Simple Method for Markerless Gene Deletion in Multidrug-Resistant Acinetobacter baumannii

The traditional markerless gene deletion technique based on overlap extension PCR has been used for generating gene deletions in multidrug-resistant Acinetobacter baumannii. However, the method is time-consuming because it requires restriction digestion of the PCR products in DNA cloning and the construction of new vectors containing a suitable antibiotic resistance cassette for the selection of A. baumannii merodiploids. Moreover, the availability of restriction sites and the selection of recombinant bacteria harboring the desired chimeric plasmid are limited, making the construction of a chimeric plasmid more difficult. We describe a rapid and easy cloning method for markerless gene deletion in A. baumannii, which has no limitation in the availability of restriction sites and allows for easy selection of the clones carrying the desired chimeric plasmid. Notably, it is not necessary to construct new vectors in our method. This method utilizes direct cloning of blunt-end DNA fragments, in which upstream and downstream regions of the target gene are fused with an antibiotic resistance cassette via overlap extension PCR and are inserted into a blunt-end suicide vector developed for blunt-end cloning. Importantly, the antibiotic resistance cassette is placed outside the downstream region in order to enable easy selection of the recombinants carrying the desired plasmid, to eliminate the antibiotic resistance cassette via homologous recombination, and to avoid the necessity of constructing new vectors. This strategy was successfully applied to functional analysis of the genes associated with iron acquisition by A. baumannii ATCC 19606 and to ompA gene deletion in other A. baumannii strains. Consequently, the proposed method is invaluable for markerless gene deletion in multidrug-resistant A. baumannii.     

Specific protein-DNA and protein-protein interaction in the hig gene system, a plasmid-borne proteic killer gene system of plasmid Rts1

The hig (host inhibition of growth) gene system of plasmid Rts1 belongs to the plasmid-encoded proteic killer gene family. Among the proteic killer genes described so far, hig is unique in that the toxin gene (higB) exists upstream of the antidote gene (higA). There are two promoters in the hig locus, Phig and PhigA, and only the former, which expresses both higB and higA genes, is negatively controlled by HigA and HigB proteins. In this study, we purified HigA protein by means of GST fusion. The electrophoretic mobility shift assay using the purified protein revealed that HigA specifically bound to the Phig region, but not to PhigA. The HigA-binding sequence was determined by DNase I footprinting assay to be a 56-bp sequence that completely covered the -35 and -10 boxes of Phig. The presence of two inverted repeats in the binding sequence and the identification of a dimer form of HigA by cross-linking experiment suggested that the protein bound to the Phig region as a dimer. HigB was purified as a GST fusion protein as well, though it was achieved only in the presence of HigA. HigA and GST-HigB formed a highly stable complex where the two proteins were present in an equimolar ratio.     

Bacterial Toxin HigB Associates with Ribosomes and Mediates Translation-dependent mRNA Cleavage at A-rich Sites

Most pathogenic Proteus species are primarily associated with urinary tract infections, especially in persons with indwelling catheters or functional/anatomic abnormalities of the urinary tract. Urinary tract infections caused by Proteus vulgaris typically form biofilms and are resistant to commonly used antibiotics. The Rts1 conjugative plasmid from a clinical isolate of P. vulgaris carries over 300 predicted open reading frames, including antibiotic resistance genes. The maintenance of the Rts1 plasmid is ensured in part by the HigBA toxin-antitoxin system. We determined the precise mechanism of action of the HigB toxin in vivo, which is distinct from other known toxins. We demonstrate that HigB is an endoribonuclease whose enzymatic activity is dependent on association with ribosomes through the 50 S subunit. Using primer extension analysis of several test mRNAs, we showed that HigB cleaved extensively across the entire length of coding regions only at specific recognition sequences. HigB mediated cleavage of 100% of both in-frame and out-of-frame AAA sequences. In addition, HigB cleaved ∼20% of AA sequences in coding regions and occasionally cut single As. Remarkably, the cleavage specificity of HigB coincided with one of the most frequently used codons in the AT-rich Proteus spp., AAA (lysine). Therefore, the HigB-mediated plasmid maintenance system for the Rts1 plasmid highlights the intimate relationship between host cells and extrachromosomal DNA that enables the dynamic acquisition of genes that impart a spectrum of survival advantages, including those encoding multidrug resistance and virulence factors.Toxin-antitoxin (TA)³/addiction/suicide modules typically include an autoregulated operon encoding a labile antitoxin and a more stable toxic protein (1). TA toxins facilitate stress survival (chromosomal) or plasmid maintenance and post-segregational killing (extrachromosomal; reviewed in Refs. 1, 2). Most chromosomal TA toxins inhibit cell growth by reversibly targeting either protein translation or DNA replication; their cognate antitoxins prevent toxin activity during periods of optimal growth but enable finely tuned control of TA module toxicity during relatively short periods of environmental stress. However, prolonged stress leads to a point of no return and cell death (3–5).There are six confirmed chromosomal TA loci in Escherichia coli K12 cells: dinJ-yafQ, relBE, yefM-yoeB, mazEF, chpBI-BK, and hipBA. The toxins MazF and ChpBK are sequence-specific endoribonucleases that cleave free mRNA (6–10). The RelE toxin interacts with the ribosome and induces mRNA cleavage with a preference for the UAG stop codon (11–13). The YafQ toxin is a ribosome-associated endoribonuclease that cleaves in-frame AAA codons that are followed by either an A or G in the subsequent codon (14). The YoeB toxin inhibits translation at the initiation step, apparently by destabilization of the initiation complex (15). HipA toxin is a kinase whose mechanism of action is not known (16, 17).Although the mechanism of action of many E. coli chromosomal and plasmid-derived toxins has been determined, the precise function of the HigB toxin has not been characterized. The higBA TA module is not present in E. coli K12; it resides on the Rts1 plasmid that typically replicates in Proteus spp. and imparts kanamycin resistance as well as temperature-sensitive post-segregational killing at 42 °C (18, 19). Interestingly, one or more chromosomal counterparts of higBA have been reported for several pathogens, including Vibrio cholerae, Streptococcus pneumoniae, E. coli CFT073, and E. coli O157:H7 (20). Some characterization of the two V. cholerae HigBA modules has been performed. First, one of the two higBA modules was shown to possess the general characteristics of TA systems by demonstration of toxin-antitoxin interaction, module organization/regulation, HigB toxicity, and rescue of toxicity with the cognate HigA antitoxin (21). Overexpression of HigB derived from two individual higBA modules encoded in V. cholerae or from Rts1 leads to inhibition of protein synthesis through translation-dependent mRNA cleavage in a manner similar to, but distinct from, RelE (22).HigB is a member of the RelE family of toxins, including RelE, YafQ, and YoeB (20). In this study, we have identified the precise mode of action of HigB from Rts1. HigB associated with the 50 S ribosomal subunit, and this HigB-ribosome complex cleaved within mRNA coding regions at all AAA triplet sequences, both in-frame and out-of-frame. HigB appeared to be responsible for the mRNA cleavage activity of the HigB-ribosome complex because a HigB H92Q mutant lacked mRNA cleavage activity but remained associated with the ribosome. Finally, the cleavage specificity of HigB on plasmid Rts1 coincided with the sequence (AAA, lysine) of either the most abundant or the second most abundant codon in its Proteus host.     

Diversity of Acinetobacter baumannii in Four French Military Hospitals, as Assessed by Multiple Locus Variable Number of Tandem Repeats Analysis

Background

Infections by A. calcoaceticus-A. baumannii (ACB) complex isolates represent a serious threat for wounded and burn patients. Three international multidrug-resistant (MDR) clones (EU clone I-III) are responsible for a large proportion of nosocomial infections with A. baumannii but other emerging strains with high epidemic potential also occur.

Methodology/Principal Findings

We automatized a Multiple locus variable number of tandem repeats (VNTR) analysis (MLVA) protocol and used it to investigate the genetic diversity of 136 ACB isolates from four military hospitals and one childrens hospital. Acinetobacter sp other than baumannii isolates represented 22.6% (31/137) with a majority being A. pittii. The genotyping protocol designed for A.baumannii was also efficient to cluster A. pittii isolates. Fifty-five percent of A. baumannii isolates belonged to the two international clones I and II, and we identified new clones which members were found in the different hospitals. Analysis of two CRISPR-cas systems helped define two clonal complexes and provided phylogenetic information to help trace back their emergence.

Conclusions/Significance

The increasing occurrence of A. baumannii infections in the hospital calls for measures to rapidly characterize the isolates and identify emerging clones. The automatized MLVA protocol can be the instrument for such surveys. In addition, the investigation of CRISPR/cas systems may give important keys to understand the evolution of some highly successful clonal complexes.     

Isolation and Characterization of Antimicrobial Compounds in Plant Extracts against Multidrug-Resistant Acinetobacter baumannii

The number of fully active antibiotic options that treat nosocomial infections due to multidrug-resistant Acinetobacter baumannii (A. baumannii) is extremely limited. Magnolia officinalis, Mahonia bealei, Rabdosia rubescens, Rosa rugosa, Rubus chingii, Scutellaria baicalensis, and Terminalia chebula plant extracts were previously shown to have growth inhibitory activity against a multidrug-resistant clinical strain of A. baumannii. In this study, the compounds responsible for their antimicrobial activity were identified by fractionating each plant extract using high performance liquid chromatography, and determining the antimicrobial activity of each fraction against A. baumannii. The chemical structures of the fractions inhibiting >40% of the bacterial growth were elucidated by liquid chromatography/mass spectrometry analysis and nuclear magnetic resonance spectroscopy. The six most active compounds were identified as: ellagic acid in Rosa rugosa; norwogonin in Scutellaria baicalensis; and chebulagic acid, chebulinic acid, corilagin, and terchebulin in Terminalia chebula. The most potent compound was identified as norwogonin with a minimum inhibitory concentration of 128 µg/mL, and minimum bactericidal concentration of 256 µg/mL against clinically relevant strains of A. baumannii. Combination studies of norwogonin with ten anti-Gram negative bacterial agents demonstrated that norwogonin did not enhance the antimicrobial activity of the synthetic antibiotics chosen for this study. In conclusion, of all identified antimicrobial compounds, norwogonin was the most potent against multidrug-resistant A. baumannii strains. Further studies are warranted to ascertain the prophylactic and therapeutic potential of norwogonin for infections due to multidrug-resistant A. baumannii.     

Identification of Novel Vaccine Candidates against Multidrug-Resistant Acinetobacter baumannii

Acinetobacter baumannii is an emerging opportunistic bacterium associated with nosocomial infections in intensive care units. The alarming increase in infections caused by A. baumannii is strongly associated with enhanced resistance to antibiotics, in particular carbapenems. This, together with the lack of a licensed vaccine, has translated into significant economic, logistic and health impacts to health care facilities. In this study, we combined reverse vaccinology and proteomics to identify surface-exposed and secreted antigens from A. baumannii. Using in silico prediction tools and comparative genome analysis in combination with in vitro proteomic approaches, we identified 42 antigens that could be used as potential vaccine targets. Considering the paucity of effective antibiotics available to treat multidrug-resistant A. baumannii infections, these vaccine targets may serve as a framework for the development of a broadly protective multi-component vaccine, an outcome that would have a major impact on the burden of A. baumannii infections in intensive care units across the globe.     

Comparative analysis of surface-exposed virulence factors of Acinetobacter baumannii

Background

Acinetobacter baumannii is a significant hospital pathogen, particularly due to the dissemination of highly multidrug resistant isolates. Genome data have revealed that A. baumannii is highly genetically diverse, which correlates with major variations seen at the phenotypic level. Thus far, comparative genomic studies have been aimed at identifying resistance determinants in A. baumannii. In this study, we extend and expand on these analyses to gain greater insight into the virulence factors across eight A. baumannii strains which are clonally, temporally and geographically distinct, and includes an isolate considered non-pathogenic and a community-acquired A. baumannii.

Results

We have identified a large number of genes in the A. baumannii genomes that are known to play a role in virulence in other pathogens, such as the recently studied proline-alanine-alanine-arginine (PAAR)-repeat domains of the type VI secretion systems. Not surprising, many virulence candidates appear to be part of the A. baumannii core genome of virulent isolates but were often found to be insertionally disrupted in the avirulent A. baumannii strain SDF. Our study also reveals that many known or putative virulence determinants are restricted to specific clonal lineages, which suggests that these virulence determinants may be crucial for the success of these widespread common clones. It has previously been suggested that the high level of intrinsic and adaptive resistance has enabled the widespread presence of A. baumannii in the hospital environment. This appears to have facilitated the expansion of its repertoire of virulence traits, as in general, the nosocomial strains in this study possess more virulence genes compared to the community-acquired isolate.

Conclusions

Major genetic variation in known or putative virulence factors was seen across the eight strains included in this study, suggesting that virulence mechanisms are complex and multifaceted in A. baumannii. Overall, these analyses increase our understanding of A. baumannii pathogenicity and will assist in future studies determining the significance of virulence factors within clonal lineages and/or across the species.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1020) contains supplementary material, which is available to authorized users.     

Complete Nucleotide Sequences of Virulence-Resistance Plasmids Carried by Emerging Multidrug-Resistant Salmonella enterica Serovar Typhimurium Isolated from Cattle in Hokkaido,Japan

In the present study, we have shown that virulence-resistance plasmids from emerging multidrug-resistant isolates of Salmonella enterica serovar Typhimurium were derived from a virulence-associated plasmid, essential for systematic invasiveness of S. Typhimurium in mice (pSLT), through acquisition of a large insert containing a resistance island flanked by IS1294 elements. A bla _CMY-2-carrying plasmid from a cefotaxime-resistant isolate comprised a segment of Escherichia coli plasmid pAR060302 and the replication region (IncFIB) of a virulence-resistance plasmid. These results provide insights into the evolution of drug resistance in emerging clones of S. Typhimurium.     

Toxin-Antitoxin Systems on the Large Defense Plasmid pSYSA of Synechocystis sp. PCC 6803

Bacterial toxin-antitoxin (TA) systems are genetic elements, which are encoded by plasmid as well as chromosomal loci and mediate plasmid and genomic island maintenance through post-segregational killing mechanisms. TA systems exist in surprisingly high numbers in all prokaryotes, but cyanobacterial TA systems have been only very poorly experimentally characterized so far. Cyanobacteria are the only prokaryotes that perform oxygenic photosynthesis. As such, cyanobacteria are of high ecological importance and are considered promising for the production of biofuels. Here, we present the molecular characterization of the sll7003/ssl7004 TA system encoded on plasmid pSYSA of the model cyanobacterium Synechocystis sp. PCC 6803 as involving a Mg²⁺-dependent RNA endonuclease activity targeting single-stranded RNA regions and demonstrate the functionality of four more TA systems encoded on this 100,749-bp plasmid. Furthermore, one additional type I, one additional type II, and three freestanding TA system components are predicted on pSYSA, all of which appear active judged by their expression. By harboring at least seven simultaneously active TA systems, pSYSA appears as the plasmid most strongly selected for among all plasmids studied in this respect thus far. These results point to a high biological relevance of pSYSA, whose coding capacity is 75% devoted to three distinct clustered regularly interspaced short palindromic repeats (CRISPR) systems mediating antiviral defense.     

Genome Sequences of Two Multidrug-Resistant Acinetobacter baumannii Strains Isolated from a Patient before and after Treatment with Tigecycline

Acinetobacter baumannii is a Gram-negative bacterium which emerged as a significant nosocomial pathogen worldwide. To investigate the molecular basis of the tigecycline-resistant mechanism, we determined the genome sequences of two multidrug-resistant A. baumannii strains isolated from a patient before and after treatment with tigecycline.     

Genome Sequence of Acinetobacter baumannii AC12, a Polymyxin-Resistant Strain Isolated from Terengganu,Malaysia

Acinetobacter baumannii is a major cause of nosocomial infection worldwide. We report the draft genome sequence of A. baumannii AC12, a multidrug-resistant nosocomial strain with additional resistance to carbapenems and polymyxin. The genome data will provide insights into the genetic basis of antimicrobial resistance and its adaptive mechanism.     

The endolysin of the Acinetobacter baumannii phage vB_AbaP_D2 shows broad antibacterial activity

The emergence and rapid spread of multidrug-resistant bacteria has induced intense research for novel therapeutic approaches. In this study, the Acinetobacter baumannii bacteriophage D2 (vB_AbaP_D2) was isolated, characterized and sequenced. The endolysin of bacteriophage D2, namely Abtn-4, contains an amphipathic helix and was found to have activity against multidrug-resistant Gram-negative strains. By more than 3 log units, A. baumannii were killed by Abtn-4 (5 µM) in 2 h. In absence of outer membrane permeabilizers, Abtn-4 exhibited broad antimicrobial activity against several Gram-positive and Gram-negative bacteria, such as Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumonia, Enterococcus and Salmonella. Furthermore, Abtn-4 had the ability to reduce biofilm formation. Interestingly, Abtn-4 showed antimicrobial activity against phage-resistant bacterial mutants. Based on these results, endolysin Abtn-4 may be a promising candidate therapeutic agent for multidrug-resistant bacterial infections.     

Exploration of piperazine-derived thioureas as antibacterial and anti-inflammatory agents. In vitro evaluation against clinical isolates of colistin-resistant Acinetobacter baumannii

A. baumannii is one of the most important multidrug-resistant microorganisms in hospital units. It is resistant to many classes of antibiotics and the development of new therapeutic strategies is necessary. The aim of this study was to evaluate the antibacterial activity of a set of piperazine-derived thioureas against 13 clinical strains of colistin-resistant A. baumannii. Six derivatives were identified to inhibit bacterial growth of 46% of the A. baumannii strains at low micromolar concentrations (Minimum Inhibitory Concentration from 1.56 to 6.25 μM). A common structural feature in most active compounds was the presence of a 3,5-bis-trifluoromethyl phenyl ring at the thiourea function. In addition, the ability of the compounds to inhibit production of nitric oxide (NO) was examined in RAW 264.7 murine macrophages, highlighting the potential of piperazine-derived thioureas as promising scaffolds for the design of new combined anti-bacterial/anti-inflammatory agents.