The Effect of Establishing Escherichia coli on Balb/C Rats as A Model of Preterm Labor

Introduction: The prevalence of preterm labor in Indonesia is estimated at 7-14%, which is around 459,200-900,000 babies per year. The mechanism of infection is the biggest cause of preterm labor. The bacteria that often causes infection is Escherichia coli . This study aims to see the effect of giving Escherichia coli to produce premature models of mice. Material and Methods: This study used a true experimental method with a post-test only control group design approach. The independent variables were preterm labor & cervical tissue culture, amniotic membranes, and placenta. The dependent variable of the study was the administration of Escherichia coli 10 9 CFU to the cervix. This research will be tested with a comparative test. Results: The treatment group were given Escherichia coli experienced parturition with the fastest time of 38 hours 22 minutes and the longest time of 42 hours 12 minutes. Escherichia coli colonies on Eosin Methylene Blue (EMB) culture media with a glowing green image was obtained. Cervical culture, amniotic membranes and placenta from the treatment group of Balb/C mice showed the growth of Escherichia coli colonies. Neutrophil infiltration and edema in the cervical stroma was found to be more severe in Balb/C mice that received treatment with Escherichia coli 10 9 CFU/ml. Conclusion: Escherichia coli shortens the time of delivery and increases the bacterial load in pregnant Balb/C mice.


INTRODUCTION
Premature delivery can lead to complications. Maternal complications such as increased risk of postpartum hemorrhage and infection. While neonatal complications can cause morbidity and mortality in infants. Therefore, the prediction and prevention of preterm labor is one of the main priorities in health services [15]. In 2018, WHO noted that there were 675,700 premature deliveries in Indonesia, there has been a decline in the number of preterm deliveries, the decline tends to slow down in the last 10 years. The prevalence of preterm labor in Indonesia is estimated at 7-14%, which is around 459,200-900,000 babies per year [68].
The mechanism of infection is the biggest cause of preterm labor, which is about 25-40% and ascending vaginal infection is the most common route of infection. The bacteria that often causes infection is Escherichia coli [9,10]. In preterm labor, infection/ inflammation (chorioamnionitis and uteroplacental insufficiency) causes changes in the architecture of the collagen fibrils resulting in rupture of the amniotic membrane and remodeling of the cervix which is necessary for labor [1114]. This study aims to see the effect of giving Escherichia coli to the cervix on preterm labor so that it can produce premature models of mice which can later be used as research models for premature labor.

Study Design
This study used a true experimental method with a post-test only control group design approach which was carried out from September to October 2021. This study consisted of 2 stages, firstly determining the dose of Escherichia coli used in Balb/C mice to produce preterm labor and then evaluating the average parturition of Balb/C mice after being given treatment. Balb/C pregnant mice were obtained from Laboratorium Penelitian dan Hewan Coba (LPHC) Fakultas Kedokteran Universitas Brawijaya Malang and Escherichia coli cultures are made in the microbiology laboratory of Fakultas Kedokteran Universitas Brawijaya Malang, while the histopathological calculation of Escherichia coli was carried out in the Anatomical Pathology Laboratory. Inclusion criteria were male and female Balb/C mice aged 2 months, average weight 20-25 grams, healthy condition characterized by active movement, bright and unblemished eyes, Balb/C mice with their first pregnancy. The exclusion criteria were previously for other experiments. The independent variables were preterm labor & cervical tissue culture, amniotic membranes, and placenta. The dependent variable of the study was the administration of Escherichia coli 10 9 CFU to the cervix. This research will be tested with a comparative test.

Data Analysis
From this treatment it was proven that giving Escherichia coli 109 CFU/ml caused pregnant Balb/C mice to experience premature parturition. It was proven by examining the microbiological culture of Escherichia coli on Eosin Methylene Blue media, then calculating the bacterial load and histopathological examination of anatomical pathology to see neutrophil infiltration in the cervical epithelium and an appearance of edema in the cervical stroma. Table 1, 32 pregnant Balb/C mice were divided into 2 groups, namely 16 control group and 16 treatment group. Based on the observation of the control group of pregnant Balb/C mice, 16 mices experienced labor over 19 days, with the fastest time is 19 days 20 hours and the longest time is 19 days 30 hours ( Table 2).

Based on
While the treatment group of pregnant Balb/C mice on the 15th day were given Escherichia coli 10 9 CFU/ ml on the cervix, 16 mices experienced parturition with the In the treatment group, after it was proven that the growth of Escherichia coli colonies on Eosin Methylene Blue (EMB) culture media with a glowing green image was obtained, then the number of colonies that grew (Bacterial load) was calculated (Fig.1).
The results of cervical culture, amniotic membranes and placenta from the treatment group of Balb/C mice showed the growth of Escherichia coli colonies. In normal parturition, there is a picture of neutrophil infiltration in the cervical epithelium and edema in the cervical stroma. The description of neutrophil infiltration and edema in the cervical stroma was found to be more severe in Balb/C mice that received treatment with Escherichia coli 10 9 CFU/ml on the cervix which indicated inflammation (Table 3).

DISCUSSION
Giving Escherichia coli 10 9 CFU/ml to the cervix of pregnant Balb/C mice on the 15th day caused Balb/C mice to have premature labor. This is in accordance with research conducted by Ronald McCarthy et al. that showed several types of mice and rats by comparing the administration of Escherichia coli and the administration of LPS to induce preterm labor. One of the results is that in Balb/C mice the administration of Escherichia coli performed intracervical will result in 92% preterm labor at a dose of 2-10 x 10 5 CFU/ml (16). In this study, the dose of Escherichia coli was 10 9 CFU/ml, the difference in the dose of Escherichia coli given was due to the different virulence of Escherichia coli in accordance with the statement of Grohmann et al in 2018 that Escherichia coli is a genus with only one species having several hundred antigenic types. This type is characterized by different combinations. O antigen (somatic) which is heat-resistant or thermostable, consisting of lipopolysaccharide containing glucosamine and present in the cell wall of gram-negative bacteria plays a role in determining the virulence level of Escherichia coli [17]. This dose corresponds to the type of Escherichia coli in the Microbiology laboratory of Fakultas Kedokteran Universitas Brawijaya.
In accordance with the statement of Mondal et al, 2022 that good growth at an optimal temperature of 37 o C in media containing 1% peptone as a carbon and nitrogen source. Escherichia coli ferments lactose and produces indole which is used to identify bacteria in culture media, can survive up to 60 o C for 15 minutes or at 55 o C for 60 minutes. Escherichia coli grows well at temperatures between 8°-46°C and an optimum temperature of 37°C. If bred below the minimum temperature or slightly above the maximum temperature, it will not die immediately but is in a state of dormancy. Escherichia coli only recognizes one type of reproduction, namely by sexual or vegetative means of rapid reproduction [1819].
Escherichia coli infection is most commonly found in the cervix, followed by the amnion and placenta. This is in accordance with research conducted by Elovitz et al, 2004 that the pathogenesis of preterm labor caused by infection causes bacterial colonization of the vagina during pregnancy to ascend through the uterine cervix and then due to virulence or due to a deficiency of genetic or immunological responses from the mother, the bacteria then replicate in the amniotic membrane, at this point, the mother's inflammatory response will be induced, so that the bacteria continue to reproduce and enter the placenta, then enter the fetal circulation through the umbilical cord. At this stage, the fetal inflammatory response will be induced. Bacteria can also enter the amniotic cavity through the amniotic membrane [20].
The description of neutrophil infiltration and edema in the cervical stroma was found to be more severe in Balb/C mice that received treatment with Escherichia coli 10 9 CFU/ml on the cervix [2122]. In accordance with the results of the bacterial load that the number of Escherichia coli bacteria was found to be the highest in the cervix, followed by the amnion and placenta, the anatomical pathology picture showed that inflammation was more severe in the cervix than the amnion and placenta.

CONCLUSION
Escherichia coli shortens the time of delivery and increases the bacterial load in pregnant Balb/C mice.

ACKNOWLEDGMENT
We thank anonymous referees for their valuable suggestions.