Multidrug-resistant New Delhi metallo-ß-lactamase 1 (NDM-1)-producing bacteria have spread globally and become a major clinical and public health threat. We report here the draft genome sequence of the Klebsiella pneumoniae clinical isolate 303K, harboring an NDM-1 coding sequence.
Faster growing and more virulent strains of methicillin resistant Staphylococcus aureus (MRSA) are increasingly displacing highly resistant MRSA. Elevated fitness in these MRSA is often accompanied by decreased and heterogeneous levels of methicillin resistance; however, the mechanisms for this phenomenon are not yet fully understood. Whole genome sequencing was used to investigate the genetic basis of this apparent correlation, in an isogenic MRSA strain pair that differed in methicillin resistance levels and fitness, with respect to growth rate. Sequencing revealed only one single nucleotide polymorphism (SNP) in the diadenylate cyclase gene dacA in the faster growing but less resistant strain. Diadenylate cyclases were recently discovered to synthesize the new second messenger cyclic diadenosine monophosphate (c-di-AMP). Introduction of this mutation into the highly resistant but slower growing strain reduced resistance and increased its growth rate, suggesting a direct connection between the dacA mutation and the phenotypic differences of these strains. Quantification of cellular c-di-AMP revealed that the dacA mutation decreased c-di-AMP levels resulting in reduced autolysis, increased salt tolerance and a reduction in the basal expression of the cell wall stress stimulon. These results indicate that c-di-AMP affects cell envelope-related signalling in S. aureus. The influence of c-di-AMP on growth rate and methicillin resistance in MRSA indicate that altering c-di-AMP levels could be a mechanism by which MRSA strains can increase their fitness levels by reducing their methicillin resistance levels.
HIV-1 coreceptor tropism assays are required to rule out the presence of CXCR4-tropic (non-R5) viruses prior treatment with CCR5 antagonists. Phenotypic (e.g., Trofile™, Monogram Biosciences) and genotypic (e.g., population sequencing linked to bioinformatic algorithms) assays are the most widely used. Although several next-generation sequencing (NGS) platforms are available, to date all published deep sequencing HIV-1 tropism studies have used the 454™ Life Sciences/Roche platform. In this study, HIV-1 co-receptor usage was predicted for twelve patients scheduled to start a maraviroc-based antiretroviral regimen. The V3 region of the HIV-1 env gene was sequenced using four NGS platforms: 454™, PacBio® RS (Pacific Biosciences), Illumina®, and Ion Torrent™ (Life Technologies). Cross-platform variation was evaluated, including number of reads, read length and error rates. HIV-1 tropism was inferred using Geno2Pheno, Web PSSM, and the 11/24/25 rule and compared with Trofile™ and virologic response to antiretroviral therapy. Error rates related to insertions/deletions (indels) and nucleotide substitutions introduced by the four NGS platforms were low compared to the actual HIV-1 sequence variation. Each platform detected all major virus variants within the HIV-1 population with similar frequencies. Identification of non-R5 viruses was comparable among the four platforms, with minor differences attributable to the algorithms used to infer HIV-1 tropism. All NGS platforms showed similar concordance with virologic response to the maraviroc-based regimen (75% to 80% range depending on the algorithm used), compared to Trofile (80%) and population sequencing (70%). In conclusion, all four NGS platforms were able to detect minority non-R5 variants at comparable levels suggesting that any NGS-based method can be used to predict HIV-1 coreceptor usage.
Advances in DNA sequencing technology have improved our ability to characterize most genomic diversity. However, accurate resolution of large structural events is challenging because of the short read lengths of second-generation technologies. Third-generation sequencing technologies, which can yield longer multikilobase reads, have the potential to address limitations associated with genome assembly. Here we combine sequencing data from second- and third-generation DNA sequencing technologies to assemble the two-chromosome genome of a recent Haitian cholera outbreak strain into two nearly finished contigs at >99.9% accuracy. Complex regions with clinically relevant structure were completely resolved. In separate control assemblies on experimental and simulated data for the canonical N16961 cholera reference strain, we obtained 14 scaffolds of greater than 1 kb for the experimental data and 8 scaffolds of greater than 1 kb for the simulated data, which allowed us to correct several errors in contigs assembled from the short-read data alone. This work provides a blueprint for the next generation of rapid microbial identification and full-genome assembly.
Liberibacter crescens BT-1, a Gram-negative, rod-shaped bacterial isolate, was previously recovered from mountain papaya to gain insight on Huanglongbing (HLB) and Zebra Chip (ZC) diseases. The genome of BT-1 was sequenced at the Interdisciplinary Center for Biotechnology Research (ICBR) at the University of Florida. A finished assembly and annotation yielded one chromosome with a length of 1,504,659 bp and a G+C content of 35.4%. Comparison to other species in the Liberibacter genus, L. crescens has many more genes in thiamine and essential amino acid biosynthesis. This likely explains why L. crescens BT-1 is culturable while the known Liberibacter strains have not yet been cultured. Similar to Candidatus L. asiaticus psy62, the L. crescens BT-1 genome contains two prophage regions.
This month’s Genome Watch describes the impact of next-generation sequencing on the ‘real-time’ analysis of pathogen genomes during outbreaks.
Myroides odoratimimus (M. odoratimimus) has been gradually implicated as an important nosocomial pathogen that poses a serious health threat to immunocompromised patients owing to its multi-drug resistance. However, the resistance mechanism is currently unclear. To clarify the antibiotic resistance and infectivity mechanisms of M. odoratimimus, whole genome sequencing was performed on the multi-drug-resistant M. odoratimimus strain PR63039. The genome sequence was completed with single molecule real-time (SMRT) technologies. Then, annotation was performed using RAST and IMG-ER. A number of databases and software programs were used to analyze the genomic characteristics, including GC-Profile, ISfinder, CG viewer, ARDB, CARD, ResFinder, the VFDB database, PHAST and Progressive Mauve. The M. odoratimimus PR63039 genome consisted of a chromosome and a plasmid. The genome contained a large number of resistance genes and virulence factors. The distribution of the resistance genes was distinctive, and a resistance region named MY63039-RR was found. The subsystem features generated by RAST indicated that the annotated genome had 108 genes that were potentially involved in virulence, disease and defense, all of which had strong associations with resistance and pathogenicity. The prophage analysis showed two incomplete prophages in the genome. The genomic analysis of M. odoratimimus PR63039 partially clarified its antibiotic resistance mechanisms and virulence factors. Obtaining a clear understanding of its genomic characteristics will be conducive to the management of multidrug-resistant M. odoratimimus.
From early 2012, a novel clone of vancomycin resistant Enterococcus faecium (assigned the multi locus sequence type ST796) was simultaneously isolated from geographically separate hospitals in south eastern Australia and New Zealand. Here we describe the complete genome sequence of Ef_aus0233, a representative ST796 E. faecium isolate. We used PacBio single molecule real-time sequencing to establish a high quality, fully assembled genome comprising a circular chromosome of 2,888,087 bp and five plasmids. Comparison of Ef_aus0233 to other E. faecium genomes shows Ef_aus0233 is a member of the epidemic hospital-adapted lineage and has evolved from an ST555-like ancestral progenitor by the accumulation or modification of five mosaic plasmids and five putative prophage, acquisition of two cryptic genomic islands, accrued chromosomal single nucleotide polymorphisms and a 80 kb region of recombination, also gaining Tn1549 and Tn916, transposons conferring resistance to vancomycin and tetracycline respectively. The genomic dissection of this new clone presented here underscores the propensity of the hospital E. faecium lineage to change, presumably in response to the specific conditions of hospital and healthcare environments.
Klebsiella pneumoniae causes severe lung and bloodstream infections that are difficult to treat due to multidrug resistance. We hypothesized that antimicrobial resistance can be reversed by targeting chromosomal non-essential genes that are not responsible for acquired resistance but essential for resistant bacteria under therapeutic concentrations of antimicrobials. Conditional essentiality of individual genes to antimicrobial resistance was evaluated in an epidemic multidrug-resistant clone of K. pneumoniae (ST258). We constructed a high-density transposon mutant library of >430,000 unique Tn5 insertions and measured mutant depletion upon exposure to three clinically relevant antimicrobials (colistin, imipenem or ciprofloxacin) by Transposon Directed Insertion-site Sequencing (TraDIS). Using this high-throughput approach, we defined three sets of chromosomal non-essential genes essential for growth during exposure to colistin (n?=?35), imipenem (n?=?1) or ciprofloxacin (n?=?1) in addition to known resistance determinants, collectively termed the “secondary resistome”. As proof of principle, we demonstrated that inactivation of a non-essential gene not previously found linked to colistin resistance (dedA) restored colistin susceptibility by reducing the minimum inhibitory concentration from 8 to 0.5?µg/ml, 4-fold below the susceptibility breakpoint (S?=?2?µg/ml). This finding suggests that the secondary resistome is a potential target for developing antimicrobial “helper” drugs that restore the efficacy of existing antimicrobials.
Polymyxin antibiotics are used as last-resort therapies to treat infections caused by multidrug-resistant Gram-negative bacteria. The plasmid-mediated colistin resistance determinant MCR-1 has been identified in Enterobacteriaceae in China. We did this study to investigate the prevalence of the mcr-1 gene in clinical isolates from patients with bloodstream infections in China.Clinical isolates of Escherichia coli and Klebsiella pneumoniae were collected from patients with bloodstream infections at 28 hospitals in China, then screened for colistin resistance by broth microdilution and for the presence of the mcr-1 gene by PCR amplification. We subjected mcr-1-positive isolates to genotyping, susceptibility testing, and clinical data analysis. We established the genetic location of mcr-1 with Southern blot hybridisation, and we analysed plasmids containing mcr-1 with filter mating, electroporation, and DNA sequencing.2066 isolates, consisting of 1495 E coli isolates and 571 K pneumoniae isolates were collected. Of the 1495 E coli isolates, 20 (1%) were mcr-1-positive, whereas we detected only one (<1%) mcr-1-positive isolate among the 571 K pneumoniae isolates. All mcr-1-positive E coli and K pneumoniae isolates were resistant to colistin, with minimum inhibitory concentrations values in the range of 4-32 mg/L, except for one E coli isolate that had a minimum inhibitory concentration less than or equal to 0·06 mg/L. All 21 mcr-1-positive isolates were susceptible to tigecycline and 20 isolates (95%) were susceptible to the carbapenem and ß-lactamase inhibitor combination piperacillin and tazobactam. One mcr-1-positive E coli isolate also produced NDM-5, which confers resistance to beta-lactam antibiotics. The 21 mcr-1-positive isolates were clonally diverse and carried mcr-1 on two types of plasmids, a 33 kb IncX4 plasmid and a 61 kb Inc12 plasmid. The 30 day mortality of the patients with bloodstream infections caused by mcr-1-positive isolates was zero.mcr-1-positive isolates from bloodstream infections were rare, sporadic, and remained susceptible to many antimicrobial agents. E coli, rather than K pneumoniae, was the main host of the mcr-1 gene. Further studies are needed to clarify the clinical impact of this novel resistance gene.National Natural Science Foundation of China. Copyright © 2017 Elsevier Ltd. All rights reserved.
The hepatitis viruses represent a major public health problem worldwide. Procedures for characterization of the genomic composition of their populations, accurate diagnosis, identification of multiple infections, and information on inhibitor-escape mutants for treatment decisions are needed. Deep sequencing methodologies are extremely useful for these viruses since they replicate as complex and dynamic quasispecies swarms whose complexity and mutant composition are biologically relevant traits. Population complexity is a major challenge for disease prevention and control, but also an opportunity to distinguish among related but phenotypically distinct variants that might anticipate disease progression and treatment outcome. Detailed characterization of mutant spectra should permit choosing better treatment options, given the increasing number of new antiviral inhibitors available. In the present review we briefly summarize our experience on the use of deep sequencing for the management of hepatitis virus infections, particularly for hepatitis B and C viruses, and outline some possible new applications of deep sequencing for these important human pathogens. Copyright © 2016 Elsevier B.V. All rights reserved.
Serious bacterial infections in immunocompromised patients require highly effective antibacterial therapy for cure, and thus, this setting may reveal novel mechanisms by which bacteria circumvent antibiotics in the absence of immune pressure. Here, an infant with leukemia developed vancomycin-resistant Enterococcus faecium (VRE) bacteremia that persisted for 26 days despite appropriate antibiotic therapy. Sequencing of 22 consecutive VRE isolates identified the emergence of a single missense mutation (L152F) in relA, which constitutively activated the stringent response, resulting in elevated baseline levels of the alarmone guanosine tetraphosphate (ppGpp). Although the mutant remained susceptible to both linezolid and daptomycin in clinical MIC testing and during planktonic growth, it demonstrated tolerance to high doses of both antibiotics when growing in a biofilm. This biofilm-specific gain in resistance was reflected in the broad shift in transcript levels caused by the mutation. Only an experimental biofilm-targeting ClpP-activating antibiotic was able to kill the mutant strain in an established biofilm. The relA mutation was associated with a fitness trade-off, forming smaller and less-well-populated biofilms on biological surfaces. We conclude that clinically relevant relA mutations can emerge during prolonged VRE infection, causing baseline activation of the stringent response, subsequent antibiotic tolerance, and delayed eradication in an immunocompromised state.The increasing prevalence of antibiotic-resistant bacterial pathogens is a major challenge currently facing the medical community. Such pathogens are of particular importance in immunocompromised patients as these individuals may favor emergence of novel resistance determinants due to lack of innate immune defenses and intensive antibiotic exposure. During the course of chemotherapy, a patient developed prolonged bacteremia with vancomycin-resistant Enterococcus faecium that failed to clear despite multiple front-line antibiotics. The consecutive bloodstream isolates were sequenced, and a single missense mutation identified in the relA gene, the mediator of the stringent response. Strains harboring the mutation had elevated baseline levels of the alarmone and displayed heightened resistance to the bactericidal activity of multiple antibiotics, particularly in a biofilm. Using a new class of compounds that modulate ClpP activity, the biofilms were successfully eradicated. These data represent the first clinical emergence of mutations in the stringent response in vancomycin-resistant entereococci. Copyright © 2017 Honsa et al.
Here, we report the complete genome sequence of the triclosan- and multidrug-resistant Pseudomonas aeruginosa strain B10W, obtained from municipal wastewater in Hawaii. The bacterium has a 6.7-Mb genome, contains 6,391 coding sequences and 78 RNAs, with an average G+C content of 66.2 mol%. Copyright © 2017 Zhong et al.
Acinetobacter baumannii has been becoming a great challenge to clinicians due to their resistance to almost all available antibiotics. In this study, we sequenced the genome from a multiple antibiotics resistant Acinetobacter baumannii stain which was named A. baumannii-1isolated from China by SMRT sequencing technology to explore its potential mechanisms to antibiotic resistance. We found that several mechanisms might contribute to the antibiotic resistance of Acinetobacter baumannii. Specifically, we found that SNP in genes associated with nucleotide excision repair and ABC transporter might contribute to its resistance to multiple antibiotics; we also found that specific genes associated with bacterial DNA integration and recombination, DNA-mediated transposition and response to antibiotics might contribute to its resistance to multiple antibiotics; Furthermore, specific genes associated with penicillin and cephalosporin biosynthetic pathway and specific genes associated with CHDL and MBL ß-lactamase genes might contribute to its resistance to multiple antibiotics. Thus, the detailed mechanisms by which Acinetobacter baumannii show extensive resistance to multiple antibiotics are very complicated. Such a study might be helpful to develop new strategies to control Acinetobacter baumannii infection. Copyright © 2017 Elsevier B.V. All rights reserved.
The amount of antibiotics needed to counteract frequent piscirickettsiosis outbreaks is a major concern for the Chilean salmon industry. Resistance to antibiotics may contribute to this issue. To understand the genetics underlying Piscirickettsia salmonis-resistant phenotypes, the genome of AY3800B, an oxytetracycline-resistant isolate bearing a multidrug resistance plasmid, is presented here. Copyright © 2017 Bohle et al.
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