Filipa Grosso

Molecular Characterization of a New Class 3 Integron in Klebsiella pneumoniae

ABSTRACT Klebsiella pneumoniae FFUL 22K was isolated in April 1999 from the urine of an intensive care unit patient in Portugal. The strain showed an extended-spectrum cephalosporin resistance profile. A typical synergistic effect between cefotaxime or cefepime and clavulanic acid was observed. An Escherichia coli transformant displayed a similar resistance phenotype and harbored a ca. 9.4-kb plasmid (p22K9). Cloning experiments revealed that the extended-spectrum β-lactamase was encoded by blaGES-1, previously described in class 1 integrons from K. pneumoniae ORI-1 and Pseudomonas aeruginosa Pa695. Further sequence analysis demonstrated that the blaGES-1 gene cassette was located on a new class 3 integron. The integron was 2,863 bp long and consisted of an intI3 integrase gene, an attI3 recombination site, two promoter regions, and two gene cassettes. The IntI3 integrase was 98.8% identical to that of Serratia marcescens AK9373. The blaGES-1 gene cassette was inserted at the attI3 site. The second gene cassette was the result of a fusion event between blaOXA-10-type and aac(6′)-Ib gene cassettes and conferred resistance to kanamycin. This is the second class 3 integron reported and the first time that the blaGES-1 gene cassette has been found on an integron belonging to this class, highlighting the considerable heterogeneity of their genetic environment and the spread of gene cassettes among different classes of integrons.

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).

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.

Outbreak of GES-1 β-Lactamase-Producing Multidrug-Resistant Klebsiella pneumoniae in a University Hospital in Lisbon, Portugal

Plasmid-located extended-spectrum β-lactamase genes are mostly found in Klebsiella pneumoniae (4), which is an important cause of nosocomial infections (1). In this study, we report the existence of K. pneumoniae clinical isolates producing the Ambler class A enzyme GES-1. This enzyme has been reported in Europe from K. pneumoniae (4) and Pseudomonas aeruginosa (2). Between February 1999 and 2001, 30 K. pneumoniae clinical isolates were collected from different patients, distributed among several wards (surgery and medical services and in different intensive care units) at the Hospital de Santa Maria, Lisbon, Portugal. Twenty-four isolates were identified from urine, four were identified from respiratory tract samples (three from sputum and one from bronchial exudate), and the remaining two isolates were found in blood and pus. Antibiotic susceptibility testing by disk diffusion (3) suggested the presence of an extended-spectrum β-lactamase. Synergies were observed among clavulanic acid-amoxicillin, cefotaxime, aztreonam, and cefepime. All isolates were resistant to clavulanic acid, ceftazidime, cefuroxime, gentamicin, kanamycin, netilmicin, nalidixic acid, and norfloxacin. They were susceptible to imipinem and cefepime and presented reduced susceptibilities to amikacin, cefotaxime, and aztreonam. The analysis of genomic DNA, digested with XbaI and resolved by pulsed-field gel electrophoresis (1), revealed the same macrorestriction pattern among all isolates, classified as indistinguishable according to the work of Tenover et al. (6). On the isoelectric focusing gel two β-lactamase activities with pIs of 5.9 and 7.6 were detected. The β-lactamase activity of pI 7.6 corresponds to chromosomal SHV penicil-linase, and the pI value of 5.9 represented the GES-1 β-lactamase. Plasmid extraction, performed according to the alkaline lysis method (5), revealed plasmids with molecular sizes ranging from 3 to 23 kb. From five selected K. pneumoniae strains we obtained Escherichia coli DH5α transformants more resistant to ceftazidime than to cefotaxime and aztreonam and harboring a plasmid with a molecular size of ca. 9 kb. Under standard PCR conditions, plasmid DNA preparations from K. pneumoniae and E. coli DH5α transformants were used as templates for amplification of the blaGES-1 gene with primers GES-1A and GES-1B (4). All isolates revealed an 864-bp PCR product. The resulting amplicon was cloned into the SmaI site of pBK-CMV. The E. coli TOP10 harboring pMFA-62 was selected for subsequent analysis and sequencing. MICs of β-lactam antibiotics were determined with E-test strips (AB Biodisk, Solna, Sweden). For the K. pneumoniae clinical isolates, E. coli DH5α transformants, and E. coli TOP10 harboring recombinant plasmid pMFA-62, the cefuroxime and ceftazidime MICs were >256 μg/ml, and the MIC ranges (in micrograms per milliliter) were 3 to 0.75 for aztreonam and 6 to 8 for cefotaxime (Table ​(Table1).1). The nucleotide sequence of the cloned fragment revealed 100% identity with blaGES-1 from P. aeruginosa Pa695 (2) and differs by a single silent mutation at position 591 from blaGES-1 described elsewhere for K. pneumoniae ORI-1 (4). TABLE 1. MICs of β-lactams for K. pneumoniae clinical isolates, E. coli DH5α transformants, E. coli TOP10 harboring recombinant plasmid pMFA-62, and reference strains E. coli DH5α and E. coli TOP10 with pBK-CMV The same macrorestriction pattern by pulsed-field gel electrophoresis indicated that an endemic K. pneumoniae strain producing GES-1 β-lactamase was presenting in different wards in the Hospital de Santa Maria. The persistence of these multiresistant microorganisms in the hospital may be associated with the existence of other resistance genes, inserted in multidrug-resistant integrons and/or plasmids.

Identification of carbapenem-resistant Acinetobacter baumannii clones using infrared spectroscopy.

In this work we assessed the discriminatory ability of Fourier-transform Infrared Spectroscopy (FTIR) in 22 representative isolates from a collection of 318 carbapenem-hydrolyzing class D β -lactamases (CHDL)-producing Acinetobacter spp. (5 hospitals; 2001-2008) previously characterized by DNA-based typing methods. FTIR spectra were acquired with a Bruker spectrometer and analyzed with support of several chemometric tools. The results showed that FTIR spectroscopy was able to distinguish the main CHDL-producing Acinetobacter baumannii lineages causing infection in Portugal, the ST103 carrying blaOXA-58 , ST98 carrying blaOXA-24/40 and ST92 carrying blaOXA-23 . Moreover, this study revealed distinctive phenotypic features of A. baumannii lineages causing infections that might justify different epidemic potential. Spectroscopy may arise as a low cost and easily to perform alternative for typing A. baumannii isolates.

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

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Successful application of the DiversiLab repetitive-sequence-based PCR typing system for confirmation of the circulation of a multiresistant Pseudomonas aeruginosa clone in different hospital wards

The applicability of the repetitive-sequence-based PCR (rep-PCR)-based DiversiLab system was tested compared with the pulsed field gel electrophoresis (PFGE) to type a phenotypically similar subset of a large collection of multiresistant Pseudomonas aeruginosa strains isolated during a 17-month period from patients treated in different wards including 4 intensive care units (ICUs). Five environmental P. aeruginosa isolates obtained from one of the ICUs were also included. The DiversiLab system and the PFGE demonstrated the genetic relationship among the isolates with the same efficacy. One of the environmental isolates had the same rep-PCR type as the circulating clone. Multilocus sequence typing of one of the clinical isolates of the circulating clone proved that it is a member of a clonal complex of P. aeruginosa that has not been previously described in clinical samples.

Snapshot on carbapenemase-producing Pseudomonas aeruginosa and Acinetobacter baumannii in Bucharest hospitals reveals unusual clones and novel genetic surroundings for blaOXA-23

OBJECTIVES The present study was designed to provide a snapshot on carbapenemase-producing Pseudomonas aeruginosa (n=11) and Acinetobacter baumannii (n=7) isolates in hospitalized patients (November 2011, January-March 2012) from two main hospitals in Bucharest, south Romania. METHODS Clonality among isolates was established by PFGE, MLST and Fourier transform infrared spectroscopy. Carbapenemases were screened by the Blue-Carba test, PCR and sequencing. Transferability of blaOXA-23 was tested by conjugation and plasmid typing (number, size and identity) was assessed by S1-PFGE, replicon typing, hybridization and PCR mapping. RESULTS All P. aeruginosa isolates carried chromosomally located blaVIM-2, associated with a common class 1 integron (aacA7-blaVIM-2) or an atypical configuration (aacA7-blaVIM-2-dfrB5-tniC). These isolates belonged to unusual lineages; mostly ST233 disseminated in one hospital unit, with ST364 and ST1074 also being detected. A. baumannii isolates carried blaOXA-23 in Tn2008, which was found truncating a TnaphA6 transposon located in a common 60 kb GR6 (aci6) pABKp1-like conjugative plasmid in highly related CC92 clones (ST437, ST764 and ST765), where CC stands for clonal complex. CONCLUSIONS Our results show the spread of VIM-2-producing P. aeruginosa and OXA-23-producing A. baumannii clinical isolates in two hospitals from Bucharest and highlight a peculiar population structure in this Eastern European country. Also, we demonstrate the dissemination of a common and conjugative aci6 pABKp1-like plasmid scaffold in different A. baumannii clones and we report the first known identification of Tnaph6-carrying pACICU2-like plasmids in Europe.