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  • 1
    Online Resource
    Online Resource
    Chemical Synergy between Ionophore PBT2 and Zinc Reverses Antibiotic Resistance
    American Society for Microbiology ; 2018
    In:  mBio Vol. 9, No. 6 ( 2018-12-21)
    Details
    In: mBio, American Society for Microbiology, Vol. 9, No. 6 ( 2018-12-21)
    Abstract: The World Health Organization reports that antibiotic-resistant pathogens represent an imminent global health disaster for the 21st century. Gram-positive superbugs threaten to breach last-line antibiotic treatment, and the pharmaceutical industry antibiotic development pipeline is waning. Here we report the synergy between ionophore-induced physiological stress in Gram-positive bacteria and antibiotic treatment. PBT2 is a safe-for-human-use zinc ionophore that has progressed to phase 2 clinical trials for Alzheimer’s and Huntington’s disease treatment. In combination with zinc, PBT2 exhibits antibacterial activity and disrupts cellular homeostasis in erythromycin-resistant group A Streptococcus (GAS), methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococcus (VRE). We were unable to select for mutants resistant to PBT2-zinc treatment. While ineffective alone against resistant bacteria, several clinically relevant antibiotics act synergistically with PBT2-zinc to enhance killing of these Gram-positive pathogens. These data represent a new paradigm whereby disruption of bacterial metal homeostasis reverses antibiotic-resistant phenotypes in a number of priority human bacterial pathogens. IMPORTANCE The rise of bacterial antibiotic resistance coupled with a reduction in new antibiotic development has placed significant burdens on global health care. Resistant bacterial pathogens such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus are leading causes of community- and hospital-acquired infection and present a significant clinical challenge. These pathogens have acquired resistance to broad classes of antimicrobials. Furthermore, Streptococcus pyogenes , a significant disease agent among Indigenous Australians, has now acquired resistance to several antibiotic classes. With a rise in antibiotic resistance and reduction in new antibiotic discovery, it is imperative to investigate alternative therapeutic regimens that complement the use of current antibiotic treatment strategies. As stated by the WHO Director-General, “On current trends, common diseases may become untreatable. Doctors facing patients will have to say, Sorry, there is nothing I can do for you.”
    Type of Medium: Online Resource
    ISSN: 2161-2129 , 2150-7511
    URL: Article
    DOI: 10.1128/mBio.02391-18
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2018
    detail.hit.zdb_id: 2557172-2
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  • 2
    Online Resource
    Online Resource
    Identification and Characterization of PtlC, an Essential Component of the Pertussis Toxin Secretion System
    American Society for Microbiology ; 1999
    In:  Infection and Immunity Vol. 67, No. 2 ( 1999-02), p. 754-759
    Details
    In: Infection and Immunity, American Society for Microbiology, Vol. 67, No. 2 ( 1999-02), p. 754-759
    Abstract: PtlC is a member of a set of proteins necessary for the secretion of pertussis toxin (PT) from Bordetella pertussis . Using polyclonal antibodies specific for PtlC, we identified PtlC as a protein with an apparent molecular weight of 85,000 that localizes to the membrane fraction of bacterial cell extracts. We found that a mutant strain of B. pertussis that contains an in-frame deletion in ptlC is unable to secrete PT and that PT secretion is fully restored by expressing ptlC in trans , indicating that PtlC is essential for transport of PT across the bacterial membrane(s). PT secretion was inhibited in wild-type B. pertussis after introduction of a plasmid expressing a mutant ptlC altered in the putative nucleotide-binding region, suggesting that this region of PtlC is essential for proper function. Moreover, the observed dominant negative phenotype suggests that PtlC either functions as a multimer or interacts with some other component(s) necessary for secretion of PT.
    Type of Medium: Online Resource
    ISSN: 0019-9567 , 1098-5522
    URL: Article
    DOI: 10.1128/IAI.67.2.754-759.1999
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 1999
    detail.hit.zdb_id: 1483247-1
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  • 3
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    Online Resource
    Latent Herpes Simplex Virus Infection of Sensory Neurons Alters Neuronal Gene Expression
    American Society for Microbiology ; 2003
    In:  Journal of Virology Vol. 77, No. 17 ( 2003-09), p. 9533-9541
    Details
    In: Journal of Virology, American Society for Microbiology, Vol. 77, No. 17 ( 2003-09), p. 9533-9541
    Abstract: The persistence of herpes simplex virus (HSV) and the diseases that it causes in the human population can be attributed to the maintenance of a latent infection within neurons in sensory ganglia. Little is known about the effects of latent infection on the host neuron. We have addressed the question of whether latent HSV infection affects neuronal gene expression by using microarray transcript profiling of host gene expression in ganglia from latently infected versus mock-infected mouse trigeminal ganglia. 33 P-labeled cDNA probes from pooled ganglia harvested at 30 days postinfection or post-mock infection were hybridized to nylon arrays printed with 2,556 mouse genes. Signal intensities were acquired by phosphorimager. Mean intensities ( n = 4 replicates in each of three independent experiments) of signals from mock-infected versus latently infected ganglia were compared by using a variant of Student's t test. We identified significant changes in the expression of mouse neuronal genes, including several with roles in gene expression, such as the Clk2 gene, and neurotransmission, such as genes encoding potassium voltage-gated channels and a muscarinic acetylcholine receptor. We confirmed the neuronal localization of some of these transcripts by using in situ hybridization. To validate the microarray results, we performed real-time reverse transcriptase PCR analyses for a selection of the genes. These studies demonstrate that latent HSV infection can alter neuronal gene expression and might provide a new mechanism for how persistent viral infection can cause chronic disease.
    Type of Medium: Online Resource
    ISSN: 0022-538X , 1098-5514
    URL: Article
    DOI: 10.1128/JVI.77.17.9533-9541.2003
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2003
    detail.hit.zdb_id: 1495529-5
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  • 4
    Online Resource
    Online Resource
    The Role of Herpes Simplex Virus ICP27 in the Regulation of UL24 Gene Expression by Differential Polyadenylation
    American Society for Microbiology ; 1998
    In:  Journal of Virology Vol. 72, No. 10 ( 1998-10), p. 7709-7714
    Details
    In: Journal of Virology, American Society for Microbiology, Vol. 72, No. 10 ( 1998-10), p. 7709-7714
    Abstract: Herpes simplex virus specifies two sets of transcripts from the UL24 gene, short transcripts (e.g., 1.4 kb), processed at the UL24 poly(A) site, and long transcripts (e.g., 5.6 kb), processed at the UL26 poly(A) site. The 1.4- and 5.6-kb transcripts initiate from the same promoter but are expressed with early and late kinetics, respectively. Measurements of transcript levels following actinomycin D treatment of infected cells revealed that the 1.4- and 5.6-kb UL24 transcripts have similar stabilities, consistent with UL24 transcript kinetics being regulated by differential polyadenylation rather than by differential stabilities. Although the UL24 poly(A) site, which gives rise to short transcripts, is encountered first during processing, long transcripts processed at the UL26 site are equally or more abundant; thus, operationally, the UL24 site is weak. Using a series of viral ICP27 mutants, we investigated whether ICP27, which has been suggested to stimulate the usage of weak poly(A) sites, stimulates 1.4-kb transcript accumulation. We found that accumulation of 1.4-kb transcripts did not require ICP27 during viral infection. Rather, ICP27 was required for full expression of 5.6-kb transcripts, and the decrease in 5.6-kb transcripts relative to 1.4-kb transcripts was not due solely to reduced DNA synthesis. Our results indicate that temporal expression of UL24 transcripts can be regulated by differential polyadenylation and that although ICP27 is not required for processing at the operationally weak UL24 poly(A) site, it does modulate 5.6-kb transcript levels at a step subsequent to transcriptional initiation.
    Type of Medium: Online Resource
    ISSN: 0022-538X , 1098-5514
    URL: Article
    DOI: 10.1128/JVI.72.10.7709-7714.1998
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 1998
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  • 5
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    Online Resource
    Copy Number Heterogeneity, Large Origin Tandem Repeats, and Interspecies Recombination in Human Herpesvirus 6A (HHV-6A) and HHV-6B Reference Strains
    American Society for Microbiology ; 2018
    In:  Journal of Virology Vol. 92, No. 10 ( 2018-05-15)
    Details
    In: Journal of Virology, American Society for Microbiology, Vol. 92, No. 10 ( 2018-05-15)
    Abstract: Quantitative PCR is a diagnostic pillar for clinical virology testing, and reference materials are necessary for accurate, comparable quantitation between clinical laboratories. Accurate quantitation of human herpesvirus 6A/B (HHV-6A/B) is important for detection of viral reactivation and inherited chromosomally integrated HHV-6A/B in immunocompromised patients. Reference materials in clinical virology commonly consist of laboratory-adapted viral strains that may be affected by the culture process. We performed next-generation sequencing to make relative copy number measurements at single nucleotide resolution of eight candidate HHV-6A and seven HHV-6B reference strains and DNA materials from the HHV-6 Foundation and Advanced Biotechnologies Inc. Eleven of 17 (65%) HHV-6A/B candidate reference materials showed multiple copies of the origin of replication upstream of the U41 gene by next-generation sequencing. These large tandem repeats arose independently in culture-adapted HHV-6A and HHV-6B strains, measuring 1,254 bp and 983 bp, respectively. The average copy number measured was between 5 and 10 times the number of copies of the rest of the genome. We also report the first interspecies recombinant HHV-6A/B strain with a HHV-6A backbone and a 〉 5.5-kb region from HHV-6B, from U41 to U43, that covered the origin tandem repeat. Specific HHV-6A reference strains demonstrated duplication of regions at U1/U2, U87, and U89, as well as deletion in the U12-to-U24 region and the U94/U95 genes. HHV-6A/B strains derived from cord blood mononuclear cells from different laboratories on different continents with fewer passages revealed no copy number differences throughout the viral genome. These data indicate that large origin tandem duplications are an adaptation of both HHV-6A and HHV-6B in culture and show interspecies recombination is possible within the Betaherpesvirinae. IMPORTANCE Anything in science that needs to be quantitated requires a standard unit of measurement. This includes viruses, for which quantitation increasingly determines definitions of pathology and guidelines for treatment. However, the act of making standard or reference material in virology can alter its very accuracy through genomic duplications, insertions, and rearrangements. We used deep sequencing to examine candidate reference strains for HHV-6, a ubiquitous human virus that can reactivate in the immunocompromised population and is integrated into the human genome in every cell of the body for 1% of people worldwide. We found large tandem repeats in the origin of replication for both HHV-6A and HHV-6B that are selected for in culture. We also found the first interspecies recombinant between HHV-6A and HHV-6B, a phenomenon that is well known in alphaherpesviruses but to date has not been seen in betaherpesviruses. These data critically inform HHV-6A/B biology and the standard selection process.
    Type of Medium: Online Resource
    ISSN: 0022-538X , 1098-5514
    URL: Article
    DOI: 10.1128/JVI.00135-18
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2018
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  • 6
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    Online Resource
    TraG from RP4 and TraG and VirD4 from Ti Plasmids Confer Relaxosome Specificity to the Conjugal Transfer System of pTiC58
    American Society for Microbiology ; 2000
    In:  Journal of Bacteriology Vol. 182, No. 6 ( 2000-03-15), p. 1541-1548
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 182, No. 6 ( 2000-03-15), p. 1541-1548
    Abstract: Plasmid conjugation systems are composed of two components, the DNA transfer and replication system, or Dtr, and the mating pair formation system, or Mpf. During conjugal transfer an essential factor, called the coupling protein, is thought to interface the Dtr, in the form of the relaxosome, with the Mpf, in the form of the mating bridge. These proteins, such as TraG from the IncP1 plasmid RP4 (TraG RP4 ) and TraG and VirD4 from the conjugal transfer and T-DNA transfer systems of Ti plasmids, are believed to dictate specificity of the interactions that can occur between different Dtr and Mpf components. The Ti plasmids of Agrobacterium tumefaciens do not mobilize vectors containing the oriT of RP4, but these IncP1 plasmid derivatives lack the trans -acting Dtr functions and TraG RP4 . A. tumefaciens donors transferred a chimeric plasmid that contains the oriT and Dtr genes of RP4 and the Mpf genes of pTiC58, indicating that the Ti plasmid mating bridge can interact with the RP4 relaxosome. However, the Ti plasmid did not mobilize transfer from an IncQ relaxosome. The Ti plasmid did mobilize such plasmids if TraG RP4 was expressed in the donors. Mutations in traG RP4 with defined effects on the RP4 transfer system exhibited similar phenotypes for Ti plasmid-mediated mobilization of the IncQ vector. When provided with VirD4, the tra system of pTiC58 mobilized plasmids from the IncQ relaxosome. However, neither TraG RP4 nor VirD4 restored transfer to a traG mutant of the Ti plasmid. VirD4 also failed to complement a traG RP4 mutant for transfer from the RP4 relaxosome or for RP4-mediated mobilization from the IncQ relaxosome. TraG RP4 -mediated mobilization of the IncQ plasmid by pTiC58 did not inhibit Ti plasmid transfer, suggesting that the relaxosomes of the two plasmids do not compete for the same mating bridge. We conclude that TraG RP4 and VirD4 couples the IncQ but not the Ti plasmid relaxosome to the Ti plasmid mating bridge. However, VirD4 cannot couple the IncP1 or the IncQ relaxosome to the RP4 mating bridge. These results support a model in which the coupling proteins specify the interactions between Dtr and Mpf components of mating systems.
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.182.6.1541-1548.2000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2000
    detail.hit.zdb_id: 1481988-0
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  • 7
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    Online Resource
    If You're Not Confused, You're Not Paying Attention: Ochrobactrum Is Not Brucella
    American Society for Microbiology ; 2023
    In:  Journal of Clinical Microbiology
    Details
    In: Journal of Clinical Microbiology, American Society for Microbiology
    Abstract: Bacteria of the genus Brucella are facultative intracellular parasites that cause brucellosis, a severe animal and human disease. Recently, a group of taxonomists merged the brucellae with the primarily free-living, phylogenetically related Ochrobactrum spp. in the genus Brucella . This change, founded only on global genomic analysis and the fortuitous isolation of some opportunistic Ochrobactrum spp. from medically compromised patients, has been automatically included in culture collections and databases. We argue that clinical and environmental microbiologists should not accept this nomenclature, and we advise against its use because (i) it was presented without in-depth phylogenetic analyses and did not consider alternative taxonomic solutions; (ii) it was launched without the input of experts in brucellosis or Ochrobactrum ; (iii) it applies a non-consensus genus concept that disregards taxonomically relevant differences in structure, physiology, population structure, core-pangenome assemblies, genome structure, genomic traits, clinical features, treatment, prevention, diagnosis, genus description rules, and, above all, pathogenicity; and (iv) placing these two bacterial groups in the same genus creates risks for veterinarians, medical doctors, clinical laboratories, health authorities, and legislators who deal with brucellosis, a disease that is particularly relevant in low- and middle-income countries. Based on all this information, we urge microbiologists, bacterial collections, genomic databases, journals, and public health boards to keep the Brucella and Ochrobactrum genera separate to avoid further bewilderment and harm.
    Type of Medium: Online Resource
    ISSN: 0095-1137 , 1098-660X
    URL: Article
    DOI: 10.1128/jcm.00438-23
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2023
    detail.hit.zdb_id: 1498353-9
    SSG: 12
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  • 8
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    Online Resource
    Molecular Analysis of Asymptomatic Bacteriuria Escherichia coli Strain VR50 Reveals Adaptation to the Urinary Tract by Gene Acquisition
    American Society for Microbiology ; 2015
    In:  Infection and Immunity Vol. 83, No. 5 ( 2015-05), p. 1749-1764
    Details
    In: Infection and Immunity, American Society for Microbiology, Vol. 83, No. 5 ( 2015-05), p. 1749-1764
    Abstract: Urinary tract infections (UTIs) are among the most common infectious diseases of humans, with Escherichia coli responsible for 〉 80% of all cases. One extreme of UTI is asymptomatic bacteriuria (ABU), which occurs as an asymptomatic carrier state that resembles commensalism. To understand the evolution and molecular mechanisms that underpin ABU, the genome of the ABU E. coli strain VR50 was sequenced. Analysis of the complete genome indicated that it most resembles E. coli K-12, with the addition of a 94-kb genomic island (GI-VR50- pheV ), eight prophages, and multiple plasmids. GI-VR50- pheV has a mosaic structure and contains genes encoding a number of UTI-associated virulence factors, namely, Afa (afimbrial adhesin), two autotransporter proteins (Ag43 and Sat), and aerobactin. We demonstrated that the presence of this island in VR50 confers its ability to colonize the murine bladder, as a VR50 mutant with GI-VR50- pheV deleted was attenuated in a mouse model of UTI in vivo . We established that Afa is the island-encoded factor responsible for this phenotype using two independent deletion (Afa operon and AfaE adhesin) mutants. E. coli VR50 afa and VR50 afaE displayed significantly decreased ability to adhere to human bladder epithelial cells. In the mouse model of UTI, VR50 afa and VR50 afaE displayed reduced bladder colonization compared to wild-type VR50, similar to the colonization level of the GI-VR50- pheV mutant. Our study suggests that E. coli VR50 is a commensal-like strain that has acquired fitness factors that facilitate colonization of the human bladder.
    Type of Medium: Online Resource
    ISSN: 0019-9567 , 1098-5522
    URL: Article
    DOI: 10.1128/IAI.02810-14
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2015
    detail.hit.zdb_id: 1483247-1
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  • 9
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    Genetically and Antigenically Divergent Influenza A(H9N2) Viruses Exhibit Differential Replication and Transmission Phenotypes in Mammalian Models
    American Society for Microbiology ; 2020
    In:  Journal of Virology Vol. 94, No. 17 ( 2020-08-17)
    Details
    In: Journal of Virology, American Society for Microbiology, Vol. 94, No. 17 ( 2020-08-17)
    Abstract: Low-pathogenicity avian influenza A(H9N2) viruses, enzootic in poultry populations in Asia, are associated with fewer confirmed human infections but higher rates of seropositivity compared to A(H5) or A(H7) subtype viruses. Cocirculation of A(H5) and A(H7) viruses leads to the generation of reassortant viruses bearing A(H9N2) internal genes with markers of mammalian adaptation, warranting continued surveillance in both avian and human populations. Here, we describe active surveillance efforts in live poultry markets in Vietnam in 2018 and compare representative viruses to G1 and Y280 lineage viruses that have infected humans. Receptor binding properties, pH thresholds for HA activation, in vitro replication in human respiratory tract cells, and in vivo mammalian pathogenicity and transmissibility were investigated. While A(H9N2) viruses from both poultry and humans exhibited features associated with mammalian adaptation, one human isolate from 2018, A/Anhui-Lujiang/39/2018, exhibited increased capacity for replication and transmission, demonstrating the pandemic potential of A(H9N2) viruses. IMPORTANCE A(H9N2) influenza viruses are widespread in poultry in many parts of the world and for over 20 years have sporadically jumped species barriers to cause human infection. As these viruses continue to diversify genetically and antigenically, it is critical to closely monitor viruses responsible for human infections, to ascertain if A(H9N2) viruses are acquiring properties that make them better suited to infect and spread among humans. In this study, we describe an active poultry surveillance system established in Vietnam to identify the scope of influenza viruses present in live bird markets and the threat they pose to human health. Assessment of a recent A(H9N2) virus isolated from an individual in China in 2018 is also reported, and it was found to exhibit properties of adaptation to humans and, importantly, it shows similarities to strains isolated from the live bird markets of Vietnam.
    Type of Medium: Online Resource
    ISSN: 0022-538X , 1098-5514
    URL: Article
    DOI: 10.1128/JVI.00451-20
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2020
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  • 10
    Online Resource
    Online Resource
    Multiple Bactericidal Mechanisms of the Zinc Ionophore PBT2
    American Society for Microbiology ; 2020
    In:  mSphere Vol. 5, No. 2 ( 2020-04-29)
    Details
    In: mSphere, American Society for Microbiology, Vol. 5, No. 2 ( 2020-04-29)
    Abstract: Globally, more antimicrobials are used in food-producing animals than in humans, and the extensive use of medically important human antimicrobials poses a significant public health threat in the face of rising antimicrobial resistance (AMR). The development of novel ionophores, a class of antimicrobials used exclusively in animals, holds promise as a strategy to replace or reduce essential human antimicrobials in veterinary practice. PBT2 is a zinc ionophore with recently demonstrated antibacterial activity against several Gram-positive pathogens, although the underlying mechanism of action is unknown. Here, we investigated the bactericidal mechanism of PBT2 in the bovine mastitis-causing pathogen, Streptococcus uberis . In this work, we show that PBT2 functions as a Zn 2+ /H + ionophore, exchanging extracellular zinc for intracellular protons in an electroneutral process that leads to cellular zinc accumulation. Zinc accumulation occurs concomitantly with manganese depletion and the production of reactive oxygen species (ROS). PBT2 inhibits the activity of the manganese-dependent superoxide dismutase, SodA, thereby impairing oxidative stress protection. We propose that PBT2-mediated intracellular zinc toxicity in S. uberis leads to lethality through multiple bactericidal mechanisms: the production of toxic ROS and the impairment of manganese-dependent antioxidant functions. Collectively, these data show that PBT2 represents a new class of antibacterial ionophores capable of targeting bacterial metal ion homeostasis and cellular redox balance. We propose that this novel and multitarget mechanism of PBT2 makes the development of cross-resistance to medically important antimicrobials unlikely. IMPORTANCE More antimicrobials are used in food-producing animals than in humans, and the extensive use of medically important human antimicrobials poses a significant public health threat in the face of rising antimicrobial resistance. Therefore, the elimination of antimicrobial crossover between human and veterinary medicine is of great interest. Unfortunately, the development of new antimicrobials is an expensive high-risk process fraught with difficulties. The repurposing of chemical agents provides a solution to this problem, and while many have not been originally developed as antimicrobials, they have been proven safe in clinical trials. PBT2, a zinc ionophore, is an experimental therapeutic that met safety criteria but failed efficacy checkpoints against both Alzheimer’s and Huntington’s diseases. It was recently found that PBT2 possessed potent antimicrobial activity, although the mechanism of bacterial cell death is unresolved. In this body of work, we show that PBT2 has multiple mechanisms of antimicrobial action, making the development of PBT2 resistance unlikely.
    Type of Medium: Online Resource
    ISSN: 2379-5042
    URL: Article
    DOI: 10.1128/mSphere.00157-20
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2020
    detail.hit.zdb_id: 2844248-9
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