Selective Pressure of Antibiotic Pollution on Bacteria of Importance to Public Health

Background: Many bacteria of clinical importance survive and may grow in different environments. Antibiotic pollution may exert on them a selective pressure leading to an increase in the prevalence of resistance. Objectives: In this study we sought to determine whether environmental concentrations of antibiotics and concentrations representing action limits used in environmental risk assessment may exert a selective pressure on clinically relevant bacteria in the environment. Methods: We used bacterial inhibition as an assessment end point to link antibiotic selective pressures to the prevalence of resistance in bacterial populations. Species sensitivity distributions were derived for three antibiotics by fitting log-logistic models to end points calculated from minimum inhibitory concentration (MIC) distributions based on worldwide data collated by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). To place bacteria represented in these distributions in a broader context, we performed a brief phylogenetic analysis. The potentially affected fraction of bacterial genera at measured environmental concentrations of antibiotics and environmental risk assessment action limits was used as a proxy for antibiotic selective pressure. Measured environmental concentrations and environmental risk assessment action limits were also directly compared to wild-type cut-off values. Results: The potentially affected fraction of bacterial genera estimated based on antibiotic concentrations measured in water environments is ≤ 7%. We estimated that measured environmental concentrations in river sediments, swine feces lagoons, liquid manure, and farmed soil inhibit wild-type populations in up to 60%, 92%, 100%, and 30% of bacterial genera, respectively. At concentrations used as action limits in environmental risk assessment, erythromycin and ciprofloxacin were estimated to inhibit wild-type populations in up to 25% and 76% of bacterial genera. Conclusions: Measured environmental concentrations of antibiotics, as well as concentrations representing environmental risk assessment action limits, are high enough to exert a selective pressure on clinically relevant bacteria that may lead to an increase in the prevalence of resistance.

1967; Legendre and Legendre 1998) were used to explore the correlation between evolutionary distances and pairwise differences in median MICs between taxa for each antibiotic dataset. Figure 1 shows Mantel correlograms for each antibiotic, along with the Mantel correlation coefficient for the entire dataset in the upper left hand side of each graph (i.e., R M ). Grouping data from co -generic species to minimize the lack of independence before SSD derivation seemed advisable for the ciprofloxacin dataset, whereas there did not seem to be a statistical ground for doing so in the erythromycin and tetracycline datasets. In this study we decided to group co-generic species in all datasets in order to apply a consistent methodology in the derivation of SSDs for all three antibiotics that would facilitate the subsequent interpretation and discussion of results.
Supplemental Material, Figure S1. Mantel correlograms descrbing the correlation between MIC 50 and evolutionary distance for ciprofloxacin (a), erythromycin (b) and tetracycline (c). Solid dots represent a significant correlation at the corresponding evolutionary distance (α = 0.05). Significance tests are based on 5000 permutations.

Supplemental Material, Table S1
Taxa represented in EUCAST MIC datasets. Cross (+) and dash (-) symbols indicate presence or absence of each entry in the given EUCAST MIC antibiotic dataset, respectively.
The All-species Living Tree Project names of the subset of species which were represented in the LTP 16S rRNA database are given in the 'LTP Name' column. MIC data from cogeneric species in each dataset were pooled to derive SSDs.

Evidence for growth of selected genera in the environment
There is evidence to suggest that several bacteria of importance to public health may, under certain conditions, grow in different environments. Hendricks (1972), for eample, showed that some Enterobacteriaceae could grow in water collected downstream of a municipal sewage facility at temperatures as low as 5°C. Gibbs et al. (1997) reported years later the regrowth of faecal coliforms and Salmonella in biosolids and soil ammended with biosolids.
The populations of E. coli in manured soils can be very dynamic (Topp et al. 2003) andInglis et al. (2010) recently showed that Campylobacter can persist for long periods of time in compost, which may suggest cryptic growth. Enterococci have been shown to grow in municipal oxidation ponds (Moriarty et al. 2008), and the facultative intracellular pathogen Listeria monocytogenes has been shown to be widespread in certain catchments (Lyautey et al. 2007) and able to grow in soil suspensions characteristic of certain organic fractions (Sidorenko et al. 2006). Staphylococci, including Staphylococcus aureus, have been isolated from marine water samples (Gunn and Colwell 1983), and methicillin -resistant Staphylococcus aureus was recently isolated from marine water and intertidal sand from beaches on the west coast of the USA (Soge et al. 2009). S. aureus has also been shown to be capable of growth in sterile soil (Liang et al. 1982), suggesting that it might be possible for it to grow in this environment under conditions of low competition. Ayyadurai et al. (2008) recently demonstrated that Yersinia pestis remained viable and fully virulent in humid sand for 40 weeks. Clostridium difficile is widely distributed in the environment (Al Saif and Brazier 1996), and it is not unreasonable to speculate that there may be niches that could support its sporulation. Collectively, these studies highlight there is a potential for growth in bacteria of clinical relevance in different environments and under varying biological and physicochemical conditions, even if only during a short temporal window. In the presence of antibiotics, wild-type populations may be inhibited to various extents, increasing the prevalence of resistance. Full references upon which we based our decision to include genera for SSD derivation are given in Table S2.

Supplemental Material, Table S2
Bacterial genera represented in the SSDs. References provide either direct evidence of growth in the environment or evidence that suggests that under certain conditions it is possible for growth to occur.