Abstract
We investigated the prevalence of plasmid-mediated quinolone resistance (PMQR) determinants and examined the association of these determinants with extended-spectrum β-lactamases (ESBLs) and/or plasmid-mediated AmpC β-lactamases (pAmpCs) in Serratia marcescens isolates in China. In this study, the presence of PMQR determinants was significantly related to the coproduction of ESBLs and/or pAmpCs (CTX-M-14, SHV-5, DHA-1, and ACT-1), among which CTX-M-14 was the most common gene type.
TEXT
Serratia marcescens is a prominent opportunistic pathogen responsible for serious infections in immunocompromised individuals. For many Gram-negative bacteria, including S. marcescens, production of chromosomally encoded AmpC-type β-lactamase is the intrinsic mechanism of resistance to β-lactam antibiotics. S. marcescens generally has inducible expression of this enzyme and has been thought to be a natural chromosomal AmpC producer (9). In China, quinolones and β-lactam antibiotics are widely used in the treatment of many bacterial infections, including infection caused by S. marcescens. Three kinds of plasmid-mediated quinolone resistance (PMQR) determinants [qnrA, qnrB, qnrS, qnrC, qnrD, aac(6′)-Ib-cr, and qepA] conferring low-level resistance to quinolones by different mechanisms [quinolone resistance (Qnr) proteins, AAC(6′)-Ib-cr, and QepA efflux] have been detected(8). The existence of these enzymes is of great concern because they not only are capable of conferring resistance against most clinically important quinolones but also are often associated with extended-spectrum β-lactamases (ESBLs) and/or plasmid-mediated AmpC β-lactamases (pAmpCs) (4). Although ESBLs have been identified in S. marcescens isolates repeatedly, there are few reports of pAmpCs being found in S. marcescens. To our knowledge, there was only one case report of an S. marcescens isolate harboring a pAmpC in a study carried out in Spain in 2010 (5). The aims of this study were to investigate the prevalence of PMQR determinants and to examine the association of these determinants with ESBLs and/or pAmpCs in S. marcescens.
A total of 146 nonduplicate S. marcescens isolates were collected from 2005 to 2011 at 34 hospitals in Anhui, China. Species identification was performed with the Vitek 2 system (bioMérieux, Marcy l'Étoile, France) and confirmed with API 20E (bioMérieux). The isolates were from respiratory specimens (69.6%), wounds (8.7%), blood (5.8%), urine (2.9%), body fluids (3.9%), and other specimens (9.1%). The MICs of piperacillin (PIP), cefotaxime (CTX), ceftazidime (CAZ), cefepime (FEP), cefoxitin (FOX), aztreonam (ATM), imipenem (IMP), meropenem (MEM), ciprofloxacin (CIP), levofloxacin (LVX), gatifloxacin (GAT), gentamicin (GM), and amikacin (AMK) (Oxoid) were determined by agar dilution method in accordance with the recommendations of the Clinical and Laboratory Standards Institute (CLSI) (3). ESBL detection was based on a double-disk synergy test (10). The modified Hodge test (15) was used to screen AmpC β-lactamase-producing strains.
For the ESBL screen-positive isolates, a search for the blaTEM, blaSHV, blaCTX-M, and blaOXA genes was performed by PCR amplification as previously described (1). For the isolates with cefoxitin MICs of ≥16 mg/liter, plasmid DNA was extracted using a Qiagen plasmid purification kit (Qiagen, Hilden, Germany), and ampC amplification was carried out using multiplex PCR, which can detect various types (MOX, CMY, LAT, DHA, ACC, MIR-1, ACT-1, and FOX) of pAmpCs (7). Conjugation experiments were carried out for all ESBL gene- and/or pAmpC gene-positive isolates with sodium azide-resistant Escherichia coli J53 as the recipient, as previously described (1). Transconjugants were selected on Luria-Bertani (LB) agar plates supplemented with sodium azide (100 mg/liter) (Sigma Chemical Co., St. Louis, MO) and FOX (16 mg/liter) or CAZ (2 mg/liter). Plasmid DNA was extracted from donors and transconjugants by using a Qiagen plasmid purification kit. The transconjugants were examined for the presence of β-lactamase genes by PCR using plasmid DNA as the template and tested for susceptibility as described above for the wild strains. For all 146 S. marcescens isolates, a search for the presence of qnrA, qnrB, qnrS, qnrC, qnrD, aac(6′)-Ib-cr, and qepA was performed by PCR using the methods described previously (2, 6, 11, 13, 14). All the purified PCR products were sequenced on an ABI Prism 3730 sequence analyzer (Applied Biosystems, Foster City, CA). Sequence alignment was compared with the GenBank nucleotide database using the nucleotide BLAST program.
Of the 146 S. marcescens isolates, 25 (17.1%) were CTX resistant (MICs ≥ 64 mg/liter) and 32 (21.9%) were CAZ resistant (MICs ≥ 32 mg/liter) according to the CLSI guidelines. In addition, 11 (7.5%) isolates showed some potentiation of the inhibitory zones of the β-lactams by clavulanic acid, suggesting the presence of ESBL activity. In these 11 putatively ESBL-positive isolates, the structural genes for CTX-M-14 (n = 5) and SHV-5 (n = 3) were found either alone or in combination in 7 strains (Table 1). Also, blaTEM-1 and blaOXA-1 were detected either alone or in combination with ESBL or pAmpC genes in 6 and 4 strains, respectively. Of the 7 ESBL gene-positive strains, 6 (85.7%) were resistant to ciprofloxacin and levofloxacin. The rate of ciprofloxacin resistance was higher in the ESBL gene-positive strains (85.7%) than in the ESBL nonproducers (27/135, 20.0%) (P < 0.05). All of the ESBL gene-positive strains were resistant to gentamicin, and 4 (57.1%) of them were resistant to amikacin. However, all the ESBL gene-positive strains were susceptible to carbapenems.
Table 1.
Profiles of 8 ESBL- and/or pAmpC-gene positive S. marcescens isolates and their transconjugants
| Isolate | Specimen | β-Lactamase(s) | PMQR determinant | MIC (mg/liter) |
||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| PIP | CTX | CAZ | FEP | FOX | ATM | IMP | MEM | CIP | LVX | GAT | GM | AMK | ||||
| GN0199 | Sputum | blaCTX-M-14, blaSHV-5, blaOXA-1,a blaACT-1 | aac(6′)-Ib-cr | 512 | 32 | 64 | 8 | >256 | 128 | 0.5 | <0.25 | >32 | 32 | 8 | >128 | 16 |
| T0199d | blaCTX-M-14, blaSHV-5, blaACT-1 | aac(6′)-Ib-cr | 256 | 16 | 32 | 2 | 256 | 32 | <0.25 | <0.25 | 1 | 0.5 | 0.5 | 8 | 2 | |
| GN0885 | Wound | blaCTX-M-14, blaTEM-1,a blaOXA-1 | aac(6′)-Ib-cr | 512 | 256 | >256 | 2 | 256 | >256 | 1 | <0.25 | >32 | >64 | 16 | >128 | >512 |
| T0885 | blaCTX-M-14, blaTEM-1, blaOXA-1 | aac(6′)-Ib-cr | 128 | 32 | 32 | 0.5 | 4 | 32 | <0.25 | <0.25 | 1 | 0.5 | 0.5 | <0.5 | <0.5 | |
| GN0480 | Sputum | blaSHV-5, blaOXA-1, blaDHA-1 | —b | 512 | 32 | 256 | 4 | 64 | >256 | 0.5 | <0.25 | >32 | 32 | 16 | >128 | >512 |
| T0480 | blaSHV-5, blaDHA-1 | — | 128 | 8 | 16 | 0.25 | 32 | 32 | <0.25 | <0.25 | <0.06 | <0.125 | <0.125 | 1 | 1 | |
| GN0818 | Wound | blaCTX-M-14, blaTEM-1 | qnrB6 | 512 | 256 | 32 | 0.5 | 256 | 64 | 0.5 | <0.25 | >32 | >64 | >64 | >128 | 16 |
| T0818 | blaCTX-M-14, blaTEM-1 | qnrB6 | 128 | 32 | 8 | 0.25 | 4 | 16 | <0.25 | <0.25 | 2 | 1 | 1 | 0.5 | 0.25 | |
| GN1237 | Sputum | blaCTX-M-14, blaDHA-1 | aac(6′)-Ib-cr | 256 | 32 | 32 | 32 | 128 | 256 | 1 | <0.25 | 8 | 8 | 16 | >128 | >512 |
| T1237 | blaCTX-M-14, blaDHA-1 | aac(6′)-Ib-cr | 64 | 8 | 8 | 1 | 128 | 16 | <0.25 | <0.25 | 1 | 1 | 0.5 | 0.25 | 0.25 | |
| GN1249 | Sputum | blaCTX-M-14, blaDHA-1 | qnrS2, aac(6′)-Ib-cr | 512 | 128 | 32 | 4 | 256 | >256 | 1 | 0.25 | >32 | >64 | 32 | >128 | >512 |
| T1249 | blaCTX-M-14, blaDHA-1 | qnrS2, aac(6′)-Ib-cr | 64 | 8 | 8 | <0.25 | 128 | 32 | <0.25 | <0.25 | 2 | 1 | 1 | 0.5 | 0.25 | |
| GN1382 | Sputum | blaDHA-1 | aac(6′)-Ib-cr | 256 | 64 | 128 | 16 | 256 | 128 | 1 | <0.25 | 4 | 8 | 8 | >128 | >512 |
| T1382 | blaDHA-1 | aac(6′)-Ib-cr | 64 | 8 | 8 | 1 | 128 | 16 | <0.25 | <0.25 | 0.5 | 0.25 | 0.25 | <0.5 | <0.5 | |
| J53c | — | — | 1 | <0.5 | <0.5 | <0.25 | <0.5 | <0.5 | <0.25 | <0.25 | <0.06 | <0.125 | <0.125 | <0.5 | <0.5 | |
| GN0889 | Sputum | blaSHV-5, blaOXA-1 | — | 256 | 256 | 32 | 16 | 512 | >256 | 2 | 0.25 | 0.5 | 0.125 | 0.125 | >128 | 4 |
| GN0413 | Wound | blaTEM-1 | — | 256 | 32 | 16 | 2 | 32 | 8 | 0.5 | <0.25 | 2 | 1 | 0.5 | >128 | 32 |
| GN0447 | Urine | blaTEM-1 | — | 512 | 128 | 128 | 0.5 | 64 | 32 | 0.5 | <0.25 | >32 | >64 | 32 | >128 | 4 |
| GN0132 | Urine | blaTEM-1 | aac(6′)-Ib-cr | 256 | 32 | >256 | 8 | 64 | >256 | 0.5 | <0.25 | >32 | 8 | 8 | >128 | >512 |
| GN0526 | Blood | blaTEM-1 | — | 512 | 512 | 128 | 32 | 256 | >256 | 0.5 | <0.25 | >32 | 16 | 4 | >128 | 4 |
blaTEM-1 and blaOXA-1 are not ESBL genes.
—, no gene was detected.
Sodium azide-resistant Escherichia coli J53.
Strains whose designation begin with “T” are transconjugants.
Of the 146 S. marcescens isolates, 30 were not susceptible to cefoxitin (MICs ≥ 16 mg/liter). Among these 30 isolates, 6 had a positive reaction according to the modified Hodge test. PCR and sequencing analysis revealed that 5 of them harbored pAmpC genes (4 contained blaDHA-1, 1 contained blaACT-1) (Table 1), but in one isolate no pAmpC gene was detected. pAmpC gene-harboring isolates also harbored blaCTX-M-14, blaSHV-5, and blaOXA-1 either alone or in combination.
Of the total of 146 isolates, 15 (10.3%) showed high-level resistance (MIC > 32 mg/liter) to both ciprofloxacin and levofloxacin. Five of the ESBL gene- and/or pAmpC gene-positive strains harbored the aac(6′)-Ib-cr variant, and one strain coexpressed qnrS2. One strain harbored qnrB6 alone. None harbored a qnrA, qnrC, qnrD, or qepA gene. In this study, the rate of PMQR determinants was significantly higher in the ESBL gene-positive strains (5/7, 71.4%) than in ESBL nonproducers (12/135, 8.9%) (P < 0.05). PMQR determinants were detected in 4 pAmpC gene-positive strains (4/5, 80.0%). The presence of PMQR determinants was significantly related to the coproduction of ESBLs and/or pAmpCs, among which CTX-M-14 was the most common (Table 1). This finding corroborates the previous reports that the PMQR determinants are frequently associated with ESBLs and/or pAmpCs and they are cotransferred by conjugation (4). Wang et al. (12) reported that several qnrA-positive strains produced SHV-7 and CTX-M-9. In our study, CTX-M-14 and DHA-1 were common among PMQR determinant-positive strains.
Among the 8 ESBL gene- and/or pAmpC gene-positive strains, 7 had genes that were successfully transferred on transferable plasmids to the recipients. An increase in the MICs of quinolones and β-lactam was detected in the transconjugants compared to the recipients (Table 1). It is suggested that the dissemination of the PMQR determinants and pAmpC is mostly due to the transmission of plasmids by horizontal exchange.
In conclusion, we verified that PMQR determinants are widespread among quinolone-resistant S. marcescens isolates in Anhui, China. More importantly, we also determined that PMQR determinants are closely related to various ESBLs and pAmpCs. Due to the multidrug resistance in S. marcescens isolates, prudent antibiotic use and accurate detection of resistance are urgently needed to prevent the spread of these organisms.
ACKNOWLEDGMENTS
We thank all the contributing hospitals that provided isolates for this study.
This work was supported by a research grant from the National Natural Science Foundation of China (no. 30972631,81101288) and by the Natural Science Foundation of Anhui Province, China (no. 11040606Q23).
Footnotes
Published ahead of print 4 June 2012
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