Plasmid-mediated azithromycin resistance in non-typhoidal Salmonella recovered from human infections

Abstract

Objectives

Mechanisms of non-typhoidal Salmonella (NTS) resistance to azithromycin have rarely been reported. Here we investigate the epidemiology and genetic features of 10 azithromycin-resistant NTS isolates.

Methods

A total of 457 NTS isolates were collected from a tertiary hospital in Guangzhou. We performed antimicrobial susceptibility tests, conjugation experiments, efflux pump expression tests, whole-genome sequencing and bioinformatics analysis to conduct the study.

Results

The results showed that 10 NTS isolates (2.8%) were resistant to azithromycin with minimum inhibitory concentration values ranging from 128 to 512 mg/L and exhibited multidrug resistance. The phylogenetic tree revealed that 5 S. London isolates (AR1–AR5) recognized at different times and departments were closely related [3–74 single-nucleotide polymorphisms (SNPs)] and 2 S. Typhimurium isolates (AR7 and AR8) were clones (<3 SNPs) at 3-month intervals. The azithromycin resistance was conferred by mph(A) gene found on different plasmids, including IncFIB, IncHI2, InFII, IncC and IncI plasmids. Among them, IncFIB, InFII and IncHI2 plasmids carried different IS26-class 1 integron (intI1) arrangement patterns that mediated multidrug resistance transmission. Conjugative IncC plasmid encoded resistance to ciprofloxacin, ceftriaxone and azithromycin. Furthermore, phylogenetic analysis demonstrated that mph(A)-positive plasmids closely related to 10 plasmids in this study were mainly discovered from NTS, Escherichia coli, Klebsiella pneumonia and Enterobacter hormaechei. The genetic environment of mph(A) in 10 NTS isolates was IS26-mph(A)-mrx(A)-mphR(A)-IS6100/IS26 that co-arranged with intI1 harbour multidrug-resistant (MDR) gene cassettes on diverse plasmids.

Conclusions

These findings highlighted that the dissemination of these plasmids carrying mph(A) and various intI1 MDR gene cassettes would seriously restrict the availability of essential antimicrobial agents for treating NTS infections.

Introduction

Non-typhoidal Salmonella (NTS) is a common bacterial pathogen that causes many severe cases of foodborne illness in humans. It is estimated that NTS causes 93.8 million infectious gastroenteritis and 155 000 deaths globally each year.¹ NTS is composed of more than 2600 serotypes and nearly 1600 serotypes classified as Salmonella enterica subspecies that are correlated with gastrointestinal diseases worldwide.² In many developing countries, NTS infections are common, resulting in diarrheal disease and posing a serious threat to children, the elderly and the immunocompromised.^3,4 The FDA has approved three different antimicrobial agents for the treatment of NTS infections in the USA: ciprofloxacin, ceftriaxone and azithromycin.^5–7 However, the resistance rate of NTS to ciprofloxacin and ceftriaxone has increased dramatically in recent years in China.^8–11 As an alternative to ciprofloxacin and ceftriaxone, azithromycin is a significant antimicrobial agent against multidrug-resistant (MDR) NTS.¹²

Owing to its superior permeability, enhanced pharmacokinetics and metabolic stability, the macrolide antibiotic azithromycin could be used for treating clinical infections caused by foodborne pathogens including Escherichia coli and NTS.^13–17 However, azithromycin-resistant NTS are on the rise due to the increasing use of azithromycin.¹⁸ Several mechanisms of resistance to macrolides had been described in Enterobacteriaceae, including drug-modifying enzymes, the activity of efflux pumps, target mutations and methylation of 23S rRNA.^19–23 In a UK-based retrospective investigation, it was found that 16 out of 667 NTS (2.3%) showed resistance to azithromycin, with the efflux pump gene mef(B) and phosphorylase genes mph(A) and mph(B) being responsible for this resistance.²⁴ A total of 76 azithromycin-resistant NTS isolates were found among 2431 NTS isolates (3.1%) from 2017 to 2018 in Taiwan, with the antimicrobial resistance mediated by methylase genes erm(B) and erm(42), phosphorylase gene mph(A) and efflux pump-regulating gene ramAp.²⁵ At present, the prevalence and resistance mechanisms of NTS with azithromycin resistance in northern Guangzhou are unknown. In this study, we described the prevalence and genetic characterization of 10 azithromycin-resistant NTS isolates from a tertiary hospital in Guangzhou, China.

Materials and methods

Bacterial isolations

From 21 May 2020 to 22 December 2021, 469 NTS strains isolated from different patients were identified at the Fifth Affiliated Hospital of the Southern Medical University, Guangzhou, China, using the Advanced Expert System of the VITEK-2 COMPACT automated microbial identification system (bioMerieux, Marcy-l’Étoile, France). E. coli ATCC 25922 and S. Typhimurium ATCC 14028 were used as the quality control strains.

Antimicrobial susceptibility testing

The minimum inhibitory concentration (MIC) values of 10 azithromycin-resistant NTS isolates for azithromycin (AZM), ceftriaxone (CRO), ciprofloxacin (CIP), amoxicillin-clavulanic acid (AMC), piperacillin-tazobactam (TZP), cefoxitin (FOX), cefuroxime (CXM), ceftazidime (CAZ), cefepime (FEP), levofloxacin (LEV), amikacin (AK), tigecycline and sulfamethoxazole (SXT) were determined by broth microdilution. The MIC values of azithromycin-resistant 10 NTS isolates for ampicillin (AMP) and chloramphenicol (DHL) were determined by the Kirby Bauer disk diffusion method. The interpretation of the results was determined based on the CLSI breakpoints and azithromycin MIC of ≥32 mg/L was considered to be resistant (ci S1, available as Supplementary data at JAC Online).²⁶

Whole-genome sequencing and bioinformatics analysis

The genomic DNA of 10 NTS isolates was extracted by the bacterial genomic DNA extraction kit (Tiangen, Beijing, China). Whole-genome sequencing was performed on the Nanopore PromethION platform (Oxford Nanopore Technologies Ltd, Oxford, UK) and Illumina NavoSeq PE150 (Illumina, SanDiego, CA, USA) at the Novogene company (Tianjin, China). It was done with R9.4 flowcells and high accuracy was used as a basecalling model. The quality of the raw sequence reads was evaluated by the interactive program FastQC,²⁷ assembled by Unicycler 0.4.8²⁸ and annotated by Prokka 1.13.²⁹ MLST 2.18.0³⁰ was used for analysing sequence types serotypes of NTS, and SISTR 1.1.1³¹ was used for confirming serotypes. Staramr 0.5.1³² was performed to screen bacterial genome contigs. The ResFinder 4.1,³³ PlasmidFinder 2.1³⁴ and ISfinder³⁵ were used to identify resistance genes, plasmid incompatible type (Inc type) and insertion sequences, respectively. Core-genome single-nucleotide polymorphism (SNP) was generated by Parsnp³⁶ and the phylogenetic tree of core-genome SNP was constructed with MEGA X.³⁷ The BLASTn algorithm³⁸ was used to screen 1133 plasmids carrying mph(A) gene from the PLSDB database.³⁹ Phylogenetic trees were built by selecting plasmids with the same replicon as the five plasmid Inc types in this study and with orthologous genes identified by OrthoFinder,⁴⁰ including IncFIB (n = 482), IncFII (435), IncHI2 (100), IncC (111) and IncI1 (15). A total of 35 mph(A)-positive plasmids obtained from PLSDB database, with coverage >87% compared with the plasmid in this study (reference), were selected for sequence comparison using BLASTn and visualized with BRIG (Table S2).⁴¹

Conjugation experiments and PCR assays

The transferability of IncC and IncI plasmids carrying mph(A) sanger gene was determined by conjugation experiments using rifampicin-resistant E. coli C600 as the recipient strain. Transconjugants were selected on Luria–Bertani agar plates containing rifampicin (100 mg/L) and azithromycin (32 mg/L). The transconjugants carrying mph(A) gene were then identified by PCR sequencing and primers were mph(A)-F:5′-ATGCGTGCACTACGCAAAG-3′ and mph(A)-R: 5′-CGAGCGGGCTATATCG AC-3′.⁴² PCR conditions were 30 cycles at 95°C for 1 min, 56°C for 1 min and 72°C for 3 min, with a final extension at 72°C for 10 min. PCR products were verified by sanger sequencing (BGI Tech Solutions Co., Limited, Beijing, China) and subsequently analysed by comparing to mph(A) gene sequences of AR6 and AR10 using the DNAMAN software (Lynnon BioSoft, Quebec, Canada). The antimicrobial susceptibility of the transconjugants was confirmed by broth microdilution. The conjugation experiments were performed twice.

RT-qPCR analysis of efflux pump genes

Different serotypes of 10 NTS isolates were selected as representatives (AR1, AR6, AR7, AR9 and AR10) for measuring the relative expression of efflux pump genes (acrB, acrD, acrF, mdsB, mdtB, mdfA, emrB, mdtJ, macB, tolC, ompF, ompC, ompA and ompS2). Of these, five NTS isolates were incubated at 37°C and 180 rpm until the density of 1.5 × 10⁸ CFU/mL. The total RNA of five NTS isolates was extracted by Bacterial RNA Kit (Omega, Guangzhou, China) and then RNA was reverse-transcribed to cDNA using a cDNA synthesis kit (Takara). cDNA was amplified by RT-PCR using Taq SYBR Green qPCR Premix (Innovagene, Changsha, China) as described in the manufacturer’s instructions on Nucleic Acid Amplification. After a 10 min activation of the modified Taq polymerase at 95°C, 40 cycles of 15 s at 95°C, 30 s at 60°C and 30 s at 72°C were performed. Data acquisition was done at 60°C and 72°C. The relative gene expression for pump gene transcripts was calculated against the internal control 16S rRNA gene. The relative expression of efflux pump genes in five NTS isolates was calculated against S. Typhimurium ATCC 14028 (expression = 1), which served as the control to evaluate overexpressed pump genes. The primer sequences were listed in Table S3. The 2^−ΔΔCT method was used to calculate the relative expression level of pump genes.⁴³ All reactions were performed in triplicate.

Data availability

The complete whole genome sequences of 10 azithromycin-resistant NTS isolates had been deposited at GenBank under the accession ID listed in Table 1.

Table 1.

Clinical information and genomic characterization of 10 azithromycin-resistant NTS isolates

Isolates	Date of isolation	Department	Salmonella serovar	ST	Vehicle of resistance genes	Resistance genesa	GenBank accession no.
AR1	2020/7/31	Physical Examination	London	155	Chromosome	aac(6′)-Iaa	JAVAIX000000000
					IncFIB	qnrB6,aac(6′)-Ib-cr,bla TEM-1 ,mph(A),floR,tet(A),aph(6)-Id,aph(3″)-Ib,aac(3)-IId,sul1,sul2,aadA16,dfrA27,arr-3
AR2	2021/10/5	Oncology	London	155	Chromosome	aac(6′)-Iaa	JAVAIW000000000
					IncFIB	qnrB6,aac(6′)-Ib-cr,bla TEM-1 ,floR,tet(A),mph(A),aph(6)-Id, aph(3″)Ib,aac(3)IId,catA2,sul1,sul2,aadA16,dfrA27,arr-3
AR3	2021/7/26	Pediatrics	London	155	Chromosome	aac(6′)-Iaa	JAVAIV000000000
					IncFIB	qnrB6,aac(6′)-Ib-cr,bla TEM-1 ,mph(a),flor,tet(A),aph(6)-Id, aph(3″)-Ib,aac(3)-IId,sul1,sul2,catA2,aadA16,dfrA27,arr-3
AR4	2021/12/4	Dispensary	London	155	Chromosome	aac(6′)-Iaa	JAVAIU000000000
					IncFII	qnrB6,aac(6′)-Ib-cr,bla TEM-1 ,bla CTX-M-24 ,floR,tet(A),mph(A),aac(3)-IId,aph(6)-Id,aph(3″)-Ib,aac(6′)-Iaa,ant(3″)-Ia,sul1,sul2,floR,catA2,arr-3,dfrA27,aadA16
AR5	2020/7/20	Physical Examination	London	155	Chromosome	aac(6′)-Iaa	JAVAIT000000000
					IncFIB	qnrB6, aac(6′)-Ib-cr, bla TEM-1 ,mph(A),floR,tet(A),aph(6)-Id,aph(3″)-Ib,aac(3)-IId,sul1,sul2,catA2,aadA16,dfrA27,arr-3
AR6	2020/9/9	Physical Examination	Thompson	26	Chromosome	aac(6′)-Iaa	JAVAIS000000000
					IncC	qepA8,qnrS1,bla TEM-1 ,bla CMY-2 ,mph(A),dfrA12,aadA2,sul1,sul2,aph(3″)-Ib,aph(6)-Id,tet(A),floR
AR7	2021/7/28	Paediatrics	Typhimurium	34	Chromosome	bla TEM-1 ,tet(B),sul2,aph(3')-Ib,aph(6)-Id,aac(6′)-Iaa	JAVAIR000000000
					IncHI2	qnrS1,bla CTX-M-55 ,bla LAP-2 ,bla TEM-1 ,mph(A),ant(3″)-Ia,aac(3)-IId,lnu(F),dfrA14,aadA16,sul3
					IncHI1&IncR	arr-3,dfrA27,aadA16,sul1,floR
AR8	2021/9/25	Paediatrics	Typhimurium	34	Chromosome	bla TEM-1 ,tet(B),sul2,aph(3″)-Ib,aph(6)-Id,aac(6′)-Iaa	JAVAIQ000000000
					IncHI2	qnrS1,bla CTX-M-55 ,bla LAP-2 ,bla TEM-1 ,mph(A),lnu(F),dfrA14,aadA16,sul3,arr-3,ant(3″)-Ia,aac(3)-IId
					IncHI1&IncR	arr-3,dfrA27,aadA16,sul1,floR
AR9	2020/6/26	Pediatrics	Saintpaul	27	Chromosome	floR,aac(6′)-Iaa	JAVAIP000000000
					IncHI2	qnrS1,bla LAP-2 ,bla TEM-1 ,bla CTX-M-55 ,mph(A),aph(3')-Ia,aph(6)-Id, floR,arr-2,dfrA14,tet(A)
AR10	2021/2/20	Pediatrics	Rissen	469	Chromosome	qnrS1,bla TEM-1 ,sul2,floR,dfrA12,aadA2,cmlA1,aadA1,sul3,tet(A),tet(M),aac(6′)-Iaa,ant(3″)-Ia	JAVAIO000000000
					IncI1	bla CTX-M-123 ,mph(A),fosA3

^aAzithromycin-resistant gene is indicated in bold.

Results

Azithromycin-resistant NTS isolates

A total of 10 azithromycin-resistant NTS isolates (2.63%) were identified from 457 NTS clinical isolates in a tertiary hospital in northern Guangzhou, with MIC values of 128 mg/L (n = 7), 256 mg/L (2), and 512 mg/L (1) (Table 1). The 10 NTS isolates were mostly detected between June and October (n = 8) and were identified in departments including paediatrics (n = 5), physical examination (3), oncology (1) and pharmacy (1). The serotypes of 10 NTS isolates consisted of S. London ST155 (n = 5), S. Typhimurium ST34 (2), S. Saintpaul ST27 (1), S. Thompson ST26 (1) and S. Rissen ST469 (1).

Phylogenetic tree analysis and antimicrobial resistance phenotypes

SNPs phylogenetic tree revealed that 10 azithromycin-resistant NTS isolates were divided into two genetically distinct clades (Figure 1a). S. Rissen AR10 was found in a clade and other NTS isolates (n = 9) were located in another clade. Among them, 5 S. London isolates identified at different times and sites were closely related with 3–74 SNPs, which encoded resistance to ampicillin, chloramphenicol, sulfamethoxazole and ciprofloxacin (AR5 was susceptible to chloramphenicol). Furthermore, two S. Typhimurium isolates AR7 and AR8 discovered in paediatrics 2 months apart were clones (two SNPs) and possessed identical resistance phenotype with S. Saintpaul AR9. S. Thompson AR6 and S. Rissen AR10 were in different clades but both exhibited resistance to first-line drugs ciprofloxacin, ceftriaxone, azithromycin, ampicillin, chloramphenicol and sulfamethoxazole.

Figure 1.

Phylogeny tree and the heatmap of antimicrobial resistance genes of 10 azithromycin-resistant NTS isolates in this study and NTS isolates from the NCBI database. (a) Phylogenetic tree relationships and antimicrobial resistance phenotypes of the 10 azithromycin-resistant NTS isolates in this study. (b) SNP distances and the spectrum of resistance genes in NTS isolates with different serotypes. The blue circles were added to represent these NTS isolates in this study. The resistance genes for diverse antibiotics are marked by coloured boxes and blank boxes reflect the resistance genes that were not found. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

The whole-genome sequence of 6 S. London ST34, 4 S. Thompson ST26, 4 S. Typhimurium ST34, 3 S. Saintpaul ST27 and 4 S. Rissen ST469 isolates with close phylogenetic relationship to the 10 NTS isolates in this study were extracted from the NCBI database (Table S4). The phylogenetic analysis of the 10 azithromycin-resistant NTS isolates indicated the following: (i) 5 S. London isolates (AR1–AR5, ST155) and 6 S. London isolates identified in China (Chengde, Huaian and Taiwan) were closely related and had similar resistance genes profiles;³⁰ (ii) S. Thompson AR6 (ST26) carried identical resistance genes with three S. Thompson isolates detected in China (Hefei, Shanghai and Taiwan); (iii) two S. Typhimurium isolates that closely phylogenetically related to S. Typhimurium AR7 and AR8 (ST34) were detected in China (Guangzhou and Hangzhou) with their resistance gene profiles are different; (iv) S. Saintpaul AR9 (ST27) and ASM2069170v1 (Guangzhou, China, 2014) were clones (no SNPs) and harboured the same resistance genes profiles. (v) S. Rissen AR10 (ST469) had 63–76 SNP distances with four S. Rissen strains derived in Shenzhen and AR10 additionally carried mph(A), bla_CTXM-123 and fosA that were located on the IncI1 plasmid (Figure 1b).

Genetic and phylogenetic analysis of mph(A)-positive plasmids

mph(A) gene encoding azithromycin phosphotransferase was found on various Inc types of plasmids in 10 azithromycin-resistant NTS isolates, including IncFIB (4/10), IncHI2 (3/10), IncFII (1/10), IncC (1/10) and IncI1(1/10) (Table 1). To evaluate the homology and genomic relationship of mph(A)-positive plasmids from this study and different bacterial species, 1119 mph(A)-harbouring plasmids were extracted from the PLSDB database. The 1119 mph(A)-positive plasmids were frequently found in E. coli (47.98%), Klebsiella pneumoniae (30.78%) and Salmonella (5.17%), with others including Vibrio spp., Citrobacter spp., Aeromonas spp., Proteus spp. and Leclercia spp. (Table S5). These plasmids from the 10 NTS isolates in this study were included for constructing phylogenetic trees of IncFIB (Figure 2a), IncFII (Figure 2b), IncHI2 (Figure 2c), IncC (Figure 2d) and IncI1 (Figure 2e) plasmids, respectively.

Figure 2.

Phylogenetic analysis of IncFIB, IncFII, IncHI2, IncC and IncI1 plasmids, respectively. OrthoFinder was used to predict gene families and FigTree was used to construct the phylogenetic trees. (a) Phylogenetic tree was built using 482 mph(A)-harbouring IncFIB plasmids. (b) Phylogenetic tree was built using 435 mph(A)-positive IncFII plasmids. (c) Phylogenetic tree was built using 100 mph(A)-carrying IncHI2 plasmids. (d) Phylogenetic tree was built using 111 mph(A)-positive IncC plasmids. (e) Phylogenetic tree was built using 15 mph(A)-harbouring IncI1 plasmids. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

For the phylogenetic tree of IncFIB plasmids, four IncFIB plasmids of four S. London isolates (AR1, AR2, AR3 and AR5) in this study formed a clade (shown in red) with other six IncFIB plasmids that were all recovered from NTS (Figure 2a). Among them, AR2p exhibited the highest sequence coverage (91%–98%) to five IncFIB plasmids in China (Shenzhen, Hongkong and Huaian districts), especially to the plasmid pSa76-CIP (accession: CP047116; 98% coverage) isolated from Hong Kong in China (Table S2). These plasmids mostly carried MDR regions flanked by IS26 that contained 8 resistance genes (mph(A), arr-3, dfrA27, aadA16, qnrB6, aac(6′)-Ib-cr, floR and sul1), Class 1 integron (intI1) and insertion sequences (IS26, ISCR1, IS6100) (Figure 3a).

Figure 3.

Circle Sequence comparison analysis of IncFIB, IncFII, IncHI2, IncC and IncI1 plasmids, respectively. (a) Sequence comparison between IncFIB plasmid of S. London AR2 (AR2p, reference) and four closely related IncFIB plasmids. (b) Sequence comparison between IncFII plasmid of S. London AR4 (reference) and four closely related IncFII plasmids. (c) Sequence comparison between IncHI2 plasmid of S. Typhimurium AR7 (AR7p, reference) and seven closely related IncHI2 plasmids. (d) Sequence comparison between IncC plasmid of S. Thompson AR6 (reference) and eight closely related IncC plasmids. (e) Sequence comparison between IncI1 plasmid of S. Rissen AR10 (reference) and five closely related IncI1 plasmids. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

In the phylogenetic tree of IncFII plasmids, the clade marked in red suggested relatedness between the IncFII plasmid of S. London AR4 (AR4p) from NTS in our research and the other 19 IncFII plasmids from different bacterial species (Figure 2b). Among them, Genome mapping showed AR4p exhibited a high level of sequence coverage (88%–90%) to five IncFII plasmids (containing two from Klebsiella, one from E. coli, one from E. hormaechei) discovered from Thailand and China (Nanchang, Meishan, Chengdu and Henan regions), all of which harboured similar backbones and differentiated in resistance gene region (Figure 3b and Table S2). In addition, AR4p carried the high similar MDR region as four IncFIB plasmids from this study.

The phylogenetic tree of IncHI2 plasmids displayed that 3 IncHI2 plasmids of 2 S. Typhimurium isolates (AR7 and AR8) and S. Saintpaul AR9 in that study and other 14 IncHI2 plasmids were detected in the same clade labelled in red (Figure 2c). Among them, AR7p exhibited high sequence coverage (89%–100%) to 12 IncHI2 plasmids (containing 3 from NTS, 6 from Escherichia and 1 from E. hormaechei) from different countries, especially to the plasmid pCFSAN086836 (accession: CP039440.1; 100% coverage) from S. Kentucky in Guangzhou (Table S2). All of these IncHI2 plasmids shared 54 kb complex intI1-IS26 arrangements that included 14 antimicrobial resistance genes [mph(A), dfrA14, arr-2, cmlA5, qnrS1, bla_TEM-2, bla_CTX-M-55, bla_LAP-2, aac(3′)-IId, lnu(F), aph(3″)-Ia and sul3] interspersed with ISs (IS26, intI1, IS26, Tn3, ISEcp1 and IS1), integron (intI1) and transposons (Tn3) (Figure 3c).

The mph(A)-positive IncC plasmids are widely detected in diverse bacterial genera, and the IncC plasmid of S. Thompson AR6 (AR6p) had a close phylogenetic relationship with 15 IncC plasmids (containing 6 from Salmonella, 9 from other Enterobacteriaceae) in the same cluster presented in red (Figures 2d and 3d). Among them, AR6p exhibited a high level of sequence coverage (95%–100%) to four IncC plasmids isolated from Salmonella in China (Guangzhou, Hefei, Shanghai and Guangdong regions), containing the plasmid pSal009 (accession: CP050832.1; 100% coverage) from pork in China. These plasmids harboured the same backbones with various antimicrobial resistance genes.

The phylogenetic tree of IncI1 plasmids showed the IncI1 plasmid of S. Rissen AR10 (AR10p) in our study formed a clade (present in red) with other four IncI1 plasmids that were recovered from E. coli in China, Japan and Korea (Figure 2e). Sequence comparison revealed that AR10p carrying mph(A) and bla_CTX-M-123 exhibited highest degree of sequence similarity (92%–100% coverage) to them (Figure 3e), especially to the plasmid pHNAH4-1(accession: NC_024955.2; 100% coverage) from Hong Kong in China and plasmid pCTXM123_C0996 (accession: NZ_KP198616.1; 99% coverage) from Anhui in China (Table S2).

Genetic environment of plasmid-mediated mph(A) gene

Two structures of mph(A) clusters were characterized from 10 mph(A)-positive plasmids in this study and named Types I and II (Figure 4). Type I was the IS26-mph(A)-mrx(A)-mphR(A)-IS6100 cluster found on IncFIIB, IncFII and IncC plasmids. In addition, Class 1 integron (IntI1) gene cassette arrangements were located upstream or downstream of the cluster, including IS26-intI1-aac(6′)Ib-cr-arr-3-dfrA27-aadA16-ISCR1 (IncFIB and IncFII plasmids) and IS6100-intI1-qepA2-dfrA12-sul1-TnAs1 (IncC plasmid). Type II located on the IncI1 and IncHI2 plasmids was IS26-mph(A)-mrx(A)-mphR(A)-IS26 that IS6100 was replaced by IS26 compared with Type I. IntI1 gene cassette array IS26-intI1-dfrA14-arr-2-cmlA5-IS26 was discovered upstream of the mph(A) cluster on 3 IncHI2 plasmids.

Figure 4.

Genetic environment of mph(A) gene on different plasmids. Coloured arrows represent open reading frames, and the direction of the arrowheads indicates the direction of transcription. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Conjugation experiments

Conjugation experiments indicated that the IncC and IncI1 plasmids could be transferred to the recipient E. coli C600 determined by PCR amplification, while the IncFIB, IncHI2 and IncFII plasmids failed to perform conjugation assays due to carrying rifampicin resistance. The transconjugant AR6TC exhibited resistance to azithromycin, ciprofloxacin, ceftriaxone, ampicillin, chloramphenicol, and sulfamethoxazole, which was mediated by mph(A), qnrS1, qepA8, bla_TEM-1, bla_CMY-2, sul1, sul2 and floR that were located on the IncC plasmid (Table 2). Acquisition of the IncI1 plasmid rendered the AR10TC resistant to azithromycin and ceftriaxone with MIC values of 64 and 32 mg/L, respectively.

Table 2.

MIC values of E. coli C600 that had acquired mph(A) gene by transformation

			MIC (mg/L)
			AZM	AMC	TZP	FOX	CXM	CAZ	CRO	FEP	AK	CIP	LEV	TGC	SXT
Strain	Organism	Plasmid Inc Type
C600	E. coli	\	<4	4	≤4	8	8	0.5	≤0.25	≤0.12	≤2	0.25	0.5	≤0.5	≤20
AR6	S. Thompson	IncC	128	32	≤4	32	32	32	32	≤0.12	≤2	16	4	1	≥320
AR6TC	E. coli	IncC	64	32	≤4	32	16	32	32	≤0.12	≤2	4	4	≤0.5	≥320
AR10	S. Rissen	IncI1	512	16	≤4	≤4	≥64	≥64	≥64	≥32	≤2	4	1	1	≥320
AR10TC	E. coli	IncI1	64	16	≤4	8	≥64	≥64	≥64	16	≤2	0.25	0.5	≤0.5	≤20

AZM, azithromycin; AMC, amoxicillin-clavulanic acid; TZP, piperacillin-tazobactam; FOX, cefoxitin; CXM, cefuroxime; CAZ, ceftazidime; CRO, ceftriaxone; FEP, cefepime; AK, amikacin; CIP, ciprofloxacin; LEV, levofloxacin; TGC, tigecycline; SXT, sulfamethoxazole.

Discussion

NTS often invades human sterile parts beyond the gut and causes severe diseases including sepsis and meningitis, with mortality incidences of up to 15% in Asia.⁴⁴ Azithromycin is the last resort for the treatment of life-threatening salmonellosis caused by MDR and extensively drug-resistant Salmonella.⁴⁵ In this study, we discovered 10 azithromycin-resistant NTS isolates (2.63%) from a tertiary care hospital in Guangzhou between 2020 and 2021, whose resistance rate was higher than the reports in Australia (0.9%) and Europe (0.5%).^46,47 Previous studies have demonstrated that MIC values of NTS resistance to azithromycin were 16–128 mg/L in Latin America, the United Kingdom, and France.^19,48,49 Our study showed that the 10 NTS isolates had high levels of resistance to azithromycin and were resistant to the regular first-line drugs including ampicillin (100%), sulfamethoxazole (100%), chloramphenicol (90%), ciprofloxacin (60%) and ceftriaxone (50%), suggesting that more attention should be focused on the development of antimicrobial resistance in NTS. Phylogenetic tree analysis revealed that five S. London isolates (AR1–AR5) derived from different times and departments were closely related and two S. Typhimurium clones were discovered in paediatrics at 2-month intervals. Moreover, 21 NTS isolates with close phylogenetic relationships to the 10 NTS isolates in this study were also found in several regions of China. These results underlined the necessity to concern the epidemic dissemination of NTS in different geographic regions.

Azithromycin resistance of 10 NTS isolates was mediated by mph(A) gene located on IncFIB(4/10), IncFII(1/10), IncHI2(3/10), IncC(1/10) and IncI1(1/10) plasmids, and the reports of mph(A) gene carried by these plasmids have been described.^7,42 Among these plasmids, four IncFIB and IncFII plasmids carried highly comparable 18 kb MDR regions that IS26 played a key role in transmission.⁵⁰ Three IncHI2 plasmids all carried a 54 kb IS26-intI1 arrangement involving a variety of resistance genes and are widely disseminated in the IncHI2 plasmids of Enterobacteriaceae.^51,52 Conjugative IncC plasmid that encoded resistance to azithromycin, ciprofloxacin, ceftriaxone, ampicillin, chloramphenicol and sulfamethoxazole significantly constrained the therapeutic options of NTS infections.^49,53,54 Additionally, phylogenetic analysis revealed that other mph(A)-positive plasmids that showed a close genetic relationship with plasmids in this study were widely recovered from diverse bacterial species, including (i) IncFIB and IncC plasmids which were recovered from NTS in China;³⁰ (ii) the IncFII plasmids were isolated from K. pneumoniae, E. coli and E. hormaechei;⁵⁵ (iii) IncHI2 plasmids were mostly distributed in NTS and E. coli;^56,57 (iv) IncI1 plasmids were identified in E. coli. These findings underlined the importance of monitoring the epidemiology of mph(A)-positive plasmids in different hosts.

By investigating the genetic environment of the plasmid-mediated mph(A) gene, it was noted that the mph(A) cluster “IS26-mph(A)-mrx(A)-mphR(A)-IS6100/IS26” was the most common and had been widely discovered in many bacterial species.^58,59 The IS26 and IS6100, which are classified as IS6 family transposons, played an essential role in the transmission of the mph(A) cluster.⁵⁰ Meanwhile, we found that the mph(A) cluster and intI1 harbouring various resistance gene cassettes are co-arranged on different plasmids in the 10 NTS isolates. Sequence analysis revealed that these different mph(A)-IntI1 arrangements are also frequently detected in NTS and E. coli. The arrangement patterns of mph(A) gene with intI1 variable resistance gene cassettes would result in the prevalence of MDR NTS and pose a huge challenge to public health.^60,61 In addition, we observed no changes in the expression of efflux pumps and outer membrane proteins (Table S6). No azithromycin resistance mutation was discovered in the 23S rRNA gene or the L22 and L4 protein sequences.

In summary, 10 NTS isolates with a high degree of azithromycin resistance were identified from patients and exhibited regional cloning epidemics. mph(A) gene associated with azithromycin resistance located on various plasmids and the close phylogenetic relationship of mph(A)-harbouring plasmids were discovered in Enterobacteriaceae. More importantly, mph(A) gene in the form of cluster IS26-mph(A)-mrx(A)-mphR(A)-IS6100/IS26 co-arranged with intI1 variable multidrug resistance gene cassettes on diverse plasmids contributed to the emergence and prevalence of MDR NTS. Further work is required to monitor the transmission and evolution of these plasmids in bacterial pathogens.

Funding

This project was funded by the Guangdong Basic and Applied Basic Research Fund Provincial Enterprise Joint Fund (2021A1515220153), Basic and Applied Basic Research Foundation of Guangdong Province Natural Science Foundation (2022A1515012481), and President Foundation of The Fifth Affiliated Hospital, Southern Medical University (No. YZ2022ZX01).

Transparency declarations

The authors all declare they have no conflict of interest.

Supplementary data

Tables S1–S6 are available as Supplementary data at JAC Online.