Clinical Isolates Of Staphylococci Research Articles

VALIDATION OF MULTIPLEX PCR STRATEGY FOR SIMULTANEOUS DETECTION AND IDENTIFICATION OF METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS

Multiplex polymerase chain reaction (PCR) strategy is described for rapid identification of clinically relevant methicillin resistant Staphylococcus aureus (MRSA) that targets mecA and coagulase genes. In this study, 150 staphylococcal clinical isolates were used that included 40 isolates of MRSA, 55 isolates of methicillin susceptible S. aureus (MSSA), 44 isolates of methicillin susceptible coagulase negative Staphylococcus spp. (MS-CoNS) and 11 isolates of methicillin resistant coagulase negative Staphylococcus spp. (MR-CoNS). Out of 55 S. aureus strains, three strains demonstrated mecA gene, which appeared to be oxacillin sensitive by disc diffusion. When (MS-CoNS) were evaluated, 10 isolates classified as oxacillin sensitive phenotypically, yielded positive results in PCR method. The results for mecA detection by PCR were more consistent with disk susceptibility tests in case of MRSA (100%) and MSSA (95%) isolates. In contrast to above results with MRSA and MSSA, mecA detection by PCR in MS-CoNS showed less correlation with disk susceptibility tests (77%). The results for coag detection by PCR were consistent with phenotypic tests in all isolates.

CcpA Mediates the Catabolite Repression of tst in Staphylococcus aureus

Some clinical isolates of Staphylococcus aureus produce the superantigenic toxic shock syndrome toxin 1 (TSST-1), encoded by tst, located on pathogenicity islands. The expression of tst is complex and is influenced by environmental conditions such as pH, CO(2), and glucose. We identified a putative catabolite-responsive element (cre) in the promoter regions of all known tst genes, indicating that tst transcription may be regulated by the catabolite control protein CcpA. By introducing tst genes under the control of their native promoters or tst promoter-reporter gene fusions in wild-type strain Newman, we showed that glucose was able to repress tst transcription and TSST-1 production, whereas glucose repression was abolished in the corresponding Delta ccpA mutant. Stabilizing the pH ruled out a pH effect due to acid production during glucose catabolism. CcpA thus directly regulates tst transcription, linking carbohydrate utilization to virulence gene expression in S. aureus.

In Vitro Evaluation of CBR-2092, a Novel Rifamycin-Quinolone Hybrid Antibiotic: Microbiology Profiling Studies with Staphylococci and Streptococci

We present data from antimicrobial assays performed in vitro that pertain to the potential clinical utility of a novel rifamycin-quinolone hybrid antibiotic, CBR-2092, for the treatment of infections mediated by gram-positive cocci. The MIC(90)s for CBR-2092 against 300 clinical isolates of staphylococci and streptococci ranged from 0.008 to 0.5 mug/ml. Against Staphylococcus aureus, CBR-2092 exhibited prolonged postantibiotic effects (PAEs) and sub-MIC effects (SMEs), with values of 3.2, 6.5, and >8.5 h determined for the PAE (3x MIC), SME (0.12x MIC), and PAE-SME (3x MIC/0.12x MIC) periods, respectively. Studies of genetically defined mutants of S. aureus indicate that CBR-2092 is not a substrate for the NorA or MepA efflux pumps. In minimal bactericidal concentration and time-kill studies, CBR-2092 exhibited bactericidal activity against staphylococci that was retained against rifampin- or intermediate quinolone-resistant strains, with apparent paradoxical cidal characteristics against rifampin-resistant strains. In spontaneous resistance studies, CBR-2092 exhibited activity consistent with balanced contributions from its composite pharmacophores, with a mutant prevention concentration of 0.12 mug/ml and a resistance frequency of <10(-12) determined at 1 mug/ml in agar for S. aureus. Similarly, CBR-2092 suppressed the emergence of preexisting rifamycin resistance in time-kill studies undertaken at a high cell density. In studies of the intracellular killing of S. aureus, CBR-2092 exhibited prolonged bactericidal activity that was superior to the activities of moxifloxacin, rifampin, and a cocktail of moxifloxacin and rifampin. Overall, CBR-2092 exhibited promising activity in a range of antimicrobial assays performed in vitro that pertain to properties relevant to the effective treatment of serious infections mediated by gram-positive cocci.

Identification of Factors Contributing to T-Cell Toxicity of Staphylococcus aureus Clinical Isolates

We examined the ability of 206 clinical isolates of Staphylococcus aureus to lyse T cells and found differences between Agr groups. We found that the beta and delta hemolysins are involved and that methicillin-resistant S. aureus strains are less toxic than methicillin-susceptible S. aureus strains.

Multiplex PCR assay to identify methicillin-resistantStaphylococcus haemolyticus

Staphylococcus haemolyticus is the most frequently coagulase-negative Staphylococcus species associated with antimicrobial resistance isolated from nosocomial infections. We developed an accurate and simple multiplex PCR assay to identify methicillin-resistant S. haemolyticus (MRSH) isolates. We designed species-specific primers of the mvaA gene that encodes a 3-hydroxy-3-methylglutaryl coenzyme A involved in the mevalonate pathway of the microorganism. Simultaneously, mecA gene primers of methicillin resistance were also used. The PCR assay was established using 16 strains of different reference Staphylococcus species and validated with a collection of 147 clinical staphylococcal isolates that were also phenotypically characterized. Reliable results for the detection of MRSH isolates were obtained for 100% of the strains evaluated, showing that this PCR assay can be used for the routine microbiology laboratories. This is the first report using species-specific multiplex PCR to detect a single segment of S. haemolyticus associated with a segment of mecA gene.

Macrolide–lincosamide–streptogramin B resistance phenotypes in clinical staphylococcal isolates

The prevalence of macrolide–lincosamide–streptogramin B (MLS B) resistance as well as the MLS B resistance phenotypes were investigated by the double-disk diffusion test among 532 clinical staphylococci isolates in a Turkish university hospital. The activity of other antimicrobials, including trimethoprim/sulfamethoxazole, telithromycin, quinupristin/dalfopristin, linezolid, gentamicin, chloramphenicol, ciprofloxacin and vancomycin, was also evaluated. Of 532 isolates, 38.5% were resistant to MLS B antibiotics; 63.9% of the resistant isolates exhibited a constitutive phenotype (cMLS B) whereas 36.1% expressed an inducible resistance phenotype (iMLS B). MLS B resistance was more prevalent among coagulase-negative staphylococci (CoNS) strains. Oxacillin-resistant strains exhibited significantly higher MLS B resistance rates compared with oxacillin-susceptible strains ( P < 0.0001). The most frequently detected resistance phenotype among the total staphylococcal isolates was the constitutive type and this phenotype was more frequently encountered among oxacillin-resistant strains. With the exception of the fully active agents such as vancomycin, linezolid and quinupristin/dalfopristin, the most effective antibiotics were telithromycin and chloramphenicol among all isolates. Susceptibility rates to other antibiotics tested were higher among isolates without MLS B resistance than isolates with MLS B resistance. The detection of a considerable rate (43.5%) of iMLS B resistance among erythromycin-resistant/clindamycin-susceptible strains suggests that the true percentage of clindamycin resistance may be underestimated if testing for inducible resistance is not performed.

Distributions of Macrolide-Lincosamide-Streptogramin B Resistance Phenotypes in Clinical Isolates of Staphylococi

Background: Increased resistance rates to macrolide-lincosamide-streptogramin B (MLSB) antibiotics among clinical isolates of staphylococci are considered as a consequence of an expanded use of these antibiotics in the treatment of Gram-positive infections. The proportion of MLSB resistance phenotypes of staphylococci is quite different by geographical variations and study periods. The aim of the present study was to determine the distribution of MLSB resistance phenotypes among clinical isolates of staphylococci in a university hospital. Methods: The MLSB resistance phenotypes of clinical isolates of staphylococci were investigated by the double-disk diffusion test using erythromycin and clindamycin disks. Results: Of 7,916 isolates, 55.7% exhibited a constitutive resistance phenotype (cMLSB) whereas 8.1% expressed an inducible resistance phenotype (iMLSB). Among 3,419 coagulase-negative staphylococci (CNS), 32.6% and 10.0% exhibited cMLSB and iMLSB resistance phenotypes, respectively. Of 4,497 Staphylococcus aureus isolates, 73.1% and 6.8% were cMLSB and iMLSB resistance phenotypes, respectively. cMLSB was detected among 90.2% of methicillin-resistant S. aureus (MRSA), 46.5% of methicillin-resistant CNS (MRCNS), 3.2% of methicillin-susceptible CNS (MSCNS), and 2.2% of methicillin-susceptible S. aureus (MSSA). iMLSB was detected among 16.5% of MSSA, 11.5% of MRCNS, 6.7% of MSCNS, and 4.4% of MRSA. Conclusion: MLSB resistance was more prevalent among S. aureus isolates than CNS strains. Although cMLSB was the most frequently detected resistance phenotype among the total staphylococcal isolates, methicillin-susceptible strains exhibited somewhat higher iMLSB resistance rates compared with methicillin-resistant strains. (Korean J Clin Microbiol 2008; 11:78-83)

Validation of multiplex PCR strategy for simultaneous detection and identification of methicillin resistantStaphylococcus aureus

Multiplex polymerase chain reaction (PCR) strategy is described for rapid identification of clinically relevant methicillin resistant Staphylococcus aureus (MRSA) that targets mecA and coagulase genes. In this study, 150 staphylococcal clinical isolates were used that included 40 isolates of MRSA, 55 isolates of methicillin susceptible S. aureus (MSSA), 44 isolates of methicillin susceptible coagulase negative Staphylococcus spp. (MS-CoNS) and 11 isolates of methicillin resistant coagulase negative Staphylococcus spp. (MR-CoNS). Out of 55 S. aureus strains, three strains demonstrated mecA gene, which appeared to be oxacillin sensitive by disc diffusion. When (MS-CoNS) were evaluated, 10 isolates classified as oxacillin sensitive phenotypically, yielded positive results in PCR method. The results for mecA detection by PCR were more consistent with disk susceptibility tests in case of MRSA (100%) and MSSA (95%) isolates. In contrast to above results with MRSA and MSSA, mecA detection by PCR in MS-CoNS showed less correlation with disk susceptibility tests (77%). The results for coag detection by PCR were consistent with phenotypic tests in all isolates.

In Vitro Activity of Novel Rifamycins against Gram-positive Clinical Isolates

We describe novel rifamycins that have improved activity, compared with rifampin, against clinical isolates of staphylococci and streptococci, with MIC(90)s of 0.008 and 0.0005 microg/ml, respectively. This enhanced antibacterial activity, along with their potential lack of drug-drug interactions, are considerations that suggest the potential of these novel rifamycins in combination therapy to treat serious Gram-positive infections.

Use of a DNA Microarray for Simultaneous Detection of Antibiotic Resistance Genes among Staphylococcal Clinical Isolates

We developed a multiplex asymmetric PCR (MAPCR)-based DNA microarray assay for characterization of the clinically relevant antibiotic resistance genes leading to penicillin, methicillin, aminoglycoside, macrolide, lincosamide, and streptogramin B (MLS(B)) resistance in staphylococci. The DNA-based assay involves detection of specific conserved regions of the mecA, blaZ (methicillin and penicillin resistance), aac(6')-Ie-aph(2'') (aminoglycoside resistance), ermA and ermC genes (MLS(B) resistance), and the msrA gene (macrolide and streptogramin B resistance). The microarray uses a variable sequence region of the 16S rRNA gene to broadly differentiate between Staphylococcus aureus and other coagulase-negative staphylococci (CoNS). The performance of the microarray was validated with a total of 178 clinically important S. aureus and 237 CoNS isolates, with correlations of 100% for S. aureus to CoNS discrimination and more than 90% for antibiotic resistance between the genotypic analysis determined by the microarray and the phenotype determined by standard methods of species identification and susceptibility testing. The major discrepant results were 17 mecA-positive CoNS and 60 aac(6')-Ie-aph(2'')-positive CoNS isolates measured by microarray that were susceptible to the corresponding antibiotics based on disk diffusion assay. Overall, this microarray-based assay offers a simultaneous, fast (< or =5 h), and accurate identification of antibiotic resistance genes from a single colony, as well as species classification. Our extensive validation of the microarray suggests that it may be a useful tool to complement phenotypic susceptibility testing in clinical laboratories and to survey the spread of antibiotic resistance determinants in epidemiological studies.

In Vitro Activity of Arbekacin Against Clinical Isolates of Staphylococcus species and Gram-negative Bacilli

Methicillin-resistant Staphylococcus aureus (MRSA) and some gram-negative bacilli are very prevalent nosocomial pathogens, commonly causing mixed infections, and are often resistant to multiple drugs. Arbekacin is an aminoglycoside used for the treatment of MRSA infections, but is also active against some gram-negative bacilli. The aim of this study was to determine in vitro activity of arbekacin against recent clinical isolates of staphylococci and gram-negative bacilli. The strains were isolated from clinical specimens of patients at Severance Hospital in 2003. Antimicrobial susceptibility was tested by the Clinical and Laboratory Standards Institute agar dilution method. The following arbekacin breakpoints were used: susceptible, </=4 microg/mL; and resistant, >/=16 microg/mL . All isolates of staphylococci tested were inhibited by </=4 microg/mL of arbekacin, regardless of their methicillin susceptibility. The MIC90s of arbekacin, 1-4 microg/mL, were 8->32-fold and >32-128-fold lower than those of amikacin and gentamicin, respectively. The resistance rates of MRSA, methicillin-susceptible S. aureus, methicillin-resistant coagulase-negative staphylococci (CNS) and methicillin-susceptible CNS were 0% to arbekacin, 0-54% to amikacin, and 24-79% to gentamicin. The MIC90s of arbekacin for Escherichia coli and Citrobacter freundii, 1 microg/mL and 16 microg/mL, were 2-4-fold and 8-16-fold lower than those of amikacin and gentamicin, respectively. However, The MIC90s of arbekacin for other species of gram-negative bacilli, 64->128 microg/mL, were similar to those of other aminoglycosides. Arbekacin may be a useful alternative to glycopeptides for the treatment of monomicrobial methicillin-resistant staphylococcal infections, as well as mixed infections with gram-negative bacilli, as most isolates of E. coli, C. freundii and some other gram-negative bacilli were also susceptible to arbekacin.

Species-level identification of clinical staphylococcal isolates based on polymerase chain reaction–restriction fragment length polymorphism analysis of a partial groEL gene sequence

A pair of degenerate primers that amplified, by polymerase chain reaction (PCR), a partial groEL gene sequence (550 bp) was used for the identification of the 12 most common human staphylococcal pathogens. The amplified products were digested by AluI endonuclease, and distinctive PCR restriction fragment length polymorphism (RFLP) patterns for reference strains were obtained. This protocol was validated by the identification of 89 clinical staphylococcal isolates, and the results were compared with those obtained by the reference biochemical identification, showing 100% concordant results. Two species, Staphylococcus aureus and Staphylococcus lugdunensis, showed intraspecies polymorphisms on their PCR-RFLP patterns. All strains were also identified using the API Staph ID test (bioMérieux, Durham, NC) and the MicroScan WalkAway automated system (Dade Behring, West Sacramento, CA). When 17 Staphylococcus isolates were tested in a blind experiment by the PCR-RFLP of the groEL gene method, all strains were also correctly identified. We propose the PCR-RFLP of the groEL gene with AluI as a reliable and reproducible method for identification of Staphylococcus spp.

Antistaphylococcal activities of CG400549, a new bacterial enoyl-acyl carrier protein reductase (FabI) inhibitor

This study was performed to analyse in vitro and in vivo activities of CG400549, a new FabI inhibitor, against clinical isolates of staphylococci. The mode of action of CG400549 and resistance mechanism of Staphylococcus aureus against CG400549 were also investigated by genetic approaches. In vitro activity of CG400549 was evaluated by the 2-fold agar sdilution method as described by the CLSI, and compared with those of oxacillin, erythromycin, ciprofloxacin, sparfloxacin, moxifloxacin, gemifloxacin, vancomycin, linezolid and quinupristin-dalfopristin. In vivo activity of CG400549 was determined against systemic infections in mice. Time-kill curves of CG400549 were analysed at concentrations of 1 x , 2 x and 4 x MIC against S. aureus strains. CG400549 had the lowest MICs among the test compounds against 238 clinical isolates of S. aureus (MIC90, 0.25 mg/L) and 51 clinical isolates of coagulase-negative staphylococci (MIC90, 1 mg/L). The activity of CG400549 was irrespective of whether the strains were methicillin-susceptible or -resistant. Furthermore, CG400549 was effective by oral or subcutaneous administration against systemic infections in mice. In a time-kill study, CG400549 at concentrations of 1 x MIC, 2 x MIC and 4 x MIC had a bacteriostatic activity during 24 h. A FabI-overexpressing S. aureus strain gave rise to an increase in the MIC of CG400549 compared with the parental strain, while the susceptibilities of the FabI-overexpressing S. aureus strain to the other antibacterial agents such as oxacillin, erythromycin and ciprofloxacin were not affected. This result showed that the mode of action of CG400549 was via inhibition of FabI, which is involved in biosynthesis of fatty acids in bacteria. Study of the resistance mechanism of S. aureus showed that CG400549-resistant mutants of S. aureus had an alteration in FabI at Phe-204 to Leu. CG400549 had potent in vitro and in vivo activity against staphylococci, including methicillin-, ciprofloxacin- and multidrug-resistant staphylococci strains. This compound could be a good candidate for clinical development as a novel anti-MRSA drug.

P892 Genetic basis of macrolide and lincosamide resistance among staphylococcal clinical isolates in a cancer centre

P892 Genetic basis of macrolide and lincosamide resistance among staphylococcal clinical isolates in a cancer centre

P710 Baseline activity of ceftobiprole against methicillin-susceptible and -resistant staphylococcal clinical isolates from Europe collected in 2005–2006

P710 Baseline activity of ceftobiprole against methicillin-susceptible and -resistant staphylococcal clinical isolates from Europe collected in 2005–2006

Multiple Staphylococcal Cassette Chromosomes and Allelic Variants of Cassette Chromosome Recombinases in Staphylococcus aureus and Coagulase-Negative Staphylococci from Norway

We investigated the nature of the staphylococcal cassette chromosome mec (SCCmec) elements and cognate insertion sites in a collection of 42 clinical staphylococcal isolates of various species from Norway. The ccr and mec genes and the attachment sites (attL/attR) were identified by PCR, Southern blot hybridization, and DNA sequencing. We found 10 possibly new SCCmec types and one previously unreported variant of SCCmec type III (mec complex A, ccrAB3, and ccrC7) in Staphylococcus epidermidis, Staphylococcus haemolyticus, and Staphylococcus hominis. Eleven of 42 strains contained multiple copies of ccr, suggesting the presence of mosaic structures composed of multiple SCC elements. S. haemolyticus contained ccrAB2 genes identical to those in S. aureus SCCmec type IV but lacked IS1272 and mec regulators. Two new allelic ccr variants, ccrC6 and ccrC7, were identified. Also, the presumed functional version of ccrB1 was found in a mecA-positive S. hominis strain and in mecA-negative S. epidermidis and S. hominis strains. Only minor differences in direct repeats in the left and right boundaries (attR/attL) were observed, while there was more variation in the inverted repeats. Coagulase-negative staphylococci (CoNS) contained several representatives of different ccr complexes and thus seemed to harbor multiple or composite new types of SCCmec. The enormous diversity observed in the SCCmec elements implies a large SCCmec reservoir in CoNS.

Mapeamento do perfil de resistência e detecção do gene mecA em Staphylococcus aureus e Staphylococcus intermedius oxacilina-resistentes isolados de espécies humanas e animais

Espécies de Staphylococcus spp resistentes a antimicrobianos representam um problema cosmopolita, sendo o controle de sua disseminação um importante desafio. O perfil de resistência a antimicrobianos de isolados de Staphylococcus aureus e Staphylococcus intermedius em amostras clínicas humanas e animais foi avaliado através do método de difusão em disco, no qual foi possível detectar um elevado nível de resistência à ampicilina e à penicilina. A avaliação da resistência à oxacilina, devido à heterogeneidade de resposta do gênero estudado, foi desenvolvida também através das seguintes técnicas: difusão em ágar modificado, ágar screen e microdiluição em caldo, e posteriormente correlacionada com a detecção do gene mecA, pela técnica de PCR, nas amostras consideradas resistentes em pelo menos um dos testes utilizados. A correlação entre os resultados obtidos nos testes fenotípicos com a presença do gene de resistência, considerado um método de referência, foi utilizada para validar a sensibilidade destes. De um total de 80 amostras avaliadas, 28 apresentaram resistência à oxacilina, sendo possível detectar a presença do gene de resistência mecA em 12 destas amostras. Os testes de suscetibilidade à oxacilina apresentaram sensibilidade superior a 50,0%, sendo a difusão em disco simples e o ágar screen considerados mais sensíveis, e a difusão em disco modificada, o de menor sensibilidade.

Formation and properties of in vitro biofilms of ica-negative Staphylococcus epidermidis clinical isolates

Coagulase-negative Staphylococcus epidermidis has become the leading cause of foreign-body infections due to its biofilm formation on all kinds of medical-device surfaces. The biofilm development of S. epidermidis includes two steps: the initial attachment phase and the accumulative phase. In the accumulative phase, the polysaccharide intercellular adhesin (PIA), encoded by the icaADBC locus, is the major component mediating intercellular adhesion. However, recent studies have revealed the emergence of biofilm-positive/ica-negative staphylococcal clinical isolates. In this report, two ica-negative S. epidermidis clinical strains, SE1 and SE4, exhibited their heterogeneity in biofilm architecture under static and flow conditions, compared with the biofilm-positive/ica-positive RP62A strain. Strains with this type of absence of PIA from biofilms also displayed intermediate resistance to vancomycin. More importantly, the cells of both SE1 and SE4 strains were more tolerant than those of RP62A to exposure to lysostaphin and vancomycin. Based on the results, it is suggested that the biofilm-positive/ica-negative strain represents a newly emergent subpopulation of S. epidermidis clinical strains, arising from selection by antibiotics in the nosocomial milieu, which displays a survival advantage in its host environment. Recent epidemiological data support this suggestion, by showing a tendency towards an increasing proportion of this subpopulation in staphylococci-associated infections.

Multi–drug resistance and β–lactamase production among clinical isolates of staphylococci

Thirty–eight isolates of staphylococci isolated from diseased eyes of human were distinguished as methicillin resistant S. aureus (MRSA) 11, methicillin sensitive S. aureus (MSSA) 19, and coagulase negative staphylococci (CNS) 8 isolates. Antibiotic resistance against &#946;–lactam antibiotics was exhibited by 81.81–100% of the isolates belonging to MRSA, followed by MSSA (0–89.47%) and CNS (25–87.50%). Similarly, resistance to other antibiotics like streptomycin, tetracycline, doxycycline and nalidixic acid was also higher among MRSA isolates than MSSA and/or CNS isolates. Overall resistance was higher to penicillin–G (78.95%) followed by ampicillin (73.68%), cefixime and nalidixic acid (68.42% each), cloxacillin (60.52%), streptomycin (57.89%), methicillin (39.40%) and least to tetracycline and doxycycline (21.05% each). Resistance to &#946;–lactam drugs was common and cross–resistance was observed in several strains. Multiple antibiotic resistance in staphylococci was exhibited by 73.68% of the isolates. All the MRSA strains showed resistance to multiple antibiotics. Minimum inhibitory concentrations (MIC) of &#946;–lactam antibiotics ranged from 6.25&#956;g/ml to 2000&#956;g/ml. High level of MIC value (&#8805;800&#956;g/ml) was detected against penicillin, ampicillin and cloxacillin in the 15, 7 and 21 isolates respectively. The &#946;–lactamases of few isolates showed substrate specificity, hydrolyzing one or more &#946;–lactam antibiotics. No significant difference was noticed in the production of &#946;–lactamase among the three groups of staphylococci (MRSA, MSSA and CNS). No co–relation was established between the resistance level against antibiotics and &#946;– lactamases production in the staphylococci isolates from diseased eyes. Staphylococci associated with the human eye infection showed resistance to &#946;–lactams and other antibiotics and &#946;–lactamase production is a common traits. Keywords: MRSA, &#946;–lactamase, antibiotics, antibiotic resistance, &#946;–lactamase, coagulase negative staphylococci Journal of Tropical Microbiology and Biotechnology Vol. 2(1) 2006: 18-23

A New Evolutionary Variant of the Streptogramin A Resistance Protein, Vga(A) LC , from Staphylococcus haemolyticus with Shifted Substrate Specificity towards Lincosamides

We found a new variant of the streptogramin A resistance gene, vga(A)LC, in clinical isolates of Staphylococcus haemolyticus resistant to lincomycin and clindamycin but susceptible to erythromycin and in which no relevant lincosamide resistance gene was detected. The gene vga(A)LC, differing from the gene vga(A) at the protein level by seven amino acid substitutions, was present exclusively in S. haemolyticus strains resistant to both lincosamides and streptogramin A (LS(A) phenotype). Antibiotic resistance profiles of the ATP-binding cassette (ABC) proteins Vga(A)(LC) and Vga(A) in the antibiotic-susceptible host S. aureus RN4220 were compared. It was shown that Vga(A)LC conferred resistance to both lincosamides and streptogramin A, while Vga(A) conferred significant resistance to streptogramin A only. Detailed analysis of the seven amino acid substitutions, distinguishing the two related ABC proteins with different substrate specificities, identified the substrate-recognizing site: four clustered substitutions (L212S, G219V, A220T, and G226S) in the spacer between the two ATP-binding cassettes altered the substrate specificity and constituted the lincosamide-streptogramin A resistance phenotype. A transport experiment with radiolabeled lincomycin demonstrated that the mechanism of lincosamide resistance in S. haemolyticus was identical to that of the reported macrolide-streptogramin B resistance conferred by Msr(A).