Genomic analysis of azithromycin-resistant Salmonella from food animals at slaughter and processing, and retail meats, 2011-2021, United States.

IMPORTANCE: Macrolides of different ring sizes are critically important antimicrobials for human medicine and veterinary medicine, though the widely used 15-membered ring azithromycin in humans is not approved for use in veterinary medicine. We document here the emergence of azithromycin-resistant Salmonella among the NARMS culture collections between 2011 and 2021 in food animals and retail meats, some with co-resistance to ceftriaxone or decreased susceptibility to ciprofloxacin. We also provide insights into the underlying genetic mechanisms and genomic contexts, including the first report of a novel combination of azithromycin resistance determinants and the characterization of multidrug-resistant plasmids. Further, we highlight the emergence of a multidrug-resistant Salmonella Newport clone in food animals (mainly cattle) with both azithromycin resistance and decreased susceptibility to ciprofloxacin. These findings contribute to a better understating of azithromycin resistance mechanisms in Salmonella and warrant further investigations on the drivers behind the emergence of resistant clones.

Use of Whole Genome Sequencing by the Federal Interagency Collaboration for Genomics for Food and Feed Safety in the United States.

ABSTRACT: This multiagency report developed by the Interagency Collaboration for Genomics for Food and Feed Safety provides an overview of the use of and transition to whole genome sequencing (WGS) technology for detection and characterization of pathogens transmitted commonly by food and for identification of their sources. We describe foodborne pathogen analysis, investigation, and harmonization efforts among the following federal agencies: National Institutes of Health; Department of Health and Human Services, Centers for Disease Control and Prevention (CDC) and U.S. Food and Drug Administration (FDA); and the U.S. Department of Agriculture, Food Safety and Inspection Service, Agricultural Research Service, and Animal and Plant Health Inspection Service. We describe single nucleotide polymorphism, core-genome, and whole genome multilocus sequence typing data analysis methods as used in the PulseNet (CDC) and GenomeTrakr (FDA) networks, underscoring the complementary nature of the results for linking genetically related foodborne pathogens during outbreak investigations while allowing flexibility to meet the specific needs of Interagency Collaboration partners. We highlight how we apply WGS to pathogen characterization (virulence and antimicrobial resistance profiles) and source attribution efforts and increase transparency by making the sequences and other data publicly available through the National Center for Biotechnology Information. We also highlight the impact of current trends in the use of culture-independent diagnostic tests for human diagnostic testing on analytical approaches related to food safety and what is next for the use of WGS in the area of food safety.

National Antimicrobial Resistance Monitoring System: Two Decades of Advancing Public Health Through Integrated Surveillance of Antimicrobial Resistance.

Drug-resistant bacterial infections pose a serious and growing public health threat globally. In this review, we describe the role of the National Antimicrobial Resistance Monitoring System (NARMS) in providing data that help address the resistance problem and show how such a program can have broad positive impacts on public health. NARMS was formed two decades ago to help assess the consequences to human health arising from the use of antimicrobial drugs in food animal production in the United States. A collaboration among the Centers for Disease Control and Prevention, the U.S. Food and Drug Administration, the United States Department of Agriculture, and state and local health departments, NARMS uses an integrated "One Health" approach to monitor antimicrobial resistance in enteric bacteria from humans, retail meat, and food animals. NARMS has adapted to changing needs and threats by expanding surveillance catchment areas, examining new isolate sources, adding bacteria, adjusting sampling schemes, and modifying antimicrobial agents tested. NARMS data are not only essential for ensuring that antimicrobial drugs approved for food animals are used in ways that are safe for human health but they also help address broader food safety priorities. NARMS surveillance, applied research studies, and outbreak isolate testing provide data on the emergence of drug-resistant enteric bacteria; genetic mechanisms underlying resistance; movement of bacterial populations among humans, food, and food animals; and sources and outcomes of resistant and susceptible infections. These data can be used to guide and evaluate the impact of science-based policies, regulatory actions, antimicrobial stewardship initiatives, and other public health efforts aimed at preserving drug effectiveness, improving patient outcomes, and preventing infections. Many improvements have been made to NARMS over time and the program will continue to adapt to address emerging resistance threats, changes in clinical diagnostic practices, and new technologies, such as whole genome sequencing.

Characterization of Resistance Genes and Plasmids from Outbreaks and Illness Clusters Caused by Salmonella Resistant to Ceftriaxone in the United States, 2011-2012.

Salmonella is an important cause of foodborne illness; however, quickly identifying the source of these infections can be difficult, and source identification is a crucial step in preventing additional illnesses. Although most infections are self-limited, invasive salmonellosis may require antimicrobial treatment. Ceftriaxone, an extended-spectrum cephalosporin, is commonly used for treatment of salmonellosis. Previous studies have identified a correlation between the food animal/retail meat source of ceftriaxone-resistant Salmonella and the type of resistance gene and plasmid it carries. In this study, we examined seven outbreaks of ceftriaxone-resistant Salmonella infections, caused by serotypes Typhimurium, Newport, Heidelberg, and Infantis. All isolates were positive for a plasmid-encoded blaCMY gene. Plasmid incompatibility typing identified five IncI1 and two IncA/C plasmids. Both outbreaks containing blaCMY-IncA/C plasmids were linked to consumption of cattle products. Three of five outbreaks with blaCMY-IncI1 (ST12) plasmids were linked to a poultry source. The remaining IncI1 outbreaks were associated with ground beef (ST20) and tomatoes (ST12). In addition, we examined isolates from five unsolved clusters of ceftriaxone-resistant Salmonella infections and used our plasmid-encoded gene findings to predict the source. Overall, we identified a likely association between the source of ceftriaxone-resistant Salmonella outbreaks and the type of resistance gene/plasmid it carries.

Ceftriaxone-Resistant Nontyphoidal Salmonella from Humans, Retail Meats, and Food Animals in the United States, 1996-2013.

BACKGROUND: Ceftriaxone resistance in Salmonella is a serious public health threat. Ceftriaxone is commonly used to treat severe Salmonella infections, especially in children. Identifying the sources and drivers of ceftriaxone resistance among nontyphoidal Salmonella is crucial. MATERIALS AND METHODS: The National Antimicrobial Resistance Monitoring System (NARMS) tracks antimicrobial resistance in foodborne and other enteric bacteria from humans, retail meats, and food animals. We examined NARMS data reported during 1996-2013 to characterize ceftriaxone-resistant Salmonella infections in humans. We used Spearman rank correlation to examine the relationships between the annual percentage of ceftriaxone resistance among Salmonella isolates from humans with isolates from retail meats and food animals. RESULTS: A total of 978 (2.9%) of 34,100 nontyphoidal Salmonella isolates from humans were resistant to ceftriaxone. Many (40%) ceftriaxone-resistant isolates were from children younger than 18 years. Most ceftriaxone-resistant isolates were one of three serotypes: Newport (40%), Typhimurium (26%), or Heidelberg (12%). All were resistant to other antimicrobials, and resistance varied by serotype. We found statistically significant correlations in ceftriaxone resistance between human and ground beef Newport isolates (r = 0.83), between human and cattle Typhimurium isolates (r = 0.57), between human and chicken Heidelberg isolates (r = 0.65), and between human and turkey Heidelberg isolates (r = 0.67). CONCLUSIONS: Ceftriaxone resistance among Salmonella Newport, Typhimurium, and Heidelberg isolates from humans strongly correlates with ceftriaxone resistance in isolates from ground beef, cattle, and poultry, respectively. These findings support other lines of evidence that food animals are important reservoirs of ceftriaxone-resistant Salmonella that cause human illness in the United States.

Identification and characterization of multidrug-resistant Salmonella enterica serotype Albert isolates in the United States.

Salmonella enterica is one of the most common causes of bacterial foodborne illness in the United States. Although most Salmonella infections are self-limiting, antimicrobial treatment of invasive salmonellosis is critical. The primary antimicrobial treatment options include fluoroquinolones or extended-spectrum cephalosporins, and resistance to these antimicrobial drugs may complicate treatment. At present, S. enterica is composed of more than 2,600 unique serotypes, which vary greatly in geographic prevalence, ecological niche, and the ability to cause human disease, and it is important to understand and mitigate the source of human infection, particularly when antimicrobial resistance is found. In this study, we identified and characterized 19 S. enterica serotype Albert isolates collected from food animals, retail meat, and humans in the United States during 2005 to 2013. All five isolates from nonhuman sources were obtained from turkeys or ground turkey, and epidemiologic data suggest poultry consumption or live-poultry exposure as the probable source of infection. S. enterica serotype Albert also appears to be geographically localized to the midwestern United States. All 19 isolates displayed multidrug resistance, including decreased susceptibility to fluoroquinolones and resistance to extended-spectrum cephalosporins. Turkeys are a likely source of multidrug-resistant S. enterica serotype Albert, and circulation of resistance plasmids, as opposed to the expansion of a single resistant strain, is playing a role. More work is needed to understand why these resistance plasmids spread and how their presence and the serotype they reside in contribute to human disease.

Characterization of blaCMY plasmids and their possible role in source attribution of Salmonella enterica serotype Typhimurium infections.

Salmonella is an important cause of foodborne illness; however, identifying the source of these infections can be difficult. This is especially true for Salmonella serotype Typhimurium, which is found in diverse agricultural niches. Extended-spectrum cephalosporins (ESC) are one of the primary treatment choices for complicated Salmonella infections. In Salmonella, ESC resistance in the United States is mainly mediated by blaCMY genes carried on various plasmids. In this study, we examined whether the characterization of blaCMY plasmids, along with additional information, can help us identify potential sources of infection by Salmonella, and used serotype Typhimurium as a model. In the United States, monitoring of retail meat, food animals, and ill persons for antimicrobial-resistant Salmonella is conducted by the National Antimicrobial Resistance Monitoring System. In 2008, 70 isolates (70/581; 12.0%) (34 isolates from retail meat, 23 food animal, and 13 human) were resistant to ceftriaxone and amoxicillin/clavulanic acid. All were polymerase chain reaction (PCR)-positive for blaCMY and 59/70 (84.3%) of these genes were plasmid encoded. PCR-based replicon typing identified 42/59 (71.2%) IncI1-blaCMY plasmids and 17/59 (28.8%) IncA/C-blaCMY plasmids. Isolates from chickens or chicken products with blaCMY plasmids primarily had IncI1-blaCMY plasmids (37/40; 92.5%), while all isolates from cattle had IncA/C-blaCMY plasmids. Isolates from humans had either IncA/C- blaCMY (n=8/12; [66.7%]) or IncI1- blaCMY (n=4/12 [33.3%]) plasmids. All of the IncI1-blaCMY plasmids were ST12 or were closely related to ST12. Antimicrobial susceptibility patterns (AST) and pulsed-field gel electrophoresis (PFGE) patterns of the isolates were also compared and differences were identified between isolate sources. When the source of a Typhimurium outbreak or sporadic illness is unknown, characterizing the outbreak isolate's blaCMY plasmids, AST, and PFGE patterns may help identify it.

Fluoroquinolone susceptibility testing of Salmonella enterica: detection of acquired resistance and selection of zone diameter breakpoints for levofloxacin and ofloxacin.

Fluoroquinolones (e.g., ciprofloxacin) have become a mainstay for treating severe Salmonella infections in adults. Fluoroquinolone resistance in Salmonella is mostly due to mutations in the topoisomerase genes, but plasmid-mediated quinolone resistance (PMQR) mechanisms have also been described. In 2012, the Clinical and Laboratory Standards Institute (CLSI) revised the ciprofloxacin interpretive criteria (breakpoints) for disk diffusion and MIC test methods for Salmonella. In 2013, the CLSI published MIC breakpoints for Salmonella to levofloxacin and ofloxacin, but breakpoints for assigning disk diffusion results to susceptible (S), intermediate (I), and resistant (R) categories are still needed. In this study, the MICs and inhibition zone diameters for nalidixic acid, ciprofloxacin, levofloxacin, and ofloxacin were determined for 100 clinical isolates of nontyphi Salmonella with or without resistance mechanisms. We confirmed that the new levofloxacin MIC breakpoints resulted in the highest category agreement (94%) when plotted against the ciprofloxacin MICs and that the new ofloxacin MIC breakpoints resulted in 92% category agreement between ofloxacin and ciprofloxacin. By applying the new MIC breakpoints in the MIC zone scattergrams for levofloxacin and ofloxacin, the following disk diffusion breakpoints generated the least number of errors: ≥28 mm (S), 19 to 27 mm (I), and ≤18 mm (R) for levofloxacin and ≥25 mm (S), 16 to 24 mm (I), and ≤15 mm (R) for ofloxacin. Neither the levofloxacin nor the ofloxacin disk yielded good separation of isolates with and without resistance mechanisms. Further studies will be needed to develop a disk diffusion assay that efficiently detects all isolates with acquired resistance to fluoroquinolones.

Increase in resistance to ceftriaxone and nonsusceptibility to ciprofloxacin and decrease in multidrug resistance among Salmonella strains, United States, 1996-2009.

BACKGROUND: Salmonella is a major bacterial pathogen transmitted commonly through food. Increasing resistance to antimicrobial agents (e.g., ceftriaxone, ciprofloxacin) used to treat serious Salmonella infections threatens the utility of these agents. Infection with antimicrobial-resistant Salmonella has been associated with increased risk of severe infection, hospitalization, and death. We describe changes in antimicrobial resistance among nontyphoidal Salmonella in the United States from 1996 through 2009. METHODS: The Centers for Disease Control and Prevention's National Antimicrobial Resistance Monitoring System conducts surveillance of resistance among Salmonella isolated from humans. From 1996 through 2009, public health laboratories submitted isolates for antimicrobial susceptibility testing. We used interpretive criteria from the Clinical and Laboratory Standards Institute and defined isolates with ciprofloxacin resistance or intermediate susceptibility as nonsusceptible to ciprofloxacin. Using logistic regression, we modeled annual data to assess changes in antimicrobial resistance. RESULTS: From 1996 through 2009, the percentage of nontyphoidal Salmonella isolates resistant to ceftriaxone increased from 0.2% to 3.4% (odds ratio [OR]=20, 95% confidence interval [CI] 6.3-64), and the percentage with nonsusceptibility to ciprofloxacin increased from 0.4% to 2.4% (OR=8.3, 95% CI 3.3-21). The percentage of isolates that were multidrug resistant (resistant to ≥3 antimicrobial classes) decreased from 17% to 9.6% (OR=0.6, 95% CI 0.5-0.7), which was driven mainly by a decline among serotype Typhimurium. However, multidrug resistance increased from 5.9% in 1996 to a peak of 31% in 2001 among serotype Newport and increased from 12% in 1996 to 26% in 2009 (OR=2.6, 95% CI 1.1-6.2) among serotype Heidelberg. CONCLUSIONS: We describe an increase in resistance to ceftriaxone and nonsusceptibility to ciprofloxacin and an overall decline in multidrug resistance. Trends varied by serotype. Because of evidence that antimicrobial resistance among Salmonella is predominantly a consequence of antimicrobial use in food animals, efforts are needed to reduce unnecessary use, especially of critically important agents.

Occurrence of ß-lactamase genes among non-Typhi Salmonella enterica isolated from humans, food animals, and retail meats in the United States and Canada.

Non-Typhi Salmonella cause over 1.7 million cases of gastroenteritis in North America each year, and food-animal products are commonly implicated in human infections. For invasive infections, antimicrobial therapy is indicated. In North America, the antimicrobial susceptibility of Salmonella is monitored by the U.S. National Antimicrobial Resistance Monitoring System (NARMS) and The Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS). In this study, we determined the susceptibility to cephalosporins by broth microdilution among 5,041 non-Typhi Salmonella enterica isolated from food animals, retail meats, and humans. In the United States, 109 (4.6%) of isolates collected from humans, 77 (15.7%) from retail meat, and 140 (10.6%) from food animals displayed decreased susceptibility to cephalosporins (DSC). Among the Canadian retail meat and food animal isolates, 52 (13.0%) and 42 (9.4%) displayed DSC. All isolates displaying DSC were screened for ß-lactamase genes (bla(TEM), bla(SHV), bla(CMY), bla(CTX-M), and bla(OXA-1)) by polymerase chain reaction. At least one ß-lactamase gene was detected in 74/109 (67.9%) isolates collected from humans, and the bla(CMY) genes were most prevalent (69/109; 63.3%). Similarly, the bla(CMY) genes predominated among the ß-lactamase-producing isolates collected from retail meats and food animals. Three isolates from humans harbored a bla(CTX-M-15) gene. No animal or retail meat isolates harbored a bla(CTX-M) or bla(OXA-1) gene. A bla(TEM) gene was found in 5 human, 9 retail meat, and 17 animal isolates. Although serotype distributions varied among human, retail meat, and animal sources, overlap in bla(CMY)-positive serotypes across sample sources supports meat and food-animal sources as reservoirs for human infection.

Drug-resistance mechanisms in Vibrio cholerae O1 outbreak strain, Haiti, 2010.

To increase understanding of drug-resistant Vibrio cholerae, we studied selected molecular mechanisms of antimicrobial drug resistance in the 2010 Haiti V. cholerae outbreak strain. Most resistance resulted from acquired genes located on an integrating conjugative element showing high homology to an integrating conjugative element identified in a V. cholerae isolate from India.

Characterization of bla(CMY)-encoding plasmids among Salmonella isolated in the United States in 2007.

Salmonella enterica is one of the most common bacterial causes of foodborne illness, and nontyphoidal Salmonella is estimated to cause ∼1.2 million illnesses in the United States each year. Plasmids are mobile genetic elements that play a critical role in the dissemination of antimicrobial resistance determinants. AmpC-type CMY ß-lactamases (bla(CMY)) confer resistance to extended-spectrum cephalosporins and ß-lactam/ß-lactamase inhibitor combinations and are commonly plasmid-encoded. A variety of plasmids have been shown to encode CMY ß-lactamases and certain plasmids may be associated with particular Salmonella serotypes or environmental sources. In this study, we characterized bla(CMY) ß-lactamase-encoding plasmids among Salmonella isolates. Isolates of Salmonella from specimens collected from humans in 2007 were submitted to the Centers for Disease Control and Prevention National Antimicrobial Resistance Monitoring System laboratory for susceptibility testing. Three percent (65/2161) of Salmonella isolates displayed resistance to ceftriaxone (minimum inhibitory concentration [MIC] ≥4 mg/L) and amoxicillin/clavulanic acid (MIC ≥32 mg/L), a combination associated with the presence of a bla(CMY) mechanism of resistance. Sixty-four (98.5%) isolates were polymerase chain reaction-positive for bla(CMY) genes. Transformation and conjugation studies showed that 95% (61/64) of the bla(CMY) genes were plasmid-encoded. Most of the bla(CMY)-positive isolates were serotype Typhimurium, Newport, Heidelberg, and Agona. Forty-three plasmids were replicon type IncA/C, 15 IncI1, 2 contained multiple replicon loci, and 1 was untypeable. IncI1 plasmids conferred only the bla(CMY)-associated resistance phenotype, whereas IncA/C plasmids conferred additional multi-drug resistance (MDR) phenotypes to drugs such as chloramphenicol, sulfisoxazole, and tetracycline. Most of the IncI1 plasmids (12/15) were sequence type 12 by plasmid multi-locus sequence typing. CMY ß-lactamase-encoding plasmids among human isolates of Salmonella in the United States tended to be large MDR IncA/C plasmids or single resistance determinant IncI1 plasmids. In general, IncI1 plasmids were identified among serotypes commonly associated with poultry, whereas IncA/C plasmids were more likely to be identified among cattle/beef-associated serotypes.

International spread of an epidemic population of Salmonella enterica serotype Kentucky ST198 resistant to ciprofloxacin.

National Salmonella surveillance systems from France, England and Wales, Denmark, and the United States identified the recent emergence of multidrug-resistant isolates of Salmonella enterica serotype Kentucky displaying high-level resistance to ciprofloxacin. A total of 489 human cases were identified during the period from 2002 (3 cases) to 2008 (174 cases). These isolates belonged to a single clone defined by the multilocus sequence type ST198, the XbaI-pulsed-field gel electrophoresis cluster X1, and the presence of the Salmonella genomic island 1 variant SGI1-K. This clone was probably selected in 3 steps in Egypt during the 1990s and the early 2000s and has now spread to several countries in Africa and, more recently, in the Middle East. Poultry has been identified as a potential major vehicle for infection by this clone. Continued surveillance and appropriate control measures should be implemented by national and international authorities to limit the spread of this strain.

Ciprofloxacin-resistant Salmonella enterica Serotype Typhi, United States, 1999-2008.

We report 9 ciprofloxacin-resistant Salmonella enterica serotype Typhi isolates submitted to the US National Antimicrobial Resistance Monitoring System during 1999-2008. The first 2 had indistinguishable pulsed-field gel electrophoresis patterns and identical gyrA and parC mutations. Eight of the 9 patients had traveled to India within 30 days before illness onset.

Antimicrobial susceptibility to azithromycin among Salmonella enterica isolates from the United States.

Due to emerging resistance to traditional antimicrobial agents, such as ampicillin, trimethoprim-sulfamethoxazole, and chloramphenicol, azithromycin is increasingly used for the treatment of invasive Salmonella infections. In the present study, 696 isolates of non-Typhi Salmonella collected from humans, food animals, and retail meats in the United States were investigated for antimicrobial susceptibility to azithromycin. Seventy-two Salmonella enterica serotype Typhi isolates from humans were also tested. For each isolate, MICs of azithromycin and 15 other antimicrobial agents were determined by broth microdilution. Among the non-Typhi Salmonella isolates, azithromycin MICs among human isolates ranged from 1 to 32 µg/ml, whereas the MICs among the animal and retail meat isolates ranged from 2 to 16 µg/ml and 4 to 16 µg/ml, respectively. Among Salmonella serotype Typhi isolates, the azithromycin MICs ranged from 4 to 16 µg/ml. The highest MIC observed in the present study was 32 µg/ml, and it was detected in three human isolates belonging to serotypes Kentucky, Montevideo, and Paratyphi A. Based on our findings, we propose an epidemiological cutoff value (ECOFF) for wild-type Salmonella of ≤16 µg/ml of azithromycin. The susceptibility data provided could be used in combination with clinical outcome data to determine tentative clinical breakpoints for azithromycin and Salmonella enterica.

Decreased susceptibility to ciprofloxacin among Shigella isolates in the United States, 2006 to 2009.

We characterized 20 Shigella isolates with decreased susceptibility to fluoroquinolones. Most patients (80%) from whom a travel history was obtained reported travel to South or Southeast Asia. Mutations within the quinolone resistance determining regions of gyrA and parC and plasmid-mediated resistance determinants (qnrB, qnrS, and aac(6')-Ib-cr) were identified. The rise in antimicrobial resistance among Shigella isolates may necessitate the increased use of extended-spectrum cephalosporins or macrolides in some patients.

Antimicrobial resistance among invasive nontyphoidal Salmonella enterica isolates in the United States: National Antimicrobial Resistance Monitoring System, 1996 to 2007.

Nontyphoidal salmonellae (NTS) are important causes of community-acquired bloodstream infection. We describe patterns of antimicrobial resistance among invasive NTS in the United States. We compared bloodstream NTS isolates with those from stool submitted to the National Antimicrobial Resistance Monitoring System (NARMS) from 1996 to 2007. We describe antimicrobial resistance among invasive strains by serogroup and serotype. Of the 19,302 NTS isolates, 17,804 (92.2%) were from stool or blood. Of these, 1,050 (5.9%) were bloodstream isolates. The median ages (ranges) of patients with and without bacteremia were 36 (<1 to 97) years and 20 (<1 to 105) years, respectively (P < 0.001). Males (odds ratio [OR], 1.21; 95% confidence interval [CI], 1.06 to 1.38) and those ≥65 years of age were at greater risk for invasive disease. Salmonella enterica serotypes Enteritidis, Typhimurium, and Heidelberg were the most common serotypes isolated from blood; S. enterica serotypes Dublin, Sandiego, and Schwarzengrund were associated with the greatest risk for bloodstream isolation. Of invasive isolates, 208 (19.8%) were resistant to ampicillin, 117 (11.1%) to chloramphenicol, and 26 (2.5%) to trimethoprim-sulfamethoxazole; 28 (2.7%) isolates were resistant to nalidixic acid and 26 (2.5%) to ceftriaxone. Antimicrobial resistance to traditional agents is common. However, the occurrence of nalidixic acid and ceftriaxone resistance among invasive NTS is cause for clinical and public health vigilance.