68-3

9/130

Figure 3　Genomic environments of blaIMP-1 and blaIMP-70 in clinical isolates of P. rettgeri and P. stuartii. Genes are represented as arrows, which indicate their transcription orientations and relative lengths. MBL genes, tnp genes, and truncated genes are shown as black arrows, gray arrows, and Δ, respectively. Label orf1 represents a gene encoding a hypothetical protein, and orf2 represents a gene encoding an ATP-binding protein. This figure is a modified version of FIG 1 in reference 18.(Iwata S, Tada T, Hishinuma T, et al: Emergence of Carbapenem-Resistant Providencia rettgeri and. Antimicrob Agents Chemother, 2020; 64.)Phe87Ser), had more efficient catalytic activities against carbapenems than those of IMP-1124). These results suggest that co-occurrence of two amino acid substitutions at these two positions increase the enzymatic activities of IMP-44, whereas the Phe87Val substitution did not affect the enzymatic activities of IMP-70. The substitution of Phe87 by a polar amino acid such as Ser, but not by a hydro-phobic amino acid such as Val, may affect enzy-matic activities.Biological significance of two copies of blaIMP-1 in tandemOne of the P. rettgeri isolates was found to harbor two copies of blaIMP-1, in tandem on the chromo-some, consisting of a repeat of the genetic struc-ture int1Δ-blaIMP-70-qacEΔ1-sul1 (Figure 3). To confirm the presence of the two copies of blaIMP-1, sequences were amplified by PCR using a primer set targeting the two copies. Amplification resulted in a 3.5-kbp PCR product as expected based on the whole-genome sequence, indicating that this isolate of P. rettgeri harbored two tandem copies of blaIMP-1 on the chromosome. Western blotting analysis revealed that all five isolates tested produced IMP- type MBLs (Figure 4). Of these five isolates, the P. rettgeri isolates harboring two copies of blaIMP-1 produced the largest quantities of IMP-type MBL (Figure 4), indicating these two copies of blaIMP-1 produce high amounts of IMP-1 MBL.The genus Providencia, belonging to the family Morganellaceae, consists of 10 species. Of these, three species, P. alcalifaciens, P. rettgeri and P. stuartii, are clinically important. P. alcalifaciens causes diarrhea by invading the intestinal mucosa, whereas P. stuartii and P. rettgeri have been found to cause hospital acquired urinary tract infections, as well as pneumonia, meningitis, endocarditis, wound infections, bloodstream infections, and trav-elers’ diarrhea. Clinical isolates of carbapenem- resistant P. rettgeri producing IMP-1 MBL were first identified during laboratory-based surveil-lance in 2000 in Japan. To date, there have been 16 reports of carbapenem-resistant P. rettgeri, eight of carbapenem-resistant P. stuartii and one of carbapenem-resistant P. vermicola, with most of these being clinical isolates. We recently obtained four P. rettgeri isolates and one P. stuartii isolate from urine samples of five patients. All five isolates were highly resistant to carbapenems. Three isolates harbored blaIMP-70, encoding a variant of IMP-1 MBL with two amino acid substitutions, and one each harbored blaIMP-1 and blaIMP-11. Molecular analyses of these isolates strongly suggest that Providencia species become more highly resistant to carbapenems by acquisi-tion of two copies of blaIMP-1 or by mutations in blaIMP genes that result in amino acid substitutions, such as blaIMP-70.We thank Miho Ogawa and Masahiro Shimojima employed by BML, Inc. for screening of carbapen-em-resistant clinical isolates. There are no patents, products in development, or marketed products to declare.This study was supported by grants from Japan Society for the Promotion of Science (grants 18K07120 and 19K16652) and Research Program on Emerging and Re-emerging Infectious Diseases 205ConclusionsAcknowledgementsFunding