About the Authors: Lance B. Price * E-mail: lprice@gwu.edu Affiliations Milken Institute School of Public Health, George Washington University, Washington DC, United States of America, Division of ...Pathogen Genomics, Translational Genomics Research Institute, Flagstaff, Arizona, United States of America ORCID http://orcid.org/0000-0002-8746-5307 Bruce A. Hungate Affiliations Center for Ecosystem Science and Society, Northern Arizona University, Flagstaff, Arizona, United States of America, Department of Biological Sciences, Northern Arizona University, Flagstaff, Arizona, United States of America ORCID http://orcid.org/0000-0002-7337-1887 Benjamin J. Koch Affiliations Center for Ecosystem Science and Society, Northern Arizona University, Flagstaff, Arizona, United States of America, Department of Biological Sciences, Northern Arizona University, Flagstaff, Arizona, United States of America Gregg S. Davis Affiliation: Milken Institute School of Public Health, George Washington University, Washington DC, United States of America ORCID http://orcid.org/0000-0001-7684-0426 Cindy M. Liu Affiliation: Milken Institute School of Public Health, George Washington University, Washington DC, United States of AmericaCitation: Price LB, Hungate BA, Koch BJ, Davis GS, Liu CM (2017) Colonizing opportunistic pathogens (COPs): The beasts in all of us. ...COPs lack predictable periods between colonization and infection, making their epidemiology cryptic. Because of this, COPs can cause insidious epidemics, where new clones transmit widely-even globally-among healthy populations before being recognized. ...antibiotics can select for antibiotic-resistant COPs, in addition to commensals within the host microbiome, regardless of whether or not the specific COP was the intended target of the antibiotic treatment. COPs require new active and integrative surveillance programs Enhanced surveillance of COPs can enable public health agencies to identify and control emerging COP clones more quickly than is currently possible. Because of the insidious nature of COP epidemics, active surveillance programs that monitor both COPs circulating among asymptomatic carriers in the community and COPs causing clinical infections are crucial.
Antimicrobial resistance is a major public health crisis, eroding the discovery of antimicrobials and their application to clinical medicine. There is a general lack of knowledge of the importance of ...agricultural antimicrobial use as a factor in antimicrobial resistance even among experts in medicine and public health. This review focuses on agricultural antimicrobial drug use as a major driver of antimicrobial resistance worldwide for four reasons: It is the largest use of antimicrobials worldwide; much of the use of antimicrobials in agriculture results in subtherapeutic exposures of bacteria; drugs of every important clinical class are utilized in agriculture; and human populations are exposed to antimicrobial-resistant pathogens via consumption of animal products as well as through widespread release into the environment.
We sought to determine if the prevalence of antibiotic-resistant Escherichia coli differed across retail poultry products and among major production categories, including organic, "raised without ...antibiotics", and conventional.
We collected all available brands of retail chicken and turkey-including conventional, "raised without antibiotic", and organic products-every two weeks from January to December 2012. In total, E. coli was recovered from 91% of 546 turkey products tested and 88% of 1367 chicken products tested. The proportion of samples contaminated with E. coli was similar across all three production categories. Resistance prevalence varied by meat type and was highest among E. coli isolates from turkey for the majority of antibiotics tested. In general, production category had little effect on resistance prevalence among E. coli isolates from chicken, although resistance to gentamicin and multidrug resistance did vary. In contrast, resistance prevalence was significantly higher for 6 of the antibiotics tested-and multidrug resistance-among isolates from conventional turkey products when compared to those labelled organic or "raised without antibiotics". E. coli isolates from chicken varied strongly in resistance prevalence among different brands within each production category.
The high prevalence of resistance among E. coli isolates from conventionally-raised turkey meat suggests greater antimicrobial use in conventional turkey production as compared to "raised without antibiotics" and organic systems. However, among E. coli from chicken meat, resistance prevalence was more strongly linked to brand than to production category, which could be caused by brand-level differences during production and/or processing, including variations in antimicrobial use.
Bacteria grow and transform elements at different rates, and as yet, quantifying this variation in the environment is difficult. Determining isotope enrichment with fine taxonomic resolution after ...exposure to isotope tracers could help, but there are few suitable techniques. We propose a modification to stable isotope probing (SIP) that enables the isotopic composition of DNA from individual bacterial taxa after exposure to isotope tracers to be determined. In our modification, after isopycnic centrifugation, DNA is collected in multiple density fractions, and each fraction is sequenced separately. Taxon-specific density curves are produced for labeled and nonlabeled treatments, from which the shift in density for each individual taxon in response to isotope labeling is calculated. Expressing each taxon's density shift relative to that taxon's density measured without isotope enrichment accounts for the influence of nucleic acid composition on density and isolates the influence of isotope tracer assimilation. The shift in density translates quantitatively to isotopic enrichment. Because this revision to SIP allows quantitative measurements of isotope enrichment, we propose to call it quantitative stable isotope probing (qSIP). We demonstrated qSIP using soil incubations, in which soil bacteria exhibited strong taxonomic variations in (18)O and (13)C composition after exposure to (18)Owater or (13)Cglucose. The addition of glucose increased the assimilation of (18)O into DNA from (18)Owater. However, the increase in (18)O assimilation was greater than expected based on utilization of glucose-derived carbon alone, because the addition of glucose indirectly stimulated bacteria to utilize other substrates for growth. This example illustrates the benefit of a quantitative approach to stable isotope probing.
E. coli
ST131 is an important extraintestinal pathogen that can colonize the gastrointestinal tracts of humans and food animals. Here, we combined detection of accessory traits associated with avian ...adaptation (ColV plasmids) with high-resolution phylogenetics to quantify the portion of human infections caused by ST131 strains of food animal origin. Our results suggest that one ST131 sublineage—ST131-
H
22—has become established in poultry populations around the world and that meat may serve as a vehicle for human exposure and infection. ST131-
H
22 is just one of many
E. coli
lineages that may be transmitted from food animals to humans. Additional studies that combine detection of host-associated accessory elements with phylogenetics may allow us to quantify the total fraction of human extraintestinal infections attributable to food animal
E. coli
strains.
Escherichia coli
sequence type 131 (ST131) has emerged rapidly to become the most prevalent extraintestinal pathogenic
E. coli
clones in circulation today. Previous investigations appeared to exonerate retail meat as a source of human exposure to ST131; however, these studies focused mainly on extensively multidrug-resistant ST131 strains, which typically carry allele 30 of the
fimH
type 1 fimbrial adhesin gene (ST131-
H
30). To estimate the frequency of extraintestinal human infections arising from foodborne ST131 strains without bias toward particular sublineages or phenotypes, we conducted a 1-year prospective study of
E. coli
from meat products and clinical cultures in Flagstaff, Arizona. We characterized all isolates by multilocus sequence typing,
fimH
typing, and core genome phylogenetic analyses, and we screened isolates for avian-associated ColV plasmids as an indication of poultry adaptation.
E. coli
was isolated from 79.8% of the 2,452 meat samples and 72.4% of the 1,735 culture-positive clinical samples. Twenty-seven meat isolates were ST131 and belonged almost exclusively (
n
= 25) to the ST131-
H
22 lineage. All but 1 of the 25 H22 meat isolates were from poultry products, and all but 2 carried poultry-associated ColV plasmids. Of the 1,188 contemporaneous human clinical
E. coli
isolates, 24 were ST131-
H
22, one-quarter of which occurred in the same high-resolution phylogenetic clades as the ST131-
H
22 meat isolates and carried ColV plasmids. Molecular clock analysis of an international ST131-
H
22 genome collection suggested that ColV plasmids have been acquired at least six times since the 1940s and that poultry-to-human transmission is not limited to the United States.
Administration of antibiotics to food animals may select for drug-resistant pathogens of clinical significance, such as methicillin-resistant Staphylococcus aureus (MRSA). In the United States, ...studies have examined prevalence of MRSA carriage among individuals exposed to livestock, but prevalence of multidrug-resistant S. aureus (MDRSA) carriage and the association with livestock raised with versus without antibiotic selective pressure remains unclear. We aimed to examine prevalence, antibiotic susceptibility, and molecular characteristics of S. aureus among industrial livestock operation (ILO) and antibiotic-free livestock operation (AFLO) workers and household members in North Carolina.
Participants in this cross-sectional study were interviewed and provided a nasal swab for S. aureus analysis. Resulting S. aureus isolates were assessed for antibiotic susceptibility, multi-locus sequence type, and absence of the scn gene (a marker of livestock association).
Among 99 ILO and 105 AFLO participants, S. aureus nasal carriage prevalence was 41% and 40%, respectively. Among ILO and AFLO S. aureus carriers, MRSA was detected in 7% (3/41) and 7% (3/42), respectively. Thirty seven percent of 41 ILO versus 19% of 42 AFLO S. aureus-positive participants carried MDRSA. S. aureus clonal complex (CC) 398 was observed only among workers and predominated among ILO (13/34) compared with AFLO (1/35) S. aureus-positive workers. Only ILO workers carried scn-negative MRSA CC398 (2/34) and scn-negative MDRSA CC398 (6/34), and all of these isolates were tetracycline resistant.
Despite similar S. aureus and MRSA prevalence among ILO and AFLO-exposed individuals, livestock-associated MRSA and MDRSA (tetracycline-resistant, CC398, scn-negative) were only present among ILO-exposed individuals. These findings support growing concern about antibiotics use and confinement in livestock production, raising questions about the potential for occupational exposure to an opportunistic and drug-resistant pathogen, which in other settings including hospitals and the community is of broad public health importance.
Circumcision is associated with significant reductions in HIV, HSV-2 and HPV infections among men and significant reductions in bacterial vaginosis among their female partners.
We assessed the penile ...(coronal sulci) microbiota in 12 HIV-negative Ugandan men before and after circumcision. Microbiota were characterized using sequence-tagged 16S rRNA gene pyrosequencing targeting the V3-V4 hypervariable regions. Taxonomic classification was performed using the RDP Naïve Bayesian Classifier. Among the 42 unique bacterial families identified, Pseudomonadaceae and Oxalobactericeae were the most abundant irrespective of circumcision status. Circumcision was associated with a significant change in the overall microbiota (PerMANOVA p = 0.007) and with a significant decrease in putative anaerobic bacterial families (Wilcoxon Signed-Rank test p = 0.014). Specifically, two families-Clostridiales Family XI (p = 0.006) and Prevotellaceae (p = 0.006)-were uniquely abundant before circumcision. Within these families we identified a number of anaerobic genera previously associated with bacterial vaginosis including: Anaerococcus spp., Finegoldia spp., Peptoniphilus spp., and Prevotella spp.
The anoxic microenvironment of the subpreputial space may support pro-inflammatory anaerobes that can activate Langerhans cells to present HIV to CD4 cells in draining lymph nodes. Thus, the reduction in putative anaerobic bacteria after circumcision may play a role in protection from HIV and other sexually transmitted diseases.
Fungal load quantification is a critical component of fungal community analyses. Limitation of current approaches for quantifying the fungal component in the human microbiome suggests the need for ...new broad-coverage techniques.
We analyzed 2,085 18S rRNA gene sequences from the SILVA database for assay design. We generated and quantified plasmid standards using a qPCR-based approach. We evaluated assay coverage against 4,968 sequences and performed assay validation following the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines.
We designed FungiQuant, a TaqMan® qPCR assay targeting a 351 bp region in the fungal 18S rRNA gene. Our in silico analysis showed that FungiQuant is a perfect sequence match to 90.0% of the 2,617 fungal species analyzed. We showed that FungiQuant's is 100% sensitive and its amplification efficiencies ranged from 76.3% to 114.5%, with r(2)-values of >0.99 against the 69 fungal species tested. Additionally, FungiQuant inter- and intra-run coefficients of variance ranged from <10% and <20%, respectively. We further showed that FungiQuant has a limit of quantification 25 copies and a limit of detection at 5 copies. Lastly, by comparing results from human-only background DNA with low-level fungal DNA, we showed that amplification in two or three of a FungiQuant performed in triplicate is statistically significant for true positive fungal detection.
FungiQuant has comprehensive coverage against diverse fungi and is a robust quantification and detection tool for delineating between true fungal detection and non-target human DNA.
Antibiotic-resistant pathogens increasingly threaten human health. Widespread application of antibiotics to animal populations raised for food, including chickens, cattle, and pigs, selects for ...resistance and contributes to the evolution of those pathogens. Despite a half century of research establishing the mechanisms and pathways by which antibiotic-resistant bacteria spread from food animals to people, scientists lack the appropriate data and models to estimate the public health burden of antibiotic-resistant human infections attributable to antibiotic use in food-animal production. Genomic technologies are enabling researchers to track the bidirectional transmissions of specific bacterial strains from livestock to people – and from people to livestock – that can amplify resistance traits. Concepts in ecology, which were developed to understand resource subsidies, metapopulations, and biological invasions, provide insight into the epidemiology of antibiotic resistance from genomic data. By applying ecological principles to highly resolved phylogenetic data, researchers can improve strategies for controlling antibiotic resistance.
Escherichia colisequence type 131 (ST131) has emerged globally as the most predominant extraintestinal pathogenic lineage within this clinically important species, and its association with ...fluoroquinolone and extended-spectrum cephalosporin resistance impacts significantly on treatment. The evolutionary histories of this lineage, and of important antimicrobial resistance elements within it, remain unclearly defined. This study of the largest worldwide collection (n= 215) of sequenced ST131E. coliisolates to date demonstrates that the clonal expansion of two previously recognized antimicrobial-resistant clades, C1/H30R and C2/H30Rx, started around 25 years ago, consistent with the widespread introduction of fluoroquinolones and extended-spectrum cephalosporins in clinical medicine. These two clades appear to have emerged in the United States, with the expansion of the C2/H30Rx clade driven by the acquisition of ablaCTX-M-15-containing IncFII-like plasmid that has subsequently undergone extensive rearrangement. Several other evolutionary processes influencing the trajectory of this drug-resistant lineage are described, including sporadic acquisitions of CTX-M resistance plasmids and chromosomal integration ofblaCTX-Mwithin subclusters followed by vertical evolution. These processes are also occurring for another family of CTX-M gene variants more recently observed among ST131, theblaCTX-M-14/14-likegroup. The complexity of the evolutionary history of ST131 has important implications for antimicrobial resistance surveillance, epidemiological analysis, and control of emerging clinical lineages ofE. coli These data also highlight the global imperative to reduce specific antibiotic selection pressures and demonstrate the important and varied roles played by plasmids and other mobile genetic elements in the perpetuation of antimicrobial resistance within lineages.
Escherichia coli, perennially a major bacterial pathogen, is becoming increasingly difficult to manage due to emerging resistance to all preferred antimicrobials. Resistance is concentrated within specificE. colilineages, such as sequence type 131 (ST131). Clarification of the genetic basis for clonally associated resistance is key to devising intervention strategies. We used high-resolution genomic analysis of a large global collection of ST131 isolates to define the evolutionary history of extended-spectrum beta-lactamase production in ST131. We documented diverse contributory genetic processes, including stable chromosomal integrations of resistance genes, persistence and evolution of mobile resistance elements within sublineages, and sporadic acquisition of different resistance elements. Both global distribution and regional segregation were evident. The diversity of resistance element acquisition and propagation within ST131 indicates a need for control and surveillance strategies that target both bacterial strains and mobile genetic elements.
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