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Microbial Contamination in Shaker Bottles among Members of Fitness Centers

  • Federal University of State of Rio de Janeiro (UNIRIO), Brazil

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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.
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Journal of Exercise Physiology
August 2018
Volume 21 Number 4
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
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
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
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
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.
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
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.
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).
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.
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
S. aureus
E. coli
Acinetobacter sp
Pseudomonas sp
Abscence of growth
Serratia sp
Proteus vulgaris
Figure 2. Percentage Prevalence of Isolated Microorganism from Shaker Bottles.
Table 1. Antimicrobial Resistance Profile of Isolated Strains.
Resistance Observed
Perceptual (%)
Proteus vulgaris CFO, CRO. 50%
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.
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.
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
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
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.
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:
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Background: Little is known concerning the potential ergogenic effects of p-synephrine supplementation. Therefore, the purpose of the present study was to examine the effects of supplementation with p-synephrine alone and in combination with caffeine on free-weight resistance exercise performance. Methods: Twelve healthy, college-aged men performed a control (CT) resistance exercise protocol consisting of 6 sets of squats for up to 10 repetitions per set using 80 % of their one repetition-maximum (1RM) with 2 min of rest in between sets. Each subject was randomly assigned (in double-blind, balanced manner) to a treatment sequence consisting of use of 3 supplements: p-synephrine (S; 100 mg), p-synephrine + caffeine (SCF; 100 mg of p-synephrine plus 100 mg of caffeine), or a placebo (P). For each supplement treatment (separated by 1 week), subjects consumed the supplement for 3 days prior to each protocol and the morning of each protocol, and subsequently did not consume any supplements for 3 days following (i.e. wash-out period). On each protocol day, subjects reported to the lab at a standard time, consumed a supplement, sat quietly for 45 min, performed the resistance exercise protocol, and sat quietly for 30 min post exercise. Performance (repetition number, force, velocity and power), blood lactate, and ratings of perceived exertion (RPE) data were collected during each protocol. Results: Supplements SCF and S produced a significantly (P < 0.05) greater number of repetitions performed than CT (by 11.0 ± 8.0 %) and P (by 6.0 ± 7.0 %) and a 10.6 ± 12.0 % greater increase in volume load per protocol than CT and P. Most of the differences were seen during the last 3 sets. Mean power and velocity for all 6 sets were significantly higher in SCF compared to CT and P by ~6.2 ± 8.0 %. No supplement effects were observed in RPE or blood lactate, and no adverse side effects were observed or reported. Conclusions: S and SCF augmented resistance exercise performance (total repetitions, volume load) without increasing blood lactate or RPE. The addition of caffeine in SCF increased mean power and velocity of squat performance. These results indicate supplementation with S and SCF can enhance local muscle endurance during resistance exercise.
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Staphylococcus aureus is a major human pathogen that causes a wide range of clinical infections. It is a leading cause of bacteremia and infective endocarditis as well as osteoarticular, skin and soft tissue, pleuropulmonary, and device-related infections. This review comprehensively covers the epidemiology, pathophysiology, clinical manifestations, and management of each of these clinical entities. The past 2 decades have witnessed two clear shifts in the epidemiology of S. aureus infections: first, a growing number of health care-associated infections, particularly seen in infective endocarditis and prosthetic device infections, and second, an epidemic of community-associated skin and soft tissue infections driven by strains with certain virulence factors and resistance to β-lactam antibiotics. In reviewing the literature to support management strategies for these clinical manifestations, we also highlight the paucity of high-quality evidence for many key clinical questions. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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In a comparative study, we investigated the effects of maximal eccentric or concentric resistance training combined with whey protein or placebo on muscle and tendon hypertrophy. 22 subjects were allocated into either a high-leucine whey protein hydrolysate + carbohydrate group (WHD) or a carbohydrate group (PLA). Subjects completed 12 weeks maximal knee extensor training with one leg using eccentric contractions and the other using concentric contractions. Before and after training cross-sectional area (CSA) of m. quadriceps and patellar tendon CSA was quantified with magnetic resonance imaging and a isometric strength test was used to assess maximal voluntary contraction (MVC) and rate of force development (RFD). Quadriceps CSA increased by 7.3 ± 1.0% (P < 0.001) in WHD and 3.4 ± 0.8% (P < 0.01) in PLA, with a greater increase in WHD compared to PLA (P < 0.01). Proximal patellar tendon CSA increased by 14.9 ± 3.1% (P < 0.001) and 8.1 ± 3.2% (P = 0.054) for WHD and PLA, respectively, with a greater increase in WHD compared to PLA (P < 0.05), with no effect of contraction mode. MVC and RFD increased by 15.6 ± 3.5% (P < 0.001) and 12-63% (P < 0.05), respectively, with no group or contraction mode effects. In conclusion, high-leucine whey protein hydrolysate augments muscle and tendon hypertrophy following 12 weeks of resistance training - irrespective of contraction mode.
Acinetobacter baumannii 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. Much of the success of A. baumannii can be directly attributed to its plastic genome, which rapidly mutates when faced with adversity and stress. However, fundamental virulence mechanisms beyond canonical drug resistance were recently uncovered that enable A. baumannii and, to a limited extent, other medically relevant Acinetobacter species to successfully thrive in the health-care environment. In this Review, we explore the molecular features that promote environmental persistence, including desiccation resistance, biofilm formation and motility, and we discuss the most recently identified virulence factors, such as secretion systems, surface glycoconjugates and micronutrient acquisition systems that collectively enable these pathogens to successfully infect their hosts.
Proteus spp are gram negative bacilli which can cause both community acquired and nosocomial infections. This study was conducted to note the antibiotic sensitivity pattern, production of ESBL and AmpC by clinical isolates of Proteus spp in a tertiary care center. Antibiotic sensitivity testing was done according to CLSI guidelines. The proteus isolates were sensitive to third generation cephalosporins, carbepenems and piperacillin/tazobactam. 20% of the isolates were ESBL and 28.5% were AmpC positive. 50% of the isolates were resistant to fluoroquinolones. 21 proteus isolates exhibited multidrug resistance. This may be due to the transfer of multiple genes from one bacterial population to another. Recognition of resistance pattern of bacteria is necessary from time to time to give appropriate treatment and avoid adverse clinical outcomes.
Availability of safe, pathogen-free drinking water is vital to public health; however, it is impossible to deliver sterile drinking water to consumers. Recent microbiome research is bringing new understanding to the true extent and diversity of microbes that inhabit water distribution systems. The purpose of this study was to determine how water chemistry in main distribution lines shape the microbiome in drinking water biofilms and to explore potential associations between opportunistic pathogens and indigenous drinking water microbes. Effects of disinfectant (chloramines, chlorine), water age (2.3d, 5.7d), and pipe material (cement, iron, PVC) were compared in parallel triplicate simulated water distribution systems. Pyrosequencing was employed to characterize bacteria and terminal restriction fragment polymorphism was used to profile both bacteria and eukaryotes inhabiting pipe biofilms. Disinfectant and water age were both observed to be strong factors in shaping bacterial and eukaryotic community structures. Pipe material only influenced the bacterial community structure (ANOSIM test, P<0.05). Interactive effects of disinfectant, pipe material, and water age on both bacteria and eukaryotes were noted. Disinfectant concentration had the strongest effect on bacteria, while dissolved oxygen appeared to be a major driver for eukaryotes (BEST test). Several correlations of similarity metrics among populations of bacteria, eukaryotes and opportunistic pathogens, as well as one significant association between mycobacterial and proteobacterial operational taxonomic units, provides insight into means by which manipulating the microbiome may lead to new avenues for limiting the growth of opportunistic pathogens (e.g., Legionella) or other nuisance organisms (e.g., nitrifiers).