We identified an erm42-carrying integrative and conjugative element, ICE_erm42, in 26.4% of multidrug-resistant Salmonella enterica serovar Albany isolates recovered from cases of human salmonellosis between 2014 and 2019 in Taiwan. ICE_erm42-carrying strains displayed high-level resistance to azithromycin, and the element could move into the phylogenetically distant species Vibrio cholerae via conjugation.
KEYWORDS: Salmonella enterica, antimicrobial drug resistance, multidrug resistance, mobile genetic element, integrative and conjugative element, azithromycin, whole-genome sequencing
ABSTRACT
We identified an erm42-carrying integrative and conjugative element, ICE_erm42, in 26.4% of multidrug-resistant Salmonella enterica serovar Albany isolates recovered from cases of human salmonellosis between 2014 and 2019 in Taiwan. ICE_erm42-carrying strains displayed high-level resistance to azithromycin, and the element could move into the phylogenetically distant species Vibrio cholerae via conjugation.
INTRODUCTION
Salmonella enterica serovar Albany is one of the most frequently isolated serovars among isolates from poultry, slaughterhouses, wet markets, and the environment in several Asian countries, including Taiwan (1–6). S. Albany is relatively rare in the United States, accounting for only 0.06% (297/477,861) of the isolates recovered from human salmonellosis from 2006 to 2016 (7). This serovar was more prevalent in Taiwan; it was the 5th most frequently identified serovar among the Salmonella isolates recovered from humans and was also found in isolates from pigs between 2004 and 2012 (8).
Antimicrobial resistance, in particular multidrug resistance, in nontyphoidal salmonellae has been an increasing major health care problem worldwide (9). In Taiwan, a high prevalence of multidrug resistance is present in several Salmonella serovars causing human salmonellosis, including S. Albany (10). Resistance determinants are often carried by mobile genetic elements (MGEs), including transposable elements, plasmids, integrons, and integrative and conjugative elements (ICEs), which play a central role in facilitating horizontal genetic exchange and promoting the acquisition and spread of resistance genes (11). S. Albany strains have been found to harbor a Salmonella genomic island, SGI1-F, which carries 5 resistance genes, dfrA1, floR, tetG, blaPSE-1 (blaCARB-2), and sul1, that together confer resistance to trimethoprim, chloramphenicol/florfenicol, tetracyclines, ampicillin, and sulfonamides (12). As indicated in previous studies (8, 10), the resistance patterns for the S. Albany isolates from humans and pigs suggest that all the S. Albany isolates may carry SGI1-F or its truncated variant (12, 13). Azithromycin is recommended as an alternative antimicrobial for the chemotherapy of multidrug-resistant typhoidal and nontyphoidal Salmonella infections (14, 15). Accordingly, monitoring azithromycin resistance in Salmonella isolates is essential to inform the treatment of invasive salmonellosis caused by multidrug-resistant strains. Since 2017, the Salmonella Reference Laboratory of the Taiwan Centers for Disease Control started to perform azithromycin susceptibility testing for Salmonella isolates and found that 3.1% (76/2,449) of isolates recovered from cases of human salmonellosis between 2017 and 2018 were resistant, defined as an MIC of ≥32 mg/liter. Of the 76 azithromycin-resistant isolates, 10 were S. Albany isolates. Azithromycin is a semisynthetic macrolide antimicrobial; macrolide resistance in Enterobacteriaceae may result from modifications (mutation, methylation, or pseudouridylation) of the antimicrobial targets, modifications of the antimicrobial molecules, decreases in drug uptake via activated efflux pumps, and outer membrane alterations (16). To date, several azithromycin resistance determinants, including mphA (17–19), erm42 (20), ermB (21), and mutations in the AcrB efflux pump (22–25), have been found in azithromycin-resistant Salmonella. In the present study, we investigated the epidemiology and genetic determinants for the high-level azithromycin resistance in S. Albany isolates.
RESULTS AND DISCUSSION
Epidemiology of S. Albany infections.
S. Albany ranked as the seventh most frequently identified serovar and accounted for 2.8% (1,031/37,460) of the isolates recovered from cases of human salmonellosis from 2004 to 2019; however, the rate had declined from 4.1% for the first 8 years (2004 to 2011) to 1.6% for the last 8 years (2012 to 2019) (Table 1). Antimicrobial susceptibility testing was performed on randomly selected Salmonella isolates recovered from 2004 to 2018. More than 97% of the S. Albany isolates tested were resistant to trimethoprim, chloramphenicol/florfenicol, tetracyclines, ampicillin, and sulfamethoxazole (Table 2). These antimicrobial susceptibility testing data imply that the S. Albany strains circulating in Taiwan in this period may carry SGI1-F or its truncated variant (12). The data also showed that nearly 90% of the isolates were resistant to nalidixic acid and nonsusceptible to ciprofloxacin, and 10 (28.6%) of the 35 S. Albany isolates collected from 2017 to 2018 were azithromycin resistant.
TABLE 1.
Distribution of S. Albany and ICE_erm42, by year, among Salmonella isolates recovered from cases of human salmonellosis
| Yr | No. of isolates (%) |
||
|---|---|---|---|
| All serovars | S. Albany | S. Albany with ICE_erm42 | |
| 2004 | 2,535 | 86 (3.4) | 0 (0.0) |
| 2005 | 2,326 | 109 (4.7) | 0 (0.0) |
| 2006 | 2,071 | 99 (4.8) | 0 (0.0) |
| 2007 | 3,766 | 150 (4.0) | 0 (0.0) |
| 2008 | 2,284 | 88 (3.9) | 0 (0.0) |
| 2009 | 1,924 | 75 (3.9) | 0 (0.0) |
| 2010 | 1,621 | 70 (4.3) | 0 (0.0) |
| 2011 | 743 | 26 (3.5) | 0 (0.0) |
| 2012 | 863 | 17 (2.0) | 0 (0.0) |
| 2013 | 2,247 | 38 (1.7) | 0 (0.0) |
| 2014 | 1,821 | 42 (2.3) | 5 (11.9) |
| 2015 | 3,035 | 45 (1.5) | 16 (35.6) |
| 2016 | 3,756 | 64 (1.7) | 13 (20.3) |
| 2017 | 5,153 | 87 (1.7) | 28 (32.2) |
| 2018 | 1,708 | 20 (1.2) | 7 (35.0) |
| 2019 | 1,607 | 15 (0.9) | 3 (20.0) |
| Total | 37,460 | 1,031 (2.8) | 72 (26.4)a |
The denominator is the S. Albany isolates collected between 2014 and 2019.
TABLE 2.
Antimicrobial susceptibilities of S. Albany isolates recovered from cases of human salmonellosis from 2004 to 2018
| Antimicrobial | No. of isolates tested | % resistant isolates | % intermediate isolates |
|---|---|---|---|
| Azithromycina | 35 | 28.6 | 28.6 |
| Ampicillin | 832 | 97.5 | 0.1 |
| Cefotaxime | 832 | 2.5 | 0.5 |
| Cefoxitinb | 248 | 4.4 | 8.5 |
| Ceftazidime | 746 | 2.4 | 0.3 |
| Chloramphenicol | 832 | 97.1 | 0.8 |
| Ciprofloxacin | 832 | 3.6 | 86.3 |
| Colistinb | 252 | 2.8 | 0.0 |
| Ertapenemb | 248 | 0.0 | 0.0 |
| Gentamicin | 832 | 6.6 | 1.6 |
| Nalidixic acid | 832 | 89.8 | 0.0 |
| Streptomycin | 828 | 10.1 | 28.1 |
| Sulfamethoxazole/trimethoprim | 832 | 98.6 | 0.0 |
| Sulfamethoxazole | 832 | 99.2 | 0.0 |
| Tetracycline | 832 | 97.1 | 0.2 |
Only isolates from 2017 to 2018.
Only isolates from 2009 to 2018.
Clustering of PFGE patterns.
To investigate the clonality of the azithromycin-resistant isolates, we performed a clustering analysis of the pulsed-field gel electrophoresis (PFGE) patterns for 1,282 S. Albany isolates in our Salmonella database recovered from humans (1,031 isolates) from 2004 to 2019 and food animals (28 isolates from pigs, 141 from chickens, 66 from turkeys, 12 from geese, and 4 from ducks) recovered from 2011 to 2017. The clustering analysis revealed that the 10 azithromycin-resistant isolates and the other 67 isolates (62 from humans, 3 from chickens, and 2 from pigs) were closely located in a distinct cluster, cluster A (see Fig. S1 in the supplemental material). We performed PCR testing on the isolates of cluster A using a method described previously by Rose et al. (26) and confirmed that all the isolates in the cluster carried an erm42 gene, which encodes a monomethyltransferase and confers type I MLSB (macrolide, lincosamide, and streptogramin B) resistance (27). The results indicate that the erm42-carrying strains are most likely derived from clonal expansion.
Genetic characterization of S. Albany isolates.
We conducted whole-genome sequencing (WGS) for 16 erm42-carrying isolates from cluster A and 6 azithromycin-susceptible isolates from clusters B, D, and E (Fig. S1) using the Illumina MiSeq platform and closed the complete genome sequences for 6 isolates, R17.5974, R16.0431, R16.0556, R15.2267, R17.2117, and R17.4301, using the Illumina and Oxford Nanopore sequencing platforms. R17.5974 had a chromosome of 5,024,703 bp (GenBank accession number CP060730.1) but had no plasmid. This isolate harbored 4 mobile genetic elements (MGEs), including SGI1-F (42,654 bp), the integrative and conjugative element ICE_erm42 (94,093 bp), ICE_shufflon (125,205 bp), and a novel prophage, TW2 (32,231 bp). SGI1-F carried 5 intact resistance genes, dfrA1, floR, tetG, blaCARB-2, and sul1 (12). ICE_erm42 belongs to the SXT/R391 ICE family (28) and was located in prfC. It carried 3 resistance genes (erm42, floR, and sul2) and shared 90.0% coverage and 98.0% sequence identity with a transposon, ICEValA056-1, found in Vibrio alginolyticus strain A056 (GenBank accession number KR231688.1) (Fig. 1). ICE_shufflon carried a shufflon operon, which determines the recipient specificity in bacterial conjugation (29). ICE_shufflon shared 74.0% coverage and 98.7% sequence identity with ICESb2 (101,865 bp), found in a Salmonella bongori strain (GenBank accession number FN669609.1) (Fig. S2). Bacteriophage TW2 shared 91.0% coverage and 99.7% sequence identity with a prophage in S. enterica serovar Haardt strain SEHaa3795 (GenBank accession number AP020330.1) (Fig. S3). The 4 MGEs were also found in the other 15 erm42-carrying isolates, including R16.0431 (recovered from pig) and R16.0556 (from chicken). R17.5974 shared nearly 100% sequence identity with isolates R16.0431 (GenBank accession number CP062004.1) and R16.0556 (accession number CP061929.1) (Fig. S4). R15.2267 (GenBank accession number CP065564.1) harbored 3 MGEs, including ICE_shufflon, SGI1-F, and prophage TW2, but no ICE_erm42. R17.4301 (GenBank accession number CP062795.1) carried SGI1-F and a variant of prophage TW2 but no ICE_erm42 or ICE_shufflon. R17.2117 (GenBank accession number CP063330.1) harbored only SGI1-F but not the other 3 MGEs. From our genetic analysis, we considered that all erm42-carrying isolates in cluster A (Fig. S1) harbored ICE_erm42. The demographic data for the isolates of cluster A indicated that the ICE_erm42-carrying strains first emerged in 2014 and accounted for 26.4% (72/273) of the S. Albany isolates recovered from cases of human salmonellosis between 2014 and 2019 (Table 1).
FIG 1.
Genetic maps for ICE_erm42 from Salmonella Albany strain R17.5974 (GenBank accession number CP060730.1) and ICEValA056-1 from Vibrio alginolyticus strain A056 (accession number KR231688.1), drawn using Easyfig software (https://mjsull.github.io/Easyfig/). ORF, open reading frame.
Mobility of ICE_erm42.
We conducted conjugation experiments to investigate the mobility of ICE_erm42 from 2 S. Albany strains, R17.5974 and R16.0431, with rifampin-resistant recipients, S. enterica serovar Goldcoast R14.0180_RIFr, Escherichia coli C600_RIFr, and Vibrio cholerae R18.2633_RIFr. We obtained transfer rates of 2.8 × 10−6 and 4.6 × 10−6 transconjugants/donor for the S. Goldcoast strain, 8.4 × 10−6 and 2.1 × 10−5 for the E. coli strain, and 1.8 × 10−6 and 3.7 × 10−6 for the V. cholerae strain, respectively (Table 3).
TABLE 3.
Frequency of conjugational transfer of resistance from azithromycin-resistant Salmonella Albany to azithromycin-susceptible S. Goldcoast, Escherichia coli, and Vibrio cholerae
| S. Albany donor strain | Recipient strain | Transfer rate (no. of transconjugants/donor) |
|---|---|---|
| R17.5974 | S. Goldcoast R14.0180_RIFr | 2.8 × 10−6 |
| R16.0431 | 4.6 × 10−6 | |
| R17.5974 | E. coli C600_RIFr | 8.4 × 10−6 |
| R16.0431 | 2.1 × 10−5 | |
| R17.5974 | V. cholerae R18.2633_RIFr | 1.8 × 10−6 |
| R16.0431 | 3.7 × 10−6 | |
Genetic relatedness among S. Albany isolates.
We investigated the genetic relatedness among 22 S. Albany isolates from Taiwan and 212 S. Albany isolates from the NCBI database by comparing the core-gene multilocus sequence typing (cgMLST) profiles, which were generated using the cgMLST scheme (based on 3,265 core genes) and the profiling tool provided on the cgMLST@Taiwan website (http://rdvd.cdc.gov.tw/cgMLST/). The cgMLST tree revealed that most S. Albany isolates from at least 11 countries were highly clonal; only 7 of the isolates had a distance of >200 loci from the predominant clone (Fig. S5). We characterized the genetic features of the 66 most closely related isolates from Taiwan and 5 other countries (China, Singapore, the United Kingdom, the United States, and Vietnam). Among the 66 isolates, all harbored SGI1-F or a truncated SGI-F variant; 29 harbored a prophage, TW2; 9 harbored a variant of prophage TW2; 23 harbored ICE_shufflon; and 16 harbored ICE_erm42 (Fig. 2). All ICE_shufflon-carrying and ICE_erm42-carrying isolates originated from Taiwan, although three were not recovered in Taiwan. All isolates except one had a mutation of either D87N or S83F in GyrA. These mutations could explain the resistance to nalidixic acid and reduced susceptibility to ciprofloxacin in a large proportion of S. Albany isolates collected in Taiwan (Table 2). We constructed a cgMLST tree using the neighbor-joining algorithm to investigate the phylogenetic relationships among the 66 isolates. The neighbor-joining tree indicated that the ICE_erm42-carrying isolates (group A) were very closely related to the ICE_shufflon-carrying (but not the ICE_erm42-carrying) isolates (group B) but less closely related to the prophage TW2-carrying (but not the ICE_erm42- or ICE_shufflon-carrying) isolates (group C) (Fig. 3). The isolates carrying prophage TW2 variants (group D) were situated in 3 separate lineages, each of which had a different prophage TW2 variant. The genetic relationships among the isolates indicated that the ICE_erm42-carrying isolates evolved from a group B strain (or strains) by the acquisition of ICE_erm42 and that the group B strains evolved from a group C strain (or strains).
FIG 2.
cgMLST tree showing the genetic relatedness among the 66 most closely related isolates from Taiwan and 5 other countries, accompanied by the relevant genetic traits for the isolates. The tree was constructed using the unweighted pair group method with arithmetic mean algorithm and the tool provided in BioNumerics software version 7.6 (Applied Maths, Belgium). The isolates that originated from Taiwan but were recovered in other countries are marked with asterisks. Isolates harboring 4 MGEs (SGI1-F, prophage TW2, ICE_shufflon, and ICE_erm42) are marked with red squares, those harboring 3 MGEs (SGI1-F, prophage TW2, and ICE_shufflon) are marked with orange squares, those harboring 2 MGEs (SGI1-F and prophage TW2) are marked with green squares, and those harboring SGI1-F and prophage TW2 variants are marked with blue squares. 1, <1, and 0 represent, respectively, a complete copy, a partial copy, and zero copies of SGI1-F, prophage TW2, ICE_shufflon, and ICE_erm42.
FIG 3.
cgMLST tree showing the phylogenetic relationships among the 66 most closely related isolates from Taiwan and the other 5 countries (China, Singapore, the United Kingdom, the United States, and Vietnam). The tree was constructed with cgMLST profiles using the neighbor-joining algorithm and the tool provided in BioNumerics software version 7.6 (Applied Maths, Belgium).
In conclusion, we found that azithromycin-resistant S. Albany strains carrying ICE_erm42 have emerged since 2014 and prevailed in infections of humans and food animals in Taiwan. ICE_erm42 is mobile; it can move from Salmonella hosts to the phylogenetically distant species V. cholerae and is expected to move easily among the species of the Enterobacteriaceae. ICE_erm42 integrated into the chromosome is typically immune to segregational loss so that the element can be stably maintained and ensured vertical transmission (28).
Azithromycin is recommended as a second-choice antimicrobial to treat invasive typhoidal and nontyphoidal Salmonella infections and is an effective drug in the treatment of extensively drug-resistant S. enterica serovar Typhi infection (30). Thus, the emergence of ICE-mediated azithromycin resistance in multidrug-resistant Salmonella is particularly alarming.
MATERIALS AND METHODS
Source of Salmonella isolates, serotyping, pulsed-field gel electrophoresis, and antimicrobial susceptibility testing.
We collected Salmonella isolates from hospitals and the Department of Veterinary Medicine, National Chiayi University, Taiwan; determined the serotypes of the isolates using the typing scheme developed previously by Chiou and colleagues (31); characterized the genotypes using the standardized PulseNet PFGE protocol (32); and conducted antimicrobial susceptibility testing using the broth microdilution method with custom-made 96-well Sensititre MIC panels (Trek Diagnostic Systems, Ltd., West Sussex, UK). The test procedure was performed according to the manufacturer’s instructions, and the interpretation of MIC results followed the guidelines of the Clinical and Laboratory Standards Institute (33) or European Committee on Antimicrobial Susceptibility Testing breakpoint tables, version 10.0, for colistin (34). In this study, the interpretive criteria for azithromycin and nalidixic acid resistance were MICs of ≥32 mg/liter. The genotypic and phenotypic data and the relevant metadata of the isolates were managed and analyzed using the tools provided by BioNumerics 7.6.3 (Applied Maths).
Whole-genome sequencing and sequence analysis.
We conducted whole-genome sequencing (WGS) of Salmonella isolates using the Illumina MiSeq sequencing platform with MiSeq reagent kit v3 (2 by 300 bp) and the Oxford Nanopore sequencing platform. We first performed sequencing for 16 isolates located in PFGE cluster A (see Fig. S1 in the supplemental material), of which 10 S. Albany isolates had antimicrobial susceptibility testing data and displayed high-level resistance to azithromycin (MIC ≥ 128 mg/liter), and 6 azithromycin-susceptible isolates from PFGE clusters B, D, and E using the Illumina sequencing method. Three isolates (R17.5974, R16.0431, and R16.0556) from cluster A and three isolates (R15.2267, R17.2117, and R17.4301) from each of clusters B, D, and E were selected to close the complete genomic sequences using Oxford Nanopore sequencing platforms to generate long-read sequences. We assembled Illumina sequence reads for 22 isolates using SPAdes assembler version 3.12.0 (http://cab.spbu.ru/software/spades/) and both Illumina sequence reads and Nanopore sequence reads for 6 isolates to complete the full genomic sequences using Unicycler Assembler (35) and identified antimicrobial resistance genes using AMRFinderPlus (https://www.ncbi.nlm.nih.gov/pathogens/antimicrobial-resistance/AMRFinder/). The genomic sequence of S. Albany strain ATCC 51960 (GenBank accession number CP019177.1) was used as a reference sequence for identifying large novel insertions in the 6 isolates with complete genomic sequences.
Construction of cgMLST trees for S. Albany isolates from Taiwan and the NCBI database.
We generated core-gene multilocus sequence typing (cgMLST) profiles (based on 3,265 core genes) using an in-house-developed cgMLST profiling tool, which is available on the cgMLST@Taiwan website (http://rdvd.cdc.gov.tw/cgMLST). We constructed cgMLST trees using the neighbor-joining algorithm, the minimum spanning tree algorithm, the unweighted pair group method with arithmetic mean algorithm, and the tools provided in BioNumerics software version 7.6 (Applied Maths, Belgium).
Conjugation.
We conducted conjugation experiments to determine the rate of transfer of ICE_erm42 from S. Albany strains R17.5974 and R16.0431 to azithromycin-susceptible and rifampin-resistant S. Goldcoast strain R14.0180_RIFr, E. coli strain C600_RIFr, and Vibrio cholerae strain R18.2633_RIFr (derived from strain R18.2633 [sequence type 75 {ST75}] [BioSample accession number SAMN11053875]) (36). For each conjugation experiment, we cocultured donor and recipient cells at a 1:1 ratio (∼107 cells each) in 5 ml LB broth and incubated the mixture at 37°C for 16 h with gentle shaking at 50 rpm/min. We screened the transconjugants by spreading serially diluted cells on LB agar plates amended with 100 mg/liter azithromycin and 100 mg/liter rifampin and donors on LB agar plates amended with 100 mg/liter of azithromycin.
Data availability.
The short reads for the genomic sequences of the 22 S. Albany isolates sequenced in this study were submitted to the SRA database of GenBank under accession numbers SRR12549519 to SRR12549528, SRR12560291, SRR8189353, SRR8189354, SRR8189364, SRR8189393, SRR8189405, SRR8189436, SRR8189504, SRR8189521, SRR8189534, SRR8189551, and SRR8189553. The complete genomic sequences for isolates R17.5974, R16.0431, R16.0556, R15.2267, R17.4301, and R17.2117 were deposited in GenBank under accession numbers CP060730.1, CP062004.1, CP061929.1, CP065564.1, CP062795.1, and CP063330.1, respectively.
Supplementary Material
ACKNOWLEDGMENT
This study was funded by the Ministry of Health and Welfare, Taiwan (grant number MOHW109-CDC-C-315-144406).
Footnotes
Supplemental material is available online only.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The short reads for the genomic sequences of the 22 S. Albany isolates sequenced in this study were submitted to the SRA database of GenBank under accession numbers SRR12549519 to SRR12549528, SRR12560291, SRR8189353, SRR8189354, SRR8189364, SRR8189393, SRR8189405, SRR8189436, SRR8189504, SRR8189521, SRR8189534, SRR8189551, and SRR8189553. The complete genomic sequences for isolates R17.5974, R16.0431, R16.0556, R15.2267, R17.4301, and R17.2117 were deposited in GenBank under accession numbers CP060730.1, CP062004.1, CP061929.1, CP065564.1, CP062795.1, and CP063330.1, respectively.