Sandra Quinteira

OXA-23-producing Acinetobacter baumannii: a new hotspot of diversity in Rio de Janeiro?

OBJECTIVES this study focused on the population structure of OXA-23-producing Acinetobacter baumannii clinical isolates from Rio de Janeiro, Brazil. METHODS the analysis included several genomic typing methods, including PFGE, two multilocus sequence typing (MLST) schemes, sequence group (SG) determination and bla(OXA-51-like) sequencing. The genomic context of the bla(OXA-23) gene was also evaluated using I-CeuI hybridizations and PCR assays. RESULTS congruent clustering was obtained revealing four lineages. In accordance, four new sequence types (STs) (ST131, ST132, ST133 and ST134) were obtained with the MLST-OD scheme (associated with the Oxford Database) and four (ST79, ST15 and two new allelic profiles) with the MLST-IP scheme (developed by the Institute Pasteur). Four SGs (SG1, SG4 and two new profiles) were identified, allowing the association of 70% of the isolates with European clone II. bla(OXA-51-like) sequencing revealed the presence of bla(OXA-66), bla(OXA-69), bla(OXA-95) and bla(OXA-132). CONCLUSIONS identification of new STs together with new SG profiles are findings suggestive of a local diversity hotspot that is worth exploring.

Understanding the dynamics of imipenem-resistant Acinetobacter baumannii lineages within Portugal

A recent collection of 213 imipenem-resistant Acinetobacter baumannii (IRAB) clinical isolates was characterized for the presence of acquired carbapenem-hydrolysing class D β-lactamases (CHDLs) and clonality. A population structure analysis of IRAB was also conducted, with five molecular typing methods. Three main clusters, each one associated with a specific CHDL, were observed with multilocus sequence typing. Overall, our results suggest a switch in the dominant clone, with sequence type (ST) 92, carrying bla(OXA-23) (63.4%), replacing the closely related ST98, carrying bla(OXA-24/40) (22%). In addition, ST103, an independent lineage, was associated with bla(OXA-58) -carrying isolates (14.6%).

Molecular Epidemiology of Imipenem-Resistant Acinetobacter haemolyticus and Acinetobacter baumannii Isolates Carrying Plasmid-Mediated OXA-40 from a Portuguese Hospital

Major outbreaks of multidrug-resistant Acinetobacter baumannii associated with nosocomial infections have been increasingly reported worldwide (1, 10, 12). The endemicity of an OXA-24/40-producing A. baumannii clone associated with mortality events in Portugal has been observed at numerous hospitals within the Iberian Peninsula (5, 6, 10). Inversely, Acinetobacter haemolyticus, isolated only occasionally from clinical samples (9), usually presents susceptibility to different antibiotics, including β-lactams (13). The isolation of two carbapenem-resistant A. haemolyticus strains prompted us to assess the relative contribution of clonal spread to the observed high rate of carbapenem-resistant Acinetobacter spp. in a general hospital in Porto, Portugal. Between January 2001 and October 2004, 224 imipenem-resistant Acinetobacter spp. were collected from several specimen sources and different hospital wards, where A. baumannii was associated with nosocomial infections and colonizations for several months (Table ​(Table1).1). Imipenem resistance significantly increased from 2001 to 2002 and from 2002 to 2003. Macrorestriction analysis of genomic DNA by pulsed-field gel electrophoresis (5) and 16S rRNA gene sequencing, performed for each clone and species representative, showed that, with the exception of two clonally related A. haemolyticus isolates, the remainder were A. baumannii isolates, distributed among three different pulsotypes. Clonal dissemination of two major pulsotypes (A and B), widespread throughout the hospital, contributed to the observed A. baumannii imipenem resistance, which has persisted since at least 2001 despite several elimination attempts, including the use of polymyxin. Pulsotype B was predominant from 2001 to 2002, after which clone A emerged as the dominant type (Table ​(Table1).1). This clone was found to be identical to the previously described Iberian OXA-24/40-producing clone (5). Pulsotype C, with only two isolates, seemed to represent a sporadic event within the observed prevalence of clones A and B. Antimicrobial susceptibilities varied among isolates according to clones (Table ​(Table2).2). A. haemolyticus isolates presented resistance to all β-lactams, with the exception of cefepime, ceftazidime, and aztreonam. All Acinetobacter sp. isolates were resistant to ciprofloxacin, whereas susceptibility to aminoglycosides was variable. Only 11 isolates (including the two A. haemolyticus isolates) showed a colistin MIC of ≥4 μg/ml (2). However, when the recently updated CLSI susceptible interpretative criterion of ≤2 μg/ml (3, 8) was applied, the susceptibility rate dropped from 96.1% to 92.1%. Detection of carbapenemase production, ulteriorly identified as an OXA-24/40 enzyme, was performed as previously described (5) and was positive only for clone A A. baumannii isolates and, for the first time, A. haemolyticus isolates. Hybridization assays after both S1 nuclease digestion and I-CeuI digestion, performed as previously described (7), revealed that although some clone A A. baumannii isolates showed a chromosome-positive signal (ca. 150 kb) for the blaOXA-24/40 probe, most also presented a positive hybridization in plasmidic bands of ca. 180 kb and ca. 30 kb. Similar hybridization signals were observed in the A. haemolyticus isolates. Further studies on plasmid characterization, assessing the homology among different plasmids, are ongoing. TABLE 1. Clinical data for imipenem-resistant Acinetobacter spp.a TABLE 2. In vitro susceptibilities of imipenem-resistant Acinetobacter sp. clinical isolates We describe, for the first time, the presence of an OXA-24/40 enzyme in an A. haemolyticus clinical isolate. Although the spread of OXA-24/40, both in the Iberian Peninsula and in France, has been correlated with the progressive dissemination of a single A. baumannii clone, the observation of this enzyme in a different, previously unreported, genomic species, A. haemolyticus, poses new questions on OXA-24/40 dissemination. It now seems reasonable to suspect a horizontal dissemination of the blaOXA-40 gene between different species, an ability supported by the observation of this enzyme, previously described as chromosomally encoded (7), in a plasmid. Notwithstanding, the dissemination of “successful” clones may possibly contribute to the high rates and persistence of imipenem-resistant A. baumannii isolates (4).

First isolation of blaVIM-2 in an environmental isolate of Pseudomonas pseudoalcaligenes.

Since the first description of VIM-2, in France, in a Pseudomonas aeruginosa clinical isolate ([7][1]), this enzyme has been detected in clinical Pseudomonas isolates in several countries ([6][2]), thus highlighting the need for a detailed knowledge of its spreading dynamics. In Portugal, since the

Role of Common blaOXA-24/OXA-40-Carrying Platforms and Plasmids in the Spread of OXA-24/OXA-40 among Acinetobacter Species Clinical Isolates

ABSTRACT The spread of OXA-24/OXA-40 (OXA-24/40)-producing Acinetobacter spp. in the Iberian Peninsula has been strongly influenced by clonal expansion, but the role of horizontal gene transfer has scarcely been explored. blaOXA-24/40-carrying plasmids and genetic environments were characterized in representative (n = 15) Acinetobacter species clinical isolates (obtained between 2001 and 2007) by Acinetobacter baumannii PCR-based replicon typing, sequencing, hybridization, and restriction fragment length polymorphism. Besides the identification of blaOXA-24/40 within the chromosomes of some isolates, the circulation of common blaOXA-24/40-carrying plasmids (30-kb repA_AB; 10-kb aci2) and genetic backbones among Acinetobacter spp. was demonstrated.

Characterization of In100, a New Integron Carrying a Metallo-β-Lactamase and a Carbenicillinase, from Pseudomonas aeruginosa

ABSTRACT In100, a new integron carrying a carbapenemase gene (blaVIM-2) associated with a carbenicillinase (blaP1b) and aminoglycoside resistance genes (aacA4 and aadA2), was detected in a Pseudomonas aeruginosa clinical isolate. The particular gene cassette organization of In100 seems to reflect the evolution of antibiotic usage in therapeutics.

Controlling for false positives: interpreting MBL Etest and MBL combined disc test for the detection of metallo-β-lactamases

48: 3729–35. 4. Abouzeed YM, Baucheron S, Cloeckaert A. ramR mutations involved in efflux-mediated multidrug resistance in Salmonella enterica serovar Typhimurium. Antimicrob Agents Chemother 2008; 52: 2428–34. 5. Bailey AM, Paulsen IT, Piddock LJV. RamA confers multidrug resistance in Salmonella enterica via increased expression of acrB, which is inhibited by chlorpromazine. Antimicrob Agents Chemother 2008; 52: 3604–11. 6. Chollet R, Chevalier J, Bollet C et al. RamA is an alternate activator of the multidrug resistance cascade in Enterobacter aerogenes. Antimicrob Agents Chemother 2004; 48: 2518–23. 7. Schneiders T, Amyes SGB, Levy SB. Role of AcrR and RamA in fluoroquinolone resistance in clinical Klebsiella pneumoniae isolates from Singapore. Antimicrob Agents Chemother 2003; 47: 2831–7. 8. Zheng J, Cui S, Meng J. Effect of transcriptional activators RamA and SoxS on expression of multidrug efflux pumps AcrAB and AcrEF in fluoroquinolone-resistant Salmonella Typhimurium. J Antimicrob Chemother 2009; 63: 95–102. 9. Andrews JM. BSAC standardized disc susceptibility testing method (version 7). J Antimicrob Chemother 2008; 62: 256–78. 10. http://www.bsac.org.uk/_db/_documents/version_7_1_february_ 2008.pdf (14 May 2009, date last accessed).

The complete nucleotide sequence of an IncP-2 megaplasmid unveils a mosaic architecture comprising a putative novel blaVIM-2-harbouring transposon in Pseudomonas aeruginosa

Objectives In Pseudomonas aeruginosa , bla VIM-2 has been mostly associated with a chromosomal location and rarely with a plasmid backbone. Until now, only three complete bla VIM-2 -carrying plasmid sequences have been described in this species. Here we explore the modular structure of pJB37, the first bla VIM-2 -carrying megaplasmid described in P. aeruginosa . Methods The complete nucleotide sequence of plasmid pJB37 was determined with an Illumina HiSeq, with de novo assembly by SPAdes, annotation by RAST and searching for antimicrobial resistance genes and virulence factors. Conjugation assays were conducted. Results Megaplasmid pJB37 (464 804 bp long and GC content of 57.2%) comprised: an IncP-2 repA-oriV-parAB region; a conjugative transfer region ( traF , traG , virD2 and trbBCDEJLFGI genes); Tn 6356 , a new putative bla VIM-2 -carrying transposon; heavy metal (mercury and tellurite) resistance operons; and an arsenal of virulence genes. Plasmid pJB37 was transferable by conjugation to a spontaneous rifampicin-resistant mutant of P. aeruginosa PAO1. Here, a bla VIM-2 -harbouring In58 integron was associated with a new complex transposable structure, herein named Tn 6356 , suggesting that In58 was most likely acquired by insertion of this element. Conclusions The mosaic arrangement exhibited by the pJB37 IncP-2 megaplasmid, which highlights the vast assembly potential of distinct genetic elements in a Pseudomonas widespread plasmid platform, gives new insights into bacterial adaptation and evolution.

Aquacultures as reservoirs of pathogenic bacteria and clinically relevant antibiotic resistance genes

The efflux pump QepA confers decreased susceptibility to hydrophilic fluoroquinolones (e.g., norfloxacin, ciprofloxacin, and enrofloxacin). In this study, we characterized the third variant, named qepA3, collected from an Escherichia coli isolate in Portugal. INSRA6015 was isolated in 2005 from the urine of a 77-year-old female patient hospitalized at the Hospital Fernando Fonseca, Portugal. Susceptibility testing was performed by disk diffusion and MIC methods, (SFM and EUCAST guidelines, respectively). PCR and sequencing were used to screen and identify bla (bla TEM , bla SHV , bla OXA , bla CTX-M and plasmid-mediated ampC ) genes, as well as plasmid-mediated quinolone resistance ( qnrA , qnrB , qnrC , qnrD , qnrS , qepA and aac(6’)Ib-cr ), and the quinolone resistance-determining regions (QRDR: gyrA , gyrB , parC , and parE ) genes. PCR-mapping was used to characterize the genetic environment of the new qepA3 gene. Transfer of resistance of the QepA3 determinant, was performed through electroporation, using the E. coli TOP10 as recipient. Plasmid content was characterized by PCR-based replicon typing. Molecular characterization of INSRA6015 showed the presence of bla TEM-1, bla CMY-2 and a new variant of qepA possessing two nucleotide substitutions, leading to Phe85Leu and Val134Ile changes. This variant, named QepA3, conferred a similar phenotype to that of the QepA1 and QepA2 determinants. Sequencing of the QDRD detected substitutions Ser83Leu and Asp87Asn in the GyrA subunit and Glu84Lys in the ParC subunit, which are consistent with the high resistance to ciprofloxacin observed in the MICs. Sequence analysis of qepA3 genetic environment revealed that the gene was located inside a genetic structure identical to that of previously described for qepA1 and qepA2 . It is noteworthy that qepA3 gene, as qepA2 , was not associated with the rmtB gene encoding an aminoglycoside ribosomal methylase, contrarily to qepA1. PCR-based replicon typing indicated the presence of the IncF plasmid. We have identified and characterized a new variant of the plasmid-mediated efflux pump QepA, which is responsible for the increased levels of resistance to several clinically important quinolones, such as ciprofloxacin, and norfloxacin. This is, at our knowledge, the first description of the co-production of QepA and CMY-2. The study highlights the need of surveillance of this resistance mechanism and reinforces a more careful use of quinolones.

Two decades of blaVIM-2-producing Pseudomonas aeruginosa dissemination: an interplay between mobile genetic elements and successful clones

Objectives Information on clonal lineages and genetic platforms involved in the mobilization of carbapenemases between Pseudomonas aeruginosa strains in Portugal is scarce. Here, we outline the genetic drivers contributing to the occurrence of blaVIM-2-producing P. aeruginosa over two decades. Methods A collection of carbapenem-resistant P. aeruginosa clinical isolates (n = 263, 1995-2014) was screened for carbapenemase production by Blue-Carba and PCR. Antimicrobial susceptibility testing was performed according to EUCAST and clonal analysis by MLST. Nine isolates representing different integrons and STs were selected for WGS, followed by bioinformatics. Results Twenty-seven blaVIM-2-producing P. aeruginosa belonging to 10 STs were identified, with ST179 and ST111 being the most prevalent and persistent clones. blaVIM-2 was associated with seven class I integrons frequently co-harbouring aminoglycoside resistance genes. In58 was commonly identified, followed by derivatives and In100. blaVIM-2-harbouring transposons of the Tn3 and Tn402 families were linked to different plasmids or integrative conjugative elements of the clc family. Conclusions The dissemination of blaVIM-2 carrying integrons is associated with a complex interplay between different mobile genetic elements, including the overlooked integrative conjugative elements, and successful spread of particular clones.