Study On The Resistance Mechanisms And Epidemiology Of Fosfomycin Resistant KPC-producing Klebsiella Pneumoniae

Since it was first reported in2001, Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae (KPC-KP) has spread globally, posing an alarming threat to public health. KPC-KP strains are usually resistant to almost all β lactams including carbapenems, as well as many antimicrobials of other classes, leaving few antibiotic-based treatments available. Recenly, fosfomyicn has been reintroduced into clinic due to its potential in vitro activity against multiresistant pathogens. However, in our previous study,43.4%KPC-KP strains were found to retain susceptibility to fosfomycin, and for rmtB-positive KPC-KP isolates, the susceptibility rate was as low as8.5%. The mechanisms underlying fosfomycin resistance in KPC-KP were still unclear then. In this study, the resistance mechanisms and epidemiology of these fosfomycin-resistant KPC-KP strains were investigated.In the former part of our study, a total of97KPC-KP strains were selected between January2010and March2013. Of them,57were resistant to fosfomycin, including44fosA3-ecoding strains and one fosA-encoding strain. The remaining12isolates were negative for all fos genes screened. All fosfomycin-resistant KPC-KP isolates were multidrug resistant (Table1). However,100.0%and94.7%of them were susceptible to colistin and tigecycline, respectively.50of the57strains carried rmtB gene, which constituted the main mechanism underlying aminoglycoside resistance among them. No significant differences were observed in the resistance phenotype between fosA3-encoding group and fos-negative group. However, the fosA-encoding isolate showed a milder phenotype, and remained susceptible to amikacin and ciprofloxacin except for tigecycline and colistin. Two sequence types (STs) and five pulsetypes (PTs) were identified in total, with56strains belonging to ST11and only1to ST494. All44fosA3-encoding isolates were assigned to ST11-PTA, indicating clonal spread of fosA3-encoding KPC-KP in our hospital.fosA-encoding strain belonged to ST11-PTE. The genetic background of fos-negative isolates seemed more complex, including ST11-PTA (6), ST11-PTB (4), ST11-PTC (1), and ST494-PTD (1). As for12fos-nonencoding strains, none mutations were detected in murA, the target gene of fosfomyicn. All these strains and4of them could grow on M9minimal medium agar supplemented only with G-6-P or G-3-P as single carbon source, respectively, suggesting functional UhpT transport system and GlpT transport system in all and four of them, respectively. Among the remaining8strains that couldn’t grow on G-3-P, one strain harbored an amino acid substitution Cys309Phe in GlpT, and two had DNA mutations△477A in glpT gene. The mutation△477A in glpT resulted in premauter stop codon and truncated GlpT, which would have lead to dysfunction of GlpT transport system and fosfomycin resistance. The other6strains didn’t have any mutations in their glpT genes, indicating that fosfomycin resistance might be attributed to down-regulated expression of their glpT genes.Fosfomyicn resistance in the representative isolate KP1034couldn’t be conjugated to the recipient cells. The E. coli transformant of KP1034remained susceptible only to tigecycline, colistin and ciprofloxaxin. It carried a～136kb plsmid encoding blaKPC-2, fosA3and rmtB, which was consistent with its resistance profile.In the latter part of our study, the fosA3-encoding plasmid pKP1034from the representative strain KP1034was fully sequenced and analyzed.pKP1034is136,848bp in length with a GC content of54.5%and harbors191predicted open reading frames. It has multiple replicons and belonged to IncR-F33:A-:B-by PCR based replicon typing (PBRT) and replicon sequence typing schemes. The overall structure of the plasmid is highly mosaic and encompassing three genetically distinct modules (Figure1):a composite transposon carrying resistance determinant catA2(chloramphenicol) flanked by two copies of IS26in opposite orientation; a54.7kb module essentially homologous to that from the epidemic fosA3-enconding plasmid pHN7A8reported in mainland China; and a78.0kb region closely related to the blaKPC-encoding plasmid pKPC-LK30from Taiwan. pKP1034could have evolved from several recombination events of these two closely related plasmids. Firstly, in plasmid pHN7A8, a composite transposon carrying catA2flanked by two copies of IS26in opposite orientations and a copy of IS26in the same orientation as the IS26at the boundary of catA2were inserted and truncated the genes tral and traB, respectively. Secondly, homologous recombination (HR) between the two directly oriented copies of IS26happened deleting a circular molecule containing the region between the two IS26plus one1S26element. Afterward, the deleted circular molecule was inserted into pKPC-LK30plasmid via another HR event between its IS26element and the one located downstream of blaSHV-11.Lastly, insertion of an extra IS1element between repB gene and resD gene followed by HR between the two directly oriented IS1copies lead to the generation of pKP1034-like plasmid.The MRRs contents on pKP1034could be divided into three parts. The first one carried catA2in a composite transposon flanked by two copies of IS26in opposite directions. The second region encoding fosA3, blaCTX-M-65, blaTEM-1, and rmtB evolved from the MRR on plasmid pHN7A8. They are almost identical except that on pKP1034an IS26was inserted into the IS1294downstream of fosA3and homologous recombination occurred afterward between the newly inserted IS26and the other IS26located upstream of fosA3, contributing to the inversion of in-between genetic contents. The third region carried blaKPC and blaSHV-12and was highly related the MRR on plasmid pKPC-LK30. However, blaSHV-12instead of blaSHV-11was located on pKP1034, suggesting that the extended β-lactamase blaSHV-12might have evolved from blaSHV-11after transfer to the pKPC-LK30-like plasmid. The immediate genetic surroundings of resistance gene blaKPC evolved from the typical Tn3-Tn4401composite structure reported in China, however, on plasmid pKP1034, the Tn3resolvase was truncated, probably by homologous recombination between directly oriented copies of IS26.Conclusion; The fosfomycin resistance was quite common (58.74%) in KPC-KP strains in the first affiliated hospital of Zhejiang University, which was mainly attributed to resistance gene fosA3(44,77.2%). One fosfomycin resistant isolate encoded fosA. These strains were highly resistant to almost all antibiotics tested and remained susceptible only to colistin and tigecycline. The high incidence of fosA3was due to clonal dissemination of the ST11-PTA clone. The overall structure of the representative plasmid pKP1034is highly mosaic, and could have evolved from several recombination events of two closely related plasmids pHN7A8and pKPC-LK30.