Macaques (Macaca mulatta), obtained from a United States-based vendor in 2005, underwent physical examination and routine diagnostic evaluations during quarantine and thereafter at quarterly intervals. Macaques were routinely pair-housed and maintained in an AAALAC International-accredited animal facility. They were fed specified amounts of primate chow (Purina Lab Diet® 5038, St. Louis, MO) twice daily and provided water ad libitum. Housing conditions were maintained between 20 and 22°C, 30–70% humidity, 10–15 non-recirculated air changes per hour, and a light cycle of 12 h light, 12 h of dark per day.

During a quarterly physical and diagnostic evaluation, macaque 05-18 presented with fresh blood in its feces. Fecal samples were collected directly from the rectum and submitted for culture and antibiotic sensitivity. Shortly after the initial fecal sample was collected, macaque 05-18 and its cagemate underwent antibiotic treatment with enrofloxacin (5 mg/kg IM every 24 h for 10 days) following research-related surgical procedures. S. flexneri variant Y (strain 06-2384) was cultured from the original submission and found sensitive to trimethoprim-sulfamethoxazole, but resistant to ampicillin, amoxicillin-clavulanic acid, cephalothin, gentamicin, and enrofloxacin using Kirby-Bauer antibiotic testing. The two clinically normal monkeys were placed in quarantine together and a set of rectal and gingival swabs were submitted for culture. The second culture sample, as well as fecal samples collected twice more at 2-week intervals, were negative for Shigella spp. Following treatment with enrofloxacin, multiple follow-up fecal cultures were negative for Shigella spp.

Fecal samples were collected from all macaques (N = 24) and submitted for culture and antibiotic susceptibility testing during the same time interval the shigella-positive macaque, 05-18, was found. Two additional macaques, 05-15 and 05-16, housed in the adjacent cage to macaque 05-18, had fecal cultures positive for S. flexneri type 5b, designated 06-2835 and 06-2836, respectively. This serotype also showed multiple-antibiotic resistance, with intermediate sensitivity to enrofloxacin and sensitivity to trimethoprim-sulfamethoxazole. The second pair was moved to quarantine and treated with enrofloxacin (5 mg/kg IM every 24 h for 10 days). However, follow-up fecal cultures from the cage pan were still positive for S. flexneri type 5b, designated strain 06-3102. Subsequently, the pair was treated with trimethoprim-sulfadiazine (30 mg/kg SQ every 24 h for 10 days); all subsequent cultures were negative for Shigella spp.

Cage pan feces and rectal swabs collected from macaques were swabbed onto XLD (xylose, lysine, deoxycholate) and HE (hektoen-enteric) agar media, and the samples were then placed into selenite broth (Remel, Inc., Lenexa, KS.). All media were incubated at 37°C for 18 to 24 h, after which the selenite broth was sub-cultured onto the same media and re-incubated. Isolates that were transparent on the XLD and HE agars were sub-cultured on BAP (Trypticase Soy Agar with 5% Sheep Blood; Remel) and identified as Shigella spp. by API 20E (bioMerieux, Inc., Hazelwood, MO). Isolates were submitted to the Texas Department of State Health Services lab for serotyping.

Antibiotics used for broth microdilution assays (ampicillin, azithromycin, cefazolin, cefoxitin, ceftazidime, ceftriaxone, cephalothin, ciprofloxacin, clarithromycin, enrofloxacin, gentamicin, imipenem, levofloxacin, nalidixic acid, and tetracycline powders) were purchased from the Sigma Chemical Company (St. Louis, MO). Amoxicillin-clavulanic acid and trimethoprim-sulfamethoxazole were obtained as E-test strips from bioMerieux (Durham, NC.). Disks of ampicillin (10 μg), cephalothin (30 μg), enrofloxacin (5 μg), nalidixic acid (30 μg), tetracycline (30 μg), amoxicillin-clavulanic acid (30 μg), and trimethoprim-sulfamethoxazole (25 μg) were obtained from Remel (Lenexa, KS.).

Antimicrobial susceptibility tests were initially performed by disk diffusion in accordance with the Clinical Laboratory Standards Institute (2010). In addition, to determine the level of sensitivity, antibiotic mean inhibitory concentrations (MICs) were determined by the broth microdilution method using 2-fold serial dilutions, as recommended by CLSI (Clinical Laboratory Standards Institute, 2012, 2014). MIC is defined as the lowest concentration of an antimicrobial agent that inhibits visible growth. Escherichia coli (ATCC25922) was used as the standard reference strain. Experiments were performed in triplicate from two or more independent experiments.

Whole DNA (includes both chromosomal and plasmid DNA) from the five S. flexneri isolates was extracted using the High Pure PCR Template Preparation Kit (Roche Molecular Biochemicals, Indianapolis, IN). Plasmid DNA was extracted using the QIAprep Spin Miniprep Kit (QIAGEN Inc. Valencia, CA).

Screening for the qnrA, qnrB, and qnrS genes was performed on whole and plasmid DNA using the primers and PCR conditions described previously (Robicsek et al., 2006). PCR products were concentrated and purified with the QIAquick PCR purification kit (Qiagen, Valencia, CA) for sequencing with an ABI Prism cycle sequencing kit (BigDye Terminator cycle sequencing kit) on an ABI 3100 genetic analyzer (Applied Biosystems, Foster City, CA). Sequences were analyzed by BLAST hosted by the National Center for Biotechnology Information (NCBI) to identify homologous genes and calculate percent identity.

Conjugation experiments were performed using the S. flexneri isolates as donors and E. coli J53 Az^r as the recipients (Wang et al., 2004). Cultures of donor and recipient cells were grown in 3 mL of fresh Luria Bertani (LB) broth (BBL®) at 37°C with shaking for 3 h. The conjugation assay was then performed in 3 mL of fresh LB broth using the donor and recipient cells in logarithmic phase (0.5 mL each) and incubated at 37°C for 3 h without shaking. Transconjugants were selected on trypticase soy agar plates (TSA; BBL®) containing sodium azide (100 μg/mL: Sigma-Aldrich Corporation, St. Louis, Mo.) for counter-selection, and ampicillin (50 μg/mL; Sigma-Aldrich Corp.) for selection of plasmid-encoded resistance (Fox, 1975; Mammeri et al., 2005; Cattoir et al., 2007). Colonies were also plated on TSA plates containing sodium azide (100 μg/mL) and nalidixic acid (16 μg/mL). The transconjugants were plated onto MacConkey agar plates, and their identities were reconfirmed as E. coli using with API 20E.

For transformation experiments, 5 μL of plasmid DNA from four donor strains was heat shocked at 42°C for 30 s into chemically component E. coli Top10 cells. The cells were then incubated in 250 μL of S.O.C. (super optimal broth with catabolic repressor) for 1 h at 37°C, followed by overnight growth on LB plates supplemented with ampicillin (50 μg/mL; Sigma-Aldrich Corp.) for selection of antibiotic-resistant transformants. Colonies were screened by PCR for the qnrS gene. MICs for the donors, recipient, transconjugants, and transformants were measured as described above.

Whole DNA was extracted from two representative S. flexneri isolates, 06-2384 and 06-3102, using the High Pure PCR Template Preparation Kit (Roche Molecular Biochemicals, Indianapolis, IN). These isolates were chosen in order to represent all infected animals (06-2384 cultured from monkey 05-18; 06-3102 cultured from co-housed monkeys 05-15 and 05-16) as well as the potential genetic diversity of both serotypes (variant Y and 5b, corresponding to 06-2384 and 06-3102, respectively). DNA was then prepared into libraries using the SMRTbell Template Prep Kit 1.0 and the DNA/Polymerase Binding Kit P6 v2 (Pacific Biosciences, Menlo Park, CA). DNA libraries were size-selected for fragments ≥~5kb and then were sequenced on a single SMRT cell per genome using a Pacific Biosciences RSII sequencer at the University of Massachusetts Deep Sequencing Core Facility (Worchester, MA). Sequencing reads were quality filtered and trimmed for de novo assembly using the Hierarchical Genome Assembly Process (HGAP 3.0) workflow hosted on the SMRT Portal 2.3. Resulting contigs were annotated using RASTtk (Aziz et al., 2008) hosted by Pathosystems Resource Integration Center (PATRIC) (Wattam et al., 2014, 2017). VirulenceFinder 1.5 (Joensen et al., 2014), ResFinder 2.1 (Zankari et al., 2012), and PlasmidFinder 1.3 (Carattoli et al., 2014) were used to identify virulence factors, antibiotic resistance genes, and plasmids, respectively. In silico multi-locus sequencing type (MLST) was determined using MLST-1.6 (Larsen et al., 2012) against the E. coli #1 allelic profile database. Average nucleotide identity (ANI) was calculated with OrthoANIu (Lee et al., 2016), and digital DNA-DNA hybridization (dDDH) was calculated with Genome-to-Genome Distance Calculator (GGDC) 2.1 (Auch et al., 2010). Graphical circular maps of chromosome and plasmid sequences were generated using the CGView Server (Grant and Stothard, 2008).

S. flexneri isolates were identified using selective agar and API 20E strips and were also serotyped by the Texas Department of State Health Services. S. flexneri variant Y strain 06-2384 was isolated in the index case (i.e., monkey 05-18). S. flexneri type 5b strains 06-2835 R1 and 06-2835 R2, which had different colony morphologies and API codes, were isolated from monkey 05-15. Strain 06-2836 was cultured from monkey 05-16. Following unsuccessful enrofloxacin treatment of paired monkeys 05-15 and 05-16, feces were collected from the cage pan and strain 06-3102 was cultured. These S. flexneri isolates were then analyzed for antibiotic susceptibility, the presence of qnr genes by PCR, and the ability to transfer plasmid-encoded antibiotic resistance genes to E. coli recipients by conjugation and transformation.

PCR detected qnrS, but not qnrA or qnrB, in both whole and plasmid DNA from all five S. flexneri isolates (Supplementary Figure 1). BLAST analysis of three representative qnrS PCR product sequences against the nr/nt database determined they were 100% identical to the plasmid-encoded qnrS1 genes found in other Enterobacteriaceae species, including pAH0376 from Shigella flexneri 2b and pVQS1 from Salmonella enterica, indicating these novel S. flexneri strains harbor the qnrS1 variant.

Antimicrobial susceptibility testing of four S. flexneri isolates by microdilution indicated multi-drug resistance to ampicillin, cephalothin, ceftazidime, ceftriaxone, cefazolin, gentamicin (except strain 06-2835), nalidixic acid, and tetracycline and decreased susceptibility to amoxicillin-clavulanic acid, ciprofloxacin, enrofloxacin, and levofloxacin compared to the E. coli control strain ATCC25922 (Table 1). The S. flexneri isolates were in general susceptible to azithromycin, imipenem, cefoxitin, trimethoprim-sulfamethoxazole (Table 1).

Plasmid DNA from these S. flexneri isolates were successfully conjugated and transformed into the E. coli recipient strains J53 Az^r and Top10, respectively. Plasmid DNA isolated from all the transconjugants and transformants were PCR-positive for the qnrS gene (Supplementary Figure 2). Likewise, plasmid profiles were similar between the donor S. flexneri isolates and the E. coli transconjugants and transformants, indicating successful transfer of plasmid DNA to the recipients (Figure 1). The S. flexneri isolates have several bands in the plasmid profile, indicating they may harbor multiple different plasmids.

Compared to the naïve E. coli recipient strains J53 Az^r and Top10, the transconjugants and transformants demonstrated increased resistance to all quinolone antibiotics tested (Table 1). The MIC of the fluoroquinolones was 8- to 32-fold higher in the transconjugants and transformants, while nalidixic acid showed a 2- to 8-fold increase versus their naïve E. coli recipient strains (Table 1). The transconjugants and transformants also had increased resistance to amoxicillin-clavulanic acid as compared to the naïve E. coli recipient strains. All transconjugants and transformants were highly resistant to ampicillin and several cephalosporins, characteristic of beta-lactamase activity. The transconjugants and transformants demonstrated similar susceptibility to azithromycin, clarithromycin, gentamicin, tetracycline, and trimethoprim-sulfamethoxazole compared to their naïve E. coli recipient strains. Interestingly, when compared to the S. flexneri donor isolates, the transconjugants and transformants in general had about 2- to 8-fold lower MIC levels for some of the quinolones tested. This result suggests the novel S. flexneri isolates may have additional mechanisms to resist quinolones not harbored on the plasmid(s) transferred to the transconjugants and transformants.

To corroborate antibiotic resistance phenotypes, whole genome sequences of two representative S. flexneri isolates, 06-2384 and 06-3102, were obtained (summarized in Table 2 and Supplementary Table 1). The genomes for S. flexneri 06-2384 and 06-3102 were assembled into 5 and 8 contigs, respectively. The largest contig for both isolates was ~4.7 Mb with a G+C content of ~50.9%, which is consistent with the chromosomal sequence of other S. flexneri genomes (Jin et al., 2002; Wei et al., 2003; Shen et al., 2017). Using PlasmidFinder-1.2, plasmid replicon sequences were detected in the remaining contigs, suggesting they are plasmids. Additionally, the top 5 BLAST hits for these contigs corresponded to plasmids sequences from Shigella spp., E. coli, S. enterica, K. pneumoniae, and Citrobacter freundii (Supplementary Table 2). Thus, S. flexneri 06-2384 and 06-3102 strains both appear to harbor several different plasmids in addition to their chromosome. This agrees with Figure 1 in that several bands were apparent after separating plasmid DNA. Despite representing the serotype variants Y and 5b, both 06-2384 and 06-3102 were identified as ST245 by in silico MLST and had an ANI of 99.95% and a dDDH of 99.80%, indicating substantial genetic similarity. However, the difference in serotypes as well as the number/size of plasmids (Figure 1 and Table 2) suggests more than one strain variant was present in the NHP colony.

ResFinder-2.1 identified several antibiotic resistance genes in the chromosome and plasmid sequences (Table 2). Aminoglycosides (aadA1, aac(3)-IId), beta-lactams (blaOXA-1), quinolones (oqxA, oqxB), chloramphenicol (catA), and tetracycline (tet(B)) resistance genes were detected in the chromosomes of both strains. In both genomes, oqxA, oqxB, and aac(3)-IId and aadA1, blaOXA-1, catA1, and tet(B) clustered together in separate regions. Both of these gene clusters were flanked by several mobile element protein, transposase, and integrase gene annotations. Mutations in the genes for DNA gyrase and topoisomerase subunits A and B were not identified in either of the genomes. Colistin resistance genes (mcr-1, mcr-2, and mcr-3) were also not identified.

The qnrS gene and beta-lactam resistance gene blaTEM-1B were detected on the 60 and 52 kb plasmid contigs for S. flexneri 06-2384 and 06-3102, respectively. BLAST analysis found these plasmid contigs were homologous to the completely-sequenced ~40 kb, conjugative plasmid pVQS1 from S. enterica (Karczmarczyk et al., 2012) and the partially-sequenced ~47 kb, conjugative plasmid pAH0376 from Shigella flexneri 2b (Hata et al., 2005), both of which harbor functional blaTEM-1B and qnrS1 resistance genes. The annotated qnrS genes from the novel S. flexneri isolates showed 100% sequence identity and coverage to the qnrS1 genes found in pAH0376 and pVQS1. In general, the resistance genes and flanking mobile element protein genes from S. flexneri 06-2384 and 06-3102 showed >98% identity and similar synteny to pVQS1 and pAH0376, except for the transposase genes tinR vs. tnpA, which had 31%, identify homology (Figure 2). Bidirectional BLASTP analysis using PATRIC's proteome comparison tool indicated >90% of the annotated genes in pVQS1 shared >97% identity to those of the S. flexneri 06-2384 and 06-3102 plasmid sequences, including homologs for replication and conjugation genes. This suggests these plasmid sequences from S. flexneri 06-2384 and 06-3102 have the potential for self-replication and ability to transfer to new hosts. The S. flexneri 06-2384 and 06-3102 plasmid sequences were identical to each other, except 06-3102 appears to contain a duplication of the region encoding qnrS and associated mobile element proteins. This second qnrS gene is truncated into a predicted 106 amino acid product and is likely non-functional as a result.

S. flexneri 06-3102 also contained the beta-lactam resistance gene blaCTX-M-14 on a ~86 kb plasmid contig. BLAST analysis of this plasmid contig detected >99% identity to the ~74 kb IncFII2 plasmid pAC2901 from C. freundii strain AC290 that also encodes the blaCTX-M-14 resistance gene. The remaining plasmid contig sequences from S. flexneri 06-2384 and 06-3102 showed BLAST homology to plasmids from Enterboacteriaceae species (Supplemental Table 2). These plasmid sequences were typed as ColRNAI using PlasmidFinder1.2, primarily contained hypothetical proteins, and some encoded phage-related gene annotations. Interestingly, the 15 and 19 kb plasmids from S. flexneri 06-2384 and 06-3102, respectively, were enriched for ferric enterobactin uptake protein FepE (8/17 and 9/25 annotations for S. flexneri 06-2384 and 06-3102, respectively), suggesting a role in iron acquisition.

Using VirulenceFinder1.5, virulence factor genes were detected on the chromosome and ~210 kb plasmid sequences from both isolates (Table 2). Additional potential virulence factor genes were also identified by BLASTP from the VFDB, Victors, and PATRIC_VF databases using the specialty genes feature hosted by PATRIC. This included 998 and 1,009 virulence factor genes detected in S. flexneri 06-2384 and 06-3102, respectively, with functions ranging from host adherence and invasion to biofilm formation and iron acquisition (Supplemental Table 3).

Of particular note, several serine protease autotransporters of Enterobacteriaceae (SPATE) enterotoxin genes were identified, which are implicated in diarrheal illness and gastrointestinal cytotoxicity by pathogenic Shigella spp. and E. coli infection (Dautin, 2010; Mattock and Blocker, 2017). For both S. flexneri 06-2384 and 06-3102, these include the chromosomally-encoded Shigella enterotoxin 1 (ShET1), protease involved in intestinal colonization (Pic), and Shigella extracellular protein A (SigA) genes, which have been shown to exert cytotoxicity to epithelial cells, mediate serum resistance, induce fluid secretion, and degrade mucus/mucin (Dautin, 2010; Faherty et al., 2012; Mattock and Blocker, 2017). Two paralogs for ShET2 along with type III secretion system (T3SS) components and conjugative transfer genes were identified on the ~210 kb, IncFII plasmids in both S. flexneri 06-2384 and 06-3102. These ShET2 paralogs are T3SS effector proteins that induce secretory activity by the gastrointestinal epithelium to cause fluid-laden diarrhea (Farfan et al., 2011; Faherty et al., 2012; Mattock and Blocker, 2017).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.