Reservoirs of Antibiotic Resistance Network -- ROAR  
ROAR -- Reservoirs of Antibiotic Resistence Network
ROAR Projects





ROAR I Prior to 2002

Probe Hybridization Array Typing:   Creating a High Throughput E coli Gene Presence/Absence Genotyping Method Suitable for Phylogenetic Analysis
Betsy Foxman
University of Michigan

Foxman plans to develop a rapid, high-throughput technique, probe hybridization array typing (PHAT), to genotype E. coli. To type E. coli, PHAT screens for the presence or absence of variable loci. Unlike multi-locus sequence typing (MLST), PHAT can provide phylogenetic information regarding genetic lineages as MLST does.  However, the rapid gain and loss of the PHAT genes provides information on the same time scale that the gain and loss of antibiotic resistance genes occurs. This technique will be used to assess the propensity of acquiring resistance in commensal and uropathogenic E. coli. [top]

Population Phylogenetic Framework of Antibiotic Resistance Emergence in Commensal Escherichia coli Isolates.
Antoine Andremont*, David Skurnik*,  Sylvain Brisse**, Erick Denamur***
*Universite Paris 7
**Institut Pasteur
***INSERM U722, Paris, France

This group proposes to examine a collection of commensal E. coli isolates exposed to varying levels of antibiotic selective pressure (animals in tropical rainforests versus mammals in high-intensity agricultural settings).  This project will use multi-locus sequence typing to assess resistance gene transfer among these isolates, compare virulence in sensitive and resistant isolates, and attempt to correlate the mechanism of tetracycline resistance with strain phylogeny and isolate host. [top]

Antibiotic Resistance and Genetic Relationship of E. coli from Australia.
David M. Gordon
The Australian National University

Gordon will determine if certain phylogenetic groups of E. coli are more prone to develop resistance and if there is a tradeoff between resistance and virulence.  To address these hypotheses, a collection of 375 commensal E. coli isolates obtained from endo- and ectothermic hosts isolated from different Australian habitats isolates will undergo antibiotic susceptibility testing, multi-locus sequence typing, bacteriocin typing, and virulence factor typing.  Preliminary data suggest that isolates from native Australian mammals are resistant to fewer antibiotics than those found associated with humans.  Thus, this collection may yield insights into what the population biology of antibiotic resistance in the pre-antibiotic era. [top]

High Throughput Molecular Genotyping of Staphylococci Carrying Class I Integrons.
Anne O. Summers
University of Georgia

The Summers project aims to elucidate whether staphylococci from humans and their companion animals and from poultry litter carry integrons and other resistance and virulence markers and, if so, whether they are on plasmids or the chromosome. Summers utilizes a novel method to examine a set of 100 integrase-positive Staphylococci from three sources: poultry litter (commensals), human clinical isolates, and companion animals. This study advances an inexpensive, efficient adaptation of microarray technology— “bead-based flow cytometry arrays”— in order to detect antimicrobial resistance markers associated with int11.  The experiments are designed to elucidate which genes are linked to intl1 genes, whether intl1 genes reside on plasmids and/or chromosomes, and to determine the diversity of mobile genetic elements in the integrase-positive staphylococci. Strains are independently assessed phenotypically and genotypically using standard agar diffusion, biochemical and PFGE techniques. [top]

Streptococcus mitis biovar 1 and its Potential as a Reservoir for S. pneumoniae.Susan K. Hollingshead
University of Alabama at Birmingham

Hollingshead hypothesizes that Streptococcus mitis biovar1, an abundant colonizer of the human nasopharynx, is a critical reservoir for breeding transferable antibiotic resistance genes which impact S. pneumoniae, a significant “commensal-like” pathogen which commonly colonizes the nasopharynx, particularly in young children. This study examines a collection of 400 S. mitis and normal, as well as atypical, S. pneumoniae from multiple, diverse global origins. In addition to serotyping and antibiotic susceptibility testing, the strains are undergoing traditional MLST, which will be supplemented with the sequencing of seven additional antibiotic resistance gene loci (associated with penicillin, cephalosporin, fluoroquinolone and macrolide resistance) that have alleles on both susceptible and resistant strains. Macrolide genes are identified by PCR. Comparative genome hybridization (CGH) is applied to selected isolates to test for both neutral and antibiotic resistance gene flow. [top]

Escherichia coli of Poultry: A Plasmid-mediated Reservoir of Resistance Genes?
Lisa K. Nolan and Timothy Johnson
Iowa State University

Nolan and Johnson are interested in the evolution of R plasmids, the impact of production animal practices on plasmid evolution, and the role these plasmids may play as a reservoir of antimicrobial resistance genes for human pathogens. In order to test their hypothesis that poultry production practices have selected for plasmid-containing avian E. coli (AEC) that may serve as reservoirs of resistance and virulence genes for human pathogenic E. coli, they have created and used multiple multiplex PCR protocols to 1) detect plasmid-associated resistance and virulence genes in order to determine if plasmid-containing AEC are emergent and if the constitution of their plasmids has changed over time, and 2) compare recently isolated fecal commensal E. coli of healthy birds (AFEC) and humans (HFEC) to pathogenic E. coli from human UTIs (UPEC) and avian colibacillosis (APEC) in order to determine if AEC might serve as a reservoir of plasmid-linked antimicrobial-resistance and virulence genes for UPEC. 966 APEC spanning three decades were analyzed for the presence of 68 virulence-associated genes, 29 resistance genes, and 17 plasmid replicons. Selected preliminary results show that several plasmid-linked virulence and resistance genes occurred more often in post-1990s APEC than in pre-1990s APEC. An increase in certain plasmid replicon types among APEC was seen over time, including the FIIA and FIB plasmids, which encode multidrug resistance and contribute to virulence, respectively. Data analysis is in progress. [top]

Exploring Transfer, Diversity, and Distribution of Antibiotic Resistance Genes Residing in Soil.
James M. Tiedje and Carlos Rodriguez-Minguela
Michigan State University

Tiedje et al. applied a PCR-based, culture-independent approach to the detection, characterization, tracking, and quantification of antibiotic resistance determinants and integrons in total DNA extracted from a variety of environmental samples. Four classes of tetracycline resistance genes (tet(M), tet(O), tet(Q), tet(36)) encoding ribosomal protection proteins, two new putative classes of RPP genes, and a novel mosaic gene were detected in DNA from agricultural soil supplemented with swine manure from swine fed tetracycline as a growth promoter. No tetracycline resistance genes were detected in non-agricultural reference soils. Nine variants (one new) of aminoglycoside nucleotydiltransferases and three genotypes (one new) for resistance to quaternary ammonium compounds were detected in association with class 1 integrons. Integrons were prevalent and diverse among all tested soils, but integrons encoding ABR genes were only detected in manured soils. Integron-encoded ABR genes persisted in manured soil after one month of manure application, while RPP determinants became less prevalent. In addition, samples from marine sediments, permafrost, and subtropical soils were screened for the presence of integron-encoded integrases. A total of 126 novel integrases were uncovered. Integrons were not detected in permafrost samples. Tiedje and Rodriguez-Minguela’s findings indicate that PCR-based techniques are effective tools for the detection and tracking of novel resistance loci in environmental DNA and that the integron module is highly diverse, prevalent in the environment, and not restricted to only bacteria recovered from the clinical setting. [top]

Profiling of mef and erm Resistance Genes in Oral Pediatric Isolates.
Marilyn C. Roberts
University of Washington
Antimicrobial Agents and Chemo. 48:9(2004)
FEMS Micro Lett 238 (2004)

Roberts et al. characterized 13 genera of commensal gram-negative oral and urine isolates from healthy children with low antibiotic exposure in Lisbon, Portugal for the presence of seven macrolide-lincosamide-streptogramin resistance genes, mef(A), erm(B), ere(a), ere(B), mph(A), mph(B), and mph(D). The genes were found in a variety of new genera.  Selected gram-negative isolates from the collection were shown to carry mobile mef(A)-msr(D) elements that could be transferred to both E. coli and Enterococcus faecalis recipients, with all transconjugates receiving and maintaining the gene. This work was presented at the 43rd and 44th ICAAC, the 104th Annual Meeting of ASM, and the 82nd General Session of the International Association for Dental Research. [top]

Characterization of Chloramphenicol and Florfenicol Resistance in Escherichia coli Associated with Bovine Diarrhea.
David G. White (ROAR I)
U.S. Food and Drug Administration (formerly of North Dakota State University)
J Clin Microbiol 38:12 Dec 2000

White et al. determined the mechanisms of florfenicol and chloramphenicol resistance in 48 E. coli isolated from diarrheic calves. Antimicrobial resistance profiles were determined and isolates were assayed for the related efflux genes flo and cmlA, (which convey resistance to both chloramphenicol and florfenicol and only chloramphenicol, respectively). 42 of 44 isolates with decreased chloramphenicol and florfenicol susceptibility were positive for flo, while only12 of the 48 isolates were positive for cmlA. DNA sequence analyses showed high homology with flo genes from the pathogen Salmonella Typhimurium DT104 and the environmental commensal, Photobacterium piscicida, suggesting horizontal gene transfer from these organisms. While most flo genes were found on a plasmid, some may be chromosomal. The findings demonstrated a wider distribution of the flo gene than previously believed, suggesting that the emerging chloramphenicol resistance phenotype observed in bovine E. coli is due to the flo gene. [top]

Antibiotic Resistance in Oral Commensal Streptococci from Healthy Mexicans and Cubans: Resistance Prevalence Does Not Mirror Antibiotic Usage
Carlos F. Amábile-Cuevas (ROAR I)
Fundación LUSARA, Mexico, D.F.
FEMS MICRO LETT 217 (2002)

Amábile-Cuevas et al. compared the frequencies of antibiotic resistance to seven drugs in 205 oral commensal streptococci from healthy Mexicans and Cubans not taking antibiotics. Despite marked differences in antibiotic usage between the two countries, resistance frequency did not differ significantly, suggesting that other selection pressures help to maintain the resistance phenotypes. Unlike the evidence compiled from gut Enterobacteriaceae, the evidence in oral streptococci suggests no significant association between antibiotic resistance and the number of dental amalgams or the mercury resistance phenotype. [top]




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ROAR is funded by grant AI50139 from the National Institute of Allergy and Infectious Diseases