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Identification of proteins is one of the most computationally intensive steps in genomics studies. It usually relies on aligners that do not accommodate rich information on proteins and require additional pipelining steps for protein identification. We introduce kAAmer, a protein database engine based on amino-acid k-mers that provides efficient identification of proteins while supporting the incorporation of flexible annotations on these proteins. Moreover, the database is built to be used as a microservice, to be hosted and queried remotely.

An article published in Microbiome in July 2018 uses incorrect definitions of integron integrase IntI1 and of class 1 integrons that affect the interpretation of the data.

We agree with Che et al. (1) that understanding how mobile genetic elements (MGEs) spread antimicrobial resistance (AMR) is important. However, decades of research have already characterized a diverse toolbox of MGEs involved in the emergence of AMR, including conjugative plasmids and insertion sequences (ISs), and their interactions. “Mobile” AMR generally arises following rare capture of a chromosomal gene by a particular “intracellularly mobile” MGE, e.g., an IS or a transposon (Tn), and translocation to “intercellularly mobile” MGEs [plasmids, phage, integrative elements (2, 3)]. Tools for systematic analysis of AMR gene−MGE interactions in mounting sequence data are needed, but incorporating existing biological knowledge into their design is vital, as are representative benchmarking datasets and recognition of data biases and resulting limitations. Che et al. screened bacterial plasmid sequences for markers to classify them as nonmobilizable (no relaxase), mobilizable (relaxase), or conjugative (relaxase+). Relying on the absence of a predicted feature is risky, and Plascad’s poor sensitivity in many species was not recognized, due to a taxonomically inadequate benchmark dataset. Numerous conjugative plasmids, and close relatives, from low-GC gram-positive organisms (dataset S1 in ref. 1; e.g., Staphylococcus, pSK41, pWBG4, pWBG749; Clostridium, pCW3, pCP13; Enterococcus/Streptococcus , pAMβ1, pCF10, pRE25, pMG1, pIP501) (3⇓–5 … [↵][1]1To whom correspondence may be addressed. Email: sally.partridge{at}health.nsw.gov.au or neville.firth{at}sydney.edu.au. [1]: #xref-corresp-1-1

A rod-shaped, motile anaerobic bacterium, designated CCRI-22567T, was isolated from a vaginal sample of a woman diagnosed with bacterial vaginosis and subjected to a polyphasic taxonomic study. The novel strain was capable of growth at 30–42 °C (optimum, 42 °C), at pH 5.5–8.5 (optimum, pH 7.0–7.5) and in the presence of 0–1.5 % (w/v) NaCl (optimally at 0.5 % NaCl). The phylogenetic trees based on 16S rRNA gene sequences showed that strain CCRI-22567T forms a distinct evolutionary lineage independent of other taxa in the family Peptostreptococcaceae . Strain CCRI-22567T exhibited 90.1 % 16S rRNA gene sequence similarity to Peptoanaerobacter stomatis ACC19aT and 89.7 % to Eubacterium yurii subsp. schtitka ATCC 43716. The three closest organisms with an available whole genome were compared to strain CCRI-22567T for genomic relatedness assessment. The genomic average nucleotide identities (OrthoANIu) obtained with Peptoanaerobacter stomatis ACC19aT, Eubacterium yurii subsp. margaretiae ATCC 43715 and Filifactor alocis ATCC 35896T were 71.8, 70.3 and 69.6 %, respectively. Strain CCRI-22567T contained C18 : 1 ω9c and C18 : 1 ω9c DMA as the major fatty acids. The DNA G+C content of strain CCRI-22567T based on its genome sequence was 33.8 mol%. On the basis of the phylogenetic, chemotaxonomic and other phenotypic properties, strain CCRI-22567T is considered to represent a new genus and species within the family Peptostreptococcaceae , for which the name Criibacterium bergeronii gen. nov., sp. nov., is proposed. The type strain of Criibacterium bergeronii is CCRI-22567T (=LMG 31278T=DSM 107614T=CCUG 72594T).

Background: With the advent of metagenomics, many large studies have been conducted with the quest of better understanding gut microbiota changes in relation to varying health conditions. Significant findings have been made for diseases such as cirrhosis, colorectal cancers, inflammatory bowel diseases and others, yet one that stands out is obesity for which conflicting results have been reported in the literature. Methods: Here, we built and analyzed a cross-study dataset of healthy and obese individuals looking for major changes in the the taxonomic and functional composition of their metagenomes. Results: Our results suggest that the overweight and normal subjects have no strong dissimilarity in their metagenomes composition. Significant differences were observed when comparing the obese and the non-obese individuals in their functional and taxonomic profiles. Conclusion: In this study, we report the most significant changes that we observed and discuss their potential implication in the obesity condition.

Serratia marcescens SCH909 is a multidrug resistant strain isolated in 1988 harboring three class 1 integrons. We wondered if these integrons were retained over time and if there were other antimicrobial resistant determinants contributing to its multidrug resistant profile. Genomic analysis showed a fourth multidrug resistance integron, a Tn7 transposon with dfrA1-sat2-ybeA-ybfA-ybfB-ybgA gene cassettes in the variable region. Insertion sequences were involved in the genesis of novel composite transposons in the L4 subtype plasmid pSCH909, such as Tn6824 carrying an arsenic regulon and two head to head class 1 integrons surrounded by two complete IS1. Remarkably, a novel chromosomal genomic island, SmaR, was identified, closely related to Multiple Antimicrobial Resistance Regions (MARR), usually found in AbaR0-type and AbGRI2-0 from global clones of Acinetobacter baumannii, and in M-type plasmids circulating in Enterobacteriaceae. Maintenance studies showed that the three class 1 integrons were maintained over 1 month without antimicrobial pressure. Since S. marcescens is considered a relevant nosocomial pathogen that can have a wide range of niches – human, plant, animal, soil and inanimate surfaces, our findings support the ability of this species to capture, maintain and spread a broad variety of antimicrobial resistance elements.

Identification of proteins is one of the most computationally intensive steps in genomics studies. It usually relies on aligners that don’t accommodate rich information on proteins and require additional pipelining steps for protein identification. We introduce kAAmer, a protein database engine based on amino-acid k-mers, that supports fast identification of proteins with complementary annotations. Moreover, the databases can be hosted and queried remotely.

Bacterial genomics studies are getting more extensive and complex, requiring new ways to envision analyses. Using the Ray Surveyor software, we demonstrate that comparison of genomes based on their k-mer content allows reconstruction of phenetic trees without the need of prior data curation, such as core genome alignment of a species. We validated the methodology using simulated genomes and previously published phylogenomic studies of Streptococcus pneumoniae and Pseudomonas aeruginosa. We also investigated the relationship of specific genetic determinants with bacterial population structures. By comparing clusters from the complete genomic content of a genome population with clusters from specific functional categories of genes, we can determine how the population structures are correlated. Indeed, the strain clustering based on a subset of k-mers allows determination of its similarity with the whole genome clusters. We also applied this methodology on 42 species of bacteria to determine the correlational significance of five important bacterial genomic characteristics. For example, intrinsic resistance is more important in P. aeruginosa than in S. pneumoniae, and the former has increased correlation of its population structure with antibiotic resistance genes. The global view of the pangenome of bacteria also demonstrated the taxa-dependent interaction of population structure with antibiotic resistance, bacteriophage, plasmid, and mobile element k-mer data sets.

We report here the complete genome sequence of Klebsiella pneumoniae CCRI-22199, isolated from a patient from India treated in Quebec City, Canada. Genes encoding beta-lactamases NDM-1 and CTX-M-15 were identified on two distinct plasmids. While the chromosome is similar to that of strain BAA-2146, CCRI-22199 provides a further example of rearrangements in plasmids.

Sequencing of the bla IMP-4 -carrying C. freundii B38 using the PacBio SMRT technique revealed that the genome contained a chromosome of 5,134,500 bp and three plasmids, pOZ172 (127,005 bp), pOZ181 (277,592 bp), and pOZ182 (18,467 bp). Plasmid pOZ172 was identified as IncFIIY, like pP10164-NDM and pNDM-EcGN174. It carries a class 1 integron with four cassettes ( bla IMP-4 - qacG2 - aacA4 - aphA15 ) and a complete hybrid tni module ( tniR - tniQ - tniB - tniA ). The recombination of tniR from Tn 402 (identical) with tniQBA from Tn 5053 (99%) occurred within the res site of Tn 402 / 5053 . The Tn 402/5053 -like integron, named Tn 6017 , was inserted into Tn 1722 at the res II site. The replication, partitioning, and transfer systems of pOZ181 were similar to those of IncHI2 plasmids (e.g., R478) and contained a sul1 -type class 1 integron with the cassette array orf-dfrA1-orf-gcu37-aadA5 linked to an upstream Tn 1696 tnpA-tnpR and to a downstream 3′ conserved sequence (3′-CS) and IS CR1 . A Tn 2 transposon encoding a bla TEM-1 β-lactamase was identified on pOZ182. Other interesting resistance determinants encoded on the B38 chromosome included multidrug resistance (MDR) efflux pumps, an AmpC β-lactamase, and resistances to Cu, Ag, As, and Zn. This is the first report of a complete tni module linked to a bla IMP-4 -carrying class 1 integron, which, together with other recently reported non- sul1 integrons, represents the emergence of a distinct evolutionary lineage of class 1 integrons lacking a 3′-CS ( qacEΔ1 - sul1 ). The unique cassette array, complete tni module of Tn 6017 , and incompatibility group of pOZ172 suggest a bla IMP-4 evolutionary pathway in C. freundii B38 different from that for other bla IMP-4 genes found in Gram-negative bacteria in the Western Pacific region.

Glutaminyl-tRNAGln in Helicobacter pylori is formed by an indirect route requiring a noncanonical glutamyl-tRNA synthetase and a tRNA-dependent heterotrimeric amidotransferase (AdT) GatCAB. Widespread use of this pathway among prominent human pathogens, and its absence in the mammalian cytoplasm, identify AdT as a target for the development of antimicrobial agents. We present here the inhibitory properties of three dipeptide-like sulfone-containing compounds analogous to the transamidation intermediates, which are competitive inhibitors of AdT with respect to Glu-tRNAGln . Molecular docking revealed that AdT inhibition by these compounds depends on π-π stacking interactions between their aromatic groups and Tyr81 of the GatB subunit. The properties of these inhibitors indicate that the 3'-terminal adenine of Glu-tRNAGln plays a major role in binding to the AdT transamidation active site.

Microbiome studies have demonstrated the high inter-individual diversity of the gut microbiota. However, how the initial composition of the microbiome affects the impact of antibiotics on microbial communities is relatively unexplored. To specifically address this question, we administered a second-generation cephalosporin, cefprozil, to healthy volunteers. Stool samples gathered before antibiotic exposure, at the end of the treatment and 3 months later were analysed using shotgun metagenomic sequencing. On average, 15 billion nucleotides were sequenced for each sample. We show that standard antibiotic treatment can alter the gut microbiome in a specific, reproducible and predictable manner. The most consistent effect of the antibiotic was the increase of Lachnoclostridium bolteae in 16 out of the 18 cefprozil-exposed participants. Strikingly, we identified a subgroup of participants who were enriched in the opportunistic pathogen Enterobacter cloacae after exposure to the antibiotic, an effect linked to lower initial microbiome diversity and to a Bacteroides enterotype. Although the resistance gene content of participants' microbiomes was altered by the antibiotic, the impact of cefprozil remained specific to individual participants. Resistance genes that were not detectable prior to treatment were observed after a 7-day course of antibiotic administration. Specifically, point mutations in beta-lactamase blaCfxA-6 were enriched after antibiotic treatment in several participants. This suggests that monitoring the initial composition of the microbiome before treatment could assist in the prevention of some of the adverse effects associated with antibiotics or other treatments.

We report here the complete genome sequence of a panresistant Pseudomonas aeruginosa strain, isolated from a patient with respiratory failure in Canada. No carbapenemase genes were identified. Carbapenem resistance is attributable to a frameshift in the oprD gene; the basis for colistin resistance remains undetermined.

We report here the complete genome sequence of a panresistant Pseudomonas aeruginosa strain, isolated from a patient with respiratory failure in Canada. No carbapenemase genes were identified. Carbapenem resistance is attributable to a frameshift in the oprD gene; the basis for colistin resistance remains undetermined.

Pseudomonas aeruginosa PA96 is a clinical isolate from Guangzhou, China, that is multiresistant to antibiotics. We previously described the 500-kb IncP-2 plasmid, pOZ176 that encodes many resistance genes including the IMP-9 carbapenemase. Whole-genome sequencing of PA96 enabled characterization of its genomic islands, virulence factors, and chromosomal resistance genes. We filled gaps using PCR and used optical mapping to confirm the correct contig order. We automatically annotated the core genome and manually annotated the genomic islands. The genome is 6 444 091 bp and encodes 5853 ORFs. From the whole-genome sequence, we constructed a physical map and constructed a phylogenetic tree for comparison with sequenced P. aeruginosa strains. Analysis of known core genome virulence factors and resistance genes revealed few differences with other strains, but the major virulence island is closer to that of DK2 than to PA14. PA96 most closely resembles the environmental strain M18, and notably shares a common serotype, pyoverdin type, flagellar operon, type IV pilin, and several genomic islands with M18.

Criibacterium bergeronii gen. nov., sp. nov., CCRI-22567 is the type strain of the new genus Criibacterium . The strain was isolated from a woman with bacterial vaginosis. The genome assembly comprised 2,384,460 bp, with 34.4% G+C content. This is the first genome announcement of a strain belonging to the genus Criibacterium .

Successful treatment of a Candida infection relies on 1) an accurate identification of the pathogenic fungus and 2) on its susceptibility to antifungal drugs. In the present study we investigated the level of correlation between phylogenetical evolution and susceptibility of pathogenic Candida spp. to antifungal drugs. For this, we compared a phylogenetic tree, assembled with the concatenated sequences (2475-bp) of the ATP2, TEF1, and TUF1 genes from 20 representative Candida species, with published minimal inhibitory concentrations (MIC) of the four principal antifungal drug classes commonly used in the treatment of candidiasis: polyenes, triazoles, nucleoside analogues, and echinocandins. The phylogenetic tree revealed three distinct phylogenetic clusters among Candida species. Species within a given phylogenetic cluster have generally similar susceptibility profiles to antifungal drugs and species within Clusters II and III were less sensitive to antifungal drugs than Cluster I species. These results showed that phylogenetical relationship between clusters and susceptibility to several antifungal drugs could be used to guide therapy when only species identification is available prior to information pertaining to its resistance profile. An extended study comprising a large panel of clinical samples should be conducted to confirm the efficiency of this approach in the treatment of candidiasis.

Glutaminyl-tRNA

Integrons are genetic elements that incorporate mobile gene cassettes by site-specific recombination and express them as an operon from a promoter (Pc) located upstream of the cassette insertion site. Most gene cassettes found in integrons contain only one gene followed by an attC recombination site. We have recently shown that a specific lineage of group IIC introns, named group IIC-attC introns, inserts into the bottom strand sequence of attC sites. Here, we show that S.ma.I2, a group IIC-attC intron inserted in an integron cassette array of Serratia marcescens, impedes transcription from Pc while allowing expression of the following antibiotic resistance cassette using an internal outward-oriented promoter (P(out)). Bioinformatic analyses indicate that one or two putative P(out), which have sequence similarities with the Escherichia coli consensus promoters, are conserved in most group IIC-attC intron sequences. We show that P(out) with different versions of the -35 and -10 sequences are functionally active in expressing a promoterless chloramphenicol acetyltransferase (cat) reporter gene in E. coli. P(out) in group IIC-attC introns may therefore play a role in the expression of one or more gene cassettes whose transcription from Pc would otherwise be impeded by insertion of the intron.

Integrons are natural expression vectors in which gene cassettes are integrated downstream of a promoter region by a site-specific recombinase. Gene cassettes usually consist of a single gene followed by a recombination site designatedattC. A major unanswered question is how a gene becomes associated with anattCsite. Here, we investigate the potential role of a specific lineage of group IIC introns, named group IIC-attC, in cassette formation. Group IIC-attCintrons preferentially targetattCwhile retaining the ability to target transcriptional terminators. We show using a PCR-based mobility assay withEscherichia colithat theS.ma.I2 intron from the genome of a clinical isolate ofSerratia marcescenscan target bothattCsite and putative terminator motifs of resistance genes. Quantitative results showed thatS.ma.I2 is more efficient in targeting variousattCsequences than three group IIC-attCintrons (54 to 64% sequence identity) from the genomes of environmental isolates. We also show that purified group IIC-attCintron-encoded reverse transcriptases have both RNA-dependent and DNA-dependent DNA polymerase activities in vitro. These data permit us to suggest a new model for gene cassette formation, in which a group IIC-attCintron targets separately a transcriptional terminator adjoining a gene and an isolatedattC, joins the gene and theattCby homologous recombination, and then splices and reverse transcribes a gene-attCRNA template, leading to the formation of a cassette.

Integrons are mobile genetic elements that can integrate and disseminate genes as cassettes by a site-specific recombination mechanism. Integrons contain an integrase gene ( intI ) that carries out recombination by interacting with two different target sites; the attI site in cis with the integrase and the palindromic attC site of a cassette. The plasmid-specified IntI1 excises a greater variety of cassettes (principally antibiotic resistance genes), and has greater activity, than chromosomal integrases. The aim of this study was to analyze the capacity of the chromosomal integron integrase SamIntIA of the environmental bacterium Shewanella amazonensis SB2BT to excise various cassettes and to compare the properties of the wild type with those of mutants that substitute consensus residues of active integron integrases. We show that the SamIntIA integrase is very weakly active in the excision of various cassettes but that the V206R, V206K, and V206H substitutions increase its efficiency for the excision of cassettes. Our results also suggest that the cysteine residue in the β-5 strand is essential to the activity of Shewanella -type integrases, while the cysteine in the β-4 strand is less important for the excision activity.

In many organisms, the formation of asparaginyl-tRNA is not done by direct aminoacylation of tRNA Asn but by specific tRNA-dependent transamidation of aspartyl-tRNA Asn . This transamidation pathway involves a nondiscriminating aspartyl-tRNA synthetase (AspRS) that charges both tRNA Asp and tRNA Asn with aspartic acid. Recently, it has been shown for the first time in an organism ( Pseudomonas aeruginosa PAO1) that the transamidation pathway is the only route of synthesis of Asn-tRNA Asn but does not participate in Gln-tRNA Gln formation. P. aeruginosa PAO1 has a nondiscriminating AspRS. We report here the identification of two residues in the anticodon recognition domain (H31 and G83) which are implicated in the recognition of tRNA Asn . Sequence comparisons of putative discriminating and nondiscriminating AspRSs (based on the presence or absence of the AdT operon and of AsnRS) revealed that bacterial nondiscriminating AspRSs possess a histidine at position 31 and usually a glycine at position 83, whereas discriminating AspRSs possess a leucine at position 31 and a residue other than a glycine at position 83. Mutagenesis of these residues of P. aeruginosa AspRS from histidine to leucine and from glycine to lysine increased the specificity of tRNA Asp charging over that of tRNA Asn by 3.5-fold and 4.2-fold, respectively. Thus, we show these residues to be determinants of the relaxed specificity of this nondiscriminating AspRS. Using available crystallographic data, we found that the H31 residue could interact with the central bases of the anticodons of the tRNA Asp and tRNA Asn . Therefore, these two determinants of specificity of P. aeruginosa AspRS could be important for all bacterial AspRSs.

A 761-bp portion of the tuf gene (encoding the elongation factor Tu) from 28 clinically relevant streptococcal species was obtained by sequencing amplicons generated using broad-range PCR primers. These tuf sequences were used to select Streptococcus -specific PCR primers and to perform phylogenetic analysis. The specificity of the PCR assay was verified using 102 different bacterial species, including the 28 streptococcal species. Genomic DNA purified from all streptococcal species was efficiently detected, whereas there was no amplification with DNA from 72 of the 74 nonstreptococcal bacterial species tested. There was cross-amplification with DNAs from Enterococcus durans and Lactococcus lactis . However, the 15 to 31% nucleotide sequence divergence in the 761-bp tuf portion of these two species compared to any streptococcal tuf sequence provides ample sequence divergence to allow the development of internal probes specific to streptococci. The Streptococcus -specific assay was highly sensitive for all 28 streptococcal species tested (i.e., detection limit of 1 to 10 genome copies per PCR). The tuf sequence data was also used to perform extensive phylogenetic analysis, which was generally in agreement with phylogeny determined on the basis of 16S rRNA gene data. However, the tuf gene provided a better discrimination at the streptococcal species level that should be particularly useful for the identification of very closely related species. In conclusion, tuf appears more suitable than the 16S ribosomal RNA gene for the development of diagnostic assays for the detection and identification of streptococcal species because of its higher level of species-specific genetic divergence.

We have characterized the variable region of three integrons found in the multiresistant strain Enterobacter cloacae SCH88040794, using polymerase chain reaction and direct sequencing of amplicons. The first integron has the gene cassette arrangement dfrAI-aadA1 in its variable region and the second aadB-oxa9. The third integron is In40 (aacA4-qacF-cmlB-oxa9), which has recently been described. The multiresistance expressed by this strain to amikacin, gentamicin, trimethoprim, sulfonamides, oxacillin, chloramphenicol and quaternary ammonium compounds is due, at least in part, to these three integrons.

Examination of the bla CTX-M-2 gene in plasmid pMAR-12 by sequencing and PCR analysis revealed that the bla gene and the surrounding DNA, which is closely related (99% homology) to the Kluyvera ascorbata chromosomal DNA that contains the bla KLUA-1 gene, are located in a complex sul1 -type integron, termed In35, that includes Orf513. It is possible that bla CTX-M-2 was acquired by plasmid pMAR-12 through an uncharacterized recombinational event in which Orf513 could be involved.

We analyzed the role of integrons in the dissemination of antibiotic resistance in a recent multiresistant clinical isolate, Serratia marcescens SCH88050909 (SCH909). This isolate harbors three integrons, all on a 60-kb conjugative plasmid. By PCR, hybridization, and sequencing analyses, we found that integron 1 has the dfrA1 and ant ( 3 ") -Ia cassettes. The first cassette in integron 2 contains the ant ( 2 ") -Ia gene, separated from its attC site (59-base element) by a 1,971-bp insert containing a group II intron; this intron codes for a putative maturase-reverse transcriptase on the complementary strand and is the first such intron to be found associated with an integron. The attC site is followed by a novel aminoglycoside resistance gene, ant ( 3 ") -Ii-aac ( 6 ′) -IId , which has been characterized for its bifunctional ANT(3")-I and AAC(6′)-II activities. DNA sequence analysis of this fused cassette suggests that insertion and excision due to the integrase activity could have an important role in the evolution of aminoglycoside resistance genes. This gene is followed by an unknown open reading frame with a typical attC site and a partial cassette composed of the beginning of the bla OXA-10 cassette interrupted by IS 1 . The sequence downstream of IS 1 revealed that the bla OXA-10 cassette is incomplete and that the 3′ conserved segment of this integron is absent. Integron 3 is in a Tn 1696 -like transposon with the aac ( 3 ) -Ia cassette followed by three unknown cassettes and ant ( 3 ") -Ia . The presence of the group II intron and the relationship of group II introns in eubacteria with mobile elements suggest a possible role of this element in events such as cassette formation and/or plasmid evolution.

Integrons are gene capture systems that can acquire and disseminate elements known as gene cassettes. These systems are implicated in the dissemination of antibiotic resistance genes. The essential components of the integron are found within the 5′-conserved segment and include an integrase gene, intI, an adjacent recombination site, attI, and a promoter region from which integrated cassettes are expressed (4). The gene cassettes are located within the variable region and are integrated in tandem at the attI site. These cassettes contain a structural gene and an imperfect palindromic integrase-specific recombination site known as an attC site or 59-base element. The cassettes are mobile, nonreplicating, and generally lack a promoter region (10). Several classes of integrons have been reported, all of which contain distinct but related integrase genes (12). Class 1 integrons are the most widespread, and most contain a 3′-conserved segment that contains a qacEΔ1 gene encoding resistance to quaternary ammonium compounds and a sulI gene encoding resistance to sulfonamides (Fig. ​(Fig.1)1) (13, 14). Classes 2 and 3, which are rarer, are, like class 1, plasmid borne (9). The class 4 integron is a first example of a chromosomal superintegron and is located in the genome of Vibrio cholerae (5). Several other classes have been identified, but they are not fully characterized yet (7, 12). FIG. 1. General structure of class 1 integrons. Cassettes are inserted in the variable region by the integrase using a site-specific recombination mechanism. The attI and attC sites are shown by a black and a grey oval, respectively, and promoters are denoted ... The integron integrase is a member of the tyrosine recombinase family, a superfamily of site-specific recombination proteins (3). Among this family, there are also the well-known XerC/XerD, Cre, Flp, and λ integrase (2, 8). However, there is an extra domain, composed of an additional 36 amino acids near Patch III that distinguishes integron integrases from other tyrosine recombinases and which is implicated in the recognition of the attI and attC sites (6).

In the last few years Acinetobacter baumannii has emerged worldwide as an important nosocomial pathogen in medical institutions. Here, we present the draft genome sequence of the international clonal lineage 1 (ICL1) A. baumannii strain A144 that was isolated in a hospital in Buenos Aires City in the year 1997. The strain is susceptible to carbapenems and resistant to trimethoprim and gentamicin. Fil: Vilacoba, Elisabet. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina Fil: Déraspe, Maxime. Laval University; Canadá Fil: Traglia, German Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina Fil: Roy, Paul H.. Laval University; Canadá Fil: Ramirez, Maria Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina. California State University; Estados Unidos Fil: Centron, Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina

Many clinical isolates of Pseudomonas aeruginosa cause infections that are difficult to eradicate due to their resistance to a wide variety of antibiotics. Key genetic determinants of resistance were identified through genome sequences of 390 clinical isolates of P. aeruginosa , obtained from diverse geographic locations collected between 2003 and 2012 and were related to microbiological susceptibility data for meropenem, levofloxacin, and amikacin. β-Lactamases and integron cassette arrangements were enriched in the established multidrug-resistant lineages of sequence types ST111 (predominantly O12) and ST235 (O11). This study demonstrates the utility of next-generation sequencing (NGS) in defining relevant resistance elements and highlights the diversity of resistance determinants within P. aeruginosa . This information is valuable in furthering the design of diagnostics and therapeutics for the treatment of P. aeruginosa infections.

We report the complete sequence and characterization of plasmid pDCPR1 harboring a blaVIM-2 gene cassette in a Tn402-type class 1 integron, which was isolated from two extensively drug-resistant strains: P. aeruginosa 802 (from a burn patient at the Hospital Municipal de Quemados, Argentina, 2005) and S. marcescens 68313 (Sanatorio Sagrado Corazón, Argentina, 2012)., Instituto de Biotecnologia y Biologia Molecular

Integrons are genetic elements capable of integrating genes by a site-specific recombination system catalyzed by an integrase. Integron integrases are members of the tyrosine recombinase family and possess the four invariant residues (RHRY) and conserved motifs (boxes I and II and patches I, II, and III). An alignment of integron integrases compared to other tyrosine recombinases shows an additional group of residues around the patch III motif. We have analyzed the DNA binding and recombination properties of class I integron integrase (IntI1) variants carrying mutations at residues that are well conserved among all tyrosine recombinases and at some residues from the additional motif that are conserved among the integron integrases. The well-conserved residues studied were H277 from the conserved tetrad RHRY (about 90% conserved), E121 found in the patch I motif (about 80% conserved in prokaryotic recombinases), K171 from the patch II motif (near 100% conserved), W229 and F233 from the patch III motif, and G302 of box II (about 80% conserved in prokaryotic recombinases). Additional IntI1 mutated residues were K219 and a deletion of the sequence ALER215. We observed that E121, K171, and G302 play a role in the recombination activity but can be mutated without disturbing binding to DNA. W229, F233, and the conserved histidine (H277) may be implicated in protein folding or DNA binding. Some of the extra residues of IntI1 seem to play a role in DNA binding (K219) while others are implicated in the recombination activity (ALER215 deletion).

We have developed a PCR-based assay which allows the detection of staphylococci at the genus level by targeting the tuf gene, which encodes the elongation factor Tu. Degenerate PCR primers derived from consensus regions of several tuf genes were used to amplify a target region of 884 bp from 11 representative staphylococcal species. Subsequently, the entire nucleotide sequence of these amplicons was determined. The analysis of a multiple alignment of these sequences revealed regions conserved among staphylococci but distinct from those of other gram-positive bacteria genetically related to staphylococci. PCR primers complementary to these regions could amplify specifically and efficiently a DNA fragment of 370 bp for all of 27 different staphylococcal species tested. There was no amplification with genomic DNA prepared from 53 nonstaphylococcal species tested to verify the specificity of the assay (20 gram positive and 33 gram negative). Furthermore, this assay amplified efficiently all 27 American Type Culture Collection (ATCC) staphylococcal reference strains as well as 307 clinical isolates of staphylococci from the Québec City region. Analysis of the multiple sequence alignment for the 884-bp fragment for the 11 staphylococcal species as well as comparison of the sequences for the 370-bp amplicon from five unrelated ATCC and clinical strains for each of the species S. aureus, S. epidermidis, S. haemolyticus, S. hominis , and S. saprophyticus demonstrated sufficient interspecies polymorphism to generate genus- and species-specific capture probes. This sequence information allowed the development of Staphylococcus -specific and species-specific (targeting S. aureus, S. epidermidis, S. haemolyticus, S. hominis , or S. saprophyticus ) capture probes hybridizing to the 370-bp amplicon. In conclusion, this PCR assay is suitable for detection of staphylococci at both genus and species levels.

The elongation factor Tu, encoded bytufgenes, is a GTP binding protein that plays a central role in protein synthesis. One to threetufgenes per genome are present, depending on the bacterial species. Most low-G+C-content gram-positive bacteria carry only onetufgene. We have designed degenerate PCR primers derived from consensus sequences of thetufgene to amplify partialtufsequences from 17 enterococcal species and other phylogenetically related species. The amplified DNA fragments were sequenced either by direct sequencing or by sequencing cloned inserts containing putative amplicons. Two differenttufgenes (tufAandtufB) were found in 11 enterococcal species, includingEnterococcus avium,Enterococcus casseliflavus,Enterococcus dispar,Enterococcus durans,Enterococcus faecium,Enterococcus gallinarum,Enterococcus hirae,Enterococcus malodoratus,Enterococcus mundtii,Enterococcus pseudoavium, andEnterococcus raffinosus. For the other six enterococcal species (Enterococcus cecorum,Enterococcus columbae,Enterococcus faecalis,Enterococcus sulfureus,Enterococcus saccharolyticus, andEnterococcus solitarius), only thetufAgene was present. Based on 16S rRNA gene sequence analysis, the 11 species having twotufgenes all have a common ancestor, while the six species having only one copy diverged from the enterococcal lineage before that common ancestor. The presence of one or two copies of thetufgene in enterococci was confirmed by Southern hybridization. Phylogenetic analysis oftufsequences demonstrated that the enterococcaltufAgene branches with theBacillus,Listeria, andStaphylococcusgenera, while the enterococcaltufBgene clusters with the generaStreptococcusandLactococcus. Primary structure analysis showed that four amino acid residues encoded within the sequenced regions are conserved and unique to the enterococcaltufBgenes and thetufgenes of streptococci andLactococcus lactis. The data suggest that an ancestral streptococcus or a streptococcus-related species may have horizontally transferred atufgene to the common ancestor of the 11 enterococcal species which now carry twotufgenes.

Multiresistant staphylococci (82 Staphylococcus aureus and 114 coagulase-negative staphylococci) were characterized by testing with rapid multiplex polymerase chain reaction (PCR) assays for species identification and detection of associated antibiotic resistance genes. These 196 staphylococci were isolated from 149 adult patients who developed wound infection after elective coronary artery bypass grafts and/or valve surgery. The multiplex PCR assays allowed identification of the most common staphylococcal species with S. aureus- and Staphylococcus epidermidis-specific primers as well as the detection of the erythromycin resistance genes ermA, ermB, ermC and msrA, the aminoglycoside resistance gene aac(6')-aph(2"), the oxacillin resistance gene mecA and the penicillin resistance gene blaZ. There was a very good correlation between the genotypic analysis by PCR and the phenotype determined by standard methods of susceptibility testing and identification of staphylococcal species: 100% for erythromycin resistance, 98.0% for gentamicin resistance, 99.0% for oxacillin resistance, 100% for penicillin resistance and 100% for S. aureus and S. epidermidis species identification. This study suggests that the incidence and distribution of the tested clinically relevant antibiotic resistance genes in staphylococci associated with infections after cardiac surgery do not differ from those in strains from other infections. These multiplex PCR assays may be used as diagnostic tools to replace or complement standard methods of susceptibility testing and identification of staphylococci.

Group B streptococcal infections are an important cause of neonatal morbidity and mortality. A rapid method for the detection of this organism in pregnant women at the time of delivery is needed to allow early treatment of neonates.We studied the efficacy of two polymerase-chain-reaction (PCR) assays for routine screening of pregnant women for group B streptococci at the time of delivery. We obtained anal, vaginal, and combined vaginal and anal specimens from 112 pregnant women; in 57 women, specimens were obtained before and after the rupture of the amniotic membranes. The specimens were tested for group B streptococci by culture in a standard selective broth medium, with a conventional PCR assay, and with a new fluorogenic PCR assay.Among the 112 women, the results of the culture of the combined vaginal and anal specimens were positive for group B streptococci in 33 women (29.5 percent). The two PCR assays detected group B streptococcal colonization in specimens from 32 of these 33 women: the one negative PCR result was in a sample obtained after the rupture of membranes. As compared with the culture results, the sensitivity of both PCR assays was 97.0 percent and the negative predictive value was 98.8 percent. Both the specificity and the positive predictive value of the two PCR assays were 100 percent. The length of time required to obtain results was 30 to 45 minutes for the new PCR assay, 100 minutes for the conventional PCR assay, and at least 36 hours for culture.Colonization with group B streptococci can be identified rapidly and reliably by a PCR assay in pregnant women in labor both before and after the rupture of membranes.

Background: Group B streptococci (GBS), or Streptococcus agalactiae, are the leading bacterial cause of meningitis and bacterial sepsis in newborns. Currently available rapid methods to detect GBS from clinical specimens are unsuitable for replacement of culture methods, mainly because of their lack of sensitivity.Methods: We have developed a PCR-based assay for the rapid detection of GBS. The cfb gene encoding the Christie-Atkins-Munch-Petersen (CAMP) factor was selected as the genetic target for the assay. The PCR primers were initially tested by a conventional PCR method followed by gel electrophoresis. The assay was then adapted for use with the LightCyclerTM. For this purpose, two fluorogenic adjacent hybridization probes complementary to the GBS-specific amplicon were designed and tested. In addition, a rapid sample-processing protocol was evaluated by colony-forming unit counting and PCR. A total of 15 vaginal samples were tested by both standard culture method and the two PCR assays.Results: The conventional PCR assay was specific because it amplified only GBS DNA among 125 bacterial and fungal species tested, and was able to detect all 162 GBS isolates from various geographical areas. This PCR assay allowed detection of as few as one genome copy of GBS. The real-time PCR assay was comparable to conventional PCR assay in terms of sensitivity and specificity, but it was more rapid, requiring only ∼30 min for amplification and computer-based data analysis. The presence of vaginal specimens had no detrimental effect on the sensitivity of the PCR with the sample preparation protocol used. All four GBS-positive samples identified by the standard culture method were detected by the two PCR assays.Conclusion: These assays provide promising tools for the rapid detection and identification of GBS.

Clinical isolates of Staphylococcus aureus (a total of 206) and S. epidermidis (a total of 188) from various countries were tested with multiplex PCR assays to detect clinically relevant antibiotic resistance genes associated with staphylococci. The targeted genes are implicated in resistance to oxacillin ( mecA ), gentamicin [ aac (6′)- aph (2")], and erythromycin ( ermA , ermB , ermC , and msrA ). We found a nearly perfect correlation between genotypic and phenotypic analysis for most of these 394 strains, showing the following correlations: 98% for oxacillin resistance, 100% for gentamicin resistance, and 98.5% for erythromycin resistance. The discrepant results were (i) eight strains found to be positive by PCR for mecA or ermC but susceptible to the corresponding antibiotic based on disk diffusion and (ii) six strains of S. aureus found to be negative by PCR for mecA or for the four erythromycin resistance genes targeted but resistant to the corresponding antibiotic. In order to demonstrate in vitro that the eight susceptible strains harboring the resistance gene may become resistant, we subcultured the susceptible strains on media with increasing gradients of the antibiotic. We were able to select cells demonstrating a resistant phenotype for all of these eight strains carrying the resistance gene based on disk diffusion and MIC determinations. The four oxacillin-resistant strains negative for mecA were PCR positive for blaZ and had the phenotype of β-lactamase hyperproducers, which could explain their borderline oxacillin resistance phenotype. The erythromycin resistance for the two strains found to be negative by PCR is probably associated with a novel mechanism. This study reiterates the usefulness of DNA-based assays for the detection of antibiotic resistance genes associated with staphylococcal infections.

Staphylococcus saprophyticus is one of the most frequently encountered microorganisms associated with acute urinary tract infections (UTIs) in young, sexually active female outpatients. Conventional identification methods based on biochemical characteristics can efficiently identify S. saprophyticus, but the rapidities of these methods need to be improved. Rapid and direct identification of this bacterium from urine samples would be useful to improve time required for the diagnosis of S. saprophyticus infections in the clinical microbiology laboratory. We have developed a PCR-based assay for the specific detection ofS. saprophyticus. An arbitrarily primed PCR amplification product of 380 bp specific for S. saprophyticus was sequenced and used to design a set of S. saprophyticus-specific PCR amplification primers. The PCR assay was specific for S. saprophyticus when tested with DNA from 49 gram-positive and 31 gram-negative bacterial species. This assay was also able to amplify efficiently DNA from all 60 strains of S. saprophyticus from various origins tested. This assay was adapted for direct detection from urine samples. The sensitivity levels achieved with urine samples was 19 CFU with 30 cycles of amplification and 0.5 CFU with 40 cycles of amplification. This PCR assay for the specific detection of S. saprophyticus is simple and rapid (approximately 90 min, including the time for urine specimen preparation).

Integrons are genetic elements that are able to capture genes by a site-specific recombination mechanism. Integrons contain a gene coding for a lambda-like integrase that carries out site-specific recombination by interacting with two different target sites; the attI site and the palindromic sequence attC (59 base element). Cassette integrations usually involve the attI site, while cassette excisions use attC . Therefore, the integrase should bind both sites to cleave DNA and perform site-specific recombination reactions. We have used purified maltose-binding protein fused with the integrase (MBP-IntI1) and native IntI1 protein and gel retardation assays with fragments containing the complete and partial attI1 site to show formation of four complexes in this region. Chemical modification of specific nucleotides within the attI1 site was used to investigate their interference with binding of the integrase protein. We attribute IntI1 specific binding to four regions in the attI1 site and a GTTA consensus sequence is found in three of the four regions. Interference by modified guanine and thymine residues in the DNA major groove and adenine residues in the minor groove were observed, indicating that the integrase interacts with both sides of the helix. Binding of IntI1 to attC is also discussed.

Staphylococcus aureus is the cause of serious infections in humans, including endocarditis, deep-seated abscesses, and bacteremia, which lead to toxic and septic shock syndromes. Rapid and direct identification of this bacterium specifically and ubiquitously directly from clinical specimens would be useful in improving the diagnosis of S. aureus infections in the clinical microbiology laboratory. A wide variety of kits based on biochemical characteristics efficiently identify S. aureus , but the rapidity and the accuracy of each of these methods combined with testing of clinically relevant antibiotic resistance genes need to be improved. On the basis of hybridization assays with randomly selected clones from an S. aureus genomic library, we have identified a chromosomal DNA fragment which is specific for S. aureus and which detected all 82 S. aureus isolates tested. This 442-bp fragment was sequenced and was used to design a set of PCR amplification primers. The PCR assay was also specific and ubiquitous for the identification from bacterial cultures of 195 clinical strains of S. aureus isolated from a variety of anatomical sites and obtained from hospitals throughout the world. The PCR assay that we have developed is simple and can be performed in about 1 h. This DNA-based test provides a novel diagnostic tool for the diagnosis of S. aureus infections.

The beta-lactamase genes of Klebsiella oxytoca were previously divided into two main groups: bla(OXY-1) and bla(OXY-2). The two beta-lactamase groups were each represented by beta-lactamases with four different pIs. In each group, one form of beta-lactamase is more frequent than the others combined. The beta-lactamase gene of each representative beta-lactamase with a different pI that was not yet sequenced (pIs 5.7, 6.8 [OXY-2], 7.1, 8.2, and 8.8 [OXY-1]) was cloned and sequenced. The susceptibility patterns as well as relative rates and kinetic parameters for beta-lactam hydrolysis revealed that OXY-2 enzymes hydrolyzed several of the beta-lactams that were examined (carbenicillin, cephalothin, cefamandole, ceftriaxone, and aztreonam) at a greater rate than the OXY-1 enzymes did. Comparison of K. oxytoca beta-lactamases with plasmid-mediated extended-spectrum beta-lactamases MEN-1 and TOHO-1 implied that the threonine at position 168 present in OXY-2 beta-lactamase instead of the alanine in OXY-1 could be responsible for its modified substrate hydrolysis. In each group, the beta-lactamase with a variant pI differs from the main form of beta-lactamase by one to five amino acid substitutions. The substrate profile and the 50% inhibitory concentrations revealed that all substitutions differing from the main form of beta-lactamase were neutral except one difference in the OXY-1 group. This substitution of an Ala to a Gly at position 237 increases the hydrolysis of some beta-lactams, particularly aztreonam; decreases the hydrolysis of benzylpenicillin, cephaloridine, and cefamandole, and decreases the susceptibility to clavulanic acid (fivefold increase in the 50% inhibitory concentration).

Pseudomonas aeruginosa 96 (PA96) was isolated during a multicenter surveillance study in Guangzhou, China, in 2000. Whole-genome sequencing of this outbreak strain facilitated analysis of its IncP-2 carbapenem-resistant plasmid, pOZ176. The plasmid had a length of 500,839 bp and an average percent G+C content of 57%. Of the 618 predicted open reading frames, 65% encode hypothetical proteins. The pOZ176 backbone is not closely related to any plasmids thus far sequenced, but some similarity to pQBR103 of Pseudomonas fluorescens SBW25 was observed. Two multiresistant class 1 integrons and several insertion sequences were identified. The bla IMP-9 -carrying integron contained aacA4 → bla IMP-9 → aacA4 , flanked upstream by Tn 21 tnpMRA and downstream by a complete tni operon of Tn 402 and a mer module, named Tn 6016 . The second integron carried aacA4 → catB8a → bla OXA-10 and was flanked by Tn 1403 -like tnpRA and a sul1 -type 3′ conserved sequence (3′-CS), named Tn 6217 . Other features include three resistance genes similar to those of Tn 5 , a tellurite resistance operon, and two pil operons. The replication and maintenance systems exhibit similarity to a genomic island of Ralstonia solanacearum GM1000. Codon usage analysis suggests the recent acquisition of bla IMP-9 . The origins of the integrons on pOZ176 indicated separate horizontal gene transfer events driven by antibiotic selection. The novel mosaic structure of pOZ176 suggests that it is derived from environmental bacteria.

The chromosomally encoded beta-lactamase gene (blaOXY-2) of the wild-type Klebsiella oxytoca SL911 was cloned and sequenced. Its nucleotide sequence similarity with the previously sequenced K. oxytoca beta-lactamase gene (blaOXY-1) (Y. Arakawa, M. Ohta, N. Kido, M. Mori, H. Ito, T. Komatsu, Y. Fujii, and N. Kato, Antimicrob. Agents Chemother. 33:63-70, 1989) is 87.3%, and its amino acid similarity is 89.7%. This group of K. oxytoca beta-lactamases is related to chromosomal beta-lactamases of Citrobacter diversus, Proteus vulgaris, and Yersinia enterocolitica and to the plasmid-mediated extended-spectrum beta-lactamases MEN-1 and Toho-1. By colony hybridization with 86 strains susceptible and resistant to aztreonam, isolated in six countries, K. oxytoca beta-lactamase genes hybridized with either a specific blaOXY-1 DNA probe (668 bp) or a blaOXY-2 DNA probe (723 bp). Thus, beta-lactamase genes could be divided into two groups: blaOXY-1 (47% of the strains) and blaOXY-2 (53% of the strains). A study of isoelectric points confirmed the great variability reported in the literature. However, the two beta-lactamase groups were each represented by four different pIs: for OXY-2, 5.2, 5.7, 6.4, and 6.8, with the 5.2 form representing 59% of all OXY-2 enzymes, and for OXY-1, 7.1, 7.5, 8.2, and 8.8, with the 7.5 form representing 88% of all OXY-1 enzymes.

We have cloned and sequenced the accIRM genes from Weeksella zoohelcum (the original identification of this strain as Acinetobacter calcoaceticus was incorrect). Our sequence differs in the coding regions from a previously published sequence by the addition of three nucleotides near the 3' end of the DNA methyltransferase-encoding gene (accIM). We have sequenced approx. 3 kb beyond this operon. Two genes were found, convergently transcribed with the R-M operon. The first of these genes encodes a protein which shows significant similarity to the recombinases of the phage integrase family. The W. zoohelcum recombinase may function as a transposon resolvase, as in Tn4430. The recombinase-encoding gene is followed by a putative transposase (Tnp), which is in turn followed by a terminator which is predicted to be Rho-dependent for the recombinase-Tnp operon and Rho-independent for the convergent R-M operon. Since the G + C content of the two operons is notably different, it is possible that the terminator is at the extremity of the mobile element and serves to protect it from incoming transcription.

The integron is a new type of mobile element which has evolved by a site-specific recombinational mechanism. Integrons consist of two conserved segments of DNA separated by a variable region containing one or more genes integrated as cassettes. Oligonucleotide probes specific for the conserved segments have revealed that integrons are widespread in recently isolated clinical bacteria. Also, by using oligonucleotide probes for several antibiotic resistance genes, we have found novel combinations of resistance genes in these strains. By using PCR, we have determined the content and order of the resistance genes inserted between the conserved segments in the integrons of these clinical isolates. PCR mapping of integrons can be a useful epidemiological tool to study the evolution of multiresistance plasmids and transposons and dissemination of antibiotic resistance genes.