Two decades of blaVIM-2-producing Pseudomonas aeruginosa dissemination: an interplay between mobile genetic elements and successful clones

Information on clonal lineages and genetic platforms involved in the mobilization of carbapenemases between Pseudomonas aeruginosa strains in Portugal is scarce. Here, we outline the genetic drivers contributing to the occurrence of blaVIM-2-producing P. aeruginosa over two decades.



A collection of carbapenem-resistant P. aeruginosa clinical isolates (n = 263, 1995-2014) was screened for carbapenemase production by Blue-Carba and PCR. Antimicrobial susceptibility testing was performed according to EUCAST and clonal analysis by MLST. Nine isolates representing different integrons and STs were selected for WGS, followed by bioinformatics.



Twenty-seven blaVIM-2-producing P. aeruginosa belonging to 10 STs were identified, with ST179 and ST111 being the most prevalent and persistent clones. blaVIM-2 was associated with seven class I integrons frequently co-harbouring aminoglycoside resistance genes. In58 was commonly identified, followed by derivatives and In100. blaVIM-2-harbouring transposons of the Tn3 and Tn402 families were linked to different plasmids or integrative conjugative elements of the clc family.



The dissemination of blaVIM-2 carrying integrons is associated with a complex interplay between different mobile genetic elements, including the overlooked integrative conjugative elements, and successful spread of particular clones.

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title: Epidemiology and carbapenem resistance mechanisms of carbapenem-non-susceptible Pseudomonas aeruginosa collected during 2009-11 in 14 European and Mediterranean countries.
abstract: To evaluate the genetic relatedness and carbapenem resistance mechanisms among carbapenem-non-susceptible Pseudomonas aeruginosa collected during 2009-11 in 14 European and Mediterranean countries.



Doripenem-non-susceptible (MIC >2 mg/L) isolates were tested for susceptibility to imipenem, meropenem, doripenem, aztreonam, ceftazidime and cefepime with and without phenyl-arginine-β-naphthylamide (PAβN) (efflux inhibitor) and/or cloxacillin (AmpC inhibitor). Carbapenemase screening was performed by PCR and sequencing. Expression of chromosomal ampC, mexA, mexC, mexE and mexX was determined by quantitative real-time PCR using P. aeruginosa PAO1 or a group of susceptible isolates as baseline. Clonality was evaluated by PFGE and multilocus sequence typing.



Among 529 (25.6% overall) carbapenem-non-susceptible P. aeruginosa, 106 were positive for metallo-β-lactamase (MβL) genes encoding VIM-2 (76 strains), VIM-4 (14), VIM-1 (7) and VIM-5 (5). IMP-15 and three new MβLs (IMP-33, VIM-36 and VIM-37) were detected in one strain each. An increasing prevalence of MβL producers was noted in 2011 (30.6%) compared with previous years (13.4% and 12.3% in 2009 and 2010, respectively). Isolates displayed high genetic diversity, with 401 unique profiles detected. CC235 and ST111 were detected among MβL-producing clusters. The PAβN/cloxacillin effect ranged from 90.0% to 56.5%/from 1.3% to 21.2%. OprD decrease/loss was the most prevalent intrinsic mechanism and was detected among 94.9% of the P. aeruginosa, followed by AmpC (44.4%) and MexAB-OprM (20.1%). When using the susceptible group of isolates as baseline, MexAB-OprM became as prevalent as OprD decrease/loss.



Increasing MβL prevalence is worrisome in various European countries; however, intrinsic resistance mechanisms in a highly genetically diverse population of carbapenem-non-susceptible P. aeruginosa are probably a matter for greater concern in these countries.

title: The increasing threat of Pseudomonas aeruginosa high-risk clones.
abstract: The increasing prevalence of chronic and hospital-acquired infections produced by multidrug-resistant (MDR) or extensively drug-resistant (XDR) Pseudomonas aeruginosa strains is associated with significant morbidity and mortality. This growing threat results from the extraordinary capacity of this pathogen for developing resistance through chromosomal mutations and from the increasing prevalence of transferable resistance determinants, particularly those encoding carbapenemases or extended-spectrum β-lactamases (ESBLs). P. aeruginosa has a nonclonal epidemic population structure, composed of a limited number of widespread clones which are selected from a background of a large quantity of rare and unrelated genotypes that are recombining at high frequency. Indeed, recent concerning reports have provided evidence of the existence of MDR/XDR global clones, denominated high-risk clones, disseminated in hospitals worldwide; ST235, ST111, and ST175 are likely those more widespread. Noteworthy, the vast majority of infections by MDR, and specially XDR, strains are produced by these and few other clones worldwide. Moreover, the association of high-risk clones, particularly ST235, with transferable resistance is overwhelming; nearly 100 different horizontally-acquired resistance elements and up to 39 different acquired β-lactamases have been reported so far among ST235 isolates. Likewise, MDR internationally-disseminated epidemic strains, such as the Liverpool Epidemic Strain (LES, ST146), have been noted as well among cystic fibrosis patients. Here we review the population structure, epidemiology, antimicrobial resistance mechanisms and virulence of the P. aeruginosa high-risk clones. The phenotypic and genetic factors potentially driving the success of high-risk clones, the aspects related to their detection in the clinical microbiology laboratory and the implications for infection control and public health are also discussed.

title: Dominance of international 'high-risk clones' among metallo-β-lactamase-producing Pseudomonas aeruginosa in the UK.
abstract: Carbapenem-resistant isolates of Pseudomonas aeruginosa producing metallo-β-lactamases (MBLs) are increasingly reported worldwide and often belong to particular 'high-risk clones'. This study aimed to characterize a comprehensive collection of MBL-producing P. aeruginosa isolates referred to the UK national reference laboratory from multiple UK laboratories over a 10 year period.



Isolates were referred to the UK national reference laboratory between 2003 and 2012 for investigation of resistance mechanisms and/or outbreaks. MBL genes were detected by PCR. Typing was carried out by nine-locus variable-number tandem repeat (VNTR) analysis and MLST.



MBL-producing P. aeruginosa isolates were referred from 267 source patients and 89 UK laboratories. The most common isolation sites were urine (24%), respiratory (18%), wounds (17%) and blood (13%). VIM-type MBLs predominated (91% of all MBLs found), but a few IMP- and NDM-type enzymes were also identified. Diverse VNTR types were seen, but 86% of isolates belonged to six major complexes. MLST of representative isolates from each complex showed that they corresponded to STs 111, 233, 235, 357, 654 and 773, respectively. Isolates belonging to these complexes were received from between 9 and 25 UK referring laboratories each.



The incidence of MBL-producing P. aeruginosa is increasing in the UK. The majority of these isolates belong to several 'high-risk clones', which have been previously reported internationally as host clones of MBLs.

title: Federal funding for the study of antimicrobial resistance in nosocomial pathogens: no ESKAPE.
abstract: The discovery of potent and safe antimicrobial agents is arguably the single greatest health care advance in history. The availability of these agents rapidly reduced the morbidity and mortality associated with a host of formerly fatal diseases. In addition, the confidence that infections could be prevented or treated by antibiotics allowed major leaps forward in the treatment of noninfectious diseases, including serious heart disease, cancers, and organ failure requiring transplants. Medical care, as we now know it, could not exist without the availability of effective antibiotics. The widespread use of antibiotics has been associated with what we now know to be the predictable emergence of resistance. Early confidence that infections would eventually be conquered has given way to a greater appreciation of the genetic flexibility of common human pathogens. Moreover, we have come to appreciate the role played by microorganisms in our homeostasis. Microorganisms are an intrinsic part of us, and we would do well to learn to live with them. Where we cannot live with them is in the hospital, because patients with compromised defenses are particularly vulnerable to bacterial diseases. Although many bacteria remain susceptible to most of our antimicrobial agents, a coterie has emerged that escape the lethal action of antibiotics. In hospitals in both the developed and the developing world, this small group Enterococcus faeciumy Staphylococcus aureusy Klebsiella pneumoniaey Acinetobacter baumanni, Pseudomonas aeruginosa, and Enterobacter species, hereafter referred to as "the ESKAPE bugs" is the same. The ESKAPE bugs are extraordinarily important, not only because they cause the lion's share of nosocomial infections but also because they represent paradigms of pathogenesis, transmission, and resistance. If we learn to control these microorganisms, our hospitals will be immeasurably safer, because the lessons learned could be applied to virtually any species that attempts to take their place. Unfortunately, the ESKAPE bugs are increasingly prevalent in our hospitals and increasingly resistant to many of our antimicrobial agents. In this issue of the Journal Peters et al.  detail the research agenda of the National Institute of Allergy and Infectious Diseases (NIAID) for antimicrobial resistance. As the primary federal agency for conducting and supporting medical research, the National Institutes of Health (NIH) is the standard-bearer for the federal government's commitment to health research. NIAID manages most, but certainly not all, of the work performed by the NIH in the areas of antimicrobial resistance and infectious diseases. As such, the NIAID agenda defines the weight of federal government efforts in the area of infectious diseases. One need look no further than the pivotal role played by NIAID in the enormous success of the AIDS research effort over the past 2 decades to understand the profound impact this institute's agenda can have on the growth and success of individual research areas. Peters et al. indicate that NIAID funding of antimicrobial research has grown considerably over the past decade, now totaling more than $800 million annually. In considering this very large number, it is important to realize that it represents NIAID's total commitment to all areas defined as being related to antimicrobial therapy. This category includes research on antibacterial, antifungal, antiparasitic, and antiviral therapies, whether related to the treatment of diseases or to their prevention through the use of vaccines. It is therefore difficult to get a firm grip on what level of support is devoted to antibacterial therapy and resistance, particularly in reference to the ESKAPE bugs. Regarding research specific to issues involving antimicrobial resistance, Peters Received 26 December 2007; accepted 3 January 2008; electronically published 7 March 2008. Potential conflicts of interest: none reported. Reprints or correspondence: Dr. Louis Rice, Medical Service 111(W), Louis Stokes Cleveland VA Medical Center.  East Blvd.. Cleveland, OH 44106 (louis.rice@va.gov).

title: Conjugative and mobilizable genomic islands in bacteria: evolution and diversity.
abstract: Horizontal transfer of genomic islands (GEIs), that is, chromosomal regions encoding functions that can be advantageous for the host, plays a key role in bacterial evolution, but their mechanisms of transfer remained elusive for a long time. Recent data suggest that numerous GEIs belong to noncanonical classes of mobile genetic elements (MGEs) that can transfer by conjugation. Among them, the integrative and conjugative elements encode their own excision, conjugative transfer, and integration, whereas the integrative mobilizable elements are autonomous for excision and integration but require the conjugation machinery of helper elements to transfer. Others can self-transfer but require the recombination machinery of the recipient cell to integrate. All these MGEs evolve by acquisition, deletion, or exchange of modules, that is, groups of genes involved in the same function. Moreover, composite GEIs can result from the insertion of a MGE within another or from the site-specific integration of an incoming MGE into one of the recombination sites flanking a resident GEI (tandem accretion). Tandem accretion enables the cis-conjugative mobilization of highly degenerated and nonautonomous GEIs, the cis-mobilizable elements. All these mechanisms contribute to the plasticity and complex evolution of GEIs and explain the highly diverse tableau revealed by more and more genome comparisons.

title: Integrative and Conjugative Elements (ICEs): What They Do and How They Work.
abstract: Horizontal gene transfer plays a major role in microbial evolution, allowing microbes to acquire new genes and phenotypes. Integrative and conjugative elements (ICEs, a.k.a. conjugative transposons) are modular mobile genetic elements integrated into a host genome and are passively propagated during chromosomal replication and cell division. Induction of ICE gene expression leads to excision, production of the conserved conjugation machinery (a type IV secretion system), and the potential to transfer DNA to appropriate recipients. ICEs typically contain cargo genes that are not usually related to the ICE life cycle and that confer phenotypes to host cells. We summarize the life cycle and discovery of ICEs, some of the regulatory mechanisms, and how the types of cargo have influenced our view of ICEs. We discuss how ICEs can acquire new cargo genes and describe challenges to the field and various perspectives on ICE biology.

title: Integrative and conjugative elements: mosaic mobile genetic elements enabling dynamic lateral gene flow
abstract: Integrative and conjugative elements (ICEs) are a diverse group of mobile genetic elements found in both Gram-positive and Gram-negative bacteria. These elements primarily reside in a host chromosome but retain the ability to excise and to transfer by conjugation. Although ICEs use a range of mechanisms to promote their core functions of integration, excision, transfer and regulation, there are common features that unify the group. This Review compares and contrasts the core functions for some of the well-studied ICEs and discusses them in the broader context of mobile-element and genome evolution.

title: Detecting genomic islands using bioinformatics approaches
abstract: Bacterial genomes contain clusters of genes that are acquired by horizontal transfer, called genomic islands (GIs). GIs are frequently associated with microbial adaptations that are of medical and environmental interest, and they have had a substantial impact on bacterial evolution. Therefore, there is growing interest in efficiently identifying GIs in newly sequenced bacterial genomes. Several computational methods for detecting GIs have been developed recently, presenting researchers with a myriad of choices. Here, we discuss the limitations and benefits of the main approaches that are available and present guidelines to aid researchers in effectively identifying these important genomic regions.

title: The Accessory Genome of Pseudomonas aeruginosa
abstract: Pseudomonas aeruginosa strains exhibit significant variability in pathogenicity and ecological flexibility. Such interstrain differences reflect the dynamic nature of the P. aeruginosa genome, which is composed of a relatively invariable “core genome” and a highly variable “accessory genome.” Here we review the major classes of genetic elements comprising the P. aeruginosa accessory genome and highlight emerging themes in the acquisition and functional importance of these elements. Although the precise phenotypes endowed by the majority of the P. aeruginosa accessory genome have yet to be determined, rapid progress is being made, and a clearer understanding of the role of the P. aeruginosa accessory genome in ecology and infection is emerging.

title: Multiresistant Gram-negative bacteria: the role of high-risk clones in the dissemination of antibiotic resistance.
abstract: Multilocus sequence typing reveals that many bacterial species have a clonal structure and that some clones are widespread. This underlying phylogeny was not revealed by pulsed-field gel electrophoresis, a method better suited to short-term outbreak investigation. Some global clones are multiresistant and it is easy to assume that these have disseminated from single foci. Such conclusions need caution, however, unless there is a clear epidemiological trail, as with KPC carbapenemase-positive Klebsiella pneumoniae ST258 from Greece to northwest Europe. Elsewhere, established clones may have repeatedly and independently acquired resistance. Thus, the global ST131 Escherichia coli clone most often has CTX-M-15 extended-spectrum β-lactamase (ESBL), but also occurs without ESBLs and as a host of many other ESBL types. We explore this interaction of clone and resistance for E. coli, K. pneumoniae, Acinetobacter baumannii- a species where three global lineages dominate--and Pseudomonas aeruginosa, which shows clonal diversity, but includes the relatively 'tight' serotype O12/Burst Group 4 cluster that has proved adept at acquiring resistances--from PSE-1 to VIM-1 β-lactamases--for over 20 years. In summary, 'high-risk clones' play a major role in the spread of resistance, with the risk lying in their tenacity--deriving from poorly understood survival traits--and a flexible ability to accumulate and switch resistance, rather than to constant resistance batteries.

title: Population Structure of Pseudomonas aeruginosa from Five Mediterranean Countries: Evidence for Frequent Recombination and Epidemic Occurrence of CC235
abstract: Several studies in recent years have provided evidence that Pseudomonas aeruginosa has a non-clonal population structure punctuated by highly successful epidemic clones or clonal complexes. The role of recombination in the diversification of P. aeruginosa clones has been suggested, but not yet demonstrated using multi-locus sequence typing (MLST). Isolates of P. aeruginosa from five Mediterranean countries (n = 141) were subjected to pulsed-field gel electrophoresis (PFGE), serotyping and PCR targeting the virulence genes exoS and exoU. The occurrence of multi-resistance (≥3 antipseudomonal drugs) was analyzed with disk diffusion according to EUCAST. MLST was performed on a subset of strains (n = 110) most of them had a distinct PFGE variant. MLST data were analyzed with Bionumerics 6.0, using minimal spanning tree (MST) as well as eBURST. Measurement of clonality was assessed by the standardized index of association (IA S). Evidence of recombination was estimated by ClonalFrame as well as SplitsTree4.0. The MST analysis connected 70 sequence types, among which ST235 was by far the most common. ST235 was very frequently associated with the O11 serotype, and frequently displayed multi-resistance and the virulence genotype exoS −/exoU +. ClonalFrame linked several groups previously identified by eBURST and MST, and provided insight to the evolutionary events occurring in the population; the recombination/mutation ratio was found to be 8.4. A Neighbor-Net analysis based on the concatenated sequences revealed a complex network, providing evidence of frequent recombination. The index of association when all the strains were considered indicated a freely recombining population. P. aeruginosa isolates from the Mediterranean countries display an epidemic population structure, particularly dominated by ST235-O11, which has earlier also been coupled to the spread of ß-lactamases in many countries.

title: Dissemination of High-Risk Clones of Extensively Drug-Resistant Pseudomonas aeruginosa in Colombia
abstract: The ability of Pseudomonas aeruginosa to develop resistance to most antimicrobials represents an important clinical threat worldwide. We report the dissemination in several Colombian hospitals of two predominant lineages of extensively drug-resistant (XDR) carbapenemase-producing P. aeruginosa strains. These lineages belong to the high-risk clones sequence type 111 (ST111) and ST235 and harbor blaVIM-2 on a class 1 integron and blaKPC-2 on a Tn4401 transposon, respectively. Additionally, P. aeruginosa ST1492, a novel single-locus variant of ST111, was identified. Clonal dissemination and the presence of mobile genetic elements likely explain the successful spread of XDR P. aeruginosa strains in Colombia.

title: Characterization of the pJB12 Plasmid from Pseudomonas aeruginosa Reveals Tn6352, a Novel Putative Transposon Associated with Mobilization of the blaVIM-2-Harboring In58 Integron
abstract: The blaVIM-2-carrying In58 integron has been linked to a chromosomal location in different bacterial species, including Pseudomonas aeruginosa. This work reports the first fully sequenced In58-harboring plasmid, which is significantly different from the two previously identified blaVIM-2-carrying plasmids in P. aeruginosa. blaVIM-2 might have been acquired by transposition of Tn6352, a novel transposon composed of the In58 and ISPa17 elements. The recognition of similar inverted repeat (IR) sites by ISPa17 reveals a common mobilization process associated with acquisition of the blaVIM-2 and blaVIM-1 genes.

title: The complete nucleotide sequence of an IncP-2 megaplasmid unveils a mosaic architecture comprising a putative novel blaVIM-2-harbouring transposon in Pseudomonas aeruginosa
abstract: In Pseudomonas aeruginosa , bla VIM-2 has been mostly associated with a chromosomal location and rarely with a plasmid backbone. Until now, only three complete bla VIM-2 -carrying plasmid sequences have been described in this species. Here we explore the modular structure of pJB37, the first bla VIM-2 -carrying megaplasmid described in P. aeruginosa .



The complete nucleotide sequence of plasmid pJB37 was determined with an Illumina HiSeq, with de novo assembly by SPAdes, annotation by RAST and searching for antimicrobial resistance genes and virulence factors. Conjugation assays were conducted.



Megaplasmid pJB37 (464 804 bp long and GC content of 57.2%) comprised: an IncP-2 repA-oriV-parAB region; a conjugative transfer region ( traF , traG , virD2 and trbBCDEJLFGI genes); Tn 6356 , a new putative bla VIM-2 -carrying transposon; heavy metal (mercury and tellurite) resistance operons; and an arsenal of virulence genes. Plasmid pJB37 was transferable by conjugation to a spontaneous rifampicin-resistant mutant of P. aeruginosa PAO1. Here, a bla VIM-2 -harbouring In58 integron was associated with a new complex transposable structure, herein named Tn 6356 , suggesting that In58 was most likely acquired by insertion of this element.



The mosaic arrangement exhibited by the pJB37 IncP-2 megaplasmid, which highlights the vast assembly potential of distinct genetic elements in a Pseudomonas widespread plasmid platform, gives new insights into bacterial adaptation and evolution.

title: Characterization of a new genetic environment associated with GES-6 carbapenemase from a Pseudomonas aeruginosa isolate belonging to the high-risk clone ST235.
abstract: Sir, Pseudomonas aeruginosa is an opportunistic pathogen commonly associated with nosocomial infections. The spread of P. aeruginosa strains resistant to carbapenems constitutes an emerging threat worldwide. The GES family of enzymes has been identified in several Gram-negative bacteria, such as P. aeruginosa, Klebsiella pneumoniae and Escherichia coli, being responsible for an extended resistance spectrum to several b-lactams. To date, according to the Lahey Clinic web site (http://www.lahey.org/Studies/), 24 variants of GES have been identified with some presenting carbapenemase activity, which is a matter of great concern. Few GES variants have been identified in Portugal, all of them found in Enterobacteriaceae. In Portugal, carbapenemases associated with P. aeruginosa include VIM-2 (the most frequent) and also IMP-5. Here, we report the first known worldwide description of the blaGES-6 gene in P. aeruginosa strains associated with a new genetic environment and also the first description of GES-producing P. aeruginosa in Portugal. In June 2012, a carbapenem-resistant P. aeruginosa isolate was recovered from bronchial secretions of a patient with pneumonia admitted to the intensive care unit of a Portuguese hospital. Antibiotic susceptibility assessed by standard disc diffusion, agar dilution and Etest methods demonstrated that the isolate was XDR, being non-susceptible to imipenem (MIC .32 mg/L), meropenem (MIC .32 mg/L), ceftazidime, cefepime, aztreonam, piperacillin/tazobactam, gentamicin, netilmicin, amikacin, tobramycin and ciprofloxacin, but susceptible to colistin (MIC1⁄42 mg/L). Carbapenemase production was confirmed by UV spectrophotometric assay and Blue-Carba. Screening of bla genes encoding metallo-b-lactamases (MBLs) gave negative results. The occurrence of ESBLs (blaBEL, blaVEB, blaPER and blaGES) was searched by PCR, revealing the presence of blaGES, which by sequencing was identified as blaGES-6. The nucleotide sequence differed from blaGES-6 (GenBank accession no. AY494718) by only one nucleotide (G instead of A at position 54), corresponding to a silent mutation. GES-6 is a class A b-lactamase with carbapenemase activity, differing from GES-1 by two amino acid substitutions (E104K and G170S), that was only described previously in Greece in a K. pneumoniae clinical isolate with reduced susceptibility to carbapenems. Association of blaGES-6 with class I integrons was performed by PCR and sequencing. A new class I integron structure of 5000 bp named In1076 by INTEGRALL (http://integrall.bio.ua.pt/) revealed an array of two gene cassettes attached to an IS  GenBank accession no. KM210290), with the blaGES-6 gene in the first position. The start codon (ATG) of blaGES-6 was preceded by two putative promoter regions [with a strong promoter, Pc; 235 (TTGACA) and 210 (TAAACT)] and a P2 promoter in its inactive form. An aacA7 gene encoding a type I aminoglycoside acetyltransferase [AAC(6′)-II], which confers resistance to amikacin, netilmicin and tobramycin, was found immediately downstream of the blaGES-6 gene. The 59 base element of the aacA7 gene cassette was interrupted at position 13 by a novel IS of 1543 bp, belonging to the IS110 family, IS1111 group, named ISPa55 and deposited in the IS finder database (https://www-is.biotoul.fr/) . Members of this group do not originate direct flanking target repeats and frequently encode a single ORF flanked by relatively long non-coding sequences (ISPa55 presented 78 and 330 bp at the left and right ends, respectively). The subterminal inverted repeats (IRs) (typical of the IS1111 group) were 5′-GAGTAAAAAGGAGACTTCCCG-3′ (left IR) and 5′-CGGGAAGCTCCTTATG-3′ (right IR). ISPa55 encodes a transposase related to the IS110 family, sharing 64% amino acid identity with that of ISPa34 identified in P. aeruginosa (GenBank accession no. ADC38937). The N-terminal regions of transposases of this family and the PIV/Moov family of DNA recombinases share a similar DEDD motif (D11E55D94D97 in ISPa55), suggesting they may use similar catalytic mechanisms. The junction promoter (Pjunc) originated by circularization of ISPa55 [TTGACG (17 bp) TAAAAA] is similar to the consensus promoters for representative IS1111 family members and is expected to increase the expression of the transposase transcript. ISPa55 disrupts the attC site of the aacA7 gene, as occurs with other IS110 family members that can insert themselves by site-specific recombination, which may compromise the excision of the corresponding gene cassettes. The genetic location of the blaGES-6 gene was determined by I-CeuI-PFGE followed by hybridization with probes specific for blaGES-6 and 16S rRNA genes. Both probes hybridized in the same band, revealing that the genetic platform bearing blaGES-6 was chromosomally located, although blaGES genes have been more frequently associated with plasmids. MLST was performed according to the P. aeruginosa database (http://pubmlst.org/paeruginosa/), revealing that the isolate belonged to the worldwide-disseminated high-risk ST235 clone associated with multidrug resistance and disseminated in Portuguese hospitals (J. Botelho, F. Grosso, C. Sousa and L. Peixe, unpublished results). Interestingly, ST235 has been previously associated with GES-1 and GES-5 in Spain. In summary, we present the first known worldwide report of the blaGES-6 carbapenemase gene in P. aeruginosa belonging to

title: Multiniche Screening Reveals the Clinically Relevant Metallo-β-Lactamase VIM-2 in Pseudomonas aeruginosa Far from the Hospital Setting: an Ongoing Dispersion Process?
abstract: A screening study of the presence of metallo-β-lactamases (IMP and VIM types and SPM-1) in isolates from different nonhospital sources was conducted, and it revealed the presence of blaVIM-2, associated with the In58 class 1 integron, in two unrelated Pseudomonas aeruginosa strains from aquatic habitats. The results suggest that the hospital setting was the possible origin of these blaVIM-2-carrying strains.

title: Metallo-beta-lactamase VIM-2 in clinical isolates of Pseudomonas aeruginosa from Portugal.
abstract: Resistance to carbapenems is emerging, and it is a great problem to therapeutics. Three isolates of Pseudomonas aeruginosa from a Portuguese hospital identified in urine and sputum, in 1995, presented a high-level resistance to imipenem (> 32 mg/L). Afterward, one isolate of P. aeruginosa recovered from urine of an ambulatory patient in 1998 showed high resistance to imipenem and meropenem. The resistance to carbapenems in these strains was associated with the production of a class B beta-lactamase, as was demonstrated by imipenem hydrolysis and inhibition by EDTA. Using primers described for bla(IMP) and bla(VIM), the amplification of the latter was observed in all isolates and a VIM-2 metallo-enzyme was identified. The pulsed-field gel electrophoresis (PFGE) patterns of these isolates were indistinguishable, suggesting dissemination to the community of this VIM-2 producer.

title: High-level expression of IMP-5 carbapenemase owing to point mutation in the -35 promoter region of class 1 integron among Pseudomonas aeruginosa clinical isolates.
abstract: Eight strains of Pseudomonas aeruginosa producing IMP-5 carbapenemases were collected from three Portuguese hospitals. All isolates were epidemiologically unrelated. The bla(IMP-5) gene was inserted into a class 1 integron previously reported in Acinetobacter baumannii 65FFC. Expression of the the bla(IMP-5) gene in P. aeruginosa has been shown to be driven by the P(1) promoter [TTGATA] in which the cytosine was replaced by thymine, which caused an increase in transcription of bla(IMP-5) that was confirmed by site-directed mutagenesis. The minimum inhibitory concentrations (MICs) of imipenem for Escherichia coli pGMLA-1 (the recombinant from A. baumannii 65FFC) and E. coli pATG-2 (the recombinant from P. aeruginosa isolates) were 0.5 mg/L and >32 mg/L, respectively. This study reports the spread of a class 1 integron In76 with a new point mutation in the P(1) promoter sequence leading to overproduction of IMP-5.

title: Blue-Carba, an Easy Biochemical Test for Detection of Diverse Carbapenemase Producers Directly from Bacterial Cultures
abstract: Quick, simple, and reliable methods are needed for laboratory detection of carbapenemases that are widely disseminated among Gram-negative bacteria, in order to improve the detection and surveillance of these clinically relevant bacteria in an epidemiological context ([1][1], [2][2]). Recently, a

title: Validation of carbapenemase and extended-spectrum β-lactamase multiplex endpoint PCR assays according to ISO 15189.
abstract: To validate and accredit a set of three multiplex endpoint PCR assays, targeting the most important carbapenemase and minor extended-spectrum β-lactamase (ESBL) resistance genes, according to the international ISO 15189 particular requirements for the quality and competence of medical laboratories.



Specific primers targeting ESBLs and carbapenemases were collected from the literature or designed internally. The multiplex PCRs were validated for sensitivity, specificity, intra- and inter-run reproducibility and accuracy by means of external quality control (EQC) using a collection of 137 characterized and referenced isolates. For each multiplex PCR assay, the presence of an extraction control ruled out false-negative results due to PCR inhibition or extraction faults. Amplicons were separated by capillary electrophoresis (QIAxcel system, Qiagen). The protocols and validation files were reviewed in the setting of an external audit conducted by the Belgian organization for accreditation (BELAC).



Sensitivity, specificity and reproducibility for each targeted gene were 100%. All isolates from the three EQC panels were correctly identified by each PCR assay (accuracy 100%). The validation files were controlled by BELAC, and the PCR protocols were accepted as accredited according to ISO 15189.



Three home-made multiplex PCRs targeting the major carbapenemases and four minor class A ESBL genes encountered in Gram-negative bacteria were accredited according to the ISO 15189 standards. This validation scheme could provide a useful model for laboratories aiming to accredit their own protocols.

title: NCBI prokaryotic genome annotation pipeline
abstract: Recent technological advances have opened unprecedented opportunities for large-scale sequencing and analysis of populations of pathogenic species in disease outbreaks, as well as for large-scale diversity studies aimed at expanding our knowledge across the whole domain of prokaryotes. To meet the challenge of timely interpretation of structure, function and meaning of this vast genetic information, a comprehensive approach to automatic genome annotation is critically needed. In collaboration with Georgia Tech, NCBI has developed a new approach to genome annotation that combines alignment based methods with methods of predicting protein-coding and RNA genes and other functional elements directly from sequence. A new gene finding tool, GeneMarkS+, uses the combined evidence of protein and RNA placement by homology as an initial map of annotation to generate and modify ab initio gene predictions across the whole genome. Thus, the new NCBI's Prokaryotic Genome Annotation Pipeline (PGAP) relies more on sequence similarity when confident comparative data are available, while it relies more on statistical predictions in the absence of external evidence. The pipeline provides a framework for generation and analysis of annotation on the full breadth of prokaryotic taxonomy. For additional information on PGAP see https://www.ncbi.nlm.nih.gov/genome/annotation_prok/ and the NCBI Handbook,

title: SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing
abstract: The lion's share of bacteria in various environments cannot be cloned in the laboratory and thus cannot be sequenced using existing technologies. A major goal of single-cell genomics is to complement gene-centric metagenomic data with whole-genome assemblies of uncultivated organisms. Assembly of single-cell data is challenging because of highly non-uniform read coverage as well as elevated levels of sequencing errors and chimeric reads. We describe SPAdes, a new assembler for both single-cell and standard (multicell) assembly, and demonstrate that it improves on the recently released E+V-SC assembler (specialized for single-cell data) and on popular assemblers Velvet and SoapDeNovo (for multicell data). SPAdes generates single-cell assemblies, providing information about genomes of uncultivatable bacteria that vastly exceeds what may be obtained via traditional metagenomics studies. SPAdes is available online ( http://bioinf.spbau.ru/spades ). It is distributed as open source software.

title: Antibacterial-Resistant Pseudomonas aeruginosa: Clinical Impact and Complex Regulation of Chromosomally Encoded Resistance Mechanisms
abstract: Treatment of infectious diseases becomes more challenging with each passing year. This is especially true for infections caused by the opportunistic pathogen Pseudomonas aeruginosa, with its ability to rapidly develop resistance to multiple classes of antibiotics. Although the import of resistance mechanisms on mobile genetic elements is always a concern, the most difficult challenge we face with P. aeruginosa is its ability to rapidly develop resistance during the course of treating an infection. The chromosomally encoded AmpC cephalosporinase, the outer membrane porin OprD, and the multidrug efflux pumps are particularly relevant to this therapeutic challenge. The discussion presented in this review highlights the clinical significance of these chromosomally encoded resistance mechanisms, as well as the complex mechanisms/pathways by which P. aeruginosa regulates their expression. Although a great deal of knowledge has been gained toward understanding the regulation of AmpC, OprD, and efflux pumps in P. aeruginosa, it is clear that we have much to learn about how this resourceful pathogen coregulates different resistance mechanisms to overcome the antibacterial challenges it faces.

title: Metallo-beta-lactamase-producing Pseudomonas putida as a reservoir of multidrug resistance elements that can be transferred to successful Pseudomonas aeruginosa clones.
abstract: To study the prevalence, nature, involved genetic elements and epidemiology of metallo-beta-lactamase (MBL)-producing Pseudomonas aeruginosa and Pseudomonas putida isolated in a Spanish hospital between 2005 and 2008.



Etests were used for susceptibility testing and screening for MBLs, confirmed through bla(VIM) PCRs and sequencing. Clonal relatedness was evaluated by PFGE and multilocus sequence typing (MLST). MBL-carrying plasmids were characterized by restriction fragment length polymorphism, Southern blot and electroporation. MBL genetic elements were studied by cloning and sequencing.



MBL-producing P. putida was detected in eight patients (one clone each; two harbouring bla(VIM-1) and six harbouring bla(VIM-2)), representing 14% of all the infections by the P. putida/fluorescens group. MBLs were detected in only 0.3% of P. aeruginosa infections (11 patients) during the same period. PFGE revealed four P. aeruginosa clones: one producing bla(VIM-13) (two patients); and three producing bla(VIM-2) (two patients, six patients and one patient, respectively). MLST indicated that the VIM-13 clone was the internationally spread sequence type (ST)235, while the major VIM-2 lineage corresponded to ST179, which is associated with chronic respiratory infections. The VIM-1 integron was shown to have both plasmid and chromosomal location, while the VIM-13 integron was only chromosomal. The VIM-2 integron was located in the same transposon (Tn402/Tn5053-like) in all P. aeruginosa and P. putida isolates, suggesting its crucial role in the dissemination of VIM-2.



The high diversity and proportion of MBL-positive P. putida suggests an environmental reservoir of these resistance determinants. Dissemination of these multidrug resistance elements to successful P. aeruginosa clones presents a major epidemiological and clinical threat.

title: Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Identifies Pseudomonas aeruginosa High-Risk Clones
abstract: We show here that matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) accurately and quickly identified the five high-risk clones of Pseudomonas aeruginosa sequence type 111 (ST111), ST175, ST235, ST253, and ST395. The use of this screening technique by clinical microbiology laboratories may tackle the spread of high-risk clones by the quick implementation of hygiene control procedures for relevant patients.

title: Faecal carriage of Pseudomonas aeruginosa in healthy humans: antimicrobial susceptibility and global genetic lineages.
abstract: The aim of this study was to analyse the Pseudomonas aeruginosa faecal carriage rate in 98 healthy humans and to perform the phenotypic and genotypic characterization of recovered isolates. The genetic relatedness among the isolates was analysed by pulsed-field gel electrophoresis and multilocus sequence typing that was compared with worldwide epidemic clones. Pseudomonas aeruginosa was isolated from eight healthy individuals (8.2%), and two of them remained colonized after 5 months (in one case by the same clone). All 10 isolates (one/sample) were susceptible to 14 tested antipseudomonal agents and lacked integron structures. Six pulsed-field gel electrophoresis patterns and six sequence types (ST245, ST253, ST254, ST274, ST663 and the new one, ST1059) were identified among them. Four groups of OprD alterations were detected based on mutations and deletions related to PAO1 reference strain in our carbapenem-susceptible strains. This is the first study focused on P. aeruginosa from faecal samples of healthy humans that provides additional insights into the antimicrobial resistance and genetic diversity of P. aeruginosa. Although the isolates were antimicrobial susceptible, most of the sequence types detected were genetically related to Spanish epidemic clones or globally spread sequence types, such as ST274 and ST253.

title: Pseudomonas aeruginosa Exhibits Frequent Recombination, but Only a Limited Association between Genotype and Ecological Setting
abstract: Pseudomonas aeruginosa is an opportunistic pathogen and an important cause of infection, particularly amongst cystic fibrosis (CF) patients. While specific strains capable of patient-to-patient transmission are known, many infections appear to be caused by unique and unrelated strains. There is a need to understand the relationship between strains capable of colonising the CF lung and the broader set of P. aeruginosa isolates found in natural environments. Here we report the results of a multilocus sequence typing (MLST)-based study designed to understand the genetic diversity and population structure of an extensive regional sample of P. aeruginosa isolates from South East Queensland, Australia. The analysis is based on 501 P. aeruginosa isolates obtained from environmental, animal and human (CF and non-CF) sources with particular emphasis on isolates from the Lower Brisbane River and isolates from CF patients obtained from the same geographical region. Overall, MLST identified 274 different sequence types, of which 53 were shared between one or more ecological settings. Our analysis revealed a limited association between genotype and environment and evidence of frequent recombination. We also found that genetic diversity of P. aeruginosa in Queensland, Australia was indistinguishable from that of the global P. aeruginosa population. Several CF strains were encountered frequently in multiple ecological settings; however, the most frequently encountered CF strains were confined to CF patients. Overall, our data confirm a non-clonal epidemic structure and indicate that most CF strains are a random sample of the broader P. aeruginosa population. The increased abundance of some CF strains in different geographical regions is a likely product of chance colonisation events followed by adaptation to the CF lung and horizontal transmission among patients.

title: Molecular Epidemiology of Metallo-β-Lactamase-Producing Pseudomonas aeruginosa Isolates from Norway and Sweden Shows Import of International Clones and Local Clonal Expansion
abstract: Scandinavia is considered a region with a low prevalence of antimicrobial resistance. However, the number of multidrug-resistant (MDR) Gram-negative bacteria is increasing, including metallo-β-lactamase (MBL)-producing Pseudomonas aeruginosa. In this study MBL-producing P. aeruginosa isolates identified in Norway (n = 4) and Sweden (n = 9) from 1999 to 2007 were characterized. Two international clonal complexes (CC), CC111 (n = 8) and CC235 (n = 2), previously associated with MBL-producing isolates, were dominant. CC111 isolates (ST111/229; serotype O12; blaVIM-2) included clonally related isolates identified in Skåne County, Sweden (n = 6), and two isolates associated with importation from Greece and Denmark. In all CC111 isolates, blaVIM-2 was located in integron In59.2 or In59 variants. The two CC235 isolates (ST235/ST230; serotype O11; blaVIM-4) were imported from Greece and Cyprus, were possibly clonally related, and carried blaVIM-4 in two different integron structures. Three isolates imported from Ghana (ST233; serotype O6; blaVIM-2), Tunisia (ST654; serotype O11; blaVIM-2), and Thailand (ST260; serotype O6; blaIMP-14) were clonally unrelated. ST233 was part of a new CC (CC233) that included other MBL-producing isolates, while ST654 could also be part of a new CC associated with MBL producers. In the isolates imported from Ghana and Tunisia, blaVIM-2 was part of unusual integron structures lacking the 3′ conserved segment and associated with transposons. The blaVIM gene was found to be located on the chromosome in all isolates. Known risk factors for acquisition of MBL were reported for all patients except one. The findings suggest that both import of successful international clones and local clonal expansion contribute to the emergence of MBL-producing P. aeruginosa in Scandinavia.

title: Detection of blaVIM-7 in an extensively drug-resistant Pseudomonas aeruginosa isolate belonging to ST1284 in Brazil.
abstract: We described for the first time an extensively drug-resistant Pseudomonas aeruginosa isolate belonging to ST1284 carrying a plasmid-mediated blaVIM-7 in Brazil. The blaVIM-7 was harbored by an integron that also carried aacA4 and blaOXA-46. Multiple virulence factors were also detected.

title: Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance.
abstract: Many different definitions for multidrug-resistant (MDR), extensively drug-resistant (XDR) and pandrug-resistant (PDR) bacteria are being used in the medical literature to characterize the different patterns of resistance found in healthcare-associated, antimicrobial-resistant bacteria. A group of international experts came together through a joint initiative by the European Centre for Disease Prevention and Control (ECDC) and the Centers for Disease Control and Prevention (CDC), to create a standardized international terminology with which to describe acquired resistance profiles in Staphylococcus aureus, Enterococcus spp., Enterobacteriaceae (other than Salmonella and Shigella), Pseudomonas aeruginosa and Acinetobacter spp., all bacteria often responsible for healthcare-associated infections and prone to multidrug resistance. Epidemiologically significant antimicrobial categories were constructed for each bacterium. Lists of antimicrobial categories proposed for antimicrobial susceptibility testing were created using documents and breakpoints from the Clinical Laboratory Standards Institute (CLSI), the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the United States Food and Drug Administration (FDA). MDR was defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories, XDR was defined as non-susceptibility to at least one agent in all but two or fewer antimicrobial categories (i.e. bacterial isolates remain susceptible to only one or two categories) and PDR was defined as non-susceptibility to all agents in all antimicrobial categories. To ensure correct application of these definitions, bacterial isolates should be tested against all or nearly all of the antimicrobial agents within the antimicrobial categories and selective reporting and suppression of results should be avoided.

title: Tn5090-like class 1 integron carrying bla(VIM-2) in a Pseudomonas putida strain from Portugal.
abstract: Three Pseudomonas putida strains containing bla(VIM-2) were isolated from an inanimate surface of a female ward sanitary facility in the Hospital Infante D. Pedro, Aveiro. A novel class 1 integron was found in strain Pp2 (aacA4/bla(VIM-2)/aac6'-IIc disrupted by an insertion sequence IS1382), and strain Pp1 was found to carry a class 1 integron (aacA7/bla(VIM-2)/aacC1/aacA4), which is described for the first time in this species. Strain PF1 carries a class 1 integron associated with a Tn5090-like transposon, constituting the first finding of this type of arrangement in a strain from Portugal. This association highlights further dissemination of bla(VIM-2) in environmental hospital isolates.

title: Identical Miniature Inverted Repeat Transposable Elements Flank Class 1 Integrons in Clinical Isolates of Acinetobacter spp
abstract: Miniature inverted repeat transposable elements (MITEs) have been identified flanking class 1 integrons. We have identified and characterized a 439-bp MITE-like structure in seven Acinetobacter species isolates from Portugal and Brazil. The complete sequence similarity of the elements and flanking regions suggests that MITEs may act as mobilizable vectors for the dissemination of integrons.

title: First Isolation of blaVIM-2 in an Environmental Isolate of Pseudomonas pseudoalcaligenes
abstract: Since the first description of VIM-2, in France, in a Pseudomonas aeruginosa clinical isolate ([7][1]), this enzyme has been detected in clinical Pseudomonas isolates in several countries ([6][2]), thus highlighting the need for a detailed knowledge of its spreading dynamics. In Portugal, since the

title: A spontaneous point mutation in the aac(6')-Ib' gene results in altered substrate specificity of aminoglycoside 6'-N-acetyltransferase of a Pseudomonas fluorescens strain.
abstract: The aac(6')-Ib' gene from Pseudomonas fluorescens BM2687, encoding an aminoglycoside 6'-N-acetyltransferase type II which confers resistance to gentamicin but not to amikacin, was characterized. Nucleotide sequence determination indicated total identity between aac(6')-Ib' and the aac(6')-Ib gene from Pseudomonas aeruginosa BM2656 [1] with the exception of a C-to-T transition that results in a serine to leucine substitution at position 83 of the deduced polypeptide. The aac(6')-Ib gene specifies a type I enzyme which confers resistance to amikacin but not to gentamicin . It thus appears that the point mutation detected is responsible for enzymic altered substrate specificity.

title: Mechanisms of stabilization of integrative and conjugative elements.
abstract: Integrative and conjugative elements (ICEs) are nearly ubiquitous in microbial genomes and influence their evolution by providing adaptive functions to their host and by enhancing genome plasticity and diversification. For a long-time, it has been assumed that by integrating into the chromosome of their host, these self-transmissible elements were passively inherited in subsequent generations. Recent findings point to a much more complex story that includes multiple strategies used by ICEs to leverage maintenance in cell populations such as transient replication, active partition of the excised circular intermediate or disassembly into multiple parts scattered in the chromosome. Here I review these diverse mechanisms of stabilization in the general context of ICEs belonging to diverse families.

title: The Repertoire of ICE in Prokaryotes Underscores the Unity, Diversity, and Ubiquity of Conjugation
abstract: Horizontal gene transfer shapes the genomes of prokaryotes by allowing rapid acquisition of novel adaptive functions. Conjugation allows the broadest range and the highest gene transfer input per transfer event. While conjugative plasmids have been studied for decades, the number and diversity of integrative conjugative elements (ICE) in prokaryotes remained unknown. We defined a large set of protein profiles of the conjugation machinery to scan over 1,000 genomes of prokaryotes. We found 682 putative conjugative systems among all major phylogenetic clades and showed that ICEs are the most abundant conjugative elements in prokaryotes. Nearly half of the genomes contain a type IV secretion system (T4SS), with larger genomes encoding more conjugative systems. Surprisingly, almost half of the chromosomal T4SS lack co-localized relaxases and, consequently, might be devoted to protein transport instead of conjugation. This class of elements is preponderant among small genomes, is less commonly associated with integrases, and is rarer in plasmids. ICEs and conjugative plasmids in proteobacteria have different preferences for each type of T4SS, but all types exist in both chromosomes and plasmids. Mobilizable elements outnumber self-conjugative elements in both ICEs and plasmids, which suggests an extensive use of T4SS in trans. Our evolutionary analysis indicates that switch of plasmids to and from ICEs were frequent and that extant elements began to differentiate only relatively recently. According to the present results, ICEs are the most abundant conjugative elements in practically all prokaryotic clades and might be far more frequently domesticated into non-conjugative protein transport systems than previously thought. While conjugative plasmids and ICEs have different means of genomic stabilization, their mechanisms of mobility by conjugation show strikingly conserved patterns, arguing for a unitary view of conjugation in shaping the genomes of prokaryotes by horizontal gene transfer.

title: Full characterization of the integrative and conjugative element carrying the metallo-β-lactamase bla SPM-1 and bicyclomycin bcr1 resistance genes found in the pandemic Pseudomonas aeruginosa clone SP/ST277.
abstract: This study aimed to characterize the genomic context of the bla SPM-1 gene in Brazilian strains belonging to the pandemic Pseudomonas aeruginosa clone SP/ST277.



WGS of clone SP/ST277 strains was performed using a Nextera paired-end library in an Illumina HiSeq 2500 sequencer. bla SPM-1 context was assessed by de novo assembly and gene prediction and annotation tools. bla SPM-1 was screened in P. aeruginosa genomes through BlastN, and comparative genomics were performed.



The metallo-β-lactamase bla SPM-1 has been disseminated by the pandemic Brazilian P. aeruginosa clone SP/ST277. In spite of its association with the CR4 element and with the Tn4371 element, the overall bla SPM-1 genomic context remains uncharacterized and its determination is valuable to understanding gene dispersion dynamics and the consequent emergence of carbapenem resistance. In this study, bla SPM-1 and its surrounding sequences (CR4-groEL-bla SPM-1-CR4-groEL) were found in the variable region of an ICE-like element resembling Tn4371 (where ICE stands for integrative and conjugative element). This element, named ICETn4371 6061, had 46 ORFs, including the bicyclomycin resistance bcr1 gene. An integrase gene and a set of conjugative transfer genes were identified. Gene content and order were shared with other Tn4371-ICEs, presenting remarkable amino acid identities. bla SPM-1 and surrounding sequences were missing in ICETn4371 6061 of PS600-MA, another isolate belonging to clone SP/ST277, indicating their mobilization. Eight/nine P. aeruginosa genomes assigned to clone SP/ST277, by in silico MLST, harboured bla SPM-1 inserted into ICETn4371 6061.



The presence of bla SPM-1 in a Tn4371-ICE with intact integration/conjugation modules demonstrated that, besides gene dispersion by clonal expansion of the pandemic SP/ST277 lineage, bla SPM-1 may be spread through ICE conjugation.

title: Unravelling the genome of a Pseudomonas aeruginosa isolate belonging to the high-risk clone ST235 reveals an integrative conjugative element housing a blaGES-6 carbapenemase
abstract: In Pseudomonas aeruginosa, the blaGES-6 carbapenemase gene was previously associated with an In1076 class I integron. Here, we conducted a genome-based analysis and explored the genetic platform associated with the mobility of this gene.



WGS of a blaGES-6-harbouring P. aeruginosa isolate (FFUP_PS_690) was performed with Illumina HiSeq, de novo assembly was performed using SPAdes and subsequent bioinformatic analysis was performed concerning antibiotic resistance genes, virulence features and mobile genetic elements.



The FFUP_PS_690 isolate belongs to the ST235 high-risk clone and houses a novel integrative conjugative element (ICE), hereby named ICEPae690. This clc-like ICE comprises the blaGES-6-harbouring In1076 integron and specific modules. An ExoU island A variant was also identified.



The presence of a 'hitch-hiking' blaGES-6-harbouring In1076 integron in an ICE and an exoU-carrying genomic island highlight the potential spread of these elements through conjugation and/or clonal expansion of the ST235 lineage.

title: Combinatorial events of insertion sequences and ICE in Gram-negative bacteria.
abstract: The emergence of antibiotic and antimicrobial resistance in Gram-negative bacteria is incremental and linked to genetic elements that function in a so-called 'one-ended transposition' manner, including ISEcp1, ISCR elements and Tn3-like transposons. The power of these elements lies in their inability to consistently recognize one of their own terminal sequences, while recognizing more genetically distant surrogate sequences. This has the effect of mobilizing the DNA sequence found adjacent to their initial location. In general, resistance in Gram-negatives is closely linked to a few one-off events. These include the capture of the class 1 integron by a Tn5090-like transposon; the formation of the 3' conserved segment (3'-CS); and the fusion of the ISCR1 element to the 3'-CS. The structures formed by these rare events have been massively amplified and disseminated in Gram-negative bacteria, but hitherto, are rarely found in Gram-positives. Such events dominate current resistance gene acquisition and are instrumental in the construction of large resistance gene islands on chromosomes and plasmids. Similar combinatorial events appear to have occurred between conjugative plasmids and phages constructing hybrid elements called integrative and conjugative elements or conjugative transposons. These elements are beginning to be closely linked to some of the more powerful resistance mechanisms such as the extended spectrum β-lactamases, metallo- and AmpC type β-lactamases. Antibiotic resistance in Gram-negative bacteria is dominated by unusual combinatorial mistakes of Insertion sequences and gene fusions which have been selected and amplified by antibiotic pressure enabling the formation of extended resistance islands.

title: Revised nomenclature for transposable genetic elements.
abstract: Transposable DNA elements occur naturally in the genomes of nearly all species of prokaryotes. A proposal for a uniform transposable element nomenclature was published prominently in the 1970s but is not, at present, available online even in abstract form, and many of the newly discovered elements have been named without reference to it. We propose here an updated version of the original nomenclature system for all of the various types of prokaryotic, autonomous, transposable elements excluding insertion sequences, for which a nomenclature system already exists. The use of this inclusive and sequential Tn numbering system for transposable elements, as described here, recognizes the ease of interspecies spread of individual elements, and allows for the naming of mosaic elements containing segments from two or more previously described types of transposons or plasmids. It will guard against any future need to rename elements following changes in bacterial nomenclature which occurs constantly with our increased understanding of bacterial phylogenies and taxonomic groupings. It also takes into account the increasing importance of metagenomic sequencing projects and the continued identification of new mobile elements from unknown hosts.

title: Clonal Dissemination of Pseudomonas aeruginosa Sequence Type 235 Isolates Carrying blaIMP-6 and Emergence of blaGES-24 and blaIMP-10 on Novel Genomic Islands PAGI-15 and -16 in South Korea
abstract: A total of 431 Pseudomonas aeruginosa clinical isolates were collected from 29 general hospitals in South Korea in 2015. Antimicrobial susceptibility was tested by the disk diffusion method, and MICs of carbapenems were determined by the agar dilution method. Carbapenemase genes were amplified by PCR and sequenced, and the structures of class 1 integrons surrounding the carbapenemase gene cassettes were analyzed by PCR mapping. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were performed for strain typing. Whole-genome sequencing was carried out to analyze P. aeruginosa genomic islands (PAGIs) carrying the blaIMP-6, blaIMP-10, and blaGES-24 genes. The rates of carbapenem-nonsusceptible and carbapenemase-producing P. aeruginosa isolates were 34.3% (148/431) and 9.5% (41/431), respectively. IMP-6 was the most prevalent carbapenemase type, followed by VIM-2, IMP-10, and GES-24. All carbapenemase genes were located on class 1 integrons of 6 different types on the chromosome. All isolates harboring carbapenemase genes exhibited genetic relatedness by PFGE (similarity > 80%); moreover, all isolates were identified as sequence type 235 (ST235), with the exception of two ST244 isolates by MLST. The blaIMP-6, blaIMP-10, and blaGES-24 genes were found to be located on two novel PAGIs, designated PAGI-15 and PAGI-16. Our data support the clonal spread of an IMP-6-producing P. aeruginosa ST235 strain, and the emergence of IMP-10 and GES-24 demonstrates the diversification of carbapenemases in P. aeruginosa in Korea.

title: Sequence Analysis of the Mobile Genome Island pKLC102 of Pseudomonas aeruginosa C
abstract: The Pseudomonas aeruginosa plasmid pKLC102 coexists as a plasmid and a genome island in clone C strains. Whereas the related plasmid pKLK106 reversibly recombines with P. aeruginosa clone K chromosomes at one of the two tRNALys genes, pKLC102 is incorporated into the tRNALys gene only close to the pilA locus. Targeting of the other tRNALys copy in the chromosome is blocked by a 23,395-bp mosaic of truncated PAO open reading frames, transposons, and pKLC102 homologs. Annotation and phylogenetic analysis of the large 103,532-bp pKLC102 sequence revealed that pKLC102 is a hybrid of plasmid and phage origin. The plasmid lineage conferred oriV and genes for replication, partitioning, and conjugation, including a pil cluster encoding type IV thin sex pili and an 8,524-bp chvB glucan synthetase gene that is known to be a major determinant for host tropism and virulence. The phage lineage conferred integrase, att, and a syntenic set of conserved hypothetical genes also observed in the tRNAGly-associated genome islands of P. aeruginosa clone C chromosomes. In subgroup C isolates from patients with cystic fibrosis, pKLC102 was irreversibly fixed into the chromosome by the insertion of the large 23,061-bp class I transposon TNCP23, which is a composite of plasmid, integron, and IS6100 elements. Intramolecular transposition of a copy of IS6100 led to chromosomal inversions and disruption of plasmid synteny. The case of pKLC102 in P. aeruginosa clone C documents the intraclonal evolution of a genome island from a mobile ancestor via a reversibly integrated state to irreversible incorporation and dissipation in the chromosome.

title: Antibiotic Resistance Determinants in a Pseudomonas putida Strain Isolated from a Hospital
abstract: Environmental microbes harbor an enormous pool of antibiotic and biocide resistance genes that can impact the resistance profiles of animal and human pathogens via horizontal gene transfer. Pseudomonas putida strains are ubiquitous in soil and water but have been seldom isolated from humans. We have established a collection of P. putida strains isolated from in-patients in different hospitals in France. One of the isolated strains (HB3267) kills insects and is resistant to the majority of the antibiotics used in laboratories and hospitals, including aminoglycosides, ß-lactams, cationic peptides, chromoprotein enediyne antibiotics, dihydrofolate reductase inhibitors, fluoroquinolones and quinolones, glycopeptide antibiotics, macrolides, polyketides and sulfonamides. Similar to other P. putida clinical isolates the strain was sensitive to amikacin. To shed light on the broad pattern of antibiotic resistance, which is rarely found in clinical isolates of this species, the genome of this strain was sequenced and analysed. The study revealed that the determinants of multiple resistance are both chromosomally-borne as well as located on the pPC9 plasmid. Further analysis indicated that pPC9 has recruited antibiotic and biocide resistance genes from environmental microorganisms as well as from opportunistic and true human pathogens. The pPC9 plasmid is not self-transmissible, but can be mobilized by other bacterial plasmids making it capable of spreading antibiotic resistant determinants to new hosts.