Deaths from diarrhea in children are still a health challenge worldwide, with particular concerns in low- and middle-income countries . E.coli play an important role in the death of children by diarrhea, being involved in more than 120,000 deaths annually of children under 5 years old [17, 18]. The treatment for less severe diarrhea is often to replace lost fluids and salts with oral rehydration saline solutions to reduce the risk of dehydration, while patients with severe diarrhea are often treated with antibiotics. However, antibiotic resistance is rapidly increasing due to their commonly use, and new and alternative treatment antibacterial drugs such as azithromycin have been proposed [9, 19]. In fact, azithromycin treatment may cause E. coli to reduce Stx production in vivo and in vitro . According to current researches and reports, E. coli resistance to azithromycin is generally less than 30%, and most results show that the resistance rate is between 10% and 20% . However, in this study, we reported that a high proportion (approximately 86.7%) of azithromycin-resistant ETEC O6 strains was prevalent in Shanghai, China. To our knowledge, this is the first study to report a high proportion of azithromycin resistance in a particular ETEC serogroup due to a specific plasmid carrying mphA.
Shanghai is an important municipality of China with 6340.5 square kilometers and a population of 24.278 million. According to previous research, infectious diarrhea caused by ETEC in Shanghai mainly occurs in adults 20 to 60 years old, and July to August is the peak period of ETEC infection . We investigated adults aged 20–60 who were infected with ETEC and had symptoms of diarrhea in July or August. From the regional perspective, azithromycin-resistant ETEC O6 strains appeared in six distinct districts. In terms of time, the resistance rates of azithromycin in strains isolated in different years were 50.0% (3/6) in 2016, 100.0% (8/8) in 2017 and 93.8% (15/16) in 2018, which increased significantly in 2017 and 2018. These indicate that azithromycin-resistant ETEC O6 strains have become widespread in Shanghai in recent years. Phylogenetic analysis showed that 28 of the 30 strains self-tested were closely clustered to form a new branch, indicating that a new dominant clone had been formed locally. Of these strains, 26/28 carried mphA except two isolated in 2016, indicating the mphA-carrying part of the clone’s rapid spread in local areas.
Besides azithromycin, the vast majority of these strains are also resistant to nalidixic acid and ampicillin, and a few resistant to tetracycline, gentamicin, streptomycin, trimethoprim and sulfisoxazole. This is consistent with the results of other studies [7, 22]. It is worrying that ampicillin is still employed as one of the first line treatments in most low and middle-income countries [19, 23]. Only one of these strains is sensitive to 14 different antibiotic agents, which indicates the severity of antibiotic resistance of ETEC strains in China. The analysis of antibiotic resistance genes showed that 26 strains resistant to azithromycin all carried the mphA gene, and none carried the ere family genes and the erm family genes, indicating that mphA is an important reason of these strains resistance to azithromycin. The macrolide-resistant phosphotransferase-encoding mphA gene is the most common azithromycin resistance gene detected in E. coli . Studies have shown that the presence of the mphA is currently the most important mechanism for high resistance to azithromycin, and 93% of the strains with MIC values above 32 mg/L .
The mphA can exist on chromosomes and on plasmids, but E. coli is often carried in the form of plasmids, allowing it to spread widely across this species and species closely related to its phylogenetic evolution. The mphA has been reported to be located on various plasmids . In this study, mphA was located on a 103 kb plasmid. This plasmid is a relatively new plasmid. Compared with other plasmid data in the NCBI database, other plasmids can cover only up to 79% of the plasmid. Compared with other plasmids in the NCBI database, other plasmids can only be up to 79% of the plasmid. The plasmid carries two resistance genes, mphA and blaTEM-1, which confer resistance to azithromycin and ampicillin in strains carrying the plasmid. The most common plasmid carrying mphA is in the form of IS26-mphA-mrx-mphR(A)-IS6100 units, but it is present in the form of IS15DIV_aa1-mphA units in this plasmid [26, 27]. Our research suggested that a new plasmid carries the mphA gene in a novel way, and this plasmid appeared in large numbers of ETEC O6 stains in Shanghai in a short period.