Genetic analysis of methicillin-resistant Staphylococcus aureus expressing high- and low-level mupirocin resistance

Clinical strains of methicillin-resistant Staphylococcus aureus (MRSA) expressing high- and low-level mupirocin resistance were studied to determine the genetic location of mupirocin and other resistance determinants. Mupirocin resistance was confirmed by MIC determination with E-test strips. Curing and transfer experiments were used to establish the genetic location of the resistance determinants and the PCR with mupA-specific primers was used to detect the presence of mupA genes. High-level mupirocin-resistant isolates had MICs >1024 mg/L, whereas the low-level resistant isolates had MICs of 32-128 mg/L. The isolates carried plasmids ranging from 2.8 to 38 kb in size. All of them carried 26- and 3.0-kb plasmids, but only the high-level mupirocin-resistant isolates carried a 38-kb plasmid. Curing and transfer experiments revealed that the 26-kb plasmid encoded resistance to cadmium, mercuric chloride, propamidine isethionate and ethidium bromide and the 38-kb plasmid was a conjugative plasmid encoding high-level mupirocin resistance. One isolate, IBN287, carried both plasmid-borne high-level and chromosomal low-level mupirocin resistance. The mupA gene was detected on the 38-kb plasmid DNA but not in the genomic DNA of the low-level mupirocin-resistant isolates. The genomic DNA of strain IBN287 cured of the 38-kb mupirocin resistance plasmid did not contain mupA. The results suggest that different genes encoded low- and high-level mupirocin resistance in these isolates.     

Diversity of staphylococci exhibiting high-level resistance to mupirocin

Plasmids mediating high-level resistance to mupirocin (MIC greater than 1000 mg/L) in staphylococci from various sources were studied by restriction endonuclease cleavage. Several patterns were obtained but six plasmids isolated from various Staphylococcus aureus and S. epidermidis strains were indistinguishable. The diversity and spread of these plasmids is illustrated.     

Emergence of high-level mupirocin resistance in coagulase-negative staphylococci associated with increased short-term mupirocin use

In our hospital, mupirocin has increasingly been used for peri-operative decolonization of Staphylococcus aureus. The target for mupirocin is isoleucyl tRNA synthetase (ileS). High-level resistance to mupirocin is conferred by acquisition of plasmids expressing a distinct ileS gene (ileS2). Here we evaluated the longitudinal trends in high-level mupirocin resistance in coagulase-negative staphylococci (CoNS) and linked this to the presence of ileS2 genes and mupirocin use. We assessed mupirocin resistance in CoNS bloodstream isolates from 2006 to 2011 tested by Phoenix automated testing (PAT). We evaluated the reliability of PAT results using Etest. PAT species determination was confirmed by MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) mass spectrometry. We investigated the presence of ileS2 in the first 100 consecutive CoNS bloodstream isolates of each year using RT-PCR. Mupirocin use increased from 3.6 kg/year in 2006 to 13.3 kg/year in 2010 and correlated with the increase in the percentage of CoNS isolates carrying ileS2 (8% in 2006 to 22% in 2011; Spearman's rho, 0.137; P = 0.01). The sensitivity and specificity of PAT for detecting high-level mupirocin resistance were 0.97 and 0.97, respectively. ileS2 was detected in 81 of 82 phenotypically highly mupirocin-resistant strains and associated with resistance to ciprofloxacin, erythromycin, and clindamycin. In conclusion, we found a rapid increase in high-level resistance to mupirocin and resistance to other antibiotics in CoNS associated with an increase in mupirocin use. The associated resistance to other antibiotics may result in a reduction of oral antibiotic options for prolonged treatment of prosthetic infections with CoNS.     

Transfer of plasmid-borne aminoglycoside-resistance determinants in staphylococci

Aminoglycoside-resistance determinants in staphylococci are borne on conjugative and non-conjugative plasmids. The conjugative plasmids were found in methicillin-resistant strains of Staphylococcus aureus isolated recently in Darwin and Sydney, Australia and in Houston, Texas, USA. These plasmids and the class-2 conjugative plasmid reported by Archer and Johnston (1983) had similar patterns of EcoR1 restriction-endonuclease fragments, encoded resistance to gentamicin, kanamycin and neomycin, transferred to a non-lysogenic recipient in conditions that promoted close cell-to-cell contact and mobilised a small, non-conjugative plasmid. A further plasmid, pWG14, encoding resistance to kanamycin, neomycin, streptomycin, erythromycin and lincomycin, also displayed conjugative properties but did not mobilise the small, non-conjugative plasmid. The transfer frequency of all conjugative plasmids was stimulated by the addition of polyethylene glycol, particularly at concentrations above 20%, to mixtures of donor and recipient broth cultures. Polyethylene glycol appeared to promote close cell-to-cell contact between donor and recipient cells. A representative of the most common aminoglycoside-resistance plasmids in Australian isolates of methicillin-resistant S. aureus was non-conjugative and transferred by a bacteriophage-mediated system to a lysogenic recipient. With the exception of plasmid pWG14, the conjugative plasmids were also transferred by a bacteriophage-mediated system. Furthermore, cultural conditions that favoured conjugative transfer of plasmids inhibited bacteriophage-mediated transfer and vice versa. The efficacy of the two transfer systems for analysing the plasmids of gentamicin-resistant, methicillin-resistant isolates of S. aureus has been compared.     

Molecular characterization of resistance to mupirocin in methicillin-susceptible and -resistant isolates of Staphylococcus aureus from nasal samples

A total of 15 of 101 (14.8%) nasal methicillin-resistant Staphylococcus aureus (MRSA) isolates exhibited mupirocin resistance (Mup(r)) compared with 1 of 154 (0.6%) methicillin-susceptible Staphylococcus aureus isolates. A total of 14 (93%) isolates exhibiting high-level Mup(r) belonged to a single clone. Horizontal plasmid transfer and transmission of Mup(r) strains contribute to a high incidence of Mup(r) MRSA at our institution.     

High-Level Mupirocin Resistance among Spanish Methicillin-Resistant Staphylococcus aureus

 Twelve strains of methicillin-resistant Staphylococcus aureus with high-level resistance to mupirocin were studied. The MIC of methicillin was 16 to 128 mg/l and the MIC of mupirocin ≥512 mg/l. All of the isolates carried a plasmid of about 30 MDa, and one strain lost resistance to mupirocin when the plasmid was cured. Three different resistance patterns were found: six strains were resistant to ciprofloxacin, kanamycin, and 10 mg/l of cadmium sulfate; two strains were resistant to these agents as well as to gentamicin, erythromycin, clindamycin, mercury chloride, and ethidium bromide; and four strains were resistant to the previously named agents as well as to rifampicin and tetracycline. The use of this valuable topical antibiotic, especially in long-term-care facilities, should be monitored to avoid dissemination of resistance.     

Genetic and molecular characterisation of resistance determinants in methicillin-resistant Staphylococcus-aureus.

A genetic analysis of resistance to antibiotics in methicillin-resistant Staphylococcus aureus was performed. Demonstration of plasmid-specific DNA either in transductants that had received antibiotic-resistance markers from multiply-resistant strains, or in segregants of methicillin-resistant strains that had lost unstable determinants except the one under study, indicated that markers of resistance to penicillin, chloramphenicol and neomycin are present on separate, mutually compatible plasmids. Absence of covalently closed circular DNA was demonstrated in transductants that were resistant to methicillin, tetracycline, erythromycin and streptomycin, as well as in segregants that had lost the penicillinase, chloramphenicol and neomycin plasmid, but were still resistant to methicillin, tetracycline, erythromycin, streptomycin and the sulphonamides. Analysis of plasmid DNA either in a 5-20% neutral sucrose gradient or by electron microscopy revealed the presence of three readily distinguishable plasmids. The molecular weights of these plasmids were estimated by comparing the sedimentation rate constants with those of known reference plasmids and by contour-length measurements. The molecular weight of the penicillinase plasmid was estimated to be 20 X 10(6) daltons, that of the chloramphenicol plasmid 3 X 10(6) daltons and that of the plasmid carrying the neomycin resistance marker 37 X 10(6) daltons.     

High frequencies of clindamycin and tetracycline resistance in methicillin-resistant Staphylococcus aureus pulsed-field type USA300 isolates collected at a Boston ambulatory health center

Individual or multiple resistance to clindamycin, tetracycline, erythromycin, levofloxacin, or mupirocin was detected in a large proportion of methicillin-resistant Staphylococcus aureus pulsed-field type USA300 isolates collected at an ambulatory health center in Boston. The clindamycin, tetracycline, and mupirocin resistance genes identified in these isolates are commonly associated with plasmids.     

Susceptibility to antimicrobial agents and analysis of plasmids in gentamicin- and methicillin-resistant Staphylococcus aureus from Dublin hospitals

Methicillin- and gentamicin-resistant Staphylococcus aureus (MGRSA) strains isolated from Dublin Hospitals were classified into two groups (phenotypes). Phenotype-I strains expressed high level resistance to gentamicin and were susceptible to fusidic acid; strains resistant to tetracycline harboured a 3 X 10(6)-mol. wt plasmid. Strains in phenotype II usually expressed low level resistance to gentamicin, were resistant to fusidic acid and often harboured a (22-24) X 10(6)-mol. wt plasmid that specified resistance to ethidium bromide, tetracycline, kanamycin, neomycin and trimethoprim, or to combinations of these markers. A few phenotype-II strains expressed higher levels of resistance to gentamicin and other aminoglycosides. All MGRSA strains carried a 21 X 10(6)-mol. wt plasmid conferring resistance to penicillin, ethidium bromide, cadmium and mercury. Gentamicin resistance was invariably chromosomal and all strains carried chromosomal resistance to methicillin, erythromycin, streptomycin and spectinomycin. Several methicillin-resistant S. aureus (MRSA) strains isolated before the emergence of gentamicin resistance harboured a 21 X 10(6)-mol. wt penicillinase plasmid with the same restriction endonuclease profile as that from some MGRSA strains. Some MRSA strains carried other plasmids related to those found in MGRSA strains.     

Antibiotic resistances and plasmids in Staphylococcus aureus from Italian hospitals

A total of 473 Staphylococcus aureus isolates from six Italian hospitals was examined for susceptibility to several antimicrobial agents and for plasmid content. Methicillin-resistant S. aureus (MRSA) were characterised by a plasmid of mol. wt (10(6)) 18-22 or 25 that carried the determinants for penicillinase production, resistance to cadmium ions and resistance to tetracycline. MRSA isolates usually harboured other smaller plasmids of mol. wt (10(6)) 2.8, 2.6 and 1.65 that encoded resistance to tetracycline, chloramphenicol and erythromycin, respectively, and cryptic plasmids of mol. wt (10(6)) c. 2 and 1 were found frequently. Methicillin-sensitive S. aureus (MSSA) that produced penicillinase often carried plasmids of mol. wt (10(6)) 11 or 13. No particular difference was found in plasmid patterns of strains from the various sources. Analysis of plasmids by EcoRI digestion showed that plasmids of similar mol. wt and phenotypic characteristics may have different restriction patterns, but often share one or more fragments in common.     

Molecular Characterization and Transfer Among Staphylococcus Strains of a Plasmid Conferring High-Level Resistance to Mupirocin

 In this work, mupirocin resistance was correlated with the presence of plasmids in methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in the Rio de Janeiro Federal University Hospital in Brazil, where topical mupirocin has been used extensively since 1990. Of 19 strains studied, those exhibiting high-level resistance carried a large and relaxable plasmid of about 35 kb. Mupirocin-sensitive derivatives, obtained by growth at 42  °C of a strain exhibiting high-level resistance, were devoid of the large plasmid, which was designated pMG1. Mupirocin resistance was transferred to strain RN8411 during overnight filter-matings at low frequencies (7.0×10^–9/donor). The pMG1 plasmid was shown to be responsible for high-level mupirocin resistance in our isolates and to be incompatible with pGO1. Hybridization experiments suggested that mupirocin resistance in pMG1 is due to the presence of the ileS-2 gene. The pMG1 plasmid was successfully and bidirectionally transferred from Staphylococcus aureus to Staphylococcus epidermidis, suggesting that the latter may be a reservoir of this resistance plasmid. No transfer was detected to Staphylococcus haemolyticus. The development of self-transferable high-level mupirocin resistance should be considered when using mupirocin to control the spread of MRSA in hospitals.     

Molecular analysis of isoleucyl-tRNA synthetase mutations in clinical isolates of methicillin-resistant Staphylococcus aureus with low-level mupirocin resistance

Emergence and spread of low-level mupirocin resistance in staphylococci have been increasingly reported in recent years. The aim of this study was to characterize missense mutations within the chromosomal isoleucyl-tRNA synthetase gene (ileS) among clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) with low-level mupirocin resistance. A total of 20 isolates of MRSA with low-level mupirocin resistance (minimal inhibitory concentration, 16-64 microg/mL) were collected from 79 patients in intensive care units for six months. The isolates were analyzed for isoleucyl-tRNA synthetase (IleS) mutations that might affect the binding of mupirocin to the three-dimensional structure of the S. aureus IleS enzyme. All isolates with low-level mupirocin resistance contained the known V588F mutation affecting the Rossman fold, and some of them additionally had previously unidentified mutations such as P187F, K226T, F227L, Q612H, or V767D. Interestingly, Q612H was a novel mutation that was involved in stabilizing the conformation of the catalytic loop containing the KMSKS motif. In conclusion, this study confirms that molecular heterogeneity in ileS gene is common among clinical MRSA isolates with low-level mupirocin resistance, and further study on clinical mutants is needed to understand the structural basis of low-level mupirocin resistance.     

Interpretive criteria for mupirocin susceptibility testing of Staphylococcus spp. using CLSI guidelines

Mupirocin is an antimicrobial agent commonly used to treat staphylococcal infection or to eliminate persistent carriage. To date, interpretive criteria have not been established to define susceptibility or resistance when performing mupirocin susceptibility testing. In this evaluation, using CLSI guidelines, a total of 502 staphylococci comprising 219 methicillin-sensitive Staphylococcus aureus, 222 methicillin-resistant S. aureus and 61 coagulase-negative staphylococci are tested by broth microdilution, disc diffusion and E-test. Disc diffusion using 5 microg mupirocin discs was found to be a reliable method to distinguish susceptible and resistant strains. Minimum inhibitory concentration (MIC) determination was required to differentiate low-level and high-level resistance to mupirocin. E-test was found to be an accurate alternative to broth microdilution for the routine determination of MIC values of staphylococci to mupirocin. Broth microdilution and disc-diffusion results were plotted on a scattergram, and error rates were calculated. No errors were found using susceptibility criteria of < 4 microg/mL (MIC) and > 19 mm (zone diameter).     

Antimicrobial properties of Pseudomonas strains producing the antibiotic mupirocin

Mupirocin is a polyketide antibiotic with broad antibacterial activity. It was isolated and characterized about 40 years ago from Pseudomonas fluorescens NCIMB 10586. To study the phylogenetic distribution of mupirocin producing strains in the genus Pseudomonas a large collection of Pseudomonas strains of worldwide origin, consisting of 117 Pseudomonas type strains and 461 strains isolated from different biological origins, was screened by PCR for the mmpD gene of the mupirocin gene cluster. Five mmpD⁺ strains from different geographic and biological origin were identified. They all produced mupirocin and were strongly antagonistic against Staphylococcus aureus. Phylogenetic analysis showed that mupirocin production is limited to a single species.Inactivation of mupirocin production leads to complete loss of in vitro antagonism against S. aureus, except on certain iron-reduced media where the siderophore pyoverdine is responsible for the in vitro antagonism of a mupirocin-negative mutant. In addition to mupirocin some of the strains produced lipopeptides of the massetolide group. These lipopeptides do not play a role in the observed in vitro antagonism of the mupirocin producing strains against S. aureus.     

Strategies for molecular characterisation of methicillin- and gentamicin-resistant Staphylococcus aureus in a Canadian nosocomial outbreak

Sixteen methicillin-resistant Staphylococcus aureus (MRSA) isolates, from a single nosocomial outbreak, were tested for molecular and phenotypic relationships. Two of the 16 outbreak strains were gentamicin resistant (Gmr) and the plasmids that they carried were characterised by reverse field electrophoresis, restriction endonuclease analysis and gene hybridisation. The gentamicin-resistant (Gmr) strains harboured two plasmids, a Gmr plasmid of 36.5 kb and a cryptic plasmid of 25.4 kb, whereas the other 14 isolates contained only the cryptic plasmid. Gentamicin resistance was encoded by a 2.5-kb HindIII fragment of the 32.8-kb plasmid and is similar to the 2.5-kb HindIII fragment also described for S. aureus Gmr plasmids from Australia and the USA. The Gmr plasmid was non-conjugal and was cured by ethidium bromide at a frequency of 4%. Two MRSA strains isolated subsequently from the same hospital were also Gmr and had identical plasmid and restriction endonuclease profiles to the two Gmr strains studied initially. Two other S. aureus isolates from the original carrier detected in this study and from his son were methicillin and gentamicin susceptible and had novel profiles. Since large plasmids show anomalous migration in agarose gels, more definitive analyses than simple plasmid identification should be considered when studying nosocomial outbreaks.     

Multiple antibiotic resistance in Staphylococcus aureus and Staphylococcus epidermidis: plasmids in strains associated with nosocomial infection

The plasmid DNA profiles were compared to phenotypically-similar, antibiotic-resistant strains of Staphylococcus aureus and Staphylococcus epidermidis associated with nosocomial infections in a Melbourne hospital. Whereas resistance to gentamicin, tobramycin and kanamycin was encoded by one of 3 plasmids [pSK1, 18 megadalton (Md); pSK4, 22 Md; pSK9, 17 Md] in S. aureus, no similar plasmids were detected in S. epidermidis. Mediated exclusively by the chromosome in S. aureus, tetracycline resistance was encoded either by the chromosome or by a 2.8 Md plasmid in strains of S. epidermidis. The inability to detect common resistance plasmids in strains of S. aureus and S. epidermidis recovered from this outbreak is in contrast to recent observations with staphylococci from other geographic areas; nevertheless, on the basis of restriction endonuclease analyses of 3 Md chloramphenicol resistance plasmids, it is suggested that a common gene pool does exist within isolates of S. aureus and S. epidermidis from Melbourne hospitals.     

Low concentrations of mupirocin in the pharynx following intranasal application may contribute to mupirocin resistance in methicillin-resistant Staphylococcus aureus

We describe a patient with methicillin-resistant Staphylococcus aureus (MRSA) colonizing the pharynx. The MIC of mupirocin was 0.25 microg/ml before treatment and increased after treatment to 8 microg/ml. Using pulsed-field gel electrophoresis, we confirmed that the genotypes of MRSA that colonized the pharynx before and after the use of mupirocin were identical. We measured the delivery of mupirocin to the pharynx in three normal volunteers and two patients. Low concentrations of mupirocin were present in the pharynx in all cases 10 min to 3 days after intranasal application. Our data suggested that low concentrations of the drug in the pharynx after intranasal application of mupirocin ointment might explain the selection of mupirocin resistance in MRSA.     

A new streptomycin-resistance plasmid from Staphylococcus hyicus and its structural relationship to other staphylococcal resistance plasmids

A small plasmid of 4.4 kb encoding resistance to streptomycin (Smr) was detected in a multiresistant Staphylococcus hyicus culture from a piglet with exudative epidermitis. The plasmid-encoded properties were determined by interspecies protoplast transformation experiments. This plasmid was further characterised by restriction endonuclease analysis and a preliminary restriction map was constructed. The plasmid from S. hyicus that conferred streptomycin resistance was designated as pSAI-1. It showed some structural homology with the streptomycin-chloramphenicol resistance plasmid pSK68 from S. aureus of human origin. The MIC of streptomycin in resistance mediated by pSAI-1 was about 10 times higher than the MICs in resistance mediated by Smr plasmids from human S. aureus strains.     

Expression of a cloned Staphylococcus aureus alpha-hemolysin determinant in Bacillus subtilis and Staphylococcus aureus.

A DNA sequence encoding Staphylococcus aureus alpha-hemolysin, which had been previously cloned and mapped in Escherichia coli K-12, was introduced into Bacillus subtilis BD170 and several strains of S. aureus by using plasmid vectors, some of which could replicate in all three organisms. The determinant was cloned on a 3.3-kilobase pair DNA fragment into B. subtilis by using the vector plasmid pXZ105 to form the hybrid plasmid pXZ111. B. subtilis cells harboring pXZ111 produced large zones of alpha-hemolysis after 18 h of growth at 37 degrees C on rabbit blood agar plates, and alpha-hemolysin activity was detected in supernatants prepared from growing cultures of this strain. The alpha-hemolysin was apparently secreted across the B. subtilis cell envelope. Polypeptides of molecular weights 34,000 and 33,000 were precipitated with anti-alpha-hemolysin serum from lysates prepared from BD170 cells harboring pXZ111. A hybrid replicon which could replicate in both E. coli and S. aureus was constructed in E. coli by ligating a HindIII fragment encoding the replication functions and chloramphenicol resistance genes of S. aureus plasmid pCW59 to the pBR322 alpha-hemolysin hybrid plasmid pDU1150. The DNA of this plasmid, pDU1212, was prepared in E. coli and used to transform protoplasts prepared from a non-alpha-hemolytic, nonrestricting strain of S. aureus RN4220. Some of the transformants contained plasmids which had suffered extensive deletions. Some plasmids, however, were transformed intact into RN4220. Such plasmids were subsequently maintained in a stable manner. pDU1212 DNA was prepared from RN4220 and transformed into alpha-hemolytic S. aureus 8325-4 and two mutant derivatives defective in alpha-hemolysin synthesis. All three strains expressed alpha-hemolysin when harboring pDU1212.     

Characterization of plasmid-mediated aphA-3 kanamycin resistance in Campylobacter jejuni

A total of 254 isolates of Campylobacter jejuni and three isolates of Campylobacter coli, isolated from Sweden, Canada, and Egypt, were screened for kanamycin resistance. Eight strains of C. jejuni contained large plasmids that carried the aphA-3 kanamycin-resistance marker. In six plasmids, the aphA-3 gene was located downstream of an apparent insertion sequence, designated IS607*, which showed a considerable similarity to IS607, characterized on the chromosome of some Helicobacter pylori strains. In contrast, the other plasmids carried the aphA-3 gene as a part of a resistance cluster. This included three resistance markers encoding 6'-adenylyltransferase (aadE), streptothricin acetyltransferase (sat), and 3'-aminoglycoside phosphotransferase type III (aphA-3). The genetic organization of this resistance cluster suggests that it has been acquired by C. jejuni from a Gram-positive organism. The IS607* element was also observed in kanamycin-susceptible strains of C. jejuni on plasmids mediating tetracycline resistance. The kanamycin-resistance phenotype transferred along with tetracycline resistance by conjugation from four representative C. jejuni strains to a recipient strain of C. jejuni. The kanamycin-resistance determinant (aphA-3) was stably transferred from one of the four C. jejuni strains to a recipient strain of Escherichia coli. However, the C. jejuni plasmid, which also carries the tetO gene, was not maintained in E. coli. Pulsed-field gel electrophoresis revealed the integration of approximately 50 kb of the plasmid into the chromosome of the E. coli recipient.