Dynamics of gut microbiota in qualified female boxers

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Anastasiia M. Kaliga1

, Oksana L. Palladina1

, Olena A. Dulo2

, Svitlana A. Burmei 2,3,Dmytro V. Shtanagei1,

Nadiya V. Boyko2,3

1

NATIONAL UNIVERSITY OF UKRAINE ON PHYSICAL EDUCATION AND SPORT, KYIV, UKRAINE

2

UZHHOROD NATIONAL UNIVERSITY, UZHHOROD, UKRAINE

3

EDIENS LLC, VELYKI LAZY, UKRAINE

ABSTRACT

Aim: To analyze changes in the gut microbiota of qualified female boxers at different stages of the training cycle, influenced by physical activity of different

intensity.

Materials and Methods: The study involved nine qualified female boxers, who performed high-intensity training sessions. The study of changes in the state

of the microbiota, namely its diversity, was carried out in two phases of the training cycle. A microbiological quantitative method was used, in-depth with

species identification and detection of anaerobes, the unit of measurement of which was colony-forming units per gram (CFU/g). The quantitative assessment

of microorganisms was performed using arithmetic mean values expressed and log-transformed values (log10 CFU/g).

Results: High-intensity training loads were associated with microbiota changes indicative of dysbiosis. It is noticeable that the concentration of typical patho-

genic genera (Candida albicans, Klebsiella pneumoniae, Streptococcus) significantly increased during the post-competition period. At the same time, classic

“beneficial” bacteria (Lactobacillus and Bifidobacterium) remained within normal limits, although Bifidobacterium showed some growth. Notably, Escherichia

coli (a normal representative of the microbiome) exceeded the norm during the post-competition period.

Conclusions: Increased training intensity in qualified female boxers resulted in alterations in gut microbiota composition, most notably a significant rise in

Escherichia coli and other opportunistic microbes, while the levels of beneficial Lactobacillus and Bifidobacteria species remained relatively stable. These changes

suggest early signs of dysbiosis, consistent with current evidence on the impact of extreme physical exertion on microbial health.

KEY WORDS: microbiota profile, high-intensity training, female athletes

Wiad Lek. 2025;78(9):1765-1770. doi: 10.36740/WLek/212509 DOI

INTRODUCTION 

Recent studies have demonstrated a complex, dose-de- pendent effect of exercise on gut microbiota composi- tion and function. A meta-analysis of 25 studies includ- ing 1044 participants showed that exercise significantly  increased gut microbiota alpha diversity, including the Shannon index [1]. A systematic review of 28 studies  confirmed that moderate- or vigorous-intensity phys- ical activity performed for 30–90 min, more than 3  times per week (or 150–270 min weekly) for a period of 8 weeks can induce changes in the gut microbiota [2]. Intensive physical activity has been associated with  an increase in butyrate- and succinate-producing bac- teria, which affects the metabolic homeostasis of the  host and has a potentially positive effect on the state of the immune system [4]. Professional soccer players have been shown to have an increased diversity of gut microbiota compared to amateurs, particularly during periods of intensive training.  A study of professional martial artists revealed a signif- icant pattern: higher-level athletes exhibit significantly  greater gut microbiota diversity compared to lower-level athletes [5]. Shannon (p = 0.019) and Simpson (p = 0.001) indices were significantly higher in highly skilled athletes  [5]. This is particularly important for female boxers, as in- creased microbial diversity correlates with better immu- nity, reduced susceptibility to respiratory infections, and  lower BMI values [6]. Gender is an important modulator of the microbiota response to physical exercise. Women tend to show greater changes in Shannon index and observed operational taxonomic units [5]. The gut microbiota may significantly influence the  physical performance of female boxers through multi- ple mechanisms, from energy metabolism to immune  regulation. Increased microbial diversity, enrichment with specific beneficial taxa, and optimized microbial  function are positively correlated with athletic perfor- mance [7].

AIM

The aim was to analyze changes in the gut microbiota

of qualified female boxers at different stages of the

training cycle, influenced by physical activity of differ-

ent intensity.

MATERIALS AND METHODS

The study involved nine qualified female boxers from

the Kyiv City School of Higher Sports Mastery (Kyiv,

Ukraine), a specialized sports training center. The

average age of the boxers was 22.00 ± 0.89 years.

The study was conducted during the preparation

period and immediately after the competitive period

(post-competition period), during which the athletes

performed high-intensity training sessions lasting 90

minutes, 6 times per week [8]. During the scientific

study, all recommendations to ethics committee rec-

ommendations on biomedical research were followed.

All participants provided written informed consent for

the use of their data in scientific research. To effectively

address the goal of the scientific study, a combination

of sport science, microbiological, and medical methods

was applied: microbiota research, leukocyte formula

analysis to verify the inflammatory process; methods

of mathematical statistics. To study the features and

diversity of the intestinal microflora in female boxers, an

in-depth study of the state of the intestinal microbiota

was conducted with the identification of commensal

and opportunistic microorganisms to the species with

the determination of the diagnostic ratio. For this

purpose, a microbiological quantitative method was

used, in-depth with species identification (biochemical

automated test systems) and detection of anaerobes,

the unit of measurement of which was colony-forming

units per gram (CFU/g). The quantitative assessment of

microorganisms was performed using arithmetic mean

values (М±m) expressed and log-transformed values

(log10 CFU/g). Statistical significance of differences was

determined using Student’s t-test. Data analysis was carried out with Statistical 10.0 Software (StatSoft, Inc., USA) and Microsoft Excel.  RESULTS The study of changes in the state of the microbiota, namely its diversity, was carried out in two phases. The first one was during the preparatory period, during which the athletes engaged in moderate-intensity physical activity. In order to monitor whether changes occur in the microbiota profile during high-intensity  training, a follow-up analysis was conducted con- ducted after 8 weeks, in the post-competition period.  The obtained test results are presented in Table 1 and Fig. 1. In the preparation period, the average value of Escherichia coli was (1.48 ± 0.98)×108 CFU/g (log10 8.17 ± 1.20), and in the post-competition period – (2.86 ±  1.55)×1010 CFU/g (log10 10.46 ± 1.17). Thus, the con- centration of E. coli in the post-competition period  increased nearly 200-fold. The log10 value also increased, but the difference did not reach statistical significance (p > 0.05). Both average values exceed the upper limit  of the norm (108 CFU/g), suggesting excessive coloni- zation – potentially a sigh of intestinal dysbiosis due to  intense training load. Enterococcus faecalis increased approximately 6-fold during the post-competition period. According to the t-test, this increase was statistically significant (t=6.41, p < 0.01). Specifically its concentration rose from (2.36 ± 1.32)×106  CFU/g (log10 6.37 ± 1.32), and in the post-competition pe- riod the concentration was (1.47 ± 0.14)×107  CFU/g (log10 7.17 ± 0.51). Both values are within the physiological norm.  Notably, the decrease in standard error (±0.51 vs ±1.32) in- dicates a more stable level in the post-competition period.  This may reflect an adaptive intestinal response to changes in metabolism or diet during and after the competition. The concentration of Lactobacillus spp was (1.66 ± 1.14)×107  CFU/g (log10 7.22 ± 1.39) during the prepa- ration period and (1.43 ± 0.14)×107  CFU/g (log10 7.16  ± 0.58) after competition. A slight decrease of ap- proximately 0.2×107 is observed, but this change is  statistically insignificant (t=0.2, p > 0.05). Such a minor reduction during intense training may be a typical sign of dysbiosis, but remained within the normal range and lacked statistical relevance. Regarding the concentration of Bifidobacterium spp in the preparation period the observed value  Table 1. Gut microbiota profile of trained female boxers by training intensity, n=9 (М±m), Log10 Microorganism  Preparation period Post-competition period Reference Concentration of CFU/g range М±m CFU/g log10 CFU/g М±m CFU/g log10 CFU/g CFU/g  Escherichia coli, lac+(n=9) (1,48±0,98)×108 8,17±1,20 (2,86±1,55)×1010 10,46±1,17  106 – 108  t-test 1,84 Р > 0,05 Enterococcus faecalis (n=9) (2,36±1,32)×106 6,37±1,32 (1,47±0,14)×107 7,17±0,51  105 – 108  t-test 6,41 Р < 0,01 Lactobacillus spp (n=8) (1,66±1,14)×107 7,22±1,39 (1,43±0,14)×107 7,16±0,58 106 – 108 t-test 0,2 Р > 0,05 Bifidobacterium spp (n=9) (3,49±1.48)×106 6,54±1,05 (5,03±2,22)×107 7,70±0,62  107 – 108  t-test 2,23 Р < 0,05 Candida albicans (n=6) 106 - (1,67±0,192)×108 8,22±0,91 <104 Klebsiella pneumoniae (n=4) 104 - (7,53±2,47)×105 5,88±0,50 <104 Streptococcus agalactiae (n=2) 106 - (5,00±5,00)×109 9,70±3,0 <104 Note: t – Student’s test; p – significance of the difference in different periods of the training cycle Source: compiled by the authors of this study  Anastasiia M. Kaliga et al.  1768  richia coli (a normal representative of the microbiome)  exceeded the norm during the post-competition peri- od. Taken together, these findings indicate a shift in the  gut microbiota balance: on the one hand, the number of commensals increased, while on the other, atypical  pathogens emerged, a combination that is character- istic of excessive physiological stress. These findings  align with previous studies reporting that excessive or extreme physical exertion may lead to a decrease in  beneficial barrier bacteria and an overgrowth of po- tentially pathogenic species, i.e., dysbiosis, reflecting  the adverse effects of overly intensive athletic training. DISCUSSION  A critical consideration in this context is the distinction be- tween moderate and intense physical activity, as they have  different effects on the gut microbiota, Clauss M. et al. [3].  Moderate exercise has been associated with re- duced inflammation, improved body composition,  and favorable changes in gut microbial diversity and composition. In contrast, high-intensity exercise may  increase the permeability of the gastrointestinal ep- ithelial barrier and reduce the thickness of the intes- tinal mucus layer, potentially allowing pathogens to  enter the bloodstream and contributing to increased  inflammation [3]. In a study of military personnel under- going intense physical training, intestinal permeability  increased by 62±57%, accompanied by alterations in the gut microbiota including increased alpha-diversity and changes in the relative abundance of over 50% of  identified genera, Grosicki, G. J. et al. [9]. These find- ings suggest that sustained physical stress negatively  affects intestinal barrier integrity while simultaneously altering microbial composition. Similarly, Barton W. et al. studying elite athletes revealed specific functional  adaptations of the gut microbiota. In particular, in mar- athon runners after the race, an increase in the relative  abundance of Veillonella atypica, a bacterium capable of metabolizing lactate as its sole carbon source, was observed [10]. Experimental administration of this strain to mice significantly prolonged treadmill running time to exhaustion, which demonstrates a direct relationship between specific microbes and sports performance. A pivotal discovery was the identification of Veilonella as a bacterium that enhances endurance through a metabolic conversion of exercise-induced lactate into propionate [10]. A study using radiolabeled isotopes showed that serum lactate crosses the epithelial barrier  into the intestinal lumen, and intrarectal administra- tion of propionate is sufficient to reproduce increased  endurance performance by 133% and reduce muscle fatigue [10].  was (3.49±1.48)×106  CFU/g (log10 6.54 ± 1.05), and in the post-competition period it increased to (5.03 ± 2.22)×107  CFU/g (log10 7.70 ± 0.62). The content of bifidobacteria in the post-competition period increased approximately 15 times. The difference is statistically  significant (p < 0.05). The reference range is approxi- mately (107  – 108  CFU/g), so the values of the prepa- ration period were somewhat underestimated, and  in the post-competition period they approached the lower threshold of the reference range. Bifidobacteria are commensal intestinal microorganisms with known  anti-inflammatory properties. Their increased abun- dance following intense physical activity may reflect a  beneficial adaptive response. In the preparation period, a level of 106  CFU/g (i.e.,  which is close to the detection limit) for Candida al- bicans was detected, in the post-competition period  it was (1.67 ± 0.192)×108  CFU/g (log10 8.22 ± 0.91). The normal upper limit for Candida albicans is <104 CFU/g, indicating a pathologically elevated level in the post-competition period. No statistical comparison was performed (it was present in the preparation period in only 1 athlete), but the fact that it appeared in 6 out of 9 people with such a high concentration indicates a significant disturbance of the microbiota. The presence of Klebsiella pneumoniae 104 CFU/g in the microbiota of the examined female boxers in the preparation period is also noteworthy. However, in the post-competition period, the level significantly exceeded the normal range (7.53 ± 2.47)×105 CFU/g (log10 5.88±0.50). Klebsiella pneumoniae is a facultative pathogen; its appearance in 4 out of 9 female athletes in significant quantities indicates the presence of dysbiosis. Also, in two female athletes, we observed the appearance of Streptococcus agalactiae during the post-competition period (5.00±5.00)×109  CFU/g (log10  9.70±3.0) and between competitions 106  CFU/g, with an  approximate norm of <104  CFU/g, which represents an unfavorable finding, since Streptococcus agalactiae is a potentially pathogenic species, the presence of which indicates the presence of dysbiosis.  High-intensity training loads were associated with mi- crobiota changes indicative of dysbiosis. It is noticeable  that the concentration of typical pathogenic genera (Candida albicans, Klebsiella pneumoniae, Streptococcus)  significantly increased during the post-competition pe- riod. This is consistent with the observations that high  physiological tension and stress can reduce the barrier functions of the intestine and promote the excessive growth of opportunistic microorganisms. At the same  time, classic “beneficial” bacteria (Lactobacillus and Bi- fidobacterium) remained within normal limits, although  Bifidobacterium showed some growth. Notably, Esche-  Dynamics of gut microbiota in qualified female boxers  1769 Current research demonstrates a complex, non-linear  relationship between exercise intensity and gut micro- biota. The “inverted U-curve” model best describes this  association: low exercise frequency and duration result in minimal changes, moderate exercise (30-90 minutes 3-5 times a week) causes the greatest positive changes, whereas excessively intense exercise (>90 minutes, >5 times a week) may elicit detrimental effects. The clinical significance of these findings lies in the possibility of developing personalized training programs that incorporate individual microbiota profiles. Such approaches may not only enhance athletic performance but also support the long-term health of athletes.  Future research should aim to elucidate the mech- anisms underlying the bidirectional interactions  between physical activity and the gut microbiota.  Moreover, standardized methodologies and longitu- dinal study designs are needed to better understand  the sustained effects of various training modalities on microbial health.  CONCLUSIONS Increased training intensity in qualified female boxers resulted in alterations in gut microbiota composition, most notably a significant rise in Escherichia coli and other opportunistic microbes, while the levels of  beneficial Lactobacillus and Bifidobacteria species re- mained relatively stable. These changes suggest early  signs of dysbiosis, consistent with current evidence on the impact of extreme physical exertion on microbial health. To enhance athletes ‘ physical performance and improve competitive outcomes, future interventions  may consider probiotic supplementation and the de- velopment of personalized nutritional strategies based  on individual microbial profiles.  Short-chain fatty acids (SCFAs) such as propionate, acetate and butyrate are key metabolites that link the gut microbiota to host metabolism. Athletes typically  exhibit increased microbial diversity and a composition- al shift towards bacterial taxa involved in amino acid  biosynthesis and carbohydrate/fiber metabolism, which leads to enhanced SCFA production, Clauss M. et al. [3].  According to Bonomini-Gnutzmann et al., exces- sively intense or prolonged physical activity can lead  to increased intestinal permeability, contributing to exercise-induced gastrointestinal disturbances and systemic inflammation [11]. Paradoxically, among elite athletes, despite a modest increase in diversity, in some cases a decrease in microbiota diversity is observed, compared to individuals performing moderate physical activity Li Y. et al. [12]. However, elite athletes still have high levels of fecal SCFA, which play a crucial role in host energy metabolism. A multi-cohort study of 543 samples from athletes participating in various sports disciplines (aerobics, wrestling, rowing) revealed the presence of sport-specific gut microbiota profiles [12]. Through the application of Latent Dirichlet Allocation (LDA), 10 microbial subgroups were identified and were associated with specific inflammation markers, dietary patterns, and anaerobic performance indicators. A review of the current literature reveals inconsistent findings regarding the impact of moderate-intensity  and vigorous-intensity exercise on gut microbiota di- versity and composition. Some studies have reported  negative effects of aerobic exercise on gut microbiota, such as elevated intestinal fatty acid–binding protein  I-FABP, gastrointestinal discomfort and adverse micro- bial shifts [11, 13]. However, the majority of evidence  supports beneficial outcomes of endurance training on gut microbiota, including increased microbial diversity. 

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