Low-Level Antimicrobials in the Medicinal Leech Select for Resistant Pathogens That Spread to Patients

Abstract

Fluoroquinolones (FQs) and ciprofloxacin (Cp) are important antimicrobials that pollute the environment in trace amounts. Although Cp has been recommended as prophylaxis for patients undergoing leech therapy to prevent infections by the leech gut symbiont Aeromonas, a puzzling rise in Cp-resistant (Cp^r) Aeromonas infections has been reported. We report on the effects of subtherapeutic FQ concentrations on bacteria in an environmental reservoir, the medicinal leech, and describe the presence of multiple antibiotic resistance mutations and a gain-of-function resistance gene. We link the rise of Cp^rAeromonas isolates to exposure of the leech microbiota to very low levels of Cp (0.01 to 0.04 µg/ml), <1/100 of the clinical resistance breakpoint for Aeromonas Using competition experiments and comparative genomics of 37 strains, we determined the mechanisms of resistance in clinical and leech-derived Aeromonas isolates, traced their origin, and determined that the presence of merely 0.01 µg/ml Cp provides a strong competitive advantage for Cp^r strains. Deep-sequencing the Cp^r-conferring region of gyrA enabled tracing of the mutation-harboring Aeromonas population in archived gut samples, and an increase in the frequency of the Cp^r-conferring mutation in 2011 coincides with the initial reports of Cp^rAeromonas infections in patients receiving leech therapy.IMPORTANCE The role of subtherapeutic antimicrobial contamination in selecting for resistant strains has received increasing attention and is an important clinical matter. This study describes the relationship of resistant bacteria from the medicinal leech, Hirudo verbana, with patient infections following leech therapy. While our results highlight the need for alternative antibiotic therapies, the rise of Cp^r bacteria demonstrates the importance of restricting the exposure of animals to antibiotics approved for veterinary use. The shift to a more resistant community and the dispersion of Cp^r-conferring mechanisms via mobile elements occurred in a natural setting due to the presence of very low levels of fluoroquinolones, revealing the challenges of controlling the spread of antibiotic-resistant bacteria and highlighting the importance of a holistic approach in the management of antibiotic use.

Keywords: Aeromonas; antibiotic resistance; ciprofloxacin; genomics; leech therapy; microbiome.

FIG 1

Resistance of Aeromonas isolates to Cp. Resistance MICs are plotted for the leech control group, which consists of pre-1999 strains from the supply chain prior to the contamination concern and one strain from a 2012 noncontaminated supplier. MICs are also shown for isolates from the main supply chain and from patients treated with leeches in 2012 to 2015. The Aeromonas strains from the main supply chain were significantly more resistant to Cp, based on the Kruskal-Wallis test using Dunn’s multiple-comparison test of sample mean ranks indicated by different lowercase letters. MICs of isolates from pre-1999 leeches differed significantly from those of leech supplies and of patient isolates in 2012 to 2015 (P = 0.0005 and <0.0001, respectively).

FIG 2

Competitiveness of Cp^r clinical (CA-13-1) and leech-derived (Hv13-B-13b) Aeromonas isolates in the presence and absence of ciprofloxacin. Competitive index (CI) values above 10⁰ indicate that the Cp^r strain outcompetes the susceptible pre-1999 leech control isolate, Hm21. (A and C) Leeches from another supplier (in which FQs were not detected) were fed blood meal containing Cp concentrations of 0, 0.0025, 0.007, and 0.01 µg/ml, and intraluminal fluid (ILF) was sampled 72 h postfeeding. (B and D) The same competition assays done in vitro (Blood) with the respective conditions. The Cp^s strain outcompetes CA-13-1 and Hv13-B-13b in 0.01-µg/ml-Cp-fed leeches and in blood, but this is reversed at lower Cp concentrations. Statistical analyses were performed with the Kruskal-Wallis test using Dunn’s multiple-comparison test, treatment groups which differ significantly from each other (P value of <0.05) are indicated with lowercase a and b. Error bars show the median within the interquartile range.

FIG 3

Abundance of the gyrA (S83) mutation in leeches over time. Leech crop content was sampled from leeches supplied by the main FDA-approved distributor (D) or farm (F) in 2009, 2011, two shipments in 2013, and one in 2014. We determined the percent relative abundance of gyrA (S83I) in total reads (a) and A. hydrophila-specific reads (b). The mean and standard deviation are shown as error bars. An asterisk indicates values for individual leech samples for which there were zero sequencing reads of gyrA (S83I).

FIG 4

Maximum likelihood reconstruction of 16 single-copy housekeeping genes. Bootstrap support values between 80 and 100% are represented in the tree by variously sized blue triangles (80%, small triangles, to 100%, large triangles). Resistance levels are indicated by colored boxes: highly susceptible, ≤1 µg/ml (light gray); susceptible, 1 µg/ml (dark gray); intermediate, 2 µg/ml (yellow); resistant, 4 µg/ml (orange), 8 to 16 µg/ml (red), and 16 to 32 µg/ml (dark red). The presence of Cp^r-conferring chromosomal mutations is shown by filled black squares, while the presence of resistance plasmid genes is shown by filled blue squares. For example, MO-11-1 has a black square for ParC^S80r to represent that it has an S-80-R mutation. The names of strains derived from clinical isolates are colored in dark red. Names of clinically associated isolates, such as those from leech aquaria, are colored orange.

FIG 5

Phylogenetic reconstruction of the AvCp group. This cladogram is a maximum likelihood reconstruction generated from whole-genome alignments. Bootstrap support values are represented by dots: dark green, 90%+ bootstraps; light green, 80%+; khaki, 70%+. Posterior probabilities of 95%+ from a Bayesian inference are represented by blue dots, and branch lengths do not carry meaning. Resistance levels are indicated by colored boxes: susceptible, ≤1 µg/ml (gray); intermediate, 2 µg/ml (yellow); resistant, 4 µg/ml (orange), 8 µg/ml (dark orange), 16 µg/ml (red), and 32 µg/ml (dark red). The presence of Cp^r-conferring genes and mutations is shown by filled black squares. The names of taxa derived from clinical isolates are colored in orange.