Chai, Jiyun

Hou, Jie

Lin, Longhua

Ma, Caiping

Schwarz, Stefan

Xu, Qiu

Zhang, Wanjiang

Zhou, Longyu

Zhu, Yao

English

Institutional Repository of the Freie Universität Berlin

Journal of Integrative Agriculture 2025, 24(7): 2881–2884Available online at ScienceDirectJournal of Integrative AgricultureLETTERIdentification of a multidrug-resistant porcine Proteus vulgaris harbouring a plasmid-borne cfr geneJie Hou1*, Qiu Xu1*, Stefan Schwarz2, 3, Longyu Zhou1, Jiyun Chai1, Longhua Lin1, Caiping Ma1, Yao Zhu1#, Wanjiang Zhang1#1 State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China2 Institute of Microbiology and Epizootics, Centre for Infection Medicine, School of Veterinary Medicine, Freie Universität Berlin, Berlin 14163, Germany3 Veterinary Centre for Resistance Research (TZR), School of Veterinary Medicine, Freie Universität Berlin, Berlin 14163, GermanyHighlights● A novel conjugative plasmid pHJ90-cfr carrying the multiresistance gene cfr was characterized in Proteus vulgaris.● A new IS5-family member, ISPmi4, was identified for the first time.● Both plasmids and ICEs were vital mobile genetic elements for horizontal transmission of cfr gene in Proteus species.Received 28 July 2024; Received in revised form 2 September 2024; Accepted 24 September 2024 Available online 6 November 2024#Correspondence Wanjiang Zhang, Tel: +86-451-51051734, E-mail: zhangwanjiang@caas.cn; Yao Zhu, E-mail: zhuyao01@caas.cn*These authors contributed equally to this study.© 2025 CAAS. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co. Ltd.  This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).  Peer review under responsibility of Editorial Board of Journal of Integrative Agriculture.doi: 10.1016/j.jia.2024.11.021Oxazolidinones, such as linezolid, represent the ‘last resort’ antimicrobial agents used to treat life-threatening human infections caused by multidrug-resistant (MDR) Gram-positive bacteria pathogens, such as Methicillin-resistant Staphylococcus aureus (MRSA) and Vancomycin-resistant enterococci (VRE) (Brenciani et al. 2022).  However, the emergence of transferable oxazolidinone resistance genes including cfr, optrA and poxtA, has led to the widely spread of linezolid-resistant bacteria among healthcare, animal and environmental settings, posing a serious threat to public health.  The cfr gene encodes an RNA methyltransferase that targets an adenine residue at position 2,503 of 23S rRNA.  However, this adenine residue is the binding region for phenicols, lincosamides, oxazolidinones, pleuromutilins and streptogramin A antibiotics.  Thus, additional methylation at A2503 mediated by the Cfr protein prevents the binding of the different classes of antimicrobials mentioned above to the ribosome, thereby generating resistance to all of these antimicrobials (Kehrenberg et al. 2005; Long et al. 2006).  Since the first description of the cfr gene in a bovine Staphylococcus sciuri isolate from Germany in 2000 (Schwarz et al. 2000), the cfr gene has been found in a wide variety of different Gram-positive and Gram-negative bacteria, including Staphylococcus, Enterococcus, Bacillus, Macrococcus, Jeotgalicoccus, Streptococcus, Escherichia, 2882 Jie Hou et al.  Journal of Integrative Agriculture  2025, 24(7): 2881–2884Proteus, Pasteurella, Morganella, Providencia, Vibrio and Leuconostoc (Brenciani et al. 2022).  Proteus vulgaris is an important clinical opportunistic pathogen and widely exists in the soil, sewage and intestinal tracts of human and animals. Proteus vulgaris is closely associated with clinical diseases and usually causes urinary tract infections, wound and burn infections, and respiratory tract infections (Dutton and Ralston 1957; Kippaz 1957; Scott 1960).  Herein, we identified a MDR P. vulgaris strain HJ90 isolated from the lung sample of a pig from Heilongjiang province, China, and characterised a cfr-carrying plasmid pHJ90-cfr.  To gain insight into the potential risks posed by the strain HJ90, the genetic background and transferability of the cfr gene located on it was elaborated.Isolate HJ90 was identified as P. vulgaris by 16S rRNA gene sequencing.  Antimicrobial susceptibility testing showed that P. vulgaris HJ90 was resistant to chloramphenicol, florfenicol, gentamicin, sulfamethoxazole, ampicillin and tetracycline according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI 2022).  Whole genome sequencing (WGS) was performed using both Illumina NovaSeq and Nanopore MinION platforms.  The short- and long-read data were de novo hybrid assembled using Unicycler 0.4.3 (Wick et al. 2017) and then annotated using the online RAST server (https://rast.nmpdr.org/) (Aziz et al. 2008) and BLASTP/BLASTN (https://blast.ncbi.nlm.nih.gov/Blast.cgi).  The complete genome of isolate HJ90 was obtained successfully and deposited in the GenBank database under accession numbers (CP150645-CP150647).  The genome of P. vulgaris HJ90 harbored a circular chromosome (4,166,418 bp) and two plasmids, designated pHJ90-1(93,273 bp with an average G+C content of 47%) and pHJ90-cfr (30,447 bp with an average G+C content of 35%), respectively.  Acquired antimicrobial resistance genes (ARGs) were predicted using ResFinder 4.4.3 on the Center for Genomic Epidemiology (CGE) server (http://genomicepidemiology.org/services/) (Bortolaia et al. 2020). The result showed that three ARGs, including aadA1, catB2 and dfrA1 were present in the chromosome.  In addition, multiple ARGs were detected on plasmid pHJ90-1, including aph(4)-la, aadA16, aph(6)-ld, aph(3´´)-la,aph(3´´)-lb, aac(3)-IV, aac(6´)-lb-cr, blaOXA-1, floR, catB3, arr-3, sul1, sul2, tet(B) and dfrA27, and no ARGs was detected on plasmid pHJ90-cfr except cfr.   Plasmid pHJ90-cfr consisted of 41 predicted ORFs with an average G+C content of 35% (Fig. 1-A).  BLAST analysis revealed that no plasmid with significant homology to pHJ90-cfr was found except pJPM35-2 (GenBank accession no. CP053900).  Plasmid pHJ90-cfr displayed 99.98% identity with 96% query coverage to plasmid pJPM35-2, which was recovered from Proteus mirabilis strain YPM35 of duck origin in China in 2021 (Zheng et al. 2021).  The plasmid replicon of pHJ90-cfr was identified using PlasmidFinder 2.1 (https://cge.food.dtu.dk/services/PlasmidFinder/), but could not be assigned to any known Inc group in the database. Interestingly, sequence analysis showed the rep gene encoding plasmid replication initiation protein was absent on pHJ90-cfr.  A plasmid segregation protein-encoding gene parM was present on plasmid pHJ90-cfr.  A number of conjugative transfer genes were found on pHJ90-cfr, including virB4, virB5, virB8, virB9, virB10, tfc2, virB1, trbl, virB11 and virB3.  To evaluate the transferability of plasmid pHJ90-cfr, conjugative transfer assay was carried out using filter mating as previously described (Yang et al. 2023).  The result indicated that pHJ90-cfr was able to transfer into the recipient Escherichia coli C600 (streptomycin resistant), which was consistent with the previous report (Zheng et al. 2021).  Additionally, antimicrobial susceptibility testing showed that the positive transconjugant displayed resistance to florfenicol and chloramphenicol.  Genetic environment analysis suggested that the cfr gene, together with three hypothetical protein genes hp, were flanked by two copies of IS26 elements located in the same orientation (IS26-hp-hp-hp-cfr-IS26).  BLASTN search showed that the segment bracketed by IS26 displayed >99% nucleotide sequence identity to the corresponding region of plasmid pJZ27-cfr from P. mirabilis isolated from chicken meat in China (Fig. 1-B).  It is suggested that the IS26-flanking segment may have the ability to spread widely across bacterial species, especially among Proteus spp.  Based on previous reports (Harmer et al. 2014; Zhu et al. 2021), a circular translocatable unit (TU) can form when two copies of insertion sequence (IS) elements belonging to IS6 family orientated in the same direction and flanking resistance genes.  To test whether such a TU was generated, a PCR assay was conducted and the result demonstrated that a circular TU mediated by IS26 was formed.  Sequence analysis showed that this TU contained an IS26 copy and the central region between two IS26 copies.  Since the sequences of two IS26 copies were identical, it is impossible to determine which one was included in this TU.  This finding further supports the view that IS26 plays a vital role in promoting the dissemination of cfr gene in Gram-negative bacteria (Wang et al. 2012; Harmer et al. 2014; Liu et al. 2022; Mei et al. 2022).  Furthermore, sequence alignment revealed that compared with plasmid pHJ90-cfr, pJPM35-2 carried an extra ~4-kb fragment, which was flanked by two IS5-like elements copies orientated in the same direction (Fig. 1-B).  Sequence analysis showed that IS5-like element was 853 bp in length and contained a 756 bp ORF for a transposase of 251 amino acids (aa). Blast searches showed that this transposase shared 57% aa similarity with that of ISAba31, a member of IS5 family. Therefore, it is reasonable to speculate that the IS5-like element was new IS5-family member.  This new IS element 2883Jie Hou et al.  Journal of Integrative Agriculture  2025, 24(7): 2881–2884was submitted to the ISfinder database and received the designation ISPmi4.  Immediately up- and downstream of the fragment bounded by ISPmi4, 2-bp direct repeat sequences (5´-TA-3´) were found, which was a typical characteristic of classical composite transposons.  This suggested that ISPmi4-flanking segment was inserted into the precursor of plasmid pJPM35-2, resulting in the structure seen on pJPM35-2.  A search on PubMed database revealed that all identified cfr-carrying Proteus spp. were found in food-producing animals or retail meat in China until now (Appendix A), which implied that Proteus spp. from food-producing animals may be a vital reservoir for cfr gene in China. Additionally, the cfr gene was found mainly located on two kinds of mobile genetic elements, namely plasmids and integrative and conjugative elements (ICEs) in Proteus.  The above result suggested that plasmids and ICEs were key vectors for horizontal transmission of cfr gene in Proteus spp.In summary, a novel conjugative plasmid pHJ90-cfr Fig. 1  A, circular map of the cfr-carrying plasmid pHJ90-cfr.  The Δ symbol indicates a truncated gene.   B, comparison of pHJ90-cfr and pJPM35-2 and the genetic context of cfr.  Arrows indicate the direction of transcription.  Genes with different functions are displayed by different colors.  Regions of >99% homology are marked by gray shading.ISPmi4ΔtnpΔtnp IS26IS26pHJ90-cfrProteus vulgarisPigpJZ27-cfrProteus mirabilisChicken meatON553414.1 pJPM35-2Proteus mirabilisDuckCP053900.1virB11virB3 virB430 kbppHJ90-cfr30,447 bp28 kbp26 kbp24 kbp22 kbp20 kbp18 kbp16 kbp 14 kbp12 kbp10 kbp8 kbp6 kbp4 kbp2 kbpvirB5GC contentABGC skewGC skew (–)GC skew (+)pHJ90-cfrvirB8virB9virB1tfc2trblΔtnpIS26cfrIS26parMvirB4virB5virB8virB4virB5virB8virB9virB1trblIS26IS26ISPmi4parMTA TAcfrvirB10tfc2virB9virB10virB1tfc2trblIS26IS26cfrparMvirB11virB3virB11virB32884 Jie Hou et al.  Journal of Integrative Agriculture  2025, 24(7): 2881–2884from P. vulgaris was characterized, which harbored the multiresistance gene cfr.  In addition, a new IS5-family member, ISPmi4, was identified.  The presence of cfr on plasmids, especially on conjugative plasmids, deserves our attention.  Our findings highlight that the prevalence of cfr gene in Gram-negative bacteria should be further monitored.AckowledgementsThis work was supported by the National Key Research and Development Program of China (2022YFF0710505) and the Central Public-interest Scientific Institution Basal Research Fund, China (1610302022001).  Declaration of competing interest The authors declare that they have no conflict of interest.Ethical statementsAll applicable international, national and institutional guidelines for the care and use of animals were followed.Appendix associated with this paper is available at https://doi.org/10.1016/j.jia.2024.11.021ReferencesAziz R K, Bartels D, Best A A, DeJongh M, Disz T, Edwards R A, Formsma K, Gerdes S, Glass E M, Kubal M, Meyer F, Olsen G J, Olson R, Osterman A L, Overbeek R A, McNeil L K, Paarmann D, Paczian T, Parrello B, Pusch G D, et al. 2008. The RAST Server: Rapid annotations using subsystems technology. 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Identification of a multidrug-resistant porcine Proteus vulgaris harbouring a plasmid-borne cfr gene

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Identification of a multidrug-resistant porcine Proteus vulgaris harbouring a plasmid-borne cfr gene

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