Gram Negative Bacteria (Escherichia coli) Win Against Gram Positive Bacteria (Staphylococcus aureus) in The Same Media

Article history: Received 23 August 2021 Received in revised form 15 September 2021 Accepted 2 Oktober 2021 Available online 30 October 2021


Introduction
Indonesia is a tropical country, hence it has warm temperature and high humidity. This condition is very beneficial for bacterial growth. This causes Indonesia to have a wide variety of bacteria, including Gram positive and Gram negative bacteria. This also causes the variety of types of bacteria and types of infection caused by bacteria in Indonesia.
Of all cases of infection, it was found 53.5% were GI tract infections, 20.9% were urinary tract infections, 9.5% were bacteremia cases, 0.6% were central nerve infections, of which 94.9% were caused by Gram positive bacteria and 5.1% were caused by Gram negative bacteria. 1 As humans we may have infections in two different places with two different types of bacteria. This condition can cause bacteremia, without knowing which bacteria type was its main cause. Hence, after culturing bacteria from patients who experience bacteremia, we may find Gram positive and Gram negative bacteria in one bacterial culture, one example is Staphylococcus aureus and Escherichia coli. On the other hand, the treatment of these two types of bacteria are also very different.
Based on the reasons above, we conducted this study to determine the doubling time of Gram positive and Gram negative bacteria when these two types of bacteria are grown in the same culture, as well as to know the types of bacteria that will be first to eliminated.

Methods
This study is an analytic observational study conducted at the Laboratory of Clinical Microbiology Installation, Diagnostic Center Building, Dr. Soetomo General Academic Hospital from August 2018 -November 2018. Staphylococcus aureus and Escherichia coli bacteria were used as samples in this study. Pure cultures of both types of bacteria were planted into nutrient broth to form bacterial suspensions with 0.5 OD turbidity, then the two bacterial suspensions were mixed and homogenized using vortex and then incubated in incubator at a temperature of 35˚C. Every one hour within 24 hour, 0.01 ml of bacterial culture was taken then diluted. It was planted in nutrient agar plate quantitatively using a droplet technique, then it was stroke using osse just once. After that, the colony that grown was calculated based on colony forming unit per ml (CFU / ml) to time, so then the doubling time can be calculated. The calculation results were compared between the Staphylococcus aureus and Escherichia coli group. Data were analyzed using two tailed t-test for normally distributed data to obtain a significant value.

Results
In incubation results, the colonies from Staphylococcus aureus and Escherichia coli can be distinguished. (Figure 1) Staphylococcus aureus bacterial colonies had a golden yellow color, whereas Escherichia coli bacterial colonies had no color, so the number of colonies to time can be calculated.  The growth suppression phenomena in the same media might due to the bacteria is not only eat nutrients contained in the media but also "eat" the other bacteria. When there are severe nutritional stresses and there is competition to get nutrients, the bacteria will make intercellular communication to produce a signal molecule so that bacteria will produce toxins or antimicrobials that will kill the other bacteria. The cells that are attacked will release nutrients that can be used as nutrients for other bacteria. To avoid suicide due to toxins or antimicrobials released by the bacteria itself, the bacteria will produce proteins that protect the bacteria themselves from the toxins or antimicrobials they release. 11 Bacteria with faster doubling time (E. coli) will have a larger amount of CFU/ml while bacteria with slower doubling time (S. aureus) will have a smaller amount of CFU/ml as well. With this difference in number, there is also a difference in the amount of toxin or antimicrobials that will be produced. The more bacteria in there, the more toxins or antimicrobials that will be produced by certain groups of bacteria so that the number of bacteria destroyed by a group of bacteria will also be greater. This is what causes the suppression of the S. aureus by E. coli. An elongation of lag phase phase was acquired. The lag phase which usually only lasted about 3 hours extended to 12 hours. 5 This might be due to the need for longer adaptation times. Even an intervention in the media can lead to elongation of the bacterial lag phase. 6 In addition, lengthening of the lag phase may caused by differences in the types of initial media (mac conkey and blood agar) and growth media in this study (nutrient broth) so that the longer adaptation time is needed. 5 This study samples that had been stored in the refrigerator were also one of the factors that cause elongation of the lag phase. Nutrient broth that used in this study was also a simple type of media so the nutrients contained are very minimal which also contributes to the elongation of the lag phase which is the adaptational phase.
The stationary phase lasted 2-4 hours where the number of bacteria is relatively constant; this was because the number of bacteria that grown is the same as the number of bacteria that die, which was caused by lack of nutrients or accumulation of toxic metabolic products. 12 The decline phase was only recorded for 2-4 hours in this study, this was due to the extension of the lag phase which causes the decline phase to occur more than 24 hours. This cell death was due to the accumulation of bacterial metabolites in the form of acidic lactic acid which is not suitable for bacterial growth, without being balanced by the growth of more bacteria because of the amount of nutrients that have been exhausted. 5 In the case of bacterial infection, practioner are using antibiotics as the main therapy, however overuse or inappropriate use of antibiotic can lead to resistance. 13 Our result indicates that in which source of infection are from two different Gram and antibiograms types of bacteria (S. aureus and E. coli), it is necessary to consider treating the E. coli first. This is based on the results of this study where Judging from faster doubling time of E. coli that has faster doubling time turned out to be more competitive in the same concentration than S. aureus, so it is most likely that the bacteremia that occurred was caused more by the E. coli.
The limitation of this study is that the results of the culture which were contaminated caused the researchers to have to retest the study. In addition, the bacteria used in this study was bacteria that had been stored in the refrigerator, not a fresh bacteria.

Conclusion
When grown in the same media, Gram-positive bacteria (S. aureus) had slower doubling time than Gram-negative bacteria (E. coli) as much as 1.32 times. A faster doubling time bacteria could produce higher toxins that can kill the competitors.

Conflict of Interest
There is no conflict of interest of this study.