Spread of GES-5 carbapenemase-producing Pseudomonas aeruginosa clinical isolates in Japan due to clonal expansion of ST235

The first outbreak in Japan of GES-5 carbapenemase-producing Pseudomonas aeruginosa occurred in a long-term care facility in 2014. To assess the spread of GES-5 producing P. aeruginosa clinical isolates in medical settings in Japan, 1,476 carbapenem-resistant P. aeruginosa isolates obtained from 2012 to 2016 were characterized. Of these 1,476 isolates, 104 (7.0%) harbored blaGES-5. Southern blotting revealed that the blaGES-5 was located on the chromosome. The isolation rates of these GES-5 producers increased significantly every year, from 2.0% (6 of 295) in 2012 to 2.8% (8 of 283) in 2013 to 5.3% (16 of 303) in 2014 to 9.7% (29 of 300) in 2015 to 15.3% (45 of 295) in 2016. Of the 104 GES-5 producers, 102 belonged to clonal complex (CC) 235, including 99 belonging to ST235 and three belonging to ST2233). Whole genome sequence analysis revealed that CC235 P. aeruginosa harboring blaGES-5 spread in a clonal manner. These results indicate that these GES-5 producing CC235 P. aeruginosa clinical isolates have spread in medical settings throughout Japan.


Whole genome sequencing
Genomic DNAs of bla GES-5 -positive isolates were extracted using DNeasy Blood and Tissue kits (Qiagen, Tokyo, Japan) and sequenced by a next generation sequencer (MiSeq; Illumina, San Diego, CA).

Phylogenetic analysis
To identify single nucleotide polymorphisms (SNPs) throughout the entire genomes of all 104 bla GES-5 -positive isolates, all reads of each isolate were aligned against P. aeruginosa NCGM2. S1 (Gen Bank accession no. AP 012280) using CLC genomics workbench version 8.0.2 (CLC bio, Tokyo, Japan) [17]. SNP concatenated sequences were aligned using MAFFT (http:// mafft.cbrc.jp/alignment/server/). Models and parameters used for the phylogenetic analyses were computed using j-Model Test-2.1.4. A maximum-likelihood phylogenetic tree was constructed from SNP alignment with PhyML 3.0 [18].

Drug resistance genes and MLST
Genes associated with resistance to β-lactams, aminoglycosides and quinolones were detected using ResFinder 3.0 (https://cge.cbs.dtu.dk/services/ResFinder/). Fluoroquinolone resistance has been associated with mutations in the quinolone resistance determining region, which includes the gyrA and parC genes that encode DNA gyrase and topoisomerase IV, respectively [19]. Multilocus sequence types (MLSTs) were deduced as described (http://pubmlst.org/ paeruginosa/). Clonal complexes (CC) were determined by eBURST version 3 (http://eburst. mlst.net). design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the 'author contributions' section.

Competing interests:
We have the following interests. Dr. Masahiro Shimojima is employed by BMI Inc. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Statistical analysis
The yearly proportions of multidrug-resistant P. aeruginosa isolates positive for bla GES-5 were analyzed by the chi-square test.
All 104 isolates harbored bla PAO. Of them, 102 (98.0%) harbored bla OXA-488 , 1 had bla OXA-396 and 1 had bla OXA-486 (Table 1). Of the 104 bla GES-5 -positive isolates, 102 (98.0%) harbored aac(6')-Ib and the other two harbored aac(6')-Ia (Table 1). The all 104 isolates were found to have point mutations in the quinolone-resistance-determining regions of gyrA and parC, consisting of the amino acid substitutions S83I in GyrA and S87L in ParC (Table 1). Of them, a quinolone-sensitive strain had the same mutations as the remaining quinolone-resistant strains, but it had amino acid substitutions in the efflux systems, including P363L in OprJ, R112Q in OprN, in addition to an OprD in-frame deletion. In-frame deletions of OprD were found in 54 of 104 isolates tested. The 99 isolates belonging to CC235 (ST235 and ST2233) harbored virulence genes, exoT, exoU and exoY, although the remaining two belonging ST274 and ST1342 did exoS, exoT and exoY (Table 1).
PFGE and Southern blotting showed that two bla GES-5 -positive isolates belonging to CC235 (ST235 and ST2233) had no plasmid harboring bla GES-5 (data not shown).

Discussion
This study showed that carbapenem-resistant bla GES-5 -positive P. aeruginosa isolates, first isolated in Japan in 2012, rapidly spread throughout Japan over the next several years. Outbreaks of bla GES-5 -positive isolates occurred among hospitals in a region (Chiba prefecture), indicating that bla GES-5 -positive isolates started to expand in a clonal manner within a specific region in Japan. Our previous study on whole genome analysis of 136 clinical isolates of ST235 multidrug-resistant P. aeruginosa producing IMP-type MBLs showed that these isolates fell within seven subclades, with each subclade having a characteristic genetic background confined to a geographic location. The previous study indicated that P. aeruginosa ST235 has become prevalent worldwide due to the antibiotics-selective pressures through mutations and acquisition of resistant-elements among local populations [2]. Our results suggest that clonal expansion is the driving force in generating the population structure of ST235 P. aeruginosa [22].
The primary reason for the emergence and spread of carbapenem-resistant bla GES-5 -positive P. aeruginosa isolates in medical settings in Japan is the inability of microbiological laboratories in Japan, even those in tertiary hospitals, to detect bla GES-5 -positive isolates. P. aeruginosa strains that produce Class B metallo-β-lactamases can be routinely detected in microbiological laboratories using several methods [23][24][25]. These methods, however, are unable to detect bacterial strains producing class A carbapenemases. The cross-transmission of bla GES-5 will be caused by the clonal nature of the outbreaks in medical settings in Japan. Microbiological laboratories therefore require methods to easily detect GES-5 producers, such as the Blue Carba test [26,27], the CIMTris [28] and a systematic bla GES PCR method.
Virulent factor ExoU will be associated with the antibiotic resistance and spreading of CC235 P. aeruginosa. Previous study reported that ExoU-positive P. aeruginosa strains had higher resistance to β-lactams and quinolones than ExoS-positive strains, because the ExoUpositive P. aeruginosa had more mutations in genes that were associated with β-lactams resistance and quinolone resistance [29]. The height of the mutation rate caused by ExoU may help to enhance the adaptability of CC235 P. aeruginosa to the environment.
GES-5 producing P. aeruginosa may spread in medical settings worldwide. A P. aeruginosa isolate producing GES-5 was first obtained in 2004 from the blood of a burn patient in China [16]. GES-5 producing P. aeruginosa strains have been isolated from patients in Brazil [30], Japan [14], Lithuania [31], South Africa [32], Spain [33] and Turkey [34]. Thus, it is important to monitor for GES-5 producing P. aeruginosa in medical settings worldwide.