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

Abstract

The widespread occurrence of carbapenem-resistant organisms has garnered significant public attention. Arthropods, 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 blaNDM genes in these three isolates varied. The blaNDM-5 gene in E. coli was located on an IncHI2/HI2A multidrug-resistant plasmid, which was conjugatively transferable. The blaNDM-1 gene in P. mirabilis resided on the pPM14-NDM_123k-like nonconjugative plasmid. The blaNDM-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.

Main text

Carbapenem-resistant organisms (CROs) are a significant concern in the global landscape of infectious diseases and affect both humans and animals. Surveillance 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. A total of four isolates were isolated using China Blue Agar plates supplemented with 0.3 μg/mL meropenem (Zhang et al. 2022). Species identification was performed using matrix-assisted laser desorption and ionization–time-of-flight mass spectrometry (MALDI-TOF MS, Bruker Daltonics, Bremen, Germany). Among the four isolates, 36-1-1, 36-1-2, 36-2-1 and 36-2-2 were identified as Escherichia coli (E. coli), Providencia manganoxydans (P. manganoxydans), Myroides odoratimimus (M. odoratimimus) and Proteus mirabilis (P. mirabilis), respectively. The minimum inhibitory concentrations (MICs) for the four isolates were determined by broth microdilution with E. coli ATCC 25922 as the control, and the results were interpreted according to CLSI standards (Clinical & Laboratory Standards Institute 2020). All four isolates were resistant to imipenem, meropenem, cefmetazole, ceftazidime, cefotaxime, piperacillin-tazobactam, cefoperazone-sulbactam, cefepime and ceftazidime-avibactam. All isolates, except for E. coli 36-1-1, were resistant to polymyxin B (Table 1).

Table 1 Minimum inhibitory concentration (µg/mL) profiles of isolates in this study

Genomes of the four isolates were sequenced using the Illumina HiSeq 2500 platform with a 2 × 150 bp paired-end strategy. De novo sequence assembly was performed using SPAdes V.3.15.5 (Bankevich et al. 2012). M. odoratimimus exhibited intrinsic resistance to carbapenem, partly due to the production of the chromosome-encoded β-lactamase MUS (Yang et al. 2020). In the M. odoratimimus isolate 36-2-1, only one antibiotic resistance gene (ARG), blaMUS-1, was identified. Among the other three isolates, 10-14 had acquired ARGs conferring resistance to various classes of antibiotics. Carbapenem resistance in the E. coli 36-1-1, P. manganoxydans 36-1-2, and P. mirabilis 36-2-2 isolates was associated with the production of NDM-5, NDM-1 and NDM-1, respectively (Fig. 1A).

Fig. 1
figure 1

A 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 blaNDM-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

Genome typing with ClermonTyping, mlst, and ECTyper suggested that E. coli 36-1-1 belongs to phylogroup A, which represents a novel sequence type (MLST alleles: adk (10)-fumC (7)-gyrB (5)-icd (8)-mdh (8)-purA (18)-recA (2)) and serotype O128:H19 (Seemann 2016; Beghain et al. 2018; Bessonov et al. 2021). Genetic alignment suggested that the blaNDM-5 gene in E. coli 36-1-1 was located on a pNDM33-1-like multidrug-resistant plasmid. pNDM33-1 (GenBank accession: MN915011) is an IncHI2/HI2A plasmid that is 266,777 bp in length and encodes 330 ORFs with a G+C content of 47.1% (Fig. 1B). In addition to blaNDM-5, pNDM33-1 also carried other ARGs, which were detected in the genome of E. coli 36-1-1, including aph(3’)-I, aph(4)-I, blaOXA-10, sul3, lnu(F), aac(3)-IVa, aadA2, dfrA14, and floR.

In P. mirabilis 36-2-2, the blaNDM-1 gene was located on the pPM14-NDM_123k-like plasmid. The pPM14-NDM_123k plasmid (GenBank accession: CP137087) is 123,188 bp in length and encodes 136 ORFs with a G+C content of 40.6% (Fig. 2). It also harbors ARGs, including dfrA1, mph(E), msr(E), sul, aadA1, and lnu(F).

Fig. 2
figure 2

Circular alignments of the reference plasmid sequence pPM14-NDM_123k (GenBank accession: CP137087) with homologous blaNDM-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

The blaNDM-1 gene in P. manganoxydans 36-1-2 was located on an 8.9 kb contig that also carried ARGs such as sul1, blaOXA-1, catB3, aac(6’)Ib-cr and arr-3. This contig was 100% identical to the corresponding chromosome fragment bordered by the mobile elements IS26 and ISCR1 from P. mirabilis XH1653 (GenBank accession: CP065039), with 98% coverage. The genetic context of blaNDM-1 in P. manganoxydans 36-1-2, qacE1-sul1-ISCR1-hp-blaNDM-1-ISAba125, was similar to that in P. mirabilis, which was qacE1-sul1-ISCR1-hp-hp-blaNDM-1-ISAba125 (Fig. 3). This evidence collectively suggested the mobility of such a blaNDM-1-carrying fragment.

Fig. 3
figure 3

Genetic context of blaNDM-1 in P. mirabilis F36-2-2 and P. manganoxydans F36-1-2. The cyan, blue and orange arrows represent resistance genes, mobile genetic elements and other proteins, respectively

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 were selected on agar plates containing 0.5 μg/mL meropenem. The carbapenem resistance of E. coli 36-1-1 was transferable, whereas that of the other three isolates was nonconjugative. The nonconjugative nature of the three isolates suggested that there are other mechanisms involved in the transmission of carbapenem resistance in these bacteria.

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 blaNDM-5 gene in E. coli was plasmid-borne and conjugatively transferable. The blaNDM-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.

Availability of data and materials

The assembled genome sequences of isolates in this study have been deposited in the National Center for Biotechnology Information (NCBI) database under Bioproject PRJNA1039261.

Abbreviations

CRO:

Carbapenem-resistant organism

CHINET:

China Antimicrobial Surveillance Network

MALDI-TOF MS:

Matrix-assisted laser desorption and ionization–time-of-flight mass spectrometry

CLSI:

Clinical and Laboratory Standards Institute

ARG:

Antibiotic resistance gene

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Acknowledgements

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Funding

This study was funded by the National Natural Science Foundation of China (Grant No. 32300156) and the Natural Science Foundation of Jiangsu Province (Grant No. BK20220493).

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HW performed strain isolation and phenotypic characterization and wrote the first draft. HZ and GC performed the genomic characterization. ND conceived the study, participated in its design and coordination, and edited the manuscript. All authors read and approved the final manuscript.

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Correspondence to Ning Dong.

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Wang, H., Zhou, H., Chen, G. et al. Isolation of four carbapenem-resistant gram-negative species from a single fly. Animal Diseases 4, 4 (2024). https://doi.org/10.1186/s44149-024-00110-5

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