Danesh Moradigaravand

The essential mycobacterial amidotransferase GatCAB is a modulator of specific translational fidelity.

Although regulation of translation fidelity is an essential process1–7, diverse organisms and organelles have differing requirements of translational accuracy8–15, and errors in gene translation serve an adaptive function under certain conditions16–20. Therefore, optimal levels of fidelity may vary according to context. Most bacteria utilize a two-step pathway for the specific synthesis of aminoacylated glutamine and/or asparagine tRNAs, involving the glutamine amidotransferase GatCAB21–25, but it had not been appreciated that GatCAB may play a role in modulating mistranslation rates. Here, by using a forward genetic screen, we show that the mycobacterial GatCAB enzyme complex mediates the translational fidelity of glutamine and asparagine codons. We identify mutations in gatA that cause partial loss of function in the holoenzyme, with a consequent increase in rates of mistranslation. By monitoring single-cell transcription dynamics, we demonstrate that reduced gatCAB expression leads to increased mistranslation rates, which result in enhanced rifampicin-specific phenotypic resistance. Consistent with this, strains with mutations in gatA from clinical isolates of Mycobacterium tuberculosis show increased mistranslation, with associated antibiotic tolerance, suggesting a role for mistranslation as an adaptive strategy in tuberculosis. Together, our findings demonstrate a potential role for the indirect tRNA aminoacylation pathway in regulating translational fidelity and adaptive mistranslation.

Recent independent emergence of multiple multidrug-resistant Serratia marcescens clones within the United Kingdom and Ireland

Serratia marcescens, a member of the Enterobacteriaceae family, is a Gram-negative bacterium responsible for a wide range of nosocomial infections. The emergence of multidrug-resistant strains is an increasing danger to public health. To design effective means to control the dissemination of S. marcescens, an in-depth analysis of the population structure and variation is required. Utilizing whole-genome sequencing, we characterized the population structure and variation, as well as the antimicrobial resistance determinants, of a systematic collection of antimicrobial-resistant S. marcescens associated with bloodstream infections in hospitals across the United Kingdom and Ireland between 2001 and 2011. Our results show that S. marcescens is a diverse species with a high level of genomic variation. However, the collection was largely composed of a limited number of clones that emerged from this diverse background within the past few decades. We identified potential recent transmissions of these clones, within and between hospitals, and showed that they have acquired antimicrobial resistance determinants for different beta-lactams, ciprofloxacin, and tetracyclines on multiple occasions. The expansion of these multidrug-resistant clones suggests that the treatment of S. marcescens infections will become increasingly difficult in the future.

Whole-genome sequencing of multidrug-resistant Mycobacterium tuberculosis isolates from Myanmar

Highlights • Drug-resistant tuberculosis (TB) is a major health threat in Myanmar.• The first whole-genome sequencing study of drug-resistant TB from Myanmar.• Introduction of second-line drug susceptibility testing as part of routine diagnosis in Myanmar is needed.

Evolution of the Staphylococcus argenteus ST2250 Clone in Northeastern Thailand Is Linked with the Acquisition of Livestock-Associated Staphylococcal Genes

ABSTRACT Staphylococcus argenteus is a newly named species previously described as a divergent lineage of Staphylococcus aureus that has recently been shown to have a global distribution. Despite growing evidence of the clinical importance of this species, knowledge about its population epidemiology and genomic architecture is limited. We used whole-genome sequencing to evaluate and compare S. aureus (n = 251) and S. argenteus (n = 68) isolates from adults with staphylococcal sepsis at several hospitals in northeastern Thailand between 2006 and 2013. The majority (82%) of the S. argenteus isolates were of multilocus sequence type 2250 (ST2250). S. aureus was more diverse, although 43% of the isolates belonged to ST121. Bayesian analysis suggested an S. argenteus ST2250 substitution rate of 4.66 (95% confidence interval [CI], 3.12 to 6.38) mutations per genome per year, which was comparable to the S. aureus ST121 substitution rate of 4.07 (95% CI, 2.61 to 5.55). S. argenteus ST2250 emerged in Thailand an estimated 15 years ago, which contrasts with the S. aureus ST1, ST88, and ST121 clades that emerged around 100 to 150 years ago. Comparison of S. argenteus ST2250 genomes from Thailand and a global collection indicated a single introduction into Thailand, followed by transmission to local and more distant countries in Southeast Asia and further afield. S. argenteus and S. aureus shared around half of their core gene repertoire, indicating a high level of divergence and providing strong support for their classification as separate species. Several gene clusters were present in ST2250 isolates but absent from the other S. argenteus and S. aureus study isolates. These included multiple exotoxins and antibiotic resistance genes that have been linked previously with livestock-associated S. aureus, consistent with a livestock reservoir for S. argenteus. These genes appeared to be associated with plasmids and mobile genetic elements and may have contributed to the biological success of ST2250. IMPORTANCE In this study, we used whole-genome sequencing to understand the genome evolution and population structure of a systematic collection of ST2250 S. argenteus isolates. A newly identified ancestral species of S. aureus, S. argenteus has become increasingly known as a clinically important species that has been reported recently across various countries. Our results indicate that S. argenteus has spread at a relatively rapid pace over the past 2 decades across northeastern Thailand and acquired multiple exotoxin and antibiotic resistance genes that have been linked previously with livestock-associated S. aureus. Our findings highlight the clinical importance and potential pathogenicity of S. argenteus as a recently emerging pathogen. IMPORTANCE In this study, we used whole-genome sequencing to understand the genome evolution and population structure of a systematic collection of ST2250 S. argenteus isolates. A newly identified ancestral species of S. aureus, S. argenteus has become increasingly known as a clinically important species that has been reported recently across various countries. Our results indicate that S. argenteus has spread at a relatively rapid pace over the past 2 decades across northeastern Thailand and acquired multiple exotoxin and antibiotic resistance genes that have been linked previously with livestock-associated S. aureus. Our findings highlight the clinical importance and potential pathogenicity of S. argenteus as a recently emerging pathogen.

What Is Resistance? Impact of Phenotypic versus Molecular Drug Resistance Testing on Therapy for Multi- and Extensively Drug-Resistant Tuberculosis.

ABSTRACT Rapid and accurate drug susceptibility testing (DST) is essential for the treatment of multi- and extensively drug-resistant tuberculosis (M/XDR-TB). We compared the utility of genotypic DST assays with phenotypic DST (pDST) using Bactec 960 MGIT or Löwenstein-Jensen to construct M/XDR-TB treatment regimens for a cohort of 25 consecutive M/XDR-TB patients and 15 possible anti-TB drugs. Genotypic DST results from Cepheid GeneXpert MTB/RIF (Xpert) and line probe assays (LPAs; Hain GenoType MTBDRplus 2.0 and MTBDRsl 2.0) and whole-genome sequencing (WGS) were translated into individual algorithm-derived treatment regimens for each patient. We further analyzed if discrepancies between the various methods were due to flaws in the genotypic or phenotypic test using MIC results. Compared with pDST, the average agreement in the number of drugs prescribed in genotypic regimens ranged from just 49% (95% confidence interval [CI], 39 to 59%) for Xpert and 63% (95% CI, 56 to 70%) for LPAs to 93% (95% CI, 88 to 98%) for WGS. Only the WGS regimens did not contain any drugs to which pDST showed resistance. Importantly, MIC testing revealed that pDST likely underestimated the true rate of resistance for key drugs (rifampin, levofloxacin, moxifloxacin, and kanamycin) because critical concentrations (CCs) were too high. WGS can be used to rule in resistance even in M/XDR strains with complex resistance patterns, but pDST for some drugs is still needed to confirm susceptibility and construct the final regimens. Some CCs for pDST need to be reexamined to avoid systematic false-susceptible results in low-level resistant isolates.

Evolution and Epidemiology of Multidrug-Resistant Klebsiella pneumoniae in the United Kingdom and Ireland

ABSTRACT Klebsiella pneumoniae is a human commensal and opportunistic pathogen that has become a leading causative agent of hospital-based infections over the past few decades. The emergence and global expansion of hypervirulent and multidrug-resistant (MDR) clones of K. pneumoniae have been increasingly reported in community-acquired and nosocomial infections. Despite this, the population genomics and epidemiology of MDR K. pneumoniae at the national level are still poorly understood. To obtain insights into these, we analyzed a systematic large-scale collection of invasive MDR K. pneumoniae isolates from hospitals across the United Kingdom and Ireland. Using whole-genome phylogenetic analysis, we placed these in the context of previously sequenced K. pneumoniae populations from geographically diverse countries and identified their virulence and drug resistance determinants. Our results demonstrate that United Kingdom and Ireland MDR isolates are a highly diverse population drawn from across the global phylogenetic tree of K. pneumoniae and represent multiple recent international introductions that are mainly from Europe but in some cases from more distant countries. In addition, we identified novel genetic determinants underlying resistance to beta-lactams, gentamicin, ciprofloxacin, and tetracyclines, indicating that both increased virulence and resistance have emerged independently multiple times throughout the population. Our data show that MDR K. pneumoniae isolates in the United Kingdom and Ireland have multiple distinct origins and appear to be part of a globally circulating K. pneumoniae population. IMPORTANCE Klebsiella pneumoniae is a major human pathogen that has been implicated in infections in healthcare settings over the past few decades. Antimicrobial treatment of K. pneumoniae infections has become increasingly difficult as a consequence of the emergence and spread of strains that are resistant to multiple antimicrobials. To better understand the spread of resistant K. pneumoniae, we studied the genomes of a large-scale population of extensively antimicrobial-resistant K. pneumoniae in the United Kingdom and Ireland by utilizing the fine resolution that whole-genome sequencing of pathogen genomes provides. Our results indicate that the K. pneumoniae population is highly diverse and that, in some cases, resistant strains appear to have spread across the country over a few years. In addition, we found evidence that some strains have acquired antimicrobial resistance genes independently, presumably in response to antimicrobial treatment. IMPORTANCE Klebsiella pneumoniae is a major human pathogen that has been implicated in infections in healthcare settings over the past few decades. Antimicrobial treatment of K. pneumoniae infections has become increasingly difficult as a consequence of the emergence and spread of strains that are resistant to multiple antimicrobials. To better understand the spread of resistant K. pneumoniae, we studied the genomes of a large-scale population of extensively antimicrobial-resistant K. pneumoniae in the United Kingdom and Ireland by utilizing the fine resolution that whole-genome sequencing of pathogen genomes provides. Our results indicate that the K. pneumoniae population is highly diverse and that, in some cases, resistant strains appear to have spread across the country over a few years. In addition, we found evidence that some strains have acquired antimicrobial resistance genes independently, presumably in response to antimicrobial treatment.

Some synonymous and non-synonymous gyrA mutations in Mycobacterium tuberculosis lead to systematic false-resistance results to fluoroquinolones with the Hain GenoType MTBDRsl assays.

ABSTRACT In this study, using the Hain GenoType MTBDRsl assays (versions 1 and 2), we found that some nonsynonymous and synonymous mutations in gyrA in Mycobacterium tuberculosis result in systematic false-resistance results to fluoroquinolones by preventing the binding of wild-type probes. Moreover, such mutations can prevent the binding of mutant probes designed for the identification of specific resistance mutations. Although these mutations are likely rare globally, they occur in approximately 7% of multidrug-resistant tuberculosis strains in some settings.

dfrA thyA Double Deletion in para-Aminosalicylic Acid-Resistant Mycobacterium tuberculosis Beijing Strains

This is the final version of the article. It first appeared from the American Society for Microbiology via http://dx.doi.org/10.1128/AAC.00253-16

The dissemination of multidrug-resistant Enterobacter cloacae throughout the UK and Ireland.

Enterobacter cloacae is a clinically important Gram-negative member of the Enterobacteriaceae, which has increasingly been recognized as a major pathogen in nosocomial infections. Despite this, knowledge about the population structure and the distribution of virulence factors and antibiotic-resistance determinants of this species is scarce. In this study, we analysed a systematic collection of multidrug-resistant E. cloacae isolated between 2001 and 2011 from bloodstream infections across hospitals in the UK and Ireland. We found that the population is characterized by the presence of multiple clones formed at widely different time periods in the past. The clones exhibit a high degree of geographical heterogeneity, which indicates extensive dissemination of these E. cloacae clones across the UK and Ireland. These findings suggest that a diverse, community-based, commensal population underlies multidrug-resistant E. cloacae infections within hospitals.

Wild-Type and Non-Wild-Type Mycobacterium tuberculosis MIC Distributions for the Novel Fluoroquinolone Antofloxacin Compared with Those for Ofloxacin, Levofloxacin, and Moxifloxacin

ABSTRACT Antofloxacin (AFX) is a novel fluoroquinolone that has been approved in China for the treatment of infections caused by a variety of bacterial species. We investigated whether it could be repurposed for the treatment of tuberculosis by studying its in vitro activity. We determined the wild-type and non-wild-type MIC ranges for AFX as well as ofloxacin (OFX), levofloxacin (LFX), and moxifloxacin (MFX), using the microplate alamarBlue assay, of 126 clinical Mycobacterium tuberculosis strains from Beijing, China, of which 48 were OFX resistant on the basis of drug susceptibility testing on Löwenstein-Jensen medium. The MIC distributions were correlated with mutations in the quinolone resistance-determining regions of gyrA (Rv0006) and gyrB (Rv0005). Pharmacokinetic/pharmacodynamic (PK/PD) data for AFX were retrieved from the literature. AFX showed lower MIC levels than OFX but higher MIC levels than LFX and MFX on the basis of the tentative epidemiological cutoff values (ECOFFs) determined in this study. All strains with non-wild-type MICs for AFX harbored known resistance mutations that also resulted in non-wild-type MICs for LFX and MFX. Moreover, our data suggested that the current critical concentration of OFX for Löwenstein-Jensen medium that was recently revised by the World Health Organization might be too high, resulting in the misclassification of phenotypically non-wild-type strains with known resistance mutations as wild type. On the basis of our exploratory PK/PD calculations, the current dose of AFX is unlikely to be optimal for the treatment of tuberculosis, but higher doses could be effective.