Eleonora Giovanetti

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.

Genetic Elements Responsible for Erythromycin Resistance in Streptococci

During the last two decades, growing rates of erythromycin resistance have been reported in many countries among both Streptococcus pyogenes (29) and Streptococcus pneumoniae (63) clinical isolates. This trend was partly due to a further spread of the conventional methylase-mediated target site modification mechanism of resistance, but to an even greater extent it reflected the emergence of an active efflux-mediated mechanism of erythromycin resistance. Besides less common mutations in 23S rRNA or ribosomal proteins, target site modification consists in posttranscriptional methylation of an adenine residue in 23S rRNA; this is caused by erm class gene-encoded methylases and usually results in coresistance to macrolide, lincosamide, and streptogramin B antibiotics (MLS phenotype) (62, 63). The traditional and widely predominant erm determinant in streptococci, erm(B), can be expressed either constitutively or inducibly and is usually associated with high-level resistance (62). A more recently described methylase gene, erm(TR) (92), an erm(A) subclass (84), is normally inducible (46, 55) and is widely distributed in S. pyogenes isolates (55, 58), whose resistance level appears to depend on the contribution of a drug efflux pump (51). erm(TR) has been detected in other beta-hemolytic Streptococcus species (59, 67, 110), whereas it is quite uncommon in S. pneumoniae (12, 34, 41, 43, 97, 106). erm(T), characterized by a very low G+C content (ca. 25%), was detected in inducibly erythromycin-resistant isolates of group D streptococci in Taiwan (99) and subsequently in the United States (40). Very recently, it has been detected in U.S. invasive isolates of inducibly resistant S. pyogenes that were negative for conventional erythromycin resistance determinants (111). Efflux-mediated erythromycin resistance is associated, in streptococci, with a low-level resistance pattern affecting, among MLS antibiotics, only 14- and 15-membered macrolides (M phenotype) (96). Active efflux is encoded by mef-class genes, which include several variants. mef(A), the first mef gene to be discovered, was originally described in S. pyogenes (22) and was subsequently found to be widespread in this species, but it is also common in S. pneumoniae and other streptococcal species (60). mef(E), detected in S. pneumoniae shortly afterward (98), was found in a variety of other Streptococcus species (60), although it has only exceptionally been reported in S. pyogenes (4, 6, 87). Less common mef genes have been detected in S. pneumoniae [mef(I) (28)] and S. pyogenes [mef(O) (87)], and mef(B) and mef(G) new alleles have recently been described in group B (15) and group G (5, 15) beta-hemolytic streptococci, respectively. An msr class gene with homology to msr(A)—an ATP-binding cassette gene associated with macrolide efflux in Staphylococcus aureus (85)—is located immediately downstream of the mef gene. This msr gene is usually designated msr(D), even though different variants are associated with different mef genes. Studies carried out in pneumococci demonstrated that mef and msr(D) are cotranscribed, suggesting that the proteins encoded by the two genes may act as a dual efflux system (48), inducible by erythromycin (3). It has also been suggested that the msr(D)-encoded pump is capable of functioning independently of the one encoded by mef (3, 33). Macrolide inactivation due to a phosphotransferase encoded by the mph(B) gene, formerly described only in gram-negative bacteria, has lately been detected in Streptococcus uberis, where the inactivation mechanism, however, conferred only resistance to spiramycin (1). Until a decade ago, knowledge about the genetic elements responsible for erythromycin resistance in streptococci was virtually confined to a few plasmids or transposons carrying erm(B), then called ermAM or simply erm (56, 65). Such transposons mainly included Tn917, detected in Enterococcus faecalis when it was still regarded as a Streptococcus species (94, 101, 102), and Tn1545, detected in S. pneumoniae and also encoding resistance, besides tetracycline, to erythromycin and kanamycin (31, 32). Remarkably, Tn1545 was related to Tn916 (47), the prototype of a family of broad-host-range conjugative transposons conferring tetracycline resistance via the tet(M) gene (24, 81). Other Tn916-related erm(B)-carrying transposons early described in Streptococcus species (81) ceased to be reported in later studies. During the last decade, the discovery of the above-mentioned variety of erythromycin resistance genes in streptococci has been closely followed by the identification and characterization of a variety of genetic elements responsible for the resistance and its possible spread via intra- and interspecific transfer. Different erythromycin resistance genes are carried by different elements: in the case of mef genes, such close gene-element association was a major argument for recommending that mef(A), mef(E), and any future mef variants continue to be discriminated and kept apart (60) as opposed to being collected in a single class, mef(A), due to their high degree of similarity (84). This minireview is aimed at presenting such new knowledge about the genetic elements responsible for erythromycin resistance in streptococci. Elements and their essential characteristics are summarized in Table ​Table11. TABLE 1. Essential characteristics of established genetic elements responsible for erythromycin resistance in streptococci

Presence of the tet(O) Gene in Erythromycin- and Tetracycline-Resistant Strains of Streptococcus pyogenes and Linkage with either the mef(A) or the erm(A) Gene

ABSTRACT Sixty-three recent Italian clinical isolates of Streptococcus pyogenes resistant to both erythromycin (MICs ≥ 1 μg/ml) and tetracycline (MICs ≥ 8 μg/ml) were genotyped for macrolide and tetracycline resistance genes. We found 19 isolates carrying the mef(A) and the tet(O) genes; 25 isolates carrying the erm(A) and tet(O) genes; and 2 isolates carrying the erm(A), tet(M), and tet(O) genes. The resistance of all erm(A)-containing isolates was inducible, but the isolates could be divided into two groups on the basis of erythromycin MICs of either >128 or 1 to 4 μg/ml. The remaining 17 isolates included 15 isolates carrying the erm(B) gene and 2 isolates carrying both the erm(B) and the mef(A) genes, with all 17 carrying the tet(M) gene. Of these, 12 carried Tn916-Tn1545-like conjugative transposons. Conjugal transfer experiments demonstrated that the tet(O) gene moved with and without the erm(A) gene and with the mef(A) gene. These studies, together with the results of pulsed-field gel electrophoresis experiments and hybridization assays with DNA probes specific for the tet(O), erm(A), and mef(A) genes, suggested a linkage of tet(O) with either erm(A) or mef(A) in erythromycin- and tetracycline-resistant S. pyogenes isolates. By amplification and sequencing experiments, we detected the tet(O) gene ca. 5.5 kb upstream from the mef(A) gene. This is the first report demonstrating the presence of the tet(O) gene in S. pyogenes and showing that it may be linked with another gene and can be moved by conjugation from one chromosome to another.

Association between erythromycin resistance and ability to enter human respiratory cells in group A streptococci

BACKGROUND An increase in erythromycin resistance rates among group A streptococci has been reported in some European countries. These bacteria, long thought to be extracellular pathogens, can be efficiently internalised by, and survive within, human cells of respiratory-tract origin. Macrolide antibiotics enter eukaryotic cells, whereas beta-lactams are essentially confined to the extracellular fluid. A protein encoded by gene prtF1 is required for efficient entry of group A streptococci into epithelial cells. We investigated isolates of group A streptococci from children with pharyngitis in Italy for the presence of prtF1 and cell-invasion efficiency. METHODS We investigated 74 erythromycin-resistant and 52 erythromycin-susceptible isolates collected throughout Italy in 1997-98 from children with pharyngitis. Erythromycin-resistance phenotypes (constitutive, inducible, and M) were assessed by the triple-disc test and resistance determinants (ermB, ermTR, and mefA) by PCR. All strains were examined for the presence of prtF1 by PCR and for their ability to enter cultured human respiratory cells. FINDINGS The proportion of prtF1-positive strains was significantly higher among erythromycin-resistant than susceptible strains (66 [89%] vs 11 [21%]; difference 68% [95% CI 52-84]). All erythromycin-resistant strains without prtF1 were of the M phenotype. The proportion of highly cell-invasive isolates (invasion efficiency >10%) was significantly higher among erythromycin-resistant than among susceptible strains (59 [80%] vs five [10%]; difference 70% [57-83]). INTERPRETATIONS The unsuspected association between erythromycin resistance and cell invasiveness in group A streptococci raises serious concern. Strains combining erythromycin resistance and ability to enter human respiratory-tract cells may be able to escape both beta-lactams by virtue of intracellular location and macrolides by virtue of resistance.

Genetic Elements Carrying erm(B) in Streptococcus pyogenes and Association with tet(M) Tetracycline Resistance Gene

ABSTRACT This study was directed at characterizing the genetic elements carrying the methylase gene erm(B), encoding ribosome modification-mediated resistance to macrolide, lincosamide, and streptogramin B (MLS) antibiotics, in Streptococcus pyogenes. In this species, erm(B) is responsible for MLS resistance in constitutively resistant isolates (cMLS phenotype) and in a subset (iMLS-A) of inducibly resistant isolates. A total of 125 erm(B)-positive strains were investigated, 81 iMLS-A (uniformly tetracycline susceptible) and 44 cMLS (29 tetracycline resistant and 15 tetracycline susceptible). Whereas all tetracycline-resistant isolates carried the tet(M) gene, tet(M) sequences were also detected in most tetracycline-susceptible isolates (81/81 iMLS-A and 7/15 cMLS). In 2 of the 8 tet(M)-negative cMLS isolates, erm(B) was carried by a plasmid-located Tn917-like transposon. erm(B)- and tet(M)-positive isolates were tested by PCR for the presence of genes int (integrase), xis (excisase), and tndX (resolvase), associated with conjugative transposons of the Tn916 family. In mating experiments using representatives of different combinations of phenotypic and genotypic characteristics as donors, erm(B) and tet(M) were consistently cotransferred, suggesting their linkage in individual genetic elements. The linkage was confirmed by pulsed-field gel electrophoresis and hybridization studies, and different elements, variably associated with the different phenotypes/genotypes, were detected and characterized by amplification and sequencing experiments. A previously unreported genetic organization, observed in all iMLS-A and some cMLS isolates, featured an erm(B)-containing DNA insertion into the tet(M) gene of a defective Tn5397, a Tn916-related transposon. This new element was designated Tn1116. Genetic elements not previously described in S. pyogenes also included Tn6002, an unpublished transposon whose complete sequence is available in GenBank, and Tn3872, a composite element resulting from the insertion of the Tn917 transposon into Tn916 [associated with a tet(M) gene expressed in some cMLS isolates and silent in others]. The high frequency of association between a tetracycline-susceptible phenotype and tet(M) genes suggests that transposons of the Tn916 family, so far typically associated solely with a tetracycline-resistant phenotype, may be more widespread in S. pyogenes than currently believed.

Differentiation of Resistance Phenotypes among Erythromycin-Resistant Pneumococci

ABSTRACT Laboratory differentiation of erythromycin resistance phenotypes is poorly standardized for pneumococci. In this study, 85 clinical isolates of erythromycin-resistant (MIC ≥ 1 μg/ml)Streptococcus pneumoniae were tested for the resistance phenotype by the erythromycin-clindamycin double-disk test (previously used to determine the macrolide resistance phenotype inStreptococcus pyogenes strains) and by MIC induction tests, i.e., by determining the MICs of macrolide antibiotics without and with pre-exposure to 0.05 μg of erythromycin per ml. By the double-disk test, 65 strains, all carrying the erm(AM) determinant, were assigned to the constitutive macrolide, lincosamide, and streptogramin B resistance (cMLS) phenotype, and the remaining 20, all carrying the mef(E) gene, were assigned to the recently described M phenotype; an inducible MLS resistance (iMLS) phenotype was not found. The lack of inducible resistance to clindamycin was confirmed by determining clindamycin MICs without and with pre-exposure to subinhibitory concentrations of erythromycin. In macrolide MIC and MIC-induction tests, whereas homogeneous susceptibility patterns were observed among the 20 strains assigned to the M phenotype by the double-disk test, two distinct patterns were recognized among the 65 strains assigned to the cMLS phenotype by the same test; one pattern (n = 10; probably that of the true cMLS isolates) was characterized by resistance to rokitamycin also without induction, and the other pattern (n = 55; designated the iMcLS phenotype) was characterized by full or intermediate susceptibility to rokitamycin without induction turning to resistance after induction, with an MIC increase by more than three dilutions. A triple-disk test, set up by adding a rokitamycin disk to the erythromycin and clindamycin disks of the double-disk test, allowed the easy differentiation not only of pneumococci with the M phenotype from those with MLS resistance but also, among the latter, of those of the true cMLS phenotype from those of the iMcLS phenotype. While distinguishing MLS from M resistance in pneumococci is easily and reliably achieved, the differentiation of constitutive from inducible MLS resistance is far more uncertain and is strongly affected by the antibiotic used to test inducibility.

Φm46.1, the Main Streptococcus pyogenes Element Carrying mef(A) and tet(O) Genes

ABSTRACT Φm46.1, the recognized representative of the most common variant of mobile, prophage-associated genetic elements carrying resistance genes mef(A) (which confers efflux-mediated erythromycin resistance) and tet(O) (which confers tetracycline resistance) in Streptococcus pyogenes, was fully characterized. Sequencing of the Φm46.1 genome (55,172 bp) demonstrated a modular organization typical of tailed bacteriophages. Electron microscopic analysis of mitomycin-induced Φm46.1 revealed phage particles with the distinctive icosahedral head and tail morphology of the Siphoviridae family. The chromosome integration site was within a 23S rRNA uracil methyltransferase gene. BLASTP analysis revealed that the proteins of Φm46.1 had high levels of amino acid sequence similarity to the amino acid sequences of proteins from other prophages, especially Φ10394.4 of S. pyogenes and λSa04 of S. agalactiae. Phage DNA was present in the host cell both as a prophage and as free circular DNA. The lysogeny module appears to have been split due to the insertion of a segment containing tet(O) (from integrated conjugative element 2096-RD.2) and mef(A) (from a Tn1207.1-like transposon) into the unintegrated phage DNA. The phage attachment sequence lies in the region between tet(O) and mef(A) in the unintegrated form. Thus, whereas in this form tet(O) is ∼5.5 kb upstream of mef(A), in the integrated form, tet(O), which lies close to the right end of the prophage, is ∼46.3 kb downstream of mef(A), which lies close to the left end of the prophage.

A Novel Efflux System in Inducibly Erythromycin-Resistant Strains of Streptococcus pyogenes

ABSTRACT Streptococcus pyogenes strains inducibly resistant (iMLS phenotype) to macrolide, lincosamide, and streptogramin B (MLS) antibiotics can be subdivided into three phenotypes: iMLS-A, iMLS-B, and iMLS-C. This study focused on inducibly erythromycin-resistant S. pyogenes strains of the iMLS-B and iMLS-C types, which are very similar and virtually indistinguishable in a number of phenotypic and genotypic features but differ clearly in their degree of resistance to MLS antibiotics (high in the iMLS-B type and low in the iMLS-C type). As expected, the iMLS-B and iMLS-C test strains had the erm(A) methylase gene; the iMLS-A and the constitutively resistant (cMLS) isolates had the erm(B) methylase gene; and a control M isolate had the mef(A) efflux gene. mre(A) and msr(A), i.e., other macrolide efflux genes described in gram-positive cocci, were not detected in any test strain. With a radiolabeled erythromycin method for determination of the intracellular accumulation of the drug in the absence or presence of an efflux pump inhibitor, active efflux of erythromycin was observed in the iMLS-B isolates as well as in the M isolate, whereas no efflux was demonstrated in the iMLS-C isolates. By the triple-disk (erythromycin plus clindamycin and josamycin) test, performed both in normal test medium and in the same medium supplemented with the efflux pump inhibitor, under the latter conditions iMLS-B and iMLS-C strains were no longer distinguishable, all exhibiting an iMLS-C phenotype. In conjugation experiments with an iMLS-B isolate as the donor and a Rifr Fusr derivative of an iMLS-C isolate as the recipient, transconjugants which shared the iMLS-B type of the donor under all respects, including the presence of an efflux pump, were obtained. These results indicate the existence of a novel, transferable efflux system, not associated with mef(A) or with other known macrolide efflux genes, that is peculiar to iMLS-B strains. Whereas the low-level resistance of iMLS-C strains to MLS antibiotics is apparently due to erm(A)-encoded methylase activity, the high-level resistance of iMLS-B strains appears to depend on the same methylase activity plus the new efflux system.

SmaI macrorestriction analysis of Italian isolates of erythromycin-resistant Streptococcus pyogenes and correlations with macrolide resistance phenotypes.

High rates of erythromycin resistance among Streptococcus pyogenes strains have been reported in Italy in the last few years. In this study, 370 erythromycin-resistant (MIC, > or = 1 microg/mL) Italian isolates of this species obtained in 1997-1998 from throat swabs from symptomatic patients were typed by analyzing SmaI macrorestriction fragment patterns by pulsed-field gel electrophoresis (PFGE). Among the typable isolates (n = 341; the genomic DNA of the remaining 29 isolates was not restricted by SmaI), 48 distinct PFGE types were recognized, of which 31 were recorded in only one isolate (one-strain types). Fifty-two percent of typable isolates fell into three type clusters and 75% into six, suggesting that erythromycin-resistant group A streptococci circulating in Italy are polyclonal, but the majority of them probably derives from the spread of a limited number of clones. In parallel experiments, the 370 test strains were characterized for the macrolide resistance phenotype: 80 were assigned to phenotype cMLS, 89 to phenotype iMLS-A, 33 to phenotype iMLS-B, 11 to phenotype iMLS-C, and 157 to phenotype M. There was a close correlation between these phenotypic data and the genotypic results of PFGE analysis, the vast majority of the isolates assigned to individual PFGE classes belonging usually to a single phenotype of macrolide resistance. All of the 29 untypable isolates belonged to the M phenotype. Further correlations were observed with tetracycline resistance.

Detection in Italy of two clinical Enterococcus faecium isolates carrying both the oxazolidinone and phenicol resistance gene optrA and a silent multiresistance gene cfr

Sir, In a century in which the issue of emerging antibiotic resistance is being dominated by severe concerns chiefly regarding Gram-negative organisms, the multiresistance gene cfr is probably the greatest emerging problem in Gram-positive pathogens, particularly staphylococci and enterococci. The concern over this problem is motivated not only by the fact that the resistance involves linezolid—widely used in serious infections caused by MDR Gram-positive organisms, often as a last-resort drug—but also, and critically, by the fact that the frequent location of cfr on conjugative plasmids makes the resistance transferable. Now, the report in China of a second plasmid-borne transferable gene, optrA, conferring efflux-mediated oxazolidinone (including second-generation tedizolid) and phenicol resistance in enterococcal isolates adds further to the concern. As soon as the first report, with the sequence of an Enterococcus faecalis plasmid (pE349) carrying optrA (accession no. KP399637), became available as an Advance Access article in the Journal of Antimicrobial Chemotherapy, we decided to test for the optrA gene in 81 Enterococcus isolates from blood samples, which make up the first batch of an enterococcal collection we had recently started for a study including cfr screening. Identification at the species level was performed using VITEK 2 (bioMérieux, Marcy-l’Étoile, France). The cfr and optrA genes were sought by PCR using primer pairs internal to either gene: respectively, the known pair cfr-fw and cfr-rv, yielding a 746 bp amplicon, and the specially designed pair optrA-fw and optrA-rv, which yielded a 422 bp amplicon (Table S1, available as Supplementary data at JAC Online). Two Enterococcus faecium isolates were positive for cfr and two were positive for optrA. Much to our surprise, there were in fact only two positive isolates (E20818 and E35048), since each carried both optrA and cfr. The antibiotic MICs and other features for the two isolates are reported in Table 1. Both isolates had a relatively low linezolid MIC, 4 mg/L, a value that is regarded as ‘susceptible’ according to EUCAST and ‘intermediate’ according to CLSI. Both had a tedizolid MIC of 2 mg/L; breakpoints for resistance have recently been established by EUCAST for staphylococci and b-haemolytic streptococci (.0.5 mg/L) and for viridans group streptococci (.0.25 mg/L). The two isolates were also examined for mutations in 23S ribosomal RNA (not detected) and for the phenicol exporter genes fexA and fexB (not detected). The two isolates exhibited closely related SmaI-PFGE profiles; one (E35048) was investigated for molecular traits. Sequencing demonstrated that optrA and cfr displayed high-level DNA identities (98% and 99%, respectively) to the respective reference sequences (accession numbers KP399637 and AJ57936). Three amino acid changes were detected in the protein sequence of cfr and 21 (4 of which were already reported in Chinese isolates) in the protein sequence of optrA compared with the respective reference sequences. The results of long PCR assays seeking a possible linkage between optrA and cfr were negative. The genetic contexts of both genes proved capable of undergoing excision in circular form, and were completely sequenced. The sequence of the minicircle containing optrA (3350 bp), deposited under accession no. KT892063, included a transposase gene downstream of optrA. This transposase gene exhibited 70% DNA identity and 65% amino acid identity to a chromosomal transposase from Clostridium sticklandii (accession no. FP565809). The minicircle (3405 bp) containing the cfr gene and one intact IS, ISEnfa5, was almost identical to a cfr genetic context described in Staphylococcus lentus (accession no. KF049005). Considering the low MICs of linezolid, florfenicol and chloramphenicol (Table 1), in spite of the prezsence of two resistance genes acting by different mechanisms (cfr perturbing the ribosome function and optrA providing for active efflux), RT–PCR experiments were performed to check the actual transcription of the two genes (Figure S1). We found that optrA was transcribed, whereas cfr was not. Although the exact mechanism of nontranscription is still being investigated, preliminary data indicate a 52 bp deletion in the regulatory region upstream of cfr. Interestingly, a cfr gene failing to mediate resistance to oxazolidinones and phenicols has been described in a porcine E. faecalis isolate in China. Our collection of Enterococcus blood isolates is still in progress, and the overall results of the survey will be assessed and