Use of critically important antimicrobial classes early in life may adversely impact bacterial resistance profiles during adult years: potential co-selection for plasmid-borne fluoroquinolone and macrolide resistance via extended-spectrum beta-lactam use in dairy cattle.

The transfer of antimicrobial resistance genes commonly occurs via vertical and horizontal gene transfer, as such genes are often found on the same mobile genetic element. This occurrence can lead to the co-selection of resistance to antimicrobials without their application. Dairy cattle located in the south-western United States were enrolled in a matched-pair longitudinal study to evaluate the effects of a two-dose ceftiofur treatment for metritis on levels of third-generation cephalosporin resistance among faecal Escherichia coli temporally. Escherichia coli chosen for further investigation were isolated on selective media, harboured extended-spectrum beta-lactam, fluoroquinolone and macrolide resistance genes. This combination has previously been unreported; importantly, it included genes encoding for resistance to antibiotics that can only be used in dairy cattle less than 20 months of age. Fluoroquinolones, macrolides and third and higher generation cephalosporins are considered critically important and highest priority for human medicine by the World Health Organization.

Macrolide-susceptible probiotic Enterococcus faecium ST296 exhibits faecal-environmental-oral microbial community cycling among beef cattle in feedlots.

Enterococci are included in the United States National Antimicrobial Resistance Monitoring System to track antibiotic resistance among commensal Gram-positive enteric bacteria, largely due to their high abundance in food animals and in retail meat. In the U.S. cattle industry, macrolides are used to prevent and control liver abscesses, which cause significant economic losses. Previous studies have suggested that feeding tylosin and the intensity of the pen environment, both expand and sustain respectively the prevalence of multidrug resistance among enterococci in feedlot cattle. This has led to research into alternative feed supplements and improved stewardship practices. In a randomized controlled trial, we measured the impact of a probiotic and an altered pen environment on antimicrobial resistance among faecal Enterococcus spp. in cattle fed tylosin. Supplementing cattle with an Enterococcus faecium and Saccharomyces cerevisiae-based probiotic yielded the isolation of E. faecium of the probiotic sequence type (ST296) from faecal and environmental samples in treatment groups, as well as from cattle and the manure pack in nearby pens. Of importance, the probiotic strain also was found in a desiccated and milled manure pack sample taken 120 days after the initial trial ended. Phylogenetic and SNP analyses revealed clonal survival and spread compatible with faecal-environmental-oral recycling of the probiotic strain within and among cattle and pens. The increase in prevalence of the ST296 strain occurred concomitant with a decrease in ST240, the dominant sequence type associated with ermB and tet(M) resistance genes in this trial. SIGNIFICANCE AND IMPACT OF THE STUDY: We demonstrate that a macrolide-susceptible probiotic Enterococcus faecium ST296 strain fed to beef cattle becomes fully embedded in the microbial community cycling of bacteria via faecal-environmental-oral transmission within and among feedlot pens. An initial investment in feeding the probiotic is thereby leveraged into expanding numbers of susceptible bacteria in cattle and their environment, even among those cattle fed tylosin.

Serotype Diversity and Antimicrobial Resistance among Salmonella enterica Isolates from Patients at an Equine Referral Hospital.

Although Salmonella enterica can produce life-threatening colitis in horses, certain serotypes are more commonly associated with clinical disease. Our aim was to evaluate the proportional morbidity attributed to different serotypes, as well as the phenotypic and genotypic antimicrobial resistance (AMR) of Salmonella isolates from patients at an equine referral hospital in the southern United States. A total of 255 Salmonella isolates was obtained from clinical samples of patients admitted to the hospital between 2007 and 2015. Phenotypic resistance to 14 antibiotics surveilled by the U.S. National Antimicrobial Resistance Monitoring System was determined using a commercially available panel. Whole-genome sequencing was used to identify serotypes and genotypic AMR. The most common serotypes were Salmonella enterica serotype Newport (18%), Salmonella enterica serotype Anatum (15.2%), and Salmonella enterica serotype Braenderup (11.8%). Most (n = 219) of the isolates were pansusceptible, while 25 were multidrug resistant (≥3 antimicrobial classes). Genes encoding beta-lactam resistance, such as blaCMY-2, blaSHV-12, blaCTX-M-27, and blaTEM-1B, were detected. The qnrB2 and aac(6')-Ib-cr genes were present in isolates with reduced susceptibility to ciprofloxacin. Genes encoding resistance to gentamicin (aph(3')-Ia, aac(6')-IIc), streptomycin (strA and strB), sulfonamides (sul1), trimethoprim (dfrA), phenicols (catA), tetracyclines [tet(A) and tet(E)], and macrolides [ere(A)] were also identified. The main predicted incompatibility plasmid type was I1 (10%). Core genome-based analyses revealed phylogenetic associations between isolates of common serotypes. The presence of AMR Salmonella in equine patients increases the risk of unsuccessful treatment and causes concern for potential zoonotic transmission to attending veterinary personnel, animal caretakers, and horse owners. Understanding the epidemiology of Salmonella in horses admitted to referral hospitals is important for the prevention, control, and treatment of salmonellosis.IMPORTANCE In horses, salmonellosis is a leading cause of life-threatening colitis. At veterinary teaching hospitals, nosocomial outbreaks can increase the risk of zoonotic transmission, lead to restrictions on admissions, impact hospital reputation, and interrupt educational activities. The antimicrobials most often used in horses are included in the 5th revision of the World Health Organization's list of critically important antimicrobials for human medicine. Recent studies have demonstrated a trend of increasing bacterial resistance to drugs commonly used to treat Salmonella infections. In this study, we identify temporal trends in the distribution of Salmonella serotypes and their mechanisms of antimicrobial resistance; furthermore, we are able to determine the likely origin of several temporal clusters of infection by using whole-genome sequencing. These data can be used to focus strategies to better contain the dissemination and enhance the mitigation of Salmonella infections and to provide evidence-based policies and guidelines to steward antimicrobial use in veterinary medicine.

Comparative microbiome analysis of beef cattle, the feedyard environment, and airborne particulate matter as a function of probiotic and antibiotic use, and change in pen environment.

Introduction: Intensive beef cattle production systems are frequently implicated as a source of bacteria that can be transferred to nearby humans and animals via effluent water, manure used as fertilizer, or airborne particulate matter. It is crucial to understand microbial population dynamics due to manure pack desiccation, antibiotic usage, and antibiotic alternatives within beef cattle and their associated feedyard environment. Understanding how bacterial communities change in the presence of antibiotics can also improve management practices for reducing the spread of foodborne bacteria. Methods: In this study, we aimed to compare the microbiomes within cattle feces, the feedyard environment and artificially produced airborne particulate matter as a function of pen change and treatment with tylosin or probiotics. We utilized 16S rRNA sequencing to compare bacterial communities among sample types, study days, and treatment groups. Results: Bacterial community diversity varied as a function of sampling day and pen change (old or new) within fecal and manure pack samples. Manure pack samples from old pens and new pens contained diverse communities of bacteria on days 0 and 84; however, by day 119 of the study these taxonomic differences were less evident. Particulate matter samples exhibited significant differences in community diversity and predominant bacterial taxa compared to the manure pack they originated from. Treatment with tylosin did not meaningfully impact bacterial communities among fecal, environmental, or particulate matter samples; however, minor differences in bacterial community structure were observed in feces from cattle treated with probiotics. Discussion: This study was the first to characterize and compare microbial communities within feces, manure pack, and airborne particulate matter from the same location and as a function of tylosin and probiotic treatment, and pen change. Although fecal and environmental samples are commonly used in research studies and other monitoring programs to infer public health risk of bacteria and antimicrobial resistance determinants from feedyard environments, our study suggests that these samples may not be appropriate to infer public health risk associated with airborne particulate matter.

Corrigendum: Comparative microbiome analysis of beef cattle, the feedyard environment, and airborne particulate matter as a function of probiotic and antibiotic use, and change in pen environment.

[This corrects the article DOI: 10.3389/fmicb.2024.1348171.].

Prevalence and Antimicrobial Resistance of Nontyphoidal Salmonella enterica from Head Meat and Trim for Ground Product at Pork Processing Facilities.

ABSTRACT: Pork head meat may harbor Salmonella and contaminate other carcass by-products during harvest and fabrication. A large pork processing plant in the United States was sampled bimonthly for 11 months to determine the concentration, prevalence, seasonality, serotype diversity, and antimicrobial susceptibility of Salmonella enterica isolated from cheek meat and head trim of swine carcasses. Each collection consisted of 25 samples on two consecutive days in the morning and afternoon shifts, for a total of 100 cheek meat and 100 head trim samples each month. Tissues were cultured for Salmonella by using restrictive media and enrichment techniques, and a subset of isolates was serotyped, analyzed for antimicrobial susceptibility, and genome sequenced. Salmonella postenrichment prevalence did not differ (P = 0.20) between cheek meat (63%) and head trim (66%). Postenrichment prevalence differed (P < 0.05) by month (January, 94%; March, 80%; May, 54%; July, 59%; September, 47%; and November, 55%) and by processing shift (morning, 68%; afternoon, 62%). The subset (n = 618) of isolates selected for serotyping yielded 21 distinct serotypes: Typhimurium (49%), Infantis (10%), Heidelberg (8%), I 4,[5],12:i:- (8%), and 17 other types (≤5%). In total, 407 multidrug-resistant (MDR; resistance to three or more antibiotic classes) isolates were identified. There were 120 isolates that exhibited the penta-resistant ACSSuT phenotype. In addition, 113 isolates exhibited decreased susceptibility to ciprofloxacin (MIC ≥ 0.12 µg/mL). Resistance genes blaCARB, blaSHV, blaTEM, aac(6')-Ib-cr,qnrB, sul2, and dfrA were expressed in numerous MDR Salmonella isolates. The data herein suggest that pork products from the head, compared with data reported for carcasses, may have a relatively high prevalence of Salmonella with diverse serotypes and MDR.

Varied prevalence of Clostridium difficile in an integrated swine operation.

The objectives of this study were to compare the prevalence of Clostridium difficile (Cd) among different age and production groups of swine in a vertically integrated swine operation in Texas in 2006 and to compare our isolates to other animal and human isolates. Results are based on 131 Cd isolates from 1008 swine fecal samples and pork trim samples (overall prevalence of 13%). The prevalence (number positive/number tested in production type) of Cd was different between the groups (P

Identification of a Plasmid-Mediated Quinolone Resistance Gene in Salmonella Isolates from Texas Dairy Farm Environmental Samples.

A recent increase in plasmid-mediated quinolone resistance (PMQR) has been detected among Salmonella isolated from humans in the United States, and it is necessary to determine the sources of human infection. We had previously isolated Salmonella from dairy farm environmental samples collected in Texas, and isolates were tested for anti-microbial susceptibility. Two isolates, serotyped as Salmonella Muenster, showed the discordant pattern of nalidixic acid susceptibility and intermediate susceptibility to ciprofloxacin. For this project, whole-genome sequencing of both isolates was performed to detect genes associated with quinolone resistance. The plasmid-mediated qnrB19 gene and IncR plasmid type were identified in both isolates. To our knowledge, this is the first report of PMQR in Salmonella isolated from food animals or agricultural environments in the United States.

Transferability of antimicrobial resistance from multidrug-resistant Escherichia coli isolated from cattle in the USA to E. coli and Salmonella Newport recipients.

OBJECTIVES: This study aimed to evaluate conjugative transfer of cephalosporin resistance among 100 strains of multidrug-resistant Escherichia coli (MDRE) to Salmonella enterica serotype Newport and E. coli DH5α recipients. METHODS: Phenotypic and genotypic profiles were determined for MDRE as well as for Salmonella Newport (trSN) and E. coli DH5α (trDH) transconjugants. RESULTS: Of 95 MDRE donor isolates, 26 (27%) and 27 (28%) transferred resistance to trSN and trDH recipients, respectively. A total of 27 MDRE (27%) were confirmed as extended-spectrum ß-lactamase (ESBL)-producers based on the double-disk synergy assay and whole-genome sequencing (WGS). WGS was performed on 25 of the ESBL-producing isolates, showing that 2 isolates carried blaCTX-M-6, 22 possessed blaCTX-M-32 and 1 was negative for blaCTX-M genes. Fourteen of the ESBLs sequenced were qnrB19. Differential transfer of IncA/C and IncN from MDRE32 was observed between trSN32 and trDH32. IncN-positive trDH32 displayed an ESBL phenotype, whereas IncA/C-positive trSN32 displayed an AmpC phenotype. The rate of ESBL transfer to trSN and trDH recipients was 11% and 96%, respectively. CONCLUSIONS: Twenty-seven MDRE were phenotypically identified as ESBL-producers. WGS of 25 MDRE revealed that 2 and 22 isolates carried blaCTX-M-6 and blaCTX-M-32, respectively. One multidrug-resistant isolate exhibited conversion from an AmpC phenotype to an ESBL phenotype with the transfer of only the IncN plasmid. The rate of resistance transfer to Salmonella or E. coli recipients was nearly identical. However, the ESBL phenotype was transferred with significantly greater prevalence to E. coli compared with Salmonella Newport (96% and 11%, respectively).

Supercritical fluid extraction and quantitative determination of organophosphorus pesticide residues in wheat and maize using gas chromatography with flame photometric and mass spectrometric detection.

An automated method using supercritical CO2 and clean-up by solid-phase extraction (SPE) using graphitized carbon black, has been developed for the quantitative determination of organophosphorus pesticide (OPP) residues in wheat and maize. Recoveries were as good as, or better than, those obtained using liquid extraction (LE) and gel permeation chromatography (GPC) for 10 OPP's spiked at levels equivalent to 0.05 and 0.50 microg/g. Lower limits of detection were possible using supercritical fluid extraction (SFE). Incurred residues were found in wheat and maize samples, and good agreement was obtained using SFE+SPE and LE+GPC. The SFE+SPE method required less analyst time and organic solvent, and hazardous waste was reduced.

Gas chromatography-mass spectrometry determination of phosphine residues in stored products and processed foods.

A gas chromatography-mass spectrometry (GC-MS) method was used for the quantitative confirmation of phosphine residues in stored products and processed foods. An established extraction technique was utilized for the preparation of headspace samples, which were analyzed by GC-MS and gas chromatography-nitrogen-phosphorus detection (GC-NPD). Wheat, oats, maize, white rice, brown rice, cornflakes, tortilla cornchips, groundnuts, and raisins were validated, showing excellent agreement between detectors when spiked at levels equivalent to 0.001 and 0.01 mg/kg phosphine and for samples containing incurred residues. The GC-MS method was reproducible and accurate when compared to the GC-NPD method and allowed five samples to be quantified in a working day. Subambient GC-MS oven temperatures were most suitable for phosphine residues ranging from 0.001 to 0.005 mg/kg, and a GC oven temperature of 100 degrees C was appropriate for residues >0.005 mg/kg. The method was sufficiently robust to be evaluated for other similar commodities as the need arises.

Design and development of a Virtual Dolphinarium for children with autism.

The recent proliferation of virtual reality (VR) technology applications in the autism therapy to promote learning and positive behavior among such children has produced optimistic results in developing a variety of skills and abilities in them. Dolphin-assisted therapy has also become a topic of public and research interest for autism intervention and treatment. This paper will present an innovative design and development of a Virtual Dolphinarium for potential autism intervention. Instead of emulating the swimming with dolphins, our virtual dolphin interaction program will allow children with autism to act as dolphin trainers at the poolside and to learn (nonverbal) communication through hand gestures with the virtual dolphins. Immersive visualization and gesture-based interaction are implemented to engage children with autism within an immersive room equipped with a curved screen spanning a 320(°) and a high-end five-panel projection system. This paper will also report a pilot study to establish trial protocol of autism screening to explore the participants' readiness for the virtual dolphin interaction. This research will have two potential benefits in the sense of helping children with autism and protecting the endangered species.

Determination of tetrachloroethylene in olive oil by automated headspace gas chromatography.

A rapid screening method for detecting low levels of tetrachloroethylene (perchloroethylene, PCE) in olive oils has been developed using headspace capillary gas chromatography. Modification of this method allows quantitative results to be obtained down to 0.001 mg kg-1. Results obtained show that olive oil samples received in this laboratory over a two year period have not contained PCE residues in excess of the European Commission guidelines.