Emergence of mobile tigecycline resistance mechanism in Escherichia coli strains from migratory birds in China

ABSTRACT Plasmid-mediated antimicrobial resistance has emerged as one of the principal global issues, posing significant threats to public health. Herein, we reported a mobile tigecycline resistance mechanism Tet(X4) on both plasmid and chromosome in Escherichia coli strains from migratory birds in China. Besides tigecycline, these tet(X4)-positive strains also exhibited elevated MICs to the FDA newly approved tetracycline antibiotics, eravacycline (4 µg/ml) and omadacycline (8 µg/ml). Worrisomely, the tet(X4)-carrying plasmids and chromosome also shared high homology with the plasmids from human. Taken together, Tet(X4) represents another emerging antimicrobial threat and collective efforts from different sectors are needed to control its further spread.

Recent studies have showed that wild birds, especially migratory birds, may play an important role in the emergence and transmission of antimicrobial resistance and infectious diseases [1]. Meanwhile, the tigecycline resistance has also sporadically occurred (although not in migratory birds), primarily due to overexpression of efflux pumps and the newly identified tigecycline-inactivating mechanisms Tet(X3) and Tet(X4) [2][3][4]. Here we reported the tet(X4) gene in Escherichia coli strains from migratory birds in China, on both plasmid and chromosome, which raised a serious public health concern.
In 2018, three tigecycline-resistant E. coli strains, namely 2FT39, 2FT38-2, and 2ZN37-2, were isolated from stool samples of the little egrets (Egretta garzetta) from Guangdong, China, during a wild bird antimicrobial resistance surveillance study (Table S1). Susceptibility testing results showed that they were all resistant to tigecycline, tetracycline, florfenicol, and sulfamethoxazole-trimethoprim (Table S2). In addition, E. coli 2FT39 was also resistant to ciprofloxacin and E. coli 2FT38-2 was resistant to gentamicin. Interestingly, the three E. coli strains also exhibited significantly higher minimal inhibitory concentrations (MICs) to the FDA newly approved tetracycline antibiotics, eravacycline (4 µg/ml) and omadacycline (8 µg/ml), in comparison to the results from previous surveillance studies [5,6].
Among them, p2FT39-3 was 68.714 kb in size and harbored 75 open reading frames (ORFs), including tet(X4), erm(42) and floR (Figure 1(A)). Plasmid structural analysis showed that it belonged to the F-: A18:B-plasmid group but lacked the entire conjugative transfer region, which could explain its failure in the conjugation experiment. Blast analysis showed that p2FT39-3 shared similar plasmid backbone (70% query coverage and 99% average sequence identity) against some other plasmids deposited in GenBank, such as p14EC001c (CP024130) from a clinical E. coli strain and p3_W5-6 (CP032995) from an E. coli strain isolated from a contaminated waterway of wild birds.
The tet(X4) gene in E. coli 2ZN37-2 was identified in a 194.2 kb genomic island on its chromosome (4931.759 kb), along with some other antimicrobial resistance genes, including aadA22, bla TEM−1B , qnrS1, lnu(G), and floR (Figure 1(B)). This genomic island was flanked by two copies of IS26 and inserted into a heavy metal resistance gene cusA, with high homology (99%) to the sequence of p2FT38-2-1, likely as a result of IS26-mediated genetic element mobilization. Similar insertion sequence-mediated chromosome integrations have also been described in other resistance genes, including aphA1, tet(D) and bla SHV [12].
We then examined the tet(X4)-neighboring genetic elements among the three strains, and found that tet (X4) was located upstream of ISCR2 and downstream of a hydrolase gene catD (Figure 1(C)). Unlike other insertion sequences, a single copy of ISCR2 could utilize a rolling-circle transposition process to transpose adjacent DNA sequences [13]. It has been found to be associated with the acquisition of diverse antimicrobial resistance genes, including bla VEB−1 [14]. Interestingly, the catD-tet(X4) element in p2FT39-3 was found be flanked by two copies of ISCR2, which strongly suggested that ISCR2 was associated with the mobilization of tet(X4). It was likely that an intact ISCR2 copy originally mobilized catD-tet(X4) by a rolling-circle transposition process and that a secondary process of homologous recombination between two ISCR2 copies led to the deletion of one copy of ISCR2. In combination with the conjugability observed in p2FT38-2-1, our results also suggested that the tet(X4) gene might be able to transfer into other plasmid vectors and spread into other bacterial strains.
In summary, we reported the emergence of tet(X4)encoding tigecycline resistance mechanism in E. coli strains from migratory birds, further highlighting that migratory birds may serve as a reservoir for the dissemination of emerging antimicrobial resistance. Worrisomely, the emergence of tet(X4) challenges the effectiveness of the entire family of tetracycline antibiotics, including the FDA newly approved eravacycline and omadacycline. Further spread of tet(X4) into clinical multidrug-resistant pathogens may create extensively drug-resistant or pandrug-resistant strains, and result in untreatable infections. A continuous surveillance of tet(X4) in humans, animals, and their environments should be considered for understanding and tackling the dissemination of tigecycline resistance.