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During surveillance of Salmonella infections in the Yulin population in 2020, 979 Salmonella strains were obtained. Among them, one strain (Sa17155) tested positive for the blaNDM-1 gene through PCR analysis. This strain was isolated from a rectal swab of a healthy food worker (a chef) who exhibited no symptoms of Salmonella infection, such as fever, diarrhea, or abdominal pain. Upon further investigation, the isolate was identified as S. Typhimurium using a serum agglutination assay. Antimicrobial Susceptibility Testing (AST) revealed that Sa17155 displayed resistance to carbapenem antibiotics, including meropenem (MIC = 4 mg/L), imipenem (MIC = 8 mg/L), and ertapenem (MIC > 2 mg/L), as well as to several other antimicrobials, such as third-generation cephalosporins, quinolones, and tetracycline. Notably, it was classified as a Multi-Drug Resistant (MDR) bacterium, although it remained susceptible to colistin, tigecycline, aztreonam, and nitrofurantoin (Table 1).
Table 1. Minimum inhibitory concentrations (mg/L) determined via antimicrobial susceptibility testing
Antimicrobial Sa17155 E. coli J53 transconjugants-E. coli J53 AMK ≤ 8 ≤ 8 ≤ 8 AMC > 32/16 ≤ 8/4 > 32/16 SAM > 16/8 ≤ 4/2 > 16/8 ATM ≤ 2 ≤ 2 ≤ 2 CFZ > 16 ≤ 2 > 16 FEP > 16 ≤ 1 > 16 SCP > 32/8 ≤ 0.5/8 > 32/8 FOX > 16 ≤ 4 > 16 CAZ > 32 ≤ 1 > 32 CRO > 32 ≤ 1 > 32 CXM > 16 ≤ 4 > 16 CHL > 16 ≤ 4 ≤ 4 CIP 1 ≤ 0.5 ≤ 0.5 COL ≤ 1 ≤ 1 ≤ 1 EIP > 2 ≤ 0.25 > 2 FOS ≤ 16 ≤ 16 ≤ 16 GEN > 8 ≤ 2 ≤ 2 IPM 8 ≤ 0.25 8 LVX ≤ 1 ≤ 1 ≤ 1 MEM 4 ≤ 0.125 4 MIN > 16 ≤ 1 ≤ 1 MXF 1 ≤ 0.5 ≤ 0.5 NIT ≤ 16 ≤ 16 ≤ 16 NOR ≤ 2 ≤ 2 ≤ 2 TZP > 64/4 ≤ 4/4 > 64/4 TET > 8 ≤ 2 ≤ 2 TGC ≤ 1 ≤ 1 ≤ 1 TOB > 8 ≤ 2 ≤ 2 SXT > 4/76 ≤ 1/19 ≤ 1/19 Note. MK, amikacin; AMC, amoxicillin-clavulanate; SAM, ampicillin-sulbactam; ATM, aztreonam; CFZ, cefazolin; FEP, cefepime; SCP, cefoperazone-sulbactam; FOX, cefoxitin; CAZ, ceftazidime; CRO, ceftriaxone; CXM, cefuroxime; CHL, chloramphenicol; CIP, ciprofloxacin; COL, colistin; EIP, ertapenem; FOS, fosfomycin w/g6p; GEN, gentamicin; IPM, imipenem; LVX, levofloxacin; MEM, meropenem; MIN, minocycline; MXF, moxifloxacin; NIT, nitrofurantoin; NOR, norfloxacin; TZP, piperacillin-tazobactam; TGC, tetracycline; TOB, tigecycline; TOB, tobramycin; SXT, trimethoprim-sulfamethoxazole. -
Whole-genome sequencing and de novo genome assembly of strain Sa17155 were conducted using both short- and long-read sequencing techniques. The genome consisted of a circular chromosome (5,041,065 bp, 52.11% GC content) and a circular hybrid plasmid, pSa17155 (198,957 bp, 48.10% GC content). Within the chromosome, multiple resistance genes were identified, including aph(6)-Id, aac(6′)-Iaa, aph(3′′)-Ib, sul2, blaTEM-1B, and tet(B). These findings were consistent with its MDR phenotype, and Sa17155 was designated as ST34. We retrieved 261 Chinese S. Typhimurium ST34 genomes from the EnteroBase database and an additional 100 genomes from a published study[20]. These ST34 S. Typhimurium isolates were sourced from various locations, including humans (197 isolates), livestock farms (43 isolates), poultry (18 isolates), food (13 isolates), wild animals (two isolates), and environmental samples (one isolate). The phylogenetic relationships among these strains, originating from different sources, were analyzed based on 158 recombination-free Single Nucleotide Polymorphisms (SNPs) (See Figure 1A). Notably, 23 strains closely resembled Sa17155, forming a subclade with core genome SNP differences ranging from 25 to 68 (Refer to Figure 1B). These 23 S. Typhimurium strains were isolated from humans (10 strains), food (11 strains), and animals (two strains) across various provinces in China. Upon comparison of resistance gene repertoires and plasmid types, it was observed that two isolates carried blaNDM-5, while blaNDM-1 was exclusively detected in Sa17155 (Figure 1A).
Figure 1. Phylogenetic tree and distribution of published drug-resistance genes and plasmid types of S. Typhimurium type ST34 in strain Sa17155 and China. (A) Phylogenetic analysis of ST34 isolates from China; the rings from inside to outside along the tree represent the geographical origins of the isolates, year of isolation, source of the isolates, and presence of blaNDM-1 and blaNDM-5 genes. (B) Phylogenetic tree depicting the branch in which strain Sa17155 is located, and phylogenetic tree representing distribution of drug-resistance genes and plasmid types of Salmonella ST34 isolated from China in the public database. Phylogenetic tree is shown on the left; heat map of drug-resistance gene carriage is shown in the middle (red indicates carriage of the gene); and distribution of plasmid types is shown on the right (blue indicates presence of plasmid type).
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The complete sequence of the blaNDM-1-positive plasmid, designated pSa17155, revealed 370 open reading frames. Analysis of its two replicons, IncHI2 and IncHI2A, indicated that pSa17155 is a hybrid plasmid. This hybrid plasmid harbors a diverse array of genes conferring resistance to various antibiotics, including beta-lactams (blaNDM-1), aminoglycosides (aac(3)-IId, aadA16, aac(6')-Ib-cr, aph(4)-Ia, aac(3)-IV, aph(3')-Ia, bleO, aadA2b, and aadA1), rifamycin (arr-3), quinolones (oqxA and oqxB), tetracycline (tet(A)), folate pathway antagonists (sul3, sul2, sul1, and dfrA27), macrolides (mph(A)), quaternary ammonium compounds (qacE and qacL), and amphenicol (catB3, cmlA1, and floR). The plasmid's Type IV secretion system (T4SS) includes trbI, traI, traN, traU, traW, traF, traC, traV, traB, traK, traE, traL, and traA, all belonging to type F (Figure 2).
Figure 2. Structure of pSa17155. From inside to outside: ring 1 indicates G+C Content; ring 2 indicates G+C Skew; ring 3 represents pSa17155 ring structure; ring 4 red: drug-resistance gene; green: insertion sequence; purple: T4SS; maroon: plasmid replication.
Comparison of the IncHI2/IncHI2A plasmid pSa17155 with the NCBI database via BLASTn revealed significant similarity (> 90% concordance and > 70% coverage) with 36 plasmids sharing a similar backbone but derived from various host bacteria, predominantly Enterobacteriaceae. The majority of these plasmids were isolated from Salmonella (29 strains) and E. coli (seven strains) (Figure 3). Most strains originated from China, with one isolated in the UK. Notably, the blaNDM-1-harboring plasmid pSa17155 exhibited high homology and identity with plasmid NZ_CP047530 from S. Typhimurium strain SJTUF10640 (92% coverage with 98% identity) isolated from human feces in Shanghai, China, and NZ_CP047379 from E. coli in swine in Hunan, China (90% coverage with 98% identity). Although the replicon types of these three plasmids were identical, variable sequences were observed at the positions of the resistance genes, suggesting potential homologous recombination events (Figure 4). Additionally, IS26 was frequently found flanking the antibiotic resistance genes (ARGs), and the genetic context of blaNDM-1 was IS26-mph(A)-IS6100-sul1-qacE-arr-3-catB3-blaNDM-1-sul1-qacE-aadA16-dfrA27-arr-3-aac(6')-Ib-cr-xerD-IS26. The differences between plasmid pSa17155 and the other two plasmids (NZ_CP047530 and NZ_CP047379) mainly involved the insertion of ARGs mediated by IS26, particularly blaNDM-1 carried by pSa17155 (Figures 2 and 4).
Figure 4. Comparison of plasmids pSa17155, NZ_CP047530, and NZ_CP047379. Detailed sequence comparison of the three plasmids, with the light blue shaded area indicating >99% homology. Triangles and arrows on the outer circle represent genes of different functional categories (red: antimicrobial resistance; green: integrase, recombinase, transposase; purple: transfer associated; blue: plasmid replication; grey: other functions).
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The plasmid pSa17155, carrying the blaNDM-1 gene, was effectively transferred to recipient E. coli J53 with notable conjugation frequencies ranging from 5.0 × 10−6 to 1.5 × 10−5 per recipient strain under various experimental conditions (Figure 5). Particularly, the conjugation frequency was significantly higher when incubated at 37 °C for 4 hours with a donor/recipient bacteria ratio of 1:2 compared to other experimental setups. Furthermore, the conjugants displayed significantly heightened resistance to carbapenems in comparison to the E. coli strain J53. Notably, the meropenem MIC of the conjugants increased by 32-fold (from 0.125 to 4 mg/L). Additionally, transconjugants exhibited acquired resistance to cephalosporins, quinolones, and penicillin (Table 1).
Figure 5. Conjugation frequency (conjugant colony forming units [CFUs]/recipient CFUs) under different experimental conditions. Conjugation experiments were performed at two temperatures (30 °C and 37 °C) for two contact times (4 hours and 20 hours) with donor and recipient broth cultures mixed at ratios of 1:1, 1:2, and 1:4.
doi: 10.3967/bes2024.104
blaNDM-1 Carried by a Transferable Plasmid in a Salmonella Strain Isolated from Healthy Individuals
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Abstract:
Objectives Our study aimed to conduct genomic characterization of Salmonella strains carrying the blaNDM-1 gene in the intestinal tract of healthy individuals. The objectives were to underscore the importance of genomic surveillance for drug resistance in both commensal and pathogenic bacteria among healthy populations, and to establish protocols for regulating drug resistance plasmids based on the completion of a comprehensive map of drug resistance plasmid genomes. Methods We performed antimicrobial susceptibility testing and employed second- and third-generation sequencing techniques to analyze Salmonella strains harboring the blaNDM-1 gene, to surveil drug-resistant bacteria in the intestines of healthy subjects. Sequence comparison was conducted using both core- and pan-genome approaches. Concurrently, conjugation experiments were carried out to assess the efficiency of plasmid transfer. Results We isolated a carbapenem-resistant Salmonella enterica serovar Typhimurium strain from a healthy food worker in China. This strain harbored an IncHI2/IncHI2A plasmid carrying blaNDM-1 along with multiple antibiotic resistance genes (ARGs). Our findings highlight the potential for asymptomatic carriers to facilitate the transmission of ARGs. Pan-genomic analysis revealed that blaNDM-1-positive plasmids could traverse bacterial species barriers, facilitating cross-host transmission. Conclusions This study marks the first detection of blaNDM-1 in Salmonella strains isolated from healthy individuals. We underscore the risk associated with the transmission of conjugative hybrid plasmids carrying blaNDM-1, which have the potential to be harbored and transmitted among healthy individuals. Enhanced surveillance of drug-resistant pathogens and plasmids in the intestinal microbiota of healthy individuals could provide insights into the risk of ARG transmission and pathways for population-wide dissemination via ARG transfer factors. -
Key words:
- Carbapenems /
- NDM-1 /
- IncHI2/IncHI2A plasmid /
- Salmonella Typhimurium /
- Healthy individual
The authors declare they have no competing interests.
&These authors contributed equally to this work.
注释:1) AUTHORS’ CONTRIBUTIONS: 2) COMPETING INTERESTS: -
Figure 1. Phylogenetic tree and distribution of published drug-resistance genes and plasmid types of S. Typhimurium type ST34 in strain Sa17155 and China. (A) Phylogenetic analysis of ST34 isolates from China; the rings from inside to outside along the tree represent the geographical origins of the isolates, year of isolation, source of the isolates, and presence of blaNDM-1 and blaNDM-5 genes. (B) Phylogenetic tree depicting the branch in which strain Sa17155 is located, and phylogenetic tree representing distribution of drug-resistance genes and plasmid types of Salmonella ST34 isolated from China in the public database. Phylogenetic tree is shown on the left; heat map of drug-resistance gene carriage is shown in the middle (red indicates carriage of the gene); and distribution of plasmid types is shown on the right (blue indicates presence of plasmid type).
Figure 4. Comparison of plasmids pSa17155, NZ_CP047530, and NZ_CP047379. Detailed sequence comparison of the three plasmids, with the light blue shaded area indicating >99% homology. Triangles and arrows on the outer circle represent genes of different functional categories (red: antimicrobial resistance; green: integrase, recombinase, transposase; purple: transfer associated; blue: plasmid replication; grey: other functions).
Figure 5. Conjugation frequency (conjugant colony forming units [CFUs]/recipient CFUs) under different experimental conditions. Conjugation experiments were performed at two temperatures (30 °C and 37 °C) for two contact times (4 hours and 20 hours) with donor and recipient broth cultures mixed at ratios of 1:1, 1:2, and 1:4.
Table 1. Minimum inhibitory concentrations (mg/L) determined via antimicrobial susceptibility testing
Antimicrobial Sa17155 E. coli J53 transconjugants-E. coli J53 AMK ≤ 8 ≤ 8 ≤ 8 AMC > 32/16 ≤ 8/4 > 32/16 SAM > 16/8 ≤ 4/2 > 16/8 ATM ≤ 2 ≤ 2 ≤ 2 CFZ > 16 ≤ 2 > 16 FEP > 16 ≤ 1 > 16 SCP > 32/8 ≤ 0.5/8 > 32/8 FOX > 16 ≤ 4 > 16 CAZ > 32 ≤ 1 > 32 CRO > 32 ≤ 1 > 32 CXM > 16 ≤ 4 > 16 CHL > 16 ≤ 4 ≤ 4 CIP 1 ≤ 0.5 ≤ 0.5 COL ≤ 1 ≤ 1 ≤ 1 EIP > 2 ≤ 0.25 > 2 FOS ≤ 16 ≤ 16 ≤ 16 GEN > 8 ≤ 2 ≤ 2 IPM 8 ≤ 0.25 8 LVX ≤ 1 ≤ 1 ≤ 1 MEM 4 ≤ 0.125 4 MIN > 16 ≤ 1 ≤ 1 MXF 1 ≤ 0.5 ≤ 0.5 NIT ≤ 16 ≤ 16 ≤ 16 NOR ≤ 2 ≤ 2 ≤ 2 TZP > 64/4 ≤ 4/4 > 64/4 TET > 8 ≤ 2 ≤ 2 TGC ≤ 1 ≤ 1 ≤ 1 TOB > 8 ≤ 2 ≤ 2 SXT > 4/76 ≤ 1/19 ≤ 1/19 Note. MK, amikacin; AMC, amoxicillin-clavulanate; SAM, ampicillin-sulbactam; ATM, aztreonam; CFZ, cefazolin; FEP, cefepime; SCP, cefoperazone-sulbactam; FOX, cefoxitin; CAZ, ceftazidime; CRO, ceftriaxone; CXM, cefuroxime; CHL, chloramphenicol; CIP, ciprofloxacin; COL, colistin; EIP, ertapenem; FOS, fosfomycin w/g6p; GEN, gentamicin; IPM, imipenem; LVX, levofloxacin; MEM, meropenem; MIN, minocycline; MXF, moxifloxacin; NIT, nitrofurantoin; NOR, norfloxacin; TZP, piperacillin-tazobactam; TGC, tetracycline; TOB, tigecycline; TOB, tobramycin; SXT, trimethoprim-sulfamethoxazole. -
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