Experimental study of the survival and growth of Pseudomonas aeruginosa in water affected by temperature, storage time and type of water.
MATERIALS AND METHODS
A multifactorial design was used to investigate the effects of temperature, inoculation dose, and type of water as a nutrient on the growth of P.aeruginosa. At first, a microbial experiment was conducted to investigate the existence of P.aeruginosa in the water samples. This experiment was conducted through 3-tube MPN1 method according to existing standards, both by Durham tube to analyze coliform bacteria and without Durham tube for general microbial analysis (14). The result of the experiment showed no opacity in the analyzed tubes. The same experiments were conducted on inoculated water samples and the results were recorded. In order to prepare the first inoculation of the bacterium, the standard pure colony of P.aeruginosa with ATCC 27853 was taken to 10 ml BHI broth and was incubated at 30 C[degrees] and after 24 hours, 1ml of the first broth was taken to the second 10 ml broth and was incubated at 30 C[degrees], and after 24 hours the bacterial count was conducted and full growth of the bacterium (1.5 x 109 cfu) was achieved and then, standard dilution was conducted (14). In order to achieve the inoculation dose of 1.5 x [10.sup.4] /ml, 0.1 CC from -2 dilution tube which contained [10.sup.7] cfu of the bacterium was carried to 100 CC of each of the water samples. In the end, the inoculation dose of the bacterium in each of the water samples was 1.5 x [10.sup.4] /ml. Then, in order to examine the growth and to draw the growth curve of the bacterium in samples at different times under sterilized and nonsterilized conditions, mineral and tap waters were sampled, and after preparing a dilution by surface plate count in BHI Agar, it was incubated at 30 C[degrees] for 24 hours (14). After counting the colonies in plates, the results were recorded at different times (day zero, 3, 6, 12, 24, and 48). In order to achieve a higher precision, each of the stages and the combined factors were cultured in Cetrimid Agar as well as BHI Agar and the bacterial growth curve was drawn for the two media. There were 8 types of water samples in this study: sterilized bottled mineral water, non-sterilized bottled mineral water, sterilized tap water, non-sterilized tap water, each of which was stored at two storage temperatures of 7 and 22 C[degrees]. Considering the objective of the study, the culture was conducted at certain days (day zero, 3, 12, 24, 48), and the results were recorded.
The results of bacterial counts in different dilutions of different types of water in BHI Agar and Cetrimide Agar were recorded. A specific dilution was chosen for each experiment and the logarithmic analysis of the data is presented in table 1.
This study was conducted by a 2 (4) factorial experiment with 16 treatments without repetition. The bacterial count results were changed into logarithm to base 10 before any statistical analysis. The methods REG, GLM, and ENTROPY and software SAS (version 9, 1) were used to analyze the data (SAS Institute Inc., 2002-2003). All the main and interactive effects were analyzed with the statistical p value set at p < 0.05.
The survival and growth of bacteria in water
On day zero (after the inoculation of the bacterium) there was no statistically significant difference between the effects of the factors on the growth of the bacterium and the arithmetic means of the logarithm of the bacterial count at the given time were 3.911 and 4.166 for mineral water and tap water. The storage temperature of water had a big effect on the bacterial count three days after the beginning of the experiment and the other factors (in main and interactive effects) didn't have a noticeable effect on this feature. Considering the variance analysis results for this feature, storage temperature of 22 C[degrees] with the mean of 6.031 meaningfully (P<0.01) increased the number of the bacteria compared to temperature storage of 7 C[degrees] with the mean of 3.922. Six days after the beginning of the experiment the main effective factor in the growth of the bacteria was storage temperature too. Also, analyzing the bacterial count on day twelve showed the storage temperature of 22 C[degrees] compared to that of 7 C[degrees] (with corresponding means of 5.607 and 3.436, respectively) had a statistically meaningful effect on the growth of the bacteria (P<0.01) . On day twenty four also the main effective factor was storage temperature followed by the sanitary condition of water. In this regard, storage temperature of 22 C[degrees] compared to that of 7 C[degrees] (with corresponding means of 5.691 and 3.924, P<0.01, P<0.01) and using sterilized water compared to non-sterilized tap water (with corresponding means of5.308 and 4.307, P<0.01, P<0.05) were more effective in the growth of the bacteria. On day forty eight, storage temperature of 22 C[degrees] (compared to that of 7 C[degrees], with corresponding means of 5.686 and 4.233), using bottled mineral water (compared to tap water, with corresponding means of 5.409 and 4.510) and using sterilized water (compared to non-sterilized water with corresponding means of 5.453 and 4.466) significantly increased the multiplication of the colonies.
In this study the effect of storage time, temperature of water, sanitary condition of water on the growth of P.aeruginosa in drinking water (mineral and tap water) was investigated. The results showed that P.aeruginosa grows well in water and among all factors studied here, temperature of water proved the most effective on the growth and survival of the bacterium. The most bacterial count was in 22 C[degrees] water (P<0.01). However, due to being cold loving, as more time passes, the bacterium may grow as much as it does in 22 C[degrees]. Also, the bacterium continues to survive in water after 48 days. On day zero (after inoculation) no factor was effective on the growth of the bacterium, but after 3 days, temperature of water had a great effect on the growth of the bacterium. The effect of temperature continues to exist from day 3 until day 48 and the effect of 22 C[degrees] water was more than that of 7 C[degrees] water and this is because generation time of the bacterium is shorter at higher temperature. From day 12 on, the interactive effect of temperature (22 C[degrees]) and the sanitary condition of water was significantly meaningful and the sanitary condition of water (sterilized and non-sterilized) from day 12 until day 48 had a positive effect on the growth of the bacterium i.e. the growth of the bacterium was more in sterilized than in non-sterilized water (P<0.05). on day 24, the most effective factor was temperature (22 C[degrees]) followed by sanitary condition of water i.e. the growth of the bacterium was more in sterilized than in non-sterilized water and this could be due to the interruption of normal flora of water by sterilization and the lack of microbial competition for the growth of P aeruginosa. 48 days after the storage of waters, type of water also was an effective factor i.e. the growth of the bacterium in mineral water was more than that in tap water (P<0.01) and this could be due to the abundance of mineral contents required for the growth of the bacterium in mineral water compared to those in tap water. Water temperature (22C[degrees]) and sanitary condition of water (sterilized) were effective and caused an increase in the growth of the bacterium. Generally, the effect of temperature was the most effective factor throughout the study.
Manaia et al. (2008) studied 15 brands of noncarbonated bottled mineral water and isolated P.aeruginosa. The results of their study, too, showed the positive effect of type of water (mineral) on the growth of P.aeruginosa (7). Blance et al. (2004) mentioned faucets of intensive care units of hospitals as an endemic source of P.aeruginosa. Studying persistence of the bacterium was part of this study and the bacterium was shown to exist in water after 48 days (3). Legnani et al. (1999) in a 5-year study on two-tailed growth curve of P aeruginosa inoculated at the density of [10.sup.2] cfu/ ml in samples of natural mineral water found that the number of bacteria increased by 3 Log after 4-5 days. The results of this study too, showed the positive effects of type of water (bottled mineral water) and storage time on the growth of P.aeruginosa (6). Bagheri et al. (2013) isolated P.aeruginosa from the spa pools in Sarein. The results of this study also showed the effect of type of water (mineral) and storage time on the growth of P.aeruginosa (2). Baghal Asghari et al. (2013) by analyzing Pseudomonas Infections found in hospitals concluded that detecting the source of pollution is a high priority in preventing such infections in hospitals. This study also found type of water as a positive factor on the growth of P.aeruginosa (1).
Analyzing the survival and growth of P.aeruginosa in bottled mineral waters (sterilized and non-sterilized) and tap waters (sterilized and non-sterilized) at temperatures of 22 and 1 C[degrees] in storage time of 48 days after the inoculation shows that the bacterium has a noticeable growth in water and storage temperature (22 C[degrees]) (P<0.01) and sanitary condition of water (sterilized) (P<0.05) and type of water (mineral) (P<0.01) have a positive effect on the growth of P.aeruginosa. All in all, water temperature was the most effective factor in the growth and survival of the bacterium i.e. the bacterium had the greatest growth at 22 C[degrees] (P<0.01). Considering the survival of the bacterium 48 days after the inoculation, it is suggested that its growth be investigated after 96 days after inoculation at 31 C[degrees] which is the desired temperature for the growth of the bacterium.
(1.) Baghal Asghari, F. Nik Ayin, M. The importance of Bio films of water systems in infection's spread of Pseudomonas aeroginosa in hospital 2013.
(2.) Bagheri Ardebilian, P. Sadeghi, H. Fazlzadeh davil, M. Rostami, R. Poureshgh Y. Pseudomonas aeroginosa in Public Hot Spring Pools of Sareyn country 2013.
(3.) Blanc, D.S. Nahimana, I. Petignat, C. Wenger, A. Bille, J. Francio, P., Faucets as a reservoir of endemic Pseudomonas aeruginosa colonization/ infections in intensive care units, 2004; (10). pp 1964-1968.
(4.) Jawetz, Melnick and Adelberg's Medical microbiology, Twenty. Fifth Edition- LANGE Basic Science.
(5.) Jay, J.M., Modern Food microbiology, Fourth Edition, chapman and Hall, Newyurk, 1992; pp: 4695-469.
(6.) Legnani, P. Leoni, E. Rapuano, S. Turin, D. Valenti, C., Survival and growth of Pseudomonas aeroginosa in natural mineral water: a 5- year study, International Journal of Food Microbiology, 53(2-3): Pages 153-158.
(7.) Manaia, Celia. Nunes, Olga C. Morais, Paula V. and Dacosta, M. Heterotrophic Plate counts and the isolation of bacteria From mineral waters on selective and enrichment media, 1990; 69: 871-876.
(8.) Nabizadeh, R. Faezi Razi, D. Drinking water quality guidelines 1996.
(9.) Nami Fard, Zahra. Water borne diseases 2010.
(10.) Noroozi, J. Pathogenic bacteria 2008.
(11.) Razavilar, V. Pathogenic Microorganisms in Foods and Epidemiology Food Poisoning 2002.
(12.) Razavilar. V. and C. Lenigeorgis. Interactive effect of trmperature, atmosphere and storage time on the Probability of colony formation on blood agar by four listeria Species. J. Food. Prot, 1992; 55: 88-92.
(13.) Razavilar, V Hazard analysis critical control point (HACCP) and Modeling In: Proc., Food Safety sym posium. Univ Of medical Sciences of Tehran, Iran 1997.
(14.) Speck, marvin L. Compendium of methods for the microbiological examination of foods. Prepared by the APHA intersociety Agency committee on microbiological methods for foods 1984.
(15.) Tamagnin, L.M and Gonzalez, r.D. Bacteriological Stability and growth kinetics of Pseudomonas aeruginosa in bottled water, Article First published online: 25 NOV 2003, Society for Applied Bacteriology, 1997; 83: 91-94.
(16.) Trautmann, Matthias. Lepper, Philipp M. Haller, Mathias. Ecology of Pseudomonas aeruginosa in the intensive care unit and the evolving role of water outlets as a reservoir of the organism, 2005; 33(5): Supplement, Pages S41-S49.
(17.) Trautmann, Matthias. Michalsky, Thomas. Wiedeck, Heidemarie. Radosavljevic, Vladan. Ruhnke, Markus. Tap Water Colonization With Psmdomonas aeruginosa in a Surgical Intensive Care Unit (ICU) and Relation to Psmdomonas Infections of ICU Patients, 2001; : 49-52.
(18.) Varnan, A.H., E vans, M.G. (2005). Food borne Pathogens, an illustrated text. 3th edition, manson publications.
(19.) Walls, I. and scotl, V.N. Use of predictive microbiology in microbial food safety risk assessment. Int. J. Food Microbial, 1997; 36 (2-3): 97-102.
Azin Takalou , Vadood Razavilar  * and Mohammad Reza Abedini 
 Departmant of Hygiene, Faculty of Veterinary Sciences, Science and Research Branch, Islamic Azad university, Tehran, Iran.
 Departmant of Animal Sciences, Agriculture College, Varamin Branch, Islamic Azad university, Varamin, Iran.
(Received: 10 December 2015; accepted: 20 January 2016)
* To whom all correspondence should be addressed. E-mail: firstname.lastname@example.org
Caption: Diagram 1. The effect of temperature of waters at different times of the experiment
Caption: Diagram 2. The effect of type of water at different times of the experiment
Caption: Diagram 3. The effect of sanitary condition of waters at different times of the experiment
Table 1. The results of the logarithmic bacterial growth in a suitable dilution for each type of water Observation Day zero Day 3 Day 6 Type of water Mineral sterilized 4 6.471292 6.278754 22[degrees] Normal form 4 6.326336 6.093422 Mineral sterilized 3.778151 4 3.778151 7[degrees] Normal form 3.954243 4.633468 3.30103 Tap water sterilized 4 6.808886 6.614897 22[degrees] Normal form 4.845098 5.60206 5.30103 Tap water sterilized 4.544068 3.954243 3.477121 7[degrees] Normal form 3.845098 4 4.380211 Mineral sterilized 4 4.357935 6.423246 22[degrees] Normal form 4 6.50515 5.139879 Mineral sterilized 3.60206 3.477121 3.778151 7[degrees] Normal form 3.954243 3.845098 3 tap water sterilized 4 6.634477 6.487138 22[degrees] Normal form 4.113943 5.544068 5.240549 Tap water sterilized 3.832509 3.623249 2.30103 7[degrees] Normal form 4.146128 3.845098 3.30103 Observation Day 12 Day 24 Type of water Mineral sterilized 6.257679 6.448706 22[degrees] Normal form 5.812913 6.176091 Mineral sterilized Lab.Error 5.758912 7[degrees] Normal form 3 3.30103 Tap water sterilized 6.363612 6.523746 22[degrees] Normal form 4.278754 3.60206 Tap water sterilized 3 3.69897 7[degrees] Normal form 4.69897 4.380211 Mineral sterilized 6.303196 6.429752 22[degrees] Normal form 5.50515 6.217484 Mineral sterilized 3.30103 3.30103 7[degrees] Normal form 3.30103 3.778151 tap water sterilized 6.334454 6.429752 22[degrees] Normal form 4 3.69897 Tap water sterilized 3.146128 3.869232 7[degrees] Normal form 3.60206 3.30103 Observation Day 48 Type of Type of water culture medium Mineral sterilized 6.41162 BHI Agar 22[degrees] Normal form 6.462398 BHI Agar Mineral sterilized 4.322219 BHI Agar 7[degrees] Normal form 4.778151 BHI Agar Tap water sterilized 6.518514 BHI Agar 22[degrees] Normal form 4.079181 BHI Agar Tap water sterilized 4.845098 BHI Agar 7[degrees] Normal form 3 BHI Agar Mineral sterilized 6.346353 Cetrimid Agar 22[degrees] Normal form 6.060698 Cetrimid Agar Mineral sterilized 4.146128 Cetrimid Agar 7[degrees] Normal form 4.748188 Cetrimid Agar tap water sterilized 6.307496 Cetrimid Agar 22[degrees] Normal form 3.30103 Cetrimid Agar Tap water sterilized 4.725912 Cetrimid Agar 7[degrees] Normal form 3.30103 Cetrimid Agar
|Printer friendly Cite/link Email Feedback|
|Author:||Takalou, Azin; Razavilar, Vadood; Abedini, Mohammad Reza|
|Publication:||Journal of Pure and Applied Microbiology|
|Date:||Mar 1, 2016|
|Previous Article:||Importance of Picoa spp. as desert truffles fungi.|
|Next Article:||Biophysical removal of some toxic heavy metals by Aeromonas strains.|