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Effect of mixed probiotics on pulmonary flora in patients with mechanical ventilation: an exploratory randomized intervention study

European Journal of Medical Research volume 29, Article number: 473 (2024) Cite this article

Abstract

Objective

The study objective was to investigate the effect of mixed probiotics on the diversity of the pulmonary flora in critically ill patients requiring mechanical ventilation by analysing the changes in lung microbes.

Methods

24 adult critically ill patients who needed mechanical ventilation in our hospital were randomly divided into a probiotic group and a control group. Then, the probiotic group was given Live Combined Bifidobacterium, Lactobacillus and Enterococcus Capsules, Oral (Bifico) by nasal feeding within 24 h after mechanical ventilation. Bronchoalveolar lavage fluid (BALF) and venous blood were collected within 24 h after mechanical ventilation and on the 5th day after mechanical ventilation, and the treatment status of patients (mechanical ventilation time, 28-day survival), measured cytokine levels (IL-1 β, IL-6, IL-8, IL-17A) and changes in pulmonary microorganisms were observed.

Results

The microbial diversity of BALF samples decreased in the control group, and there was no significant difference in the probiotic group. Species difference analysis showed that among the three probiotics (Bifidobacterium, Lactobacillus, Enterococcus) used for intervention, Lactobacillus caused significant differences in BALF in the control group. Clinical factor association analysis displayed significant associations with IL-17A levels in both blood and BALF.

Conclusion

Mechanical ventilation can cause a decline in pulmonary microbial diversity, which can be improved by administering mixed probiotics.

Introduction

There are trillions of bacteria living on the skin and mucous membrane of the human body [1]. These microbes play an important role in a variety of physiological states of the host, including metabolism, antibacterial capability, immune regulation and stability of the internal environment [2, 3]. These microbiota change in accordance with the host's lifestyle, diet or disease. The microbiota of patients in the intensive care unit (ICU) fluctuates greatly due to the acute disease status related to critical diseases and common interventions (such as mechanical ventilation, antibacterial drugs, acid inhibitors and enteral nutrition). The literature shows that the pulmonary flora of patients with respiratory failure who need mechanical ventilation decreased with the intubation time, and the imbalance of pulmonary flora is the most serious in patients with ventilator-associated pneumonia [4, 5]. Probiotics have been proven to have health benefits [6], such as enhancing immunity, maintaining the structural balance of intestinal flora, inhibiting intestinal inflammation, and protecting the intestinal mucosal barrier. Some studies have confirmed that [7] through the "gut–lung axis", changes in intestinal flora biodiversity can affect pulmonary flora diversity. At present, the relevant studies mostly adopt preventive application of probiotics and the cross-sectional comparison of the flora after the intervention. The individual differences in microbial flora are relatively large, and the vertical changes in the flora over time and with treatment can better explain its impact on individuals than the cross-sectional results. In this study, an exploratory randomized intervention study method was adopted. The study involved administering mixed probiotics as adjuvant treatment to critical patients who needed mechanical ventilation and analysing the changes in pulmonary microorganisms to observe the impact of mixed probiotics on the diversity of pulmonary flora.

Method

Patient selection

Critically ill patients admitted to the First Hospital of Hebei Medical University, Department I of Critical Care Medicine, were selected. The inclusion criteria were as follows: (1) adult patients (≥ 18 years old) and (2) an estimated duration of mechanical ventilation of more than 72 h. The exclusion criteria included (1) a length of mechanical ventilation < 5 days; (2) inability or contraindication of enteral nutrition; (3) immunosuppression (human immunodeficiency virus (HIV), use of immunosuppressive drugs, organ or bone marrow transplantation in the past, absolute neutrophil count < 500 cells/μl); (4) history of factors that increase the occurrence of blood borne infections (rheumatic heart disease, congenital heart disease, mechanical valves, endocarditis, intravascular grafts or implantation of permanent intravascular devices such as haemodialysis catheters, pacemakers or defibrillators). This study was approved by the Medical Ethics Committee of our hospital (approval number: 20210741). In this study, all participants signed an informed consent form. After the patient was admitted to the ICU, if the patient was conscious, we explained to him/her and obtained his/her consent. If the patient was unconscious, we explained the purpose of the study and possible risks to his or her relatives, obtained their consent, and signed the informed consent form.

Experimental groups

The simple randomization grouping method was applied in which patients were numbered according to the order of visits, beginning with the first column of the sixth row of the random numbers table [8], and two digits were sequentially read and recorded under the number as a random number. Then, all random numbers were sorted from small to large and sequentially used to divide the patients into the control group and the probiotic group in ordinal order. The group designations were concealed from the patients and clinicians. The probiotic group was given Bifico (Shanghai Shinji Pharmaceutical Factory Co., Ltd., Sinopharm quasizu s10950032, specification: 210 mg/grain) by nasogastric tube within 24 h of receiving mechanical ventilation at 2 grains each twice a day until leaving the ICU, and the control group received conventional treatment only. Enteral nutrition was administered in a continuous on-pump fashion throughout the study period. The control group and the probiotic group used antibiotics according to the same principles. During the mechanical ventilation of patients, we monitored the cuff pressure of the tracheal tube per 6h and kept it at 25-30cmH2O. Patients' general conditions were recorded, including their age, sex, weight, Acute Physiology and Chronic Health Evaluation (APACHE II) score, Sequential Organ Failure Assessment (SOFA) score, Nutrition Risk in Critically ill (NUTRIC) score, duration of mechanical ventilation, 28-day survival, average cost per day during ICU stay and average cost per day during hospital stay. BALF, stool, and venous blood were collected within 24 h of the initiation of mechanical ventilation and on the 5th day after mechanical ventilation was applied.

BALF

Before taking the BALF samples, we routinely confirm the pressure of the cuff. The tip of a fibre optic bronchoscope was wedged into the bronchial opening at the lingual segment of the left lung. If there was lung infection at the site, the middle lobe of the right lung was chosen. From the biopsy hole, sterilized normal saline was injected quickly at 37 ℃, and the liquid was recovered by negative pressure suction at 6.66 ~ 13.3 kPa at 4 ~ 6 ml of each injection for a total volume of 30 ml or 15 ml for 16S rDNA gene sequencing for lung flora analysis and 15 ml for cytokine detection.

The lavage fluid was filtered with a double layer of sterile gauze to remove mucus and larger impurities, collected into sterile centrifuge tubes, placed in ice water (0 ~ 4 ℃) and sent to the laboratory. After centrifugation at 400 × g for 10 min at room temperature, the supernatant was taken and centrifuged at 10,000 × g for 10 min, and the supernatant was discarded. The bottom of the high-speed centrifuge tube was rinsed thoroughly with 1 ml of sterile saline, and the pellet (containing microorganisms) was resuspended. The entire wash solution was transferred to a 1.5-ml EP tube, labelled appropriately, placed in a −80 ℃ freezer and stored until 16S rDNA gene sequencing.

16S rDNA gene sequencing

According to the E.Z.N.A.® soil DNA kit (Omega Biotek, Norcross, GA, U.S.) instructions, the total DNA was extracted from microbial communities. One percent agarose gel electrophoresis was used to examine the extraction quality of DNA. DNA concentration and purity were determined using a NanoDrop2000. The v3–v4 variable region of 16S rRNA was PCR amplified using primers 338F (5’-ACTCCTACGGGAGGCAGCAG-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’) using an ABI GeneAmp® 9700 PCR machine. PCR products from the same sample were mixed and recovered using a 2% agarose gel. The AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) was utilized for recovery product purification. Two percent agarose gel electrophoresis was performed, and the recovered products were detected and quantified using the Quantus™ Fluorometer (Promega, USA). The NEXTFLEX Rapid DNA-Seq Kit was used to generate libraries, and Illumina's MiSeq PE300 platform was used for sequencing (Shanghai Meiji Biomedical Technology Co., Ltd.).

Cytokine measurements

Plasma from venous blood samples and supernatant from BALF were collected after centrifugation, and a capture microsphere mixture (containing polystyrene microspheres and mouse anti-human monoclonal antibodies against IL-1β, IL-6, IL-8, and IL-17A) and fluorescent detection reagents (containing PE-labelled detection antibodies) were added, which were purchased from Jiangxi Saiji Biotechnology Co., Ltd. After the samples were vortexed and mixed thoroughly, the cells were incubated in the dark at room temperature for 2.5 h, and then a Beckman Coulter Navios flow cytometer was used for fluorescence detection.

Statistics

Statistical analysis was performed using SPSS 25.0 statistical software, and the measurement data are presented as the mean ± standard deviation. The measurement data were tested for normality, the t test was used for comparisons between groups with data that conformed to a normal distribution, and the Wilcoxon rank-sum test was used for data that did not conform to a normal distribution. Results with an α level of ± 0.05, P < 0.05 were considered significantly different. As no corresponding references were identified for reference, a priori sample size estimation was not performed in this study, but we conducted test power calculations based on the Shannon index of trial outcomes using PASS 2021 21.0.

Results

Participants

A total of 813 people, including 652 mechanically ventilated patients, were admitted to the First Department of Critical Care Medicine at the First Hospital of Hebei Medical University between September 2021 and July 2022. Among them, 45 met the inclusion criteria, and 24 were finally enrolled in the study, all managed by experienced full-time ICU doctors. Among them, one BALF sample was removed from the samples obtained from each of the control and probiotic groups during the PCR amplification phase on day 1 of mechanical ventilation because the bands of PCR products were too weak or not detectable for subsequent experiments. The corresponding BALF samples of the patient on day 5 of mechanical ventilation when tested for between-group differences were similarly rejected. During the period of this study, the incidence of constipation was recorded and 16SrDNA gene sequencing of intestinal microbes was not performed because the subjects had different degrees of constipation, which resulted in a large difference in retention of faecal samples between the two groups and lack of comparability. The flowchart is shown in Fig. 1.

Fig. 1
figure 1

Flowchart of participant samples

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Patient demographic characteristics, disease severity, clinical characteristics, antimicrobial use, and outcomes

There were no significant differences between the two groups in terms of population characteristics, severity, nutritional status, or antimicrobial use. Both groups started enteral nutrition within 24 h of mechanical ventilation and reached the target amount of 30 kcal/kg/d and protein 1.0–1.2 g/kg/d within 72 h. There was a greater proportion of trauma patients in the control group and a greater proportion of heart failure patients in the probiotic group, but none of the differences were statistically significant. The duration of mechanical ventilation and the 28-day mortality rate in the probiotic group were better than those in the control group, but there was no statistically significant difference. Constipation was significantly less frequent in the probiotic group than in the control group (P = 0.035). It follows that this study showsagain that probiotics improve intestinal symptoms in critically ill patients (Table 1).

Table 1 Baseline characteristics, management and outcomes of critically ill patients
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There are 22 beds in this ICU. All patients were Han Chinese and had no history of hospitalization, antibiotics, probiotics, or PPIs use in the past 3 months. A control group of 5 patients and a probiotics group of 6 patients were admitted to the emergency department. The other study subjects were transferred from the corresponding department, and their hospitalization time in the corresponding department was less than 3 days. Antimicrobial strategy: In this study, antibiotics were used according to the following strategies: trauma patients with suspected contaminated wounds were given second-generation cephalosporins or quinolones. Patients with pneumonia complicated with respiratory failure were given third-generation cephalosporins or penicillin plus enzyme inhibitors. During hospitalization, patients with fever, elevated white blood cells, C-reactive protein, procalcitonin, cough and sputum with chest X-ray suggestive of exudative shadow, abdominal pain with abdominal ultrasound or CT suggestive of inflammatory lesions, urinary symptoms with elevated urinary white blood cells, were given the third-generation cephalosporins or penicillins plus enzyme inhibitors or quinolones. Antibiotics were determined by the clinician. The dosage of antibiotics was given according to the drug instructions. If the symptoms and auxiliary examinations did not improve after 3–5 days of treatment, the antibacterial drugs would be upgraded. If the treatment was effective, the treatment course would be 10–12 days. Causes of mortality: In the probiotic group, one died due to withdrawal, one died due to multivisceral failure caused by heart failure. In the control group, two died due to multivisceral failure caused by pneumonia, one died due to ARDS, one died due to irreversible cerebral lesions. We used protective ventilation only in patients who were considered to have ARDS, while we used conventional mechanical ventilation support in other patients. In this study, patients with ARDS had tidal volumes 6-8 ml/kgPBW and plateau pressures < 30 cm H2O, because patients with tidal volumes was 6 ml/kgPBW had unacceptable hypercapnia (PaCO2 > 60 cm H2O, pH < 7.25). Patients receiving conventional mechanical ventilation had tidal volumes > 8 ml/kg and plateau pressures < 35 cmH2O, which can ensure minute ventilation volume and prevent hypercapnia. The PEEP in patients with ARDS was 10.80 ± 0.84 cmH2O and the FiO2 was 84.00 ± 8.22%. PEEP was 6.37 ± 1.17 cmH2O and FiO2 was 50.00 ± 7.27 in patients receiving conventional mechanical ventilation. Significant differences were observed in tidal volume (t = −4.429, p = 0.000), PEEP(t = 7.926, p = 0.000), and FiO2(t = 9.084, p = 0.000). Constipation is defined as infrequent passage of stools or difficulty with evacuation of stools. Fewer than 3 bowel movement per week, or no bowel movements for more than 3 days. During the study from admission to day 5, we also monitored the gastric residual volume, abdominal distension, emesis and diarrhoea of the patients, there was no significant difference between the two groups, except for constipation.

Microbial flora

Operational taxonomic unit (OTU) analysis

A total of 32 species at the phylum level were identified in this study, and the top 5 species were Proteobacteria, Firmicutes, Bacteroidota, Actinobacteriota, and Fusobacteriota. The lung flora on the first day of mechanical ventilation was consistent with the above findings. On the fifth day of mechanical ventilation, Proteobacteria abundance increased in the lung flora of the control group, whereas Actinobacteriota was more abundant in the probiotic group (Fig. 2).

Fig. 2
figure 2

OTU taxonomic analysis. BALF flora samples from the control and probiotic groups on days 1st and 5th of mechanical ventilation were analysed at the phylum level. cgbd1: BALF flora samples from control group on the 1st day of mechanical ventilation. cgbd5: BALF flora samples from control group on the 5th day of mechanical ventilation. pgbd1: BALF flora samples from probiotics group on the 1st day of mechanical ventilation. pgbd5: BALF flora samples from probiotics group on the 5th day of mechanical ventilation

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Alpha-diversity analysis

In this study, diversity analysis was conducted using BALF flora samples from both the control and probiotic groups on the 1st and 5th days of mechanical ventilation. The results showed that there was no significant difference in the alpha-diversity index (sobs, ace, Shannon) between the control group and the probiotic group on the first day of mechanical ventilation. The sobs (196 ± 46.76 vs 70.55 ± 74.59, p = 0.003), ace (215.97 ± 49.46 vs 105.42 ± 75.22, p = 0.001), and Shannon (2.38 ± 0.41 vs 0.67 ± 0.98, p = 0.001) indices of the BALF in the control group showed significant differences between the 1st and 5th days of mechanical ventilation. However, there was no significant difference in Alpha-diversity indices between the 1st and 5th days of mechanical ventilation in the probiotic group. There were significant differences in alpha-diversity index (sobs, ace, Shannon) between the control group and probiotics group on the 5th day of mechanical ventilation. The result showed that mechanical ventilation was related to the reduction of microbial diversity in the lung, while administration of mixed probiotics could improve the above situation (Fig. 3).

Fig. 3
figure 3

Alpha-diversity index intergroup difference test. Alpha-diversity is used to appreciate the number of OTUs in a single sample. A greater alpha-diversity indicates a higher number of OTUs. Main alpha-diversity index used are the sobs, ace and Shannon index. The Shannon index of BALF flora samples from the control group and probiotics group on the 1st and 5th day of mechanical ventilation were tested by Wilcoxon rank-sum test. cgbd1: BALF flora samples from control group on the 1st day of mechanical ventilation. cgbd5: BALF flora samples from control group on the 5th day of mechanical ventilation. pgbd1: BALF flora samples from probiotics group on the 1st day of mechanical ventilation. pgbd5: BALF flora samples from probiotics group on the 5th day of mechanical ventilation. **P ≤ 0.01

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Using the Shannon index results obtained from alveolar lavage fluid from the control group, the efficacy of the analysis was verified in this study, which showed that group sample sizes of 11 and 11 achieved 99.851% power to reject the null hypothesis of equal means when the population mean difference was μ1—μ2 = 2.377–0.67 = 1.707 with standard deviations of 0.411 for group 1 and 0.977 for group 2 and with a significance level (alpha) of 0.05 using a two-sided two-sample unequal-variance t test.

To determine the influence of pressure and oxygen concentration on lung microbiota, we divided patients into the ARDS group (AG, n = 5) and the conventional mechanical ventilation group (NG, n = 19) based on whether protective mechanical ventilation was used. The results showed significant differences in the Shannon index among all groups except for the first day's results, further suggesting that hyperoxia and high pressure can affect lung microbiota diversity (Table 2).

Table 2 The Shannon index of AG and NG
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Sample comparison analysis

According to the results of principal coordinate analysis (PCoA), there was no significant difference in lung microbiota between the control group and the probiotic group on the 1st day of mechanical ventilation (R = 0.0376, P = 0.755). The lung flora of the control group on the 1st and 5th day of mechanical ventilation were significantly different (R = 0.2448, P = 0.004). The lung flora of the probiotic group on the 1st and 5th day of mechanical ventilation were not significantly different (R = 0.0109, P = 0.514), and the lung flora of the control group and the probiotic group on the 5th day of mechanical ventilation were significantly different (R = 0.1296, P = 0.035) (Fig. 4A). Using this analysis, we again showed that mixed probiotics ameliorated the decreased microbial community diversity in the lungs due to mechanical ventilation.

Fig. 4
figure 4

PCoA analysis. Beta diversity is used to describe the differences in OTUs diversity between samples. A greater beta-diversity indicates higher differences of OTUs between samples. Widely used beta-diversity index are Bray–Curtis distance. Investigating the microbial community composition at different durations of mechanical ventilation at the genus level by principal coordinate analysis (PCoA) based on Bray–Curtis distances. cgbd1: BALF flora samples from control group on the 1st day of mechanical ventilation. cgbd5: BALF flora samples from control group on the 5th day of mechanical ventilation. pgbd1: BALF flora samples from probiotics group on the 1st day of mechanical ventilation. pgbd5: BALF flora samples from probiotics group on the 5th day of mechanical ventilation. PC: principal component. The red and blue circles represented the confidence ellipse at a confidence level of 95%, respectively. Spots of different colours represented samples with different groupings, and the closer the two spots were, the more similar the species composition of the two samples was

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Analysis of species differences

The analysis of species differences showed that the microbial communities significantly differed between the lungs of the control and probiotic groups at the genus level during different durations of mechanical ventilation. The microbiota of each site was not the same, and among the three probiotics used for intervention (Bifidobacterium, Lactobacillus, Enterococcus), only Lactobacillus showed a significant difference in the BALF of the control group (P = 0.009692). Lactobacillus abundance was more susceptible to change due to mechanical ventilation compared to the other two species, while probiotic supplementation did not cause an increase in the abundance of specific microbes, but rather improved the diversity of the overall microbial community (Fig. 5).

Fig. 5
figure 5

Analysis of species differences. The Wilcoxon rank-sum test was used to perform hypothesis testing on lung samples from the control and probiotic groups at various mechanical ventilation times, assessing the significance of species abundance differences at the genus level, and identifying significant differences in species abundance between the groups. cgbd1: BALF flora samples from control group on the 1st day of mechanical ventilation. cgbd5: BALF flora samples from control group on the 5th day of mechanical ventilation. pgbd1: BALF flora samples from probiotics group on the 1st day of mechanical ventilation. pgbd5: BALF flora samples from probiotics group on the 5th day of mechanical ventilation

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Analysis of associations with clinical factors

In this study, the measurement of IL-1β, IL-6, IL-8 and IL-17A levels in BALF (Fig. 6) and blood (Fig. 7) indicated that in the control group, the IL-1β, IL-6, IL-8 levels in BALF and blood increased over time, and decreased in the probiotic group. In the control group, the IL-17A levels in BALF and blood decreased over time, and decreased in the probiotic group. The differences were all statistically significant. Probiotics can affect the inflammatory response of critically ill patients. In order to determine whether changes in cytokines are affected by the primary disease, we divided patients into a death group (DG) and a survival group (SG) based on their prognosis, and compared the changes in cytokines. The results showed that there was no statistical difference in each cytokine between the groups (Tables 3, 4). This result further strengthens our hypothesis.

Fig. 6
figure 6

Cytokine levels in BALF. CG: control group. PG: probiotics group. IL-1βd1: IL1β results on 1st day. IL-1βd5: IL1β results on 5th day. IL-6d1: IL6 results on 1st day. IL-6d5: IL6 results on 5th day. IL-6d1: IL6 results on 1st day. IL-6d5: IL6 results on 5th day. IL-17Ad1: IL17A results on 1st day. IL-17Ad5: IL17A results on 5th day

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Fig. 7
figure 7

Cytokine levels in blood. CG: control group. PG: probiotics group. IL-1βd1: IL1β results on 1st day. IL-1βd5: IL1β results on 5th day. IL-6d1: IL6 results on 1st day. IL-6d5: IL6 results on 5th day. IL-6d1: IL6 results on 1st day. IL-6d5: IL6 results on 5th day. IL-17Ad1: IL17A results on 1st day. IL-17Ad5: IL17A results on 5th day

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Table 3 IL-1β, IL-6, IL-8, IL-17A in BALF of DG and SG groups
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Table 4 IL-1β, IL-6, IL-8, IL-17A in blood of DG and SG groups
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According to analysis of associations with clinical factors, the results showed that in the control group, there was a significant correlation between the levels of pulmonary microbes and IL-17A levels (r2 = 0.4584, p = 0.003) in BALF and IL-17A levels (r2 = 0.3772, p = 0.01) in the blood. In the probiotic group, there was a significant correlation between the levels of pulmonary microbes and IL-17A (r2 = 0.2776, p = 0.042) and IL-6 (r2 = 0.2861, p = 0.04) levels in BALF and IL-17A levels (r2 = 0.3755, p = 0.017) in the blood (Fig. 8).

Fig. 8
figure 8

Clinical factor association analysis. To investigate the relationship between each cytokine and the lung microbial flora, in this study, the correlation between the levels of lung microbes at the genus level and cytokine was analysed by db-RDA based on the Bray‒Curtis distance algorithm. cgbd1: BALF flora samples from control group on the 1st day of mechanical ventilation. cgbd5: BALF flora samples from control group on the 5st day of mechanical ventilation. pgbd1: BALF flora samples from probiotics group on the 1st day of mechanical ventilation. pgbd5: BALF flora samples from probiotics group on the 5st day of mechanical ventilation. CAP: canonical analysis of principal coordinates. Arrows represent clinical factors. balil1: IL-1 levels in BALF. balil6: IL-6 levels in BALF. balil8: IL-8 levels in BALF. balil17: IL-17A levels in BALF. bloil1: IL-1 levels in blood. bloil6: IL-6 levels in blood. bloil8: IL-8 levels in blood. bloil17: IL-17A levels in blood. The spots of different colours represented samples of different groups, the distance between the spots represented the similarity and difference in the composition of the sample microbiota, the red arrow represented quantitative clinical factors, and the length of the clinical factor arrow represented the degree of influence of clinical factors on species data

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Discussion

Probiotics are defined by the Food and Agriculture Organization (FAO) / World Health Organization (WHO) as live microorganisms that, when administered in moderate amounts, produce health benefits to the host [9]. When the normal natural flora is disturbed, exogenously supplemented probiotics can temporarily colonize the gut and stabilize the flora composition, thereby restoring important physiological functions. Animal trials have shown [10, 11] that the application of probiotics improves the survival of mice infected by viruses or bacteria. In the present study, the application of probiotics did not improve patients' duration of mechanical ventilation or 28-day mortality, but patients' incidence of constipation was improved. Vieira AT et al. [10] showed that under the action of probiotics, TNF-α and IL-6 increased due to infection were significantly reduced. In this study, the levels of IL-1β, IL-6 and IL-8 in the probiotic group were lower than those in the control group on day 5, and there was no statistical difference in cytokines between the SG and the DG, indicating that probiotics can affect the inflammatory response in critically ill patients.

Emonet et al. [12] showed that the proportion of Proteobacteria increased and the proportion of Firmicutes decreased in VAP patients over time, which may be related to the changes caused by mechanical ventilation [5]. During the study from admission to day 5, one VAP occurred in the control group, and no VAP occurred in the probiotic group whose microbiota did not show any of the above changes. This was consistent with the above research.

The impact of probiotics does not reside in their ability to transplant in the microbiota, but in supporting the challenged microbiota by producing specific metabolites or cellular components and sharing genes [13]. In this study, among the probiotics we used for intervention, Bifidobacterium belongs to Actinobacteria, which increased most significantly in the probiotics group, and Lactobacillus belongs to Firmicutes, which also increased in the probiotics group. Coyte KZ et al. [14] found that increasing cooperation within colonies leads to a decrease in the stability of the microbiota, as cooperation leads to positive feedback between species. This means that when one species helps another species survive and replicate, a decrease in the abundance of one species will lower the abundance of another species, thereby disrupting overall stability. Relatively, competition can improve the stability of microbial communities, while the key is the introduction of new species and the weakening of inter species interactions. The probiotics we used were intervened at the genus level, while the OTU analysis we did was at the phylum level, which may be affected by the changes of other bacteria. We will increase the sample size in the follow-up study to strengthen the research in this area.

Nasogastric tubes are commonly used to provide nutrition and drugs to patients in the ICU, and several studies [15,16,17,18,19] have indicated that Bifico nasogastric tube administration can promote the recovery of intestinal function, improve intestinal barrier function, promote neurological recovery, and improve intestinal immune function. Nasogastric feeding can allow Bifico to function normally, and the present study used a continuous pumping mode to administer enteral nutrition, which could achieve the same bioavailability as oral nutrition.

Mechanical ventilation, as a common therapeutic measure in the ICU, can be used to treat respiratory failure and improve patient outcomes and is the most effective means of respiratory support in critically ill patients due to its effects on improving oxygenation, maintaining ventilation, and reducing the work of respiratory muscles [20, 21]. However, it was also found to be accompanied by ventilator-associated lung injury, decreased cardiac output, increased intracranial pressure, oxygen toxicity, ventilator-associated pneumonia and other complications [22, 23]. It has been confirmed in several studies [24, 25] that mechanical ventilation can induce a decrease in the diversity of the lower respiratory tract microbial flora in patients. In this study, the alpha-diversity index, which reflects the richness and diversity of pulmonary microbial communities, decreased in the control group. Then, sample comparison analysis showed that over time, there was a significant difference in beta-diversity between the two groups of patients. The reason for this may be related to hyperoxia and high pressure [26, 27].

Lactobacillus, unlike Bifidobacterium and Enterococcus, decreased significantly in the control group. We did not find any research on the survival of Lactobacillus, Bifidobacterium and Enterococcus in the lung. Oxygen, pH, blood perfusion and the number of inflammatory cells and other factors can affect the changes of bacterial flora [28]. A study used different substrates to simulate the gastrointestinal environment in vitro for Lactobacillus and Bifidobacterium, and the results showed that the survival rate of Bifidobacterium was higher than that of Lactobacillus [29]. Similarly, another study used dynamic computer-controlled in vitro model (TIM-1) to study the survival rate of Lactobacillus and Bifidobacterium, and obtained the same results [30]. Thus, the reduction of Lactobacillus in the control group may be related to its low survival rate.

Studies have shown that disruption of microbial community diversity in humans may cause disturbances in host metabolism, immunity, and even neurocognition [4], and the decreased diversity of lung flora is associated with acute respiratory distress syndrome and ventilator-associated pneumonia (VAP) [4]. Animal experiments have confirmed that prophylactic application of probiotics improves the alpha-diversity of the microflora [31] and increases the survival rate [32]. The present study, in which probiotics were administered after patients had received mechanical ventilation, showed that alpha-diversity was not reduced by mechanical ventilation in patients in the probiotics group and, similarly, did not show a significant difference during sample comparisons. This finding illustrates that administration of probiotics ameliorates the decline in flora diversity due to mechanical ventilation. Species difference analysis indicated that the decreased abundance of Lactobacillus was improved by probiotic supplementation, while Bifidobacterium and Enterococcus did not show significant differences in abundance although they were also used for intervention. Since probiotics can competitively inhibit pathogenic microorganisms by occupying adhesion sites, consuming nutrients and producing antimicrobial substances, etc. [33], and regulate the diversity of microbial flora, the supplementation of probiotics does not simply increase the number of specific microbes, but rather improve the diversity of the flora overall; therefore, this study also provides new evidence for the safety of probiotic use.

At present, the link between the gut and the lung is mainly explained through the gut–lung axis, but the specific mechanisms are not fully understood, and the migration of immune cells and cytokines through the circulatory system is one of the mechanisms [34,35,36]; components of microorganisms are recognized by transmembrane toll-like receptors (TLRs), and the downstream effects of TLR signalling lead to the activation of innate immune cells, including the differentiation of CD4 + T cells into Th17 cells, which promotes IL-17 expression, which in turn regulates immune responses to pathogenic microorganisms. In the clinical factor association analysis, both blood and BALF IL-17A levels showed significant correlations, indicating the involvement of cytokines in the gut–lung axis.

In this study, IL-17 showed inconsistent changes with other cytokines. IL-17A was shown to fortify tight junction formation between epithelial cells by stimulating mucin production by intestinal epithelial cells, thereby increasing the integrity of the intestinal barrier [37]. It was also revealed that murine DSS(dextran sulfate sodium)-induced colitis was worsened in IL-17A-knockout mice but was substantially improved in IL-17F-knockout mice [38]. IL-17 is a cytokine known to directly activate intestinal cells, leading to the secretion of antimicrobial peptides and the recruitment of neutrophils [35]. Segmented filamentous bacteria (SFB), which play a major role in the maturation of the intestinal immune system, promote the differentiation of Th17 CD4 T cells under the effect of IL-17, and induce Th17 cells to prevent the intestinal colonization of pathogens [39]. Under inflammatory conditions, the metabolites produced by different probiotics with different prebiotics have different immune regulatory properties on immune cells [40].

There are still certain limitations to this study. First, there was a failure to analyse the gut microbiota in the study. Due to the inability to obtain suitable stool samples, we cannot detect the intestinal flora and provide the most direct evidence of changes in the digestive tract flora. Second, microbial metabolites were not analysed, which will provide better elucidation of the mechanisms underlying the gut–lung axis. Third, it is necessary to evaluate the number of organisms in order to demonstrate the beneficial effects of probiotics on the host. Fourth, our trial was a single-centre study, not double-blinded, with a small sample size and may have had many confounding factors that may have affected the results. We will refine this in future designs.

In conclusion, the present study showed that mechanical ventilation induced a decline in microbial diversity in the lungs, which can be improved by the administration of a mixed probiotic.

Availability of data and materials

All data generated and/or analysed during this study are included in this published article.

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Authors and Affiliations

  1. Department of Emergency Medicine, Hebei Medical University, Shijiazhuang, Hebei, China

    Peng Lu

  2. Department of Intensive Care Unit, Hebei General Hospital, Shijiazhuang, Hebei, China

    Peng Lu, Huawei Wang & Heling Zhao

  3. Department of Intensive Care Unit I, The First Hospital of Hebei Medical University, Shijiazhuang, Hebei, China

    Peng Lu, Dongliang Li, Jie Zhang & Zhitao Zhao

  4. Department of Chest Surgery, The First Hospital of Hebei Medical University, Shijiazhuang, Hebei, China

    Qing Tian

Authors
  1. Peng Lu
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  2. Dongliang Li
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  3. Qing Tian
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  4. Jie Zhang
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  5. Zhitao Zhao
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  6. Huawei Wang
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  7. Heling Zhao
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Contributions

Peng Lu, and Heling Zhao contributed to the conception and design of the research; Dongliang Li and Qing Tian contributed to the acquisition and analysis of the data; Jie Zhang and Zhitao Zhao contributed to the acquisition of the data; Huawei Wang contributed to the analysis and interpretation of the data.

Corresponding author

Correspondence to Heling Zhao.

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The study was registered on the China Clinical Trial Registry website: www.chictr.org.cn (ChiCTR2300068452) and authorized by the First Hospital of Hebei Medical University 's Ethics Committee (approval number: 20210741).

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The authors declare no competing interests.

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Lu, P., Li, D., Tian, Q. et al. Effect of mixed probiotics on pulmonary flora in patients with mechanical ventilation: an exploratory randomized intervention study. Eur J Med Res 29, 473 (2024). https://doi.org/10.1186/s40001-024-02059-0

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  • DOI: https://doi.org/10.1186/s40001-024-02059-0

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European Journal of Medical Research

ISSN: 2047-783X

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