Antimicrobial susceptibility profiles and tentative epidemiological cutoff values of Legionella pneumophila from environmental water and soil sources in China

Abstract

Legionnaires' disease (LD), caused by Legionella, including the most prevalent Legionella pneumophila, has been treated primarily with antibiotics. Environmental water and soil are the reservoirs for L. pneumophila. Studying antimicrobial susceptibility using a large number of isolates from various environmental sources and regions could provide an unbiased result. In the present study, antimicrobial susceptibility of 1464 environmental L. pneumophila isolates that were derived from various environmental water and soil sources of 12 cities in China to rifampin (RIF), erythromycin (ERY), clarithromycin (CLA), azithromycin (AZI), ciprofloxacin (CIP), moxifloxacin (MOX), levofloxacin (LEV), and doxycycline (DOX) was investigated, and minimum inhibitory concentration (MIC) data were obtained. We show that regarding macrolides, ERY was least active (MIC₉₀ = 0.5 mg/L), while CLA was most active (MIC₉₀ = 0.063 mg/L). A total of three fluoroquinolones have similar MICs on L. pneumophila. Among these antimicrobials, RIF was the most active agent, while DOX was the most inactive one. We observed different susceptibility profiles between serogroup 1 (sg1) and sg2-15 or between water and soil isolates from different regions. The ECOFFs were ERY and AZI (0.5 mg/L), RIF (0.002 mg/L), CIP, CLA and MOX (0.125 mg/L), LEV (0.063 mg/), and DOX (32 mg/L). Overall, two fluoroquinolone-resistant environmental isolates (0.14%) were first documented based on the wild-type MIC distribution. Not all azithromycin-resistant isolates (44/46, 95.65%) harbored the lpeAB efflux pump. The MICs of the ERY and CLA on the lpeAB + isolates were not elevated. These results suggested that the lpeAB efflux pump might be only responsible for AZI resistance, and undiscovered AZI-specific resistant mechanisms exist in L. pneumophila. Based on the big MIC data obtained in the present study, the same defense strategies, particularly against both CLA and RIF, may exist in L. pneumophila. The results determined in our study will guide further research on antimicrobial resistance mechanisms of L. pneumophila and could be used as a reference for setting clinical breakpoints and discovering antimicrobial-resistant isolates in the clinic, contributing to the antibiotic choice in the treatment of LD.

Keywords: Legionella pneumophila; antimicrobial susceptibility; azithromycin; clarithromycin; epidemiological cut-off values; fluoroquinolones; lpeAB; rifampin.

FIGURE 1

Average minimum inhibitory concentrations (MICs) among the eight antibiotics against L. pneumophila. Data are shown as floating bars with the max, min, and mean values.

FIGURE 2

Antimicrobial susceptibility profiles of L. pneumophila belonging to different sgs or from different environmental sources or regions in China. (A) Antibiotic susceptibility profiles of isolates belonging to different sgs. (B) Antibiotic susceptibility profiles of isolates from different sources. W, water sources; S, soil sources. (C) PCA biplot of MICs of the isolates. Isolates are shown as dots and colored by groups based on the sources and sgs. Indices are given as lines with arrows and colored. The configuration of indices in the biplot represents the relationship between variables and principal components. The gray shadow indicates 95% confidence for sg2-15 isolates from water sources. A dashed ellipse indicates significantly correlated variables. (D) Antimicrobial susceptibility profiles of isolates belonging to different sgs and from different sources. W1, sg1 isolates from water sources; W2-15, sg2-15 isolates from water sources; S1, isolates from soil sources; S2-15, sg2-15 isolates from water sources. Data are shown as floating bars with the max and min and mean values. Dotted lines indicate that log MICs are different in W2-15 isolates when compared with the other three types of isolates. (E) Regions of China where the tested isolates were obtained are shown. Cities where the isolates were obtained in each region are shown. (F) Antibiotic susceptibility profiles of isolates from different regions of China. Colored regions indicate the provinces where the isolates are from. We defined those MICs were 1.25 times higher/lower (log MIC gap > 0.969) than the contrast with P < 0.05 as significant. ****P < 0.0001.

FIGURE 3

Principal component analysis and correlation matrices for the minimum inhibitory concentrations (MICs) were obtained from 1464 L. pneumophila isolates. (A) PCA of indices of the isolates. Isolates are shown as dots and colored red. Indices are shown as lines with arrows and colored. The configuration of indices in the plot represented the relationship between variables and principal components. (B) Correlation matrix for isolates’ indices (MICs). WPC, weak positive correlation, 0.2 < r ≤ 0.3; MPC, moderate positive correlation, 0.3 < r ≤ 0.5; SPC, strong positive correlation, r > 0.5. NC, no correlation. The largest r was found between CLA and ERY (r = 0.51). The dotted lines indicate that these antibiotics (in the dotted lines with the same color) belong to the same class.

FIGURE 4

Wild-type cutoff (COWT) of the eight antibiotics against L. pneumophila using the ECOFFinder method. (A–H) MIC distributions of the eight antibiotics, fitted curves, and ECOFF were obtained by the ECOFFinder. (I) Relevant data table obtained by the ECOFFinder. Asterisks indicate the possible largest WT MICs.

FIGURE 5

Design of lepAB primers with genomic sequences from L. pneumophila harboring the lpeAB efflux, and multiplex PCR to detect lpeAB genes and their spatial arrangement. (A) Arrangement of the lpeAB is shown using L. pneumophila str. Paris as a reference. Primers and DNA sequences of lpeAB, and GenBank accession numbers are shown, and the blue italics indicate the gene location in the selected sequences of the strains. (B) PCR amplification conditions. (C) Representative results for detection of lpeAB efflux in L. pneumophila isolates. DNA electrophoresis shows specific lpeA targets (green arrow, 152 bp), lpeB targets (blue arrow, 499 bp), and lpeA-lpeB combined targets (red arrow, 1009 bp), which indicate the right arrangement of the two genes. Lanes 1-9 show positive for lpeAB efflux; lanes 10-11 show negative for lpeAB efflux; n.c. indicates negative control which used sterile water as a template.

FIGURE 6

Presence of lpeAB efflux in L. pneumophila and its distribution in the isolates belonging to different sgs or from different sources and regions in China. (A) Presence of lpeAB efflux in AZI-sensitive and –resistant isolates. (B) PCA biplot of indices of the isolates harboring or not harboring lpeAB efflux. Isolates are shown as dots and colored by groups based on the presence of lpeAB efflux. Indices showed as lines with arrows and colored. The configuration of indices in the biplot represented the relationship between variables and principal components. The red shadow indicates 95% confidence for lpeAB + isolates. (C) Average MICs between isolates with or without lpeAB efflux. Data are shown as floating bars with the max and min and mean values. (D). Different distribution patterns of lpeAB in the isolates belonging to different sgs or from different sources and regions of China. *** P < 0.001, ****P < 0.0001.