Co-integrate Col3m bla NDM-1-harboring plasmids in clinical Providencia rettgeri isolates from Argentina

Abstract

The first cases of bla _NDM in Argentina were detected in three Providencia rettgeri (Pre) recovered from two hospitals in Buenos Aires city in 2013. The isolates were genetically related, but the plasmid profile was different. Here, we characterized the bla _NDM-1-harboring plasmids of the first three cases detected in Argentina. Hybrid assembly obtained from short- and long-read sequencing rendered bla _NDM-1 in Col3M plasmids of ca. 320 kb (p15268A_320) in isolate PreM15268, 210 kb (p15758B_210) in PreM15758, and 225 kb (p15973A_225) in PreM15973. In addition, PreM15758 harbored a 98-kb circular plasmid (p15758C_98) flanked by a putative recombination site (hin-TnAs2), with 100% nucleotide ID and coverage with p15628A_320. Analysis of PFGE/S1-nuclease gel, Southern hybridization with bla _NDM-1 probe, hybrid assembly of short and long reads suggests that pM15758C_98 can integrate by homologous recombination. The three bla _NDM-1-plasmids were non-conjugative in vitro. Moreover, tra genes were incomplete, and oriT was not found in the three bla _NDM-1-plasmids. In two isolates, bla_NDM-1 was embedded in a partially conserved structure flanked by two ISKox2. In addition, all plasmids harbored aph(3')-Ia, aph(3')-VI, and qnrD1 genes and aac(6´)Ib-cr, bla _OXA-1, catB3, and arr3 as part of a class 1 integron. Also, p15268A_320 and p15973A_225 harbored bla _PER-2. To the best of our knowledge, this is the first report of clinical P. rettgeri harboring bla_NDM-1 in an atypical genetic environment and located in unusual chimeric Col3M plasmids. The study and continuous surveillance of these pathogens are crucial to tracking the evolution of these resistant plasmids and finding solutions to tackle their dissemination. IMPORTANCE Infections caused by carbapenem hydrolyzing enzymes like NDM (New Delhi metallo-beta-lactamase) represent a serious problem worldwide because they restrict available treatment options and increase morbidity and mortality, and treatment failure prolongs hospital stays. The first three cases of NDM in Argentina were caused by genetically related P. rettgeri recovered in two hospitals. In this work, we studied the genetic structure of the plasmids encoding bla _NDM in those index cases and revealed the enormous plasticity of these genetic elements. In particular, we found a small plasmid that was also found inserted in the larger plasmids by homologous recombination as a co-integrate element. We also found that the bla _NDM plasmids were not able to transfer or move to other hosts, suggesting their role as reservoir elements for the acquisition of resistance genes. It is necessary to unravel the dissemination strategies and the evolution of these resistant plasmids to find solutions to tackle their spread.

Keywords: ISKox2; NDM-1; Providencia rettgeri; TnAs2; plasmid.

Fig 1

(A) PFGE of S1-nuclease digested genomic DNA of PreM15628, PreM15758, and PreM15793. The S1-PFGE shows an estimated number of plasmids and sizes (in kilobases), as bands indicated with arrows. (B) Southern blot hybridization membrane with bla _NDM-1 probe. Black bands on the membrane indicated with colored arrows show positive hybridization signal. The table below summarizes the number of plasmids seen in each isolate and the estimated size by S1-nuclease. Those bands that hybridized with bla _NDM-1 probe were indicated with the same color code throughout panels A, B, and C. (C) BRIG alignment of bla _NDM-1-harboring Col3M plasmids. Each plasmid ring was colored according to the bands seen in panels A and B except for p15758C_98, which was not seen in panel A or B, as described in the text. The comparisons were obtained relative to the largest plasmid p15628A_320 (inner ring). At the center of the ring, color levels indicate BLAST result with a matched degree of shared regions. The outermost ring depicts genes as arrows in the corresponding transcription orientation as follows: red, resistance genes; green, mobile genetic elements; orange, transfers genes; brown, toxin/antitoxin system; purple, arsenic and mercury operons; gray, hypothetical proteins. Tra modules and bla _NDM genetic environment are also indicated.

Fig 2

Partial sequence alignment of the genetic structure of p15628A_320, p15758C_98, p15793A_225, and p15758B_210. The nucleotide sequence of the three longer plasmids was uniformly shortened in silico starting in ISKox2 and aligned against p15758C_98. Genes are shown as arrows in their transcription orientation. The color code used was: red, resistance genes; green, mobile genetic elements (integrases, insertion sequences, or transposons); orange, plasmid transfer genes; purple, mercury resistance operon; brown, toxin/antitoxin system; gray, hypothetical proteins. Light blue shadow blocks represent 100% nucleotide ID between sequences, and the thin black arrows indicate inversions.