Publications by authors named "Raghavendra G Amachawadi"

34 Publications

A systematic review and -analysis on the prevalence of infectious diseases of Duck: A world perspective.

Saudi J Biol Sci 2021 Sep 24;28(9):5131-5144. Epub 2021 May 24.

Department of Sciences, Amrita School of Arts and Sciences, Amrita Vishwa Vidyapeetham, Mysuru Campus, Mysuru, Karnataka 570 026, India.

The Indian poultry industry is one of the fast-growing sectors of which duck farming plays an important role. Duck population in India is 33.51 million that is concentrated towards north-east and southern parts of the country who rears mainly for eggs and meat. Duck diseases are of great concern as they badly affect the financial status of the small, landless farmers. Databases such as Google Scholar, PubMed, J gate were used to search articles between 2000 and 2019 that showed the prevalence of viral, bacterial, and parasitic duck diseases. R open source software was used to derive forest plots by statistical analysis. Pooled prevalence estimates of duck diseases worldwide was found to be 20% (95%-CI:15-26). Also, continent-wise analysis of all duck diseases has revealed highest prevalence in North America, followed by Asia, Africa, Europe,Oceania and South America. This prevalence of data would be helpful to the policymakers to develop appropriate intervention strategies to prevent and control diseases in their respective locations.
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http://dx.doi.org/10.1016/j.sjbs.2021.05.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8381006PMC
September 2021

Genomic Characterization of Fluoroquinolone-Resistant Thermophilic Strains Isolated from Layer Chicken Feces in Gangneung, South Korea by Whole-Genome Sequencing.

Genes (Basel) 2021 Jul 25;12(8). Epub 2021 Jul 25.

Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506-5606, USA.

Thermophilic species of poultry origin have been associated with up to 80% of human campylobacteriosis cases. Layer chickens have received less attention as possible reservoirs of species. Initially, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of two archived isolates ( strain 200605 and strain 200606) from layer chickens to five antimicrobials (ciprofloxacin, nalidixic acid, erythromycin, tetracycline, and gentamicin) were determined using broth microdilution while the presence of selected antimicrobial resistance genes was performed by polymerase chain reaction (PCR) using specific primers. Whole-genome sequencing (WGS) was performed by the Illumina HiSeq X platform. The analysis involved antimicrobial resistance genes, virulome, multilocus sequence typing (MLST), and phylogeny. Both isolates were phenotypically resistant to ciprofloxacin (MIC: 32 vs. 32 µg/mL), nalidixic acid (MIC: 128 vs. 64 µg/mL), and tetracycline (MIC: 64 vs. 64 µg/mL), but sensitive to erythromycin (MIC: 1 vs. 2 µg/mL) and gentamicin (MIC: 0.25 vs. 1 µg/mL) for strain 200605 and strain 200606, respectively. WGS confirmed C257T mutation in the A gene and the presence of ABC complex conferring resistance to FQs in both strains. Both strains also exhibited (O) genes associated with tetracycline resistance. Various virulence genes associated with motility, chemotaxis, and capsule formation were found in both isolates. However, the analysis of virulence genes showed that strain 200605 is more virulent than strain 200606. The MLST showed that strain 200605 belongs to sequence type ST-5229 while strain 200606 belongs to ST-5935, and both STs are less common. The phylogenetic analysis clustered strain 200605 along with other strains reported in Korea (CP028933 from chicken and CP014344 from human) while strain 200606 formed a separate cluster with (CP007181) from turkey. The WGS confirmed FQ-resistance in both strains and showed potential virulence of both strains. Further studies are recommended to understand the reasons behind the regional distribution (Korea, China, and Vietnam) of such rare STs.
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http://dx.doi.org/10.3390/genes12081131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8391547PMC
July 2021

Assessment of Chemopreventive Potential of the Plant Extracts against Liver Cancer Using HepG2 Cell Line.

Molecules 2021 Jul 29;26(15). Epub 2021 Jul 29.

Division of Molecular Biology, Faculty of Life Science, JSS Academy of Higher Education & Research, Mysuru 570015, India.

The edible parts of the plants , and were extensively used in ancient practices such as Ayurveda, owing to their potent biomedical significance. They are very rich in secondary metabolites such as polyphenols, which are very good antioxidants and exhibit anti-carcinogenic properties. This study aims to evaluate the anti-cancerous properties of these plant crude extracts on human liver cancer HepG2 cells. The leaves of , and the seeds of were collected and methanolic extracts were prepared. Then, these extracts were subjected to DPPH, α- amylase assays to determine the antioxidant properties. A MTT assay was performed to investigate the viability of the extracts of HepG2 cells, and the mode of cell death was detected by Ao/EtBr staining and flow cytometry with PI Annexin- V FITC dual staining. Then, the protein expression of BAX and BCl2 was studied using fluorescent dye to determine the regulation of the BAX and BCl2 genes. We observed that all the three extracts showed the presence of bioactive compounds such as polyphenols or phytochemicals. The bioactive compounds were found to have the highest anti-proliferative activity on human liver cancer cells.
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http://dx.doi.org/10.3390/molecules26154593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8348645PMC
July 2021

Shiga Toxin-Producing in Wheat Grains: Detection and Isolation by Polymerase Chain Reaction and Culture Methods.

Foodborne Pathog Dis 2021 Jun 28. Epub 2021 Jun 28.

Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas, USA.

Shiga toxin-producing (STEC) are major foodborne pathogens and seven serogroups, O26, O45, O103, O111, O121, O145, and O157, often called top-7 STEC, account for the majority of the STEC-associated human illnesses in the United States. Two Shiga toxins, Shiga toxins 1 and 2, encoded by 1 and 2 genes, are major virulence factors that are involved in STEC infections. Foodborne STEC infections have been linked to a variety of foods of both animal and plant origin, including products derived from cereal grains. In recent years, a few STEC outbreaks have been linked to contaminated wheat flour. The microbiological quality of the wheat grains is a major contributor to the safety of wheat flour. The objective of the study was to utilize polymerase chain reaction (PCR)- and culture-based methods to detect and isolate STEC in wheat grains. Wheat grain samples ( = 625), collected from different regions of the United States, were enriched in modified buffered peptone water with pyruvate (mBPWp) or (EC) broth, and they were then subjected to PCR- and culture-based methods to detect and isolate STEC. Wheat grains enriched in EC broth yielded more samples positive for genes (1.6% vs. 0.32%) and STEC serogroups (5.8% vs. 2.4%) than mBPWp. The four serogroups of top-7 detected and isolated were O26, O45, O103, and O157 and none of the isolates was positive for the Shiga toxin genes. A total of five isolates that carried the 2 gene were isolated and identified as serogroups O8 (0.6%) and O130 (0.2%). The EC broth was a better medium to enrich wheat grains than mBPWp for the detection and isolation of STEC. The overall prevalence of virulence genes and STEC serogroups in wheat grains was low. The 2-positive serogroups isolated, O8 and O130, are not major STEC pathogens and have only been implicated in sporadic infections in animals and humans.
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http://dx.doi.org/10.1089/fpd.2021.0013DOI Listing
June 2021

Antimicrobial Activity of Sorghum Phenolic Extract on Bovine Foodborne and Mastitis-Causing Pathogens.

Antibiotics (Basel) 2021 May 17;10(5). Epub 2021 May 17.

Department of Diagnostic Medicine/Pathobiology, Kansas State University, Manhattan, KS 66506, USA.

Antimicrobial resistance in bacterial pathogens associated with bovine mastitis and human foodborne illnesses from contaminated food and water have an impact on animal and human health. Phenolic compounds have antimicrobial properties and some specialty sorghum grains are high in phenolic compounds, and the grain extract may have the potential as a natural antimicrobial alternative. The study's objective was to determine antimicrobial effects of sorghum phenolic extract on bacterial pathogens that cause bovine mastitis and human foodborne illnesses. Bacterial pathogens tested included , Typhimurium, , , , , , , and . Antibacterial activities of sorghum phenolic extracts were determined by agar-well diffusion assay. Sorghum phenolic extract was added to the wells in concentrations of 0, 100, 200, 500, 1000, or 4000 µg/mL. The control wells did not receive phenolic extract. Plates were incubated for 18-24 h, and the diameter of each zone of inhibition was measured. The results indicated that sorghum phenolic extract had inhibitory effects on , , , and .
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http://dx.doi.org/10.3390/antibiotics10050594DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8156376PMC
May 2021

A systematic review on ethnopharmacology, phytochemistry and pharmacological aspects of Linn.

Heliyon 2021 May 18;7(5):e07054. Epub 2021 May 18.

Departments of Clinical Sciences, Kansas State University, Manhattan, KS, 66506-5606, USA.

Linn. is a medicinal and culinary herb from the Southern European region known for its anti-infective, cardioprotective, gastroprotective, anti-inflammatory, and immunomodulatory activities since the Egyptian era. The reported pharmacological activities of L. include antibacterial, antioxidant, anti-inflammatory, antiviral, and anti-cancerous activities. In this review, a comprehensive approach is put forth to scrutinize and report the available data on phytochemistry, ethnopharmacology, pharmacology, and toxicology of the plant. The different extracts and essential oil obtained from the plant have been assessed and reported to treat ailments like microbial infections, inflammation, non-communicable diseases like cancer, and sexually transmitted diseases like HIV-1 and Herpes. The literature review has also indicated the use of volatile oils, phenolic acids, terpenoids, flavonoids, saponins, steroids, tannins, alkaloids, and polysaccharides in pharmacotherapy. Applications of these compounds including antidiabetic, anti-Alzheimer's, cardio, neuro and hepatoprotective, anti-osteoporosis, sedative, immunomodulatory, antioxidant, anti-tyrosinase, antispasmodic, antinociceptive, gastroprotective, anticonvulsant, antihypertensive, antidepressant, anti-amnesia, and anti-helminthic activities have been mentioned. Further, based on research gaps, recommendations have been provided to evaluate L. systematically to develop plant-based drugs, nutraceuticals, and to evaluate their clinical efficiency and safety.
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http://dx.doi.org/10.1016/j.heliyon.2021.e07054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8141878PMC
May 2021

Draft Genome Sequences of Salmonella enterica subsp. Serotype IIIb_61:I,v:1,5,(7) Strains Isolated from Wheat Grains.

Microbiol Resour Announc 2021 May 20;10(20). Epub 2021 May 20.

Department of Diagnostic Medicine and Pathobiology, Kansas State University, Olathe, Kansas, USA.

subsp. serotypes are primarily involved in reptile-associated salmonellosis in humans. Here, we report the draft genome sequences of three subsp. strains belonging to the serotype IIIb_61:1,v:1,5,(7), isolated from wheat grains collected at the time of harvest. Strains of serotype IIIb_61:1,v:1,5,(7) have been isolated from feces of reptiles, cattle, and sheep and from infections in humans.
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http://dx.doi.org/10.1128/MRA.00035-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8188352PMC
May 2021

An Understanding of the Global Status of Major Bacterial Pathogens of Milk Concerning Bovine Mastitis: A Systematic Review and Meta-Analysis (Scientometrics).

Pathogens 2021 Apr 30;10(5). Epub 2021 Apr 30.

Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USA.

In this study, the major mastitis pathogen prevalence in the cattle and buffalo of the world was estimated by a meta-analysis. (S) species, (St) species, and (Ec) prevalence studies reported during 1979-2019 were collected using online databases, and offline resources. A meta-analysis of these data was done with the meta package in R-Software. The was the major mastitis pathogen, mostly causing subclinical mastitis, Ec causing clinical mastitis and St causing subclinical and clinical mastitis. The pooled prevalence estimates of S, St, and Ec were 28%, 12%, and 11% in the world from 156, 129, and 92 studies, respectively. The S, St, and Ec prevalences were high in Latin America (51%), Oceania (25%), and Oceania (28%), respectively. Higher S, St, and Ec prevalences were observed by molecular methods, signifying high sensitivity and usefulness for future studies. Among bacterial species, (25%) followed by coagulase-negative species (20%), (11%), (9%),   (9%) were the important pathogens present in the milk of the world. We hypothesize that there is a urgent need to reduce mastitis pathogen prevalence by ensuring scientific farm management practices, proper feeding, therapeutic interventions to augment profits in dairying, and improving animal and human health.
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http://dx.doi.org/10.3390/pathogens10050545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8147236PMC
April 2021

Antimicrobial Resistance Profiles, Virulence Genes, and Genetic Diversity of Thermophilic Species Isolated From a Layer Poultry Farm in Korea.

Front Microbiol 2021 29;12:622275. Epub 2021 Mar 29.

Natural Product Informatics Research Center, KIST Gangneung Institute of Natural Products, Gangneung, South Korea.

Thermophilic species are among the major etiologies of bacterial enteritis globally. This study aimed at assessing the antimicrobial resistance (AMR) profiles, virulence genes, and genetic diversity of thermophilic species isolated from a layer poultry farm in South Korea. One hundred fifty-three chicken feces were collected from two layer poultry farms in Gangneung, South Korea. The species were isolated by cultural techniques, while PCR and sequencing were used for species confirmation. Antimicrobial susceptibility testing for six antimicrobials [ciprofloxacin (CIP), nalidixic acid (NAL), sitafloxacin (SIT), erythromycin (ERY), tetracycline (TET), and gentamicin (GEN)] was carried out by broth microdilution. Three AMR and nine virulence genes were screened by PCR. Genotyping was performed by A-restriction fragment length polymorphism (RFLP) and multilocus sequence typing (MLST). Of the 153 samples, spp. were detected in 55 (35.9%), with and being 49 (89.1%) and six (10.9%), respectively. High-level resistance was observed for CIP (100%), NAL (100%), and TET (, 93.9%; : 83.3%). No resistance was observed for SIT. The missense mutation (C257T) in A gene was confirmed by sequencing, while the (O) gene was similar to known sequences in GenBank. The rate of multidrug-resistant (MDR) strains was 8.2%, and they all belonged to . All isolates possessed five virulence genes (B, II, A, F, and J), but none possessed , while the rates for other genes (A, B, and A) ranged between 33.3 and 95.9%. The A-RFLP yielded 26 A types (: 21 and : five), while the MLST showed 10 sequence types (STs) for and three STs for , with CC-607 (STs 3611) and CC-460 (ST-460) being predominant. Among the 10 STs of , three were newly assigned. The findings of this study highlight the increased resistance to quinolones and TET, the virulence potential, and the diverse genotypes among strains isolated from the layer poultry farm.
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http://dx.doi.org/10.3389/fmicb.2021.622275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8043113PMC
March 2021

Luteolin-Fabricated ZnO Nanostructures Showed PLK-1 Mediated Anti-Breast Cancer Activity.

Biomolecules 2021 03 5;11(3). Epub 2021 Mar 5.

Department of Biotechnology and Bioinformatics, School of Life Sciences, JSS Academy of Higher Education and Research, Mysuru, Karnataka 570 015, India.

The present work describes a facile and convenient procedure for synthesizing zinc oxide nanoparticles using luteolin isolated from plant (L-ZnONPs) at room temperature. The formation of as-grown L-ZnONPs was confirmed by X-ray diffraction analysis (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HR-TEM), and selected area electron diffraction (SAED). The Wurtzite structure of ZnO was observed by its hexagonal phases in diffraction patterns. The SEM images revealed the different sizes and morphologies of L-ZnONPs, with diameters between 12 and 25 nm. The HR-TEM result showed that the inter-planar distance between two lattice fringes was 0.262 nm, which coincides with the d-spacing of (002) and (101) lattice planes of the as-obtained material. The anticancer activity of L-ZnONPs against the breast cancer cell line MCF-7 was greater as compared to that of luteolin or ZnO alone. The mechanistic evaluation of such an activity carried out using in silico methods suggested that the anti-breast cancer activity of L-ZnONPs was mediated by polo-like kinase 1 (PLK1) proteins.
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http://dx.doi.org/10.3390/biom11030385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7998981PMC
March 2021

Bacterial community analysis of purulent material from liver abscesses of crossbred cattle and Holstein steers fed finishing diets with or without tylosin.

J Anim Sci 2021 Apr;99(4)

Department of Diagnostic Medicine/Pathobiology, Kansas State University, Manhattan, KS 66506, USA.

Liver abscesses in feedlot cattle are polymicrobial infections. Culture-based studies have identified Fusobacterium necrophorum as the primary causative agent, but a number of other bacterial species are frequently isolated. The incidence of liver abscesses is highly variable and is affected by a number of factors, including cattle type. Holstein steers raised for beef production have a higher incidence than crossbred feedlot cattle. Tylosin is the commonly used antimicrobial feed additive to reduce the incidence of liver abscesses. The objective of this study was to utilize 16S ribosomal RNA amplicon sequence analyses to analyze the bacterial community composition of purulent material of liver abscesses of crossbred cattle (n = 24) and Holstein steers (n = 24), each fed finishing diet with or without tylosin. DNA was extracted and the V3 and V4 regions of the 16S rRNA gene were amplified, sequenced, and analyzed. The minimum, mean, and maximum sequence reads per sample were 996, 177,070, and 877,770, respectively, across all the liver abscess samples. Sequence analyses identified 5 phyla, 14 families, 98 genera, and 102 amplicon sequence variants (ASV) in the 4 treatment groups. The dominant phyla identified were Fusobacteria (52% of total reads) and Proteobacteria (33%). Of the top 25 genera identified, 17 genera were Gram negative and 8 were Gram positive. The top 3 genera, which accounted for 75% of the total reads, in the order of abundance, were Fusobacterium, Pseudomonas, and Bacteroides. The relative abundance, expressed as percent of total reads, of phyla, family, and genera did not differ (P > 0.05) between the 4 treatment groups. Generic richness and evenness, determined by Shannon-Weiner and Simpson's diversity indices, respectively, did not differ between the groups. The UniFrac distance matrices data revealed no clustering of the ASV indicating variance between the samples within each treatment group. Co-occurrence network analysis at the genus level indicated a strong association of Fusobacterium with 15 other genera, and not all of them have been previously isolated from liver abscesses. In conclusion, the culture-independent method identified the bacterial composition of liver abscesses as predominantly Gram negative and Fusobacterium as the dominant genus, followed by Pseudomonas. The bacterial community composition did not differ between crossbred and Holstein steers fed finishing diets with or without tylosin.
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http://dx.doi.org/10.1093/jas/skab076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8075120PMC
April 2021

Leukotoxin production by Fusobacterium necrophorum strains in relation to severity of liver abscesses in cattle.

Anaerobe 2021 Jun 12;69:102344. Epub 2021 Feb 12.

Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS, 66506, USA. Electronic address:

Fusobacterium necrophorum, a Gram-negative anaerobe, is the primary etiologic agent of liver abscesses of beef cattle. The bacterium, a member of the microbial community of the rumen, travels to the liver via portal circulation to cause abscesses. The severity of liver abscesses vary from mild with one or two small abscesses to severe with medium to large multiple abscesses. Leukotoxin, a secreted protein, is the critical virulence factor involved in the infection. Our objective was to compare leukotoxin production between strains of F. necrophorum isolated from mild and severe liver abscesses collected from slaughtered cattle. The quantification of leukotoxin was based on assays to measure cytotoxicity and protein antigen concentration. One-hundred strains, 50 from mild and 50 from severe abscesses, were utilized in the study. Cell-free supernatants were prepared from cultures grown in anaerobic broth at 9 and 24 h incubations. The leukotoxic activity was quantified by measuring cytotoxicity based on the release of lactic dehydrogenase from bovine lymphocyte cells, BL3, treated with the culture supernatant. Leukotoxin protein concentration was quantified by a sandwich ELISA assay with a leukotoxin-specific monoclonal antibody as the capture antibody. The leukotoxin activity and concentration were highly variable among the strains within each severity of liver abscesses. Although the leukotoxic activity was unaffected by incubation time, leukotoxin protein concentration was consistently higher at 24 h compared to 9 h incubation. Strains from severe liver abscesses had significantly higher leukotoxic activity and higher protein concentration compared to strains from mild liver abscesses (P < 0.0001) at both 9 and 24 h culture supernatants. Across all strains, the correlation coefficients between leukotoxic activity and leukotoxin concentration at 9 and 24 h were 0.14 (P = 0.17) and 0.47 (P < 0.0001), respectively. In conclusion, strains isolated from severe liver abscesses had significantly higher leukotoxic activities and leukotoxin protein concentrations compared to strains isolated from mild liver abscesses.
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http://dx.doi.org/10.1016/j.anaerobe.2021.102344DOI Listing
June 2021

Effects of Zinc and Menthol-Based Diets on Co-Selection of Antibiotic Resistance among and spp. in Beef Cattle.

Animals (Basel) 2021 Jan 21;11(2). Epub 2021 Jan 21.

Department of Veterinary Pathobiology, Texas A&M University, College Station, TX 77843, USA.

Antibiotic resistance represents a growing crisis in both human and veterinary medicine. We evaluated the use of antibiotic alternatives-heavy metals and essential oils-in beef cattle feeding, and their effects on Gram-negative and Gram-positive bacteria. In this randomized controlled field trial, we measured the impact of supplemental zinc and menthol on antibiotic resistance among commensal enteric bacteria of feeder cattle. Fecal suspensions were plated onto plain- and antibiotic-supplemented MacConkey and m- agar for quantification of total and antibiotic-resistant and spp., respectively. Temporal effects on overall growth were significant ( < 0.05), and menthol was associated with decreased growth on tetracycline-supplemented agar. Zinc was associated with significant increases in growth on erythromycin-supplemented m- agar. Cattle fed zinc exhibited significantly higher levels of macrolide resistance among fecal enterococci isolates.
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http://dx.doi.org/10.3390/ani11020259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7909843PMC
January 2021

Susceptibility of Strains to Selected Natural Products and Frontline Antibiotics.

Antibiotics (Basel) 2020 Nov 9;9(11). Epub 2020 Nov 9.

Natural Product Informatics Research Center, KIST Gangneung Institute of Natural Products, Gangneung 25451, Korea.

species have developed resistance to existing antibiotics. The development of alternative therapies is, therefore, a necessity. This study evaluates the susceptibility of strains to selected natural products (NPs) and frontline antibiotics. Two strains (ATCC 33560 and MT947450) and two strains (ATCC 33559 and MT947451) were used. The antimicrobial potential of the NPs, including plant extracts, essential oils, and pure phytochemicals, was evaluated by broth microdilution. The growth was measured by spectrophotometry and iodonitrotetrazolium chloride. Antibiotic resistance genes ((O) and A) were characterized at the molecular level. The minimum inhibitory concentrations (MICs) and the minimum bactericidal concentrations (MBCs) ranged from 25 to 1600 µg/mL. Cinnamon oil, (E)-Cinnamaldehyde, clove oil, eugenol, and baicalein had the lowest MIC and MBC values (25-100 µg/mL). MT947450 and MT947451 were sensitive to erythromycin and gentamicin but resistant to quinolones and tetracycline. Mutations in A and (O) genes from resistant strains were confirmed by sequencing. The findings show that NPs are effective against drug-sensitive and drug-resistant strains. The resistance to antibiotics was confirmed at phenotypic and genotypic levels. This merits further studies to decipher the action mechanisms and synergistic activities of NPs.
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http://dx.doi.org/10.3390/antibiotics9110790DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7697650PMC
November 2020

Polymerase Chain Reaction-Based Prevalence of Serogroups of Known to Carry Shiga Toxin Genes in Feces of Finisher Pigs.

Foodborne Pathog Dis 2020 12 21;17(12):782-791. Epub 2020 Aug 21.

Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas, USA.

Shiga toxin-producing (STEC) are major foodborne pathogens and seven serogroups, O26, O45, O103, O111, O121, O145, and O157, that account for the majority of the STEC-associated illness in humans. Similar to cattle, swine also harbor STEC and shed them in the feces and can be a source of human STEC infections. Information on the prevalence of STEC in swine feces is limited. Therefore, our objective was to utilize polymerase chain reaction (PCR) assays to determine prevalence of major virulence genes and serogroups of STEC. Fecal samples ( = 598), collected from finisher pigs within 3 weeks before marketing in 10 pig flows located in 8 states, were included in the study. Samples enriched in broth were subjected to a real-time PCR assay targeting three virulence genes, Shiga toxin 1 (1), Shiga toxin 2 (2), and intimin (), which encode for Shiga toxins 1 and 2, and intimin, respectively. A novel PCR assay was designed and validated to detect serogroups, O8, O20, O59, O86, O91, O100, O120, and O174, previously reported to be commonly present in swine feces. In addition, enriched fecal samples positive for Shiga toxin genes were subjected to a multiplex PCR assay targeting O26, O45, O103, O104, O111, O121, O145, and O157 serogroups implicated in human clinical infections. Of the 598 fecal samples tested by real-time PCR, 25.9%, 65.1%, and 67% were positive for 1, 2, and , respectively. The novel eight-plex PCR assay indicated the predominant prevalence of O8 (88.6%), O86 (35.5%), O174 (24.1%), O100 (20.2%), and O91 (15.6%) serogroups. Among the seven serogroups relevant to human infections, three serogroups, O121 (17.6%), O157 (14%), and O26 (11%) were predominant. PCR-based detection indicated high prevalence of Shiga toxin genes and serogroups that are known to carry Shiga toxin genes, including serogroups commonly prevalent in cattle feces and implicated in human infections and in edema disease in swine.
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http://dx.doi.org/10.1089/fpd.2020.2814DOI Listing
December 2020

Tumoricidal Potential of Novel Amino-1,10-phenanthroline Derived Imine Ligands: Chemical Preparation, Structure, and Biological Investigations.

Molecules 2020 Jun 22;25(12). Epub 2020 Jun 22.

Department of Botany and Microbiology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia.

Herein we report the synthesis and structural elucidation of two novel imine-based ligands, 2-(1,10-phenanthrolin-5-yl)imino)methyl)-5-bromophenol (PIB) and -(1,10-phenanthrolin-5-yl)-1-(thiophen-3-yl)methanimine (PTM) ligands. An in vitro cytotoxicity assay of the synthesized molecules was carried out against breast, cervical, colorectal, and prostate cancer cell lines as well as immortalized human keratinocytes. The observations indicated that both the molecules possesses dose-dependent selective cytotoxicity of cancer cells with no detrimental effect on the normal cell lines. Furthermore, the detailed computational analysis of newly synthetized ligands (PIB and PTM) has been conducted in order to identify their most important parts from the perspective of local reactivity. The IC values of PIB treatment on MCF-7, HeLa, HCT-116 and PC-3 were 15.10, 16.25, 17.88, 17.55 and 23.86 micromoles, respectively. Meanwhile, the IC values of PTM on MCF-7, HeLa, HCT-116, PC-3 and HaCat were observed to be 14.82, 15.03, 17.88, 17.28 and 21.22 micromoles, respectively. For computational analysis, we have employed the combination of Density Functional Theory (DFT) calculations and MD simulations. DFT calculations provided us with information about structure and reactivity descriptors based on the electron distribution. Surfaces of molecular electrostatic potential (MEP) and averaged local ionization energy (ALIE) indicated the sites within studied molecules that are most reactive. These results indicated the importance of nitrogen atoms and OH group. Additionally, the values of bond dissociation for hydrogen abstraction showed that both molecules, especially the PTM, are stable toward the influence of autoxidation mechanism. On the other side, MD simulations gave us an insight how ligands interact with water molecules. Namely, the radial distribution functions (RDF) indicated that the hydrogen atom of the OH group in the case of the PIB has the most pronounced interactions with water.
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http://dx.doi.org/10.3390/molecules25122865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7356530PMC
June 2020

Longitudinal Characterization of Prevalence and Concentration of Shiga Toxin-Producing Serogroups in Feces of Individual Feedlot Cattle.

Foodborne Pathog Dis 2020 10 18;17(10):631-639. Epub 2020 Mar 18.

Center for Outcomes Research and Epidemiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas, USA.

The objective of this study was to quantify the frequency, distribution, and variability of fecal shedding and super-shedding of Shiga toxin-producing (STEC) serogroups O26, O45, O103, O111, O121, O145, and O157 in feedlot cattle over time. A total of 750 fecal grab samples were collected over a 5-week period (June-July 2017) from 150 cattle housed in 10 pens at a commercial feedlot operation. Samples were subjected to culture-based methods and real-time quantitative polymerase chain reaction for STEC detection and quantification. Cumulative animal-level prevalence estimates were 9.5%, 5.2%, and 15.8% for STEC O157, non-O157 STEC serogroups only (STEC-6), and for all STEC serogroups tested (STEC-7), respectively, with the prevalence of STEC O157 and STEC-7 significantly differing between weeks ( < 0.01). Most of the variability in fecal shedding for STEC O157, STEC-6, and STEC-7 was between pens, rather than between cattle. Over the 5-week period, 10 animals (6.7%) persistently shed STEC non-O157 over 3 or more consecutive weeks, whereas 2 animals (1.3%) intermittently shed STEC non-O157 on nonconsecutive weeks. Fifteen animals (10.0%) shed multiple STEC serogroups within the same fecal sample and five animals (3.3%) shed multiple serogroups at super-shedding levels, higher than 10 CFU (colony-forming units)/g, in the same sample. The presence of a super-shedder in a pen was significantly associated with a greater within pen-level prevalence of STEC-6 ( = 0.01). This study gives further insights into intermittent and persistent shedding and super-shedding patterns of STEC serogroups in individual feedlot cattle, which can enable the development and effective application of preharvest and periharvest interventions, as well as surveillance strategies, for these pathogens.
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http://dx.doi.org/10.1089/fpd.2019.2777DOI Listing
October 2020

Impact of added copper, alone or in combination with chlortetracycline, on growth performance and antimicrobial resistance of fecal enterococci of weaned piglets.

J Anim Sci 2020 Mar;98(3)

Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS.

Studies suggest a link between added copper (Cu) and co-selection of antimicrobial resistance (AMR) in Enterococcus spp., but data are inconsistent. This study aimed to assess the impact of added Cu, alone or with a feed-grade antimicrobial, on growth performance, transferable Cu resistance gene (tcrB) prevalence, abundance of tcrB in fecal community DNA, and AMR in fecal enterococci in weaned piglets. A total of 320 barrows (DNA 200 × 400, DNA Genetics) weaned at approximately 21 d of age with 7.4 kg (7.4 ± 0.06 kg) BW were used in a 28-d study. Piglets were fed a common non-medicated diet for 7 d of acclimation. Treatments were arranged in a 2 × 2 factorial design with main effects of added Cu (0 vs. 200 mg/kg Cu from Cu sulfate) and chlortetracycline (0 vs. 440 mg/kg CTC). Growth performance and fecal samples were obtained on days 0, 14, and 28. There was no evidence (P > 0.05) for Cu and CTC interaction in growth performance. Pigs fed diets with added Cu had increased (P < 0.05) ADG and ADFI from days 0 to 14, with no evidence for differences (P > 0.05) from days 15 to 28 and 0 to 28. Pigs fed diets with CTC had improved (P < 0.01) ADG, ADFI, and G:F from days 0 to 28. Prevalence of tcrB-positive enterococci was not affected by the addition of Cu and/or CTC (P > 0.05). Prevalence of tcrB-positive enterococci was higher on day 14 than other sampling days (P = 0.002). Prevalence of tetracycline resistance gene [tet(M)]-positive enterococci was not affected by treatments or day (P > 0.05). Prevalence of macrolide resistance gene [erm(B)]-positive enterococci had a significant treatment and sampling day interaction (P = 0.021). The abundance of the tcrB gene in feces, quantified by PCR, was not affected by Cu treatment. The median Cu minimum inhibitory concentrations (MIC) of tcrB-negative and -positive isolates were 3 and 20 mM, respectively (P < 0.001). For day 0 and day 28, all Enterococcus isolates were susceptible to gentamicin, kanamycin, streptomycin, daptomycin, and tigecycline, with a majority of isolates resistant to chloramphenicol, erythromycin, lincomycin, linezolid, tetracycline, tylosin tartrate, and Synercid. In conclusion, 200 mg/kg added Cu or 440 mg/kg CTC in nursery diets improved growth performance of nursery pigs. Added Cu, with or without a selection pressure of CTC, did not increase Cu-resistant enterococci and did not co-select resistance to antibiotics.
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http://dx.doi.org/10.1093/jas/skaa003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072034PMC
March 2020

Prevalence of extended-spectrum beta-lactamase producing bacteria from animal origin: A systematic review and meta-analysis report from India.

PLoS One 2019 4;14(9):e0221771. Epub 2019 Sep 4.

Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, KS, United States of America.

Antimicrobial resistance (AMR) due to the emergence and spread of extended-spectrum beta-lactamase (ESBL) producing bacteria are becoming a serious global public health concern. This article aims to assess the overall prevalence of ESBLs among animals in India, with year-wise, zone-wise and species-wise stratification. Systematic search from PubMed, Google Scholar and J-Gate Plus was carried out and 24 eligible articles from 2013-2019 in India were retrieved. The R Open source Scripting software was used to perform statistical analysis. The overall prevalence of ESBLs among animals in India was 9%. The pooled prevalence of ESBLs in animals were 26, 11, 6 and 8% for north, east, south and central zones, respectively. The reported prevalence of ESBLs in animals were 12, 5, 8, 8, 12, 13 and 33% were reported for the years 2013, 2014, 2015, 2016, 2017, 2018, 2019 respectively. The species-wise stratified results showed a predominance of ESBL producing Klebsiella pneumoniae strains (11%) when compared to Escherichia coli and Pseudomonas spp. which were 7% and 5%, respectively. The prevalence data generated could be utilized in infection control and in antibiotic use management decisions for developing appropriate intervention strategies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0221771PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6726241PMC
March 2020

Leukotoxic activity of Fusobacterium necrophorum of cattle origin.

Anaerobe 2019 Apr 13;56:51-56. Epub 2019 Feb 13.

Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS, 66506, USA. Electronic address:

Fusobacterium necrophorum is a Gram negative, rod-shaped and aero tolerant anaerobe. In animals, it is an opportunistic pathogen frequently associated with necrotic infections, generally called necrobacillosis, such as calf diphtheria, foot rot and liver abscesses in cattle. Two subspecies exist: subsp. necrophorum and subsp. funduliforme. Among several virulence factors, leukotoxin (Lkt) is considered to be a major factor and a protective antigen. The objective of the study was to utilize BL3 cells and measure the release of lactic dehydrogenase to quantify Lkt activity of F. necrophorum. The assay was used to examine the effects of storage and handling conditions, growth media, polymyxin B addition on the cytotoxicity and evaluate Lkt activities of F. necrophorum strains isolated from bovine liver abscesses and foot rot. The Lkt activity peaked at 9 h of incubation. There was a significant decrease in the cytotoxicity measured in the samples after each freeze and thaw cycle. No difference was observed in the cytotoxicity for the samples handled aerobically versus anaerobically. Lkt activities of strains grown in anaerobic Brain-Heart Infusion broth were higher compared to Vegitone broth. A small reduction in the cytotoxicity activity was observed after the addition of polymyxin. The Lkt activity was consistently higher in strains of subsp. necrophorum than subsp. funduliforme of liver abscess origin. Among the strains isolated from cattle foot rot, Lkt activities of subsp. necrophorum strains appear to be much more variable. Use of BL3 cells in combination of lactic acid dehydrogenase assay appears to be a simple and valid assay to measure Lkt activity of F. necrophorum.
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http://dx.doi.org/10.1016/j.anaerobe.2019.02.009DOI Listing
April 2019

Effects of Tylosin Administration Routes on the Prevalence of Antimicrobial Resistance Among Fecal Enterococci of Finishing Swine.

Foodborne Pathog Dis 2019 05 24;16(5):309-316. Epub 2019 Jan 24.

4 Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas.

Antibiotics can be administered orally or parenterally in swine production, which may influence antimicrobial resistance (AMR) development in gut bacteria. A total of 40 barrows and 40 gilts were used to determine the effects of tylosin administration route on growth performance and fecal enterococcal AMR. The antibiotic treatments followed Food and Drug Administration label directions and were as follows: (1) no antibiotic (CON), (2) 110 mg tylosin per kg feed for 21 d (IN-FEED), (3) 8.82 mg tylosin per kg body weight through intramuscular injection twice daily for the first 3 d of each week for 3 weeks (IM), and (4) 66 mg tylosin per liter of drinking water (IN-WATER). Antibiotics were administered during d 0 to 21 and all pigs were then fed the CON diet from d 21 to 35. Fecal samples were collected on d 0, 21, and 35. Antimicrobial susceptibility was determined by microbroth dilution method. No evidence of route × sex interaction ( > 0.55) was observed for growth performance. From d 0 to 21, pigs receiving CON and IN-FEED had greater ( < 0.05) average daily gain (ADG) than those receiving IM, with the IN-WATER group showing intermediate ADG. Pigs receiving CON had greater ( < 0.05) gain-to-feed ratio (G:F) than IM and IN-WATER, but were not different from pigs receiving IN-FEED. Overall, enterococcal isolates collected from pigs receiving IN-FEED or IM were more resistant ( < 0.05) to erythromycin and tylosin than CON and IN-WATER groups. Regardless of administration route, the estimated probability of AMR to these two antibiotics was greater on d 21 and 35 than on d 0. In summary, IM tylosin decreased ADG and G:F in finishing pigs, which may be because of a response to the handling during injection administration. Tylosin administration through injection and feed resulted in greater probability of enterococcal AMR to erythromycin and tylosin compared with in-water treatment.
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http://dx.doi.org/10.1089/fpd.2018.2551DOI Listing
May 2019

Effects of supplemental zinc sulfate on growth performance, carcass characteristics, and antimicrobial resistance in feedlot heifers.

J Anim Sci 2019 Jan;97(1):424-436

Department of Animal Sciences and Industry, Kansas State University, Manhattan, KS.

Effects of supplemental Zn as Zn sulfate on feedlot performance, carcass traits, and antimicrobial resistance were evaluated using 480 crossbred heifers (BW = 385 kg ± 13.08) in a randomized complete block design. Heifers were blocked by BW and randomly assigned within block to diets with 0, 30, 60, or 90 mg supplemental Zn/kg DM. Heifers were housed in dirt-surfaced pens (20 animals per pen; 6 pens per treatment) equipped with fence-line feed bunks and automatic water fountains. Heifers were fed once daily to ensure ad libitum intake. Plasma was collected on day 0 from five randomly selected heifers per pen and repeated on days 63 and 115 to determine plasma Zn concentrations. Random samples of freshly voided feces were collected from the surface of each pen the day of harvest to determine antibiotic resistance. Heifers were transported on day 144 to a commercial abattoir where hot carcass weight (HCW) and incidence of liver abscesses were recorded at harvest and HCW, dressed yield, ribeye area, 12th rib fat, quality and yield grades were recorded after 36 h of refrigeration. Plasma Zn concentration increased (P = 0.02) linearly in response to increasing concentrations of dietary Zn. Final BW and ADG were unaffected by supplementation (P ≥ 0.29). Quantified levels of resistance to ceftriaxone and tetracycline among fecal Escherichia coli were not impacted (P > 0.05) by dietary zinc concentrations. Increasing Zn concentrations tended to decrease (linear effect, P = 0.07) DMI, resulting in a linear (P = 0.03) and tendency for quadratic (P = 0.12) improvement in feed efficiency with increasing Zn concentration. No differences were detected for HCW, dressed yield, ribeye area, 12th rib fat, percentages of carcasses grading Select or Choice, or yield grade (P > 0.53), but added Zn tended to affect percentage of carcasses that graded Prime, peaking at 60 mg/kg added Zn (quadratic effect, P = 0.07). In vitro fermentations were performed using ruminal fluid cultures containing 0, 30, 60, 90, 120, or 150 mg Zn/kg substrate DM to determine impact of Zn on gas production, VFA concentrations, and in vitro DM disappearance (IVDMD). There were no effects of Zn on in vitro gas production, IVDMD, or most VFA (P > 0.15), but isovalerate decreased linearly in response to added Zn (P = 0.05). Supplementing finishing heifers up to 60 mg Zn/kg diet DM improved feed efficiency compared to other treatments.
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http://dx.doi.org/10.1093/jas/sky411DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6313150PMC
January 2019

Effects of chlortetracycline alone or in combination with direct fed microbials on nursery pig growth performance and antimicrobial resistance of fecal Escherichia coli.

J Anim Sci 2018 Dec;96(12):5166-5178

Department of Clinical Sciences, Manhattan, KS.

A total of 300 nursery pigs (initially 5.9 ± 0.05 kg BW) were used in a 42-d growth trial to evaluate the effects of feeding a therapeutic level of chlortetracycline (CTC) with or without direct fed microbials (DFM) on growth performance and antimicrobial resistance (AMR) of fecal Escherichia coli. CTC is a broad-spectrum in-feed antibiotic commonly used in the swine industry. Weaned pigs (~21 d of age) were allotted to pens based on initial BW and fed a common starter diet for 4 d. Pens were then blocked by BW and allotted to dietary treatments in a completely randomized block design. Dietary treatments were arranged in a 2 × 3 factorial consisting of combinations of CTC (none vs. 400 mg/kg from days 0 to 42) and DFM (0 vs. 0.05% DFM 1 vs. 0.05% DFM 2). Fecal samples were collected from three randomly selected pigs from each pen on days 0, 21, and 42 for E. coli isolation and AMR determination. Overall, pigs fed diets containing CTC had improved (P < 0.001) ADG, ADFI, and BW compared to those not fed CTC with no evidence for any effect of either DFM 1 or DFM 2. Regardless of CTC, inclusion of DFM 2 in diets improved (P < 0.05) ADFI from days 0 to 14 and on day 14 BW compared to diets that did not include DFM 2. The addition of CTC with or without DFMs to nursery pig diets increased (P < 0.05) the probability of AMR to tetracycline and ceftiofur of fecal E. coli isolates, but this resistance generally decreased (P < 0.05) over time. A decrease (P < 0.05) in AMR to ampicillin and tetracycline (TET) throughout the trial was observed, while resistance to ceftriaxone decreased (P < 0.020) from days 0 to 21 and increased from days 21 to 42 amongst dietary treatments regardless of CTC or DFM inclusion in the diet. A CTC × DFM × day interaction (P < 0.015) was observed for streptomycin, whereby from days 21 to 42 AMR increased in diets containing either CTC or DFM 1 alone, but the combination decreased resistance. There was no evidence for any effect of DFMs on AMR of fecal E. coli isolates to any other antibiotics evaluated. In conclusion, therapeutic levels of added CTC with or without DFM inclusion improved nursery pig performance, but increased AMR of fecal E. coli isolates to TET and ceftiofur. A moderate improvement in intake and day 14 BW was observed when DFM 2 was included in the diet with or without CTC, but, except for streptomycin, there was no evidence that added dietary DFMs affected resistance of fecal E. coli to antibiotics.
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http://dx.doi.org/10.1093/jas/sky370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6276563PMC
December 2018

Distribution of the Gene Cluster and Associated Genetic Determinants among Swine from a Controlled Feeding Trial.

Genes (Basel) 2018 Oct 18;9(10). Epub 2018 Oct 18.

Department of Pathobiology, Ontario Veterinary College, University of Guelph, 50 Stone Rd. E., Guelph, ON N1G 2W1, Canada.

Copper is used as an alternative to antibiotics for growth promotion and disease prevention. However, bacteria developed tolerance mechanisms for elevated copper concentrations, including those encoded by the operon in Gram-negative bacteria. Using cohorts of weaned piglets, this study showed that the supplementation of feed with copper concentrations as used in the field did not result in a significant short-term increase in the proportion of -positive fecal . The and (silver resistance) operons were found concurrently in all screened isolates, and whole-genome sequencing showed that they were distributed among a diversity of unrelated strains. The presence of / in was not associated with elevated copper minimal inhibitory concentrations (MICs) under a variety of conditions. As found in previous studies, the / operons were part of a Tn-like structure found both on the chromosome or on plasmids in the strains investigated. Transfer of a / IncHI2 plasmid from to resulted in elevated copper MICs in the latter. may represent a reservoir of / genes transferable to other organisms such as , for which it may represent an advantage in the presence of copper. This, in turn, has the potential for co-selection of resistance to antibiotics.
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http://dx.doi.org/10.3390/genes9100504DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6211086PMC
October 2018

Effects of intermittent feeding of tylosin phosphate during the finishing period on feedlot performance, carcass characteristics, antimicrobial resistance, and incidence and severity of liver abscesses in steers.

J Anim Sci 2018 Jun;96(7):2877-2885

Department of Animal Sciences and Industry, Kansas State University, Manhattan, KS.

Liver abscesses (LA) are a source of economic loss for feedlot cattle feedlots, and the 2017 veterinary feed directive has restricted further use of tylosin phosphate to prevention and control of LA. Our objective was to evaluate effects of intermittent tylosin phosphate feeding on incidence and severity of LA in feedlot cattle and presence of total antimicrobial-resistant Enterococcus spp. Steers (n = 312, 411.4 ± 6.71 kg) were blocked by initial BW and randomly assigned to a treatment group. Treatments included a negative control group (no tylosin phosphate throughout the finishing period), a positive control group (tylosin phosphate fed continuously throughout the finishing period), and a group that received tylosin phosphate off-label by feeding the drug on a repeated intermittent basis (1 wk on, 2 wk off). Steers were housed in 24 soil-surfaced pens with 13 steers per pen. Body weights of cattle were obtained every 28 d and at the end of 119 d the steers were weighed and harvested at a commercial abattoir. Fecal samples were collected on days 0, 21, and 118 to characterize antimicrobial-resistant Enterococcus spp. Total LA percentage was greater (P = 0.012) for the no tylosin phosphate treatment compared with the other treatments, but did not differ between the continuous tylosin phosphate treatment and the intermittently fed tylosin phosphate treatment (P = 0.716). No difference was observed among treatments for ADG (P = 0.21), DMI (P = 0.28), or G:F (P = 0.75). Marbling score was lower (P = 0.022) for tylosin phosphate treatment when compared with both intermittent treatment and continuous tylosin phosphate treatment. Enterococcus spp. bacterial counts did not differ by treatment group over time (P > 0.05); however, there was a strong period effect for macrolide resistance among all groups (P < 0.01), suggesting an important environmental component as cattle were first placed in pens and then progressed through the feeding period. We conclude that feeding tylosin phosphate intermittently during the finishing phase decreases the total percentage of LA and maintains feedlot performance and carcass characteristics to the same extent as feeding tylosin phosphate throughout the finishing phase; furthermore, we hypothesize that enteric antimicrobial resistance is a result of longer term antibiotic usage in a particular environment rather than a direct short-term result of the treatment during any given feeding period.
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http://dx.doi.org/10.1093/jas/sky166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6095443PMC
June 2018

Antimicrobial resistance of Enterococcus faecium strains isolated from commercial probiotic products used in cattle and swine.

J Anim Sci 2018 Apr;96(3):912-920

Diagnostic Medicine/Pathobiology, Kansas State University, Manhattan, KS.

Probiotics, an antibiotic alternative, are widely used as feed additives for performance benefits in cattle and swine production systems. Among bacterial species contained in probiotics, Enterococcus faecium is common. Antimicrobial resistance (AMR), particularly multidrug resistance, is a common trait among enterococci because of their propensity to acquire resistance and horizontally transfer AMR genes. Also, E. faecium is an opportunistic pathogen, and in the United States, it is the second most common nosocomial pathogen. There has been no published study on AMR and virulence potential in E. faecium contained in probiotic products used in cattle and swine in the United States. Therefore, our objectives were to determine phenotypic susceptibilities or resistance to antimicrobials, virulence genes (asa1, gelE, cylA, esp, and hyl) and assess genetic diversity of E. faecium isolated from commercial products. Twenty-two commercially available E. faecium-based probiotic products used in cattle (n = 13) and swine (n = 9) were procured and E. faecium was isolated and species confirmed. Antimicrobial susceptibility testing to determine minimum inhibitory concentrations was done by micro-broth dilution method using National Antimicrobial Resistance Monitoring Systems Gram-positive Sensititre panel plate (CMV3AGPF), and categorization of strains as susceptible or resistant was as per Clinical Laboratory and Standards Institute's guidelines. E. faecium strains from 7 products (3 for swine and 4 for cattle) were pan-susceptible to the 16 antimicrobials tested. Strains from 15 products (6 for swine and 9 for cattle) exhibited resistance to at least one antimicrobial and a high proportion of strains was resistant to lincomycin (10/22), followed by tetracycline (4/22), daptomycin (4/22), ciprofloxacin (4/22), kanamycin (3/22), and penicillin (2/22). Four strains were multidrug resistant, with resistant phenotypes ranging from 3 to 6 antimicrobials or class. None of the E. faecium strains were positive for any of the virulence genes tested. The clonal relationships among the 22 E. faecium strains were determined by pulsed-field gel electrophoresis (PFGE) typing. A total of 10 PFGE patterns were observed with 22 strains and a few of the strains from different probiotic products had identical (100% Dice similarity) PFGE patterns. In conclusion, the E. faecium strains in a few commercial probiotics exhibited AMR to medically-important antimicrobials, but none contained virulence genes.
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http://dx.doi.org/10.1093/jas/sky056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6093546PMC
April 2018

Antimicrobial Susceptibility of Enteric Gram Negative Facultative Anaerobe Bacilli in Aerobic versus Anaerobic Conditions.

PLoS One 2016 18;11(5):e0155599. Epub 2016 May 18.

Department of Diagnostic Medicine/Pathobiology, Institute of Computational Comparative Medicine, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas, United States of America.

Antimicrobial treatments result in the host's enteric bacteria being exposed to the antimicrobials. Pharmacodynamic models can describe how this exposure affects the enteric bacteria and their antimicrobial resistance. The models utilize measurements of bacterial antimicrobial susceptibility traditionally obtained in vitro in aerobic conditions. However, in vivo enteric bacteria are exposed to antimicrobials in anaerobic conditions of the lower intestine. Some of enteric bacteria of food animals are potential foodborne pathogens, e.g., Gram-negative bacilli Escherichia coli and Salmonella enterica. These are facultative anaerobes; their physiology and growth rates change in anaerobic conditions. We hypothesized that their antimicrobial susceptibility also changes, and evaluated differences in the susceptibility in aerobic vs. anaerobic conditions of generic E. coli and Salmonella enterica of diverse serovars isolated from cattle feces. Susceptibility of an isolate was evaluated as its minimum inhibitory concentration (MIC) measured by E-Test® following 24 hours of adaptation to the conditions on Mueller-Hinton agar, and on a more complex tryptic soy agar with 5% sheep blood (BAP) media. We considered all major antimicrobial drug classes used in the U.S. to treat cattle: β-lactams (specifically, ampicillin and ceftriaxone E-Test®), aminoglycosides (gentamicin and kanamycin), fluoroquinolones (enrofloxacin), classical macrolides (erythromycin), azalides (azithromycin), sulfanomides (sulfamethoxazole/trimethoprim), and tetracyclines (tetracycline). Statistical analyses were conducted for the isolates (n≥30) interpreted as susceptible to the antimicrobials based on the clinical breakpoint interpretation for human infection. Bacterial susceptibility to every antimicrobial tested was statistically significantly different in anaerobic vs. aerobic conditions on both media, except for no difference in susceptibility to ceftriaxone on BAP agar. A satellite experiment suggested that during first days in anaerobic conditions the susceptibility changes with time. The results demonstrate that assessing effects of antimicrobial treatments on resistance in the host's enteric bacteria that are Gram negative facultative Anaerobe Bacilli requires data on the bacterial antimicrobial susceptibility in the conditions resembling those in the intestine.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0155599PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4871507PMC
July 2017

Genome Sequences of Salmonella enterica subsp. enterica Serovar Lubbock Strains Isolated from Liver Abscesses of Feedlot Cattle.

Genome Announc 2016 May 5;4(3). Epub 2016 May 5.

Department of Veterinary and Biomedical Sciences, South Dakota State University, Brookings, South Dakota, USA

The genome sequencing of 13 Salmonella enterica subsp. enterica serovar Lubbock strains isolated from liver abscesses of feedlot cattle is reported here. The availability of these genomes will help to further understand the etiologic role of Salmonella strains in liver abscesses of cattle and will serve as references in microbial trace-back studies to improve food safety.
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http://dx.doi.org/10.1128/genomeA.00319-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4859176PMC
May 2016

Effects of In-Feed Copper, Chlortetracycline, and Tylosin on the Prevalence of Transferable Copper Resistance Gene, tcrB, Among Fecal Enterococci of Weaned Piglets.

Foodborne Pathog Dis 2015 Aug 2;12(8):670-8. Epub 2015 Jun 2.

1 Departments of Diagnostic Medicine and Pathobiology, Kansas State University , Manhattan, Kansas.

Heavy metals, such as copper, are increasingly supplemented in swine diets as an alternative to antibiotics to promote growth. Enterococci, a common gut commensal, acquire plasmid-borne, transferable copper resistance (tcrB) gene-mediated resistance to copper. The plasmid also carried resistance genes to tetracyclines and macrolides. The potential genetic link between copper and antibiotic resistance suggests that copper supplementation may exert a selection pressure for antimicrobial resistance. Therefore, a longitudinal study was conducted to investigate the effects of in-feed copper, chlortetracycline, and tylosin alone or in combination on the selection and co-selection of antimicrobial-resistant enterococci. The study included 240 weaned piglets assigned randomly to 6 dietary treatment groups: control, copper, chlortetracycline, tylosin, copper and chlortetracycline, and copper and tylosin. Feces were collected before (day 0), during (days 7, 14, 21), and after (days 28 and 35) initiating treatment, and enterococcal isolates were obtained from each fecal sample and tested for genotypic and phenotypic resistance to copper and antibiotics. A total of 2592 enterococcal isolates were tested for tcrB by polymerase chain reaction. The overall prevalence of tcrB-positive enterococci was 14.3% (372/2592). Among the tcrB-positive isolates, 331 were Enterococcus faecium and 41 were E. faecalis. All tcrB-positive isolates contained both erm(B) and tet(M) genes. The median minimum inhibitory concentration of copper for tcrB-negative and tcrB-positive enterococci was 6 and 18 mM, respectively. The majority of isolates (95/100) were resistant to multiple antibiotics. In conclusion, supplementing copper or antibiotics alone did not increase copper-resistant enterococci; however, supplementing antibiotics with copper increased the prevalence of the tcrB gene among fecal enterococci of piglets.
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http://dx.doi.org/10.1089/fpd.2015.1961DOI Listing
August 2015

Effects of chlortetracycline and copper supplementation on the prevalence, distribution, and quantity of antimicrobial resistance genes in the fecal metagenome of weaned pigs.

Prev Vet Med 2015 May 23;119(3-4):179-89. Epub 2015 Feb 23.

Department of Animal Sciences and Industry, College of Agriculture, Kansas State University, Manhattan, KS 66506, USA.

Use of in-feed antibiotics such as chlortetracycline (CTC) in food animals is fiercely debated as a cause of antimicrobial resistance in human pathogens; as a result, alternatives to antibiotics such as heavy metals have been proposed. We used a total community DNA approach to experimentally investigate the effects of CTC and copper supplementation on the presence and quantity of antimicrobial resistance elements in the gut microbial ecology of pigs. Total community DNA was extracted from 569 fecal samples collected weekly over a 6-week period from groups of 5 pigs housed in 32 pens that were randomized to receive either control, CTC, copper, or copper plus CTC regimens. Qualitative and quantitative PCR were used to detect the presence of 14 tetracycline resistance (tet) genes and to quantify gene copies of tetA, tetB, blaCMY-2 (a 3rd generation cephalosporin resistance gene), and pcoD (a copper resistance gene), respectively. The detection of tetA and tetB decreased over the subsequent sampling periods, whereas the prevalence of tetC and tetP increased. CTC and copper plus CTC supplementation increased both the prevalence and gene copy numbers of tetA, while decreasing both the prevalence and gene copies of tetB. In summary, tet gene presence was initially very diverse in the gut bacterial community of weaned pigs; thereafter, copper and CTC supplementation differentially impacted the prevalence and quantity of the various tetracycline, ceftiofur and copper resistance genes resulting in a less diverse gene population.
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http://dx.doi.org/10.1016/j.prevetmed.2015.02.008DOI Listing
May 2015
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