Multiple Mechanisms of Tigecycline Resistance in Enterobacteriaceae from a Pig Farm, China

ABSTRACT We isolated eight tigecycline-resistant Enterobacteriaceae strains from a pig farm in Shanghai, China, including Escherichia coli (n = 1), Proteus cibarius (n = 1), and Enterobacter hormaechei (n = 6). Two of them (E. coli and P. cibarius) were positive for tet(X). E. coli SH19PTE6 contained an IncFIA18/IncFIB(K)/IncX1 hybrid plasmid pYUSHP6-tetX, highly similar to other tet(X)-bearing hybrid plasmids from E. coli in China. In P. cibarius SH19PTE4, tet(X) was located within a new chromosomal integrative and conjugative element (ICE), ICEPciChn2, belonging to the SXT/R391 ICE family. All tigecycline-resistant E. hormaechei isolates carried the tet(A) variant; cloning and transfer of this tet(A) variant into various hosts increased their MICs for tigecycline (4- to 8-fold). Tigecycline resistance observed on a pig farm is mediated by the tet(A) variant and tet(X) via a plasmid or ICE. The rational use of antibiotics such as doxycycline and surveillance of tigecycline resistance in livestock are warranted. IMPORTANCE As a last-resort antimicrobial agent to treat serious infections, the emergence and spread of tigecycline resistance in Enterobacteriaceae and Acinetobacter have raised global concerns. Multiple mechanisms mediate tigecycline resistance in Enterobacteriaceae, such as the monooxygenase Tet(X), mutations in Tet proteins, and overexpression of efflux pumps. Although tigecycline is not approved for animals, tigecycline resistance has been observed in Escherichia coli, Proteus cibarius, and Enterobacter hormaechei isolates on a pig farm, mediated by the tet(A) variant and tet(X) via a plasmid or ICE. The heavy use of tetracyclines such as doxycycline in food-producing animals in China may be the reason for the emergence and transmission of tigecycline resistance.

T igecycline is used as a last-resort antimicrobial agent for the treatment of serious infections caused by multidrug-resistant bacteria, particularly carbapenem-resistant Enterobacteriaceae (1). Tigecycline resistance in Enterobacteriaceae has been previously associated with the overexpression of efflux pumps (AcrAB-TolC and OqxAB), a ribosomal S10 protein mutation (rpsJ), and mutations in the plasmid-mediated Tet proteins [Tet(A), Tet(K), and Tet(M)] (1). Tet(X) family genes encode flavin-dependent monooxygenases that enzymatically inactivate most tetracyclines, including tigecycline (2). The chromosomally located tet(X) and its variant tet(X2), which originated from Bacteroides species, have been sporadically reported worldwide as conferring tigecycline resistance (3,4). Recently, the identification of novel plasmid-borne tet(X) genes [namely, tet(X3) and tet(X4)] conferring high level of tigecycline resistance in Enterobacteriaceae and Acinetobacter from China in 2019 is of great concern (4).
[According to the standards of the nomenclature center, http://faculty.washington .edu/marilynr/, all tet(X) variant genes at present can only be designated as tet(X).] Mobile tet(X) genes [tet(X3) and tet(X4)] have recently been identified in Acinetobacter spp., numerous Enterobacteriaceae, and six other bacterial species (3). To date, tet(X) and 14 variants [tet(X1) through tet(X14)] have been identified and confer differential degrees of tigecycline resistance (5). In 2020, a novel plasmid-mediated efflux pump gene cluster, tmexCD1-toprJ1, conferring resistance to multiple drugs, including tigecycline, was identified in Klebsiella pneumoniae strains (6). In this study, we aimed to analyze and elucidate the mechanisms of tigecycline resistance in Enterobacteriaceae in a pig farm in China.
On 23 September 2019, 128 samples, including pig feces, pig nasal swabs, feed, pig drinking water, vegetables grown on the pig farm, vegetable field soil, floor swabs from pens, and the shoe soles of workers, were collected from a pig farm in Shanghai, China (Table S1 in the supplemental material). One sample per pig was collected, and no more than five samples were collected from pigs in the same house. The samples were incubated in buffered peptone water (BPW) broth for ;18 to 24 h and then inoculated onto MacConkey agar with and without 2 mg/liter tigecycline. One isolate per plate was selected and identified using 16S rRNA gene sequencing according to a previously described method (7). A total of 74 Enterobacteriaceae isolates, including 45 E. coli, 22 Enterobacter cloacae complex, 3 Citrobacter freundii, 2 Leclercia adecarboxylata, 1 Aeromonas veronii, and 1 Proteus cibarius isolate, were obtained (Table S1 in the supplemental material). All isolates had MICs determined for tigecycline, ampicillin, cefotaxime, meropenem, gentamicin, amikacin, streptomycin, tetracycline, doxycycline, chloramphenicol, florfenicol, nalidixic acid, ciprofloxacin, colistin, fosfomycin, and sulfamethoxazole/trimethoprim using the agar dilution method or the broth microdilution method (limited to colistin and tigecycline). The results were interpreted according to EUCAST (https://www.eucast.org). Among the isolates, eight exhibited an MIC of 4 to 16 mg/liter to tigecycline, including one E. coli, one P. cibarius, and six E. hormaechei isolates obtained from different sources, and also showed resistance to multiple antimicrobial agents ( Table 1). The remaining 66 isolates were susceptible to tigecycline with MICs of 0.125 to 0.25 mg/liter. We further screened for the presence of tet(X) in tigecycline-resistant isolates by PCR and sequencing (8) and confirmed that two of them (E. coli and P. cibarius) were positive for tet(X) [former names, tet(X4) and tet(X6)] ( Table 1). All eight isolates failed to transfer tigecycline resistance to E. coli C600 using conjugation following a previously described protocol (9), but E. coli SH19PTE6 successfully transferred tet(X) to DH5a via electroporation.
The draft genome sequences of six E. hormaechei isolates were obtained. Isolate SH19PTE2, as a representative ST109 E. hormaechei strain, was further sequenced using PacBio to obtain the whole-genome sequence. We did not identify tmexCD1-toprJ1 or any genes belonging to the tet(X) family in the six E. hormaechei isolates. Compared to the E. hormaechei isolates showing susceptibility to tigecycline in this study, strain FY01 obtained from a patient in France (15), and E. cloacae NCTC9394 (GenBank accession no. FP929040), no amino acid changes within the genes ramR, ramA, marA, marR, acrA, acrB, or tolC possibly associated with tigecycline were identified (Table S2 in the supplemental material). However, we found that all tigecycline-resistant E. hormaechei isolates carried the tet(A) variant (IncFIB plasmid pYUSHP2-2), identical to that previously described in a tigecyclineresistant K. pneumoniae strain (16). This Tet(A) variant, located within the incomplete transposon Tn1721, harbored double frameshift mutations (S201A, F202S, and V203F) and mutations (I5R, V55M, I75V, and T84A), compared to the original Tet(A) in plasmid RP1 (X00006; E. coli). To further confirm its function, the full length of the tet(A) variant was amplified using PCR and cloned into the pUC57 vector. The recombinant plasmid pUC57tet(A) variant was transformed into commonly observed Enterobacteriaceae, namely, E. hormaechei, E. cloacae, E. coli, Salmonella enterica serovar Typhimurium, and K. pneumoniae. The MICs for tigecycline were increased 4-to 8-fold compared with their host strains ( Table S3 in the supplemental material). This further underlines the role of the tet(A) variant in tigecycline resistance in various hosts and may explain the tigecycline resistance in the six E. hormaechei isolates in this study.
Although tigecycline has not been approved in animals, tigecycline resistance mediated by the tet(A) variant and tet(X) via plasmid or ICE was observed on this pig farm. The use of tetracyclines such as doxycycline, one of the most used antimicrobial agents in food-producing animals in China, may be the reason for the emergence and transmission of tigecycline resistance. Appropriate measures should be taken to limit tigecycline resistance in animals.
Data availability. The draft genome sequences for the tet(X)-bearing plasmid pYUSHP6-tetX and ICEPciChn2 have been deposited in GenBank under accession no. MW423609 and MW423608. Other sequenced data have been deposited in the GenBank under accession no. PRJNA724799.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. SUPPLEMENTAL FILE 1, PDF file, 1.2 MB.

ACKNOWLEDGMENTS
This work was funded by the fifth phase of the "333 Project" scientific research project in Jiangsu Province (BRA2020002) and the Key Research and Development Program (Modern Agriculture) project of Jiangsu Province (grant no. BE2017341).