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...

The effects on cell physiology of gene order within the bacterial chromosome are poorly understood. In silico approaches have shown that genes involved in transcription and translation processes, in particular ribosomal protein (RP) genes, localize near the replication origin (oriC) in fast-growing bacteria suggesting that such a positional bias is an evolutionarily conserved growth-optimization strategy. Such genomic localization could either provide a higher dosage of these genes during fast growth or facilitate the assembly of ribosomes and transcription foci by keeping physically close the many components of these macromolecular machines. To explore this, we used novel recombineering tools to create a set of Vibrio cholerae strains in which S10-spec-α (S10), a locus bearing half of the ribosomal protein genes, was systematically relocated to alternative genomic positions. We show that the relative distance of S10 to the origin of replication tightly correlated with a reduction of S10 dosage, mRNA abundance and growth rate within these otherwise isogenic strains. Furthermore, this was accompanied by a significant reduction in the host-invasion capacity in Drosophila melanogaster. Both phenotypes were rescued in strains bearing two S10 copies highly distal to oriC, demonstrating that replication-dependent gene dosage reduction is the main mechanism behind these alterations. Hence, S10 positioning connects genome structure to cell physiology in Vibrio cholerae. Our results show experimentally for the first time that genomic positioning of genes involved in the flux of genetic information conditions global growth control and hence bacterial physiology and potentially its evolution.

Transferable quinolone resistance has not previously been reported in Argentina. Here we describe three complex class 1 integrons harboring the novel allele qnrB10 in a unique region downstream of orf513, one of them also containing aac(6)-Ib-cr within the variable region of integrons. The three arrays differed from bla CTX-M-2 -bearing integrons, which are broadly distributed in Argentina.

cWe studied a collection of 105 clinical enterobacteria with unusual phenotypes of quinolone susceptibility to analyze the occurrence of plasmid-mediated quinolone resistance (PMQR) and oqx genes and their implications for quinolone susceptibility. The oqxA and oqxB genes were found in 31/34 (91%) Klebsiella pneumoniae and 1/3 Klebsiella oxytoca isolates. However, the oqxAand oqxB-harboring isolates lacking other known quinolone resistance determinants showed wide ranges of susceptibility to nalidixic acid and ciprofloxacin. Sixty of the 105 isolates (57%) harbored at least one PMQR gene [qnrB19, qnrB10, qnrB2, qnrB1, qnrS1, or aac(6=)-Ib-cr)], belong to 8 enterobacterial species, and were disseminated throughout the country, and most of them were categorized as susceptible by the current clinical quinolone susceptibility breakpoints. We developed a disk diffusionbased method to improve the phenotypic detection of aac(6=)-Ib-cr. The most common PMQR genes in our collection [qnrB19, qnrB10, and aac(6=)-Ib-cr] were differentially distributed among enterobacterial species, and two different epidemiological settings were evident. First, the species associated with community-acquired infections (Salmonella spp. and Escherichia coli) mainly harbored qnrB19 (a unique PMQR gene) located in small ColE1-type plasmids that might constitute its natural reservoirs. qnrB19 was not associated with an extended-spectrum ␤-lactamase phenotype. Second, the species associated with hospital-acquired infections (Enterobacter spp., Klebsiella spp., and Serratia marcescens) mainly harbored qnrB10 in ISCR1-containing class 1 integrons that may also have aac(6=)-Ib-cr as a cassette within the variable region. These two PMQR genes were strongly associated with an extended-spectrum ␤-lactamase phenotype. Therefore, this differential distribution of PMQR genes is strongly influenced by their linkage or lack of linkage to integrons.

Plasmids pPAB19-1, pPAB19-2, pPAB19-3, and pPAB19-4, isolated from Salmonella and Escherichia coli clinical strains from hospitals in Argentina, were completely sequenced. These plasmids include the qnrB19 gene and are 2,699, 3,082, 2,989, and 2,702 nucleotides long, respectively, and they share extensive homology among themselves and with other previously described small qnrB19-harboring plasmids. The genetic environment of qnrB19 in all four plasmids is identical to that in these other plasmids and in transposons such as Tn2012, Tn5387, and Tn5387-like. Nucleotide sequence comparisons among these and previously described plasmids showed a variable region characterized by being flanked by an oriT locus and a Xer recombination site. We propose that this arrangement could play a role in the evolution of plasmids and present a model for DNA swapping between plasmid molecules mediated by site-specific recombination events at oriT and a Xer target site. Qnrs are pentapeptide repeat proteins that mediate resistance to quinolones by protecting type II DNA topoisomerases (14, 28). They are known since 1998 when the first qnr gene was found in the multiresistance plasmid pMG252 harbored by a Klebsiella pneumoniae strain isolated from the urine of a patient at the University of Alabama (20). Since then five qnr families (qnrA, qnrB, qnrC, qnrD, and qnrS) have been found, usually hosted in large plasmids (31). The first qnrB gene (qnrB1) was identified in a plasmid from a K. pneumoniae strain isolated in South India (16), and 38 members of the family quickly followed (http://www.lahey .org/qnrStudies/) (13). The qnrB19 gene has been found in several genera of Enterobacteriaceae isolated from humans (healthy people and clinical isolates), animals, and food of animal origin in numerous geographical regions (6,9,12,17,21,22,27). An interesting characteristic of the qnrB19 allele is that it has been found within large plasmids, associated to ISEcp1C-based transposons (6,9,27), and in small plasmids (ϳ3 kbp) lacking ISEcp1C or any other insertion sequence (12,17,22) (Table 1). However, in spite of being located in such dissimilar elements, the qnrB19 genes share a conserved genetic environment (22).We have recently analyzed a collection of clinical enterobacterial isolates with decreased quinolone susceptibility, and we found four small plasmids harboring qnrB19 (2). We describe here their molecular features and characterize their relationships with other qnrB19-harboring genetic platforms. Furthermore, we propose possible pathways of evolution of the qnrB19 environment as well as a site-specific recombination-based model for DNA modifications at a variable region found in these plasmids. MATERIALS AND METHODSBacterial strains and plasmids. The plasmids pPAB19-1, pPAB19-2, pPAB19-3, and pPAB19-4 analyzed in the present study were isolated from Salmonella enterica serovar Infantis M7849, Escherichia coli M9996, E. coli M9888, and Salmonella sp. strain M9397, respectively (Table 1). Salmonella Infantis M7849 was isolated at the ...

SummaryDam methylates GATC sequences in γ-proteobacteria genomes, regulating several cellular functions including replication. In Vibrio cholerae, which has two chromosomes, Dam is essential for viability, owing to its role in chr2 replication initiation. In this study, we isolated spontaneous mutants of V. cholerae that were able to survive the deletion of dam. In these mutants, homologous recombination and chromosome dimer resolution are essential, unless DNA mismatch repair is inactivated. Furthermore, the initiator of chr2 replication, RctB, is no longer required. We show that, instead, replication of chr2 is insured by spontaneous fusion with chr1 and piggybacking its replication machinery. We report that natural fusion of chr1 and chr2 occurred by two distinct recombination pathways: homologous recombination between repeated IS elements and site-specific recombination between dif sites. Lastly, we observed a preferential fusion of the two chromosomes in their terminus of replication.

The increasing frequency of bacteria showing antimicrobial resistance (AMR) raises the menace of entering into a postantibiotic era. Horizontal gene transfer (HGT) is one of the prime reasons for AMR acquisition. Acinetobacter baumannii is a nosocomial pathogen with outstanding abilities to survive in the hospital environment and to acquire resistance determinants. Its capacity to incorporate exogenous DNA is a major source of AMR genes; however, few studies have addressed this subject. The transformation machinery as well as the factors that induce natural competence in A. baumannii are unknown. In this study, we demonstrate that naturally competent strain A118 increases its natural transformation frequency upon the addition of Ca 2؉ or albumin. We show that comEA and pilQ are involved in this process since their expression levels are increased upon the addition of these compounds. An unspecific protein, like casein, does not reproduce this effect, showing that albumin's effect is specific. Our work describes the first specific inducers of natural competence in A. baumannii. Overall, our results suggest that the main protein in blood enhances HGT in A. baumannii, contributing to the increase of AMR in this threatening human pathogen.A cinetobacter baumannii has emerged as a severe nosocomial pathogen over the course of the last few decades, with high levels of morbidity and mortality associated with infections by this pathogen (1, 2). A. baumannii is considered to be a paradigm of multidrug resistance since it has developed resistance to almost all available antibiotics, leaving few or no treatment options left. The ability of A. baumannii to persist in the clinical setting even under desiccation and nutrient starvation, as well as its ability to accumulate several antibiotic resistance determinants, allowed its evolution as a successful pathogen in the hospital environment (3).The large number of available A. baumannii genomes (n ϭ 1,289) shows that foreign DNA is acquired at high frequencies (4-7).The transformation process has been well described for some species, such as Streptococcus pneumoniae, Vibrio cholerae, Neisseria meningitidis, and Helicobacter pylori (8-12). However, how natural competence is regulated has not been thoroughly studied, particularly for Gram-negative bacteria. Some well-characterized competence inducers are DNA damage in H. pylori (13), starvation, as was suggested for Haemophilus influenzae, and chitin metabolism in Vibrio cholerae (14,15). In most known examples, natural competence is a transitory state that is regulated by different internal and external signals. Often, these regulation networks are not completely understood. For Acinetobacter spp., most of the studies focusing on this issue were performed by using Acinetobacter baylyi strain ADP1 (16-20), a bacterium not as threatening to human health as A. baumannii (18-23). Data regarding A. baumannii and competence inducers are scarce (24-27). Wilharm et al. showed previously that several A. baumannii clinical isolates were a...

Recent works suggest that bacterial gene order links chromosome structure to cell homeostasis. Comparative genomics showed that, in fast-growing bacteria, ribosomal protein genes (RP) locate near the replication origin (oriC). We recently showed that Vibrio cholerae employs this positional bias as a growth optimization strategy: under fast-growth conditions, multifork replication increases RP dosage and expression. However, RP location may provide advantages in a dosage-independent manner: for example, the physical proximity of the many ribosomal components, in the context of a crowded cytoplasm, may favor ribosome biogenesis. To uncover putative dosage-independent effects, we studied isogenic V. cholerae derivatives in which the major RP locus, S10-spc-α (S10), was relocated to alternative genomic positions. When bacteria grew fast, bacterial fitness was reduced according to the S10 relative distance to oriC. The growth of wild-type V. cholerae could not be improved by additional copies of the locus, suggesting a physiologically optimized genomic location. Slow growth is expected to uncouple RP position from dosage, since multifork replication does not occur. Under these conditions, we detected a fitness impairment when S10 was far from oriC. Deep sequencing followed by marker frequency analysis in the absence of multifork replication revealed an up to 30% S10 dosage reduction associated with its relocation that closely correlated with fitness alterations. Hence, the impact of S10 location goes beyond a growth optimization strategy during feast periods. RP location may be important during the whole life cycle of this pathogen.