Clinical Impact of Multiplex Molecular Diagnostic Testing in Children With Acute Gastroenteritis Presenting to an Emergency Department: A Multicenter Prospective Study.

BACKGROUND: Multiplex molecular diagnostic panels have greatly enhanced detection of gastrointestinal pathogens. However, data on the impact of these tests on clinical and patient-centered outcomes are limited. METHODS: We conducted a prospective, multicenter, stepped-wedge trial to determine the impact of multiplex molecular testing at 5 academic children's hospitals on children presenting to the emergency department with acute gastroenteritis. Caregivers were interviewed on enrollment and 7-10 days after enrollment to determine symptoms, risk factors, subsequent medical visits, and impact on family members. During the pre-intervention period, diagnostic testing was performed at the clinician's discretion . During the intervention period, multiplex molecular testing was performed on all children, with results available to clinicians. The primary outcome was return visits to a healthcare provider within 10 days of enrollment. RESULTS: Potential pathogens were identified by clinician-ordered tests in 19 of 571 (3.3%) in the pre-intervention period compared with 434 of 586 (74%) in the intervention period; clinically relevant pathogens were detected in 2.1% and 15%, respectively. In the multivariate model, the intervention was associated with a 21% reduction in the odds of any return visit (odds ratio, 0.79; 95% confidence interval, .70-.90) after adjusting for potential confounders. Appropriate treatment was prescribed in 11.3% compared with 19.6% during the intervention period (P = .22). CONCLUSIONS: Routine molecular multiplex testing for all children who presented to the ED with acute gastroenteritis detected more clinically relevant pathogens and led to a 21% decrease in return visits. Additional research is needed to define patients most likely to benefit from testing. Clinical Trials Registration. NCT02248285.

A multicenter evaluation of viral bloodstream detections in children presenting to the Emergency Department with suspected systemic infection.

BACKGROUND: Fever is a common symptom in children presenting to the Emergency Department (ED). We aimed to describe the epidemiology of systemic viral infections and their predictive values for excluding serious bacterial infections (SBIs), including bacteremia, meningitis and urinary tract infections (UTIs) in children presenting to the ED with suspected systemic infections. METHODS: We enrolled children who presented to the ED with suspected systemic infections who had blood cultures obtained at seven healthcare facilities. Whole blood specimens were analyzed by an experimental multiplexed PCR test for 7 viruses. Demographic and laboratory results were abstracted. RESULTS: Of the 1114 subjects enrolled, 245 viruses were detected in 224 (20.1%) subjects. Bacteremia, meningitis and UTI frequency in viral bloodstream-positive patients was 1.3, 0 and 10.1% compared to 2.9, 1.3 and 9.7% in viral bloodstream-negative patients respectively. Although viral bloodstream detections had a high negative predictive value for bacteremia or meningitis (NPV = 98.7%), the frequency of UTIs among these subjects remained appreciable (9/89, 10.1%) (NPV = 89.9%). Screening urinalyses were positive for leukocyte esterase in 8/9 (88.9%) of these subjects, improving the ability to distinguish UTI. CONCLUSIONS: Viral bloodstream detections were common in children presenting to the ED with suspected systemic infections. Although overall frequencies of SBIs among subjects with and without viral bloodstream detections did not differ significantly, combining whole blood viral testing with urinalysis provided high NPV for excluding SBI.

Comparison of the ID Now Influenza A & B 2, Cobas Influenza A/B, and Xpert Xpress Flu Point-of-Care Nucleic Acid Amplification Tests for Influenza A/B Virus Detection in Children.

Early diagnosis of influenza (Flu) is critical for patient management and infection control. The ID Now influenza A & B 2 (ID Now) assay (Abbott Laboratories), Cobas influenza A/B nucleic acid test (LIAT; Roche Molecular Systems, Inc.), and Xpert Xpress Flu (Xpert; Cepheid) are rapid, point-of-care molecular assays for Flu virus detection. The study aim was to compare the performances of these three commercially available Clinical Laboratory Improvement Amendments (CLIA)-waived Flu virus assays. We prospectively enrolled 201 children <18 years old from January to April 2018 and collected nasopharyngeal swab specimens in viral medium. Aliquots were frozen for testing on different diagnostic platforms, as per the manufacturers' instructions. CDC Flu A/B PCR was used as a reference method to evaluate the performances of these three platforms. Among the 201 specimens tested, the CDC Flu A/B PCR assay detected Flu A/B virus in 107 samples (Flu A virus, 73 samples; Flu B virus, 36 samples; dual Flu A/B virus positive, 2 samples), while the ID Now virus detected 102 samples (Flu A virus, 69 samples; Flu B virus, 37 samples; dual Flu A/B virus positive, 4 samples; invalid rate, 1/201 [0.5%]), the LIAT detected 112 samples (Flu A virus, 74 samples; Flu B virus, 38 samples; invalid rate, 11/201 [5.5%]), and the Xpert assay detected 112 samples (Flu A virus, 76 samples; Flu B virus, 36 samples; invalid rate, 6/201 [3.0%]). The overall sensitivities for the ID Now assay, LIAT, and Xpert assay for Flu A virus detection (93.2%, 100%, and 100%, respectively) and Flu B virus detection (97.2%, 94.4%, and 91.7%, respectively) were comparable. The specificity for Flu A and B virus detection by all methods was >97%. These molecular assays had higher sensitivity than did a historical standard-of-care test from the BD Veritor antigen test (Flu A virus, 79.5%; Flu B virus, 66.7%).

Comparison of Six Sample-to-Answer Influenza A/B and Respiratory Syncytial Virus Nucleic Acid Amplification Assays Using Respiratory Specimens from Children.

The rapid and accurate detection of influenza A virus (FluA), influenza B virus (FluB), and respiratory syncytial virus (RSV) improves patient care. Sample-to-answer (STA) platforms based on nucleic acid amplification and detection of these viruses are simple, automated, and accurate. We compared six such platforms for the detection of FluA, FluB, and RSV: Cepheid GeneXpert Xpress Flu/RSV (Xpert), Hologic Panther Fusion Flu A/B/RSV (Fusion), Cobas influenza A/B & RSV (Liat), Luminex Aries Flu A/B & RSV (Aries), BioFire FilmArray respiratory panel (RP), and Diasorin Simplexa Flu A/B & RSV (Simplexa). Nasopharyngeal (NP) swab specimens (n = 225) from children previously tested by RP were assessed on these platforms. The results were compared to those of the Centers for Disease Control and Prevention (CDC)-developed real-time reverse transcription-PCR (rRT-PCR) assay for influenza A/B viruses and RSV. Subtyping for FluA and FluB was performed for discrepant analysis where applicable. The percent sensitivities/specificities for FluA detection were 100/100 (Fusion), 98.6/99.3 (Xpert), 100/100 (Liat), 98.6/100 (Aries), 98.6/100 (Simplexa), and 100/100 (RP). The percent sensitivities/specificities for FluB detection were 100/100 (Fusion), 97.9/99.4 (Xpert), 97.9/98.3 (Liat), 93.7/99.4 (Aries), 85.4/99.4 (Simplexa), and 95.8/97.7 (RP); and those for RSV detection were 98.1/99.4 (Xpert), 98.1/99.4 (Liat), 96.3/100 (Fusion), 94.4/100 (Aries), 87/94.4 (Simplexa), and 94.4/100 (RP). The 75 strains confirmed to be FluA included 29 pH1N1, 39 H3N2, 4 sH1N1, and 3 untyped strains. The 48 strains confirmed to be FluB included 33 strains of the Yamagata lineage, 13 of the Victoria lineage, 1 of both the Yamagata and Victoria lineages, and 1 of an unknown lineage. All six STA platforms demonstrated >95% sensitivity for FluA detection, while three platforms (Fusion, Xpert, and Liat) demonstrated >95% sensitivity for FluB and RSV detection.

Evaluation of the illumigene Mycoplasma Direct DNA Amplification Assay.

Mycoplasma pneumoniae is a common cause of community-acquired pneumonia. The illumigene Mycoplasma Direct (iMD) DNA amplification assay is a qualitative in vitro test utilizing loop-mediated isothermal amplification (LAMP) technology for the direct detection of M. pneumoniae DNA in respiratory specimens. The iMD assay does not require the preextraction of nucleic acids from specimens, which is a prerequisite step for the previously approved illumigene Mycoplasma (iM) assay. The aim of this prospective multicenter study was to evaluate the performance characteristics of the newly developed iMD assay, compared with the iM assay. Subjects with symptoms of upper respiratory illnesses suggesting M. pneumoniae infection were enrolled at three sites in the United States. Respiratory specimens were obtained using dual throat swabs. One swab was tested with the iMD assay at each enrollment site. Reference testing with the iM assay was performed by the manufacturer. Among 456 specimens tested, the iM reference method detected M. pneumoniae in 25 specimens (5.5%), while the iMD assay identified 34 specimens (7.5%) as M. pneumoniae positive. There were 10 false-positive results and 1 false-negative result with the iMD assay. The overall positive and negative agreement rates were 96.0% (95% confidence interval [CI], 80.5 to 99.3%) and 97.7% (95% CI, 95.8 to 98.7%), respectively. The overall agreement rate was determined to be 97.6% (95% CI, 95.7 to 98.6%). We conclude that the iMD test results were comparable to the iM assay results. The removal of the DNA extraction step for the iMD assay simplifies testing, saves time, and reduces the costs of detecting M. pneumoniae from throat swabs, compared to the iM assay.

Etiologies of bloody diarrhea in children presenting with acute gastroenteritis to US emergency departments.

Among 111 children presenting with bloody diarrhea in a multicenter study of molecular testing in US emergency departments, we found viral pathogens in 18%, bacteria in 48%, protozoa in 2%, and no pathogens detected in 38%.

Comparative in vitro antipseudomonal activity of ceftolozane/tazobactam against Pseudomonas aeruginosa isolates from children with cystic fibrosis.

This study evaluated the in vitro activity of Ceftolozane/tazobactam (C/T) vs 10 comparator agents against Pseudomonas aeruginosa isolates obtained from clinical respiratory samples from pediatric patients with cystic fibrosis at three hospitals during 2015 to 2020. Antimicrobial susceptibility testing was performed using microbroth dilution technique with custom prepared Sensititre® MIC plates. MICs were determined via Sensititre Vizion® system and results were interpreted using current CLSI and EUCAST (2022) breakpoint criteria. C/T was the most potent agent as compared with other antipseudomonal drugs against 291 isolates with MIC50 = 1 µg/mL and MIC90 = 2 µg/mL with percent susceptibility as 95.2%. C/T remained active against majority of ß-lactam non-susceptible isolates; percent susceptibility ranging from 61.2% to 80% including 65.9% ceftazidime non-susceptible isolates. C/T had high activity against P. aeruginosa from 3 geographically diverse pediatric medical centers. Study results suggest that C/T may be used as a potential therapeutic option for treating pediatric patients with CF.

Clinical Impact of Multiplex Molecular Diagnostic Testing in Children with Acute Gastroenteritis Presenting to An Emergency Department: A Multicenter Prospective Study.

Background: Multiplex molecular diagnostic panels have greatly enhanced detection of gastrointestinal pathogens. However, data on the impact of these tests on clinical and patient-centered outcomes are limited. Methods: We conducted a prospective, multicenter, stepped-wedge trial to determine the impact of multiplex molecular testing at five academic children's hospitals in children presenting to the ED with acute gastroenteritis. Caregivers were interviewed on enrollment and again 7-10 days after enrollment to determine symptoms, risk factors, subsequent medical visits, and impact on family members. During the pre-intervention period, diagnostic testing was performed at the discretion of clinicians. During the intervention period, multiplex molecular testing was performed on all children with results available to clinicians. Primary outcome was return visits to a health care provider within 10 days of enrollment. Results: Potential pathogens were identified by clinician ordered tests in 19/571 (3.3%) in the pre-intervention period compared to 434/586 (74%) in the intervention period; clinically relevant pathogens were detected in 2.1% and 15% respectively. In the multivariate model adjusting for potential confounders, the intervention was associated with a 21% reduction in the odds of any return visit (OR 0.79; 95% CI 0.70-0.90). Appropriate treatment was prescribed in 11.3% compared to 19.6% during the intervention period(P=0.22). Conclusions: Routine molecular multiplex testing for all children presenting to the ED with AGE detected more clinically relevant pathogens and led to a 21% decrease in return visits. Additional research is needed to define patients most likely to benefit from testing.

Comparison of diagnostic performance of five molecular assays for detection of SARS-CoV-2.

We compared the performance of the Abbott Real Time SARS-CoV-2 assay (Abbott assay), Aptima™ SARS-CoV-2 assay (Aptima assay), BGI Real-Time SARS-CoV-2 assay (BGI assay), Lyra® SARS-CoV-2 assay (Lyra assay), and DiaSorin Simplexa™ COVID assay for SARS-CoV-2 detection. Residual nasopharyngeal samples (n = 201) submitted for routine SARS-CoV-2 testing by Simplexa assay during June-July 2020 and January 2021 were salvaged. Aliquots were tested on other assays and compared against the CDC 2019-nCoV Real-Time RT-PCR assay. Viral load in positive samples was determined by droplet digital PCR. Among 201 samples, 99 were positive and 102 were negative by the CDC assay. The Aptima and Abbott assays exhibited the highest positive percent agreement (PPA) at 98.9% while the BGI assay demonstrated the lowest PPA of 89.9% with 10 missed detections. Negative percent agreement for all 5 platforms was comparable, ranging from 96.1% to 100%. The performance of all five assays was comparable.

Comparative in vitro effectiveness of ceftolozane/tazobactam against pediatric gram-negative drug-resistant isolates.

Ceftolozane/tazobactam (C/T), a cephalosporin/beta-lactamase inhibitor combination, was evaluated in vitro vs. 10 comparators against 299 pediatric extended-spectrum-cephalosporin-resistant or carbapenem-resistant (ESC-R/CR) Gram-negative Enterobacteriaceae from three freestanding pediatric centers. Isolates were from urine or other sterile sites of children and adolescents through 21 years of age. Susceptibilities were assayed by microbroth dilution via custom Sensititre plates (Thermo Fisher Scientific). Susceptibility was determined using the Sensititre Vizion® system (Thermo Fisher Scientific). Susceptibility breakpoint criteria were those of the Clinical and Laboratory Standards Institute (CLSI) for 2017, except for colistin (EUCAST 2019). Overall, 87.5% isolates were C/T susceptible (MIC ≤2 µg/ml; MIC50/90, 0.25/4 µg/ml). Susceptibility to C/T was detected more frequently as compared to all other antimicrobials tested except for colistin (95.4%) and meropenem (97.4%). Percent susceptibility to C/T was high for E. coli (91%) and Klebsiella spp. (73.3%). C/T demonstrated good in-vitro activity and high potency against most beta-lactam resistant pediatric Enterobacteriaceae from three geographically diverse U.S. regions.

Comparative analysis of Four sample-to-answer influenza A/B and RSV nucleic acid amplification assays using adult respiratory specimens.

BACKGROUND: The use of Sample-to-answer (STA) platforms for the detection of influenza A/B and respiratory syncytial virus (RSV) have greatly improved patient care. These diagnostic assays based on nucleic acid amplification are rapid, accurate and relatively easy to perform. OBJECTIVES: We compared four such platforms for detecting FluA, FluB, and RSV from adult respiratory specimens: Hologic Panther Fusion® Flu A/B/RSV (Fusion), Cobas® Influenza A/B & RSV (Liat), Luminex Aries® Flu A/B & RSV (Aries), and Diasorin SimplexaTM Flu A/B & RSV (Simplexa). STUDY DESIGN: Nasopharyngeal (NP) swabs (n = 224) from adults were tested on these platforms and results were compared to Center for Disease Control and Prevention recommended real-time RT-PCR assay for influenza A/B and RSV. Subtyping for FluA and FluB was performed for discrepant analysis where applicable. RESULTS: Of the 82 FluA, 26 FluB, 15 RSV-positive specimens tested, the positive and negative percentage agreements (PPA and NPA respectively) for FluA detection were 100/100 (Fusion), 95.1/100 (Liat), 92.5/100 (Aries), and 84.1/99.3 (Simplexa); PPA and NPA for FluB detection were 92.3/99.5 (Fusion), 96/99.5 (Liat), 100/99.5 (Aries), and 80.8/100 (Simplexa); and for RSV detection were 100/100 (Fusion), 100/100 (Liat), 88.6/99.5 (Aries), and 73.3/100 (Simplexa). 82 confirmed FluA included 23 pH1N1 and 57 H3N2 strains with 2 strains remaining untyped. Of the 26 confirmed FluB, 25 were of the Yamagata lineage and 1 of unknown lineage. CONCLUSION: Only 2 STA platforms demonstrated >95% PPA for the detection of all three targets while all the 4 platforms demonstrated >95% NPA for FluA, FluB and RSV.

Viral Etiology of Acute Gastroenteritis in <2-Year-Old US Children in the Post-Rotavirus Vaccine Era.

BACKGROUND: The rotavirus disease burden has declined substantially since rotavirus vaccine was introduced in the United States in 2006. The aim of this study was to determine the viral etiology of acute gastroenteritis (AGE) in US children aged <2 years. METHODS: The New Vaccine Surveillance Network (NVSN) of geographically diverse US sites conducts active pediatric population-based surveillance in hospitals and emergency departments. Stool samples were collected from children aged <2 years with symptoms of AGE (n = 330) and age-matched healthy controls (HCs) (n = 272) between January and December 2012. Samples were tested by real-time reverse-transcriptase polymerase chain reaction assays {adenovirus (type 40 and 41), norovirus, parechovirus A, enterovirus, sapovirus, and astrovirus} and an enzyme immunoassay (rotavirus). All samples that tested positive were genotyped. RESULTS: Detection rates of pathogens in children with AGE versus those of HCs were, respectively, 23.0% versus 6.6% for norovirus (P < .01), 23.0% versus 16.0% for adenovirus (P = .08), 11.0% versus 16.0% for parechovirus A (P = .09), 11.0% versus 9.0% for enterovirus (P = .34), 7.0% versus 3.0% for sapovirus (P = .07), 3.0% versus 0.3% for astrovirus (P = .01), and 3.0% versus 0.4% for rotavirus (P = .01). A high prevalence of adenovirus was detected at 1 surveillance site (49.0% for children with AGE and 43.0% for HCs). Norovirus GII.4 New Orleans was the most frequently detected (33.0%) norovirus genotype. Codetection of >1 virus was more common in children with AGE (16.0%) than in HCs (10.0%) (P = .03). CONCLUSIONS: Norovirus, astrovirus, sapovirus, and rotavirus were detected significantly more in children with AGE than in HCs, and norovirus was the leading AGE-causing pathogen in US children aged <2 years during the year 2012.

Intestinal Carriage of Third-Generation Cephalosporin-Resistant and Extended-Spectrum ß-Lactamase-Producing Enterobacteriaceae in Healthy US Children.

BACKGROUND: The epidemiology of antibiotic-resistant Enterobacteriaceae intestinal carriage in healthy US children has not been well characterized. METHODS: Children between 14 days and 14 years of age were enrolled during well-child visits in Oakland, California, Kansas City, Kansas, and Nashville, Tennessee, between December 2013 and March 2015. Data on recent antibiotic use by the child and travel and hospitalization history of all members of each child's household were obtained with a risk-factor survey. Stool specimens collected from the subjects were screened for extended-spectrum ß-lactamase-producing (ESBL-P) bacteria using CHROMagar ESBL medium. Putative ESBL-P Escherichia coli and Klebsiella colonies underwent phenotypic confirmation by double-disk synergy testing; confirmed third-generation cephalosporin-resistant (3GCR) isolates underwent additional antibiotic-susceptibility testing. RESULTS: In 519 subjects, the overall 3GCR Enterobacteriaceae carriage rate was 4.4% (n = 23) and ranged from 3.4% to 5.1% among the study sites. The ESBL-P Enterobacteriaceae carriage rate was 3.5% (n = 18). The rates of 3GCR Enterobacteriaceae carriage was highest in 1 to <2 year olds at 6.5%, and was 5.2% in <5 year-olds vs 1.7% in ≥5-year-olds (P = .11). 3GCR and ESBL-P Enterobacteriaceae carriage was associated with international travel within the previous year; 11.1% of ESBL-P Enterobacteriaceae carriers reported this history compared with 1.6% of noncarriers (P = .004). No other queried factor was found to increase risk. Of the 24 analyzed 3GCR isolates, 58% were multidrug resistant. CONCLUSIONS: The 3GCR Enterobacteriaceae carriage rate exceeds 5% in healthy US children <5 years of age. International travel within the previous year increased the risk of 3GCR and ESBL-P Enterobacteriaceae carriage. In contrast, we found no differences in the rates of hospitalization or recent antibiotic exposure between carriers and noncarriers. Young children, who have the highest prevalence of colonization, might be a sentinel population to study to gain a better understanding of community sources of antibiotic-resistant Enterobacteriaceae.

Head-to-head comparison of the diagnostic accuracies of BD Veritor™ System RSV and Quidel® Sofia® RSV FIA systems for respiratory syncytial virus (RSV) diagnosis.

BACKGROUND: Respiratory syncytial virus (RSV) is one of the most common causes of severe lower respiratory tract disease among infants and young children. BD Veritor™ System RSV (BD) and Quidel(®) Sofia(®) RSV FIA (QD) are the new generation lateral flow digital immunoassay (DIA) tests with an instrumented read for the qualitative detection of RSV viral antigens. OBJECTIVE: To compare the diagnostic accuracies of BD and QD for RSV detection using fresh nasopharyngeal aspirates and nasopharyngeal swab specimens collected in universal transport media during 2013-2014 respiratory season. STUDY DESIGN: The two DIA tests were performed simultaneously on randomly selected specimens on a weekly basis during the RSV season until 200 fresh remnant specimens were enrolled. Real-time RT-PCR assay results were used to compare and evaluate the performance of both RSV DIA assays. RESULTS: Among 200 specimens tested, RSV real-time RT-PCR assay detected RSV in 104 samples, while QD detected 84 samples and BD detected 74 samples as positive. The overall sensitivity for detection of RSV in comparison to PCR was 71.15% (61.3-79.4) for BD and 80.77% (71.6-87.6) for QD system (P=0.36). The specificity was 100% (95.2-100) for both systems. The work flow analysis revealed that the overall specimen processing time was significantly lower for BD as compared with the QD assay. CONCLUSIONS: In comparison with the real-time PCR, the QD system showed a higher sensitivity than that of the BD system, but the difference did not reach statistical significance (P=0.36). Both BD and QD systems were found comparable in terms of specificity.

Impact of treatment strategies on cephalosporin and tetracycline resistance gene quantities in the bovine fecal metagenome.

The study objective was to determine the effects of two treatment regimens on quantities of ceftiofur and tetracycline resistance genes in feedlot cattle. The two regimens were ceftiofur crystalline-free acid (CCFA) administered to either one or all steers within a pen and subsequent feeding/not feeding of therapeutic doses of chlortetracycline. A 26-day randomized controlled field trial was conducted on 176 steers. Real-time PCR was used to quantify bla(CMY-2), bla(CTX-M), tet(A), tet(B), and 16S rRNA gene copies/gram of feces from community DNA. A significant increase in ceftiofur resistance and a decrease in tetracycline resistance elements were observed among the treatment groups in which all steers received CCFA treatment, expressed as gene copies/gram of feces. Subsequent chlortetracycline administration led to rapid expansion of both ceftiofur and tetracycline resistance gene copies/gram of feces. Our data suggest that chlortetracycline is contraindicated when attempting to avoid expansion of resistance to critically important third-generation cephalosporins.

Effects of ceftiofur and chlortetracycline treatment strategies on antimicrobial susceptibility and on tet(A), tet(B), and bla CMY-2 resistance genes among E. coli isolated from the feces of feedlot cattle.

A randomized controlled field trial was conducted to evaluate the effects of two sets of treatment strategies on ceftiofur and tetracycline resistance in feedlot cattle. The strategies consisted of ceftiofur crystalline-free acid (CCFA) administered to either one or all of the steers within a pen, followed by feeding or not feeding a therapeutic dose of chlortetracycline (CTC). Eighty-eight steers were randomly allocated to eight pens of 11 steers each. Both treatment regimens were randomly assigned to the pens in a two-way full factorial design. Non-type-specific (NTS) E. coli (n = 1,050) were isolated from fecal samples gathered on Days 0, 4, 12, and 26. Antimicrobial susceptibility profiles were determined using a microbroth dilution technique. PCR was used to detect tet(A), tet(B), and bla CMY-2 genes within each isolate. Chlortetracycline administration greatly exacerbated the already increased levels of both phenotypic and genotypic ceftiofur resistance conferred by prior CCFA treatment (P<0.05). The four treatment regimens also influenced the phenotypic multidrug resistance count of NTS E. coli populations. Chlortetracycline treatment alone was associated with an increased probability of selecting isolates that harbored tet(B) versus tet(A) (P<0.05); meanwhile, there was an inverse association between finding tet(A) versus tet(B) genes for any given regimen (P<0.05). The presence of a tet(A) gene was associated with an isolate exhibiting reduced phenotypic susceptibility to a higher median number of antimicrobials (n = 289, median = 6; 95% CI = 4-8) compared with the tet(B) gene (n = 208, median = 3; 95% CI = 3-4). Results indicate that CTC can exacerbate ceftiofur resistance following CCFA therapy and therefore should be avoided, especially when considering their use in sequence. Further studies are required to establish the animal-level effects of co-housing antimicrobial-treated and non-treated animals together.