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July 7, 2019

Tigecycline resistance in clinical isolates of Enterococcus faecium is mediated by an upregulation of plasmid-encoded tetracycline determinants tet(L) and tet(M).

Tigecycline represents one of the last-line therapeutics to combat multidrug-resistant bacterial pathogens, including VRE and MRSA. The German National Reference Centre for Staphylococci and Enterococci has received 73 tigecycline-resistant Enterococcus faecium and Enterococcus faecalis isolates in recent years. The precise mechanism of how enterococci become resistant to tigecycline remains undetermined. This study documents an analysis of the role of efflux pumps in tigecycline resistance in clinical isolates of Enterococcus spp.Various tigecycline MICs were found for the different isolates analysed. Tigecycline-resistant strains were analysed with respect to genome and transcriptome differences by means of WGS and RT-qPCR. Genes of interest were cloned and expressed in Listeria monocytogenes for verification of their functionality.Detailed comparative whole-genome analyses of three isogenic strains, showing different levels of tigecycline resistance, revealed the major facilitator superfamily (MFS) efflux pump TetL and the ribosomal protection protein TetM as possible drug resistance proteins. Subsequent RT-qPCR confirmed up-regulation of the respective genes. A correlation of gene copy number and level of MIC was inferred from further qPCR analyses. Expression of both tet(L) and tet(M) in L. monocytogenes unequivocally demonstrated the potential to increase tigecycline MICs upon acquisition of either locus.Our results indicate that increased expression of two tetracycline resistance determinants, a tet(L)-encoded MFS pump and a tet(M)-encoded ribosomal protection protein, is capable of conferring tigecycline resistance in enterococcal clinical isolates.© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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July 7, 2019

Coproduction of KPC-18 and VIM-1 carbapenemases by Enterobacter cloacae: Implications for newer ß-lactam-ß-lactamase inhibitor combinations.

Enterobacter cloacae strain G6809 with reduced susceptibility to carbapenems was identified from a patient in a long-term acute care hospital in Kentucky. G6809 belonged to sequence type (ST) 88 and carried two carbapenemase genes, blaKPC-18 and blaVIM-1. Whole-genome sequencing localized blaKPC-18 to the chromosome and blaVIM-1 to a 58-kb plasmid. The strain was highly resistant to ceftazidime-avibactam. Insidious coproduction of metallo-ß-lactamase with KPC-type carbapenemase has implications for the use of next-generation ß-lactam-ß-lactamase inhibitor combinations. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

Whole genome sequence of the emerging oomycete pathogen Pythium insidiosum strain CDC-B5653 isolated from an infected human in the USA

Pythium insidiosum ATCC 200269 strain CDC-B5653, an isolate from necrotizing lesions on the mouth and eye of a 2-year-old boy in Memphis, Tennessee, USA, was sequenced using a combination of Illumina MiSeq (300 bp paired-end, 14 millions reads) and PacBio (10 Kb fragment library, 356,001 reads). The sequencing data were assembled using SPAdes version 3.1.0, yielding a total genome size of 45.6 Mb contained in 8992 contigs, N50 of 13 Kb, 57% G + C content, and 17,867 putative protein-coding genes. This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession JRHR00000000.

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July 7, 2019

Complete genome sequence of Klebsiella pneumoniae subsp. pneumoniae KP617, coproducing OXA-232 and NDM-1 carbapenemases, isolated in South Korea.

The prevalence of Klebsiella pneumoniae coproducing carbapenemase metallo-ß-lactamase 1 (NDM-1) and OXA-48 has been increasing globally since 2013. The complete genome of KP617 was sequenced and assembled into a circular chromosome and two plasmids. This sequence provides the genetic background for understanding the evolution of carbapenemase genes in K. pneumoniae KP617.

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July 7, 2019

Comparative analysis of an IncR plasmid carrying armA, blaDHA-1 and qnrB4 from Klebsiella pneumoniae ST37 isolates.

The objective of this study was to conduct a comparative analysis with reported IncR plasmids of a Klebsiella pneumoniae IncR plasmid carrying an MDR region.MDR K. pneumoniae isolates were serially identified from two inpatients at a hospital in the USA in 2014. MDR plasmid pYDC676 was fully sequenced, annotated and compared with related plasmids. Antimicrobial susceptibility testing, PFGE and MLST were also conducted.The K. pneumoniae isolates were identical by PFGE, belonged to ST37 and harboured an identical ~50 kb IncR plasmid (pYDC676). pYDC676 possessed the backbone and multi-IS loci closely related to IncR plasmids reported from aquatic bacteria, as well as animal and human K. pneumoniae strains, and carried an MDR region consisting of armA, blaDHA-1 and qnrB4, a combination that has been reported in IncR plasmids from K. pneumoniae ST11 strains in Europe and Asia. A plasmid with the identical IncR backbone and a similar MDR region containing blaDHA-1 and qnrB4 has also been reported in ST37 strains from Europe, suggesting potential dissemination of this lineage of IncR plasmids in K. pneumoniae ST37.K. pneumoniae ST37 strains with an MDR IncR plasmid carrying armA, blaDHA-1 and qnrB4 were identified in a hospital in the USA, where these resistance genes remain rare. The IncR backbone may play a role in the global dissemination of these resistance genes.© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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July 7, 2019

Multiple mechanisms responsible for strong Congo-red-binding variants of Escherichia coli O157:H7 strains.

High variability in the expression of csgD-dependent, biofilm-forming and adhesive properties is common among Shiga toxin-producing Escherichia coli. Although many strains of serotype O157:H7 form little biofilm, conversion to stronger biofilm phenotypes has been observed. In this study, we screened different strains of serotype O157:H7 for the emergence of strong Congo-red (CR) affinity/biofilm-forming properties and investigated the underlying genetic mechanisms. Two major mechanisms which conferred stronger biofilm phenotypes were identified: mutations (insertion, deletion, single nucleotide change) in rcsB region and stx-prophage excision from the mlrA site. Restoration of the native mlrA gene (due to prophage excision) resulted in strong biofilm properties to all variants. Whereas RcsB mutants showed weaker CR affinity and biofilm properties, it provided more possibilities for phenotypic presentations through heterogenic sequence mutations. Published by Oxford University Press on behalf of FEMS 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

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July 7, 2019

Whole genome sequence of Klebsiella pneumoniae U25, a hypermucoviscous, multidrug resistant, biofilm producing isolate from India.

Klebsiella pneumoniae U25 is a multidrug resistant strain isolated from a tertiary care hospital in Chennai, India. Here, we report the complete annotated genome sequence of strain U25 obtained using PacBio RSII. This is the first report of the whole genome of K. pneumoniaespecies from Chennai. It consists of a single circular chromosome of size 5,491,870-bp and two plasmids of size 211,813 and 172,619-bp. The genes associated with multidrug resistance were identified. The chromosome of U25 was found to have eight antibiotic resistant genes [blaOXA-1,blaSHV-28, aac(6′)1b-cr,catB3, oqxAB, dfrA1]. The plasmid pMGRU25-001 was found to have only one resistant gene (catA1) while plasmid pMGRU25-002 had 20 resistant genes [strAB, aadA1,aac(6′)-Ib, aac(3)-IId,sul1,2, blaTEM-1A,1B,blaOXA-9, blaCTX-M-15,blaSHV-11, cmlA1, erm(B),mph(A)]. A mutation in the porin OmpK36 was identified which is likely to be associated with the intermediate resistance to carbapenems in the absence of carbapenemase genes. U25 is one of the few K. pneumoniaestrains to harbour clustered regularly interspaced short palindromic repeats (CRISPR) systems. Two CRISPR arrays corresponding to Cas3 family helicase were identified in the genome. When compared to K. pneumoniaeNTUHK2044, a transposase gene InsH of IS5-13 was found inserted.

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July 7, 2019

Complete genome sequence of Serratia marcescens SmUNAM836, a nonpigmented multidrug-resistant strain isolated from a Mexican Patient with obstructive pulmonary disease.

Serratia marcescens SmUNAM836 is a multidrug-resistant clinical strain isolated in Mexico City from a patient with chronic obstructive pulmonary disease. Its complete genome sequence was determined using PacBio RS II SMRT technology, consisting of a 5.2-Mb chromosome and a 26.3-kb plasmid, encoding multiple resistance determinants and virulence factors. Copyright © 2016 Sandner-Miranda et al.

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July 7, 2019

Colistin-Nonsusceptible Pseudomonas aeruginosa Sequence Type 654 with blaNDM-1 Arrives in North America.

This study describes 3 different blaNDM-1 genetic platforms in 3 different species obtained from the same patient who was directly transferred to an institution in Calgary, Alberta, Canada, following a prolonged hospital stay in India. The blaNDM-1 in the Escherichia coli isolate was located on a 176-kb IncA/C plasmid contained within an ISCR1 region. The blaNDM-1 in the Providencia rettgeri isolate was located on a 117-kb IncT plasmid contained within Tn3000, while the blaNDM-1 in the Pseudomonas aeruginosa isolate was located on the chromosome within an ISCR3 region. This report highlights the plasticity of the genetic regions and environments associated with blaNDM-1. To the best of our knowledge, this is the first report of P. aeruginosa with blaNDM-1 identified in North America and the first report of blaOXA-181 in P. rettgeri. The P. aeruginosa isolate belonged to the international high-risk sequence type 654 clone and was nonsusceptible to colistin. This case emphasizes the need for the use of appropriate infection prevention and control measures and vigilant screening for carbapenem-resistant Gram-negative bacteria in patients with a history of travel to areas of endemicity, such as the Indian subcontinent. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

Rapid emergence and evolution of Staphylococcus aureus clones harbouring fusC-containing Staphylococcal cassette chromosome elements.

The prevalence of fusidic acid (FA) resistance amongst Staphylococcus aureus in New Zealand (NZ) is amongst the highest reported globally, with a recent study describing a resistance rate of approximately 28%. Three FA-resistant S. aureus clones (ST5 MRSA, ST1 MSSA and ST1 MRSA) have emerged over the past decade and now predominate in NZ, and in all three clones FA resistance is mediated by the fusC gene. In particular, ST5 MRSA has rapidly become the dominant MRSA clone in NZ, although the origin of FA-resistant ST5 MRSA has not been explored, and the genetic context of fusC in FA-resistant NZ isolates is unknown. To better understand the rapid emergence of FA-resistant S. aureus, we used population-based comparative genomics to characterise a collection of FA-resistant and FA-susceptible isolates from NZ. FA-resistant NZ ST5 MRSA displayed minimal genetic diversity, and represented a phylogenetically distinct clade within a global population model of clonal complex 5 (CC5) S. aureus. In all lineages, fusC was invariably located within staphylococcal cassette chromosome (SCC) elements, suggesting that SCC-mediated horizontal transfer is the primary mechanism of fusC dissemination. The genotypic association of fusC with mecA has important implications for the emergence of MRSA clones in populations with high usage of fusidic acid. In addition, we found that fusC was co-located with a recently described virulence factor (tirS) in dominant NZ S. aureus clones, suggesting a potential fitness advantage. This study points to the likely molecular mechanisms responsible for the successful emergence and spread of FA-resistant S. aureus. Copyright © 2016 Baines et al.

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July 7, 2019

A highly arginolytic Streptococcus species that potently antagonizes Streptococcus mutans.

The ability of certain oral biofilm bacteria to moderate pH through arginine metabolism by the arginine deiminase system (ADS) is a deterrent to the development of dental caries. Here, we characterize a novel Streptococcus strain, designated strain A12, isolated from supragingival dental plaque of a caries-free individual. A12 not only expressed the ADS pathway at high levels under a variety of conditions but also effectively inhibited growth and two intercellular signaling pathways of the dental caries pathogen Streptococcus mutans. A12 produced copious amounts of H2O2 via the pyruvate oxidase enzyme that were sufficient to arrest the growth of S. mutans. A12 also produced a protease similar to challisin (Sgc) of Streptococcus gordonii that was able to block the competence-stimulating peptide (CSP)-ComDE signaling system, which is essential for bacteriocin production by S. mutans. Wild-type A12, but not an sgc mutant derivative, could protect the sensitive indicator strain Streptococcus sanguinis SK150 from killing by the bacteriocins of S. mutans. A12, but not S. gordonii, could also block the XIP (comX-inducing peptide) signaling pathway, which is the proximal regulator of genetic competence in S. mutans, but Sgc was not required for this activity. The complete genome sequence of A12 was determined, and phylogenomic analyses compared A12 to streptococcal reference genomes. A12 was most similar to Streptococcus australis and Streptococcus parasanguinis but sufficiently different that it may represent a new species. A12-like organisms may play crucial roles in the promotion of stable, health-associated oral biofilm communities by moderating plaque pH and interfering with the growth and virulence of caries pathogens. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

Population structure and acquisition of the vanB resistance determinant in German clinical isolates of Enterococcus faecium ST192.

In the context of the global action plan to reduce the dissemination of antibiotic resistances it is of utmost importance to understand the population structure of resistant endemic bacterial lineages and to elucidate how bacteria acquire certain resistance determinants. Vancomycin resistant enterococci represent one such example of a prominent nosocomial pathogen on which nation-wide population analyses on prevalent lineages are scarce and data on how the bacteria acquire resistance, especially of the vanB genotype, are still under debate. With respect to Germany, an increased prevalence of VRE was noted in recent years. Here, invasive infections caused by sequence type ST192 VRE are often associated with the vanB-type resistance determinant. Hence, we analyzed 49 vanB-positive and vanB-negative E. faecium isolates by means of whole genome sequencing. Our studies revealed a distinct population structure and that spread of the Tn1549-vanB-type resistance involves exchange of large chromosomal fragments between vanB-positive and vanB-negative enterococci rather than independent acquisition events. In vitro filter-mating experiments support the hypothesis and suggest the presence of certain target sequences as a limiting factor for dissemination of the vanB element. Thus, the present study provides a better understanding of how enterococci emerge into successful multidrug-resistant nosocomial pathogens.

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July 7, 2019

Genomic and transcriptomic analysis of the streptomycin-dependent Mycobacterium tuberculosis strain 18b.

The ability of Mycobacterium tuberculosis to establish a latent infection (LTBI) in humans confounds the treatment of tuberculosis. Consequently, there is a need to discover new therapeutic agents that can kill M. tuberculosis both during active disease and LTBI. The streptomycin-dependent strain of M. tuberculosis, 18b, provides a useful tool for this purpose since upon removal of streptomycin (STR) it enters a non-replicating state that mimics latency both in vitro and in animal models.The 4.41 Mb genome sequence of M. tuberculosis 18b was determined and this revealed the strain to belong to clade 3 of the ancient ancestral lineage of the Beijing family. STR-dependence was attributable to insertion of a single cytosine in the 530 loop of the 16S rRNA and to a single amino acid insertion in the N-terminal domain of initiation factor 3. RNA-seq was used to understand the genetic programme activated upon STR-withdrawal and hence to gain insight into LTBI. This revealed reconfiguration of gene expression and metabolic pathways showing strong similarities between non-replicating 18b and M. tuberculosis residing within macrophages, and with the core stationary phase and microaerophilic responses.The findings of this investigation confirm the validity of 18b as a model for LTBI, and provide insight into both the evolution of tubercle bacilli and the functioning of the ribosome.

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