Adaptation of Escherichia coli ATCC 8739 to 11 % NaCl

Escherichia coli (E. coli) is a nonhalophilic microbe and used to indicate faecal contamination. Salt (sodium chloride, NaCl) is a common food additive and is used in preservatives to encounter microbial growth. e effect of how E. coli interacts with the salt present in the human diet is unclear. us, it is important to investigate this relationship. In order to adapt and survive the changes in the environment, E. colimay undergo halophilization. In this study, we observed the genetic changes and growth kinetics of E. coliATCC 8739 under 3%–11%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. Gram staining and PCR/RFLP showed that the cultures are Gram negative and the DNA pro�les of all 4 replicates to be similar, suggesting that the cultures had not been contaminated.


Introduction
Escherichia coli is a textbook example of nonhalophilic 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].is 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, ful�lling its requirement as an indicator for pathogenic bacteria.In addition, the survival of E. coli is independent of the amount of organic matter [2].is suggested that E. coli is a suitable indicator as it can survive in media of different nutritional richness.
Previous studies [3][4][5][6] on the adaptation and evolution of E. coli were carried out using antibiotics and drugs.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 [7,8] had suggested that E. coli is able to adapt to food additives over extended culture.is suggests that E. coli may be able to adapt to higher concentrations of food additives but this has yet to be studied.Doudoroff [9] demonstrated that the viable count of E. coli previously cultured in ordinary fresh water media and then transferred to saline nutrient solutions remained at a constant up to 7% NaCl concentration.e viable count dropped progressively with further increase in concentration suggesting that E. coli is nonhalophilic and 7% NaCl is bacteriostatic.Hrenovic and Ivankovic [10] reported that the growth of E. coli is optimal below 5% NaCl.e adaptation of E. coli to salt may suggest similar resistance to other preservatives and its ability to grow in saline environment may be underestimated.
is study aims to observe gradual adaptation of E. coli ATCC 8739, a fully sequenced strain, cultured in NaClsupplemented 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.Gram staining and DNA �ngerprinting by PCR/restriction fragment length polymorphism at Passage 72 showed that the cultures are Gram negative and the PCR-RFLP pro�les of all 4 replicates to be similar.is suggested that the cultures had not been contaminated with Staphylococcus aureus, which is Gram positive and the most likely contaminant as S. aureus is a salt-tolerant commensal found on human skin.

Methodology
Main culture experiment was carried out with an initial inoculum of 9.7×10 6 E. coli ATCC 8739 cells from Passage 70 of Lee et al. [8] as the �rst passage in each of the 4 replicates of 10 mL of 1X nutrient broth with a �xed concentration of NaCl (we refer to this as adaptation media) and cultured in tightly capped 15 mL conical tubes.Subculture was performed using 1% of the previous culture on every odd day except Sunday (3 subculturing per week).NaCl concentration for the passages was as follows: Passages 1 to 15 at 3% NaCl, Passages 16 to 31 at 4% NaCl, Passages 32 to 39 at 4.5% NaCl, Passages 40 to 50 at 5% NaCl, Passages 51 to 62 at 6% NaCl, Passages 63 to 74 at 7% NaCl, and Passages 75 to 80 at 8% NaCl.
Contamination monitoring was as follows.e cultures were monitored routinely for contamination using Gram staining and DNA �ngerprinting.e DNA �ngerprinting by PCR/restriction fragment length polymorphism was performed using the procedure in a previous similar adaptation study [8] where each of the 3 primers (Primer 5, CgCgCTggC; Primer 6, gCTggCggC; and Primer 7, CAggCggCg) was used as both forward and reverse primers.e PCR reaction was carried out (Hybaid Limited, PCR express) under the cycling condition of initial denaturation at 95 ∘ C for 10 minutes; 35 cycles of ampli�cation at 95 ∘ C for 1 minute, 27 ∘ C for 1 minute, and 72 ∘ C for 3 minutes; followed by a �nal extension at 72 ∘ C for 10 minutes before digestion with 1 unit of TaqI restriction endonuclease for 16 hours at 65 ∘ C before analysis on 2% (w/v) agarose gel with 1X GelRed.
Passage monitoring was as follows.e growth conditions of each passage were monitored by optical density at 600 nm wavelength (OD600 readings) using Shimadzu UV-1601 UV/light spectrophotometer.OD600 reading of each replicate was taken prior to each subculture for estimating inoculum density.OD600 readings were taken at the �h and seventh days aer inoculation for estimating cell density at stationary phase.At every third passage, 10 L of E. coli culture from each replicate (A, B, C, and D) was inoculated into 1 mL of 1X nutrient broth and OD600 readings taken at

Dataset Description
e dataset associated with this Dataset Paper consists of 5 items which are described as follows.
Dataset Item 1 (Table ).OD600 readings for each replicate taken at the day of the next subculture which can be used to estimate the inoculum density of each passage and the number of generations between each culture.e table is in long format where each row has only one measured data attribute.In this case, the measured data will be the OD600 readings.e other attributes are descriptors of the measured data attribute.is is opposed to the wide format whereby the descriptors for a particular OD600 reading are given as row and column labels.e table contains only 3 attributes: Passage, Replicate, and OD600 Reading denoting the passage number, the replicates labeled from "A" to "D", and the OD600 measurement, respectively.).OD600 readings for each replicate ta�en at the ��h and seventh days a�er inoculation for stationary phase cell density estimation.e table is in long format where each row has only one measured data attribute.In this case, the measured data will be the OD600 readings.e other attributes are descriptors of the measured data attribute.is is opposed to the wide format whereby the descriptors for a particular OD600 reading are given as row and column labels.e table contains 4 attributes: Passage, Replicate, Day, and OD600 Reading denoting the passage number, the replicates labeled from "A" to "D", whether the OD600 reading was performed on the �h or seventh day aer inoculation, and the OD600 measurement, respectively.).OD600 readings taken at inoculation (time zero or zero minutes), 2 hours aer inoculation, and at regular intervals aer the second hour.is data can be used for generation time estimation.e table is in long format where each row has only one measured data attribute.In this case, the measured data will be the OD600 readings.e other attributes are descriptors of the measured data attribute.is is opposed to the wide format whereby the descriptors for a particular OD600 reading are given as row and column labels.e table contains 4 attributes: Passage, Replicate, Minutes, and OD600 Reading denoting the passage number, the replicates labeled from "A" to "D", the amount of time in minutes aer inoculation when the OD600 readings were taken, and the OD600 measurement, respectively.).OD600 readings from MIC experiment of each colony whereby 10 random colonies were selected for each replicate (total of 40 colonies were chosen) aer single colony isolation from each of the 4 replicates.In this case, the measured data will be the OD600 readings.e other attributes are descriptors of the measured data attribute.is is opposed to the wide format whereby the descriptors for a particular OD600 reading are given as row and column labels.e table contains 5 attributes: Passage, Replicate, Colony, [NaCl], and OD600 Reading denoting the passage number, the replicates labeled from "A" to "D", the colony in 2 alphabets where the �rst alphabet denotes the replicate and the second alphabet denotes the colony, the concentration of NaCl for MIC estimation, and the OD600 measurement, respectively.For example, the colony labeled as "DA" represents colony "A" of replicate "D" (see Tables 2, 3, and 4 containing colony MIC data given in wide format for easy reference).

Concluding Remarks
Our MIC results showed an increase in OD600 readings from 11% (w/v) NaCl-supplemented nutrient media aer Passage 60 (Figure 1) suggesting that E. coli had adapted to 11% (w/v) NaCl.is is further veri�ed by MIC on randomly selected colonies at Passage 72 (Table 4).us, this study provides a set of data which may be used as benchmark for other adaptation studies.Gram staining and PCR-RFLP based on previous studies [7,8] at Passage 72 (Figure 2) showed that the cultures are Gram negative and the PCR-RFLP pro�les of all 4 replicates to be similar.is suggested that the cultures had not been contaminated.

F 2 :
PCR-RFLP and Gram staining of Passage 72.(a) Genomic DNA extraction and PCR using Lee et al. [8] and 16-hour TaqI restriction endonuclease digestion showed similar pro�le in all 4 replicates.M is Log2 DNA marker.ree sets of PCR-RFLP were performed where the same primer (Primers 5, 6, and 7) was used as forward and reverse primer.(b) Gram staining showed predominately Gram-negative cultures.ese suggest that the cultures were uncontaminated.
T 1: OD600 tabulation for minimum inhibitory concentration estimation.Representative data from each salt concentration is shown.
T 2: Colony MIC OD Readings for Passage 44 (4% NaCl).NaCl of different salt concentrations.For colony MIC, each sample of E. coli was streaked on nutrient agar and incubated overnight at 37 ∘ C. Ten colonies were randomly taken from each plate and inoculated into 1 mL of 1X nutrient T 3: Colony MIC OD Readings for Passage 53 (5% NaCl).
∘ C before taking OD600 readings.

Table ) .
OD600 readings from the MIC experiment of each replicate.e table is in long format where each row has only one measured data attribute.In this case, the measured data will be the OD600 readings.e other attributes are descriptors of the measured data attribute.is is opposed to the wide format whereby the descriptors for