On-farm soil resistome is modified after treating dairy calves with the antibiotic florfenicol.

We determined the immediate impact of exposure to antibiotic-treated animals on housing soil microbiome and resistome. Fecal (n = 36) and soil (n = 108) samples from dairy calves (n = 6) treated with and without florfenicol over 30 days were collected. There were temporary changes in the gut microbiome of antibiotic-treated calves as measured by Shannon diversity (16S rRNA gene sequencing; P = 0.03), but not in the housing soil microbiome (P > 0.05). Droplet-digital PCR demonstrated that floR gene increased by 1-log in soil exposed to treated animals (P < 0.001), but it remained relatively stable in the control soil whereby calves were not treated with antibiotic. Resistome in exposed soil was largely modified (P = 0.004) with the overall prevalence of antimicrobial resistance genes (ARGs) significantly elevated (3.8-fold increase by day 10; P = 0.01). In addition to florfenicol, enriched ARGs collectively conferring resistance to tetracyclines, aminoglycosides, sulfonamides, elfamycins, macrolides-lincosamides-streptrogramin A/B, and beta-lactams. Quantitative PCR validated that ARGs including str and tetG in soil exposed to florfenicol-treated calves had gradually increased fold-change difference relative to the control soil over time. Moreover, a greater diversity of transferrable ARGs was observed in exposed soil and these were associated with a greater diversity of bacterial species. Evaluation of on-farm effects to soil in situ after exposure to antibiotic-treated animals can help design effective managements to mitigate antibiotic resistance in food-animal production.

Medicinal foods and beverages among Maasai agro-pastoralists in northern Tanzania.

ETHNOPHARMACOLOGICAL RELEVANCE: Pastoralist Maasai populations of east Africa use several different wild plants as dietary and medicinal additives in beverages (soups and teas), yet little is known about how the plants used and the rationales for use compare and contrast across different Maasai beverages, including how gender specific dietary and health concerns structure patterns of intake. AIM OF THE STUDY: We investigated three Maasai beverages: almajani (tea or herbal infusion); motorí (traditional soup); and okiti (psychoactive herbal tea). In order to build knowledge about the cultural functions of these Maasai food-medicines and their incidence of use we also investigated use rationales and self-reported frequencies of use. We conclude by examining gender differences and the possible pharmacological antimicrobial activity of the most frequently used plants. MATERIALS AND METHODS: Research was conducted in 2015, with a population of semi-nomadic agropastoralist Maasai residing in northern Tanzania. Data were collected using key informant interviews, plant collections, n = 32 structured surveys, and n = 40 freelist interviews followed by a literature review to determine the known antimicrobial activity of the most used plants. RESULTS: We identified 20 plants that Maasai add to soup, 11 in tea, and 11 in the psychoactive tea, for a total of 24 herbal additives. Seven plant species were used in all three Maasai beverages, and these clustered with 10 common ailments. Based on self-reports, women use the beverages less frequently and in smaller amounts than men. There were also several gender differences in the plants that Maasai add to motorí and their associated use rationales. CONCLUSIONS: There are several intersections concerning the plant species used and their associated rationales for use in almajani, motori, and okiti. Moving outward, Maasai beverages and their additives increasingly involve gender specific concerns. Female use of food-medicines, relative to men, is structured by concerns over pregnancy, birth, and lactation. The frequent consumption of herbal additives, many of which contain antimicrobial compounds, potentially helps modulate infections, but could have other unintentional effects as well.

Medical pluralism and livestock health: ethnomedical and biomedical veterinary knowledge among East African agropastoralists.

BACKGROUND: Human and animal health are deeply intertwined in livestock dependent areas. Livestock health contributes to food security and can influence human health through the transmission of zoonotic diseases. In low-income countries diagnosis and treatment of livestock diseases is often carried out by household members who draw upon both ethnoveterinary medicine (EVM) and contemporary veterinary biomedicine (VB). Expertise in these knowledge bases, along with their coexistence, informs treatment and thus ultimately impacts animal and human health. The aim of the current study was to determine how socio-cultural and ecological differences within and between two livestock-keeping populations, the Maasai of northern Tanzania and Koore of southwest Ethiopia, impact expertise in EVM and VB and coexistence of the two knowledge bases. METHODS: An ethnoveterinary research project was conducted to examine dimensions of EVM and VB knowledge among the Maasai (N = 142 households) and the Koore (N = 100). Cultural consensus methods were used to quantify expertise and the level of agreement on EVM and VB knowledge. Ordinary least squares regression was used to model patterns of expertise and consensus across groups and to examine associations between knowledge and demographic/sociocultural attributes. RESULTS: Maasai and Koore informants displayed high consensus on EVM but only the Koore displayed consensus on VB knowledge. EVM expertise in the Koore varied across gender, herd size, and level of VB expertise. EVM expertise was highest in the Maasai but was only associated with age. The only factor associated with VB expertise was EVM expertise in the Koore. CONCLUSIONS: Variation in consensus and the correlates of expertise across the Maassi and the Koore are likely related to differences in the cultural transmission of EVM and VB knowledge. Transmission dynamics are established by the integration of livestock within the socioecological systems of the Maasai and Koore and culture historical experiences with livestock disease. Consideration of the nature and coexistence of EVM and VB provides insight into the capacity of groups to cope with disease outbreaks, pharmaceutical use patterns, and the development of community health interventions.

Hyperosmotic Agents and Antibiotics Affect Dissolved Oxygen and pH Concentration Gradients in Staphylococcus aureus Biofilms.

Biofilms on wound surfaces are treated topically with hyperosmotic agents, such as medical-grade honey and cadexomer iodine; in some cases, these treatments are combined with antibiotics. Tissue repair requires oxygen, and a low pH is conducive to oxygen release from red blood cells and epithelialization. We investigated the variation of dissolved oxygen concentration and pH with biofilm depth and the variation in oxygen consumption rates when biofilms are challenged with medical-grade honey or cadexomer iodine combined with vancomycin or ciprofloxacin. Dissolved oxygen and pH depth profiles in Staphylococcus aureus biofilms were measured using microelectrodes. The presence of cadexomer iodine with vancomycin or ciprofloxacin on the surface of the biofilm permitted a measurable concentration of oxygen at greater biofilm depths (101.6 ± 27.3 µm, P = 0.02; and 155.5 ± 27.9 µm, P = 0.016, respectively) than in untreated controls (30.1 µm). Decreases in pH of ∼0.6 and ∼0.4 units were observed in biofilms challenged with medical-grade honey alone and combined with ciprofloxacin, respectively (P < 0.001 and 0.01, respectively); the number of bacteria recovered from biofilms was significantly reduced (1.26 log) by treatment with cadexomer iodine and ciprofloxacin (P = 0.002) compared to the untreated control. Combining cadexomer iodine and ciprofloxacin improved dissolved oxygen concentration and penetration depth into the biofilm, while medical-grade honey was associated with a lower pH; not all treatments established a bactericidal effect in the time frame used in the experiments.IMPORTANCE Reports about using hyperosmotic agents and antibiotics against wound biofilms focus mostly on killing bacteria, but the results of these treatments should additionally be considered in the context of how they affect physiologically important parameters, such as oxygen concentration and pH. We confirmed that the combination of a hyperosmotic agent and an antibiotic results in greater dissolved oxygen and reduced pH within an S. aureus biofilm.

Soil-borne reservoirs of antibiotic-resistant bacteria are established following therapeutic treatment of dairy calves.

We determined if antibiotics residues that are excreted from treated animals can contribute to persistence of resistant bacteria in agricultural environments. Administration of ceftiofur, a third-generation cephalosporin, resulted in a ∼ 3 log increase in ceftiofur-resistant Escherichia coli found in the faeces and pen soils by day 10 (P = 0.005). This resistant population quickly subsided in faeces, but was sustained in the pen soil (∼ 4.5 log bacteria g(-1)) throughout the trial (1 month). Florfenicol treatment resulted in a similar pattern although the loss of florfenicol-resistant E. coli was slower for faeces and remained stable at ∼ 6 log bacteria g(-1) in the soil. Calves were treated in pens where eGFP-labelled E. coli were present in the bedding (∼ 2 log g(-1)) resulting in amplification of the eGFP E. coli population ∼ 2.1 log more than eGFP E. coli populations in pens with untreated calves (day 4; P < 0.005). Excreted residues accounted for > 10-fold greater contribution to the bedding reservoir compared with shedding of resistant bacteria in faeces. Treatment with therapeutic doses of ceftiofur or florfenicol resulted in 2-3 log g(-1) more bacteria than the estimated ID50 (2.83 CFU g(-1)), consistent with a soil-borne reservoir emerging after antibiotic treatment that can contribute to the long-term persistence of antibiotic resistance in animal agriculture.

Antimicrobial resistance in beef and dairy cattle production.

Observational studies of cattle production systems usually find that cattle from conventional dairies harbor a higher prevalence of antimicrobial resistant (AMR) enteric bacteria compared to organic dairies or beef-cow operations; given that dairies usually use more antimicrobials, this result is not unexpected. Experimental studies have usually verified that application of antimicrobials leads to at least a transient expansion of AMR bacterial populations in treated cattle. Nevertheless, on dairy farms the majority of antibiotics are used to treat mastitis and yet AMR remains relatively low in mastitis pathogens. Other studies have shown no correlation between antimicrobial use and prevalence of AMR bacteria including documented cases where the prevalence of AMR bacteria is non-responsive to antimicrobial applications or remains relatively high in the absence of antimicrobial use or any other obvious selective pressures. Thus, there are multi-factorial events and pressures that influence AMR bacterial populations in cattle production systems. We introduce a heuristic model that illustrates how repeated antimicrobial selection pressure can increase the probability of genetic linkage between AMR genes and niche- or growth-specific fitness traits. This linkage allows persistence of AMR bacteria at the herd level because subpopulations of AMR bacteria are able to reside long-term within the host animals even in the absence of antimicrobial selection pressure. This model highlights the need for multiple approaches to manage herd health so that the total amount of antimicrobials is limited in a manner that meets animal welfare and public health needs while reducing costs for producers and consumers over the long-term.

The streptomycin-sulfadiazine-tetracycline antimicrobial resistance element of calf-adapted Escherichia coli is widely distributed among isolates from Washington state cattle.

Association of specific antimicrobial resistance patterns with unrelated selective traits has long been implicated in the maintenance of antimicrobial resistance in a population. Previously we demonstrated that Escherichia coli strains with a specific resistance pattern (resistant to streptomycin, sulfadiazine, and tetracycline [SSuT]) have a selective advantage in dairy calf intestinal environments and in the presence of a milk supplement commonly fed to the calves. In the present study we identified the sequence of the genetic element that confers the SSuT phenotype and show that this element is present in a genetically diverse group of E. coli isolates, as assessed by macrorestriction digestion and pulsed-field gel electrophoresis. This element was also found in E. coli isolates from 18 different cattle farms in Washington State. Using in vitro competition experiments we further demonstrated that SSuT strains from 17 of 18 farms were able to outcompete pansusceptible strains. In a separate set of experiments, we were able to transfer the antimicrobial resistance phenotype by electroporation to a laboratory strain of E. coli (DH10B), making that new strain more competitive during in vitro competition with the parental DH10B strain. These data indicate that a relatively large genetic element conferring the SSuT phenotype is widely distributed in E. coli from cattle in Washington State. Furthermore, our results indicate that this element is responsible for maintenance of these traits owing to linkage to genetic traits that confer a selective advantage in the intestinal lumens of dairy calves.

Antimicrobial resistance in Salmonella enterica serovar Dublin isolates from beef and dairy sources.

Salmonella enterica serovar Dublin (S. Dublin) is a cattle-adapted Salmonella serovar, so if antimicrobial resistance in S. Dublin arises as a result of antimicrobial use this most likely occurs within the cattle reservoir without impact from antimicrobial use in humans. We tested the antimicrobial resistance of bovine-origin S. Dublin isolates from 1986 through 2004 using a standard disk diffusion method. High proportions of isolates throughout the time period were resistant to one or more antimicrobials, and a marked increase in resistance to ceftazidime occurred between 2000 and 2004. Dairy-origin isolates were more likely to be resistant to several antibiotics than were isolates from beef operations where exposure to antimicrobials is likely to be less frequent. Plasmid analysis of a subset of isolates also supported the hypothesis that antimicrobial resistance traits in the cattle-adapted serovar Dublin were acquired within the bovine host environment.

Use of a nonmedicated dietary supplement correlates with increased prevalence of streptomycin-sulfa-tetracycline-resistant Escherichia coli on a dairy farm.

We examined how a dietary supplement affects the prevalence of antibiotic-resistant Escherichia coli on a dairy farm in Washington State. Between 2001 and 2004 the prevalence of fecal E. coli strains resistant to streptomycin, sulfadiazine, and tetracycline (SSuT strains) declined from 59.2% to 26.1% in the calf population. In 2003 the dairy discontinued use of a dietary supplement, and we hypothesized that the decline in prevalence of SSuT strains was related to this change in management. To test this we established three treatments in which calves received no supplement, the dietary supplement with oxytetracycline, or the dietary supplement without oxytetracycline. Calves receiving either dietary supplement had a significantly higher prevalence of SSuT E. coli than the no-supplement control group (approximately 37% versus 20%, respectively; P = 0.03). Importantly, there was no evidence that oxytetracycline contributed to an increased prevalence of fecal SSuT E. coli. We compared the growth characteristics of SSuT and non-SSuT E. coli in LB broth enriched with either the complete dietary supplement or its individual constituents. Both the complete dietary supplement and its vitamin D component supported a significantly higher cell density of SSuT strains (P = 0.003 and P = 0.001, respectively). The dry milk and vitamin A components of the dietary supplement did not support different cell densities. These results were consistent with selection and maintenance of SSuT E. coli due to environmental components independent of antibiotic selection.

Antimicrobial drug resistance genes do not convey a secondary fitness advantage to calf-adapted Escherichia coli.

Maintenance of antimicrobial drug resistance in bacteria can be influenced by factors unrelated to direct selection pressure such as close linkage to other selectively advantageous genes and secondary advantage conveyed by antimicrobial resistance genes in the absence of drug selection. Our previous trials at a dairy showed that the maintenance of the antimicrobial resistance genes is not influenced by specific antimicrobial selection and that the most prevalent antimicrobial resistance phenotype of Escherichia coli is specifically selected for in young calves. In this paper we examine the role of secondary advantages conveyed by antimicrobial resistance genes. We tested antimicrobial-susceptible null mutant strains for their ability to compete with their progenitor strains in vitro and in vivo. The null mutant strains were generated by selection for spontaneous loss of resistance genes in broth supplemented with fusaric acid or nickel chloride. On average, the null mutant strains were as competitive as the progenitor strains in vitro and in newborn calves (in vivo). Inoculation of newborn calves at the dairy with antimicrobial-susceptible strains of E. coli did not impact the prevalence of antimicrobial-resistant E. coli. Our results demonstrate that the antimicrobial resistance genes are not responsible for the greater fitness advantage of antimicrobial-resistant E. coli in calves, but the farm environment and the diet clearly exert critical selective pressures responsible for the maintenance of antimicrobial resistance genes. Our current hypothesis is that the antimicrobial resistance genes are linked to other genes responsible for differential fitness in dairy calves.

Amplicon secondary structure prevents target hybridization to oligonucleotide microarrays.

DNA microarrays that are used as end-point detectors for PCR assays are typically composed of short (15-25 mer) oligonucleotide probes bound to glass. When designing these detectors, we have frequently encountered situations where a probe would not hybridize to its complementary, terminally labeled PCR amplicon. To determine if failures could be explained by general phenomenon such as secondary structure, we designed a microarray to detect eight regions of the Escherichia coli 16S rDNA gene. We then amplified eight amplicons of different lengths using a biotin conjugated, antisense primer. Amplicons were then hybridized to the microarray and detected using a combination of signal amplification and fluorescence. In most cases, probe sequences complementary to the 5' region of the amplified products (sense orientation) did not hybridize to their respective amplicon. We tested for positional bias and showed that a biotin conjugated sense primer mirrored the same probe failures. Nick translated products, however, hybridized to all probes. Because nick translation generates many labeled fragments of random length, we concluded that this method disrupted secondary structure that otherwise prevented the amplicons from hybridizing to their respective probes. We also show that nick translation does not compromise detector sensitivity even when used with long PCR amplicons (ca. 1.5 kbp). Despite the increased cost of the nick translation, we concluded that this labeling strategy will reduce the time needed to design new assays as well as avoid possible false negatives during field applications. Alternative labeling strategies are also discussed.

Role of calf-adapted Escherichia coli in maintenance of antimicrobial drug resistance in dairy calves.

The prevalence of antimicrobial drug-resistant bacteria is typically highest in younger animals, and prevalence is not necessarily related to recent use of antimicrobial drugs. In dairy cattle, we hypothesize that antimicrobial drug-resistant, neonate-adapted bacteria are responsible for the observed high frequencies of resistant Escherichia coli in calves. To explore this issue, we examined the age distribution of antimicrobial drug-resistant E. coli from Holstein cattle at a local dairy and conducted an experiment to determine if low doses of oxytetracycline affected the prevalence of antimicrobial drug-resistant E. coli. Isolates resistant to tetracycline (>4 microg/ml) were more prevalent in <3-month-old calves (79%) compared with lactating cows (14%). In an experimental trial where calves received diets supplemented with or without oxytetracycline, the prevalence of tetracycline-resistant E. coli was slightly higher for the latter group (P = 0.039), indicating that drug use was not required to maintain a high prevalence of resistant E. coli. The most common resistance pattern among calf E. coli isolates included resistance to streptomycin (>12 microg/ml), sulfadiazine (>512 microg/ml), and tetracycline (>4 microg/ml) (SSuT), and this resistance pattern was most prevalent during the period when calves were on milk diets. To determine if prevalence was a function of differential fitness, we orally inoculated animals with nalidixic acid-resistant strains of SSuT E. coli and susceptible E. coli. Shedding of SSuT E. coli was significantly greater than that of susceptible strains in neonatal calves (P < 0.001), whereas there was no difference in older animals (P = 0.5). These data support the hypothesis that active selection for traits linked to the SSuT phenotype are responsible for maintaining drug-resistant E. coli in this population of dairy calves.