Florfenicol-resistant, Rabbit-derived Providencia Rettgeri Isolate With 4 Copies of oR Genes in a Tandem-repeat Plasmid

Background: This work aimed to investigate the resistance mechanism to antimicrobial agents (especially orfenicol) exhibited by Providencia isolates obtained from various sources. Methods: The polymerase chain reaction (PCR) was performed to detect the distribution of the orfenicol resistance genes. Conjugation, S1-PFGE (S1 nuclease pulsed-eld gel electrophoresis), sequencing and other molecular methods were employed to analyze the transferability and structure of the resistance gene-encoding plasmids. Results: It showed that the human pathogens exhibited a wider resistance spectrum, while the animal bacteria had higher MIC values for most antibiotics analyzed. Of the seven orfenicol resistance genes (oR, fexA, fexB, optrA, estDL136, cfr and pexA) screened, only oR was identied in both animal (5/10, 50%) and human bacteria (3/13, 23.1%), and none of them was detected in freshwater sh isolates (0/22). The oR-positive strains exhibited high MIC levels for orfenicol ( ≥ 64 mg/L). The whole genome sequencing of the multidrug-resistant Providencia rettgeri strain R39 demonstrated that it carried four plasmids, with two larger plasmids encoding 5 copies of the oR genes. The largest plasmid, pR39-33 (32,936 bp) with 4 oR genes, was composed of 4 copies of the second large plasmid pR39-8 which encoded 8 ORFs (open reading frames) including a oR and two mobilization genes (mobAC) in the form of tandem repeat. It indicated that the oR gene represented the primary mechanism underlying the orfenicol resistance of the Providencia isolates. Conclusions: This report is the rst to describe an antibiotic-resistant plasmid composed of four copies of another plasmid sequence in the form of a tandem repeat.


Background
The Providencia species belong to Enterobacteriaceae; unlike many other bacteria of this family, these species are considered to be uncommon causes of infections. The genus Providencia consists of eight species that have been isolated from a wide variety of environments, such as wastewater, oceans, food animals and human clinical isolates. Providencia species have been isolated from numerous animal sources worldwide, including penguin feces in German zoos, sea turtles in the Mediterranean [1], shark mouths in Brazil [2], and so on. Among the species in the genus Providencia, P. stuartii, P. rettgeri and P. alcalifaciens are the most common human opportunistic pathogens. P. stuartii strains have been reported to cause bacterial pericarditis [3], meningitis [4] and peritonitis [5]. P. rettgeri has been isolated from patients with urinary tract infections [6], nephrocutaneous stula [7], and the bacteria has been obtained from the soft tissue of the toe of a patient suffering from diabetes [8]. P. alcalifaciens is the most frequently reported causative agent of diarrhea outbreaks in both humans [9] and animals [10][11][12]. With the overuse of antibiotics in human clinical and agricultural practice, an increasing number of multidrug-resistant Providence strains have been isolated [13][14][15][16]. Providencia stuartii was observed to simultaneously carry the bla VIM−2 , bla PER−1 and armA genes [14,17].
Florfenicol is a derivative of chloramphenicol that is utilized to treat bacterial infections in animals. However, compared with chloramphenicol, orfenicol is a special veterinary drug that chloramphenicol acetyltransferase cannot deactivate [18,19]. Florfenicol is able to bind the 50S ribosomal subunit to inhibit the protein biosynthesis of the bacteria. Because of the wide antimicrobial spectrum, good absorption, and wide distribution in the animal body with few side effects, orfenicol has been widely utilized in veterinary medicine and animal farming [18,20]. Accompanying the use of orfenicol, an increasing number of orfenicol-resistant bacteria, such as Pasteurella multocida [ However, the resistance characteristics of these bacteria, especially the molecular resistance mechanism of Providencia rettgeri to orfenicol, have not been elucidated. In this work, the orfenicol resistance of 45 Providencia isolates of different sources was analyzed, and the genome structure of Providencia rettgeri with 5 copies of oR genes was characterized.

Materials And Methods
Bacterial strains and antimicrobial susceptibility testing The bacterial isolates examined in this work are listed in Table 1 and Table S1

Polymerase chain reaction analysis
The template genomic DNA of the isolates was extracted by AxyPrep Bacterial Genomic DNA Miniprep kits (Axygen Scienti c, Union City, CA, USA). The primers for the 7 orfenicol resistance-associated genes (fexA, fexB, oR, cfr, optrA, pexA and estDL136) are presented in Table 2. The PCR products were puri ed using a MinElute PCR Puri cation kit (QIAGEN China, Shanghai, China) and sequenced by Sanger sequencing (Annoroad Gene Technology Co., Ltd., Beijing, China). The nucleotide sequences of the PCR products were analyzed and compared with those in the NCBI nucleotide database using the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST).

Genome sequencing, assembly and bioinformatic analyses
The whole genome sequence of P. rettgeri R39 DNA was sequenced by Illumina HiSeq 2500 and Paci c Bioscience sequencers at Annoroad Gene Technology Co., Ltd. (Beijing, China). The sequence contigs were obtained using SOAPdenovo software to assemble the reads. When a gap was encountered, we remapped the short reads or sequenced the PCR product of the gap. Potential open reading frames (ORFs) were predicted using Glimmer3.02 with default parameters (http://ccb.jhu.edu/software/glimmer) and annotated by BLASTX (https://blast.ncbi.nlm.nih.gov) with an e-value threshold of 1e-5. Gview (https://server.gview.ca/) was utilized to construct basic genomic features that were subsequently employed in comparative genomic analysis.
Collection and processing of plasmid sequences We compared the sequence of pR39-8 against the NCBI nucleotide database with a cutoff value (max score) of approximately 8700. The sequences with ≥ 80% identity with pR39-8 were retained as candidates for later orthologous analysis using BLASTP [33] and InParanoid [34]. The other bioinformatic tools used in this work were written with Python and Biopython scripts.

S1-PFGE and restriction enzyme digestion of plasmids
Pulsed-eld gel electrophoresis (PFGE) was performed as described previously [35]. P. stuartii genomic DNA in agarose blocks was digested with S1 nuclease (Takara, Dalian, China). A contour-clumped homogeneous electric eld (CHEF) method was used for S1-PFGE on a CHEF DRII system (Bio-Rad Laboratories, Munich, Germany). The resulting fragments were resolved in a 1.0% agarose gel run at 6 V/cm in 0.5*TBE (Tris-borate-EDTA) electrophoresis buffer at 14°C, with switch times ramping from 3 s to 30 s for 14 hours.
Plasmid DNA from P. stuartii R39 was isolated using the QIAGEN Plasmid Midi Kit (QIAGEN GmbH, Germany). Plasmid bands from agarose gels were isolated using the Omega E.Z.N.A. Ultra-Sep Gel Extraction Kit (Omega Bio-tek, Norcross, USA) according to the protocol. The ngerprinting of the isolated plasmid DNA was con rmed by digestion with the restriction enzymes BamHI (Takara, Dalian, China) and/or EcoRV (Takara, Dalian, China) at 37°C for 2 h, and agarose gel electrophoresis was performed to determine the sizes of the resulting restriction fragments.
Cloning of the open reading frame of the oR gene with its promoter region To con rm the function of the oR genes, we PCR-ampli ed and cloned the ORF of the oR gene with its predicted promoter region (http://www.softberry.com/berry.phtml? topic=bprom&group=programs&subgroup=g ndb) from R39 into the pUC18 vector. The primers ( Table 2) were designed using Primer Premier 5.0 and synthesized by Shanghai Sunny Biotechnology Co., Ltd. (Shanghai, China). The plasmid containing oR (pUC18-oR) was transformed into E. coli DH5α using the calcium chloride method, and bacterial colonies were grown on Luria-Bertani agar plates supplemented with ampicillin (100 mg/L). Recombinants (pUC18-oR/DH5α) were con rmed by sequencing and MICs against the corresponding antibiotics.
Plasmid curing P. rettgeri R39 was cultured on LB plates at 37°C overnight. Colonies were selected and cultured in 3 mL LB broth in a vibrating incubator (37°C, 200 r/min) for 16 h, and 60 μL of the cultures were then transferred to 3 mL LB broth supplemented with a sublethal concentration (16 mg/L) of sodium dodecyl sulfate (SDS) (Sigma, USA) and incubated in a vibrating incubator (43°C, 200 r/min) for 20 h. Later, 60 μL of the cultures were transferred to 3 mL of LB broth and cultured in a vibrating incubator (37°C, 200 r/min) for 16 h. The last two procedures were performed once and a second time. In every 2-3 rounds of the aforementioned procedures, the culture was checked to determine whether the plasmid was cured by the following procedures.
The cultures were diluted in 0.9% normal saline, plated onto LB plates and incubated at 37°C for 24 h. Each colony was streaked on two LB plates, of which one was supplemented with 32 mg/L orfenicol, while the other was free of antibiotics. The colonies that grew on plates free of orfenicol and failed to grow on plates with orfenicol were de ned as putative plasmid-cured derivatives. The physical loss of the plasmid in the cured derivative was further con rmed by PCR of the oR gene, antimicrobial testing, plasmid extraction and agarose gel electrophoresis.

Nucleotide sequence accession number
The nucleotide sequences for the plasmids pR39-8 and pR39-33 and the whole chromosome sequence of P. rettgeri R39 were submitted to DDBJ/EMBL/GenBank under accession numbers CP066317, CP066316 and CP066315, respectively.

Results
Antibiotic susceptibility testing and resistance gene screening of Providencia strains A total of 45 nonredundant strains of ve Providencia species were analyzed, and the strains included 29 Providencia rustigianii isolates, 5 Providencia alcalifaciens isolates, 5 Providencia stuartii isolates, 4 Providencia rettgeri isolates, and 2 Providencia vermicola isolates. The bacteria from 22 freshwater sh were all P. rustigianii isolates, while the 2 P. vermicola isolates were obtained from the animal specimens only. Thirteen isolates from patients were species of P. alcalifaciens, P. rettgeri and P. stuartii (Table S1).
The clinical isolates had a wider resistance spectrum and showed resistance to 13 antibiotics (ranging from 7.7% to 100%). The animal isolates showed resistance to 8 antibiotics (ranging from 10.0% to 80.0%), while the freshwater sh bacteria only showed resistance to 5 antibiotics (ranging from 4.5% to 50.0%). Notably, although the clinical strains showed a wider resistance spectrum, the animal strains showed higher resistance rates than the clinical strains in 5 of 9 antibiotics, which they both showed resistance to, especially to orfenicol (the animal strains with a resistance rate of 50%, while the human clinical isolates was only 23.1%) ( Table 3, Table S2).
Detection of the orfenicol resistance genes demonstrated that of the 7 genes ( oR, fexA, fexB, optrA, estDL136,cfr and pexA) screened, only the oR gene yielded positive results in both human (3/13, 23.1%) and animal bacteria (5/10, 50%). The oR-positive strains had high MIC levels for orfenicol (> 32 mg/L) and chloramphenicol, while the oR-negative isolates showed notably lower MIC levels (with all of them exhibiting MIC levels < 8 mg/L) (Table S2).
General features of the P. rettgeri R39 genome To analyze the molecular mechanism governing the orfenicol resistance of the bacteria from animal sources, the whole genome of P. rettgeri isolate R39, which exhibited the highest orfenicol MIC level (Table  S2) Fig. 1). The whole genome was observed to encode 9 resistance genes, of which 4 (aadA1, sat2, catB2 and drfA1) were located on the chromosome, while the other 5 oR genes were located on the plasmids, with 4 oR genes on pR39-33 and one oR gene on pR39-8 (Fig.  1a, Fig. 1b). According to the drug susceptibility results, the drug resistance gene pro le was consistent with the drug resistance spectrum of the bacterium (Table S2). We digested the plasmids extracted from R39 with BamHI or EcoRV alone, or with the combination of BamHI and EcoRV, and the results were obtained as predicted. When digested with BamHI or EcoRV alone, a single band of approximately 8 kb was obtained. When digested with BamHI + EcoRV, two bands of approximately 3.5 kb and 4.5 kb were observed (Fig. 2b). The digestion results of the recovered pR39-33 with BamHI or EcoRV alone or with both BamHI and EcoRV also yielded the expected results (Fig. 2c). This result con rmed that the structure of pR39-33 consisted of a tandem repeat of 4 copies of pP39-8 sequences.
Comparative genomic analysis of the oR gene encoding plasmids As mentioned above, R39 carried 2 resistant plasmids (pR39-33 and pR39-8), and pR39-33 is composed of a tandem repeat of four copies of the pR39-8 sequence. pR39-8 encoded 7 complete ORFs and a truncated insertion sequence (ΔIS91). These seven genes included a oR gene, 2 replication-related genes (repA and repB), 2 mobilization-related genes (mobA and mobC) and 2 hypothetical protein genes. The oR gene was related to a transposon-like structure (ΔIS91-hp-oR).
The ve plasmids with the highest similarity (> 90% identity and > 83% coverage) to pR39-8 were retrieved from the NCBI nucleotide database (https://www.ncbi.nlm.nih.gov/nucleotide/). These plasmids have ve or more genes that are the same as those encoded on pR39-8. The six plasmids described in this study were from bacteria of four different genera. Speci cally, both plasmid pRCADGH-1 and plasmid pCCK381 were from Pasteurella multocida, the plasmids pM3446F and unamed1 were from Actinobacillus pleuropneumoniae, pMh1405 is a Mannheimia haemolytica plasmid, and pR39-8 described in this work was from a strain of P. rettgeri. Of the ve plasmid sequences obtained from the database, pRCADGH-1 exhibited the same sequence as pR39-8, but the plasmids were from two different genera, that is, Pasteurella multocida and Providencia rettgeri, respectively (Fig. 3, Table S3).

Discussion
In this study, Providencia strains isolated from different sources showed different resistance spectra of antibiotics tested. Although human bacteria are resistant to more antibiotics than animal or freshwater sh bacteria, animal Providencia strains showed higher resistance rates against some of the antibiotics, especially quinole (nor oxacin and levo oxacin) and amphynol (chloramphenicol and orfenicol), compared with human bacteria. This phenomenon may be attributable to different antibiotic selection pressures, as these antibiotics are commonly used in veterinary medicine or animal farming [36]. Amphenol has been widely used in animals but seldom (chloramphenicol) or not ( orfenicol) used in human clinical practice [37].
The antibiotic susceptibility pro les of human clinical Providencia isolates have been reported worldwide. The isolates are generally resistant to gentamicin, tobramycin, aminopenicillins and rst-generation cephalosporins, but they are commonly susceptible to carbapenems, amikacin, aztreonam, and second-and third-generation cephalosporins [38]. Similar to previous reports, the human clinical Providencia isolates analyzed in this study were also sensitive to aztreonam and meropenem and resistant to cephalosporins, gentamicin, aminopenicillins, and imipenem to different degrees. Until recently, few publications have reported the resistance pro le of Providencia isolates obtained from animals or marine sources. A previous publication reported the antibacterial susceptibility of two Providencia alcalifaciens isolates from shark against 13 antibiotics, with the results showing that the two isolates were both sensitive to 7 antibiotics (ampicillin, amikacin, cipro oxacin, levo oxacin, ceftazidime, imipenem and aztreonam) and that only one isolate of the two showed resistance to the other 6 antibiotics (gentamicin, estreptomicin, chloramphenicol, cefalotin, amoxicillin + clavulanicacid and tetracycline) [2]. Other reports have primarily examined the treatment of animal infections caused by Providencia [10] or investigated the bacterial contamination of animal products, including retail meats, such as chicken, pork, and beef [39].
Notably, in this study, a larger plasmid was determined to be composed of four copies of a smaller plasmid sequence in the form of a tandem repeat in one host, which made the host to carry 5 copies of the oR genes.
On the other hand, in P. rettgeri R39, the plasmids carrying the oR genes were not related to the intact mobile genetic elements, although a small part of an IS91 remnant was located nearby. It has been found that the oR genes are related to a variety of mobile genetic elements, such as integrons, transposons and insertion sequences of the different structures that are encoded on chromosomes or plasmids across different species or genera of the bacteria [21,40]. The homologous plasmids (> 90% identity and > 83% coverage) of pR39-8 were distributed over several bacteria of different origins, including pRCADGH-1 from a Pasteurella multocida isolate obtained from duck in Chengdu, Sichuan Province, China, pCCK381 of Pasteurella multocida and pMH1405 in M. haemolytica, both isolated from calves in Japan, pM3446F of Actinobacillus pleuropneumoniae, obtained from a pig in Greece, and the plasmid unnamed1 of an Actinobacillus pleuropneumoniae from a pig in South Korea, while the P. rettgeri R39 described in this work was isolated from a rabbit in Wenzhou, Zhejiang Province, China. These plasmids were characteristic of encoding mobilization-related genes (all carrying mobA and mobC, with the exception of pM3446F, which carried mobA alone) [21-23, 41,42]. This nding might indicate that these plasmids are transferred mainly by a mobilization mechanism.
R39 carried 5 copies of the oR genes encoded on the two plasmids. To the best of our knowledge, no publication has documented one strain encoding more than two copies of oR genes. In addition, PCR screening for the oR gene showed that it was present not only in animal-origin Providencia strains but also in human-source isolates. Several studies have also reported the identi cation of oR genes in human clinical pathogens, such as Vibrio cholera [43] and Klebsiella pneumonia [44]. Florfenicol has been utilized only for animals. The spread of the oR gene from animal bacteria to human microbes re ects the risk to human health posed by the overuse of antibiotics in veterinary medicine or agricultural practice.

Conclusion
The results of this study indicated that the Providencia isolates from human clinical practice showed wider resistance spectra than those obtained from animal or freshwater sh sources, while the animal-obtained bacteria had higher antibiotic MIC levels than those did the bacteria from other sources. The oR gene was identi ed in both animal and human Providencia isolates. To the best of our knowledge, this report is the rst to demonstrate that a so-called tandem-repeat plasmid, pR39-33, carrying four copies of the oR gene, is composed of four copies of another plasmid sequence in one bacterium. The mobilization genes (mobA and mobC) encoded in the resistance plasmids might play roles in the horizontal transfer of the resistance genes. These ndings indicate that the widespread and heavy use of antibiotics in agricultural practice makes the emergence of high-level and multidrug-resistant bacteria increasingly serious. Dissemination of the resistance genes between animal and human bacteria may pose a severe threat to human health.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Competing Interests
The authors declare that they have no competing interests        Figure 1 Circular maps of the plasmids pR39-8 and pR39-33. (a) Circular map of pR39-8 (CP066317). The three circles, counting from inside to outside, showed the position in bp, GC skew (G-C/G+C) (a positive GC skew toward the outside and a negative GC skew toward the inside) and GC content (a G+C content of more than 50% is shown toward the outside, otherwise, inward). The remaining circle shows the genes encoded, as well as EcoRV and BamHI sites on the plasmid. (b) Circular map of pR39-33 (CP066317). The three circles, counting from inside to outside, showed the position in kb, GC skew (G-C/G+C) and GC content. The remaining circles indicate that the plasmid can be divided into four parts, and each part shares the same sequence as pR39-8.