. A tube of E. coli without PMNLs treated similarly was used as control. The plates were incubated at 37 °C for 48 hour and the extent of killing was determined by using the following formula: Colony count of control – Colony count of test %Killing = ______________________________________ × 100 Colony count of control

Statistical analysis The data were analysed using the statistical software ‘SPSS 11.0’. The means and standard deviation of each of the parameter were obtained. The Kruskal-Wallis ANOVA was used to compare between groups. A statistical signiÞcance of the comparison between groups was followed by a multiple comparison criterion to identify the group that was signiÞcantly different from the rest. Results Oxidative stress had a signiÞcant effect on the surface hydrophobicity of E. coli (Table 1). All the strains of E. coli showed a signiÞcant decrease in surface hydrophobicity when subjected to oxidative stress by exposure to hydrogen peroxide (H2O2). There was a 30 to 50% decrease in the cell surface hydrophobicity of all the three strains of E. coli when exposed to 0.2 mM of H2O2. Escherichia coli exposed to 0.3 mM H2O2 showed 70% decrease in cell surface hydrophobicity. Oxidative stress had an inhibitory effect on the adherence of E. coli (see Fig. 1). E. coli strains showed signiÞcant decrease (P < 0.001) in adherence on exposure to oxidative stress. A signiÞcant decrease (P < 0.001) in adherence was observed with exposure to increasing concentrations of H2O2 in all the four strains of E. coli (Fig. 1). Oxidative stress had a profound effect on the production of haemolysin by all the strains of E. coli (Table 2). Exposure to H2O2 (0.02 and 0.03 mM) caused a signiÞcant decrease in the amount of haemolysin produced by all the strains of E. coli. Oxidative stress caused a steep reduction in the amount of haemolysin synthesized by E. coli strain A030. No

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Hegde et al - Effect of H2O2 on E. coli

January-March 2008

Table 1: Effect of oxidative stress on surface hydrophobicity of E. coli Hydrogen peroxide (mM) 0.1 0.2 Hydrophobicity index 30.3 ± 3.5* 24.3 ± 3.5** 22.2 ± 1.4* 15.2 ± 1.4** 19.0 ± 1.1* 11.0 ± 1.1*** 33.3 ± 3.5 27.3 ± 3.5*

E. coli strains 0 MTCC 729 MTCC 723 A030 A105

34.7 ± 2.6 27.9 ± 1.2 23.4 ± 1.9 36.7 ± 2.6

0.3 12.0 ± 1.2*** 9.0 ± 0.5*** 7.0 ± 1.2*** 18.0 ± 1.2**

*P < 0.05; **P < 0.01; ***P < 0.001

Table 2: Effect of oxidative stress on haemolysin production by E. coli

Average number of bacteria/cells

18 16

E. coli strains



12 10

MTCC 729 MTCC 723 A030 A105

8 6 4 2



0.3 ND ND ND 2*

*P < 0.001

0 0

32 16 64 32

Hydrogen peroxide (mM) 0.1 0.2 Haemolytic unit50 16* 8* 8* 4* 32* 2* 16* 8*


Hydrogen Peroxide (mM) MTCC 729

MTCC 723

A 030

Table 3: Effect of oxidative stress on serum resistance of E. coli

A 105

Figure 1: Effect of oxidative stress on the adherence of E. coli to epithelial cells

hemolysin was detected in three strains of E. coli exposed to 0.3 mM H2O2. Oxidative stress caused a decrease in serum resistance of both the E. coli strains (Table 3). The decrease in serum resistance was signiÞcant in E. coli MTCC 729 on exposure to 0.02 and 0.03 mM H2O2. Exposure to 0.1 mM H2O2 did not cause a signiÞcant change in serum resistance of this strain. On the other hand, the uropathogenic E. coli A105 showed a signiÞcant decrease (P < 0.05) in serum resistance when exposed to 0.1, 0.2 and 0.3 mM H2O2. It is clear from the results that exposure to oxidative stress caused a signiÞcant decrease in serum resistance of E. coli. Exposure to oxidative stress signiÞcantly increased (P < 0.05) the susceptibility of E. coli to phagocytic killing by PMNLs (Table 4). Escherichia coli strain MTCC 729 exposed to 0.2 mM H2O2 and 0.3 mM H2O2 was signiÞcantly more (P < 0.05) susceptible to phagocytic killing. Discussion During infection, E. coli is subjected to different kinds of stress. The results of the present study clearly indicate that oxidative stress may result in changes that may inßuence the virulence of E. coli. In the present study, we observed that oxidative stress by exposure to H2O2 had an inhibitory effect on the cell surface hydrophobicity and adherence of

E. coli strains MTCC 729 MTCC 723 A030 A105

Hydrogen peroxide (mM) 0 0.1 0.2 Percentage killing 94.3 ± 0.3 94.3 ± 0.8 99.0 ± 0.4* 92.3 ± 0.7 95.7 ± 0.3 100* 87.2 ± 0.2 90.0 ± 0.3 98.2 ± 0.5* 75.3 ± 0.4 85.2 ± 0.2* 99.1 ± 0.1*

0.3 100* 100* 100* 100*

*P < 0.05

E. coli. A recent study showed that adherence and surface hydrophobicity of E. coli decreases when grown in Mueller Hinton broth containing oxidizing agents, melatonin and vitamin E.[11] The above Þnding supports the view that oxidative stress plays an important role in modifying the surface characteristics of E. coli, which could affect the micro-organism’s capacity to adhere to epithelia. Surface hydrophobicity of micro-organisms is one of the virulence factors which contributes to the adherence of microorganisms to host tissue.[12,13] Previous studies have shown a correlation between surface hydrophobicity and in vitro adherence to epithelial cells.[14-16] We observed that any change in the surface hydrophobicity of bacteria resulted in a corresponding change in bacterial adherence to epithelial cells. Oxidative stress also caused a decrease in haemolysin synthesis in both the strains of E. coli. This again could be due to repression of hly genes by H2O2. Oxidative stress caused an increase in serum susceptibility of E. coli. The present study showed that oxidative stress caused


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Indian Journal of Medical Microbiology

vol. 26, No. 1

Table 4: Effect of oxidative stress on phagocytosis of E. coli E. coli strains 0 MTCC 729 MTCC 723 A030 A105

70.8 ± 2.4 85.5 ± 2.7 88.5 ± 2.1 75.6 ± 2.0

Hydrogen peroxide (mM) 0.1 0.2 Percentage killing 81.8 ± 2.6* 93.5 ± 2.3* 88.9 ± 1.2 95.4 ± 2.3* 92.4.9 ± 1.7 95.8 ± 1.3* 80.4 ± 0.6* 94.5 ± 0.3*

0.3 98.5 ± 1.3* 96.5 ± 0.3* 99.5 ± 0.1* 98.5 ± 0.3*

*P < 0.05, All assays were done in duplicate and the results are the mean ± SD of four experiments

by exposure to H2O2 increased susceptibility of E. coli to phagocytosis. A previous study showed that catalase activity can be induced in several organisms on exposure to exogenous H2O2.[17] Catalase is thought to be an antioxidant enzyme which protects the bacteria during oxidative stress. It has been reported that gonococci pretreated with H2O2 are signiÞcantly more resistant to neutrophils than control bacteria.[17] Such resistance seems most likely to be due to increased formation of catalase. However, we observed increased susceptibility of E. coli to phagocytosis on exposure to H2O2. The difference in the result could be due to the induction of oxidative stress. Earlier workers have used more than 0.5 mM of H2O2.[18] It could be that the concentrations of H2O2 used in this study may not have been enough to induce sufÞcient catalase. Phagocytosis of micro-organisms is a major defence mechanism in humans. Resistance to phagocytosis is an important virulence mechanism of E. coli strains isolated from cases of intestinal and extra-intestinal diseases. Phagocytosis and intracellular killing of E. coli are crucially determined by the surface properties of the bacterial cell.[18] In the present study, we observed that oxidative stress in E. coli could signiÞcantly inßuence the virulence factors of the bacterium. This, in turn, may inßuence the pathogenesis of E. coli infections.

6. 7. 8. 9. 10. 11.




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