Genome-Based Characterization of Multidrug-Resistant Escherichia coli Isolated from Clinical Bovine Mastitis

Current Microbiology (2023) 80:89 https://doi.org/10.1007/s00284-023-03191-6 SHORT COMMUNICATION Genome‑Based Characterization of Multidrug‑Resistant Escherichia coli Isolated from Clinical Bovine Mastitis Taila dos Santos Alves1 · Vinícius Sanches Rosa1 · Domingos da Silva Leite1 · Simony Trevizan Guerra2 · Sâmea Fernandes Joaquim2 · Felipe Freitas Guimarães2 · José Carlos de Figueiredo Pantoja2 · Simoni Baldini Lucheis2 · Vera Lúcia Mores Rall3 · Rodrigo Tavanelli Hernandes3 · Helio Langoni2 · Márcio Garcia Ribeiro2 Received: 26 May 2022 / Accepted: 13 January 2023 © The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2023 Abstract Mastitis occurrence in dairy cows is a broad topic that involves several sectors, from antimicrobial resistance and virulence of strains to economic implications and cattle management practices. Here, we assessed the molecular characterization (antimicrobial resistance determinants, virulence genes, sequences type, serotypes, and plasmid types) of 178 Escherichia coli strains isolated from milk samples from cows with clinical mastitis using a genome-based k-mers approach. Of these, 53 (29.8%) showed multidrug resistance by disc diffusion. We selected eight multidrug-resistant mastitis-associated E. coli for whole-genome sequencing and molecular characterization based on raw data using k-mers. We assessed antimicrobial resistance genes, virulence factors, serotypes, Multilocus Sequence Typing (MLST), and plasmid types. The most antimicrobial resistance gene found were blaTEM-1B (7/8), tetA (6/8), strA (6/8), strB (6/8), and qnrB19 (5/8). A total of 25 virulence factors were detected encoding adhesins, capsule, enzymes/proteins, increased serum survival, hemolysin, colicins, and iron uptake. These virulence factors were associated with Extraintestinal Pathogenic E. coli. Three pandemic clones were found: ST10, ST101, and ST69. Two E. coli were assigned in the O117 serogroup and one in the O8:H25 serotype. The most common plasmid groups were IncFII (7/8) and IncFIB (6/8). Our findings contribute to the knowledge of virulence mechanisms, epidemiological aspects, and antimicrobial resistance determinants of E. coli strains obtained from clinical mammary infections of cows. Introduction Mastitis is a growing and serious problem in dairy farms due to the effects it has on animal health, milk quality, and losses to the dairy industry. Among the infectious agents related to mammary infections of environmental origin, Escherichia coli is the most common, followed by Klebsiella and * Taila dos Santos Alves ; 1 Department of Genetics, Evolution, Microbiology and Immunology, Institute of Biology, University of Campinas-UNICAMP, Campinas, SP 13083 862, Brazil 2 Department of Animal Production and Preventive Veterinary Medicine, School of Veterinary Medicine and Animal Sciences, São Paulo State University-UNESP, Botucatu, SP 18618 681, Brazil 3 Department of Microbiology and Immunology, São Paulo State University-UNESP, Botucatu, SP 18618 689, Brazil Enterobacter species [1]. In this context, antimicrobials have been routinely used to treat clinical mastitis cases (therapeutic use) and prevent future infections (prophylactic use) using the dry cow therapy [2]. Antimicrobial use on dairy farms has been associated with antimicrobial resistance, and these farms have been indicated as sources of antimicrobial resistance genes, acting as a dissemination hotspot together with human medicine and agriculture [3]. Classical molecular investigations use laborious and expensive methodologies for the characterization of bacteria, e.g., polymerase chain reaction (PCR) test, agglutination for serotyping, and enzyme restriction patterns. Usually, these approaches show incomplete results since they depend on the previous selection of genetic determinants. Thus, we aimed to investigate the molecular characterization of E. coli strains isolated from clinical bovine mastitis using a genome-based approach by k-mers to assess antimicrobial resistance determinants, virulence genes, Multilocus 13 Vol.:(0123456789) 89 Page 2 of 7 Sequence Typing (MLST), serotypes, and plasmid types using whole-genome sequencing (WGS). Materials and Methods Escherichia coli strains were isolated from 4,275 milk samples of cows that showed clinical signs of mastitis on ten farms located in the states of São Paulo and Minas Gerais, in Southeast Brazil, from September 2017 to March 2019 (Ethics Committee on Animal Use—CEUA, São Paulo State University, protocol No. 2015/19688-8). The calves were from medium-scale farms (20–200 hectares), with different average size of herds and breeding with similar nutrition, management, technical level, and sanitary conditions. All cows were housed in sand-bedded freestalls and were milked three times per day. Farms eligibility criteria included the following: (1) Holstein or Holstein crossbreed cows, (2) mastitis control programs with data recorded in management software, (3) somatic cell count (SCC) < 400,000 SCC/mL, (4) production > 20 L/cow/day, (5) at least 200 lactating cows in each farm, (6) mechanical milking system, and (7) history of clinical mastitis. The diagnosis of clinical mastitis was performed at every milking. The first milk streams were visually inspected in strip cup test or deposited on the floor with black rubber to identify any abnormalities. Mild (Score 1) signs of clinical cases were characterized by any abnormal appearance in milk (presence of flakes, blood, pus, or color changes). Moderate clinical cases (Score 2) were characterized by macroscopic changes of milk and udder inflammation (local pain, swelling, or redness in the affected mammary gland). Cases with additional signs such as inappetence, fever, tachypnea, tachycardia, decubitus, or abnormal ruminal motility were identified as severe (Score 3) [4]. After milking, technicians carried out the udder hygiene procedures (examination of the first milk streams, pre-dipping, and drying of the teats), the teat end was disinfected using cotton pads soaked with 70% alcohol solution. Then, the first streams of milk were discarded, and 15 mL of milk were collected in a sterile plastic vial and kept in refrigerated conditions (4–8 °C) in isothermal boxes until transport to the laboratory for further bacteriological and molecular analyses. The samples were cultivated onto MacConkey agar, and lactose-positive colonies were identified as E. coli using biochemical tests, namely, glucose fermentation, urea hydrolysis, hydrogen sulfide production, deamination of tryptophan, motility, lysine decarboxylase and indole production, and Simmons citrate utilization [5]. Antimicrobial susceptibility was performed by disk diffusion test according to Clinical and Laboratory Standards Institute (CLSI) guidelines [6, 7] using ampicillin (10 µg), 13 T. dos Santos Alves et al. amoxicillin (10 µg), ceftiofur (30 µg), cefoperazone (30 µg), (...truncated)