A strain of S. marcescens, designated GN1384, was isolated from the urine of a 81-yr-old male patient who suffered from urinary tract infections in September 2009 and was admitted to the second hospital of Hefei, China. Species identification was performed with the Vitek2 system (bioMerieux, Marcy l'Etoile, France) and confirmed with API 20E (bioMerieux). The minimum inhibitory concentrations (MICs) of various antibiotics were determined by using the agar dilution method, and susceptibility data were interpreted by using the Clinical and Laboratory Standards Institute guidelines. The isolate showed a multiple drug resistant pattern, including some beta-lactams, all the aminoglycosides, and fluoroquinolones (Table 1).
Then, genomic and plasmid DNA of strain GN1384 was extracted. Using primers described previously, we identified the resistance genotype of this strain by detecting the following resistance determinants through PCR, based on the above resistance phenotypes: 16S rRNA methyltransferase genes (armA, rmtA, rmtB, rmtC, rmtD, rmtE, and npmA); plasmid-mediated quinolone resistance determinants (qnrA, qnrB, qnrS, qnrC, qnrD, aac (6')-Ib-cr, and qepA); chromosome mutations associated with fluoroquinolone resistance (gyrA, parC, and parE); and genes coding for beta-lactamases (TEM, CTX-M, SHV, OXA-1). The positive amplicons were subsequently sequenced, and the results indicated the presence of rmtB, aac(6')-Ib-cr, blaCTX-M-14, blaTEM-1, and blaOXA-1. In addition, two amino acid changes (Ser83Leu and Asp87Asn) in GyrA and five amino acid changes (Thr57Ser, Ser80Ile, Ala129Ser, Val131Leu, and Gly134Ser) in ParC were observed.
Conjugation experiments were performed with Escherichia coli J53 as the recipient, as previously described. The transconjugants were selected on agar plates containing sodium azide (100 mg/L) supplemented with amikacin (128 mg/L). Conjugation experiments showed that aac(6')-Ib-cr, blaTEM-1, blaCTX-M-14, and blaOXA-1 were cotransferred with rmtB to the recipient. The MIC results revealed that it simultaneously exhibited an elevated level of resistance to beta-lactams, aminoglycosides, and fluoroquinolones (Table 1).
The extremely high MICs of all aminoglycosides tested against the multidrug-resistant S. marcescens were likely due to the presence of the rmtB gene. In contrast to the rmtB plasmid isolated by Doi et al., the plasmid bearing rmtB in our study was self-transferable, as observed by Bogaerts et al.. In accordance with a previous study, the close linkage between 16S rRNA methyltransferase and beta-lactamase as well as plasmid-mediated quinolone resistance determinants revealed the significant relationship of these genes amongst Enterobacteriaceae. It was also observed that the plasmid-mediated quinolone resistance gene together with the mutations in gyrA and parC contributed to the high-level quinolone resistance in S. marcescens GN1384. Further studies are required to assess the genetic environment of these genes and the role of these new substitutions in resistance to fluoroquinolones. The continuous and widespread use of aminoglycosides, fluoroquinolones, and beta-lactams in human or animal infection is the major driving force leading to these sophisticated resistance gene linkages. The dissemination of these linked genes by horizontal transfer simultaneously is very worrisome, and continuous monitoring should be reinforced.