Baumannii Plasmids Research Articles

Genomic study of Acinetobacter baumannii strains co-harboring bla OXA-58 and bla NDM-1 reveals a large multidrug-resistant plasmid encoding these carbapenemases in Brazil.

Acinetobacter baumannii contributes significantly to the global issue of multidrug-resistant (MDR) nosocomial infections. Often, these strains demonstrate resistance to carbapenems (MDR-CRAB), the first-line treatment for infections instigated by MDR A. baumannii. Our study focused on the antimicrobial susceptibility and genomic sequences related to plasmids from 12 clinical isolates of A. baumannii that carry both the blaOXA-58 and bla NDM-1 carbapenemase genes. Whole-genome sequencing with long-read technology was employed for the characterization of an A. baumannii plasmid that harbors the bla OXA-58 and blaNDM-1 genes. The location of the bla OXA-58 and bla NDM-1 genes was confirmed through Southern blot hybridization assays. Antimicrobial susceptibility tests were conducted, and molecular characterization was performed using PCR and PFGE. Multilocus Sequence Typing analysis revealed considerable genetic diversity among bla OXA-58 and bla NDM-1 positive strains in Brazil. It was confirmed that these genes were located on a plasmid larger than 300 kb in isolates from the same hospital, which also carry other antimicrobial resistance genes. Different genetic contexts were observed for the co-occurrence of these carbapenemase-encoding genes in Brazilian strains. The propagation of bla OXA-58 and bla NDM-1 genes on the same plasmid, which also carries other resistance determinants, could potentially lead to the emergence of bacterial strains resistant to multiple classes of antimicrobials. Therefore, the characterization of these strains is of paramount importance for monitoring resistance evolution, curbing their rapid global dissemination, averting outbreaks, and optimizing therapy.

Transposons Carrying the aacC2e Aminoglycoside and blaTEM Beta-Lactam Resistance Genes in Acinetobacter.

This study examines the genetic contexts and evolutionary steps responsible for the formation of the widely spread transposon Tn6925 carrying blaTEM and aacC2e, which confers resistance to beta-lactam and aminoglycoside antibiotics in Gram-negative bacteria. The blaTEM-1 and aacC2e genes were found in several transposons. They were first observed within an IS26 bounded 3.7 kb transposon (Tn6925) on several Acinetobacter baumannii plasmids located within a 4.7 kb dif module. Truncated and expanded variations of Tn6925 were found across other A. baumannii plasmids, as well as in other Gram-negative bacteria (including Vibrio cholerae). Moreover, blaTEM-1 and aacC2e were in much larger resistance-heavy transposons including the ISAba1-bounded 24.6 kb (here called Tn6927), found in an A. baumannii chromosome. A novel ISKpn12-bounded transposon was also observed to contain blaTEM and aacC2e which was found interrupting Tn5393 along with an IS26 pseudo-compound transposon to form a 24.9 kb resistance island in an Acinetobacter pittii plasmid. Multiple mobile genetic elements are involved in the formation of transposon structures that circulate blaTEM and aacC2e. Among these, IS26 and ISAba1 appear to have played a major role in the formation and spread of these elements in the Acinetobacter species.

The Whole genome sequence for Acinetobacter baumannii strain in Iraqi Hospitals Outbreak

A study was conducted to identify specialized F plasmid genes and regulatory systems in Acinetobacter baumannii. These genes could be very important for the capacity of these species to coexist with the human host and show a wide range of diversity in genes that contribute to antibiotic resistance. Identification of 50A. baumannii isolates was carried out through morphology and culture on CHROM agar and genotypic identification was performed using blaOxa-51 gene. Out of 50, one AB isolate was chosen for whole genome sequencing (WGS) using Illumina MiSeq technology. The analysis identified specialized genes in these isolates that contribute to antibiotic stress and lipopolysaccharide barrier, including complex sets of partial and complete integrons and transposons. The recent findings, transferring of A. baumannii plasmid genes are inducing under the regulatory system to which the plasmid elements confer Adaptation such as F-plasmid genes transfer in A.baumannii induced by antibiotic stress under the control of transcriptional factor regulation system in order to antimicrobial drug resistance. The selected A. baumannii isolate, IS1- A.baumannii 32 contain five global regulator systems .Our study were provided detailed genomic picture in detected both innate and acquired plasmid-encoded AMR genes. Study aim is the role of genetic regulation to control the transcription, virulence factors and antibiotic resistance related mechanism in A.baumannii .

Examining the role of Acinetobacter baumannii plasmid types in disseminating antimicrobial resistance

Acinetobacter baumannii is a Gram-negative pathogen responsible for hospital-acquired infections with high levels of antimicrobial resistance (AMR). The spread of multidrug-resistant A. baumannii strains has become a global concern. Spread of AMR in A. baumannii is primarily mediated by the acquisition of AMR genes through mobile genetic elements, such as plasmids. Thus, a comprehensive understanding of the role of different plasmid types in disseminating AMR genes is essential. Here, we analysed the distribution of plasmid types, sampling sources, geographic locations, and AMR genes carried on A. baumannii plasmids. A collection of 813 complete plasmid entries was collated and analysed. We previously devised an Acinetobacter Plasmid Typing (APT) scheme where rep types were defined using 95% nucleotide identity and updated the scheme in this study by adding 12 new rep/Rep types (90 types in total). The APT scheme now includes 178 unique Rep variants belonging to three families: R1, R3, and RP. R1-type plasmids were mainly associated with global clone 1 strains, while R3-type plasmids were highly diverse and carried a variety of AMR determinants including carbapenem, aminoglycoside and colistin resistance genes. Similarly, RP-type and rep-less plasmids were identified as important carriers of aminoglycoside and carbapenem resistance genes. This study provides a comprehensive overview of the distribution and characteristics of A. baumannii plasmids, shedding light on their role in the dissemination of AMR genes. The updated APT scheme and findings enhance our understanding of the molecular epidemiology of A. baumannii and provide valuable insights for surveillance and control strategies.

Detection and Typing of Plasmids in Acinetobacter baumannii Using rep Genes Encoding Replication Initiation Proteins.

Plasmids found in Acinetobacter species contribute to the spread of antibiotic resistance genes. They appear to be largely confined to this genus and cannot be typed with available tools and databases. Here, a method for distinguishing and typing these plasmids was developed using a curated, non-redundant set of 621 complete sequences of plasmids from Acinetobacter baumannii. Plasmids were separated into 3 groups based on the Pfam domains of the encoded replication initiation (Rep) protein and a fourth group that lack an identifiable Rep protein. The rep genes of each Rep-encoding group (n = 13 Rep_1, n = 107 RepPriCT_1, n = 351 Rep_3) were then clustered using a threshold of >95% nucleotide identity to define 80 distinct types. Five Rep_1 subgroups, designated R1_T1 to R1-T5, were identified and a sixth reported recently was added. Each R1 type corresponded to a conserved small plasmid sequence. The RepPriCT_1 plasmids fell into 5 subgroups, designated RP-T1 to RP-T5 and the Rep_3 plasmids comprised 69 distinct types (R3-T1 to R3-T69). Three R1, 2 RP and 32 R3 types are represented by only a single plasmid. Over half of the plasmids belong to the 4 most abundant types: the RP-T1 plasmids (n = 97), which include conjugation genes and are often associated with various acquired antibiotic resistance genes, and R3-T1, R3-T2 and R3-T3 (n = 95, 30 and 45, respectively). To facilitate typing and the identification of plasmids in draft genomes using this framework, we established the Acinetobacter Typing database containing representative nucleotide and protein sequences of the type markers (https://github.com/MehradHamidian/AcinetobacterPlasmidTyping). IMPORTANCE Though they contribute to the dissemination of genes that confer resistance to clinically important carbapenem and aminoglycoside antibiotics used to treat life-threatening Acinetobacter baumannii infections, plasmids found in Acinetobacter species have not been well studied. As these plasmids do not resemble those found in other Gram-negative pathogens, available typing systems are unsuitable. The plasmid typing system developed for A. baumannii plasmids with an identifiable rep gene will facilitate the classification and tracking of sequenced plasmids. It will also enable the detection of plasmid-derived contigs present in draft genomes that are widely ignored currently. Hence, it will assist in the tracking of resistance genes and other genes that affect survival in the environment, as they spread through the population. As identical or similar plasmids have been found in other Acinetobacter species, the typing system will also be broadly applicable in identifying plasmids in other members of the genus.

Replication initiator proteins of Acinetobacter baumannii plasmids: An update note

The bioinformatic analysis that we made of 492 Acinetobacter baumannii plasmid sequences identified 418 genes encoding Replication Initiator (Rep) proteins that fell into at least fourteen groups according to the protein domains that they contained. The most abundant group of Rep proteins contained a Rep_3 superfamily domain, followed by Rep proteins containing Replicase/PriCT_1 superfamily domains, and then by Reps possessing only an HTH_MerR-SF superfamily domain. The remaining eleven groups contain only a few members. To evaluate the diversity of these Rep proteins, we classify them using the current scheme of GR homology groups, which contains 34 groups. However, we needed to create 22 additional GR homology groups to capture all the Rep protein diversity of the plasmid collection. Finally, our bioinformatic analysis suggests that a large fraction of the plasmids seem to have a restricted host range limited to Acinetobacter species, except for those belonging to GR38 that have a very wide host range. To facilitate the future analysis of the Rep proteins, we included a list of the DNA and protein sequences, in fasta format, of the representatives of each one of the GR homology groups.

Functional Analysis of the Acinetobacter baumannii XerC and XerD Site-Specific Recombinases: Potential Role in Dissemination of Resistance Genes.

Modules composed of a resistance gene flanked by Xer site-specific recombination sites, the vast majority of which were found in Acinetobacter baumannii, are thought to behave as elements that facilitate horizontal dissemination. The A. baumannii xerC and xerD genes were cloned, and the recombinant clones used to complement the cognate Escherichia coli mutants. The complemented strains supported the resolution of plasmid dimers, and, as is the case with E. coli and Klebsiella pneumoniae plasmids, the activity was enhanced when the cells were grown in a low osmolarity growth medium. Binding experiments showed that the partially purified A. baumannii XerC and XerD proteins (XerCAb and XerDAb) bound synthetic Xer site-specific recombination sites, some of them with a nucleotide sequence deduced from existing A. baumannii plasmids. Incubation with suicide substrates resulted in the covalent attachment of DNA to a recombinase, probably XerCAb, indicating that the first step in the recombination reaction took place. The results described show that XerCAb and XerDAb are functional proteins and support the hypothesis that they participate in horizontal dissemination of resistant genes among bacteria.

Structure and Evolution of Acinetobacter baumannii Plasmids.

Acinetobacter baumannii is an emergent bacterial pathogen that provokes many types of infections in hospitals around the world. The genome of this organism consists of a chromosome and plasmids. These plasmids vary over a wide size range and many of them have been linked to the acquisition of antibiotic-resistance genes. Our bioinformatic analyses indicate that A. baumannii plasmids belong to a small number of plasmid lineages. The general structure of these lineages seems to be very stable and consists not only of genes involved in plasmid maintenance functions but of gene sets encoding poorly characterized proteins, not obviously linked to survival in the hospital setting, and opening the possibility that they improve the parasitic properties of plasmids. An analysis of genes involved in replication, suggests that members of the same plasmid lineage are part of the same plasmid incompatibility group. The same analysis showed the necessity of classifying the Rep proteins in ten new groups, under the scheme proposed by Bertini et al. (2010). Also, we show that some plasmid lineages have the potential capacity to replicate in many bacterial genera including those embracing human pathogen species, while others seem to replicate only within the limits of the Acinetobacter genus. Moreover, some plasmid lineages are widely distributed along the A. baumannii phylogenetic tree. Despite this, a number of them lack genes involved in conjugation or mobilization functions. Interestingly, only 34.6% of the plasmids analyzed here possess antibiotic resistance genes and most of them belong to fourteen plasmid lineages of the twenty one described here. Gene flux between plasmid lineages appears primarily limited to transposable elements, which sometimes carry antibiotic resistance genes. In most plasmid lineages transposable elements and antibiotic resistance genes are secondary acquisitions. Finally, broad host-range plasmids appear to have played a crucial role.

Role of plasmid carrying bla NDM in mediating antibiotic resistance among Acinetobacter baumannii clinical isolates from Egypt.

We investigated antibiotic resistance levels among blaNDM-positive (n = 9) and -negative (n = 65) A. baumannii clinical isolates collected in 2010 and 2015 from Alexandria Main University Hospital, Egypt using disc diffusion and minimum inhibitory concentration (MIC) determination. Plasmids from blaNDM-positive isolates were transformed into a carbapenem-susceptible A. baumannii (CS-AB) isolate to assess the role of plasmid transfer in mediating carbapenem resistance. Imipenem, meropenem, and ertapenem MIC90 values against blaNDM-positive isolates were 128, > 256, and 256 µg/mL, respectively. Plasmid isolation and polymerase chain reaction revealed that blaNDM was plasmid mediated. The plasmids were electroporated into the cells of a CS-AB isolate at an efficiency of 1.3 × 10–8 to 2.6 × 10–7, transforming them to blaNDM-positive carbapenem-resistant cells with an imipenem MIC increase of 256-fold. In addition to carbapenem resistance, the blaNDM-positive isolates also exhibited higher levels of cephalosporins, tetracycline, aminoglycosides, fluoroquinolones, and colistin resistance than the blaNDM-negative isolates. Acquisition of blaNDM-carrying plasmids dramatically increased imipenem resistance among A. baumannii isolates. Intriguingly, blaNDM-positive isolates also showed a high degree of resistance to antibiotics of different classes. The potential co-existence of different resistance determinants on A. baumannii plasmids and their possible transfer owing to the natural competence of the pathogen are especially alarming. More effective infection control and antibiotic stewardship programs are needed to curb the spread and treat such infections in both hospital and community settings.Electronic supplementary materialThe online version of this article (10.1007/s13205-020-2157-y) contains supplementary material, which is available to authorized users.

Genetic structure of four plasmids found in Acinetobacter baumannii isolate D36 belonging to lineage 2 of global clone 1.

Four plasmids ranging in size from 4.7 to 44.7 kb found in the extensively antibiotic resistant Acinetobacter baumannii isolate D36 that belongs to lineage 2 of global clone 1 were examined. D36 includes two cryptic plasmids and two carrying antibiotic resistance genes. The smallest plasmid pD36-1 (4.7 kb) carries no resistance genes but includes mobA and mobC mobilisation genes related to those found in pRAY* (pD36-2, 6,078 bp) that also carries the aadB gentamicin, kanamycin and tobramycin resistance gene cassette. These two plasmids do not encode a Rep protein. Plasmid pRAY* was found to be mobilised at high frequency by the large conjugative plasmid pA297-3 but a pRAY* derivative lacking the mobA and mobC genes was not. The two larger plasmids, pD36-3 and pD36-4, encode Rep_3 family proteins (Pfam1051). The cryptic plasmid pD36-3 (6.2 kb) has RepAci1 and pD36-4 (44.7 kb) encodes two novel Rep_3 family proteins suggesting a co-integrate. Plasmid pD36-4 includes the sul2 sulfonamide resistance gene, the aphA1a kanamycin/neomycin resistance gene in Tn4352::ISAba1 and a mer module in a hybrid Tn501/Tn1696 transposon conferring resistance to mercuric ions. New examples of dif modules flanked by pdif sites (XerC-XerD binding sites) that are part of many A. baumannii plasmids were also identified in pD36-3 and pD36-4 which carry three and two dif modules, respectively. Homologs of three dif modules, the sup sulphate permease module in pD36-3, and of the abkAB toxin-antitoxin module and the orf module in pD36-4, were found in different contexts in diverse Acinetobacter plasmids, consistent with module mobility. A novel insertion sequence named ISAba32 found next to the pdif site in the abkAB dif module is related to members of the ISAjo2 group which also are associated with the pdif sites of dif modules. Plasmids found in D36 were also found in some other members of GC1 lineage 2.

The higBA Toxin-Antitoxin Module From the Opportunistic Pathogen Acinetobacter baumannii - Regulation, Activity, and Evolution.

Acinetobacter baumannii is one of the major causes of hard to treat multidrug-resistant hospital infections. A. baumannii features contributing to its spread and persistence in clinical environment are only beginning to be explored. Bacterial toxin-antitoxin (TA) systems are genetic loci shown to be involved in plasmid maintenance and proposed to function as components of stress response networks. Here we present a thorough characterization of type II system of A. baumannii, which is the most ubiquitous TA module present in A. baumannii plasmids. higBA of A. baumannii is a reverse TA (the toxin gene is the first in the operon) and shows little homology to other TA systems of RelE superfamily. It is represented by two variants, which both are functional albeit exhibit strong difference in sequence conservation. The higBA2 operon is found on ubiquitous 11 Kb pAB120 plasmid, conferring carbapenem resistance to clinical A. baumannii isolates and represents a higBA variant that can be found with multiple sequence variations. We show here that higBA2 is capable to confer maintenance of unstable plasmid in Acinetobacter species. HigB2 toxin functions as a ribonuclease and its activity is neutralized by HigA2 antitoxin through formation of an unusually large heterooligomeric complex. Based on the in vivo expression analysis of gfp reporter gene we propose that HigA2 antitoxin and HigBA2 protein complex bind the higBA2 promoter region to downregulate its transcription. We also demonstrate that higBA2 is a stress responsive locus, whose transcription changes in conditions encountered by A. baumannii in clinical environment and within the host. We show elevated expression of higBA2 during stationary phase, under iron deficiency and downregulated expression after antibiotic (rifampicin) treatment.

Site-Specific Recombination at XerC/D Sites Mediates the Formation and Resolution of Plasmid Co-integrates Carrying a blaOXA-58- and TnaphA6-Resistance Module in Acinetobacter baumannii.

Members of the genus Acinetobacter possess distinct plasmid types which provide effective platforms for the acquisition, evolution, and dissemination of antimicrobial resistance structures. Many plasmid-borne resistance structures are bordered by short DNA sequences providing potential recognition sites for the host XerC and XerD site-specific tyrosine recombinases (XerC/D-like sites). However, whether these sites are active in recombination and how they assist the mobilization of associated resistance structures is still poorly understood. Here we characterized the plasmids carried by Acinetobacter baumannii Ab242, a multidrug-resistant clinical strain belonging to the ST104 (Oxford scheme) which produces an OXA-58 carbapenem-hydrolyzing class-D β-lactamase (CHDL). Plasmid sequencing and characterization of replication, stability, and adaptive modules revealed the presence in Ab242 of three novel plasmids lacking self-transferability functions which were designated pAb242_9, pAb242_12, and pAb242_25, respectively. Among them, only pAb242_25 was found to carry an adaptive module encompassing an ISAba825-blaOXA-58 arrangement accompanied by a TnaphA6 transposon, the whole structure conferring simultaneous resistance to carbapenems and aminoglycosides. Ab242 plasmids harbor several XerC/D-like sites, with most sites found in pAb242_25 located in the vicinity or within the adaptive module described above. Electrotransformation of susceptible A. nosocomialis cells with Ab242 plasmids followed by imipenem selection indicated that the transforming plasmid form was a co-integrate resulting from the fusion of pAb242_25 and pAb242_12. Further characterization by cloning and sequencing studies indicated that a XerC/D site in pAb242_25 and another in pAb242_12 provided the active sister pair for the inter-molecular site-specific recombination reaction mediating the fusion of these two plasmids. Moreover, the resulting co-integrate was found also to undergo intra-molecular resolution at the new pair of XerC/D sites generated during fusion thus regenerating the original pAb242_25 and pAb242_12 plasmids. These observations provide the first evidence indicating that XerC/D-like sites in A. baumannii plasmids can provide active pairs for site-specific recombination mediating inter-molecular fusions and intra-molecular resolutions. The overall results shed light on the evolutionary dynamics of A. baumannii plasmids and the underlying mechanisms of dissemination of genetic structures responsible for carbapenem and other antibiotics resistance among the Acinetobacter clinical population.

Three Novel Acinetobacter baumannii Plasmid Replicase-Homology Groups Inferred from the Analysis of a Multidrug-Resistant Clinical Strain Isolated in Argentina

Acinetobacter baumannii is an important opportunistic pathogen responsible for a variety of nosocomial infections. Its success in the hospital environment obeys to multiple causes, among them, the ability to resist antimicrobial compounds. It can rapidly evolve Multidrug Resistance (MDR) when confronted with antibiotic therapy and in particular, the emerging resistance to last-resort carbapenems represents a major concern worldwide. The most frequent cause of carbapenem resistance in A. baumannii is represented nowadays by the acquired Carbapenem-Hydrolyzing Class D β-Lactamases (CHDL) of the OXA-23, OXA-40 and OXA-58 groups, with the respective blaOXA genes generally embedded in distinct genetic structures carried by plasmids.

Small, Enigmatic Plasmids of the Nosocomial Pathogen, Acinetobacter baumannii: Good, Bad, Who Knows?

Acinetobacter baumannii is a Gram-negative nosocomial pathogen that has become a serious healthcare concern within a span of two decades due to its ability to rapidly acquire resistance to all classes of antimicrobial compounds. One of the key features of the A. baumannii genome is an open pan genome with a plethora of plasmids, transposons, integrons, and genomic islands, all of which play important roles in the evolution and success of this clinical pathogen, particularly in the acquisition of multidrug resistance determinants. An interesting genetic feature seen in majority of A. baumannii genomes analyzed is the presence of small plasmids that usually ranged from 2 to 10 kb in size, some of which harbor antibiotic resistance genes and homologs of plasmid mobilization genes. These plasmids are often overlooked when compared to their larger, conjugative counterparts that harbor multiple antibiotic resistance genes and transposable elements. In this mini-review, we will examine our current knowledge of these small A. baumannii plasmids and look into their genetic diversity and phylogenetic relationships. Some of these plasmids, such as the Rep-3 superfamily group and the pRAY-type, which has no recognizable replicase genes, are quite widespread among diverse A. baumannii clinical isolates worldwide, hinting at their usefulness to the lifestyle of this pathogen. Other small plasmids especially those from the Rep-1 superfamily are truly enigmatic, encoding only hypothetical proteins of unknown function, leading to the question of whether these small plasmids are “good” or “bad” to their host A. baumannii.

RCH51, a multiply antibiotic-resistant Acinetobacter baumannii ST103IP isolate, carries resistance genes in three plasmids, including a novel potentially conjugative plasmid carrying oxa235 in transposon Tn6252.

To determine the identity and context of genes conferring antibiotic resistance in a sporadic multiply antibiotic-resistant Acinetobacter baumannii recovered at Royal Children's Hospital, Brisbane. The antibiotic resistance phenotype for 23 antibiotics was determined using disc diffusion or MIC determination. The whole-genome sequence of RCH51 was determined using the Illumina HiSeq platform. Antibiotic resistance determinants were identified using ResFinder. Plasmids were recovered by transformation. Isolate RCH51 belongs to the uncommon STs ST103 IP (7-3-2-1-7-1-4) and ST514 OX (1-52-29-28-18-114-7). It was found to be resistant to sulfamethoxazole, tetracycline, gentamicin, tobramycin and kanamycin and also exhibited reduced susceptibility to imipenem (MIC 2 mg/L) and meropenem (MIC 6 mg/L). RCH51 carries the oxa235 , sul2 , floR , aadB and tet39 resistance genes, all located on plasmids. The largest of the three plasmids, pRCH51-3, is 52 789 bp and carries oxa235 in the ISAba1-bounded transposon Tn 6252 , as well as sul2 and floR . pRCH51-3 represents a new A. baumannii plasmid family that is potentially conjugative as it contains several genes predicted to encode transfer functions. However, conjugation of pRCH51-3 was not detected. The aadB and tet39 resistance genes were each found in small plasmids identical to the known plasmids pRAY*-v1 and pRCH52-1, respectively. The resistance gene complement of RCH51 was found in three plasmids. pRCH51-3, which carries the oxa235 , sul2 and floR resistance genes, represents a new, potentially conjugative A. baumannii plasmid type.

A large conjugative Acinetobacter baumannii plasmid carrying the sul2 sulphonamide and strAB streptomycin resistance genes

Acinetobacter baumannii is an important nosocomial pathogen that often complicates treatment because of its high level of resistance to antibiotics. Though plasmids can potentially introduce various genes into bacterial strains, compared to other Gram-negative bacteria, information about the unique A. baumannii plasmid repertoire is limited. Here, whole genome sequence data was used to determine the plasmid content of strain A297 (RUH875), the reference strain for the globally disseminated multiply resistant A. baumannii clone, global clone 1(GC1). A297 contains three plasmids. Two known plasmids were present; one, pA297-1 (pRAY*), carries the aadB gentamicin, kanamycin and tobramycin resistance gene and another is an 8.7kb cryptic plasmid often found in GC1 isolates. The third plasmid, pA297-3, is 200kb and carries the sul2 sulphonamide resistance gene and strAB streptomycin resistance gene within Tn6172 and a mer mercuric ion resistance module elsewhere. pA297-3 transferred sulphonamide, streptomycin and mercuric ion resistance at high frequency to a susceptible A. baumannii recipient, and contains several genes potentially involved in conjugative transfer. However, a relaxase gene was not found. It also includes several genes encoding proteins involved in DNA metabolism such as partitioning. However, a gene encoding a replication initiation protein could not be found. pA297-3 includes two copies of a Miniature Inverted-Repeat Transposable Element (MITE), named MITE-297, bracketing a 77.5kb fragment, which contains several IS and the mer module.Several plasmids related to but smaller than pA297-3 were found in the GenBank nucleotide database. They were found in different A. baumannii clones and are wide spread. They all contain either Tn6172 or a variant in the same position in the backbone as Tn6172 in pA297-3. Some related plasmids have lost the segment between the MITE-297 copies and retain only one MITE-297. Others have segments of various lengths between two MITE-297 copies, and these can be derived from the region in pA297-3 via a deletion adjacent to IS related to IS26 such as IS1007 or IS1007-like. pA297-3 and its relatives represent a third type of conjugative Acinetobacter plasmid that contributes to the dissemination of antibiotic resistance in this species.

Antimicrobial susceptibility, plasmid profiles, and RAPD-PCR typing of Acinetobacter bacteria

Abstract Background: Multiple-drug resistant Acinetobacter have widely spread in the last decades imposing a serious nosocomial source of infection. Nevertheless, little knowledge was gaimed on tracing the development of antibiotic resistance in Acinetobacter species. Objectives: Explore Acinetobacter spp. via antimicrobial susceptibility, plasmid profiles, and random amplified polymorphism DNA polymerase chain reaction (RAPD-PCR) typing. Methods: One hundred twelve Acinetobacter isolates (including 66 A. baumannii and 46 non-Acinetobacter baumannii strains) were obtained from three university hospitals. The source of infection of these isolates included blood, urine, wound, and respiratory tract. Their susceptibilities to 17 antibiotics were tested and then all Acinetobacter isolates were typed by plasmid analysis and RAPD-PCR method. Results: A. baumannii isolates revealed nine different patterns of antibiotic resistance. Of those, non- A. baumannii, were associated with plasmid and RAPD-PCR typings (p <0.05). A. baumannii was more resistant to multiple antibiotics than non-A. baumannii (p <0.05). Seven different plasmid profiles were observed among 112 Acinetobacter isolates. Plasmids were found in 107 (95.5%) of the 112 isolates. Unlike in RAPD-PCR typing, there was no difference between the type of Acinetobacter, A. or non-A. baumannii strains and plasmid profiles (p >0.05). By RAPD-PCR, six profiles were found for each A. and non-A. baumannii strains. The pattern 6 was the most common pattern among the isolates. Both plasmid and RAPD-PCR typing showed no association between plasmid profiling and site of infection (p >0.05). Conclusion: There is a wide spread of multi-drug resistant Acinetobacter spp., particularly A. baumannii, in the Middle East region that can be traced efficiently by plasmid and genotyping typing of Acinetobacter. More care should be taken for tracing the development of antimicrobial resistance of Acinetobacter using precise molecular typing techniques.

Sequence and organization of pMAC, an Acinetobacter baumannii plasmid harboring genes involved in organic peroxide resistance

Acinetobacter baumannii 19606 harbors pMAC, a 9540-bp plasmid that contains 11 predicted open-reading frames (ORFs). Cloning and transformation experiments using Acinetobacter calcoaceticus BD413 mapped replication functions within a region containing four 21-bp direct repeats ( ori) and ORF 1, which codes for a predicted replication protein. Subcloning and tri-parental mating experiments mapped mobilization functions to the product of ORF 11 and an adjacent predicted oriT. Three ORFs code for proteins that share similarity to hypothetical proteins encoded by plasmid genes found in other bacteria, while the predicted products of three others do not match any known sequence. The product of ORF 8 is similar to Ohr, a hydroperoxide reductase responsible for organic peroxide detoxification and resistance in bacteria. This ORF is immediately upstream of a coding region whose product is related to the MarR family of transcriptional regulators. Disk diffusion assays showed that A. baumannii 19606 is resistant to the organic peroxide-generating compounds cumene hydroperoxide (CHP) and tert-butyl hydroperoxide ( t-BHP), although to levels lower than those detected in Pseudomonas aeruginosa PAO1. Cloning and introduction of the ohr and marR ORFs into Escherichia coli was associated with an increase in resistance to CHP and t-BHP. This appears to be the first case in which the genetic determinants involved in organic peroxide resistance are located in an extrachromosomal element, a situation that can facilitate the horizontal transfer of genetic elements coding for a function that protects bacterial cells from oxidative damage.