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ISSN: 2008-5842

Editorial Office

Editor-in-chief: Mehrdad Jalalian, M.D.

Executive Editor: Hamidreza Mahboobi, M.D.

Technical Editors:

Mohammad Esmaeil Shahrzad.

Tahereh Khorgoei.

Shekoofe Amirzadeh Shams.

International Board of Editors

Dr. Sharat Agrawal (Assistant professor, North Eastern Indira Gandhi Regional Institute of Health & Medical Sciences, India)

Dr. Erfan Ahmadi (MD and MSc. in Biology, research associate in University of Manchester, U.K)

Dr. Subramaniam Uthayathas (Ph.D., Emory University, Yerkes National Primate Research Center, U.S.A)

Dr. Parichehr Hanachi (PhD. in Molecular Biology, Associate Professor, Alzahra University, Iran)

Dr. Sanjay Kalra (MD., Bharti Hospital Karnal, India)

Dr. Viroj Wiwanitkit (MD., Professor, Faculty of Medicine, Chulalongkorn University, Thailand)

Dr. Anthonia Ogbera (MBBS, MPH, MWACP, FMCP, FACE, FACP. Lagos State University Teaching Hospital Ikea, Lagos, Nigeria)

Dr. Gan Siew Hua (Ph.D. Associate Professor & Director of Human Genome Centre, School of Medical Sciences, Universiti Sains Malaysia, Kelantan, Malaysia)

Dr. Prakash Adhikari (MD., President of Nepalese Doctors Organization. Kathmandu, Nepal)

Dr. Farooq Azam Rathore (Specialist in Rehabilitation Medicine, Armed Forces Institute of Rehabilitation Medicine (AFIRM), Rawalpindi, Pakistan)

Dr. Zafar Mahmood (MD., Sirsyed College of Medical Sciences, Karachi, Pakistan)

Dr. Rajeev Aravindhaksha (Associate professor, Oman medical college, Sohar, Oman)

Dr. Hadi Ghasemi (PhD, Mashhad University of Medical Sciences, Mashhad, Iran)

Dr. Farzam Gorouhi (MD, University of California, USA)

Dr. Ghada Khalil Al Tajir (PhD, Associate Professor, Faculty of Medicine, UAE University, Al Ain, U.A.E)

Dr. Sima Besharat (MD, Golestan University of Medical Sciences, Gorgan, Iran)

Dr. Abasalt Hosseinzdeh Colagar (PhD in Molecular and Cell Biology, Assistant Professor, Department of Biology, University of Mazandaran, Iran)

Dr. Manisha Sahay (MD. Osmania Medical College, India)

Dr. Manika Agarwal (MD., Assistant professor, North Easther Indira Gandhi Regional Institute of Health & Medical Sciences, Shillong , India)

Dr. Mojtaba Mohseni (PhD., Associate Professor, University of Mazandaran , Iran)

Dr. Shadi Sarahroodi (PhD., Assistant professor, Qom University of Medical Sciences, Qom, Iran)

Dr. Gurjeet Singh Gulati (Assistant Professor, ST. Stephens Hospital, New Delhi, India)

Dr. Leila Safaeian Naeini (PhD., Isfahan University of Medical Sciences, Iran)

Dr. Rakesh Kumar Sahay (MD., Professor, Osmania Medical College, Hyderabad, India)

Dr. Saba Zubair Abbasi (MD., Jinnah Post Graduate Medical Centre, Pakistan)

Dr. Maria Razzaq Butt (MBBS, Nawaz Sharif Social Security Hospital, Lahore. Pakistan)

Dr. Akshay Sharma (MBBS, Kasturba Medical College, India)

Dr. Navneet Agrawal (MD., Assistant Professor, Gajra Raja Medical College & J A Group of Hospitals,Gwalior , India)

Dr. Mohammadkarim Bahadori (PhD. in Health Managament System, Assistant Professor, Baqiyatallah University of Medical Science, Tehran, Iran)

Dr Ali Malekshahi Moghadam (DVM in Veterinary Medicine, Nutrition Science Researcher, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran)

Dr Mahin Jamshidi (Associate professor, Tehran University of Medical Sciences, Tehran, Iran)

Akbar Heydari, (M.A. in Sport Management, University of Tehran, Tehran)
Table of contents

Writing an eye-catching and evocative abstract for a research article: A comprehensive and practical approach ……………………...……... 520

Mehrdad Jalalian

Impact of simulated patients on teaching the Sexually Transmitted Diseases (STDs) to medical student in Iran ………………………..……….. 525

Nazanin Zia Sheikholeslami, Akram Heidari, Mariam Bahsoun

Gradual and Step-wise Halophilization Enables Escherichia coli ATCC 8739 to Adapt to 11% NaCl …………………......................................... 527

Desmond JW Goh, Jian Ann How, Joshua ZR Lim, Wei Chuan Ng, Jack SH Oon, Kun Cheng Lee, Chin How Lee, Maurice HT Ling

The role of stem cells in the improvement of brain injuries after hypoxic ischemia ..…………....... 536

Mohammad Reza Nikravesh, Mehdi Jalali, Hossein Ali Ghafaripoor, Javad Sanchooli, Darioush Hamidi, Shabnam Mohammadi, Masoomeh Seghatoleslam

Does safety climate make sense in hospitals of a developing country? ….……………………….. 544

Mahmood Nekoei-Moghadam, Mohammadreza Amiresmaili

A study of knowledge and self care practices in patients of type 2 diabetes mellitus ………..…. 551

Praveen Govinda Gowda, Vittal Balakrishna Ganjigatte

Papillary Thyroid Cancer with diffuse lung metastasis …………………………...…………. 555

Somayeh khosravi, Shirin Hasani Ranjbar, Sayeh Alizad Jahani

Evidence Based Education System (EBES): Our Achievements and way forward ………......….. 560

Suresh Kumar Rathi, Sandip Shah

Fuzzy knowledge-intensive case based classification for the detection of abnormal cardiac beats …………………………………... 565

Abdeldjalil KHELASSI, Mohamed Amin Chick

Formative evaluation of Hospital Information System According to ISO 9241-10: A case study from Iran …………..…………………………... 572

Narjes Mirabootalebi, RahelehMalaekeh, Hamidreza Mahboobi

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Writing an eye-catching and evocative abstract for a research article: A comprehensive and practical approach
Mehrdad Jalalian
Editor In-Chief, Electronic Physician Journal, Mashhad, Iran
Abstract: It is an important and difficult job to write an eye catching abstract. A large percentage of the manuscripts that are submitted to academic journals are rejected because their abstracts are poorly written. This paper provides a new and step by step approach for writing a good structured abstract.

Bibliographic Information of this article:

[Mehrdad Jalalian. Writing an eye-catching and evocative abstract for a research article: A practical approach. Electronic Physician, 2012;4(3):520-524. Available at: ]. (ISSN: 2008-5842).

Keywords: Abstract; Scientific writing; Original article
© 2009-2012 Electronic Physician

1. Why is the abstract important?

Writing an abstract properly is one of the most highly-specialized forms of academic writing. The abstract, which is a very vital part of a journal article, comes first in the article, but it is the last part to be written (1). The abstract is read more frequently than any other part of the article, and it must cover all the major points of the article (2). Since prospective readers decide whether or not to read the entire article by reading the abstract (1), the abstract functions as an advertisement for your work. Unfortunately, many researchers think that writing an abstract is an easy and trivial task, so they do not expend the time, energy, or effort required to produce a good abstract. As a result, abstracts frequently are full of errors (Table 1) and do not match the original text of the article (3). After reading an abstract, the reader should know the following:

  • Why the study was conducted.

  • What the findings of the study were.

  • How the findings can be applied.

The present paper provides a detailed discussion of the features of a good abstract. Some practical guidelines for writing a very sophisticated abstract are provided. This paper is focused on the elements of structured abstracts for research articles, because such abstracts are preferred by most journals, and they have been demonstrated to be of higher quality than alternative approaches (1).

2. A practical approach

A structured abstract for an original research article is an abstract with different, labeled parts to facilitate the reader’s comprehension. This helps the readers who wish to identify and investigate further only journal articles that are focused on a specific issue and that are valid from a methodological perspective (3, 4). This method of organizing an abstract also instructs writers in summarizing the content of their research accurately (Table 2) and facilitates the peer-review process (4, 5). Writing an effective structured abstract is difficult, but it is not an impossible task. Such an abstract must be well designed, well organized, and concise (Table 3); however, the format required for a structured abstract is not the same in all journals. Their lengths and structures vary by discipline and by the publisher’s requirements. Most journals mention the specific criteria they require for abstracts in the “Instructions for Authors” section. A widely-accepted format for structured abstracts in a research article includes the following sections: Introduction, Methods, Results, and Conclusions (3).

Table 1. Common mistakes in writing an abstract

Using an inappropriate writing style

Addressing irrelevant information that is not mentioned in the original text

Including unnecessary words or phrases

Using the first line of the Introduction as the first sentence of the abstract

Compiling the abstract by selecting some sentences from the main text

Including references in the abstract

Writing long sentences (recommended maximum = 25 words)

Writing incomplete sentences

Using inappropriate tense of verbs (and improper subject-verb agreement)

Using exaggerated, overblown and grandiose words

Using non-required synonyms and repetitive words

Using “Tables” in the abstract

Using “Figures” in the abstract

Using non-essential jargons


Grammatical mistakes

Mixing “results” and “inferences (Discussion/Conclusion)”

2.1. Writing the Introduction

This section is actually a brief summary of the Introduction of the article. It may consist of three short sentences, with the first sentence specifically mentioning the core content, the second its context i.e. the background and the third addressing the objectives of the research.

2.1.1. Writing the first and second sentences

The first sentence should provide the background of the study, address the research focus, and point out the importance of the problem or the gap in the knowledge (Table 2). Usually, a good background sentence states the problem that the study addressed, provides the context for the study, and indicates the importance of the study, each of which provides information that the reader needs to know. Make sure you address the most important aspect of the research and express the restrictions of previous studies, if possible; but BE CONCISE and encapsulate all of these components in two short sentences.

2.1.2. Writing the third sentence

Here, you should quickly address the main research question or hypothesis in the form of a research objective (Table 4). You may present the overall objective of the study or address one specific, key objective. After you have formulated your research objective, be sure to write it in the appropriate form based on the type of study (e.g., qualitative vs. quantitative) and the variables that were investigated. In expressing research objectives, it is essential to use infinitives, i.e., the combination of “to” and a verb, such as “to develop”, “to determine”, and “to assess” (Table 4).

2.2. Writing Methods

In this section, you should state the methods that were used to answer the research questions. Three to four sentences should express the research design; the study population; the subject selection process; and the instruments, measurement tools, and statistical techniques that were used. In other words, you should precisely summarize the process and the fundamental procedures you used to answer your questions. A practical approach for writing this section is to begin with an explanation of the study design and its structure (Table 2). After that, you should discuss the study population, sampling methods, and the setting (e.g., hospital, clinic, university, or company) and explain the selection procedure (e.g., the selection criteria, the number of subjects selected, and the demographic characteristics of the subjects). If you are writing about an experimental study, you should explain the characteristics of any interventions that were necessary. At the end of this part, you should mention the statistical analyses that were used in the study. It is a prudent practice to mention how was acceptable validity and reliability ensured.

Table 2. Questions that should be answered before writing each part of the abstract




1st & 2nd Sentences

(The What & Why)

Ask yourself:

What is the core content of your manuscript/thesis (about WHAT?)

Why were the problem and the results important?

What problems did you aim to solve?

What was the main gap(s) in knowledge that your research was intended to fill?

3rd Sentence

(The “What for”)

Ask yourself:

What was the general objective of the study?

What was the key, specific objective of the study?

Was there a major hypothesis?


Ask yourself (The “How”):

What was the study design?

What was the population and who were the subjects of the study?

What were the key variables?

What were the entry criteria that subjects had to meet?

What measurement tool(s) did you use?

What steps did you follow and how?

What kind of statistical analyses did you use?

What steps did you use to ensure acceptable reliability & validity?


Ask yourself (The “So What”):

What were the major results of the research?

Were the results significant? How were they significant?

Did you find any meaningful changes? If so, what were the magnitudes of the effects?

Any consideration of effect size? If so, is it statistical significance or clinical significance?


Ask yourself (The “Then What”) :

What do your findings actually mean?

What are the implications of your findings?

Can the findings be generalized to other situations?

Are the results specific and limited to a particular case or situation?

Did the research findings fill the gap(s) of knowledge identified earlier in the Introduction section?

How comparable are your findings relative to those of other studies?

Did the results point towards developing a new hypothesis?

Table 3. Main rules for developing a good abstract - a recommended “to-do” list

Structure and grammar

Follow an appropriate writing style.

Use the appropriate tense for verbs.

Use active voice rather than passive voice whenever possible.

Use complete sentences.

Use correct punctuation and spaces.

Use figures and numbers appropriately.

Use past tense to report “Method” & “Results”


Highlight the main points of the article.

Include all of the notable findings and points of the article.

Create a distinct image of the content of the article.

Reflect the purpose and content of the main text.

Include precise information.

Only report your findings; do not evaluate, review, or comment on them.

Be concise, accurate, and clear.

Never present new information; just summarize the content of the article.

Include key terms.

2.3. Writing Results

In this section, you must describe you major findings, and you can use a word count that is similar to that used in the Methods section. You should clearly define the primary outcome of your research and the key information provided in the article. Confidence intervals, P-values, odds ratios, relative risks, and effect sizes are among the most common kinds of information that authors usually present in this section. You should resist the temptation to include peripheral or irrelevant information that is not included in the main text (Table 1).

2.4. Writing Conclusions

In this section, you have the opportunity to inspire your colleagues in one or two great sentences in which you state your main conclusions and recommendations. Only new, important, and major findings and their implications should be included; but, be careful to ensure that all of your conclusions are supported fully by the findings of the research; it is essential that you avoid exaggerating your findings or making rash overgeneralizations about their significance. PLEASE, don’t flash neon lights that say “I am a beginner” in the Conclusions section by using grandiose, exaggerated, and overblown descriptions of the importance of your findings! A good approach in writing the Conclusions section is to outline the key findings (but not directly pasting phrases or sentences from the “Results”) from the research and present a rational statement about their potential for beneficial applications (Table 2).

Table 4. Examples of some bad sentences and good sentences in an abstract




This paper discusses three issues. The first issue is X. The second and thirds issues are Y and Z.

This paper discusses X, Y and Z.

Use as few words as possible.

The main hypothesis of this study was: “There is a positive relationship between X and Y.”

The purpose of this study was to determine the relationship between X and Y.

Write the “hypothesis” in “objective” form.

The purpose of this study was to investigate the relationship between X and Y.

The purpose of this study was to determine the relationship between X and Y.

Use the appropriate verbs to describe the objectives of the quantitative and qualitative research.

According to Author et al. (2009), there is a significant relation between X and Y.

There is a significant relationship between X and Y.

Do not use a reference in the abstract.

There is an extreme relationship between X and Y.

There is a significant relationship between X and Y.

Use words that are statistically meaningful, and avoid using words that have general meanings, such as extreme, extremely, very, outstanding, and huge.

There was a huge difference between the mean ages of the two groups.

There was a significant difference between the mean ages of the two groups (P < 0.05).

  1. Use P-values, confidence intervals, and Odds ratios whenever possible.

  2. Use statistically-meaningful words and avoid using words that have general meanings, such as extreme, extremely, very, outstanding, and huge.

It was examined by the study …

The study examined …

Avoid using the passive voice whenever possible.

I examined …

  1. The study examined X, Y, and Z.

  2. We examined X, Y, and Z.

  3. In the study, X, Y, and Z were examined.

Avoid using the first person whenever possible (Use the alternative form No.1). If you really need to do so then using “We” is much better than “I” (the alternative form No.2). Even using the passive voice (the alternative form No.3) is much better than using “I”.

3. Ask the expert

Writing a good succinct abstract is difficult, because your goal is to convey as much content as possible in the fewest words possible. Therefore, using scientific writing services (6) and asking an expert to review the abstract (or the entire manuscript) before submission are often good ideas. It is recommended that you add this article to the reference section of your manuscripts and place the appropriate citation in the text if you use the approach recommended in this article. For example, you can cite this article using the following sentence at the end of the Methodology section of your article: “The report of the study was prepared based on the journal format and other guidelines provided in this reference (Jalalian, 2012)”.

Corresponding Author:

Dr. Mehrdad Jalalian,

Editorial office, Electronic Physician Journal,

Mashhad, Iran

Website (Persian):

Website (English):


  1. Alain Dupuy, MD; Kiarash Khosrotehrani, MD; Céleste Lebbé, MD, PhD; Michel Rybojad, MD; Patrice Morel, MD. Quality of Abstracts in 3 Clinical Dermatology Journals. Arch Dermatol. 2003; 139(5):589-593. doi:10.1001/archderm.139.5.589

  2. Margaret A. Winker. The Need for Concrete Improvement in Abstract Quality. JAMA. 1999; 281(12):1129-1130. doi:10.1001/jama.281.12.1129

  3. Turner A. English Solutions for Engineering and Sciences Research Writing: A guide for English learners to publish in international journals. Hanyang University Center for Teaching and Learning; 2009. Accessed online on May.20.2012. Available at:

  4. Haynes RB, Mulrow CD, Huth EJ, Altman DG, Gardner MJ. More informative abstracts revisited. Ann Intern Med. 1990 Jul 1; 113(1):69-76. PubMed PMID: 2190518. Available from: .

  5. Hayward RS, Wilson MC, Tunis SR, Bass EB, Rubin HR, Haynes RB. More informative abstracts of articles describing clinical practice guidelines. Ann Intern Med. 1993 May 1; 118(9):731-7. PubMed PMID: 8460861. Available from: .

  6. Native American Editors (NAE). Editing, Scientific writing and publishing service for academic works. Available Online from:

Letter to the Editor
Impact of simulated patients on teaching the Sexually Transmitted Diseases (STDs) to medical student in Iran
Nazanin Zia Sheikholeslami 1, Akram Heidari 2, Mariam Bahsoun 3
1. Associated professor of Infectious Disease, Qom University of Medical Sciences, Qom, Iran

2. Assistant professor of Community Medicine, Qom University of Medical Sciences, Qom, Iran

3. Department of Clinical Research, Ali Ebne Abitaleb Hospital, Rafsanjan University of Medical Sciences, Rafsanjan, Iran

Bibliographic Information of this article:

[Nazanin Zia Sheikholeslami, Akram Heidari, Mariam Bahsoun. Impact of simulated patients on teaching the Sexually Transmitted Diseases (STDs) to medical student in Iran. Electronic Physician, 2012;4(3):525-526. Available at: ]. (ISSN: 2008-5842).

Keywords: Gonorrhea; Medical education; Simulated patient; Sexually Transmitted Disease (STD)

© 2009-2012 Electronic Physician

Dear Editor,

Outpatient education is one of the critical problems in educational hospitals in Iran especially on the topic of Sexually Transmitted Diseases (STDs). In addition, the STDs are among the prevalent diseases that because of the moral aspects of accessing these patients we have some difficulties in teaching of medical students. Therefore, a simulated patient has been used for educational studies in recent years. Learners believe that using simulated patient would improve the learning experience (1); so, this method of education is increasingly used in some educational clinics (2). Simulated patients are not used in a particular ward, but they can be used in all wards including internal and surgical wards (3). Education of STD could by this method of education is proved to be useful in other countries (4). In this study we used the innovative education for medical students on teaching the gonorrhea by using simulated patients for the first time in Iran. In a seven years study from 2005, 400 students and interns of Rafsanjan and Qom Medical Universities was assessed by this method. After passing the educational courses on gonorrhea, they were given a questionnaire which included 15 questions on taking the past history of gonorrheal patients, dealing with them and their families, examining, treatment and their follow up. In the next step, the students were asked to take the medical history of the simulated patient. All of the students received the feedback from their professor about their mistakes on history taking and the way they deal with the stimulated patients. Finally, an educational visual material was presented to the students. After one month, the questionnaires were administered to the students again as a re-test. Our pre-test findings showed that 69% of students did not appropriately know how to take the history of a gonorrheal patient, 75% of them did not know how to cope with gonorrheal patients and their families, 68% were not familiar with lab tests, and 60% of participants did not know how to treat gonorrhea disease. The post test figures for the above mentioned statistics were 11%, 8%, 15%, and 21% respectively. It should be mentioned that all of the students were satisfied with this method of education. It is recommended that this educational method (especially in gonorrheal diseases) be used in all universities of the country as well as in clinical practical classes. A more controlled educational experiment can be a good recommendation for future research.
Corresponding Author:

Nazanin Zia Sheikholeslami

Qom University of Medical Sciences, Qom, Iran

Tel: +98.251771351


  1. Lareau SA, Kyzer BD, Hawkins SC, McGinnis HD. Advanced Wilderness Life Support Education Using High-Technology Patient Simulation. Wilderness Environ Med. 2010 Jun;21(2):166-170.e2.

  2. Forrest K, McKimm J. Using simulation in clinical education. Br J Hosp Med (Lond). 2010 Jun 9;71(6):345-349.

  3. Bashankaev B, Baido S, Wexner SD Review of available methods of simulation training to facilitate surgical education. Surg Endosc. 2010 Jun 15. [Epub ahead of print]

  4. Russell NK, Boekeloo BO, Rafi IZ, Rabin DL. Unannounced simulated patients' observations of physician STD/HIV prevention practices. Am J Prev Med. 1992 Jul-Aug;8(4):235-40.

Original Article
Gradual and Step-wise Halophilization Enables Escherichia coli ATCC 8739 to Adapt to 11% NaCl
Desmond JW Goh1,§, Jian Ann How1,§, Joshua ZR Lim1,§, Wei Chuan Ng1, Jack SH Oon1, Kun Cheng Lee1, Chin How Lee1, Maurice HT Ling1, 2
1. School of Chemical and Life Sciences, Singapore Polytechnic, Singapore

2. Department of Zoology, The University of Melbourne, Melbourne, Australia

§.These authors have equal contributions


Introduction: Escherichia coli (E. coli) is a non-halophilic microbe and is used to indicate faecal contamination. Salt (sodium chloride, NaCl) is a common food additive and is used in preservatives to counter microbial growth. Previous studies had shown that pathogenic E. coli has a higher salt tolerance than non-pathogenic E. coli. The effect of how E. coli interacts with the salt present in the human diet is under-studied. Thus, it is important to investigate this relationship.

Methods: In this study, we observed the genetic changes and growth kinetics of E. coli ATCC 8739 under 3% - 11% NaCl over 80 passages. Growth kinetics was estimated by generation time, cell density and minimum inhibitory concentration (MIC) of NaCl.

Results: Our results suggested that E. coli was able to adapt from 1% NaCl to 11% NaCl with an increment of 1% NaCl per month. Our MIC results suggested that E. coli was able to grow at NaCl concentration of more than 7.5% based on the Area under Curve (AUC) from 5% at passage 44 (cultured in 5% NaCl) to 13% at passage 72 (cultured at 7% NaCl).

Conclusion: We conclude that E. coli ATCC 8739 can be adapted to grow in 11% NaCl by incremental adaptation.

Bibliographic Information of this article:

[Desmond JW Goh, Jian Ann How, Joshua ZR Lim, Wei Chuan Ng, Jack SH Oon, Kun Cheng Lee, Chin How Lee, Maurice HT Ling. Gradual and Step-wise Halophilization Enables Escherichia coli ATCC 8739 to Adapt to 11% NaCl. Electronic Physician, 2012;4(3):527-535. Available at: ]. (ISSN:2008-5842).

Keywords: Escherichia coli (E. coli); Halophilization; Growth kinetics; Adaptation; Sodium chloride

© 2009-2012 Electronic Physician

1. Introduction

Escherichia coli is a textbook example of non-halophilic bacteria and is an indicator organism for faecal contamination of water as E. coli is a more consistent predictor of gastrointestinal illness than other bacterial indicators in water (1). This corroborates Burton et al. (2) whom suggested that E. coli was a suitable predictor of Salmonella enterica serovar newport in various freshwater sediments and has been observed to survive as long as or longer than Salmonella spp.; thus, fulfilling its requirement as an indicator for pathogenic bacteria. In addition, the survival of E. coli is independent of the amount of organic matter (2). This suggested that E. coli is a suitable indicator as it can survive in media of different nutritional richness.

Doudoroff (3) demonstrated that the viable count of E. coli previously cultured in ordinary fresh water media then transferred to saline nutrient solutions remained at a constant up to 7% NaCl concentration. The viable count dropped progressively with further increase in concentration suggesting that E. coli is non-halophilic and 7% NaCl is bacteriostatic. It was observed that concentration of yeast autolysate, aeration and physiological conditions affected E. coli’s overall salt tolerance at high concentration of NaCl (3). The relation of viable count to NaCl concentration (3) corroborated the findings of Vaas (4) for Bacillus megatherium where the growth of E. coli in a saline environment is greatest at the early stationary phase and lowest in the logarithmic phase (3).

Previous studies (5-8) on E. coli’s adaptation and evolution were carried out using antibiotics and drugs. Recently, a study in which pigs treated with ampicillin, a common antibiotic, demonstrated a significant increase in the occurrence of ampicillin-resistant E. coli from 6% to more than 90% after a course of 7 days (9) suggesting incomplete intestinal absorption as the evolutionary pressure for intestinal bacteria such as E. coli towards antibiotic resistance (10-11). However, common food additive such as salt, which is used to preserve food and inhibit microorganisms, is less understood in terms of E. coli’s adaptive mechanism although recent studies (12-13) had suggested that E. coli is able to adapt to food additives over extended culture. This suggests that E. coli may be able to adapt to higher concentrations of food additives but this has yet to be studied. The adaptation of E. colisalt may suggest similar resistance to other preservatives and its ability to grow in saline environment may be underestimated. In addition, number of stress adaptation studies had demonstrated that the growth phases may have an impact on E. coli adaptation. Nair and Finkel (14) suggested that a non-specific DNA binding protein, dps, may confer multiple stress tolerance at stationary phase, which concur with Jolivet-Gougeon et al. (15).

We define halophilization as the adaptation of the organism to the inhibitory effects of salt (sodium chloride; NaCl) by introducing increasing concentrations of salt into the growth environment. The adaptation can either be gradual which requires more than one genetic or epigenetic change or instantaneous whereby only one genetic or epigenetic change is required. Increasing salt concentration can cause a variety of stresses (16) such as osmotic and ionic stress. Organisms that grow in a salty environment experience osmotic stress cause by the influx of salt (reduced aw) and efflux of water molecule out of the cells. This is important to water monitoring as osmotic stress is closely related to water activity (aw), defined as the amount of water that is available for growth and reproduction of an organism (17) and is inversely proportional to the solute concentration at the same temperature. Pure water has an aw of one.

The growth of E. coli is optimal when it has a higher aw which normally requires an aw of at least 0.98 which corresponds to about 5% NaCl (18) at physiological temperature (19). This corresponds to Presser et al. (20) which demonstrates the inhibitory effect of combinations of water activity and pH varied with temperature for the growth of E. coli. E. coli demonstrated prolonged lag phase when exposed to NaCl and decrease in growth rate in increasing NaCl concentration (21) resulting in the efflux of water (16). However, E. coli 0157:H7 strain had been shown to be tolerant to low pH and low water activity (22). It has been suggested that E. coli copes with the high concentration of NaCl by accumulating organic osmolytes (23-24) such as glycine, betaine, glycerol and trehalose. It has also been suggested that high levels of betaine in E. coli enables adaptation to heat stress (24). Hence, it may be plausible that salt tolerance may result in increased tolerance to other chemical stresses.

A study (25) comparing the halotolerance of E. coli to a halophilic bacterium, Vibrio spp., using carbonyl cyanide m-chlorophenylhydrazone (CCCP) and NaCl suggested that the growth-inhibitory effect of CCCP was less on E. coli compared to Vibrio spp. However, the growth-inhibition of NaCl was more pronounced for E. coli but stable for Vibrio spp. when concentration of sodium chloride increases. This suggests that the minimum inhibitory concentration (MIC) of E. coli is lower as compared to Vibrio spp. when grown on NaCl and the resistance of E. coli was dependent on the medium. Another study (26) demonstrated that E. coli O157:H7were cultured in brain heart infusion (BHI) experienced more morphological damage and alteration with increasing NaCl concentration compared to Staphylococcus aureus in 0%, 5% and 10% NaCl. This agrees with Jay (27) suggesting that E. coli O157:H7 tolerates up to 8% NaCl in culture media and up to 20% for certain strains of S. aureus.

Taken together, these studies (5, 18, 27) suggested that different strains of E. coli have different salt tolerance with the pathogenic E. coli O157:H7 strain having the highest salt tolerance. However, the reason as to why pathogenic E. coli are more salt tolerant to its non-pathogenic counterparts is not obvious although it may be hypothesized that constant low level exposure to chemicals, such as antibiotics and preservatives, during food preparation and medical treatment may provide a selective pressure towards chemical tolerance.

This study aims to observe gradual adaptation of E. coli ATCC 8739, a fully sequenced strain, cultured in NaCl supplemented medium up to 8% NaCl over 80 passages. Our results suggest that E. coli adapted to 1% increase in NaCl every month with a successful adaptation to 11% NaCl.

2. Materials and Methods
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