Inducible Expression of both ermB and ermT Conferred High Macrolide Resistance in Streptococcus gallolyticus subsp. pasteurianus Isolates in China

Streptococcus gallolyticus subsp. pasteurianus is an under-recognized pathogen and zoonotic agent causing opportunistic infections in humans. Despite increasing recognition of this subspecies as a cause for human infectious diseases, limited information is known about its antibiotic resistance mechanism. In this study, we aim to identify the molecular mechanism underlying the high macrolide resistance of six S. gallolyticus subsp. pasteurianus isolates from dead ducklings collected in several natural outbreaks in China during 2010–2013. All isolates exhibited multi-drug resistance including high macrolide resistance (MIC ≥ 1024 mg/L for erythromycin, and 512 mg/L for clarithromycin). Efflux-encoding mefA and mefE genes were not detectable in these isolates. The presence of 23S rRNA mutations in specific isolates did not significantly change macrolide MICs. No nucleotide substitutions were found in genes encoding ribosomal proteins L4 or L22. The ermB and ermT genes were found in the genomes of all isolates. These two genes were acquired independently in one highly virulent isolate AL101002, and clustered with Tn916 and IS1216, respectively. The expression of both ermB and ermT in all isolates was erythromycin inducible and yielded comparable macrolide MICs in all six isolates. Taken together, inducible expression of both ermB and ermT conferred high macrolide resistance in these S. gallolyticus subsp. pasterianus isolates. Our findings reveal new macrolide resistance features in S. gallolyticus subsp. pasteurianus by both ermB and ermT.


Expression of ermB and ermT in pET21a Vector
Using BL21DE3 host cells containing either ermB or ermT with leader peptide in pET21a vector, the levels of each protein were analyzed by Western blot analysis using a commercial monoclonal anti-His6 antibody. While empty pET21a vector did not yield any protein band recognizable by His6-tag antibody, the ermB and ermT clone in the pET21a vector gave a distinct band corresponding to 27 kD when erythromycin was supplemented. The expression level of ermB appeared to be stable with erythromycin, which was not affected by increasing erythromycin concentration. Interestingly, the ermB showed a very weak band when erythromycin was depleted. The ermT was also expressed with or without erythromycin in BL21DE3 cells. Its expression increased with higher level of erythromycin ( Figure S2). The observed ErmB and ErmT protein in BL21DE3 cells supported erythromycin-inducible expression in S. gallolyticus subsp. pasteurianus isolates. The expression of the two proteins without erythromycin could be attributed by the leaky expression off the lac promoter in pET21a vector. Figure S2. ermB and ermT expression in BL21DE3 cells. Cell lysates from BL21DE3 containing either ermB or ermT in pET21a with 0-64 mg/L of erythromycin were resolved on a 12% SDS-PAGE. ErmB and ErmT protein were blotted using a commercial monoclonal His6 antibody. Actin expression served as loading control. pET21a vector was a negative control. ERY stands for erythromycin.

Strain Identification
All isolates in this study were identified using biochemical characterization along with 16S rRNA gene sequencing [3].

Protein Expression and Purification
BL21DE3 cells harboring ermB-or ermT-containing pET28a plasmids were seeded and cultured at 37 °C overnight in LB medium supplemented with 50 μg/mL kanamycin. The overnight culture was diluted in 1L LB medium with 25 μg/mL kanamycin and grown at 37 °C to an OD600 within 0.4-0.6. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added into the culture to a final concentration of 0.8 mM. The culture was further incubated at 25 °C for 5 h. Cells were harvested and spun down at 16,000 rpm at 4 °C. Cell pellet was resuspended in a buffer containing 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5 mM β-mercaptoethanol and 2 mg/mL lysozyme, and further homogenized using sonication. The cell lysate was incubated with 1.5 mL of Ni-NTA resin (Qiagen) at 4 °C under gentle agitation. The recombinant ErmB and ErmT proteins carrying a His6-tag were eluted using imidazole in above lysis buffer in a gravity column [4,5]. The fractions were analyzed using SDS-PAGE and pooled accordingly.

Preparation of Polyclonal Antibodies against ErmB and ErmT
The polyclonal antibodies were prepared following published procedures [6]. 600 μg/mL purified recombinant ErmB and ErmT proteins were separately mixed with an equal volume of Freund's complete adjuvant (Sigma-Aldrich, St. Louis, MO, USA). Subsequently, 100 μg ErmB or ErmT in the adjuvant was injected subcutaneously into 6 week-old BALAB/c mice. Two weeks after the first injection, the mice were immunized twice at 1-week interval, using same amount of each protein. Blood was collected 7 days after the last injection and placed at room temperature for 1 h, and then at 4 °C overnight. The supernatant was collected via centrifugation at 2200× g for 10 min. The above experimental protocols were performed according to the Regulations of the Standing Committee of Hubei People's Congress and approved by the Ethical Committee of Huazhong Agricultural University (4200695757, 29 July 2005), China.

Western Blot Analysis
Monoclonal anti-Histag (1:5000) from ProteinTech was used to detect ErmB and ErmT protein levels in BL21DE3 cells containing ermB or ermT expression clones in pET21a vector. Cells were cultured in LB medium for 8 h at 37 °C with various concentration of erythromycin. One mL of culture was taken and spun down. After removing the supernatant, cell pellet was resuspended in 1× Laemmli Sample Buffer and boiled for 5 min. 20 μg of total protein was applied to 12% SDS-PAGE. After transfer to nitrocellulose membrane, ErmB and ErmT were blotted with monoclonal Histag antibody, followed by goat anti-mouse IgG-HRP secondary antibody. Actin was used as loading control. Its expression was detected using β-actin monoclonal antibody (1:2000) from ProteinTech (Wuhan, China). The images were visualized using ECL substrate kit from Seven Sea Biotech (Shanghai, China) and taken by ImageQuant LAS 4000 instrument from GE Healthcare (Beijing, China).