Maria Pia Montanari

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

Phenotypes and Genotypes of Erythromycin-Resistant Pneumococci in Italy

ABSTRACT Of 120 erythromycin-resistant pneumococci isolated in Italian hospitals, 39 (32.5%) were M-type isolates, carrying the mef gene alone. The mef gene was also detected, together with erm(AM), in one constitutively resistant isolate and in five isolates of the partially inducible phenotype. Among the 45 mef-positive isolates, 25 (55.6%) carried mef(A) and 20 (44.4%) carried mef(E) as observed from PCR-restriction fragment length polymorphism analysis of a 1,743-bp amplicon. The same result was obtained by a similar method applied to a more common 348-bp amplicon.

Phenotypic and Molecular Characterization of Tetracycline- and Erythromycin-Resistant Strains of Streptococcus pneumoniae

ABSTRACT Sixty-five clinical isolates of Streptococcus pneumoniae, all collected in Italy between 1999 and 2002 and resistant to both tetracycline (MIC, ≥8 μg/ml) and erythromycin (MIC, ≥1 μg/ml), were investigated. Of these strains, 11% were penicillin resistant and 23% were penicillin intermediate. With the use of the erythromycin-clindamycin-rokitamycin triple-disk test, 14 strains were assigned to the constitutive (cMLS) phenotype of macrolide resistance, 44 were assigned to the partially inducible (iMcLS) phenotype, 1 was assigned to the inducible (iMLS) phenotype, and 6 were assigned to the efflux-mediated (M) phenotype. In PCR assays, 64 of the 65 strains were positive for the tetracycline resistance gene tet(M), the exception being the one M isolate susceptible to kanamycin, whereas tet(K), tet(L), and tet(O) were never found. All cMLS, iMcLS, and iMLS isolates had the erythromycin resistance gene erm(B), and all M phenotype isolates had the mef(A) or mef(E) gene. No isolate had the erm(A) gene. The int-Tn gene, encoding the integrase of the Tn916-Tn1545 family of conjugative transposons, was detected in 62 of the 65 test strains. Typing assays showed the strains to be to a great extent unrelated. Of 16 different serotypes detected, the most numerous were 23F (n = 13), 19A (n = 10), 19F (n = 9), 6B (n = 8), and 14 (n = 6). Of 49 different pulsed-field gel electrophoresis types identified, the majority (n = 39) were represented by a single isolate, while the most numerous type included five isolates. By high-resolution restriction analysis of PCR amplicons with four endonucleases, the tet(M) loci from the 64 tet(M)-positive pneumococci were classified into seven distinct restriction types. Overall, a Tn1545-like transposon could reasonably account for tetracycline and erythromycin resistance in the vast majority of the pneumococci of cMLS, iMcLS, and iMLS phenotypes, whereas a Tn916-like transposon could account for tetracycline resistance in most M phenotype strains.

Further characterization of borderline methicillin-resistant Staphylococcus aureus and analysis of penicillin-binding proteins.

Eighty-nine Staphylococcus aureus strains were grouped according to their susceptibility or resistance to methicillin and oxacillin. The role of beta-lactamase in borderline methicillin resistance was confirmed by tests with beta-lactamase inhibitors, particularly when salt-supplemented medium was used. A penicillin-binding protein assay indicated that borderline methicillin-resistant S. aureus strains do not produce PBP 2a. Images

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.

erm(B)-Carrying Elements in Tetracycline-Resistant Pneumococci and Correspondence between Tn1545 and Tn6003

ABSTRACT This study investigated the genetic organization of erm(B)-carrying transposons of Streptococcus pneumoniae and their distribution in tetracycline-resistant clinical isolates. By comparatively analyzing reference pneumococci carrying erm(B)/tet(M) transposon Tn1545, Tn6003, Tn6002, or Tn3872, we demonstrated a substantial correspondence between Tn1545 and Tn6003, which have the same resistance gene combination [tet(M) (tetracycline), erm(B) (erythromycin), and aphA-3 (kanamycin)]; share the macrolide-aminoglycoside-streptothricin element, containing a second erm(B); and only differ by a ca. 1.2-kb insertion (containing a putative IS1239 insertion sequence) detected in Tn1545 from S. pneumoniae reference strain BM4200. These results enabled elucidation of the structure of Tn1545, the first erm(B)-carrying transposon described in S. pneumoniae. A collection of 83 erythromycin- and tetracycline-resistant clinical pneumococci, representative of recent Italian isolates carrying erm(B) as the sole erythromycin resistance gene, was used to investigate the distribution of the different transposons. All 83 organisms were positive for tet(M) and bore an erm(B)/tet(M) transposon that could be characterized by using a specific set of primer pairs; Tn3872 was detected in 18 isolates, Tn6002 in 59 isolates, and Tn6003 in 6 (the sole kanamycin-resistant) isolates. The genetic organization of transposon Tn1545, with its specific insertion, was not detected in any of the isolates tested. The erm(B)-carrying elements of tetracycline-resistant pneumococci substantially corresponded to those [bearing a silent tet(M) gene] recently detected in tetracycline-susceptible pneumococci. Overall, in erm(B)-positive pneumococci, Tn6003 was the least common erm(B)-carrying Tn916-related element and Tn6002 the most common.

Molecular Characterization of Pneumococci with Efflux-Mediated Erythromycin Resistance and Identification of a Novel mef Gene Subclass, mef(I)

ABSTRACT The molecular genetics of macrolide resistance were analyzed in 49 clinical pneumococci (including an “atypical” bile-insoluble strain currently assigned to the new species Streptococcus pseudopneumoniae) with efflux-mediated erythromycin resistance (M phenotype). All test strains had the mef gene, identified as mef(A) in 30 isolates and mef(E) in 19 isolates (including the S. pseudopneumoniae strain) on the basis of PCR-restriction fragment length polymorphism analysis. Twenty-eight of the 30 mef(A) isolates shared a pulsed-field gel electrophoresis (PFGE) type corresponding to the England14-9 clone. Of those isolates, 27 (20 belonging to serotype 14) yielded multilocus sequence type ST9, and one isolate yielded a new sequence type. The remaining two mef(A) isolates had different PFGE types and yielded an ST9 type and a new sequence type. Far greater heterogeneity was displayed by the 19 mef(E) isolates, which fell into 11 PFGE types, 12 serotypes (though not serotype 14), and 12 sequence types (including two new ones and an undetermined type for the S. pseudopneumoniae strain). In all mef(A) pneumococci, the mef element was a regular Tn1207.1 transposon, whereas of the mef(E) isolates, 17 carried the mega element and 2 exhibited a previously unreported organization, with no PCR evidence of the other open reading frames of mega. The mef gene of these two isolates, which did not match with the mef(E) gene of the mega element (93.6% homology) and which exhibited comparable homology (91.4%) to the mef(A) gene of the Tn1207.1 transposon, was identified as a novel mef gene variant and was designated mef(I). While penicillin-nonsusceptible isolates (three resistant isolates and one intermediate isolate) were all mef(E) strains, tetracycline resistance was also detected in three mef(A) isolates, due to the tet(M) gene carried by a Tn916-like transposon. A similar mechanism accounted for resistance in four of the five tetracycline-resistant isolates carrying mef(E), in three of which mega was inserted in the Tn916-like transposon, giving rise to the composite element Tn2009. In the fifth mef(E)-positive tetracycline-resistant isolate (the S. pseudopneumoniae strain), tetracycline resistance was due to the presence of the tet(O) gene, apparently unlinked to mef(E).

Composite Structure of Streptococcus pneumoniae Containing the Erythromycin Efflux Resistance Gene mef(I) and the Chloramphenicol Resistance Gene catQ

ABSTRACT In recent years mef genes, encoding efflux pumps responsible for M-type macrolide resistance, have been investigated extensively for streptococci. mef(I) is a recently described mef variant detected in particular isolates of Streptococcus pneumoniae instead of the more common mef(E) and mef(A). This study shows that mef(I) is located in a new composite genetic element, whose sequence was completely analyzed and the left and right junctions determined, demonstrating a unique genetic organization. The new composite structure (30,505 bp), designated the 5216IQ complex, consists of two halves: a left one (15,316 bp) formed by parts of the known transposons Tn5252 and Tn916, and a right one (15,115 bp) formed by a new fragment, designated the IQ element. While the defective Tn916 contained a silent tet(M) gene, the IQ element, ending with identical transposase genes on both sides and containing the mef(I) gene with an adjacent new msr(D) gene variant and a catQ chloramphenicol acetyltransferase gene, was completely different from the genetic elements carrying other mef genes in pneumococci. This is the first report demonstrating catQ in S. pneumoniae and showing its linkage with a mef gene. Analysis of the chromosomal region beyond the left junction revealed an organization more similar to that of S. pneumoniae strain TIGR4 than to that of strain R6. The 5216IQ complex was apparently nonmobile, with no detectable transfer of erythromycin resistance being obtained in repeated transformation and conjugation assays.

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.

In Vitro Antibacterial Activities of AF 3013, the Active Metabolite of Prulifloxacin, against Nosocomial and Community Italian Isolates

ABSTRACT AF 3013, the active metabolite of prulifloxacin, was tested to determine its inhibitory and bactericidal activities against 396 nosocomial and 258 community Italian isolates. Compared with that of ciprofloxacin, its activity (assessed in MIC and minimal bactericidal concentration tests) was generally similar or greater against gram-positive bacteria and greater against gram-negative bacteria. In time-kill assays using selected isolates, its bactericidal activity was comparable to that of ciprofloxacin.