Abstract
Antibiotics are disseminated into aquatic environments via human waste streams and agricultural run-off. Here they can persist at low, but biologically relevant, concentrations. Antibiotic pollution establishes a selection gradient for resistance and may also raise the frequency of events that generate resistance: point mutations; recombination; and lateral gene transfer. This study examined the response of bacteria to sub-inhibitory levels of antibiotics. Pseudomonas aeruginosa and Pseudomonas protegens were exposed kanamycin, tetracycline or ciprofloxacin at 1/10 the minimal inhibitory concentration (MIC) in a serial streaking experiment over 40 passages. Significant changes in rep-PCR fingerprints were noted in both species when exposed to sub-inhibitory antibiotic concentrations. These changes were observed in as few as five passages, despite the fact that the protocols used sample less than 0.3% of the genome, in turn suggesting much more widespread alterations to sequence and genome architecture. Experimental lines also displayed variant colony morphologies. The final MICs were significantly higher in some experimental lineages of P. protegens, suggesting that 1/10 the MIC induces de-novo mutation events that generate resistance phenotypes. The implications of these results are clear: exposure of the environmental microbiome to antibiotic pollution will induce similar changes, including generating newly resistant species that may be of significant concern for human health.
Highlights
Antibiotic resistance has been identified as one of the greatest threats to human health for the twenty-first century by the World Health Organisation (WHO, 2014)
One line of P. protegens PF5 that had been exposed to 1/10 the minimal inhibitory concentration (MIC) of ciprofloxacin over the serial plating experiment exhibited a 10-fold increase in MIC for ciprofloxacin (DF = 11, F = 11.94, P < 0.0001)
The role of antibiotics as environmental pollutants is attracting more attention, as more concern is being raised about their effects at sub-inhibitory concentrations (Gillings and Stokes, 2012; Andersson and Hughes, 2014)
Summary
Antibiotic resistance has been identified as one of the greatest threats to human health for the twenty-first century by the World Health Organisation (WHO, 2014). Overuse and misuse of antibiotics in the medical and agricultural sectors have contributed to the problem, and it is estimated that 70% of pathogens exhibit resistance to at least one or more antibiotics (Berdy, 2012). In the United States in 2013, there were 23,000 confirmed deaths due to Antibiotic resistance (US CDC) and Europe reports 25,000 deaths per year (2007, ECDC). Unnecessarily high dosages and over-use all promote resistance (Campoccia et al, 2010; Andersson and Hughes, 2012; Hvistendahl, 2012; Witte, 2013). It has been estimated that 50–70% of antibiotics produced in the United States of America are used in agriculture (Lipsitch et al, 2002; Berge et al, 2005)
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