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

ABSTRACT Azithromycin, a 15-membered ring macrolide, is among the recommended antimicrobials for treating invasive salmonellosis in humans. It is not approved for use in veterinary medicine. We analyzed the U.S. National Antimicrobial Resistance Monitoring System (NARMS) culture collections (~40,700) between 2011 and 2021 from food animals at slaughter and processing and retail meats, and identified 31 azithromycin-resistant Salmonella spp. with the first occurrence in 2015. These isolates belonged to 12 Salmonella serovars and possessed one or more macrolide resistance determinants: erm(42), mef(C), mph(A), mph(E), mph(G), and msr(E) or a point mutation (acrB_R717L), of which mph(A) was dominant (61.3%). Compared with azithromycin-susceptible controls, these determinants accounted for up to 256-fold MIC increases against azithromycin with MIC50 and MIC90 increased by 32- and 8-fold, respectively. We report the first detection of an mph(G)-mef(C)-mph(E)-msr(E)-containing Salmonella Agona isolate with very high-level azithromycin resistance (1,024 µg/mL) and the first detection of acrB_R717L accounting for azithromycin resistance in nontyphoidal Salmonella serovars in the United States. Plasmids of diverse replicon types were identified, with 86.2% carrying multidrug resistance including azithromycin and ceftriaxone, or decreased susceptibility to ciprofloxacin. This report also highlights an emerging mph(A)-containing (on an IncR plasmid) Salmonella Newport clone of cattle/beef origin with high-level azithromycin resistance (128 µg/mL) and decreased susceptibility to ciprofloxacin (0.25 µg/mL). Further work is needed to better understand the drivers of emerging azithromycin resistance in nontyphoidal Salmonella associated with food animal sources. 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.

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.

MIC distributions by azithromycin resistance determinants
Table 2 shows MIC distributions of the 31 azithromycin-resistant Salmonella (test group) and 14 susceptible ones (control group), and in subgroups possessing different azithromycin resistance determinant profiles.For isolates in the test group, MICs ranged from 32 to 1,024 μg/mL with MIC 50 and MIC 90 both at 128 µg/mL.Control group isolates had much lower MICs with MIC 50 and MIC 90 at 4 and 16 µg/mL, respectively.This translates to up to 256-fold MIC increases between azithromycin-susceptible and resistant ones.
Three Salmonella controls harbored ere(A), mef(B), or mph(A) but were susceptible to azithromycin.Genomic analysis showed that the ere(A) gene in S. I 4, [5],12:i:-isolate 20MO07PC10-S1 (CVM N20S0154) was truncated (missing the first 62 amino acids), whereas the promoter region of mph(A) in S. Typhimurium isolate Food Safety and Inspection Service (FSIS)12032448 was also truncated by 24 bp.Both the mef(B) gene and its promoter in S. Johannesburg isolate 19MN02PC03 (CVM N19S0223) remained intact.Eleven other Salmonella controls were initially selected based on their phenotypic resistance to azithromycin or carrying potential macrolide resistance genes.AST and WGS performed at CVM showed they differed from the original designations.This was due to the loss of plasmids containing mph(A) or ere(A), or AST test variations for borderline isolates (azithromycin MICs around 16 µg/mL).For three isolates in the latter group, no mutations in the 23S rRNA gene were found.Alternative IDs are provided for some isolates because historically different IDs were used at NCBI or in the literature for designating these isolates and corresponding plasmids.

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c Three control isolates possessed nonfunctional azithromycin resistance genes. d For Food Safety and Inspection Service isolates, those without parentheses were from product sample testing at slaughter.Cecal and lymph node samples are not destined for food.
e Breakpoints used were ≥32 mg/mL for azithromycin (a 15-membered ring macrolide) and ≥4 mg/mL for ceftriaxone (a third-generation cephalosporin).Decreased susceptibility to ciprofloxacin at ≥0.12 mg/mL was used as a marker for emerging fluoroquinolone resistance.
f CVM, Center for Veterinary Medicine.

Azithromycin-resistant plasmids
All azithromycin resistance determinants were mapped to plasmids except for the acrB_R717L mutation in two isolates.The 29 closed plasmids belonged to various incompatibility groups (IncR, n = 11; IncC, n = 3; IncHI2/IncHI2A, n = 3; IncQ1, n = 3; others, n = 9), with 8 containing 2 or more replicon types (Table 3).The sizes ranged from 10,966 bp for an IncQ1 plasmid to 326,166 bp for an IncHI1B(pNDM-CIT)/ IncHI1A(NDM-CIT) plasmid.All were MDR (resistant to ≥3 drug classes) except for three IncQ1 plasmids carrying erm (42) only and one IncP6 plasmid harboring mph(E)-msr(E).All but six plasmids contained the biocide resistance gene qacEdelta1 (one to three copies), and one also carried qacG2.Twelve plasmids contained heavy metal resistance genes, with as many as 18 genes resistant to four heavy metals in one isolate.
Figure 1A shows the genomic contexts among 20 mph(A)-containing plasmids from food animal sources, 11 of which were S. Newport IncR plasmids.Not surprisingly, mobile genetic elements such as class 1 integrons and insertion sequence (IS) elements were frequently observed.Nine out of the 20 mph(A)-containing plasmids carried the class 1 integron integrase gene intI1, 8 of which had a class 1 integron proximal to mph(A)containing MDR, biocide, and heavy metal resistance genes, and various IS elements.A BLAST query of pFSIS12106020 (S.Newport, IncR) as a representative mph(A)-containing plasmid revealed multiple matches with Escherichia coli plasmids, including CP104124 and CP043753.Of the 4 plasmids that contained the complete mph(A) cluster [IS26mph(A)-mrx-mphR-IS6100], 1 was from S. Agona (pFSIS21821150) and 3 others were from S. Newport (pFSIS12106020, pFSIS31903059-1, and pFSIS32104969).
Similar genetic backgrounds were found among three erm(42)-containing plasmids from different Salmonella serovars recovered from food animals (Fig. 1C).All were small IncQ1 plasmids with the erm (42) gene upstream from an ISVa3 element.On pF18S049, this gene was immediately downstream of another IS element, ISVch6.The presence of these IS elements again suggests the mobility of this resistance gene.

DISCUSSION
Both World Health Organization and U.S. Food and Drug Administration (FDA) have classified macrolides as critically important antimicrobials for human medicine, along with third-generation cephalosporins and quinolones/fluoroquinolones (16,17).All three drug classes are also considered critically important for veterinary medicine, though the widely used 15-membered ring macrolide azithromycin in humans is not approved for use in veterinary medicine (18).We report here the detection of Salmonella sero vars in food animals and retail meats with co-resistance to ceftriaxone (a third-genera tion cephalosporin) or decreased susceptibility to ciprofloxacin (a fluoroquinolone) and provide insights into the underlying genetic mechanisms and genomic contexts on MDR plasmids.Further, the report highlights the emergence of an MDR S. Newport clone in food animals (mainly cattle, also sheep) in the United States with azithromycin resistance and decreased susceptibility to ciprofloxacin.
Though NARMS food animal and retail meat arms started routine azithromycin AST for Salmonella in 2011, occurrence of resistance was first detected in 2015 in an S. Derby isolate from ground turkey that had the acrB_R717L mutation (Table 1).During 2016, the S. I 4, [5],12:i:-clone with mph(E)-msr(E) was first detected, in addition to one erm(42)-con taining Salmonella Meleagridis and two mph(A)-containing Salmonella Schwarzengrund isolates.In 2018, we detected the highly resistant S. Agona isolate harboring mph(G)mef(C)-mph(E)-msr(E) and additional serovars (Agona, Mbandaka, Newport, and Ohio) containing mph(A).This study clearly documents the sequential detection of azithromy cin resistance in Salmonella, including the S. Newport clone of cattle/beef origin in 2018.This trend can also be observed in the real-time display of NARMS monitoring data accessible through the NARMS Now dashboard online (19).
As noted earlier, NARMS monitoring of azithromycin resistance in nontyphoidal Salmonella was based on the CLSI's breakpoint (≥32 µg/mL) for Salmonella Typhi.Our study (Table 2) and others (unpublished data) provided further evidence that Salmonella MICs to azithromycin do not differ among serovars.This breakpoint also agrees with the European Committee on Antimicrobial Susceptibility Testing's epidemiological cut-off value, which distinguishes Salmonella likely resistant to azithromycin from a wild-type susceptible population (20).
The predominance of mph(A) in our study isolates corroborates with numerous reports on azithromycin resistance mechanisms in Enterobacterales (9,(21)(22)(23).Four S. Newport isolates (FSIS21924576, FSIS12033266, FSIS12035959, and FSIS22106147) have lost the mph(A)-containing plasmids [searching up these isolates in NCBI pathogen detection isolates browser (15) still shows the presence of this gene as those reflect original testing results], suggesting plasmid instability/mobility.The finding of one mph(A)-harboring Salmonella Typhimurium isolate with a truncated promoter region is interesting, suggesting a new mechanism accounting for the lack of azithromycin resistance phenotype in Salmonella harboring mph(A).A recent in-depth characteriza tion of two Salmonella conjugative plasmids showed the complete genetic makeup of the mph(A) cluster [IS26-mph(A)-mrx-mphR-IS6100], and the deletion of mphR restored azithromycin susceptibility (24).Four study isolates (three S. Newport and one S. Agona) had the complete mph(A) cluster and adjacent class 1 integron, suggesting the transferability of this gene cluster.
The mph(E) gene was co-located with msr(E) among all study isolates, supporting previous reports that they are part of a common operon in Gram-negative bacteria of different origins, including livestock (9,25,26).These two genes are often found on plasmids flanked by IS26 sequences associated with Tn1548::armA transposon, which facilitates the spread (25,26).Agreeably, armA was found in five (out of eight) S. I 4, [5],12:i:-containing mph(E)-msr(E).Further, we detected one S.I 4, [5],12:i:-isolate from swine containing mph(E)-msr(E) with adjacent mph(A) on an IncFIB(K) plasmid and one S. Agona isolate harboring the mph(G)-mef(C)-mph(E)-msr(E) cluster on an IncC plasmid.The MIC to azithromycin in the latter isolate was eightfold higher than those harboring mph(A) alone.A recent study reported the first detection of Shiga toxin-producing E. coli in France carrying plasmid-borne mef(C)-mph(G) (27).Another report showed that mef(C)-mph(G) was carried on various vectors including plasmids and integrative and conjugative elements (ICEs) among marine and wastewater bacteria in Asian countries (28), and yet another study reported the occurrence of mef(C)-mph(G) in bacteria from the aquatic environment (29).The latter study also showed that mef(C) alone did not influence azithromycin susceptibility of E. coli, but mph(G) alone did, with more dramatic increases when both were introduced, suggesting a synergistic effect (29).To the best of our knowledge, ours is the first report on mph(G)-mef(C)-mph(E)-msr(E)-containing Salmonella with high-level azithromycin resistance.
The contribution of erm (42) to azithromycin resistance in Salmonella has been confirmed recently by direct cloning using an IncFIB/IncHI1B plasmid from Klebsiella pneumoniae (30).This gene has been detected earlier on a type 2 IncA/C2 plasmid from two Salmonella serovars (Ohio and Senftenberg) from swine in Australia (31).Two very recent studies in Taiwan reported an erm(42)-carrying ICE in 26.4% of MDR Salmonella Albany from human salmonellosis (32) as well as in Salmonella Enteritidis and S. Typhimurium IS26 composite transposons in the chromosomes (33).To our best knowledge, the genetic context of the three Salmonella serovars containing erm (42) reported in this study (on IncQ plasmids) is novel.Mutation in the RND-type transporter gene (acrB_R717L/Q), which has been reported mostly in S. Typhi and S. Paratyphi A, has been confirmed recently to confer azithromycin resistance in E. coli by cloning (34,35).The first Arg717 substitution was predicted to have emerged around 2010 with travel-related R717Q mutant found in the United Kingdom (36).To our best knowledge, this is the first report of acrB_R717L in nontyphoidal Salmonella in the United States.
Whether, and to what degree, the wide use of other 15-membered ring macrolides such as gamithromycin and tulathromycin, and 14-membered or 16-membered ring macrolides in veterinary medicine (specifically food animals) selects for resistance or confers cross-resistance to macrolides used in human medicine, such as azithromycin, remains largely unknown.As Salmonella is intrinsically resistant to 14-and 16-membered ring macrolides, further cloning studies are needed to better evaluate the contribution of azithromycin resistance determinants such as those identified in this study to crossresistance to those macrolides.
In our study, all azithromycin resistance genes were carried on plasmids, with 86.2% being MDR.This is not surprising owing to the significant role plasmids play in the evolution and acquisition of AMR genes in Salmonella (12).The finding of an S. I 4, [5],12:i:-isolate resistant to macrolides, third-generation cephalosporins, and with decreased susceptibility to fluoroquinolones, and an emerging MDR S. Newport clone of cattle origin (also sheep) resistant to macrolides with decreased susceptibility to fluoroquinolones is concerning.This clone has been involved in a 2018-2019 outbreak linked to beef in the United States and soft cheese obtained in Mexico (37).Notably, clinical isolates dominate this NCBI SNP cluster, whereas environmental isolates are mainly from water sources.We acknowledge that this SNP analysis was limited as it was solely based on publicly available data at NCBI.To contain the evolution and spread of azithromycin resistance in Salmonella, a One Health approach focused on monitoring and infection control, and a strong commitment to better understand the drivers of azithromycin resistance in Salmonella associated with food animal sources is warranted.

Agar dilution
Besides the broth dilution mentioned above, AST against an expanded range of azithromycin concentrations (0.125-1,024 µg/mL) for all 45 Salmonella isolates was performed by agar dilution following CLSI guidelines (38).Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, and Staphylococcus aureus ATCC 29213 were used as quality control organisms, with QC ranges from CLSI where available (3,39).MIC differences between azithromycin-resistant isolates (n = 31) and susceptible controls (n = 14) were compared, including fold changes, MIC 50 , and MIC 90 .

Short-read and long-read WGS
Short-read WGS was performed at NARMS partner labs and CVM following manufactur ers' instructions.The procedure used at CVM has been described previously (40).Briefly, genomic DNA was extracted using the QIAamp 96 DNA QIAcube HT kit in the automated QIAcube HT instrument (QIAGEN, Germantown, MD).Libraries were prepared with the Nextera XT DNA library preparation kit (Illumina, San Diego, CA) in the automated Sciclone G3 liquid handling workstation (PerkinElmer, Santa Clara, CA).Sequencing was performed on the MiSeq platform using the MiSeq reagent kit v3 chemistry with the 600-cycle option (Illumina).Raw reads were de novo assembled using the CLC genomics workbench 10 (QIAGEN).
Long-read sequencing was also performed at CVM as described previously (41) with slight modifications.Briefly, genomic DNA libraries with an average insert size of 10 kb or larger were prepared using the SMRTbell template prep kit 1.0 (PacBio, Menlo Park, CA).Sequencing was performed on the Sequel platform with the Sequel sequencing kit 3.0, and reads were assembled using the microbial assembly pipeline in SMRT link v11.0 (PacBio).
For data visualization, alignments of AMR gene-encoding regions were performed using Easyfig v2.2.2 (47).Circular structures of select azithromycin-resistant plasmids were drawn with BLAST ring image generator (48).The clustering of azithromycin-resist ant Salmonella isolates to clinical and environmental Salmonella isolates deposited at NCBI was observed using the NCBI pathogen detection isolates browser (15).

a
NF stands for not found, suggesting a new multilocus sequence type.b

1 a
None of the isolates had MICs from <0.125 to 2 mg/mL or >1,024 mg/mL.MIC 50 and MIC 90 values were not calculated for subgroups with different azithromycin resistance determinants due to low numbers of isolates.b These genes were either truncated or lacking the promoter region.

FIG 3
FIG3 The structure of a representative MDR IncR plasmid (pFSIS12106020) from the emerging Salmonella Newport clone of cattle/beef origin.

TABLE 1
Salmonella (n = 45)examined in this study from U.S. food animals at slaughter and processing and retail meats

Serovar Multilocus sequence type a Isolate b c Year State Source d MIC (μg/mL) e Azithromycin Ceftriaxone Ciprofloxacin
Test group (n = 31)Agona (n = 2)

TABLE 1
Salmonella (n = 45)examined in this study from U.S. food animals at slaughter and processing and retail meats (Continued)

TABLE 2
Azithromycin MIC distributions among 45 Salmonella isolates from U.S. food animals at slaughter and processing and retail meats Group Azithromycin resistance determinant Serovar (no.

TABLE 3
Azithromycin-resistant Salmonella plasmids (n = 29) from U.S. food animals at slaughter and processing and retail meats

TABLE 3
Azithromycin-resistant Salmonella plasmids (n = 29) from U.S. food animals at slaughter and processing and retail meats (Continued) a Biocide resistance genes are underlined, whereas heavy metal resistance genes are in bold.