Phenotypic and Genotypic Characterization of Foodborne Commensals and Pathogens from Sheep and Their Abattoir Environment in North Carolina: A Serial Cross-Sectional Study

Atlaw, Nigatu Aklilu North Carolina State University ProQuest Dissertat 2021 해외박사(DDOD)

Reports of widespread dissemination of extended-spectrum beta-lactamase (ESBL) E. coli and Salmonella in food animals, retail meats, and humans are available. However, information on ESBLs in small ruminants is limited, particularly in the United States (U.S.). The objectives of these studies were: 1) to investigate the prevalence and antimicrobial resistance (AMR) of extended-spectrum beta-lactamase (ESBL) E. coli and Salmonella in sheep and their abattoir environment in North Carolina, 2) to genotypically characterize ESBL E. coli from healthy sheep and their abattoir environment, and 3) to genotypically characterize AMR determinants in Salmonella recovered from sheep and their abattoir environment. Hence, we conducted a year-round serial cross-sectional study and collected a total of 1128 samples from sheep (n=780) and their abattoir environment (n=348). Sheep samples consisted of feces, cecal contents, carcass swabs, and abattoir resting area feces. Environmental samples consisted of soil samples, lairage swab, animal feed, and drinking water for animals. ESBL E. coli and Salmonella were isolated and confirmed using standard culture methods. We selected one confirmed isolate per positive sample for these studies. Collectively, we analyzed a total of 318 ESBL E. coli (318/1128; 28.2%) and 368 Salmonella (368/1128; 32.6%) isolates. The prevalence of ESBL E. coli and Salmonella was significantly higher in environmental samples (166/348; 47.7% and 228/348; 65.5%) compared to the sheep samples (152/780; 19.5% and 140/780; 17.9%), respectively (P<0.0001). All confirmed isolates were tested against a panel of 14 antimicrobials to elucidate susceptibility profiles. More than 97% of ESBL E. coli were multidrug-resistant (MDR; resistant to ≥ 3 classes of antimicrobials). Most Salmonella isolates (77.2%) were pansusceptible, and 10.1% were MDR. We identified a total of 24 different Salmonella serotypes; the top five serotypes in decreasing order of frequency are S. Agona (19.8%), S. Typhimurium (16.2%), S. Cannstatt (13.2%), S. Reading (13.2%), and S. Anatum (9.6%). Prevalence and percent resistance of ESBL E. coli and Salmonella isolates varied significantly by season and sample type (P<0.0001). Whole-genome sequencing (WGS) (MiSeq Illumina platform) was conducted on 113 ESBL E. coli (64 from sheep and 48 from abattoir environment) and 164 Salmonella isolates (63 from sheep and 101 from abattoir environment). Bioinformatics tools were used to analyze sequence data from both pathogens. In both E. coli and Salmonella of pathogens, AMR determinants demonstrated resistance to aminoglycosides, beta-lactams, phenicols, folate pathway antagonists, tetracyclines, and quinolones. ESBL E. coli isolates distinctively carried genes that confer resistance to macrolides and lincosamides while 37% of Salmonella isolates carried fosA7, a gene known to confer Fosfomycin resistance. This report is the first observation of fosA7 in multiple serotypes of Salmonella in the U.S. Many CTX-M-type beta-lactamase genes detected in ESBL E. coli isolates included blaCTX-M-1, blaCTX-M-14, blaCTX-M-15, blaCTX-M-27, blaCTX-M-32, blaCTX-M-55, and blaCTX-M-65. Other beta-lactamase genes detected included blaCMY-2, blaTEM-1A/B/C, and blaCARB-2. Interestingly, blaTEM-1A and blaTEM-1B were the only beta-lactamase gene types detected in the Salmonella isolates. Twenty-eight and 17 different profiles of plasmid types were detected in ESBL E. coli and Salmonella, respectively. Of these, we observed that most occurred in both sheep and environmental sources of isolates. In addition, we identified 29 and 24 different sequence types (STs) of ESBL E. coli and Salmonella, respectively. The core genome phylogenetic analyses of both pathogens revealed that STs were clustered around seasonal sampling but dispersed across sample types.In conclusion, this is the first comprehensive AMR and WGS report on ESBL E. coli and Salmonella from sheep and their abattoir environment in North Carolina. Our study confirmed that sheep are important reservoirs of AMR ESBL E. coli and MDR Salmonella in the U.S. However, further studies are required to determine associated public health risks.

Novel Bacterial Strain Bacillus cereus ALT1 Up-regulates Growth Attributes, Endogenous Phytohormones and Strengthens Antioxidant System of Soybean under Cadmium Stress

Sahile, Atlaw Anbelu 경북대학교 대학원 2021 국내석사

Cadmium is one of the most toxic food contaminants and results in a significant decrease in agricultural productivity worldwide. Plant growth promoting rhizobacteria (PGPR) are eco-friendly and inexpensive tool for mitigating heavy metal stress in crops. We isolated rhizospheric bacteria from selected sand dune plants at Pohang beach, screened them for different PGP traits and Cd tolerance. Out of 55 bacterial isolates, only 7 isolates showed multiple PGP traits and were screened with different Cd concentration (0.5 mM to 2.5 mM) on LB plates. The Bacillus cereus ALT1 showed high tolerance to Cd stress, and also secreted IAA and organic acids in the culture broth. Elevated levels of Cd stress (0.7mM, 1.4mM and 2.1mM) reduced soybean shoot length (13.6-19.64%), root length (15.5-44%), root/shoot fresh weight (13.1-45.98%), root/shoot dry weight (19.77-46.89%), chlorophyll contents (12-42%), Chl A (18-80%), Chl B (38-89%) and carotenoid (19-79%); however inoculation of bacterial isolate ALT1 mitigated Cd stress and enhanced soybean shoot length (2.21-5.51%), root length (4.4-40%), root/shoot fresh weight (5.04-40.21%), root/shoot dry weight (36.14-72.00%), chlorophyll contents (16-24%), Chl A (8-43%), Chl B (13-46%) and carotenoid (14-39%) content. Furthermore, inoculation of ALT1 down-regulated abscisic acid biosynthesis (0.77 folds), enhanced salicylic acid (6-16%), and strengthened antioxidants i.e. increased total proteins (10%-20%), and superoxide dismutase (22.14-43.73%) and reduced glutathione content (56%-179%), while decreased lipid peroxidation (0.8-5 folds), peroxidase (0.8-2folds), superoxide anion (21%-68%) and polyphenol oxidase (26.38-39.18%) in soybean. In addition, ICP results showed that soybean plant treated with bacterial isolate ALT1 enhanced K+ uptake (20.74-30.26%) and reduced Cd contents (4 to 25 folds). In conclusion, the present study reveals that Cd tolerant bacterial isolate ALT1 can alleviate Cd toxicity, increase plant growth and could be used as an eco-friendly bio-fertilizer under Cd stress. 카드뮴은 매우 해로운 식품 오염 물질이며 전세계적으로 농업 생산성의 심각한 감소를 불러일으키는 물질이다. 식물 성장 촉진 근권 박테리아(PGPR)은 작물 내 중금속 스트레스를 완화하는 친환경적이며 경제적인 수단으로 이용되고 있다. 우리는 포항 해변에 있는 사구 식물로부터 근권 세균을 분리하였고 세균에서 다른 종류의 식물 성장 촉진(PGP) 특징과 카드뮴 내성을 검사하였다. 분리된 55 개의 균주 중 6 개의 균주들이 LB 배지에서 카드뮴 농도(0.5mM 에서 2.5mM 까지)에 따라 다양한 식물 성장 촉진(PGP) 특성을 보였다. Bacillus cereus ALT1 은 카드뮴 스트레스에 강한 내성을 보였고 IAA 와 유기산을 액체 배양 배지에서 분비하였다. 높은 레벨의 카드뮴 스트레스(0.7mM, 1.4mM 그리고 2.1mM)는 콩 지상부의 길이를 13.6-19.64% 줄이고 지하부의 길이를 15.5-44% 줄였으며 지하부와 지상부의 생체중을 13.1-45.98% 줄이고 지하부와 지상부의 건물 중을 19.77-46.89% 줄였다. 또한 높은 레벨의 카드뮴 스트레스는 엽록소 함량의 12-42% 줄이고 엽록소 a 함량을 18-80% 줄였으며 카로티노이드 함량을 19-79% 줄였다. ALT1 접종 시 카드뮴 스트레스는 경감되었고 콩 지상부의 길이는 2.21-5.51% 늘어났으며 지하부의 길이는 4.4-40% 증가하였다. 또한 지하부와 지상부의 38 생체중은 5.04-40.21% 증가하였고 지하부와 지상부의 건물중은 36.14- 72% 증가하였으며 엽록소 함량은 16-24% 증가하였고 엽록소 a 는 8- 43% 엽록소 b 는 13-46% 증가하였고 카로티노이드 함량은 14-39% 증가하였다. 그러나 ALT1 접종 시 ABA 생합성은 0.77 배로 하향 조절되었고 SA 는 6-16% 향상되었으며 항산화효소의 활성이 강화되었다. 즉 ALT1 접종은 총 단백질량을 10%-20% 증가시키고 SOD (Superoxide dismutase)를 22.14-43.73% 증가시켰으며 GSH 함량을 56%-179% 증가시켰다. 또한 ALT1 접종은 콩 내부 지질 과산화반응을 0.8 배에서 5 배까지 감소시켰고 과산화 효소를 0.8 배에서 2 배까지 변화시켰으며 SOD 를 21-68% 감소시켰고 폴리페놀 산화효소를 26.38-39.18% 감소시켰다. 게다가 ICP 결과에 따르면 박테리아 ALT1 를 처리한 콩은 칼륨 흡수가 20.74-30.26% 향상되었고 카드뮴 함량이 4 배에서 25 배 감소되었다. 결과적으로 본연구는 카드뮴 내성이 있는 박테리아 ALT1 이 카드뮴 독성을 경감시킬 수 있고 식물 생육을 촉진시키며 카드뮴 스트레스 아래서 친환경적 생물 비료로서 사용될 수 있다는 사실을 확인하였다.