Isolation of four carbapenem-resistant gram-negative species from a single fly

The widespread occurrence of carbapenem-resistant organisms has garnered significant public attention. Arthro-pods, including flies, are important vectors of multidrug-resistant bacteria. In this study, we reported the simultaneous carriage of four carbapenem-resistant isolates from different species, namely, Escherichia coli ( E. coli ), Providencia manganoxydans ( P. manganoxydan ), Myroides odoratimimus ( M. odoratimimus ) and Proteus mirabilis ( P. mirabilis ), from a single fly in China. These isolates were characterized through antimicrobial susceptibility testing, conjugation assays, whole-genome sequencing, and bioinformatics analysis. M. odoratimimus showed intrinsic resistance to carbapenems. The mechanisms of carbapenem resistance in E. coli , P. manganoxydans , and P. mirabilis were due to the production of NDM-5, NDM-1 and NDM-1, respectively. Genetic context of the bla NDM genes in these three isolates varied. The bla NDM-5 gene in E. coli was located on an IncHI2/HI2A multidrug-resistant plasmid, which was con-jugatively transferable. The bla NDM-1 gene in P. mirabilis resided on the pPM14-NDM_123k-like nonconjugative plasmid. The bla NDM-1 gene in P. manganoxydans was found in a nonconjugatively transferable, multidrug-resistant region. The results of this study enhance our understanding of the dissemination of carbapenem-resistant organisms and suggest the need for a more comprehensive approach to antibiotic resistance research encompassing humans, animals, and the environment.

networks such as the CHINET and dedicated pet/animal monitoring initiatives highlight the importance of CROs, emphasizing their prevalence and impact (Yang et al. 2023;Sands et al. 2021).Apart from human carriage, the transmission of multidrug-resistant bacteria, including CROs, can also occur through arthropods, particularly flies that feed on human and animal wastes (Hassan et al. 2021).In this study, we collected a fly from a sheep farm in Hubei Province, China, which carried four CROs belonging to different species.This study advocates for a more comprehensive approach to antibiotic resistance research involving not only humans but also animals and the environment.
In April 2023, a fly was collected using flypaper and subsequently homogenized by grinding in sterile saline.The homogenate was transferred into 5 mL LB broth and incubated at 35°C for 18-20 h for amplification.
To test the transferability of the carbapenem-resistant phenotype, we conducted a conjugation experiment with the filter-mating method using rifampicin-resistant E. coli EC600 as the recipient.Transconjugants

Conclusions
In summary, this study reported the simultaneous carriage of four carbapenem-resistant isolates from different species, namely, E. coli, P. manganoxydans, M. odoratimimus and P. mirabilis, from one fly in China.M. odoratimimus was intrinsically resistant to carbapenems, and the mechanism of carbapenem resistance in the other isolates involved the production of NDM carbapenemases.The bla NDM-5 gene in E. coli was plasmid-borne and conjugatively transferable.The bla NDM-1 genes in the P. manganoxydans and P. mirabilis isolates were both nontransferable.Our findings contributed valuable insights to understanding the dissemination of CRO and posed critical questions about the correlations among antibiotic resistance in livestock, flies, and humans.

Fig. 1 A
Fig.1A Heatmap of antimicrobial resistance genes carried by carbapenem-resistant isolates from a fly in this study.The horizontal axis represents the antimicrobial resistance genes, and the vertical axis represents the isolate IDs.The red boxes represent the presence of the corresponding items among the sequenced isolates, and the white boxes represent their absence.The gradient identity bar indicates the percentage similarity of related genes.B Circular alignment of the reference plasmid sequence pNDM33-1 (GenBank accession: MN915011) with homologous bla NDM-5 -carrying contigs from the E. coli isolate 36-1-1 in this study.Representative genes, such as antimicrobial resistance genes and conjugation-associated genes, are labeled in the outermost circle

Fig. 2
Fig.2Circular alignments of the reference plasmid sequence pPM14-NDM_123k (GenBank accession: CP137087) with homologous bla NDM-1 -carrying contigs from the Proteus mirabilis strain 36-2-2 in this study.Representative genes, such as antimicrobial resistance genes and conjugation-associated genes, are labeled in the outermost circle