Microbial Contamination in Shaker Bottles among Members of Fitness Centers

Abstract

Silveira MB, Scudese E, Senna GW, Ferreira AP, Dantas EHM, Ribeiro LCP, Alvares AFH, Guedes PG. Microbial Contamination in Shaker Bottles among Members of Fitness Centers. JEPonline 2018;21(4):134-142. The purpose of this study was to analyze the presence of different bacterial strains and to determine resistance to antimicrobials in used bottles (UB) from the members of fitness centers and new non-used bottles (NUB). A total of 60 shakers (30 UB and 30 NUB) were selected and submitted to microbiological analysis. The samples were collected through Swabs containing Stuart's medium and delivered to the laboratory. Gram staining and biochemical tests were performed for the identification of microorganisms and antimicrobial susceptibility. The Cochran Q test presented significant difference (P=0.001) in contamination status (UB vs. NUB). All the NUB tests showed 100% absence of contamination while 90% of the UB showed bacterial contamination. For the 60 samples investigated, we were able to isolate six species of different microorganisms. It is important to note that 16.6% had no bacterial growth. The antibacterial susceptibility test revealed a varied range of resistance profile. In conclusion, six pathogenic microorganisms were isolated from poorly sanitized bottles highlighting that the post-use hygiene must be made appropriately to avoid the proliferation of pathogenic bacteria.

Full text

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Journal of Exercise Physiology
online
August 2018
Volume 21 Number 4
Editor
-
in
-
Chief
Tommy Boone, PhD, MBA
Review Board
Todd Astorino, PhD
Julien Baker, PhD
Steve Brock, PhD
Lance Dalleck, PhD
Eric Goulet, PhD
Robert Gotshall, PhD
Alexander Hutchison, PhD
M. Knight-Maloney, PhD
Len Kravitz, PhD
James Laskin, PhD
Yit Aun Lim, PhD
Lonnie Lowery, PhD
Derek Marks, PhD
Cristine Mermier, PhD
Robert Robergs, PhD
Chantal Vella, PhD
Dale Wagner, PhD
Frank Wyatt, PhD
Ben Zhou, PhD
Official Research Journal
of the American Society of
Exercise Physiologists
ISSN 1097-9751
Official Research Journal of
the American Society of
Exercise Physiologists
ISSN 1097-9751
JEPonline
Microbial Contamination in Shaker Bottles among
Members of Fitness Centers
Matheus Baffi Silveira1, Estevão Scudese2,3, Gilmar Weber Senna2,3,
Ana Paula Ferreira1, Estélio Henrique Martin Dantas2, Luiz Cláudio
Pereira Ribero2, Alessandra Fiuza Hoelz Alvares3, Patrícia Guedes
Garcia1
1Microbiology Post-Graduation Program, Medical Sciences and
Health Faculty, Juiz de Fora, MG, Brazil, 2Nursing and Biosciences
Post-Graduation Program, Doctorate of Federal University of State
of Rio de Janeiro, Brazil, 3Sports Science and Exercise Laboratory
(LaCEE), Catholic University of Petrópolis, RJ, Brazil
ABSTRACT
Silveira MB, Scudese E, Senna GW, Ferreira AP, Dantas EHM,
Ribeiro LCP, Alvares AFH, Guedes PG. Microbial Contamination
in Shaker Bottles among Members of Fitness Centers. JEPonline
2018;21(4):134-142. The purpose of this was to analyze the
presence of different bacterial strains and to determine resistance to
antimicrobials in used bottles (UB) from the members of fitness
centers and new non-used bottles (NUB). A total of 60 shakers (30
UB and 30 NUB) were selected and submitted to microbiological
analysis. The samples were collected through Swabs containing
Stuart's medium and delivered to the laboratory. Gram staining and
biochemical tests were performed for the identification of
microorganisms and antimicrobial susceptibility. The Cochran Q test
presented significant difference (P=0.001) in contamination status
(UB vs. NUB). All the NUB tests showed 100% absence of
contamination while 90% of the UB showed bacterial contamination.
For the 60 samples investigated, we were able to isolate six species
of different microorganisms. It is important to note that 16.6% had no
bacterial growth. The antibacterial susceptibility test revealed a
varied range of resistance profile. In conclusion, six pathogenic
microorganisms were isolated from poorly sanitized bottles
highlighting that the post-use hygiene must be made appropriately to
avoid the proliferation of pathogenic bacteria.
Key Words: Bacteria, Exercise, Health, Microbiology

135
INTRODUCTION
In recent years, the general concern for health and well-being has increased significantly with
more people pursuing a more conscious lifestyle. This phenomenon is in line with the growth
in the total number of fitness centers around the world. A gym environment is a place where
individuals engage in regular exercise to improve their quality of life and health (3). Among
this population, a consensus on the importance of overall nutrition and hydration for achieving
their health and fitness goals are well known. For instance, adequate water intake is vital to
prevent dehydration and its adverse effects, such as headaches and urinary lithiasis during
exercise (1).
Nutrition is an important tool within sports practice and when well elaborated promotes the
maintenance of the health of the physical exercise practitioner (2). Previous studies have
demonstrated both acute and chronic ergogenic effects of different types of supplementation
in subjects undergoing strength training routines (10). Additionally, different substances may
require distinct intake approaches and strategies. For instance, the window of opportunity for
hypertrophy may be associated with the use of whey protein hours after training (9) as
opposed to pre-workout/stimulants (20). The fact of the matter is that independent of the
timing, many individuals carry shakers bottles along their gym routine.
The propagation of this knowledge is allied to the growth of the supplement industry that has
increased the use of shaker bottles among gym members (2). However, what is normally not
well-known is that improper use of shaker bottles may favor the growth of microorganisms on
the walls of the container. While the practice of regular exercise increases the demand of
water intake and, often times, the use of some commonly prescribed supplements (such as
branched-chain amino acids (BCAA) and Whey Protein), this practice might lead people to
use the same shaker for both hydration and supplementation.
Water quality control is a universal need, requiring attention from health authorities and
consumers in general. Specifically, regarding water intended for human consumption, there is
a concern due to its potential to become a vehicle capable of transmitting various pathogens
that threaten the well-being and health of gym members (23). Hence, if the bottle is not
adequately cleaned, instead of drinking pure water, individuals might be drinking
contaminated water that may cause symptoms in some individuals. One should also consider
the lack of hygiene of the hands as a source of contamination of the container since the
hands are essential sources of cross infection.
Thus, the purpose of the present study was to analyze the shaker bottles conditions for the
possible presence of different bacterial strains from two distinct fitness centers and to
determine its resistance to antimicrobials.
METHODS
Sample
A total of 60 bottles were selected for this study. Thirty of these bottles were purchased in
different stores and sampled as the non-used bottles (NUB). These samples were obtained
from three distinct brands, chosen for their high prevalence in the Brazilian fitness market. In
addition, different batches were chosen for each brand to minimize possible contamination

136
due to poor transportation or storage of one batch in particular. For the used shaker bottles
(UB), we randomly selected from 30 gym members of two distinct fitness centers (15 at each)
in the city of Petrópolis, RJ, Brazil, from November 2016 to January 2017. The most
commonly used dietary supplements by the UB sample were: (a) whey protein; (b)
compounds derived from meat protein; (c) carbohydrate supplements; (d) branched chain
amino acids (BCAA); and (e) creatine.
Procedures
The samples were collected in a dry and empty bottle state at the pre-use moment. To avoid
any attempt of exacerbating cleaning of the shakers, the subjects were asked to participate at
the time of the data collection, without previous knowledge about the research. Immediately
after collection, the subjects were interviewed regarding basic hygiene knowledge and bottle
usage purposes. Samples were collected by smooth friction through sterile Swabs in the
inner walls of the shakers. The Swabs were immediately introduced into the Stuart medium
and directly sent to the Microbiology Laboratory via temperature-controlled boxes.
The Swabs were inoculated into brain-heart infusion (BHI) broth and incubated at 35°C ± 1°C
for 24 hrs. Subsequently, the samples were seeded in blood agar, MacConkey agar, and
mannitol salt agar from Probac™ (Brazil), and then incubated at 35°C ± 1°C, 24 hrs in the
aerobic incubator model ECB 1.2 Digital Odontobras™. After bacterial growth, the isolated
colonies were submitted to gram staining and biochemical identification tests such as oxidase
test, Bactray 1 and 2 - Laborclin tests, for the identification of Gram-negative glucose-
fermenting bacilli and Bactray 3 for non-fermenting bacteria. In addition to the catalase
assays, the coagulase was performed for Staphylococcus aureus identification. After
identifying the bacterial genera and species, the strains isolated were submitted to the
Antimicrobial Susceptibility Test according to EUCAST (2017).
For the following bacteria: Escherichia coli; Proteus vulgaris, and Serratia sp, the following
antibiotics were tested: Ampicillin + Sulbactam, Amoxicillin + Clavulanic Acid, Piperacillin +
Tazobactam, Cefoxitin, Cefotaxime, Ceftriaxone, Imipenem, Meropenem, Ertapenem,
Aztreonam, Levofloxacin, Ciprofloxacin, Amikacin, Gentamycin, Sulfamethoxazole +
Trimethoprim, always observing the intrinsic resistance of each microorganism. The
antibiotics tested for Pseudomonas sp were: Piperacillin + Tazobactam, Cefepime,
Imipenem, Meropenem, Levofloxacin, Ciprofloxacin, Amikacin, Gentamicin. For
Staphylococcus aureus the following antibiotics were tested: Amoxicillin + Clavulanic Acid,
Oxacillin + Tazobactam, Cefadroxil, Cephalexin, Cefoxitin, Cefepime, Ceftriaxone,
Cefuroxime, Imipenem, Meropenem, Levofloxacin, Ciprofloxacin, Moxifloxacin, Amikacin,
Gentamycin, Azithromycin, Clarithromycin, Clindamycin, Erythromycin, Doxycycline,
Linezolid, Rifampicin, Sulfamethoxazole + Trimethoprim. The antibiotics tested for
Acinetobacter sp were: Imipenem, Meropenem, Ciprofloxacin, Levofloxacin, Amikacin,
Gentamycin, Sulfamethoxazole + Trimethoprim.
Statistical Analyses
All results were presented by percent delta and/or the absolute number. The Cochran Q test
was applied to verify significant differences between two sample groups for its binary
characteristics (0 or 1). The significance level was set at P≤0.05. The software used for all
statistical analyses was the SPSS, version 21.0 (IBM, Inc).

137
RESULTS
From the data obtained by Cochran Q analysis, there was a significant difference (P=0.001)
in contamination situation to compared distinct shaker bottle conditions (UB vs. NUB).
Specifically, the analysis of the NUB showed 100% absence of contamination. Conversely,
contamination (i.e., presence of microorganism) was found in 90% of the UB.
NUB UB
-20
0
20
40
60
80
100
120
Uncontaminated
Contaminated
*
Figure 1. Percentage Contamination from Used Bottles (UB) and Non-Used Shaker
Bottles (NUB). *Significant difference to UB sample.
For the 30 samples analyzed in UB, we observed bacteria growth in 25 (Δ% = 83%) of the
verified shaker bottles. Specifically, we verified that the Staphylococcus aureus (Δ% =
26.66%) and E. coli (Δ% = 16.66%) were the most frequent species. The percentage
prevalence of isolated bacteria species is shown in Figure 2.
Table 2 shows the results of the antimicrobial susceptibility test for the isolated strains,
showing the resistance profile. Briefly, most cases of contamination have demonstrated
antimicrobial resistance to different types of substances. Specifically, the Proteus vulgaris
had a 100% resistance score to most antimicrobials tested (APS, AMC, AZT, SUT, CFM);
Escherichia coli presented 100% resistance to SUT; and Serratia had 100% resistance for
APS, AMP, AMC, CFO and CFM.

138
S. aureus
E. coli
Acinetobacter sp
Pseudomonas sp
Abscence of growth
Serratia sp
Proteus vulgaris
4%
6%
8%
10%
12%
14%
16%
18%
20%
22%
24%
26%
28%
Figure 2. Percentage Prevalence of Isolated Microorganism from Shaker Bottles.
Table 1. Antimicrobial Resistance Profile of Isolated Strains.
Bacteria
Resistance Observed
Perceptual (%)
Proteus vulgaris CFO, CRO. 50%
APS, AMC, AZT, SUT, CFM. 100%
E. coli LEV, CIP. 25%
SUT. 100%
Staphylococcus aureus CFO, LEV, CIP, OXA. 12.5%
SUT. 25%
Acinetobacter sp. LEV. 25%
Pseudomonas IMI, MER. 25%
AMI, GEN. 25%
Serratia APS, CFO, CFM.
100%
APS= Ampicillin + Sulbactam; LEV = Levofloxacim; CIP = Ciprofloxacim; CFO = Cefoxitin; CRO = Ceftriaxone;
IMI = Imipenem; MER = Meropenem; OXA = Oxacillin; SUT = Sulfamethoxazole + Trimethoprim; AMC =
Amoxicillin + Clavulanic Acid; AMI = Amikacin; GEN = Gentamicin; AZT = Aztreonam; CFM = Cefuroxime.

139
DISCUSSION
Among the major findings of the present study, we observed that microbial growth was
present in 90% in the used bottles (UB). The inadequate hygiene of the container leads to the
growth of environmental microorganisms and even pathogens, which may pose a risk to
human health. Our tests conducted on the shaker bottles of gym members demonstrate the
growth of strains of antibiotic-resistant bacteria. Antimicrobial resistance has become a threat
to public health due to its association between the development of bacterial resistance (i.e.,
S. aureus, and enterococci), increases in mortality, and costs of health care. Patients with
infections due to antimicrobial resistant organisms have higher costs (i.e., $ 6,000 - $ 30,000)
than patients with antimicrobial infections. The cost difference is even greater when patients
infected with antimicrobial resistant organisms are compared with patients without infection
(5).
Specifically, the prevalence of Staphylococcus aureus strains was found in 26.6% the used
shakers and was sensible to most of the antibiotics tested. Staphylococcus aureus is a
prominent etiological agent in infections acquired both in the community and in the hospital
environment, and is considered an important pathogen due to its high capacity to cause
disease (22). In fact, the S. aureus is the most commonly isolated human bacterial pathogen.
It is an important cause of skin and soft tissue infections, endovascular infections,
pneumonia, septic arthritis, endocarditis, osteomyelitis, foreign-body infections, and sepsis
(7). This specie may colonize the anterior nares and other body sites (16). Gorwitz et al. (12)
identified a significant association between obesity and colonization with S. aureus in adults.
Similar association has been reported for adult patients who had undergone general,
cardiothoracic, or neurologic surgery (14). The reasons for this association are unclear, but
may include physical, biochemical, or hormonal factors that predispose these individuals to
colonization with S. aureus (12). The prevalence of S. aureus colonies has also been
demonstrated previously in males (14), which was our population tested. Our data portray the
need for an ideal hygiene of shakers bottles mainly for males.
Proteus species are widespread in the environment and are part of the normal flora of the
human gastrointestinal tract as well as mammals. They cause a variety of diseases acquired
in the community, including urinary tract infections, wounds, and the bloodstream (18). Drug
resistance has been increasingly reported for this genus, and the predominant mechanism for
resistance to β-lactam antibiotics is for the synthesis of β-lactamases. Among β-lactamases,
the production of extended-spectrum β-lactamases (ESBLs) and β-lactamases AmpC is more
common (21). Belonging to the same family, Serratia sp., Escherichia coli and Klebsiella sp.,
are responsible for urinary infections, being E. coli more prevalent. The Escherichia coli is
also known to be a very well adapted entero-invasive pathogenic able to enter epithelial cells
of colon, multiplicate within them, and move between adjacent cells. This pathogen is one of
the most recorded infection agents worldwide, as documented by recent outbreaks in the
industrialized countries (19).
Additionally, we found the presence of Pseudomonas species in four shakers. This pathogen
is one of the major microorganism causing hospital-acquired infections and can be more
aggressive towards immune-suppressed individuals. This agent presents a large genome,
and it can develop antibiotic resistance involving almost all classes of antibiotics. This trait
can be correlated to chromosomal mutations or by horizontal acquisition of resistant

140
determinants. The habitat of this microorganism is water and soil and can cause infections in
several body systems and parts such as urinary, skin, bone, and blood, being more severe in
the hospital environment (4).
The Acinetobacter specie was isolated in four shakers. It is also found in soil and water, most
commonly in dry environments. The Acinetobacter was originally identified in 1938. It is
ubiquitous in the environment as fresh water, vegetables, and animals (5). Several
Acinetobacter strains have been identified as causing infections in humans such as
pneumonia, sepsis, skin infections and infected wounds, according to the site affected (11).
This agent is a nosocomial pathogen that causes ventilator-associated as well as
bloodstream infections in critically ill patients, and the spread of multidrug-resistant
Acinetobacter strains is cause for concern. This microorganism is also considered
opportunistic, rarely causing community infections, except in cases of comorbidities such as
alcoholism, smoking, diabetes, and chronic obstructive pulmonary disease where much of the
success can be directly attributed to its plastic genome, which rapidly mutates when faced
with adversity and stress (13). Sporadic cases have occurred occasionally in healthy patients
exposed to environmental sources (8).
An estimated four billion cases of diarrhea annually represented 5.7% of the global disease
burden in the year 2000 (24). The scientific literature has accumulated over the decades a
good understanding of the transmission of several pathogens that cause diarrhea and other
diseases through drinking water (15). It has previously been observed that the microbiological
quality of water in domestic vessels is lower than at source, suggesting that contamination is
widespread during water collection, transport, storage, and extraction (17). We found that
most of the microorganism species that colonized the shakers were enterobacteria. Some are
present in the intestinal microbiota, which leads us to believe that not being hand sanitized or
incorrectly used may be contaminating the shakers. We must also consider the non-hygiene
practice of gym members who do not clean their shakers, thus facilitating bacterial growth.
CONCLUSIONS
The isolation of pathogenic microorganisms from shakers used by physical activity
practitioners at fitness centers such as Staphylococcus aureus, Acinetobacter sp,
Pseudomonas sp, Methicillin-resistant Staphylococcus aureus, and members of the family
Enterobacteriaceae presented a varied profile of antimicrobial resistance. It is worth
mentioning that most of the bacteria isolated in this study belong to the group of
enterobacteria, present in the intestinal microbiota and are pathogenic, emphasizing that
manipulation with contaminated hands may contribute to the colonization of the shakers. We
conclude that the best way to avoid bacterial proliferation in the shakers is make sure they
are correctly and frequently cleaned, such as daily washing with neutral soup in association
with proper hand hygiene to prevent contamination.
Address for correspondence: Gilmar Senna, Federal University of State of Rio de Janeiro,
Xavier Sigaud, 290/401, Praia Vermelha, Rio de Janeiro, RJ, Brazil, 22290-180. Email:
sennagw@gmail.com

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REFERENCES
1. American College of Sports Medicine. Position stand on exercise and fluid
replacement. Med Sci Sports Exerc. 2007;39:377-390.
2. American College of Sports Medicine. Position stand on nutrition and athletic
performance. Med Sci Sports Exerc. 2015;48:543-568.
3. American College of Sports Medicine Position stand on quantity and quality of
exercise for developing and maintaining cardiorespiratory, musculoskeletal, and
neuromotor fitness in apparently healthy adults: Guidance for prescribing exercise.
Med Sci Sports Exerc. 2011;43:1334-1359.
4. Bassetti M, Vena A, Croxatto A, Righi E, Guery B. How to manage Pseudomonas
aeruginosa infections. Drugs Context. 2018;29:212527.
5. Cosgrove SE. The relationship between antimicrobial resistance and patient
outcomes: Mortality, length of hospital stay, and health care costs. Clin Infect
Dis. 2006;42(2):S82-89.
6. Cowan ST. Unusual infections following cerebral operations. Lancet. 1938;232:1052-
1054.
7. David MZ, Daum RS. Community-associated methicillin-resistant Staphylococcus
aureus: Epidemiology and clinical consequences of an emerging epidemic. Clin
Microbiol Rev. 2010;23(3):616-687.
8. Davies, RW, Carson, BP, Jakeman, PM. The effect of whey protein supplementation
on the temporal recovery of muscle function following resistance training: A systematic
review and meta-analysis. Nutrients. 2018;10:1-10.
9. Dijkshoorn L, van der Toorn J. Acinetobacter species: Which do we mean? Clin Infect
Dis. 1992;15(4):748-749.
10. Farup J, Rahbek SK, Vendelbo MH, Matzon A, et al. Whey protein hydrolysate
augments tendon and muscle hypertrophy independent of resistance exercise
contraction mode. Scand J Med Sci Sports. 2014;24(5):788-798.
11. Giamarellou H, Antoniadou A, Kanellakopoulou K. Acinetobacter baumanii: A universal
threat to public health. Int J Antimicrob Agents. 2008;32:106-119.
12. Gorwitz RJ, Kruszon-Moran D, McAllister SK, McQuillan G, McDougal LK, Fosheim
GE, Jensen BJ, Killgore G, Tenover FC, Kuehnert MJ. Changes in the prevalence of
nasal colonization with Staphylococcus aureus in the United States, 2001-2004. J
Infect Dis. 2008;197(9):1226-1234.
13. Harding CM, Hennon SW, Feldman MF. Uncovering the mechanisms of Acinetobacter
baumannii virulence. Nat Rev Microbiol. 2018;16(2):91-102.

142
14. Herwaldt LA, Cullen JJ, French P, Hu J, et al. Preoperative risk factors for nasal
carriage of Staphylococcus aureus. Infect Control Hosp Epidemiol. 2004;25(6):481-
484.
15. Hunter PR. Does calculation of the 95th percentile of microbiological results offer any
advantage over percentage exceedance in determining compliance with bathing water
quality standards? Lett Appl Microbiol. 2002;34(4):283-286.
16. Kluytmans J, van Belkum A, Verbrugh H. Nasal carriage of Staphylococcus aureus:
Epidemiology, underlying mechanisms, and associated risks. Clin Microbiol Rev.
1997;10:505-520.
17. Lindskog RU, Lindskog PA. Bacteriological contamination of water in rural areas: An
intervention study from Malawi. Am J Trop Med Hyg. 1988;91:1-7.
18. O'Hara CM, Brenner FW, Miller JM. Classification, identification, and clinical
significance of Proteus, Providencia, and Morganella. Clin Microbiol Rev. 2000;
13(4):534-546.
19. Pasqua M, Michelacci V, Di Martino ML, Tozzoli R, Grossi M, Colonna B, Morabito S,
Prosseda G. The intriguing evolutionary journey of enteroinvasive E. coli (EIEC)
toward pathogenicity. Front Microbiol. 2017;5(8):2390.
20. Ratamess NA, Bush JA, Kang J, Kraemer WJ, Stohs SJ, Nocera VG, Leise MD,
Diamond KB, Faigenbaum AD. The effects of supplementation with P-Synephrine
alone and in combination with caffeine on resistance exercise performance. J Int Soc
Sports Nutr. 2015;12:1-11.
21. Shenoy SM, Mohit, Sinha R. Antibiotic sensitivity pattern of clinical isolates of Proteus
species with special reference to ESBL and Amp C production. Indian J Appl Res.
2013;3:293-294.
22. Tong SYC, Davis JS, Eichenberger E, Holland TL, Fowler VG, Jr. 27 May 2015.
Staphylococcus aureus infections: Epidemiology, pathophysiology, clinical
manifestations, and management. Clin Microbiol Rev. 2015;28(3):603-661.
23. Wang H, Masters S, Edwards MA, Falkinham JO 3rd, Pruden A. Effect of disinfectant,
water age, and pipe materials on bacterial and eukaryotic community structure in
drinking water biofilm. Environ Sci Technol. 2014;48:1426-1435.
24. WHO. The World Health Report 2002. World Health Organisation, Geneva.
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