Genetic Characterization of Plasmid-Borne blaOXA-58 in Distinct Acinetobacter Species

Although the blaOXA-58 gene has been infrequently described in Brazil, contrasting with other bordering South American countries, we verified the maintenance of this resistance determinant over time among carbapenem-resistant Acinetobacter species isolates, not only in nosocomial settings but also in the environment. In addition, to the best of our knowledge, this is the first study to have used WPS analysis to evaluate the genetic surroundings of blaOXA-58 in Brazil. Moreover, the A. seifertii and A. baumannii clinical strains evaluated in this study were recovered 17 years apart in hospitals located in distinct Brazilian geographic regions.

KEYWORDS Acinetobacter calcoaceticus-Acinetobacter baumannii complex, Gramnegative bacilli, carbapenem resistance, carbapenem-hydrolyzing class D ␤-lactamase, nosocomial infection A cinetobacter species are important pathogens frequently responsible for causing nosocomial infections, mainly in patients hospitalized at intensive care units (ICU) (1,2). The spread of major carbapenem-resistant A. baumannii clones has been associated with the increasing frequency of the carbapenem resistance phenotype worldwide (1). The production of carbapenem-hydrolyzing class D ␤-lactamases (CHDLs) has been reported to be the main mechanism of carbapenem resistance (3). The spread of multidrug-resistant (MDR) A. baumannii sequence type 79 (ST79) isolates carrying bla OXA-23 and, more recently, bla OXA-72 has contributed to the high carbapenem resistance rates (85%) observed in Brazil (3). In contrast, bla OXA-58 has been rarely reported in this country, contrasting with the high frequency of this CHDL-encoding gene seen in neighboring countries, mainly in Argentina (4). To date, only six OXA-58-producing Acinetobacter species strains recovered from human (5-8) and environmental (9) sources have been reported in distinct Brazilian cities since the 90s (5-9). The diverse genetic structures surrounding bla OXA-58 documented worldwide (3, 10, 11) play a major role not only in the mobilization of this resistance determinant but also in driving its expression, decisively leading to the carbapenem resistance phenotype (3,12).
The carriage of bla OXA-58 by distinct Acinetobacter species recovered over a long period of time-3 decades-suggests that this CHDL-encoding gene has been mobilized by horizontal gene transfer (HGT), although the hypothesis of clonal spread could not be completely discarded. In order to reveal the genetic environment of bla OXA-58 and the corresponding implications for the mobilization and maintenance of this resistance determinant over time, we molecularly characterize two distinct plasmids harboring bla OXA-58 obtained from A. seifertii and A. baumannii clinical strains recovered in the years 1993 and 2010, respectively, from distinct Brazilian geographic regions.
Two Re27 sequences were found adjacent to an ISAba3-bla OXA-58 -ISAba3 arrangement on pAb45063_b, with Re27-1 located upstream of 5=-ISAba3 and Re27-2 adjacent to ISAba125 located downstream of araC1 and lysE genes ( Fig. 2A), which coded for a threonine efflux protein and a transcriptional regulator, respectively. In contrast, we failed to identify Re27-like regions on pAs1069_a. In this plasmid, lysE was disrupted by TnaphA6, which harbored aminoglycoside modifying enzyme (AME)-encoding gene aphA6. This AME confers resistance to gentamicin and amikacin. aphA6 was flanked by two copies of ISAba125 in the same orientation (Fig. 2B), while TnaphA6 generates a 7-bp duplication (ATTCGCC) upon transposition (Fig. 2B). The production of aminoglycoside O-phosphotransferase AphA6 by A. seifertii Asp-1069 justified the high MICs for amikacin (256 g/ml) and gentamicin (512 g/ml) verified in such strain (Table 1). In addition, a genetic arrangement composed of two copies of IS26 and the narrowspectrum-␤-lactamase-encoding gene bla TEM-1 and the AME-encoding gene aac(3)-IIa was also found upstream of ISAba3-bla OXA-58 -ISAba3 on pAb45063_b ( Fig. 2A). AAC(3)-IIa confers a high level of resistance to gentamicin but not amikacin, justifying the phenotype observed for A. baumannii Acb-45063 (MICs of 512 and 8 g/ml for gentamicin and amikacin, respectively; Table 1). Other two plasmids (pAb45063_a and pAs1069_b) were also detected in the OXA-58-producing Acinetobacter species strains evaluated in the present study ( Fig. 1C and D). The 183,767-bp plasmid Ab45063_a carried the streptomycin resistance genes strA and strB and the sulfonamide resistance gene sul2 (Fig. 1D), contrasting with the small plasmid pAs1069_b of 13,129 bp (Fig. 1C).
Although the two OXA-58-producing Acinetobacter species strains showed similar profiles of susceptibility to ␤-lactams, A. baumannii Acb-45063 MICs were 0.06 g/ml and 64 g/ml for polymyxin B and ciprofloxacin, respectively, contrasting with those presented by A. seifertii Asp-1069 strains (MICs of 4 and 1 g/ml for polymyxin B and ciprofloxacin, respectively) ( Table 1). Distinct virulence factors were observed in all four plasmids (Table 1) as follows: outer membrane protein-encoding gene tonB, septicolysin-encoding gene spl, phosphoglucosamine mutase-encoding gene glmM, inorganic pyrophosphatase-encoding gene ppa, sulfate permease-encoding gene sulP, and methionine aminopeptidase type I-encoding gene map. In addition, distinct toxin-antitoxin system-encoding genes were also detected in the two bla OXA-58 -harboring plasmids (Fig. 1A and B), such as stbD and relE (pAs1069_a) and brnT and brnA (pAb45063_b). Although xreE was found in the large (183-kb) pAb45063_a plasmid (Fig. 1D), no toxin-antitoxin systems were found in the 13-kb pAs1069_b plasmid (Fig. 1C).

DISCUSSION
The plasmid carrying bla OXA-58 , pAs1069_a, recovered from A. seifertii, shares 99% identity with the plasmid harboring bla OXA-58 pAb242_25 described in a MDR A. baumannii clinical strain (Ab242) isolated in the city of Rosario, Argentina (4). Interestingly, Ab242 strain was recovered in 1997 (11), 4 years later than the two clonally related OXA-58-producing A. seifertii clinical strains isolated in Brazil (8). Also, Narciso and colleagues described an OXA-58-producing A. seifertii strain (Ac-12.1) recovered in 2012 from a cloaca of a black-necked swan residing in the lakes of the São Paulo Zoo (9). This strain was clonally related to both A. seifertii clinical strains-including the Asp-1069 evaluated in the present study-isolated 19 years earlier in a tertiary hospital located in the city of São Paulo (8,9). Since the genetic environment surrounding bla OXA-58 of Ac-12.1 was identical to that of the corresponding gene in the Asp-1069 strain, except for a truncated copy of 3=-ISAba3 (8,9), it reinforces the idea of the capability of rearrangement and the complexity of transposable elements among plasmid-borne bla OXA-58 genes (3).
Although the OXA-58-producing A. baumannii Acb-45063 strain was included in ST15 IP , it belongs to same clonal complex (CC15 IP /CC103 OX [Oxford scheme]) as the Ab242 ST104 OX A. baumannii strain recovered in Argentina (4). Note that the city of Porto Alegre, where the Acb-45063 strain was recovered, is located in a Brazilian state next to the Argentinian border, where bla OXA-58 is prevalent and of public health concern (4, 10). However, the plasmids carrying bla OXA-58 detected in Argentinean and Brazilian A. baumannii strains showed distinct genetic backbones. Although plasmids carrying bla OXA-58 that belonged to GR8/GR23 were found among distinct Acinetobacter species in South American countries in the 1990s, a distinct genetic backbone surrounding bla OXA-58 was found in a GR4 plasmid from a A. baumannii Acb-45063 strain recovered in Brazil at 2010. According to Ravasi and colleagues, the presence of ISAba825 upstream of bla OXA-58 , as observed in pAb45063_b (ΔISAba3/ISAba825bla OXA-58 -ISAba3), results in a hybrid promoter that overexpresses this CHDL, leading to 16-fold and 8-fold increases in the MICs for imipenem and meropenem, respectively (13). Re27-like sites found in pAb45063_b, but not in pAs1069_a, are short genomic sequences implicated in site specific recombination processes involved in the evolution of plasmids, many of them carrying CHDL-encoding genes (4,10,14,15). These sequences have been identified bordering ISAba3-like elements, allowing the occurrence of multiple recombination processes that promote different arrangements and acquisition of bla OXA-58 by Acinetobacter species (4, 10-12, 14, 15).
Although it has been previously suggested that the presence of virulence-encoding genes does not guarantee the expression of virulence factors and/or bacterial pathogenicity (16), curiously, our study revealed the presence of distinct virulence factors in all plasmids evaluated, some of which had never been described before in Acinetobacter spp. (13,(15)(16)(17). The TonB outer membrane protein is associated with iron uptake, and its expression may be related to the survival of the bacterial cell in the lungs and blood (13,16,17). The spl gene encodes a septicolysin with cytolytic activity related to the invasion of tissues or cells, while glmM codes for a phosphoglucosamine mutase that has been related as a highly sensitive predictor of several clinical outcomes (13,16,17). Other three genes found, ppa, sulP, and map, have been associated with bacterial pathogenicity (4). In addition, distinct toxin-antitoxin system-encoding genes found in three of four plasmids evaluated ensure the stability of transferable genetic elements in the bacterial host cell (4,15,16).
In conclusion, a complex and dynamic backbones were found surrounding the bla OXA-58 carried by distinct plasmids from A. seifertii and A. baumannii strains recovered 17 years apart in Brazil. Such data demonstrated that although this CHDL-encoding gene has rarely been reported in Brazil, genetic plasticity has occurred over time, composed of a variety of resistance and virulence markers associated with the stability conferred by toxin-antitoxin systems. These findings accounted in part for the success of efforts that have kept plasmids carrying bla OXA-58 from escaping nosocomial settings for a long period of time.