A surgical heater-cooler unit has been implicated as the source for Mycobacterium chimaera infections among cardiac surgery patients in several countries. We isolated M. chimaera from heater-cooler units and patient infections in the United States. Whole-genome sequencing corroborated a risk for these units acting as a reservoir for this pathogen.
Despite their ubiquity, relatively few bacteriophages have been characterized. Here, we set out to explore Caulobacter bacteriophages (caulophages) in the rhizosphere and characterized Kronos, the first caulophage isolated from the rhizosphere. Kronos is a member of the Siphoviridae family since it has a long flexible tail. In addition, an analysis of the Kronos genome indicated that many of the predicted proteins were distantly related to those of bacteriophages in the lambdoid family. Consistent with this observation, we were able to demonstrate the presence of cos sites that are similar to those found at the ends of lambdoid phage genomes. Moreover, Kronos displayed a relatively rare head and tail morphology compared to other caulophages but was similar to that of the lambdoid phages. Taken together, these data indicate that Kronos is distantly related to lambdoid phages and may represent a new Siphoviridae genus.
Talaromyces marneffei (T. marneffei) is a medically important opportunistic dimorphic fungus that infects both humans and bamboo rats. However, the mechanisms of transmission and pathogenicity of T. marneffei are poorly understood. In our study, we combined Illumina and PacBio sequencing technologies to sequence and assemble a complete genome of T. marneffei. To elucidate the transmission route and source, we sequenced three additional T. marneffei isolates using Illumina sequencing technology. Variations among isolates were used to develop a multilocus sequence typing (MLST) system comprising five housekeeping genes that can be used to discriminate between isolates derived from different sources. Our analysis revealed that human and bamboo rat share identical genotypes in these five loci. Thus, we hypothesized that T. marneffei is transmitted to humans through inhalation of spores in the surrounding environment into the lungs and that the bamboo rat can serve as an important natural reservoir for pathogens. Furthermore, we also identified temperature-dependent polyketide synthases, non-ribosomal peptide synthetases and secreted proteins as putative pathogenicity-related factors. In addition, we identified antifungal drug targets that can be investigated in future studies to elucidate the mechanisms underlying drug resistance. In summary, our study presents the basic features of the T. marneffei genome and provides insights into the transmission and pathogenicity of T. marneffei, which warrant fundamental experimental research.Copyright © 2019 Elsevier Inc. All rights reserved.
Infection by Helicobacter pylori is the primary cause of gastric adenocarcinoma. The most potent H. pylori virulence factor is cytotoxin-associated gene A (CagA), which is translocated by a type 4 secretion system (T4SS) into gastric epithelial cells and activates oncogenic signaling pathways. The gene cagY encodes for a key component of the T4SS and can undergo gene rearrangements. We have shown that the cancer chemopreventive agent a-difluoromethylornithine (DFMO), known to inhibit the enzyme ornithine decarboxylase, reduces H. pylori-mediated gastric cancer incidence in Mongolian gerbils. In the present study, we questioned whether DFMO might directly affect H. pylori pathogenicity. We show that H. pylori output strains isolated from gerbils treated with DFMO exhibit reduced ability to translocate CagA in gastric epithelial cells. Further, we frequently detected genomic modifications in the middle repeat region of the cagY gene of output strains from DFMO-treated animals, which were associated with alterations in the CagY protein. Gerbils did not develop carcinoma when infected with a DFMO output strain containing rearranged cagY or the parental strain in which the wild-type cagY was replaced by cagY with DFMO-induced rearrangements. Lastly, we demonstrate that in vitro treatment of H. pylori by DFMO induces oxidative DNA damage, expression of the DNA repair enzyme MutS2, and mutations in cagY, demonstrating that DFMO directly affects genomic stability. Deletion of mutS2 abrogated the ability of DFMO to induce cagY rearrangements directly. In conclusion, DFMO-induced oxidative stress in H. pylori leads to genomic alterations and attenuates virulence.
When choosing antibiotics to treat bacterial infections, it is assumed that the susceptibility of the target bacteria to an antibiotic is reflected by laboratory estimates of the minimum inhibitory concentration (MIC) needed to prevent bacterial growth. A caveat of using MIC data for this purpose is heteroresistance, the presence of a resistant subpopulation in a main population of susceptible cells. We investigated the prevalence and mechanisms of heteroresistance in 41 clinical isolates of the pathogens Escherichia coli, Salmonella enterica, Klebsiella pneumoniae and Acinetobacter baumannii against 28 different antibiotics. For the 766 bacteria-antibiotic combinations tested, as much as 27.4% of the total was heteroresistant. Genetic analysis demonstrated that a majority of heteroresistance cases were unstable, with an increased resistance of the subpopulations resulting from spontaneous tandem amplifications, typically including known resistance genes. Using mathematical modelling, we show how heteroresistance in the parameter range estimated in this study can result in the failure of antibiotic treatment of infections with bacteria that are classified as antibiotic susceptible. The high prevalence of heteroresistance with the potential for treatment failure highlights the limitations of MIC as the sole criterion for susceptibility determinations. These results call for the development of facile and rapid protocols to identify heteroresistance in pathogens.
Primary sclerosing cholangitis (PSC) is a chronic inflammatory liver disease and its frequent complication with ulcerative colitis highlights the pathogenic role of epithelial barrier dysfunction. Intestinal barrier dysfunction has been implicated in the pathogenesis of PSC, yet its underlying mechanism remains unknown. Here, we identify Klebsiella pneumonia in the microbiota of patients with PSC and demonstrate that K.?pneumoniae disrupts the epithelial barrier to initiate bacterial translocation and liver inflammatory responses. Gnotobiotic mice inoculated with PSC-derived microbiota exhibited T helper 17 (TH17) cell responses in the liver and increased susceptibility to hepatobiliary injuries. Bacterial culture of mesenteric lymph nodes in these mice isolated K.?pneumoniae, Proteus mirabilis and Enterococcus gallinarum, which were prevalently detected in patients with PSC. A bacterial-organoid co-culture system visualized the epithelial-damaging effect of PSC-derived K.?pneumoniae that was associated with bacterial translocation and susceptibility to TH17-mediated hepatobiliary injuries. We also show that antibiotic treatment ameliorated the TH17 immune response induced by PSC-derived microbiota. These results highlight the role of pathobionts in intestinal barrier dysfunction and liver inflammation, providing insights into therapeutic strategies for PSC.
Trypanosoma evansi is the causative agent of the animal trypanosomiasis surra, a disease with serious economic burden worldwide. The availability of the genome of its closely related parasite Trypanosoma brucei allows us to compare their genetic and evolutionarily shared and distinct biological features. The complete genomic sequence of the T. evansi YNB strain was obtained using a combination of genomic and transcriptomic sequencing, de novo assembly, and bioinformatic analysis. The genome size of the T. evansi YNB strain was 35.2 Mb, showing 96.59% similarity in sequence and 88.97% in scaffold alignment with T. brucei. A total of 8,617 protein-coding genes, accounting for 31% of the genome, were predicted. Approximately 1,641 alternative splicing events of 820 genes were identified, with a majority mediated by intron retention, which represented a major difference in post-transcriptional regulation between T. evansi and T. brucei. Disparities in gene copy number of the variant surface glycoprotein, expression site-associated genes, microRNAs, and RNA-binding protein were clearly observed between the two parasites. The results revealed the genomic determinants of T. evansi, which encoded specific biological characteristics that distinguished them from other related trypanosome species.
The gammaherpesviruses, including Epstein-Barr virus (EBV), Kaposi’s sarcoma-associated herpesvirus (KSHV), and murine gammaherpesvirus 68 (MHV68, MuHV-4, ?HV68), are etiologic agents of a wide range of lymphomas and non-hematological malignancies. These viruses possess large and highly dense dsDNA genomes that feature >80 bidirectionally positioned open reading frames (ORFs). The abundance of overlapping transcripts and extensive splicing throughout these genomes have until now prohibited high throughput-based resolution of transcript structures. Here, we integrate the capabilities of long-read sequencing with the accuracy of short-read platforms to globally resolve MHV68 transcript structures using the transcript resolution through integration of multi-platform data (TRIMD) pipeline. This approach reveals highly complex features, including: (1) pervasive overlapping transcript structures; (2) transcripts containing intra-gene or trans-gene splices that yield chimeric ORFs; (3) antisense and intergenic transcripts containing ORFs; and (4) noncoding transcripts. This work sheds light on the underappreciated complexity of gammaherpesvirus transcription and provides an extensively revised annotation of the MHV68 transcriptome. Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.
Ceftriaxone and ciprofloxacin are the drugs of choice in treatment of invasive Salmonella infections. This study discovered a novel type of plasmid, pSa44-CIP-CRO, which was recovered from a S. London strain isolated from meat product and comprised genetic determinants that encoded resistance to both ciprofloxacin and ceftriaxone. This plasmid could be resolved into two daughter plasmids and co-exist with such daughter plasmids in a dynamic form in Salmonella; yet it was only present as a single plasmid in Escherichia coli. One daughter plasmid, pSa44-CRO, was found to carry the blaCTX-M-130 gene, which encodes resistance to ceftriaxone, whereas the other plasmid, pSa44-CIP, carried multiple PMQR genes such as qnrB6-aac(6′)-Ib-cr, which mediated resistance to ciprofloxacin. These two daughter plasmids could be integrated into one single plasmid through ISPa40 mediated homologous recombination. Mouse infection and treatment experiments showed that carriage of plasmid, pSa44-CIP-CRO by S. typhimurium led to the impairment of treatment by ciprofloxacin or cefitiofur, a veterinary drug with similar properties as ceftriaxone. In conclusion, dissemination of such conjugative plasmids impairs current choices of treatment for life-threatening Salmonella infection and hence constitutes a serious public health threat.
Carbapenem-resistant Klebsiella pneumoniae (CRKP) has become a major cause of nosocomial infections and posed challenges on clinical treatments. The main objective of this study was to determinate the genetic characteristics of the NDM-19-producing CRKP strain SCM96. From 2015 to 2017, 18 CRKP strains were recovered from sputum samples of patients in respiratory medicine in 6 hospitals from 5 provinces and cities in China. Polymerase chain reaction results for carbapenem resistance genes detection showed strain SCM96 carried blaNDM-19. Three types of transconjugants harboring different plasmids were selected by conjugation experiment. The Whole Genome Sequencing (WGS) was performed using the PacBio RS platform. The genome size of SCM96 was 5,579,775?bp and composed of chromosomal DNA (5,398,745?bp) and 2 plasmids, IncFII type plasmid pSCM96-1 (134,869?bp) and IncX3 type plasmid pSCM96-2 (46,161?bp). SCM96 belonged to ST15 and K28. In addition to the 4 antibiotic resistance genes located in the chromosome, pSCM96-1 carried a complex resistance region containing 17 resistance genes and several mobile genetic elements (MGEs) like ?Tn6029, In4-like integron, and Tn3, and pSCM96-2 had only 1 blaNDM-19 gene. As far as we know, this was the first description of blaNDM-19 in K. pneumoniae. Up to 22 antibiotic resistance genes, several important MGEs, and transferable plasmids might increase the possibility of co-spreading of blaNDM-19 with other resistance genes.Copyright © 2018 Elsevier Inc. All rights reserved.
The macaque simian or simian/human immunodeficiency virus (SIV/SHIV) challenge model has been widely used to inform and guide human vaccine trials. Substantial advances have been made recently in the application of repeated-low-dose challenge (RLD) approach to assess SIV/SHIV vaccine efficacies (VE). Some candidate HIV vaccines have shown protective effects in preclinical studies using the macaque SIV/SHIV model but the model’s true predictive value for screening potential HIV vaccine candidates needs to be evaluated further. Here, we review key parameters used in the RLD approach and discuss their relevance for evaluating VE to improve preclinical studies of candidate HIV vaccines.Crown Copyright © 2019. Published by Elsevier Ltd. All rights reserved.
Oral azithromycin given during labour reduces carriage of bacteria responsible for neonatal sepsis, including Staphylococcus aureus. However, there is concern that this may promote drug resistance.Here, we combine genomic and epidemiological data on S. aureus isolated from mothers and babies in a randomized intra-partum azithromycin trial (PregnAnZI) to describe bacterial population dynamics and resistance mechanisms.Participants from both arms of the trial, who carried S. aureus in day 3 and day 28 samples post-intervention, were included. Sixty-six S. aureus isolates (from 7 mothers and 10 babies) underwent comparative genome analyses and the data were then combined with epidemiological data. Trial registration (main trial): ClinicalTrials.gov Identifier NCT01800942.Seven S. aureus STs were identified, with ST5 dominant (n?=?40, 61.0%), followed by ST15 (n?=?11, 17.0%). ST5 predominated in the placebo arm (73.0% versus 49.0%, P?=?0.039) and ST15 in the azithromycin arm (27.0% versus 6.0%, P?=?0.022). In azithromycin-resistant isolates, msr(A) was the main macrolide resistance gene (n?=?36, 80%). Ten study participants, from both trial arms, acquired azithromycin-resistant S. aureus after initially harbouring a susceptible isolate. In nine (90%) of these cases, the acquired clone was an msr(A)-containing ST5 S. aureus. Long-read sequencing demonstrated that in ST5, msr(A) was found on an MDR plasmid.Our data reveal in this Gambian population the presence of a dominant clone of S. aureus harbouring plasmid-encoded azithromycin resistance, which was acquired by participants in both arms of the study. Understanding these resistance dynamics is crucial to defining the public health drug resistance impacts of azithromycin prophylaxis given during labour in Africa. © The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.
Cronobacter sakazakii causes meningitis and necrotizing enterocolitis in premature infants. However, its virulence determinants, especially those specific for strains associated with neonate infections, remain largely unknown.In this study, we performed a comparative genomic analysis of 209 C. sakazakii genomes, and 8 clonal groups (CGs) were revealed.CG1 and CG2 were found to be significantly associated with neonate infections, and significantly prevalent genes in these 2 CGs were identified. Of these, a gene encoding the LysR-type regulator, CklR, was shown to contribute to bacterial pathogenicity based on animal experiments. We found that CklR directly binds and activates the suf Fe-S cluster biosynthesis operon, and high expression of the suf operon increases bacterial resistance to oxidative stress, which increases survival within the host. This leads to a high degree of bacteremia, which contributes to the development of meningitis.Our work revealed a novel virulence factor specific to predominant pathogenic C. sakazakii strains. © The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.
IncR, IncFII, IncpA1763-KPC, and IncN1 plasmids have been increasingly found among Enterobacteriaceae species, but plasmids with hybrid structures derived from the above-mentioned incompatibility groups have not yet been described.Plasmids p721005-KPC, p504051-KPC, and pA3295-KPC were fully sequenced and compared with previously sequenced related plasmids pHN84KPC (IncR), pKPHS2 (IncFIIK), pKOX_NDM1 (IncFIIY), pHN7A8 (IncFIIpHN7A8), and R46 (IncN1).The backbone of p721005-KPC/p504051-KPC was a hybrid of the entire 10-kb IncR-type backbone from pHN84KPC, the entire 64.3-kb IncFIIK-type maintenance, and conjugal transfer regions from pKPHS2, a 15.5-kb IncFIIY-type maintenance region from pKOX_NDM1 and a 5.6-kb IncpA1763-KPC-type backbone region from pA1763-KPC, and it contained a primary IncR replicon and two auxiliary IncpA1763-KPC and IncN1 replicons. The backbone of pA3295-KPC was a hybrid of a 7.2-kb IncFIIpHN7A8-type backbone region from pHN7A8, the almost entire 33.3-kb IncN1-type maintenance and conjugal transfer regions highly similar to R46, a 26.2-kb IncFIIK-type maintenance regions from pKPHS2, the above 15.5-kb IncFIIY-type maintenance region, and the above 5.6-kb IncpA1763-KPC-type backbone region, and it contained a primary Inc-FIIpHN7A8 replicon and two auxiliary IncpA1763-KPC and IncN1 replicons. Each of p721005-KPC, p504051-KPC, and pA3295-KPC acquired a wealth of accessory modules, carrying a range of intact and residue mobile elements (such as insertion sequences, unit transposons, and integrons) and resistance markers (such as blaKPC, tetA, dfrA, and qnr).In each of p721005-KPC, p504051-KPC, and pA3295-KPC, multiple replicons in coordination with maintenance and conjugation regions of various origins would maintain a broad host range and a stable replication at a steady-state plasmid copy number.
Natural product discovery from microorganisms provided important sources for antibiotics, anti-cancer agents, immune-modulators, anthelminthic agents, and insecticides during a span of 50 years starting in the 1940s, then became less productive because of rediscovery issues, low throughput, and lack of relevant new technologies to unveil less abundant or not easily detected drug-like natural products. In the early 2000s, it was observed from genome sequencing that Streptomyces species encode about ten times as many secondary metabolites as predicted from known secondary metabolomes. This gave rise to a new discovery approach-microbial genome mining. As the cost of genome sequencing dropped, the numbers of sequenced bacteria, fungi and archaea expanded dramatically, and bioinformatic methods were developed to rapidly scan whole genomes for the numbers, types, and novelty of secondary metabolite biosynthetic gene clusters. This methodology enabled the identification of microbial taxa gifted for the biosynthesis of drug-like secondary metabolites. As genome sequencing technology progressed, the realities relevant to drug discovery have emerged, the conjectures and misconceptions have been clarified, and opportunities to reinvigorate microbial drug discovery have crystallized. This perspective addresses these critical issues for drug discovery.
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