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The expression of efflux pump gene lde in ciprofloxacin resistant (CipR) and susceptible strains of Listeria monocytogenes collected from retail food samples was investigated. For two CipR strains, the MICs of ciprofloxacin decreased four- to eightfold in the presence of reserpine; however, no significant alterations were observed with naturally sensitive isolates. Overexpression of the lde gene induced by ciprofloxacin was observed in two resistant isolates. The present findings indicate that expression of lde and the MICs of ciprofloxacin are well correlated with the presence and absence of reserpine, suggesting that Lde might be involved in ciprofloxacin resistance of L. monocytogenes.
Listeria monocytogenes, a Gram-positive, facultative intracellular bacterium, is capable of causing serious human infections with a high fatality rate (1). Foodborne transmission is the main route of acquisition of these infections in both epidemic and sporadic cases (2, 3). Since the first multiresistant L. monocytogenes strain was identified in France (4), different antibiotic resistance patterns in environmental, food and clinical sources have been reported (5, 6, 7). Although indications for the use of fluoroquinolones do not include listeriosis, they can select resistant L. monocytogenes because of their increasing use for other pathogens (8). In general, resistance to quinolones in Gram-positive bacteria usually results from mutational alterations in the QRDRs of the intracellular targets of quinolones, DNA gyrase and topoisomerase IV, or active export of the drugs via efflux pumps (8). There are reportedly two efflux pumps in L. monocytogenes (9). One efflux pump, MdrL, can extrude macrolides, cefotaxime, heavy metals, and ethidium bromide (10). The other efflux pump is termed Lde. Although previous studies showed that efflux pump Lde is responsible for fluoroquinolone resistance and in part, for resistance to acridine orange and ethidium bromide in L. monocytogenes (8, 9, 11), little is known about the gene expression of lde in L. monocytogenes isolates kept under different conditions.
In this study, we investigated eighteen strains of L. monocytogenes isolated from retail food samples between 2005 and 2007, including fifteen ciprofloxacin resistant (CipR) strains and three susceptible strains. We identified the strains as described previously (12).
We examined alterations in QRDRs of gyrA, gyrB, parC and parE in CipR isolates of L. monocytogenes by PCR. We extracted genomic DNA using the Promega Wizard Genomic DNA purification kit (Promega, Madison, WI, USA) according to the manufacturers’ recommendations for Gram-positive bacteria. The PCR primers and conditions were as described previously (8). We sequenced the PCR products and compared the resulting DNA sequence data with GenBank database sequences using the BLAST algorithm available at the National Center for Biotechnology Information web site (www.ncbi.nlm.nih.gov).
We performed molecular typing of the isolates by PFGE as described previously (13). We determined the MICs of ciprofloxacin (Sigma-Aldrich, St. Louis, MO, USA) and levofloxacin (Sigma-Aldrich) by the broth microdilution method (2). We used the efflux pump inhibitor reserpine (final concentration, 20 μg/mL; Sigma-Aldrich) as described previously (8, 9, 14). We observed changes in MICs in both the absence and the presence of reserpine for all examined strains.
We extracted total RNA using an RNAprep pure Cell/Bacteria Kit (Dongsheng Biotech, Guangzhou, Guangdong, China), according to the manufacturer's instructions. We estimated the degree of expression of the lde gene by qRT-PCR. We retro-transcribed 200 ng of total RNA using the Reverse Transcription System (Promega) in a final volume of 20 μL. We used 23S rRNA as a reference gene. Primers for the 23S rRNA gene were 5′-GTGTCAGGTGGGCAGTTTG-3′ and 5′-CATTCTGAGGGAACCTTTGG-3′ as described by Rudi et al. (15). Primers for lde were 5′-ATCCTCATATAACTCAAGCG-3′ and 5′-CAATGGCTTTCGCACAA-3′ (9). The PCR mix consisted of 1 × RealMasterMix (Tiangen Biotech, Beijing, China), 1 × SYBR Green solution (Tiangen), 100 nm of each primer, and 1 μL of cDNA in a final volume of 20 μL. The PCR program was 95°C for 3 mins; 39 cycles of 95°C for 30 s, 53°C for 30 s, and 72°C for 20 s. As a final step, we performed melting curve analysis between 65°C and 95°C. To evaluate the effect of ciprofloxacin on lde expression in the L. monocytogenes isolates, we grew bacteria for different periods (0.5 hrs, 1 hrs, 2 hrs, 4 hrs, 6 hrs and 8 hrs) from an exponential culture in the presence of ciprofloxacin at 0.2, 0.4, 0.8 and 1 × the respective MICs. When performing the experiment, we also evaluated a parallel control of cells grown in the absence of quinolone. We performed all experiments in triplicate.
We observed no differences in the sequences of gyrA, gyrB and parE of the CipR isolates. Incidentally, we found that L28 and L45 have point mutations in parC gene (351, TA substitution); however, this did not cause changes in the amino acid sequence. In all, resistance to ciprofloxacin of these strains did not result from mutational alterations in QRDRs.
We investigated all 18 isolates of L. monocytogenes for the effect of reserpine (Table 1), a well-established inhibitor of efflux pumps that can block the activity of efflux pumps in Gram-positive microorganisms, on their MICs (16, 17, 18). The presence of reserpine did not affect the MICs of ciprofloxacin for the sensitive strains. In comparison, we observed four- to eightfold decreases in two resistant strains (L47 and L28), which belong to different serovars and have different PFGE patterns (Table 1), demonstrating that they are not clonally related. It is possible that efflux pump Lde is involved in resistance to ciprofloxacin and inhibited by reserpine in these strains. Such an inhibition could give rise to decreased MICs of ciprofloxacin. However, we found either no variation or a onefold decrease in the remaining thirteen CipR strains. All isolates were sensitive to levofloxacin and the presence of reserpine had almost no impact on the MICs.
Table 1. MICs of ciprofloxacin and levofloxacin for L. monocytogenes isolates in the presence and absence of reserpine
|Strain no.||Serotype||PFGE pattern||MIC (μg/mL)|
|CIP||CIP + reserpine||LEV||LEV + reserpine|
Since we found the lde gene by PCR in all isolates examined irrespective of their susceptibilities to ciprofloxacin (data not shown), we proposed that resistance is probably related to strong expression of lde. In order to confirm this view, we selected five L. monocytogenes isolates for examination of the effect of ciprofloxacin and inhibition by reserpine on lde expression (Table 2). In preliminary experiments, the degree of expression of lde did not vary noticeably with the exposure time (data not shown). When we treated the strains for 4 hrs, we observed the greatest degree of expression at 0.4 × MIC (data not shown). Finally, we tested the comparative effects of ciprofloxacin on lde expression at 0.4 × MIC for 4 hrs. We identified no significant differences in the basal degree of expression of the isolates. The resistant strains L28 and L47 showed significant increases in lde after treatment with ciprofloxacin (4.17-fold and 3.93-fold, respectively); however, in the presence of reserpine, lde exhibited a significant drop in degree of gene expression (1.04-fold and 0.72-fold, respectively). These results are in line with the alterations in MIC in the presence of reserpine (Table 1), which demonstrate that strong expression of efflux pump lde might be associated with ciprofloxacin resistance in these strains and that expression of lde is inhibited in the presence of reserpine. For strains L31 and L45, we observed no obvious changes in expression of lde in the presence of ciprofloxacin and reserpine. For the sensitive strain L69, ciprofloxacin did not affect lde expression. Overall, expression of the lde gene correlated well with the degree of ciprofloxacin resistance in L. monocytogenes.
Table 2. Relative gene expression data for efflux pump Lde in strains of L. monocytogenes
|Strain no.||Basal degree of expression†||Relative gene expression†|
|CIP||CIP + reserpine|
|L28||1.47 ± 0.74||4.17 ± 0.79||1.04 ± 0.45|
|L47||1.02 ± 0.50||3.93 ± 1.11||0.72 ± 0.41|
|L31||1.22 ± 0.56||1.05 ± 0.85||1.18 ± 0.96|
|L45||1.67 ± 0.78||1.88 ± 1.12||1.70 ± 0.85|
|L69||1||1.04 ± 0.75||ND|
We observed overexpression of the lde gene in only two resistant isolates. It is possible that the degree of expression of the lde gene is affected by mutation(s) in the promoter region or regulatory gene of lde. As for the other thirteen CipR isolates, we found no mutational alterations in the QRDRs and the degree of expression of lde did not change significantly in the presence of ciprofloxacin and reserpine. We suggest that overexpression of the lde gene is not the only reason for ciprofloxacin resistance. Maybe the Lde pump is not involved in ciprofloxacin resistance in these strains, other unknown pumps or resistance mechanisms could be involved.
In this study, we isolated resistant strains from food, and investigated the degree of gene expression of lde under different conditions in detail. Our results indicate that expression of efflux pump gene lde correlates well with the MICs of ciprofloxacin in the presence and absence of reserpine, suggesting that Lde might contribute to ciprofloxacin resistance in L. monocytogenes. However, additional longer-term studies are needed to prove this point. Furthermore, the regulation of lde in these strains under different conditions should be investigated in the future.