Acinetobacter baumannii A118 was isolated from a patient's blood culture. It is susceptible to several antibiotics, is naturally competent, and supports replication and stable maintenance of four plasmid replicons. A. baumannii A118 took up a fluorophore-labeled oligonucleotide analog. These characteristics make this isolate a convenient model for genetic studies. CASE REPORTAcinetobacter baumannii A118 was isolated from a blood culture of a patient admitted to an intensive care unit in a hospital in Buenos Aires, Argentina. The isolate was identified at the species level using several criteria: (i) the biochemical scheme described by Bouvet and Grimont (1); (ii) amplified ribosomal DNA restriction analysis (ARDRA) (12,22); the restriction pattern obtained with CfoI, AluI, and MboI, which is 111, characteristic for A. baumannii (http://users.ugent.be /ϳmvaneech/ARDRA/Acinetobacter.html) (Fig. 1A); (iii) amplification and sequencing of the 16S rRNA; (iv) amplification of the rpoB gene; and (v) identification and sequencing of bla OXA-51-like, a carbapenemase gene intrinsic to A. baumannii (12,21). A. baumannii A118 lacks the integrase genes intI1, intI2, and intI3 and includes the competence genes comA, comM, and pilC, as determined by PCR amplification and DNA sequencing. Determination of antibiotic susceptibilities using the agar dilution method according to the CLSI guidelines (2) indicated that A. baumannii A118 is susceptible to ceftazidime, cefepime, piperacillin, minocycline, amikacin (Amk), gentamicin, trimethoprim-sulfamethoxazole, and ciprofloxacin. Although there is no CLSI guideline for using kanamycin (Kan) on A. baumannii, we determined the MIC, 1.5 g/ml, because this antibiotic is widely used as a selective drug in laboratory experiments.Bacteria of the genus Acinetobacter have been shown to be naturally competent (4). However, while data on natural competence of A. baylyi abound, studies of natural competence, as well as stability of plasmid replicons that could be used as potential cloning vehicles for researching A. baumannii, are scarce (13). Here we determined the competency of the A. baumannii A118 isolate and the stability of several plasmid replicons, some of them widely used as genetic tools. The plasmids pJHCMW1 (16), pMET1 (17), pAADA1KN, pAADB, and pVK102 (9) (Table 1) were used in transformation and stability assays. Plasmid DNA was isolated using the QIAfilter midi kit (Qiagen). Plasmid stability assays were carried out for 40 generations, as described previously (20). Briefly, plasmid-containing cells in late log phase were diluted 10 Ϫ6 -fold in fresh nonselective medium (LB broth) and incubated at 37°C until the culture reached the same optical density (20 generations). This culture was again diluted, and the procedure was repeated to reach 40 generations. These cultures were diluted and spread on selective and nonselective plates to determine the percentage of plasmid-containing cells. The case of pAADA1KN is discussed below. Experiments were done twice; plasmid DNA was prepared f...
We analyzed the role of integrons in the dissemination of antibiotic resistance in a recent multiresistant clinical isolate, Serratia marcescens SCH88050909 (SCH909). This isolate harbors three integrons, all on a 60-kb conjugative plasmid. By PCR, hybridization, and sequencing analyses, we found that integron 1 has the dfrA1 and ant(3؆)-Ia cassettes. The first cassette in integron 2 contains the ant(2؆)-Ia gene, separated from its attC site (59-base element) by a 1,971-bp insert containing a group II intron; this intron codes for a putative maturase-reverse transcriptase on the complementary strand and is the first such intron to be found associated with an integron. The attC site is followed by a novel aminoglycoside resistance gene, ant(3؆)-Ii-aac(6)-IId, which has been characterized for its bifunctional ANT(3؆)-I and AAC(6)-II activities. DNA sequence analysis of this fused cassette suggests that insertion and excision due to the integrase activity could have an important role in the evolution of aminoglycoside resistance genes. This gene is followed by an unknown open reading frame with a typical attC site and a partial cassette composed of the beginning of the bla OXA-10 cassette interrupted by IS1. The sequence downstream of IS1 revealed that the bla OXA-10 cassette is incomplete and that the 3 conserved segment of this integron is absent. Integron 3 is in a Tn1696-like transposon with the aac(3)-Ia cassette followed by three unknown cassettes and ant(3؆)-Ia. The presence of the group II intron and the relationship of group II introns in eubacteria with mobile elements suggest a possible role of this element in events such as cassette formation and/or plasmid evolution.
Molecular evolution of multiresistance in nontyphoid Salmonella spp. was investigated with 155 isolates obtained in Argentina from 1984 to 1998. In 74 isolates obtained from 1984 to 1988 resistance was associated with the presence of Tn3, Tn9, class I (In0) and II (Tn7) integrons, and the aac(3)-IIa gene. Extended-spectrum cephalosporin (ESC) resistance in Salmonella spp. emerged in 1989, and 81 isolates resistant to at least one ESC and one aminoglycoside were collected thereafter. Among these, two patterns of antimicrobial resistance mechanisms were found: from 1989 to 1992, resistance was related to the spreading of Tn1331 and bla CTX-M-2 , in addition to the persistence of In0 and Tn7. From 1993 to 1998, several integrons were added to the first pattern and three integron groups (IG), namely, IG1 (38% of the isolates), IG2 (51%), and IG3 (11%), were identified. At least two -lactamase genes were detected in 65% of the isolates (after 1989) by PCR analysis. Furthermore, five -lactamase genes, bla CTX-M-2 , bla OXA-9 , bla OXA-2 , bla TEM-1 , and bla PER-2 , were found in two isolates. The bla CTX-M-2 gene was found in several complex sulI-type integrons with different rearrays within the variable region of class I integrons, suggesting evolution of these integrons in nontyphoid Salmonella. In conclusion, progressive acquisition and accumulation of plasmid-mediated resistance determinants occurred from 1984 to 1998 in nontyphoid Salmonella isolates of the most prevalent serovars from Argentina. It is suggested that antimicrobial resistance mechanisms in these bacteria may have been the consequence of plasmid exchange between Salmonella enterica serovar Typhimurium and Escherichia coli or Shigella flexneri and/or spreading of mobile elements from the nosocomial environment.
Examination of the bla CTX-M-2 gene in plasmid pMAR-12 by sequencing and PCR analysis revealed that the bla gene and the surrounding DNA, which is closely related (99% homology) to the Kluyvera ascorbata chromosomal DNA that contains the bla KLUA-1 gene, are located in a complex sul1-type integron, termed In35, that includes Orf513. It is possible that bla CTX-M-2 was acquired by plasmid pMAR-12 through an uncharacterized recombinational event in which Orf513 could be involved.
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