Therapeutic failures of antibiotics used to treat macrolide-susceptible Streptococcus pyogenes infections may be due to biofilm formation

Abstract

Streptococcus pyogenes infections often fail to respond to antibiotic therapy, leading to persistent throat carriage and recurrent infections. Such failures cannot always be explained by the occurrence of antibiotic resistance determinants, and it has been suggested that S. pyogenes may enter epithelial cells to escape antibiotic treatment. We investigated 289 S. pyogenes strains isolated from different clinical sources to evaluate their ability to form biofilm as an alternative method to escape antibiotic treatment and host defenses. Up to 90% of S. pyogenes isolates, from both invasive and noninvasive infections, were able to form biofilm. Specific emm types, such as emm6, appeared to be more likely to produce biofilm, although variations within strains belonging to the same type might suggest biofilm formation to be a trait of individual strains rather than a general attribute of a serotype. Interestingly, erythromycin-susceptible isolates formed a significantly thicker biofilm than resistant isolates (P < 0.05). Among resistant strains, those carrying the erm class determinants formed a less organized biofilm than the mef(A)-positive strains. Also, prtF1 appeared to be negatively associated with the ability to form biofilm (P < 0.01). Preliminary data on a selection of strains indicated that biofilm-forming isolates entered epithelial cells with significantly lower efficiency than biofilm-negative strains. We suggest that prtF1-negative macrolide-susceptible or mef(A)-carrying isolates, which are poorly equipped to enter cells, may use biofilm to escape antimicrobial treatments and survive within the host. In this view, biofilm formation by S. pyogenes could be responsible for unexplained treatment failures and recurrences due to susceptible microorganisms.

FIG. 1.

Biofilms formed by S. epidermidis ATCC 35984 and three S. pyogenes strains with different biofilm-forming abilities. The ODs obtained with the plate test are compared with the biofilm appearances by scanning electron microscopy. The biofilms formed by S. pyogenes (b and c) appear to be less homogeneous than that of S. epidermidis (a), with large bacterial aggregates embedded in an amorphous extracellular matrix (arrows), coating the plastic surface more as scattered microcolonies. Bar, 20 μm.

FIG. 2.

(a) Numbers of S. pyogenes isolates forming biofilm at an OD of <0.061 (dark-gray bars), 0.061 < OD < 0.240 (gray bars), and an OD of >0.240 (light-gray bars) under the different atmosphere conditions. NM, unmodified atmosphere; ANA, anaerobiosis. (b) Index of biofilm produced in unmodified atmosphere (dotted bars), 5% CO₂ (striped bars), or anaerobiosis (open bars) by isolates from invasive infection or throat swabs. Bars indicate the mean ODs ± SD.

FIG. 3.

Biofilm formation by S. pyogenes in relation to emm type. Symbols represent the means of at least three different determinations carried out in triplicate. Numbers along the x axis represent emm types (e.g., 1, emm1).

FIG. 4.

Biofilm formation by antibiotic-susceptible and antibiotic-resistant S. pyogenes isolates. (a) Antibiotic-susceptible strains produced significantly more biofilm than resistant strains (P < 0.05). (b) Moreover, macrolide-resistant strains carrying erm genes produced a less thick biofilm than strains resistant to macrolides by the efflux pump mef(A). Values reported here are those obtained after incubation in anaerobiosis. Symbols represent the mean ODs of three different determinations carried out in triplicate for each strain.

FIG. 5.

Biofilm formation by S. pyogenes in relation to the presence or absence of the prtF1 gene. (a) prtF1-negative strains formed a significantly thicker biofilm than prtF1-carrying strains (P < 0.01). (b) Biofilm formation in susceptible isolates or macrolide-resistant isolates containing either erm or mef and prtF1 genes is shown. Bars are the median ODs of all isolates tested in triplicate at least three times. NS, not significant.

FIG. 6.

Biofilm formation abilities of a selection of 14 S. pyogenes isolates in relation to efficiency in invading Hep-2 cells. Bars represent the mean ODs (±SD) of three different determinations carried out in triplicate for each strain. The difference in OD between the two groups was significant (P < 0.01).