Generation of persistence to β-lactam antibiotics
As expected, all GAS tested (n = 211) for MIC determinations were susceptible to penicillin, and MIC values varied from 0.0025–0.02 µg/mL. Both MIC50 and MIC90 were 0.01 µg/mL. However, under HCD conditions, all 211 GAS tested grew by forming extremely tiny hemolytic colonies on the surface of BAB plates containing penicillin concentrations as high as 8 µg/mL (800xMIC) (Fig. 1a, 1b and 1c). However, no GAS growth was detected on 8 µg/mL-penicillin plates containing 1–2 × 106 CFU/plate (Fig. 1c). To observe a possible influence of defibrinated sheep blood on persistence generation, GAS strain 37–97 was grown at HCD conditions on BAB plates or on blood agar base without blood supplementation, both containing 8 µg/mL penicillin. Persistent cells were formed in both conditions. Similar to the results obtained for penicillin, persistent cells could also be generated at HCD conditions on BAB plates containing 4 µg/mL (8xMIC) cephalexin (Fig. 1a). The average of persistent cells recovered from 8 µg/mL penicillin-plate was 1.6 ± 0.5 × 109 CFU/mL, corresponding to 2.7% of the total cell population grown at HCD condition without penicillin (6.0 ± 2.4 × 1010 CFU/mL). Likewise, the mean value for persistent cells grown on 4 µg/mL-cephalexin plates was 1.1 ± 0.1 × 109 CFU/mL, corresponding to 1.8% of the total cell population grown at HCD condition without penicillin (6.0 ± 2.4 × 1010 CFU/mL) (Fig. 1b).
Despite the persistence observed, drug susceptibility could be reverted when cells were submitted to serial passaging on BAB plates without antibiotics, with previously persistent cells returning to their original state of drug susceptibility (MIC = 0.01 µg/mL). Additionally, DNA from penicillin-persistent and susceptible cells of strain 37–97 underwent whole-genome sequencing (WGS). Both sequences were identified as S. pyogenes ST62. WGS alignments generated in MAUVE showed high identity and perfect synteny of collinear blocks (Fig. 2a). There was also no difference in the absence or presence of mobile genetic elements, genomic islands, or unique genes in the persistent cells (Acc: CP041615) compared with that of susceptible ones (Acc: CP041408). Despite some differences in SNPs observed in the WGS, these could not be confirmed by Sanger resequencing of these regions, thus mutations were not associated with the emergence of persistent cells (Fig. 2b).
A total of 79 proteins were only detected when GAS strain 37–97 was grown in LCD conditions (Supplementary Table S2 online), 61 were only detected at HCD (Supplementary Table S3 online), and 128 were found in both density conditions (Supplementary Table S4 online). The most remarkable feature was the low frequency of L ribosomal proteins (LRP) in GAS cells grown at HCD (3.3%). However, the most frequently detected proteins under LCD conditions were the LRP proteins (31.6%), which play essential roles in ribosome assembly and are crucial for protein synthesis and cell growth (Fig. 3). These data suggest a decrease in growth activity under HCD conditions, which is consistent with the tiny colonies formed by persistent cells. Some multidrug resistance (MDR) efflux pump components were only detected under HCD conditions, including a protein associated with the periplasmic component of the efflux system that belongs to the root-nodulation-cell-division (RND) family (Uniprot access: Q1J790). Multiple sugar transport ATP-binding protein MsmK (Uniprot access: Q1J4L0) and the multidrug resistant ABC transporter ATP-binding and permease protein (Uniprot access: Q1J8L9) were also observed under HCD conditions (Supplementary Table S3 online).
Efflux pump activity
A universal efflux pump substrate, EtBr, was used to compare the efflux pump activity between cells grown at HCD and LCD conditions. The MIC value of strain 37–97 for EtBr was 0.06 µg/mL. However, when bacteria were grown at HCD conditions, growth was observed at concentrations of EtBr as high as 4 µg/mL (Fig. 4a), indicating intense efflux activity under conditions that generate persistent GAS cells. The number of EtBr-refractory cells recovered at concentration of 4 µg/mL were 3.3 ± 0.9 × 109 CFU/mL, corresponding to 6% of the GAS cell population grown in BAB plates in the absence of EtBr at HCD condition without EtBr (5.5 ± 2.0 × 1010 CFU/mL) (Fig. 4b).
Because antibiotics are well-known substrates of efflux pumps, to further analyze the role of these pumps in antibiotic persistence, we also tested other types of antimicrobials under HCD conditions. The data showed that under HCD conditions, antibiotic persistence arose for all antimicrobials tested independent on the class of the drug analyzed. Persistent cells grew at MIC levels and concentrations as high as 4 µg/mL (33xMIC) erythromycin, 4 µg/mL (33xMIC) azithromycin, 1 µg/mL (100xMIC) clindamycin, 16 (16xMIC) µg/mL chloramphenicol, and 16 µg/mL (133xMIC) tetracycline (Fig. 4a). The percentage of persistent cells recovered, considering all antimicrobials tested, ranged from 1.7 ± 0.1 × 108 CFU/mL (0.32%) to 2.7 ± 1.7 × 109 CFU/mL (5.0%). In relation to the cell population (5.5 ± 2.0 × 1010 CFU/mL) grown at HCD condition without antimicrobials (Fig. 4b).
Since the resistance-nodulation-division (RND) family of efflux pumps was one of the drug/proton antiporters detected in the proteome under HCD conditions, we used the pump inhibitor CCCP to dissipate the proton-motive force. Control plates with CCCP (100 µM) without antibiotic cause no effect on bacterial growth. Despite the inhibition by CCCP of persistent cells to clindamycin and chloramphenicol (Fig. 4c), this compound did not inhibit persistence to β-lactams or other antimicrobials tested.
Gene expression analysis
Of the 15 genes analyzed that were associated with the efflux pumps, seven showed some levels of upregulation in penicillin-persistent cells compared with those of susceptible GAS cells (Fig. 5a). Among these, genes of an operon associated with efflux pumps of the RND family showed increases of ≥ 4-fold, which included MGAS10750_Spy1817 (gene product: ABC transporter ATP binding protein; (p = 0.0156)), MGAS10750_Spy1818 (gene product: ABC transporter permease protein; p = 0.0088), and MGAS10750_Spy1819 (gene product: periplasmic component of efflux system, p < 0.0001). An increase in transcripts of > 4-fold was also observed for a gene product annotated as belonging to a major facilitator superfamily, the multidrug resistance protein B (MGAS10750_Spy0495) (p = 0.04). Another gene upregulated was a homolog of the multiple sugar transport ATP-binding protein msmK (MGAS10750_Spy1776), which displayed a 2.2-fold increase in expression levels, however this value was not statistically significant (p = 0.1490). The loci MGAS10750_Spy0043 and MGAS10750_Spy1633 (norA homologue) showed about 2-fold increase (p < 0.0001 and p = 0.0031, respectively; Fig. 5a).
Because clindamycin was one of the antibiotics completely inhibited by CCCP, we also investigated the effect of antibiotic in the overexpression of MGAS10750_Spy1819, which is part of the ABC transport operon. Our data showed an increase of about 9-fold in the expression of this gene. It was observed that CCCP had simultaneously affected the transcript levels of this ABC operon and two genes homologs to ihk (MGAS10750_Spy1815) and irr (MGAS10750_ Spy1816) encoding the two-component regulatory system (TCS) Ihk/Irr, which are adjacent to and upstream this operon. Our data showed that both the operon and two-component regulator were downregulated in the presence of CCCP (ABC operon: p = 0.03, p = 0.021, p = 0.0009 and ihk/irr: p = 0.0057, p = 0.0389, respectively; Fig. 5b). Similar to the downstream genes belonging to the ABC operon, ihk/irr homologs also displayed increased levels of transcripts (4-fold) for persistent cells grown in the absence of CCCP (p = 0.0006 and p = 0.01), compared with the susceptible GAS cells grown at LCD (Fig. 5b). These data suggest that the Ihk/Irr system could be acting as a regulator of this operon. Indeed, consistent with an increase in pump activity, genes (MGAS10750_Spy1765 and MGAS10750_Spy1120) annotated as belonging to the MarR and GntR families (pump negative transcriptional regulators) were significantly downregulated in the persistent cells (p = 0.0032 and p = 0.0137; Fig. 5c).
Additionally, the expression of genes associated with protein biosynthesis and cell growth/division were evaluated. For all these genes, the transcript levels decreased, but for bcaT homolog (gene product: branched-chain-amino acid aminotransferase) this decrease was not significant. The guaA homolog (gene product: GMP synthesis [glutamine hydrolyzing]), which is involved in the GTP pathway, was 2-fold down-regulated (p = 0.016). Decreased expression was also observed for relA (gene product: GTP pyrophosphokinase; p = 0.003) and typA (gene product: GTP-binding protein TypA/BipA; p = 0.0314). Finally, the ftsA homolog, which is essential for cell division, was reduced 3-fold (p = 0.0187) (Fig. 5d).
Among the genes associated with the stress conditions studied, which includes some genes related to oxidative stress, the majority was down-regulated in penicillin-persistent GAS cells. A significant increase was only observed for a dpr homolog (gene product: hydrogen peroxide resistance regulator), which was about 2-fold (p = 0.03) more expressed compared with the susceptible GAS cells (Fig. 5e). Finally, we examined the expression of three genes homologous to toxin-antitoxin (TA) systems found in the genome of S. pyogenes strain 37–97. Increased expression was only observed for the hicA/B homologs (2.8-fold and 3.5-fold increase, respectively; p = 0.007 and p = 0.017, respectively) for persistent cells (Fig. 5f).