Primer sequences and PCR fragment size of tested MLS resistance genes.
A total of 92 genes that confer resistance to MLS antibiotics have been described to date. They can be roughly divided into three groups, depending on the mechanisms by which they confer resistance to one or all of these groups of antibiotics. Three main mechanisms of resistance to MLS antibiotics have been described: methylation of rRNA (target modification), active efflux and inactivation of the antibiotic. Target modification is achieved through the action of the protein product of one of more than 42 different erm (erythromycin rRNA methylase) genes. They confer cross resistance between macrolides, lincosamides and streptogramin B (so-called MLSB resistance) and evoke most concerns. Active efflux and inactivating enzymes (M and L) represent two additional mechanisms of resistance that are targeted only to particular antibiotics or antibiotic classes. Based on the mechanisms of resistance, various resistant phenotypes are expressed. The most prevalent phenotypes are ΜLSB (constitutive or inducible), which is associated with the presence mainly of ermA and ermC genes, followed by the MSB phenotype due to the presence of msrA gene. In livestock S. aureus strains, such as CC 398, other genes such as ermT, lnuA, lsaE and mphC genes are detected.
Resistance to macrolides-lincosamides and streptogramins B (MLSB antibiotics) is associated with three main mechanisms: (1) methylation of rRNA (target modification), (2) active efflux and (3) enzymatic inactivation. Till date, a total of 92 genes, conferring resistance to MLSB antibiotics, have been described. The most common genes are
The macrolide group of antibiotics includes natural members, prodrugs and semisynthetic derivatives. The chemical structure of macrolides is characterized by a large lactone ring containing from 12 to 16 atoms to which are attached, via glycosidic bonds, one or more sugars. Erythromycin, whose lactone ring contains 14 atoms, is the oldest molecule (1952), whereas all second-generation macrolides, like roxithromycin and clarithromycin, are hemisynthetic derivatives of erythromycin. Azithromycin is the only macrolide with 15 carbon atoms. Azithromycin, which is produced through the introduction of a nitrogen atom into the macrolide nucleus at C10, exhibits (1) improved penetration into macrophages, fibroblasts and polymorpho-neutrophils, (2) increased accumulation within acidified vacuoles and (3) extended half-life. Additionally, azithromycin shows improved activity against Gram-negative bacteria and other pathogens associated with parasitic infections. Spiramycin and josamycin are macrolides with 16 carbon atoms. All chemical modifications of macrolides were made in order that their properties and action are optimized.
Although the structure of lincosamides is different from the structure of macrolides, they present a similar action spectrum. Lincomycin, which was isolated in 1962, is a fermentation product of
Type-A streptogramin includes cyclic-poly-unsaturated macrolactones: virginiamycin M, pristinamycin IIA and dalfopristin. Type-B streptogramin consists of the cyclic hexadepsipeptide compounds virginiamycin S, pristinamycin IA and quinupristin. Until now, only three streptogramins have been marketed either for treatment or growth promotion: virginiamycin, pristinamycin and quinupristin-dalfopristin. Virginiamycin, a mixture of virginiamycin M (type A streptogramin) and virginiamycin S (type B streptogramin), has been used mainly as growth promoter feed additive in commercial animal farming in the United States and Europe. In contrast, pristinamycin has been used orally and topically in human medicine only in France. Qiunupristin-dalfopristin, in a 30:70 mixture (Synercid), was approved in 1999 for the treatment of serious infections caused by multidrug resistant Gram-positive pathogens, including vancomycin-resistant
MLSB antibiotics share a similar mode of action because they inhibit protein synthesis by targeting the peptidyl transferase center within the 50S subunit (23 s rRNA) of the bacterial ribosome . We note that the bacterial ribosomes are 70S particles comprising of two subunits, 30s and 50S, which are made of RNAs enveloped by proteins; 50S is composed of 5S, 23S rRNAs and 36 proteins (L1-L36) [6, 7].
Although the peptidyl transferase center is the main target site for many antibiotics, the exact mechanism for its activity is still unclear . Overall, the inhibitory action of antibiotics is not only determined by their interaction with specific nucleotides. MLSB could also inhibit peptidyl transferase by interfering with the proper positioning and movement of the tRNAs at the peptidyl transferase cavity [9, 10].
2. Antibacterial spectrum of MLSB
Τhe spectrum of MLSB includes mainly Gram-positive microorganisms (streptococci, staphylococci); however, some of them also have activity against Gram-negative microorganisms (
It is known that some Gram-positive species have intrinsic resistance to some of them.
3. Mechanisms of acquisition of resistance to MLSB
Staphylococci resist MLSB antibiotics in three ways: (1) through target-site modification by methylation or mutation that prevents the binding of the antibiotic to its ribosomal target, (2) through efflux of the antibiotic and (3) by drug inactivation. Modification of the ribosomal target confers broad-spectrum resistance to macrolides, lincosamides and streptogramin B, whereas efflux and inactivation affect only some of these molecules .
3.1. Ribosomal methylation
The most widespread mechanism of resistance to MLSB in Gram-positive bacteria, including both
Erm proteins, encoded by
More than 42
3.2. Antibiotic efflux
In Gram-positive organisms, acquisition of macrolide resistance by active efflux is caused by two classes of pumps, members of the ATP-binding-cassette (ABC) transporter superfamily and of the major facilitator superfamily (MFS). ABC transporters require ATP to function and are usually formed by a channel comprising two membrane-spanning domains and two ATP-binding domains located at the cytosolic surface of the membrane .
The first determinant encoding ABC transporter in staphylococci was the plasmid-borne
However, latter, the combined resistance to lincosamides, pleuromutilins and streptogramin A (SA), referred as the PLSA phenotype, was found to be associated with the presence of the ARE subfamily of class 2 ATP-binding cassette (ABC) ATPases, a class of ABC proteins made up of two homologous ABC ATPase domains separated by a flexible linker without any identifiable transmembrane domains [16, 17, 18]. The flexible linker between each ATPase domain is presumed to be the drug-binding region of the ARE proteins. The
3.3. Enzymatic inactivation
Enzymatic inactivation confers resistance to structurally related antibiotics only. Esterases and phosphotransferases, encoded by
In addition, lincosamide nucleotidyl transferases encoded by
3.4. Uncommon mechanisms of resistance
Ribosomal mutations (A2058G/U or A2059G) of 23S rRNA gene such as mutations in the
On the other hand,
4. Resistant phenotypes: expression, detection and interpretation
Depending on the mechanism of resistance and on the carriage of respective genes, staphylococci can express various MLSB resistant phenotypes. Briefly, these types are described as follows.
4.1. MLSB phenotype (
MLSB phenotype can be expressed as constitutive or inducible . Isolates with a constitutive MLSB phenotype express high level cross-resistance to macrolides, lincosamides and streptogramin B. In fact, clinical methicillin-resistant strains that are constitutively resistant to MLSB antibiotics are widespread.
On the other hand, isolates with an inducible MLSB phenotype express phenotypically only resistance to macrolides and susceptibility to lincosamides. This phenomenon is explained by the fact that, in constitutive resistance, bacteria produce an active mRNA encoding methylase, whereas in inducible resistance, bacteria produce an inactive mRNA, which is unable to encode ribosome methylases. However, in the presence of a macrolide, which acts like an inducer, the mRNA becomes active . The presence of an inducer leads to rearrangements of mRNA, which allow ribosomes to translate the methylase coding sequence.
Inducible expression of
The use of antibiotics being noninducers (such as clindamycin) for treatment of an infection due to a
According to the rules of EUCAST, if a staphylococcal isolate with an inducible MLSB phenotype is detected, it must be reported as resistant and considered adding this comment to the report “Clindamycin may still be used for short-term therapy of less serious skin and soft tissue infections as constitutive resistance is unlikely to develop during such therapy.”
4.2. MSB-phenotype (
MSB phenotype is associated with resistance only to 14- (clarithromycin, erythromycin, roxithromycin) and 15-membered ring macrolides (azithromycin) and streptogramin B, while 16-membered ring macrolides (josamycin and spiramycin) and lincosamides remain active [12, 15]. The
Isolates with this phenotype have probably decreased susceptibility to the combination of quinupristin-dalfopristin. Additional tests (see below) are required for its detection.
M-phenotype ( mphCgenotype)
M-phenotype is associated with the presence of enzymes which inactivate enzymatically only macrolides. Clinical isolates of erythromycin-resistant
PLS A -phenotype
PLSA-phenotype is associated with resistance to lincosamides, pleuromutilins and streptogramins A, while macrolides and streptogramin B remain active  . Various genes such as
4.5. L-phenotype (
L-phenotype is associated with resistance to lincomycin due to the presence of lincosamide nucleotidyl transferases encoded by
Although more than 90 genes conferring resistance to macrolides and lincosamides have been described till date, their presence has not turned out to be a successful story for Gram-positive bacteria. This observation, which is in contrast with the success of emergence of
SB-phenotype is expressed by resistance to streptogramin B due to the presence of
5. Confirmation methods of resistant phenotypes
Among the different types of resistant phenotypes, the most common are MLSB (constitutive or inducible), MSB and M-phenotypes. The clinical microbiology laboratory detects easily and reliably the MLSB constitutive phenotype: the isolates are fully resistant to macrolides and lincosamides. However, isolates with MLSB inducible, MSB and M-phenotypes share the same profile: resistance to macrolides and susceptibility to lincosamides. Therefore, additional test, the double disk diffusion test (D test) is required to be applied.
For the detection of MLSB inducible resistance, it is recommended to place the erythromycin and clindamycin disks 12–20 mm apart (edge to edge, D test). In disk-diffusion tests, a D-shaped zone, caused by induction of methylase production by erythromycin, can be observed (Figure 1). Nowadays, the automated system Vitek II (BoMerieux) has the possibility to detect it.
However, after a negative D test, the differentiation between MSB and M-phenotypes is more complicated and could be based on the MIC values of erythromycin. Isolates with M-phenotype have often lower MIC values to erythromycin, due to the weak activity of hydrolytic enzymes, than isolates with MSB-phenotype, which express fully resistance to macrolides. In addition, MSB-phenotype affects the susceptibility to quinupristin-dalfopristin, decreasing it slowly.
Finally, it is difficult to discriminate isolates with PLSA-phenotype from those with L-phenotype; both share the same profile, including resistance to lincomycin and susceptibility to erythromycin. On the other hand, pleuromutilins and streptogramins A are not included in the panel of antibiotics proposed for susceptibility testing. Probably, the values of MICs to clindamycin and quinupristin-dalfopristin, which usually are not affected by L-phenotype, can be used as indicators .
Molecular detections of the most common genes involved in MLSB resistance are an accurate method for phenotype determination (Table 1).
|Gene||Primers sequence (5′–3′)||PCR fragment size (bp)|
6. Historical background
The first report about the activity of erythromycin was confirmed in 1954 by Derek ; in 1964, Macleod et al. indicated that lincomycin was effective against
In 1971, Lai et al. demonstrated altered methylation of ribosomal RNA in a erythromycin-resistant
In 1990, Ross et al. identified
To date, a variety of genes (such as
7. Epidemiology of MLSΒ resistant staphylococci: recent data
The rate of MLSB-resistant staphylococci varies between countries and species. Unfortunately, in the last decade, data concerning the rate of MLS resistance in staphylococci are limited. Otsuka et al. reported that 97% of MRSA and 34.6% of MSSA were resistant to one or more MLSB agents in a study conducted between 2001 and 2006 . Cetin et al. in a large collection of staphylococci in a Turkish hospital have found that 38.5% were resistant to MLSB antibiotics, while Uzun et al. reported that during 2011–2012, 79% isolates were found as erythromycin-resistant in a tertiary hospital in Ismir [65, 66]. In a tertiary Greek hospital, the rate of MLSB
Regarding the distribution of resistant phenotypes, the most common are MLSB (constitutive or inducible) followed by MSB. In Japan, Otsuka et al. revealed higher incidence of the MLSB-inducible phenotype than in Europe, Turkey and the USA [41, 64, 70, 71, 72, 73]. Such differences in the incidence of phenotypes might reflect differences in the drug usage, the gene carriage and the clonality of strains.
Totally, 92 genes, which confer resistance to MLS antibiotics, have been described to date. They can be roughly divided into three groups, depending on the mechanisms by which they confer resistance to one or all of these groups of antibiotics. Data from different studies agree that the most prevalent genes are
Staphylococci and specially