Staphyloccus aureus is an opportunistic pathogen capable of causing a wide variety of infectious diseases and is usually associated with humans as commensal colonizing organisms in at least 30% of the population [1–3]. Staphylococcal infections are primarily of the skin and soft tissues; however, they are capable of causing much more serious systemic infections and death, especially when associated with methicillin resistance [4, 5]. Initially, outbreaks of methicillin resistant S. aureus (MRSA) infections were associated with hospitals and healthcare-associated exposures in compromised patients; however, since the late 1990 s with the emergence of new more aggressive community-associated MRSA (CA-MRSA), these infections are no longer limited to these settings.

Since its emergence, outbreaks of CA-MRSA infections in otherwise young healthy individuals [6] have been linked to close contact and sharing of common facilities such as locker rooms, schools and prisons [7]. These community-associated strains have made their way into the hospitals [5], being responsible for a significant number of hospital-acquired infections; and in a recent report, they have now been shown to be responsible for the majority of skin infections requiring treatment in emergency departments in multiple U.S. cities [8].

Waterborne transmission routes have not been traditionally associated with S. aureus infections. However, in some earlier studies, investigators in Hawaii reported cases of S. aureus infections associated with exposure to coastal marine waters [9, 10], with humans serving as the suspected primary source [11]. They also showed that these organisms are able to remain viable in seawater over several days [12]. Therefore, coastal marine waters used for recreation could provide a transmission pathway for both colonization and/or infection of individuals. Previous studies have also identified S. aureus in recreational marine water [12, 13], and S. aureus and MRSA in sand [14–16].

In an earlier study attempting to quantify S. aureus release by humans in marine water [17], investigators showed that humans shed greater quantities of S. aureus than the fecal indicator bacteria enterococci. However, this earlier study was limited in its methodology and criteria used to isolate and confirm S. aureus, and it did not address the potential presence of MRSA in the isolates. Furthermore, the study was also limited to an adult population, and it did not evaluate for S. aureus colonization of the human population studied.

As recreational marine waters and beaches may be commonly used by many people over the course of a short period of time, the risk of exposure to all microorganisms that are in this environment increases. Given that transmission of S. aureus (including MRSA) has been documented in settings associated with shared facilities and close contact, the use of recreational marine waters and beaches could certainly represent another possible route of exposure and transmission of these potentially pathogenic organisms and warrant investigation.

The aim of this study was to evaluate the amounts, as well as the characteristics, of S. aureus, methicillin sensitive S. aureus (MSSA), and MRSA shed by humans into recreational waters and sands. In this study, S. aureus, MSSA, and MRSA shed from adults, and for the first time children, were identified using stringent selection and identification procedures.

The study was approved by the Florida Department of Health Internal Review Board (IRB 1491; DOH IRB Number, H07164) and the University of Miami Internal Review Board (IRB 20070306). Consent forms were signed by each study participant (or parent/guardian), and participant identity was kept confidential. The field experimental design followed that of Elmir et al. [17, 18], including the use of the same study site (a sub-tropical non-point source recreational marine beach).

Bacterial isolation and identification

S. aureus was isolated from the water samples using a standard membrane filtration (MF) method [19], followed by growth on selective media, Baird Parker agar (Becton, Dickinson and Company, Sparks, MD) with Egg Yolk (EY) Tellurite Enrichment (Becton, Dickinson and Company), BP, and CHROMagar, CHR (Becton, Dickinson and Company) (see Figure 1 for process flow). MSSA and MRSA isolated from BP plates were subjected to genetic tests and compared to organisms isolated from nasal cultures. MSSA isolated from CHR plates was used for direct comparison of colony counts obtained during previous studies. Facilities used for processing samples were located within minutes from the study site, allowing for the processing of samples within one hour after collection. Volumes of the source water used for filtration were 10 ml and 100 ml; volumes of the pool water samples used for filtration prior to and after adult participant contact were 10 ml and 50 ml respectively; volumes of the water used for filtration after contact with the pediatric participants were 5 ml, 10 ml, and 50 ml. Multiple volumes were filtered in order to obtain quantifiable colony counts as the levels of bacteria in both the source water and the experimental pool water samples were unknown.

The analysis of S. aureus in sand was similar to that for water with the exception of two pre-processing steps. The first step measured the water content of sand (weight difference of sand before and after drying at 110°C for 24 h). The second step extracted bacteria from the sand particles to a predefined volume of sterile water. To accomplish this, pre-weighed un-dried sand was aseptically removed from the corresponding sample container and placed into a sterile pre-weighed jar. One hundred and ten milliliters of sterile phosphate buffer saline (PBS) were added to each jar, and the jars were shaken vigorously for 30 seconds. The samples were permitted to settle for 30 seconds, and the supernatant was subsequently used for membrane filtration. One hundred milliliters of the sand eluate samples were used for the filtration and bacterial quantification.

Following standard MF, filter membranes were placed on BP and CHR, and incubated aerobically at 37°C for a minimum of 24 h. After incubation, colonies found to be black, shiny, convex, 2-5 mm in diameter, and surrounded by clear zones (BP) or mauve (CHR), were considered presumptive S. aureus, and subjected to confirmatory tests. All presumptive positive isolates were transferred to Mannitol Salt agar (Becton, Dickinson and Company), for the determination of mannitol fermentation, and incubated aerobically at 37°C for 16-24 h. All mannitol-fermenting isolates were enriched [20] on Trypticase Soy Agar with 5% Sheep Blood (TSA II, Becton, Dickinson and Company) for determination of colony morphology and gross pigmentation, the ability to lyse red blood cells and to provide bacterial cells for latex agglutination tests for clumping factor and protein A using the Remel BactiStaph Latex Agglutination Test (Thermo Fisher Scientific, Lenexa, KS).

The analysis of the nasal swab cultures focused on detection and genetic characterization, rather than quantification. The method used was the same as that used for the water samples, except that the membrane filtration step was omitted. Utilizing standard aseptic techniques swabs were placed in 0.5 ml tripticase soy broth TSB (Becton, Dickinson and Company) supplemented with 6.5% sodium chloride, and incubated overnight at 37°C for enrichment. One hundred micro-liters of the overnight broth were transferred to Mannitol Salt agar (Becton, Dickinson and Company), and the organisms were identified and confirmed as detailed above.

Chromosomal DNA was extracted from colonies isolated from water, sand, and nasal cultures. Whole cell extracts were prepared from latex agglutination positive bacterial isolates using the Amplicor MTB Sputum Specimen Preparation Kit (Roche Molecular Systems, Inc., Indianapolis, IN) according to the manufacture's recommendations, and used as template for confirming and characterizing polymerase chain reactions (PCR) as outlined below. These DNA extracts (up to a maximum of 22 per filter) were subjected to PCR analysis of the S. aureus specific gyrA gene for S. aureus confirmation and the mecA gene for genetic MRSA confirmation. Oligonucleotide primers and thermal cycling conditions were used as described previously [21], with the minor modification that 5-µl of whole cell extract was used as template in initial PCR reactions instead of purified chromosomal DNA.

All organisms determined to be genotypic MRSA (testing positive for mecA) were re-isolated from agar plates, and grown on oxacillin resistance screening agar base media ORSAB (Remel; Thermo Fisher Scientific), a selective media for confirmation of phenotypic MRSA. All genotypic MRSA isolates from this study showed the phenotypic characteristics of MRSA. All confirmed MRSA (n = 17) and MSSA (n = 162) collected from water and sand samples and all nasal cultures were stored as stock strains at -80°C.

The number of colonies testing positive for gyrA gene (for S. aureus counts) and mecA gene (for MRSA counts) were reported. Counts were then adjusted to colony forming units per 100 ml water (CFU/100 ml) or per 100 g sand (CFU/100 g) using the volume of water applied to the filters or the weight of the sand collected from the pool. The numbers of microbes shed per person were determined by multiplying the difference in microbial concentrations measured before and after bathing in the pools by the water volumes corresponding to each person.

In these studies, we demonstrated that human bathers, both adults and toddlers in diapers, have the potential to release significant amounts of S. aureus (including MRSA) into the water column from direct shedding off their body and via sand transported on their skin. This suggests that recreational beaches may be potential exposure and transmission pathways for S. aureus (including MRSA). The authors hypothesize that the low background levels of MSSA in the off shore water was due to the residual effects from bather swimming activities from normal beach use given the potential persistence of these organisms in seawater [12]. These background levels, however, were very low in comparison to those levels observed during the small and large pool studies (which allowed for the quantification of the number of MSSA and MRSA released by the study participants).

The average quantities of S. aureus shed in this study were lower than those observed previously by Elmir et al. [17] using less stringent identification criteria. In addition to more stringent techniques, the difference in numbers may also be due to the differences in the degree to which the adults in the different studies were colonized by, and therefore shed, S. aureus. The shedding numbers reported above take into account the entire population, which included both those individuals who shed and those who did not shed bacteria. Therefore, individuals who participated in the large pool study who were not truly colonized, would not have contributed organisms to the pool water, yet were considered in the overall per person shedding calculations. However, when shedding was evaluated on an individual basis (as was done with the toddler study), the number of organisms shed could have been much higher per person if an adult bather in the group happened to have been colonized and was not detected by nares culture. This was the case in the adult Group II where no MSSA was isolated from participants directly, but MSSA was in the water during cycles 1 and 2 prior to sand exposure. This difference may also be due to variability of S. aureus shedding among different people depending upon their individual colonization status, body site colonized and quantity of organisms. Variable shedding by individuals was observed from the small pool study, where toddler shedding ranged from non-detectable levels up to values above 10⁵ CFU/person.

Direct shedding of S. aureus from the body can include release from known colonized body areas such as the nares. This was seen when the large pool sample water (for Group II) was positive for MRSA only when MRSA was found in the anterior nares of participants who bathed in that water; and the majority of these organisms were shown to have the same genetic characteristics as the colonizing MRSA. Direct shedding was also observed when the single known nasally colonized toddler shed into the water sample in the small pool study. The results reported here confirm that S. aureus are shed by colonized adults and toddlers into the water column. This is supported by the results from both adults and toddlers in the separate pool studies. In the large pool studies, MSSA and MRSA were isolated when the participants were known only to be colonized with MRSA only (Group II); however, although only 1 toddler was shown to be colonized by nares sampling method, 10 toddlers shed MSSA. As a result of these findings, we hypothesize that both adults and toddlers are likely colonized with S. aureus, in particular MSSA, in other areas of the body, and that these locations contribute to bacterial shedding when exposed to water. This observation is consistent with clinical observations showing that about one third of MRSA-infected patients were not nasally colonized [25], with alternate colonization sites including skin [26] and throat [27].

Both the large pool study and the small pool study demonstrated that sand played a relatively small role in S. aureus shedding. In the small pool study during the single bathing cycle, sand accounted for less than 1% of shedding. Elmir et al. [18] also found that sand accounted for roughly 3.7% of the enterococci contribution in the first bathing cycle for the small pool study. For the large pool study, an increase in S. aureus shedding was observed when participants were exposed to sand between the second and third bathing cycles, but the impacts were less pronounced for S. aureus as compared to enterococci shedding as observed in prior studies [18]. Increased numbers of S. aureus shed in the third cycle could be associated with sand exposures; however, the ultimate source of the S. aureus in the sand is unknown, and may be associated with naturally existing S. aureus and/or from direct shedding from humans to the sand.

Because of the differences in the designs of the large pool study (adults) and the small pool study (toddlers), direct comparison of the amount of shedding between toddlers and adults in this study is limited. Nevertheless, we compared the numbers of S. aureus shed by adult and toddlers, keeping these limitations in mind. The average of S. aureus shed by adults during the four cycles in the large pool (n = 8 composites of 10 people) was 6.3 × 10⁵ CFU/person, and by toddlers (n = 14) was 4.3 × 10⁴ CFU/person in the small pool. In this comparison, adults shed 13 times more S. aureus than toddlers on average (75 times on median). It should be noted that the estimated adult body surface areas based on height and weight [28] were approximately 3.5 times larger than the toddlers in this study. After normalizing shedding numbers by the body surface factor of 3.5, the numbers of S. aureus shed by adults were 4 times more than toddlers on average (21 times on median). Therefore, in this investigation, toddlers in diapers shed fewer organisms than the adults; however, additional studies need to be done under the same conditions to confirm these findings.

The results of this study showed that both MSSA and MRSA were shed by human populations into marine waters. The amount of shedding varied, was likely dependent upon the level of colonization of the host, and colonization was not limited to the anterior nares. In this study, the shedding of MRSA was directly dependent upon its colonization of the human host. MRSA shedding was observed intermittently, only among Group II adults and water, with the apparent lower number of humans colonized by MRSA relative to MSSA. No MRSA was observed in the sand samples as the pediatric populations evaluated in this study were apparently not colonized with MRSA. However, it is highly likely that similar studies with additional pediatric participants would result in the isolation of MRSA [29]. Future studies should focus on the collection of additional samples from human participants as the current study was limited by the restricted numbers of carriers identified. These future studies should collect samples from the skin and from other areas where S. aureus resides, in addition to samples from the anterior nares.

Once S. aureus is released from bathers, its potential for transmission is highly dependent on its persistence in the environment. Gregg and LaCroix [30] inoculated saltwater pool water with MRSA, and found very low levels after 1 hour exposure. They concluded that swimming pool water would not likely put children at risk for acquiring MRSA. However, we argue here that more research is needed to evaluate the risk of illness associated with water exposures and the potential for transmission through sand, including the residency time of these human pathogens in both recreational marine waters and beach sand [12]. Future research should be conducted with S. aureus species from actively colonized individuals, as the current study found large amounts released from individuals, and the actively growing clinical strains may survive differently in comparison to laboratory grown strains used for inoculation experiments. Experimentation should closely control environmental factors as some studies have documented growth of S. aureus under optimal environmental conditions [31].

Overall, the results from this study confirmed that both adults and toddlers can be sources of potentially pathogenic MSSA and MRSA in recreational marine waters, and support the potential for exposure and transmission of these organisms through the use of recreational beaches. More research is needed to evaluate the persistence of strains released by humans into the water column and the possibility of disease transmission at recreational beach sites.