Investigation of Stress Situation of Pediatric Nurses and Corresponding Changes of Intestinal Flora


 BACKGROUND: Sequencing the stool samples to explore the relationship between intestinal flora and the stressor of nurses. METHODS: The research take the pediatric nurses in the Third Xiangya Hospital as the research group and the internal medicine nurses as the control group. Use the "Chinese Nurse Stressor Scale"(CNSS) for general investigation and stress-related assessment. 6 in internal medicine group and 7 in pediatric group to conduct cell sequencing of intestinal flora. Gut microbiome was profiled by 16S rRNA sequencing. RESULTS: The work stressors of internal medicine and pediatric nurses includes nursing specialty and work (10.94±3.17 vs 12.25±2.78), work environment and resources (4.91±1.63 vs 5.66±2.03), patient care (15.86±4.30 vs 18.95±5.12) and management and interpersonal relationship (8.83±4.94 vs 10.86±5.01). The results were statistically different (P < 0.05). The abundance of Clostridia in internal medicine group is higher, while the abundance of Bacteroidia in pediatric group is higher. The abundance of γ-proteobacteria and β-proteobacteria also have obvious differences between two groups.CONCLUSIONS: The overall stress score of the pediatric nurse group was higher, with their stool samples having more abundance of Bacteroidia, but Clostridia, γ-Proteobacteria and β-Proteobacteria are fewer than that in internal medicine nurses.

few studies on intestinal ora and occupation-related stress, especially among pediatric nurses, so this is a topic worthy of attention.
In this experiment, for one thing, pediatric nurses were given questionnaires and their stress state was evaluated by scales. For another, their gut ora from stool samples was sequenced to seek for differences. This study aims to pay attention to the physical and mental health of pediatric nurses and analyze the current status of stressors in pediatric nurses. In this way, some corresponding measures can be proposed to improve the working atmosphere of pediatric nurses.

Participants
In this study, we used online survey questionnaires and anonymous submission of questionnaires to conduct pressurerelated surveys on pediatric and internal medicine nurses at The Third Xiangya Hospital of Central South University. All participants meet the following criteria: (1) registered nurses registered in The Third Xiangya Hospital;(2) female in good health, not pregnant or lactating; (3) known the project process of this study and been willing to cooperate with the investigation; (4) exclude those who have severe mental illness or have been diagnosed as anxiety disorder or depression anxiety; (5) no related gastrointestinal diseases or recent gastrointestinal symptoms; (6) there were no major family changes or other events that seriously affected the mood of the test subjects recently; (7) the objects have passed the relevant ethical review. (8) exclude who have history of drugs, antibiotics, probiotics or prebiotics taken within 3 months. This study was reviewed and approved by the hospital ethics committee, and informed consent was signed by all nurses investigated.

Questionnaire content
Chinese nurses' stressors scale was selected for stress-related investigation. This scale investigates 35 questions from ve aspects: nursing specialty and work problems, workload and time allocation problems, work environment and resources problems, patient care problems and management and interpersonal relationship problems. Each question is divided into four grades: 0, 1, 2 and 3. The higher the score of the answer is, the more pressure the nurse has. Therefore, the questionnaire we selected is scienti c, universal and comprehensive in content coverage. (Supplement1)

Experimental content
In order to ensure the randomness of the sample, we selected 35 and 44 nurses by random number table respectively.
Fresh feces with 7 internal medicine nurses(in control group) and 6 pediatric nurses(in experimental group) were picked out for sequencing. The sampler intercepts the middle part of the sample, distributes it to a sterilized cryopreservation tube, and stored in a -80 ℃ refrigerator for future use. Total DNA extraction was performed using a QIAamp® DNA Stool Mini Kit (QIAGEN, Tokyo, Japan) according to the manufacturer's instructions. The 16S rDNA sequencing selection region is V1-V2 variable region, and the universal primers used are 27F (5'AGAGTTTGATCCTGGCTCAG-3') and 338R (5'-TGCTGCCTCCCGTAGGAGT-3'). Products ampli ed by PCR were puri ed and quanti ed using the Qubit dsDNA BR Assay Kit.
OTU (operational taxonomic units) is a uni ed mark set for a taxon (strain, genus, species, group, etc.) in the study of phylogeny or population genetics for the convenience of analysis. In order to understand the number of species, genera and other information in the sequencing results of a sample, we need to cluster the sequence. Through the classi cation operation, the sequences are classi ed into many groups according to their similarity, each group is an OTU. In general, tags with similarity of more than 97% are clustered into an OTU. Using software usearch(v7.0.1090). Cluster the spliced tags into OTU. The main process is as follows: 1) using uparse to cluster under 97% similarity to get the representative sequence of OTU; 2) using uchime(v4.2.40). The mosaics produced by PCR were removed from the OTU representative sequences (16S compared with the existing mosaics database to remove the mosaics. 18S adopts denovo method to remove the chimera 16S chimera database: golddatabase (v20110519) its chimera database: unite (v201407 03), divided into its full length, ITS1 and ITS2, selected according to sequencing area); 3) using usearch-the global method compares all tags back to the OTU representative sequence, and obtains the OTU abundance statistical table of each sample.
PLS-DA analysis (PLS-DA) is a multivariate statistical analysis method for discriminant analysis, which is often used to determine how to classify the research objects. Compared with PCA, PLS-DA method can combine the function of principal component analysis and multiple regression to maximize the interval between components of PCA. That is to say, compared with PCA analysis, the interval between groups in PLS-DA analysis results is larger, and it is easier to see the differences between groups.

Statistical analysis
The subjects lled out questionnaires without giving their names. We summarized the results, entered the data in Excel, and conducted statistical analysis with SPSS 22.0. Quantitative variables are expressed as mean values with standard deviations. Quantitative variables are expressed in Numbers and percentages. T test was used to compare the differences in the total stress scores and the scores of each dimension among nurses in different departments. P value less than or equal to 0.05 was considered statistically signi cant.

Stressors questionnaire results
A total of 35 internal medicine nurses and 44 pediatric nurses participated in the trial. Their basic information and corresponding proportions are described in detail in Table 1. There was a signi cant difference in the number of years of service between the control group and the experimental group (66% of nurses with more than 10 years of service in internal medicine versus 32% in pediatrics). In addition, the proportion of medical nurses over 30 years old (71%) was higher than that of pediatrics (57%). The proportion of people with educational background and marriage status is relatively consistent.
The scores of internal medicine nurses and pediatric nurses in various dimensions of work stressors are shown in Table 2. In terms of nursing specialty and work (10.94 ± 3.17 vs 12.25 ± 2.78), work environment and resources (4.91 ± 1.63 vs 5.66 ± 2.03), patient care (15.86 ± 4.30 vs 18.95 ± 5.12) and management and interpersonal relationship (8.83 ± 4.94 vs 10.86 ± 5.01), there were statistically signi cant differences between the control group and the experimental group. Meanwhile, the overall score of internal medicine nurses is 50.17 ± 12.13 while pediatric nurses is 57.93 ± 13.01(P < 0.05).
The results of the objective factors of nurses' stressors in different departments were further compared, as shown in Table 3. The scores of pediatric nurses who worked for more than 10 years (63.50 ± 13.06) were higher than those of internal medicine nurses (47.96 ± 11.98). The pressure of pediatric nurses over 30 years old (59.56 ± 12.92) was also higher than that of medical nurses (48.72 ± 12.49). The stress score of the highly educated pediatric nurses (59.40 ± 14.20) was higher than that of the internal medicine nurses (45.00 ± 10.65). In addition, the stress of married pediatric nurses (59.18 ± 14.12) was higher than that of internal medicine nurses (50.68 ± 12.81). The results are statistically different(P < 0.05).

Intestinal ora analysis results
6 nurses selected randomly from internal medicine were set as the control group (group A) while 7 from pediatric were the experimental group (group B) for further analysis by 16S ampli cation. The structure and abundance of intestinal ora between the two groups showed signi cant differences. Partial least squares discrimination analysis (PLS-DA) was used to explore the differences in the structure of intestinal ora between groups A and B. The result can be seen in Fig. 1.
Species abundance heat map is a graphical display method of clustering according to the similarity of species or sample abundance by the color gradient representing the relative abundance of species. It can intuitively display the composition and proportion of each sample species, re ecting the changes of species between samples. As can be seen from the gure, the main difference between the two groups exists in Clostridia and Bacteroidia. The abundance of Clostridia in group A is higher than that in group B, whereas Bacteroidia is lower than that in group B. There are also differences in abundance of γ-Proteobacteria and β-Proteobacteria between the two groups( Figs. 2 and 3).

Discussion
It can be clearly seen from the survey results that the overall pressure of pediatric nurses is relatively high higher than internal nurses. It is mainly re ected in nursing specialty, working environment and resources, patient care, management and interpersonal relationship. Working experience, marriage condition and educational background are all factors in uencing the stress levels of pediatric nurses. Lupien et al. have shown that nurses experience high levels of test anxiety, state anxiety, and stress, which can be improved after intervention [16]. Sonia et al. point out that the effects of stress on the brain and behavior in adulthood are reversible and usually disappear when the stressor stops [17]. Thus, analysis of possible stressors is important to deal with the pressure of pediatric nurse.
One of the critical reasons is the particularity of the pediatric nurses. Children have weak immune capacity and insu cient regulatory adaptability because of immature development and imperfect system function [18]. The disease has the characteristics of rapid onset and rapid development of the disease process and the prognosis is not optimistic, which cause pediatric nurses to have heavy work content, signi cant responsibility, and greater pressure than internal medicine. Secondly, the second child policy in China in recent years generate a fertility boom, but the number of pediatric staff and the overall scale of pediatrics have not been expanded enough, which has increased the workload of pediatric medical staff. The increasing number of pediatric patients lead to the insu ciency of hospital capacity. Long waiting time for medical consultation and impatience among parents can easily spur con icts between nurses and patients, or even escalates to the workplace violence [19]. Studies have shown that emergency and pediatrics are the two most vulnerable departments for nurses to meet violence at work [20]. Thirdly, in aspects of working environment, pediatric nurses have a low status, low rewards, more onerous work and more complex doctor-patient relationship compared with other departments [19], which aggravates the loss of personnel of pediatric nurses and makes it more di cult for pediatrics to recruit medical staff. Many hospitals have experienced pediatric "employment shortage". The lack of nursing staff, the heavy workload of nurses and the harsh working environment increase the subjective anxiety and pressure of nurses, leading to the stress among pediatric nurses generally higher than that among internal medicine nurses. A lot more reasons are worthy to consider, and our experiment looks at the problem from a novel perspective.
By comparing stool samples from pediatric nurses and internal medicine nurses, we found signi cant differences in the structure and abundance of intestinal bacteria (Fig. 1). Pediatric nurses had lower abundance of H.ammodendron but higher Bacteroides than medical nurses. Meanwhile, the abundances of γ-Proteobacteria and β-Proteobacteria were also varied in the bunch (Fig. 2, Fig. 3). Consistent with this, the analysis of the stress scores did show that the total stress factor scores and four dimensions of pediatric nurses were considerably higher than that of internal medicine nurses, and the differences were statistically signi cant.
This interesting consistency suggests a role for the brain's gut axis. The stress of pediatric nurses is closely related to the psychological effects of work stress. The external stimulation generated by working pressure in uences intestinal activities through direct and indirect ways, causing changes in the release of duodenal bicarbonate, hormonal changes, and decreased mucosal immune function [21], and even intestinal diseases such as intestinal stress syndrome may occur. Enteropathy affects the central nervous system through changes in brain-gut peptide secretion. Anxiety, depression and other emotions of pediatric nurses continue to intensify, forming a vicious circle, and eventually lead to differences in the composition of intestinal bacteria between pediatric nurses and medical staff.
Existing studies have shown that biofeedback therapy and behavioral relaxation therapy can improve these symptoms [22]. Bravo et al. found that probiotics can regulate GABA receptor expression in the cerebral cortex through the vagus nerve, thereby reducing anxiety and depressive behaviors [23]. Sharon et al. demonstrated that serum 4-ethylphenyl sulfate (4EPS) concentrations were increased in autistic mice, and after treatment with B. fragilis, 4EPS concentrations were decreased and anxiety behaviors were alleviated in mice [24]. These studies suggest that due to the characteristics of bidirectional communication of the brain-gut axis, it can also act on the central nervous system through the regulation of intestinal bacteria, providing a new therapeutic idea for clinical practice. At the same time, the latest research shows that non-probiotic interventions such as resistant dextrin supplementation can achieve better results, so the effect of dietary restructuring is better than probiotic supplements [25].
Numerous studies have shown that psycho-psychological factors are closely linked to intestinal motility disorders and visceral sensitivity mechanisms [26]. Behavior and cognition can affect intestinal activity through indirect complex pathways. After feeling external stimuli, the action pathways of the brain-gut axis on the intestine include indirect pathways and direct pathways. The indirect pathway is through changes in gastrointestinal motility and secretion or changes in intestinal permeability. Direct pathways include the release of signaling molecules into the luminal lamina propria of intestinal cells (cells of enterochroma n cells, neural cells, immune cells). Changes in intestinal activity, on the other hand, can affect changes in intestinal bacteria. For example, de Magistris's et al. found that autistic patients had increased intestinal mucosal permeability and increased blood concentrations of bacterial lipopolysaccharide (LPS), indicating that brain-gut axis dysfunction can disrupt the intestinal mucosal barrier causing bacterial translocation [27]. In addition, the brain-gut axis causes irritable bowel syndrome (IBS) under stress conditions. IBS is a common functional bowel disorder which is closely related to the function of brain-gut axis. Since the bidirectional pathway of brain-gut axis also acts on the central nervous system, the generation of IBS will also have adverse effects on the patient's mood. Brilliant et al., after acupuncture and oral treatment of trimebutine maleate tablets in IBS patients, found that while IBS symptoms were relieved, brain-gut peptide secretion such as 5-HT, NPY, and CCRP was reduced, and mental stress was signi cantly reduced [28].
As a result, appropriate use of psychological biological agents, antibiotics and other drugs to regulate intestinal ora, through the intestinal and brain axis action to alleviate relatively serious psychological stress (such as anxiety, depression, etc.) [9,29,30]. Probiotics and prebiotics can be considered taken in daily work for pediatric nurses so as to moderately supply for reduced intestinal ora. From the perspective of traditional Chinese medicine, proper diet therapy can also improve the type and quantity of intestinal ora.
True, merely nding for a curable treatment to reduce psychological stress is a limited approach, which could only treat the symptoms but not the root causes. In view of this situation, pediatric nurses requires an appropriate policy and a better working environment to relieve potential stress. Hospitals should appropriately expand the scale of pediatrics to adapt to the "second-child policy" to ensure the workload of pediatric nurses within acceptable limits. Adopt more diversi ed management strategies, such as "double night shifts". Under this policy, both physical need for rest and work effectiveness can be accommodated among the pediatric nurses. Managers could care about nurses such as organizing mindful exchange meetings to eliminate the impact of work stress. For the nurses themselves, improving their professionalism, correctly balancing the relationship between family and work and nding suitable channels for stress relief are also effective ways to relieve stress.

Conclusion
The psychological stress of pediatric nurses is generally more serious than that of internal medicine nurses due to the surging number of children, large workload, poor working environment and the tension between pediatricians and patients. Considering that intestinal ora and psychological stress may interact through the brain-gut axis, we tested intestinal ora samples from people with high stress. Further analysis of the types and quantities of intestinal ora found that pediatric nurses had more Bacteroidia, but fewer Clostridia, γ-Proteobacteria and β-Proteobacteria than medical nurses. More relevant clinical intervention studies can be carried out to elucidate the relationship between intestinal bacteria regulation and pressure improvement by focusing on the regulation of intestinal bacteria through the nonprobiotic pathway in the future.

Abbreviations
All abbreviations are de ned in the manuscript.

Declarations
Ethics approval and consent to participate: This study was approved by the institutional review board of the Third Xiangya Hospital Central South University (Permission Number: 20020).

Consent for publication:
Not applicable.

Availability of data and material:
The data used to support the ndings of this study are included within the article.  Tables   Table 1 Baseline data of participants