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July 7, 2019  |  

Emerging mechanisms of antimicrobial resistance in bacteria and fungi: advances in the era of genomics.

Bacteria and fungi continue to develop new ways to adapt and survive the lethal or biostatic effects of antimicrobials through myriad mechanisms. Novel antibiotic resistance genes such as lsa(C), erm(44), VCC-1, mcr-1, mcr-2, mcr-3, mcr-4, bla KLUC-3 and bla KLUC-4 were discovered through comparative genomics and further functional studies. As well, mutations in genes that hitherto were unknown to confer resistance to antimicrobials, such as trm, PP2C, rpsJ, HSC82, FKS2 and Rv2887, were shown by genomics and transcomplementation assays to mediate antimicrobial resistance in Acinetobacter baumannii, Staphylococcus aureus, Enterococcus faecium, Saccharomyces cerevisae, Candida glabrata and Mycobacterium tuberculosis, respectively. Thus, genomics, transcriptomics and metagenomics, coupled with functional studies are the future of antimicrobial resistance research and novel drug discovery or design.

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July 7, 2019  |  

Complete genome sequence of Melissococcus plutonius DAT561, a strain that shows an unusual growth profile, obtained by PacBio sequencing.

Melissococcus plutonius is the causative agent of European foulbrood, and its isolates were believed to be remarkably genetically homogeneous. However, recent epidemiological and pathogenic studies have shown this pathogen to be more heterogeneous than expected. Herein, we present the whole-genome sequence of M. plutonius DAT561, a representative atypical strain. Copyright © 2018 Okumura et al.

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July 7, 2019  |  

Complete genome sequence of a Staphylococcus aureus sequence type 612 isolate from an Australian horse.

Staphylococcus aureus is a serious pathogen of humans and animals. Multilocus sequence type 612 is dominant and highly virulent in South African hospitals but relatively uncommon elsewhere. We present the complete genome sequence of methicillin-resistant Staphylococcus aureus strain SVH7513, isolated from a horse at a veterinary clinic in New South Wales, Australia.

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July 7, 2019  |  

Transposon insertion sequencing elucidates novel gene involvement in susceptibility and resistance to phages T4 and T7 in Escherichia coli O157.

Experiments using bacteriophage (phage) to infect bacterial strains have helped define some basic genetic concepts in microbiology, but our understanding of the complexity of bacterium-phage interactions is still limited. As the global threat of antibiotic resistance continues to increase, phage therapy has reemerged as an attractive alternative or supplement to treating antibiotic-resistant bacterial infections. Further, the long-used method of phage typing to classify bacterial strains is being replaced by molecular genetic techniques. Thus, there is a growing need for a complete understanding of the precise molecular mechanisms underpinning phage-bacterium interactions to optimize phage therapy for the clinic as well as for retrospectively interpreting phage typing data on the molecular level. In this study, a genomics-based fitness assay (TraDIS) was used to identify all host genes involved in phage susceptibility and resistance for a T4 phage infecting Shiga-toxigenic Escherichia coli O157. The TraDIS results identified both established and previously unidentified genes involved in phage infection, and a subset were confirmed by site-directed mutagenesis and phenotypic testing of 14 T4 and 2 T7 phages. For the first time, the entire sap operon was implicated in phage susceptibility and, conversely, the stringent starvation protein A gene (sspA) was shown to provide phage resistance. Identifying genes involved in phage infection and replication should facilitate the selection of bespoke phage combinations to target specific bacterial pathogens.IMPORTANCE Antibiotic resistance has diminished treatment options for many common bacterial infections. Phage therapy is an alternative option that was once popularly used across Europe to kill bacteria within humans. Phage therapy acts by using highly specific viruses (called phages) that infect and lyse certain bacterial species to treat the infection. Whole-genome sequencing has allowed modernization of the investigations into phage-bacterium interactions. Here, using E. coli O157 and T4 bacteriophage as a model, we have exploited a genome-wide fitness assay to investigate all genes involved in defining phage resistance or susceptibility. This knowledge of the genetic determinants of phage resistance and susceptibility can be used to design bespoke phage combinations targeted to specific bacterial infections for successful infection eradication. Copyright © 2018 Cowley et al.

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July 7, 2019  |  

Low-level antimicrobials in the medicinal leech select for resistant pathogens that spread to patients.

Fluoroquinolones (FQs) and ciprofloxacin (Cp) are important antimicrobials that pollute the environment in trace amounts. Although Cp has been recommended as prophylaxis for patients undergoing leech therapy to prevent infections by the leech gut symbiont Aeromonas, a puzzling rise in Cp-resistant (Cpr) Aeromonas infections has been reported. We report on the effects of subtherapeutic FQ concentrations on bacteria in an environmental reservoir, the medicinal leech, and describe the presence of multiple antibiotic resistance mutations and a gain-of-function resistance gene. We link the rise of CprAeromonas isolates to exposure of the leech microbiota to very low levels of Cp (0.01 to 0.04 µg/ml), <1/100 of the clinical resistance breakpoint for Aeromonas Using competition experiments and comparative genomics of 37 strains, we determined the mechanisms of resistance in clinical and leech-derived Aeromonas isolates, traced their origin, and determined that the presence of merely 0.01 µg/ml Cp provides a strong competitive advantage for Cpr strains. Deep-sequencing the Cpr-conferring region of gyrA enabled tracing of the mutation-harboring Aeromonas population in archived gut samples, and an increase in the frequency of the Cpr-conferring mutation in 2011 coincides with the initial reports of CprAeromonas infections in patients receiving leech therapy.IMPORTANCE The role of subtherapeutic antimicrobial contamination in selecting for resistant strains has received increasing attention and is an important clinical matter. This study describes the relationship of resistant bacteria from the medicinal leech, Hirudo verbana, with patient infections following leech therapy. While our results highlight the need for alternative antibiotic therapies, the rise of Cpr bacteria demonstrates the importance of restricting the exposure of animals to antibiotics approved for veterinary use. The shift to a more resistant community and the dispersion of Cpr-conferring mechanisms via mobile elements occurred in a natural setting due to the presence of very low levels of fluoroquinolones, revealing the challenges of controlling the spread of antibiotic-resistant bacteria and highlighting the importance of a holistic approach in the management of antibiotic use. Copyright © 2018 Beka et al.

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July 7, 2019  |  

Evolution and comparative genomics of F33:A-:B- plasmids carrying blaCTX-M-55 or blaCTX-M-65 in Escherichia coli and Klebsiella pneumoniae isolated from animals.

To understand the underlying evolution process of F33:A-:B- plasmids among Enterobacteriaceae isolates of various origins in China, the complete sequences of 17 blaCTX-M-harboring F33:A-:B- plasmids obtained from Escherichia coli and Klebsiella pneumoniae isolates from different sources (animals, animal-derived food, and human clinics) in China were determined. F33:A-:B- plasmids shared similar plasmid backbones comprising replication, leading, and conjugative transfer regions and differed by the numbers of repeats in yddA and traD and by the presence of group II intron, except that pHNAH9 lacked a large segment of the leading and transfer regions. The variable regions of F33:A-B- plasmids were distinct and were inserted downstream of the addiction system pemI/pemK, identified as the integration hot spot among F33:A-B- plasmids. The variable region contained resistance genes and mobile elements or contained segments from other types of plasmids, such as IncI1, IncN1, and IncX1. Three plasmids encoding CTX-M-65 were very similar to our previously described pHN7A8 plasmid. Four CTX-M-55-producing plasmids contained multidrug resistance regions related to that of F2:A-B- plasmid pHK23a from Hong Kong. Five plasmids with IncN and/or IncX replication regions and IncI1-backbone fragments had variable regions related to those of pE80 and p42-2. The remaining five plasmids with IncN replicons and an IncI1 segment also possessed closely related variable regions. The diversity in variable regions was presumably associated with rearrangements, insertions, and/or deletions mediated by mobile elements, such as IS26 and IS1294 IMPORTANCE Worldwide spread of antibiotic resistance genes among Enterobacteriaceae isolates is of great concern. F33:A-:B- plasmids are important vectors of resistance genes, such as blaCTX-M-55/-65, blaNDM-1, fosA3, and rmtB, among E. coli isolates from various sources in China. We determined and compared the complete sequences of 17 F33:A-:B- plasmids from various sources. These plasmids appear to have evolved from the same ancestor by mobile element-mediated rearrangement, acquisition, and/or loss of resistance modules and similar IncN1, IncI1, and/or IncX1 plasmid backbone segments. Our findings highlight the evolutionary potential of F33:A-:B- plasmids as efficient vectors to capture and diffuse clinically relevant resistance genes. Copyright © 2018 Wang et al.

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July 7, 2019  |  

Loss of RXFP2 and INSL3 genes in Afrotheria shows that testicular descent is the ancestral condition in placental mammals.

Descent of testes from a position near the kidneys into the lower abdomen or into the scrotum is an important developmental process that occurs in all placental mammals, with the exception of five afrotherian lineages. Since soft-tissue structures like testes are not preserved in the fossil record and since key parts of the placental mammal phylogeny remain controversial, it has been debated whether testicular descent is the ancestral or derived condition in placental mammals. To resolve this debate, we used genomic data of 71 mammalian species and analyzed the evolution of two key genes (relaxin/insulin-like family peptide receptor 2 [RXFP2] and insulin-like 3 [INSL3]) that induce the development of the gubernaculum, the ligament that is crucial for testicular descent. We show that both RXFP2 and INSL3 are lost or nonfunctional exclusively in four afrotherians (tenrec, cape elephant shrew, cape golden mole, and manatee) that completely lack testicular descent. The presence of remnants of once functional orthologs of both genes in these afrotherian species shows that these gene losses happened after the split from the placental mammal ancestor. These “molecular vestiges” provide strong evidence that testicular descent is the ancestral condition, irrespective of persisting phylogenetic discrepancies. Furthermore, the absence of shared gene-inactivating mutations and our estimates that the loss of RXFP2 happened at different time points strongly suggest that testicular descent was lost independently in Afrotheria. Our results provide a molecular mechanism that explains the loss of testicular descent in afrotherians and, more generally, highlight how molecular vestiges can provide insights into the evolution of soft-tissue characters.

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July 7, 2019  |  

Genetic variation of Pyrenophora teres f. teres isolates in Western Australia and emergence of a Cyp51A fungicide resistance mutation

Genome-wide, unlinked, simple sequence repeat markers were used to examine genetic variation and relationships within Pyrenophora teres f. teres, a common pathogen of barley, in Western Australia. Despite the region’s geographic isolation, the isolates showed relatively high allelic variation compared to similar studies, averaging 7.11 alleles per locus. Principal component, Bayesian clustering and distance differentiation parameters provided evidence for both regional genotypic subdivision together with juxtaposing of isolates possessing different genetic backgrounds. Genotyping of fungicide resistant Cyp51A isolates indicated a single mutation event occurred followed by recombination and long-distance regional dispersal over hundreds of kilometres. Selection of recently emergent favourable alleles such as the Cyp51A mutation and a cultivar virulence may provide an explanation, at least in part, for juxtaposed genotypes. Factors affecting genotypic composition and the movement of new genotypes are discussed in the context of grower practices and pathogen epidemiology, together with the implications for resistance breeding.

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July 7, 2019  |  

DNA sequences and predicted protein structures of prot6E and sefA genes for Salmonella ser. Enteritidis detection

Genes prot6E and sefA are used as targets for detection of Salmonella enterica subsp. enterica serovar Enteritidis (Salmonella ser. Enteritidis). We investigated variations in these genes across 64 different Salmonella ser. Enteritidis strains isolated from egg and chicken samples, then used Whole Genome Sequence (WGS) data to model the structures of their protein products. Isolates were sequenced using Illumina technologies. Based on the resulting phylogenetic tree, our isolates clustered in 2 distinct clades. All isolates carried prot6E and sefA. Comparative genomic analyses indicated two non-synonymous mutations (Glycine ? Serine and Valine ? Isoleucine) of prot6E in 11 isolates (9 egg samples, 2 chicken samples). However, SWISS-MODEL was unable to clearly model the protein structure of these two mutations. We identified one non-synonymous mutation (Valine ? Glutamic Acid) in the sefA gene in 4 isolates from egg samples. The model for the protein structure of this mutant gene was clearly different from that of the other isolates studied herein. Circular maps of plasmid genomes from two PacBio platform-sequenced Salmonella ser. Enteritidis isolates revealed prot6E gene was located on the tail of the plasmid. Based on the biosynthesis of amino acids – Reference pathway in the KEGG pathway Database, the transition of amino acid from sefA Var. was a transversion from essential amino acid to non-essential amino acid, while that of prot6E Var.1 happened between the conditionally non-essential amino acid, and prot6E Var. 2 occurred between essential amino acids. Properties of these mutated amino acids, such as side-chain polarity or charge, may contribute to the occurrence and rate of mutations in prot6E and sefA. These insights can be used to improve detection methods for Salmonella ser. Enteritidis.

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July 7, 2019  |  

Complete genome sequence of the multidrug-resistant neonatal meningitis Escherichia coli serotype O75:H5:K1 strain mcjchv-1 (NMEC-O75).

Neonatal meningitis Escherichia coli (NMEC) is the second leading cause of neonatal bacterial meningitis worldwide. We report the genome sequence of the multidrug-resistant NMEC serotype O75:H5:K1 strain mcjchv-1, which resulted in an infant’s death. The O75 serogroup is rare among NMEC isolates; therefore, this strain is considered an emergent pathogen.

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July 7, 2019  |  

Complete genome sequence of Moraxella bovis strain Epp-63 (300), an etiologic agent of infectious bovine keratoconjunctivitis.

We report here the complete closed genome sequence of Moraxella bo- vis strain Epp-63 (300) (Epp63). This strain was isolated from an infectious bovine keratoconjunctivitis (IBK) case in 1963. Since then, Epp63 has been used extensively for IBK research. Consequently, the genome sequence of Epp63 should help eluci- date IBK host-pathogen interactions.

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July 7, 2019  |  

Closed genome sequences and antibiograms of 16 Pasteurella multocida isolates from bovine respiratory disease complex cases and apparently healthy controls.

Pasteurella multocida is an animal-associated Gram-negative member of the Pasteurellaceae family. It is an opportunistic pathogen and is one of the principal bacterial species contributing to bovine respiratory disease complex (BRDC) in feedlot cattle. We present 16 closed genome sequences and antibiograms of isolates cultured from calves exhibiting clinical signs of BRDC and from control calves not showing signs of BRDC.

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July 7, 2019  |  

New variant of multidrug-resistant Salmonella enterica serovar Typhimurium associated with invasive disease in immunocompromised patients in Vietnam.

Nontyphoidal Salmonella (NTS), particularly Salmonella enterica serovar Typhimurium, is among the leading etiologic agents of bacterial enterocolitis globally and a well-characterized cause of invasive disease (iNTS) in sub-Saharan Africa. In contrast, S Typhimurium is poorly defined in Southeast Asia, a known hot spot for zoonotic disease with a recently described burden of iNTS disease. Here, we aimed to add insight into the epidemiology and potential impact of zoonotic transfer and antimicrobial resistance (AMR) in S Typhimurium associated with iNTS and enterocolitis in Vietnam. We performed whole-genome sequencing and phylogenetic reconstruction on 85 human (enterocolitis, carriage, and iNTS) and 113 animal S Typhimurium isolates isolated in Vietnam. We found limited evidence for the zoonotic transmission of S Typhimurium. However, we describe a chain of events where a pandemic monophasic variant of S Typhimurium (serovar I:4,[5],12:i:- sequence type 34 [ST34]) has been introduced into Vietnam, reacquired a phase 2 flagellum, and acquired an IncHI2 multidrug-resistant plasmid. Notably, these novel biphasic ST34 S Typhimurium variants were significantly associated with iNTS in Vietnamese HIV-infected patients. Our study represents the first characterization of novel iNTS organisms isolated outside sub-Saharan Africa and outlines a new pathway for the emergence of alternative Salmonella variants into susceptible human populations.IMPORTANCESalmonella Typhimurium is a major diarrheal pathogen and associated with invasive nontyphoid Salmonella (iNTS) disease in vulnerable populations. We present the first characterization of iNTS organisms in Southeast Asia and describe a different evolutionary trajectory from that of organisms causing iNTS in sub-Saharan Africa. In Vietnam, the globally distributed monophasic variant of Salmonella Typhimurium, the serovar I:4,[5],12:i:- ST34 clone, has reacquired a phase 2 flagellum and gained a multidrug-resistant plasmid to become associated with iNTS disease in HIV-infected patients. We document distinct communities of S Typhimurium and I:4,[5],12:i:- in animals and humans in Vietnam, despite the greater mixing of these host populations here. These data highlight the importance of whole-genome sequencing surveillance in a One Health context in understanding the evolution and spread of resistant bacterial infections. Copyright © 2018 Mather et al.

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July 7, 2019  |  

Emergence of gyrovirus 3 in commercial broiler chickens with transmissible viral proventriculitis.

Gyrovirus 3 (GyV3) has been identified in faeces from children with acute gastroenteritis. However, whether GyV3 is prevalent in poultry has not been determined to date. To the best of our knowledge, this study is the first to isolate GyV3 from commercial broiler chickens with transmissible viral proventriculitis (TVP) in China. The complete genome of the virus shares 98.4% sequence identity with the FecGy strain that causes acute gastroenteritis in children. Epidemiological investigation from 2013 to 2017 revealed that the infection rate of GyV3 reached 12.5% (42/336) in commercial broiler chickens with TVP, indicating that the infection of GyV3 was ubiquitous in chickens. The emergence of GyV3 in commercial broiler chickens should be highly concerning for public health.© 2018 Blackwell Verlag GmbH.

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