Enhancing the one health initiative by using whole genome sequencing to monitor antimicrobial resistance of animal pathogens: Vet-LIRN collaborative project with veterinary diagnostic laboratories in United States and Canada.

BACKGROUND: Antimicrobial resistance (AMR) of bacterial pathogens is an emerging public health threat. This threat extends to pets as it also compromises our ability to treat their infections. Surveillance programs in the United States have traditionally focused on collecting data from food animals, foods, and people. The Veterinary Laboratory Investigation and Response Network (Vet-LIRN), a national network of 45 veterinary diagnostic laboratories, tested the antimicrobial susceptibility of clinically relevant bacterial isolates from animals, with companion animal species represented for the first time in a monitoring program. During 2017, we systematically collected and tested 1968 isolates. To identify genetic determinants associated with AMR and the potential genetic relatedness of animal and human strains, whole genome sequencing (WGS) was performed on 192 isolates: 69 Salmonella enterica (all animal sources), 63 Escherichia coli (dogs), and 60 Staphylococcus pseudintermedius (dogs). RESULTS: We found that most Salmonella isolates (46/69, 67%) had no known resistance genes. Several isolates from both food and companion animals, however, showed genetic relatedness to isolates from humans. For pathogenic E. coli, no resistance genes were identified in 60% (38/63) of the isolates. Diverse resistance patterns were observed, and one of the isolates had predicted resistance to fluoroquinolones and cephalosporins, important antibiotics in human and veterinary medicine. For S. pseudintermedius, we observed a bimodal distribution of resistance genes, with some isolates having a diverse array of resistance mechanisms, including the mecA gene (19/60, 32%). CONCLUSION: The findings from this study highlight the critical importance of veterinary diagnostic laboratory data as part of any national antimicrobial resistance surveillance program. The finding of some highly resistant bacteria from companion animals, and the observation of isolates related to those isolated from humans demonstrates the public health significance of incorporating companion animal data into surveillance systems. Vet-LIRN will continue to build the infrastructure to collect the data necessary to perform surveillance of resistant bacteria as part of fulfilling its mission to advance human and animal health. A One Health approach to AMR surveillance programs is crucial and must include data from humans, animals, and environmental sources to be effective.

Species Diversity of Puerto Rican Heterotermes (Dictyoptera: Rhinotermitidae) Revealed by Phylogenetic Analyses of Two Mitochondrial Genes.

The goal of this study was to infer Heterotermes (Froggatt) (Dictyoptera: Rhinotermitidae) species diversity on the island of Puerto Rico from phylogenetic analyses of DNA sequence data from two mitochondrial genes, 16S rRNA and cytochrome oxidase II (COII). This termite genus is a structural pest known to be well adapted to arid environments in subtropical and tropical regions worldwide including Puerto Rico and many other Caribbean islands. Extensive sampling was accomplished across Puerto Rico, and phylogenetic analyses of individual gene sequences from these samples indicated robust datasets of congruent gene tree topologies showing three monophyletic groups: H. cardini (Snyder), H. convexinotatus (Snyder), and H. tenuis (Hagen). We found that H. cardini and H. convexinotatus were widespread in the arid coastal regions of Puerto Rico, whereas H. tenuis was uncommon and may represent a relatively new introduction. We found only H. convexinotatus on Culebra Island. We provide strong evidence that Puerto Rico may be linked to the Heterotermes in southern Florida, USA, since its GenBank 16S sequence was identical to that of seven Puerto Rican H. cardini sequences. Our study represents the first records of H. cardini from Puerto Rico and Grand Bahama.

Genomics accurately predicts antimicrobial resistance in Staphylococcus pseudintermedius collected as part of Vet-LIRN resistance monitoring.

Whole-genome sequencing (WGS) has changed our understanding of bacterial pathogens, aiding outbreak investigations and advancing our knowledge of their genetic features. However, there has been limited use of genomics to understand antimicrobial resistance of veterinary pathogens, which would help identify emerging resistance mechanisms and track their spread. The objectives of this study were to evaluate the correlation between resistance genotypes and phenotypes for Staphylococcus pseudintermedius, a major pathogen of companion animals, by comparing broth microdilution antimicrobial susceptibility testing and WGS. From 2017-2019, we conducted antimicrobial susceptibility testing and WGS on S. pseudintermedius isolates collected from dogs in the United States as a part of the Veterinary Laboratory Investigation and Response Network (Vet-LIRN) antimicrobial resistance monitoring program. Across thirteen antimicrobials in nine classes, resistance genotypes correlated with clinical resistance phenotypes 98.4 % of the time among a collection of 592 isolates. Our findings represent isolates from diverse lineages based on phylogenetic analyses, and these strong correlations are comparable to those from studies of several human pathogens such as Staphylococcus aureus and Salmonella enterica. We uncovered some important findings, including that 32.3 % of isolates had the mecA gene, which correlated with oxacillin resistance 97.0 % of the time. We also identified a novel rpoB mutation likely encoding rifampin resistance. These results show the value in using WGS to assess antimicrobial resistance in veterinary pathogens and to reveal putative new mechanisms of resistance.

Compensatory base changes illuminate morphologically difficult taxonomy.

Compensatory base changes (CBCs) in the ribosomal RNA (rRNA) internal transcribed spacer 2 (ITS2) secondary structures have been used to successfully verify the taxonomy of closely related species. CBCs have never been used to distinguish morphologically indistinct species. Under the hypothesis that CBCs will differentiate species in higher eukaryotes, novel software for CBC analysis was applied to morphologically indistinguishable insect species in the genus Altica. The analysis was species-specific for sympatric Altica beetles collected across four ecoregions and concordant with scanning electron microscopy data. This research shows that mining for CBCs in ITS2 rRNA secondary structures is an effective method for eukaryotic taxon analysis.

Evaluation of public health risks at recreational beaches in Lake Michigan via detection of enteric viruses and a human-specific bacteriological marker.

UNLABELLED: Each year the National Resource Defense Council addresses the quality of US beaches by routine bacterial indicators. In the Great Lakes region the indicator used is Escherichia coli and for 2007 more beaches were closed and impacted than ever before. In this study, water quality was addressed at two Lake Michigan Beaches over the 2004 swimming season by monitoring infectious enteric viruses by cell culture and integrated PCR and for a human sewage marker based on the Enterococcal Surface Protein (esp). Our goals for this study were to 1) examine the occurrence and variety of human enteric viruses present during peak usage of the beaches 2) determine key variables for development of predictive models for viruses; and 3) use quantitative risk assessment to estimate the potential health impact. Our results demonstrate that for both beaches predictive models of virus pollution were best described utilizing physical parameters like wind speed, wind direction and water temperature. The esp marker was not predictive of human viruses. The daily risk of acquiring a viral infection at either of the beaches ranged from 0.2 to 2.4/1000 swimmers using a quantitative microbial risk assessment model, with three swims during a day at the beach for children and over the season, the risk was 9-15/1000 swimmers using adenovirus as the model. CONCLUSIONS: Lake Michigan recreational beaches are being adversely impacted by human fecal pollution. Monitoring for the traditional indicators of water quality does not address viral risks and models can be developed and potentially used as real-time water quality forecasting tools.

DNA technology, interstate commerce, and the likely origin of formosan subterranean termite (Isoptera: Rhinotermitidae) infestation in Atlanta, Georgia.

This study was the first to use DNA sequencing data to infer the origin of a Formosan subterranean termite, Coptotermes formosanus Shiraki, infestation. Cytochrome oxidase II gene sequence was used to define eight specific maternal lineages from 14 geographic locations across the southeastern United States, Hawaii, and China. One maternal lineage, A, was unique to all the Atlanta collections and one New Orleans collection, which indicated that termites at these sites shared a common maternal ancestor. The impact of interstate commerce in terms of Formosan subterranean termite introductions is addressed as is the industry response needed to arrest the spread of these termites via commercial traffic in used railroad cross ties.

Population Genetic Baseline of the First Plataspid Stink Bug Symbiosis (Hemiptera: Heteroptera: Plataspidae) Reported in North America.

The stink bug, Megacopta cribraria, has an obligate relationship with a bacterial endosymbiont which allows it to feed on legumes. The insect is a pest of soybeans in Asia and was first reported in the Western Hemisphere in October 2009 on kudzu vine, Pueraria montana, in North Georgia, USA. By October 2010 M. cribraria had been confirmed in 80 counties in Georgia actively feeding on kudzu vine and soybean plants. Since the symbiosis may support the bug's ecological expansions, a population genetic baseline for the symbiosis was developed from mitochondrial DNA (mtDNA) and nuclear DNA (nuDNA) gene sequence collected from each insect and its primary g- proteobacterium and secondary a -proteobacterium endosymbionts. A single mitochondrial DNA haplotype was found in all insects sampled in Georgia and South Carolina identified as GA1. The GAI haplotype appears to be rapidly dispersing across Georgia and into contiguous states. Primary and secondary endosymbiont gene sequences from M. cribraria in Georgia were the same as those found in recently collected Megacopta samples from Japan. The implications of these data are discussed.

Discovering and verifying DNA polymorphisms in a mung bean [V. radiata (L.) R. Wilczek] collection by EcoTILLING and sequencing.

BACKGROUND: Vigna radiata, which is classified in the family Fabaceae, is an important economic crop and a dietary staple in many developing countries. The species radiata can be further subdivided into varieties of which the variety sublobata is currently acknowledged as the putative progenitor of radiata. EcoTILLING was employed to identify single nucleotide polymorphisms (SNPs) and small insertions/deletions (INDELS) in a collection of Vigna radiata accessions. FINDINGS: A total of 157 DNA polymorphisms in the collection were produced from ten primer sets when using V. radiata var. sublobata as the reference. The majority of polymorphisms detected were found in putative introns. The banding patterns varied from simple to complex as the number of DNA polymorphisms between two pooled samples increased. Numerous SNPs and INDELS ranging from 4-24 and 1-6, respectively, were detected in all fragments when pooling V. radiata var. sublobata with V. radiata var. radiata. On the other hand, when accessions of V. radiata var. radiata were mixed together and digested with CEL I relatively few SNPs and no INDELS were detected. CONCLUSION: EcoTILLING was utilized to identify polymorphisms in a collection of mung bean, which previously showed limited molecular genetic diversity and limited morphological diversity in the flowers and pod descriptors. Overall, EcoTILLING proved to be a powerful genetic analysis tool providing the rapid identification of naturally occurring variation.

Phylogeography illuminates maternal origins of exotic Coptotermes gestroi (Isoptera: Rhinotermitidae).

Coptotermes gestroi, the Asian subterranean termite (AST), is an economically important structural and agricultural pest that has become established in many areas of the world. For the first time, phylogeography was used to illuminate the origins of new found C. gestroi in the US Commonwealth of Puerto Rico; Ohio, USA; Florida, USA; and Brisbane, Australia. Phylogenetic relationships of C. gestroi collected in indigenous locations within Malaysia, Thailand, and Singapore as well as from the four areas of introduction were investigated using three genes (16S rRNA, COII, and ITS) under three optimality criteria encompassing phenetic and cladistic assumptions (maximum parsimony, maximum likelihood, and neighbor-joining). All three genes showed consistent support for a close genetic relationship between C. gestroi samples from Singapore and Ohio, whereas termite samples from Australia, Puerto Rico, and Key West, FL were more closely related to those from Malaysia. Shipping records further substantiated that Singapore and Malaysia were the likely origin of the Ohio and Australia C. gestroi, respectively. These data provide support for using phylogeography to understand the dispersal history of exotic termites. Serendipitously, we also gained insights into concerted evolution in an ITS cluster from rhinotermitid species in two genera.

Potential use of a host associated molecular marker in Enterococcus faecium as an index of human fecal pollution.

Several genotypic and phenotypic microbial source tracking (MST) methods have been proposed and utilized to differentiate groups of microorganisms, usually indicator organisms, for the purpose of tracking sources of fecal pollution. Targeting of host-specific microorganisms is one of the approaches currently being tested. These methods are useful as they circumvent the need to isolate individual microorganisms and do not require the establishment of reference databases. Several studies have demonstrated that the presence and distribution of Enterococcus spp. in feces seems to be influenced by the host species. Here, we present a method for detection of genetic sequences in culturable enterococci capable of identifying human sources of fecal pollution in the environment. The human fecal pollution marker designed in this study targets a putative virulence factor, the enterococcal surface protein (esp), in Enterococcus faecium. This gene was detected in 97% of sewage and septic samples but was not detected in any livestock waste lagoons or in bird or animal fecal samples. Epidemiological studies in recreational and groundwaters have shown enterococci to be useful indicators of public health risk for gastroenteritis. By identifying the presence of human fecal pollution, and therefore the possible presence of human enteric pathogens, this marker allows for further resolution of the source of this risk.