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.     

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.     

Common antibiotic resistance plasmids in Staphylococcus aureus and Staphylococcus epidermidis from human and canine infections

The plasmids of a multiresistant "canine" Staphylococcus epidermidis-culture were investigated. Two small plasmids, the 4.55 kB chloramphenicol resistance (CmR-) plasmid pSC4 and the 4.45 kB tetracycline resistance (TetR-) plasmid pST 3 could be isolated. Detailed restriction maps of pSC 4 and pST 3 were constructed by double restriction endonuclease digests. The restriction maps revealed extensive structural homologies between pSC 4 from "canine" S. epidermidis and the CmR-plasmid pC 221 from "human" S. aureus as well as between pST 3 from "canine" S. epidermidis and the TetR-plasmid pT 181 from "human" S. aureus. These data suggested that an exchange of small plasmids between S. epidermidis and S. aureus might be possible.     

Characterisation of chloramphenicol resistance plasmids of Staphylococcus aureus and S. epidermidis by restriction enzyme mapping techniques

Chloramphenicol resistance (Cmr) plasmids pSK2 and pSK5 from Staphylococcus aureus and pSK102 and pSK103 from S. epidermidis have been characterised and detailed restriction endonuclease cleavage maps constructed. TaqI digestion profiles illustrated the identity of pSK5 and pSK102 and also revealed a high degree of similarity between these four Cmr plasmids from Australian staphylococci and three Cmr plasmids from S. aureus strains of geographically unrelated origin. DNA-DNA hybridisation indicated that the chloramphenicol acetyltransferase determinant carried by pSK5/pSK102 could be found on other structurally-distinct Cmr plasmids. The role of S. epidermidis as a reservoir for Cmr plasmids found in S. aureus is discussed.     

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.     

Genetic linkage between resistance to quaternary ammonium compounds and beta-lactam antibiotics in food-related Staphylococcus spp

Little is known about the occurrence of antimicrobial resistance determinants in staphylococci isolated from food and food processing industries. Quaternary ammonium compound (QAC)-resistant coagulase-negative staphylococci (CNS) isolated from food and food-processing industries were investigated for the presence of genetic determinants (qacA/B and qacC/smr) encoding resistance to the QAC benzalkonium chloride (BC), several antibiotic resistance genes, and staphylococcal insertion sequences IS257 and IS256. Six qacA/B-harboring strains were resistant to penicillin and hybridized to a blaZ probe. The qacA/B and blaZ probes hybridized to plasmids of similar size in three isolates. Molecular and genetic characterization of the 23-kb plasmid (pST6) of Staphylococcus epidermidis St.6 revealed the presence of qacB adjacent to an incomplete beta-lactamase transposon Tn552 encoding the gene cluster blaZ, blaR, and blaI. Sequence analysis of flanking regions and the intergenic region between blaZ and qacB revealed the presence of IS257 downstream of blaZ as well as sin and binR between blaZ and qacB. In the three other BC and penicillin-resistant strains, the qacA/B and blaZ genes were located on separate plasmids. A qacC harboring S. epidermidis strain (St.17) also hybridized to tetK (tetracycline resistance) and ermB (erythromycin resistance) genes. The individual genes were located on separate plasmids, suggesting no linkage between QAC and antibiotic resistance determinants. Plasmid-free Staphylococcus aureus RN4220 allowed uptake of the pST6 plasmid DNA, indicating that the resistance genes could potentially be transferred to pathogens under selective stress. In conclusion, presence of both resistance determinants could lead to co-selection during antimicrobial therapy or disinfection in hospitals or in food industries.     

Co-transfer of plasmids in association with conjugative transfer of mupirocin or mupirocin and penicillin resistance in methicillin-resistant Staphylococcus aureus

Two distinct strains of methicillin-resistant Staphylococcus aureus (MRSA) isolated from patients in a dermatology ward were also resistant to mupirocin. The mupirocin resistance plasmids from both strains were indistinguishable by EcoRI and HindIII restriction digest analysis, except for the presence of genes apparently mediating penicillinase production in some transconjugants. Conjugative transfer of the plasmid mediating mupirocin resistance from one of these strains to a recipient S. aureus was accompanied in some cases by co-transfer of plasmids mediating resistance to tetracycline or erythromycin; in some instances a plasmid which possessed no apparent resistance markers was also transferred. The second strain demonstrated conjugative transfer of penicillin and mupirocin resistance as well as transfer of a plasmid mediating gentamicin resistance, but transfer of erythromycin resistance was not apparently plasmid-mediated.     

Variability of resistance plasmids in coagulase-negative staphylococci and their importance as a reservoir of antimicrobial resistance

Coagulase-negative staphylococci (CoNS) are an important cause of human and animal diseases. Treatment of these diseases is complicated by their common antimicrobial resistance, caused by overuse of antibiotics in hospital and veterinary environment. Therefore, they are assumed to serve as a reservoir of resistance genes often located on plasmids. In this study, we analyzed plasmid content in 62 strains belonging to 10 CoNS species of human and veterinary origin. In 48 (77%) strains analyzed, 107 different plasmids were detected, and only some of them showed similarities with plasmids found previously. In total, seven different antimicrobial-resistance genes carried by plasmids were identified. Five of the CoNS staphylococci carried plasmids identical with either those of other CoNS species tested, or a well characterized Staphylococcus aureus strain COL, suggesting plasmid dissemination through horizontal transfer. To demonstrate the possibility of horizontal transfer, we performed electroporation of four resistance plasmids among Staphylococcus epidermidis, Staphylococcus petrasii, and coagulase-positive S. aureus strains. Plasmids were transferred unchanged, were stably maintained in recipient strains, and expressed resistance genes. Our work demonstrates a great variability of plasmids in human and veterinary staphylococcal strains and their ability to maintain and express resistance plasmids from other staphylococcal species.     

Analysis of antibiotic resistance determinants inProteus penneri

The plasmid profiles of 65 strains ofProteus penneri were analyzed to determine whether resistance was determined chromosomally or by plasmids. Only seven strains harboured one to three plasmids, although these strains exhibited resistance to a wide range of antibiotics. Markers for ampicillin and tetracycline resistance could be transferred toEscherichia coli by transformation. Plasmids carried resistance to chloramphenicol in two strains and resistance to sulfonamides in one strain. The results showed that resistance is determined chromosomally rather than by plasmids, however the possibility that these bacteria may acquire resistance plasmids which change their antibiotic susceptibility pattern cannot be excluded.     

Transferable or mobilisable antibiotic resistance in Shigella dysenteriae types 1, 2, 3, 4, 6 and 7 isolated in Ethiopia during 1974-85

A total of 199 Shigella dysenteriae isolates resistant to one or more antibiotics and belonging to types 1, 2, 3, 4, 6 and 7 was examined by one-step broth mating with Escherichia coli K12 and, if non-conjugative, additionally by triparental crosses with the conjugative plasmids X and delta. Of the S. dysenteriae type 1 (Shiga's bacillus) strains, 96% harboured conjugative plasmids. During 1974-79, isolates of Shiga's bacillus carried conjugative plasmids coding for ACSSuT (ampicillin, chloramphenicol, streptomycin, sulphonamide, tetracycline) resistance that transferred at low frequencies (less than 10(-4). After 1980, however, about 50% of isolates of Shiga's bacillus with this resistance (R)-type carried conjugative plasmids that transferred at high frequencies (10 degrees-10(-2)) and that expressed the ACT determinant only. The introduction of a new clone of Shiga's bacillus into Ethiopia in 1980 is suspected. Conjugative plasmids coding for SSuT resistance were detected in S. dysenteriae types 2, 3, and 4. Non-conjugative SSu determinants in S. dysenteriae type 3 were mobilised by conjugative plasmids X and delta. R-type CSSuT in strains of types 2 and 7, and R-type ACST in type-3 strains were neither transferable nor mobilisable and are probably determined chromosomally.     

Comparison of phage-mediated and conjugative transfer of staphylococcal plasmids in vitro and in vivo

A strain of Staphylococcus aureus was constructed with which to compare transfer of resistance plasmids by the mechanisms of phage-mediated conjugation and conjugation. Transfer by each mechanism could be distinguished by the patterns of resistances transferred. Conjugation was favoured on dry absorbent surfaces, e.g., human skin, tissue and surgical gauze, whereas phage-mediated conjugation was favoured in fluids, e.g., milk and urine. The degree of hydration of the mating cells is postulated as one factor determining whether plasmids are transferred by phage-mediated conjugation or conjugation. Preliminary evidence indicates that topical creams and ointments affect the conjugative transfer of plasmids.     

Transfer of resistance determinants from a multi-resistant Staphylococcus aureus isolate

The clinical isolate Staphylococcus aureus WBG1024 was resistant to cadmium, benzyl penicillin, kanamycin, neomycin, streptomycin, tetracycline and trimethoprim and harboured a conjugative plasmid pWBG637 (34.5 kb) and non-conjugative plasmids of 23.8, 4.4, 2.8 and 1.9 kb. Transduction and mixed-culture transfer experiments demonstrated that the 4.4-kb plasmid (pWBG632) encoded resistance to tetracycline and the 23.8-kb plasmid (pWBG628) encoded resistance to cadmium, benzyl penicillin, kanamycin, neomycin and streptomycin. The conjugative plasmid pWBG637 was able to mobilise a further 4.4-kb plasmid (pWBG633) encoding streptomycin resistance and recombined with the multiresistance plasmid pWBG628 to produce transconjugantes of various resistance phenotypes.     

Pheromone cross-inhibition between Staphylococcus aureus and Staphylococcus epidermidis

Cross-inhibition by quorum-sensing pheromones between Staphylococcus aureus and Staphylococcus epidermidis was investigated using all known S. aureus agr pheromone subgroups. All S. aureus subgroups were sensitive towards the S. epidermidis pheromone, with the exception of the recently identified subgroup 4. The subgroup 4 pheromone was also the only S. aureus pheromone able to inhibit the S. epidermidis agr response. The close relation of subgroup 4 to subgroup 1 suggests that subgroup 4 might have evolved from subgroup 1 by mutation under the selective pressure of competition with S. epidermidis. The competition between S. aureus and S. epidermidis by means of quorum-sensing cross talk seems to be generally in favor of S. epidermidis, which might explain the predominance of S. epidermidis on the skin and in infections on indwelling medical devices.     

Plasmids coding for colonization factor antigen I and heat-stable enterotoxin production isolated from enterotoxigenic Escherichia coli: comparison of their properties.

We examined seven enterotoxigenic Escherichia coli strains which produced colonization factor antigen I (CFA/I). Four of these strains were from South Africa (three serotype O78:H12 and one serotype O63:H-), one was from Ethiopia (O78:H12), and two were from Bangladesh (O78:H11 and O78:H12). Plasmids coding for CFA/I were mobilized from six of these strains by using resistance or enterotoxin factors. No plasmid was mobilized from the serotype O78:H12 Bangladesh strain. The transconjugants obtained from crosses with the O78 strains also produced heat-stable enterotoxin (ST), and additional investigations showed that CFA/I and ST were coded for by a single non-autotransferring plasmid. These plasmids were fertility inhibition negative, did not restrict any of the coliphages with which they were tested, and were incompatible with each other. Four had molecular weights of approximately 60 X 10(6), and one had a molecular weight of 52 X 10(6). Like the other CFA/I plasmids, the CFA/I plasmid transferred from the O63:H- strain coded for ST, but this plasmid also coded for heat-labile enterotoxin. In most other respects the properties of this plasmid were similar to those of the CFA/I-ST plasmids previously described. The molecular weight of this plasmid was 65 X 10(6). The IncT R-factor Rtsl was marked with a transposon for tetracycline resistance and then transferred into the two Bangladesh wild-type strains. Plasmids which coded for tetracycline resistance, CFA/I, and ST were transferred from these strains. These plasmids were incompatible with Rtsl and with the CFA/I-ST plasmids described above and were recombinants between Rtsl and a CFA/I-ST plasmid. Their properties are also described.     

Genetic and molecular characterization of pR351 plasmid from enteropathogenic Escherichia coli strain

A pR351 plasmid (Tc Ap Cb) conferring drug resistance of naturally occurring E. coli strain was examined. Conjugation and transduction experiments have indicated that this plasmid is R plasmid aggregate consisting of three independent plasmids: a) conjugative plasmid pR351 A (SuTc) fi- (F) belonging to incompatibility group L, b) conjugative plasmid pR351 B (SuApCb) fi- (F) belonging to incompatibility O, and c) non-conjugative plasmid pR351 C (ApCb). The existance of these plasmids in pR351 plasmid aggregate was confirmed by the agarose gel analysis of plasmid DNA isolated from the lysates of E. coli J53 transconjugants carrying pR351 A or pR351 B plasmids and from E. coli K12 C600 transductant carrying pR351 C plasmid. Molecular mass of these plasmids was found to be 55.60 and 3 Mdal respectively. The non-conjugative pR351 C plasmid could be mobilized by Col B and F factors. Our findings have indicated that two fi- (F) R plasmids can stably coexist in R plasmid aggregate.     

Plasmids coding for drug resistance and localized adherence to HeLa cells in enteropathogenic Escherichia coli O55:H- and O55:H6.

Plasmids coding for drug resistance and localized adherence (LA) to HeLa cells were found in two enteropathogenic Escherichia coli strains belonging to serotypes O55:H- and O55:H6. Strain 49-81 HSJ (O55:H-) carries two plasmids, one coding for both ampicillin resistance (Apr) and LA (pMS49). Strain 71-82 HSJ (O55:H6) harbors only one plasmid, coding for resistance to sulfadiazine, chloramphenicol, kanamycin, ampicillin, and LA (pMS71). Plasmids pMS49 and pMS71 were transferred to E. coli K-12 711 and from this strain to E. coli K-12 J53. Curing with acridine orange of an Apr LA+ transconjugant showed that both characteristics were lost simultaneously. The plasmids have a molecular weight of approximately 55 X 10(6) and are the first naturally recombinant plasmids coding for adherence and drug resistance described in enteropathogenic E. coli.     

Detection of methicillin-resistant staphylococci by using the polymerase chain reaction.

A polymerase chain reaction (PCR)-based test was developed for the detection of mecA in staphylococci. To facilitate this process, a rapid cell lysis procedure was established for the release of DNA from staphylococcal strains. Primers based on the DNA sequence of the mecA gene from Staphylococcus aureus were used in PCRs to screen for the presence of this gene in a total of 98 staphylococcal isolates. Fifty-one isolates were mecA positive (17 S. aureus strains and 34 coagulase-negative staphylococci including S. epidermidis, S. haemolyticus, and S. simulans). Results obtained with PCRs were generally consistent with those of standard microbiological assays. PCRs designed to detect the femA gene (factor essential for methicillin resistance) revealed the presence of the gene in all S. aureus strains examined regardless of the susceptibility profiles of the strains to methicillin. In contrast, femA could not be detected in coagulase-negative staphylococci by PCR with the same primers. Low-stringency hybridization suggested the presence of a gene structurally related to femA in S. epidermidis and other coagulase-negative staphylococci examined.     

In vitro activities of eight antibiotics against methicillin-resistant S. aureus and S. epidermidis strains isolated in Korea

Staphylococcus aureus and Staphylococcus epidermidis strains isolated at eight large medical centers in Korea were examined for methicillin resistance and resistance to eight other antibiotics; cefazolin, cefamandole, cefuroxime, cefoxitin, cefotaxime, moxalactam, penicillin G and vancomycin. Methicillin resistance was found in 296 of 1225 strains (24.2%) of S. aureus and 126 of 348 strains (36.2%) of S. epidermidis. Methicillinresistant strains were isolated from all sources with the frequency of isolation ranging from 11% to 60%. From pleural effusion, throat swab and blood, methicillin-resistant strains of S. aureus were more frequently isolated with statistical significance (Chi-squared test, 95% confidence). Almost all of Methicillin-resistant S. aureus (MRSA) and S. epidermidis (MRSE) strains were multiply resistant to one or more tested eight antibiotics. However only 7(2.4%) of 296 MRSA strains and 2(1.6%) of 126 MRSE strains were resistant to vancomycin. Vancomycin was the most effective antibiotic against staphylococcal isolates as well as MRSA and MRSE.     

Detection of Staphylococcus aureus and Staphylococcus epidermidis in Clinical Samples by 16S rRNA-Directed In Situ Hybridization

Staphylococcus epidermidis and Staphylococcus aureus are the most common causes of medical device-associated infections, including septicemic loosenings of orthopedic implants. Frequently, the microbiological diagnosis of these infections remains ambiguous, since at least some staphylococci have the capacity to reduce their growth rate considerably. These strains exhibit a small-colony phenotype, and often they are not detectable by conventional microbiological techniques. Moreover, clinical isolates of S. aureus and S. epidermidis adhere to polymer and metal surfaces by the generation of thick, multilayered biofilms consisting of bacteria and extracellular polysaccharides. This study reports improved detection and identification of S. aureus and S. epidermidis by an in situ hybridization method with fluorescence-labeled oligonucleotide probes specific for staphylococcal 16S rRNA. The technique has proven to be suitable for the in situ detection of staphylococci, which is illustrated by the identification of S. epidermidis in a connective tissue sample obtained from a patient with septicemic loosening of a hip arthroplasty. We also show that this technique allows the detection of intracellularly persisting bacteria, including small-colony variants of S. aureus, and the differentiation of S. epidermidis from other clinically relevant staphylococci even when they are embedded in biofilms. These results suggest that the 16S rRNA in situ hybridization technique could represent a powerful diagnostic tool for the detection and differentiation of many other fastidious microorganisms.     

Presence of icaA and icaD genes and slime production in a collection of staphylococcal strains from catheter-associated infections

Both Staphylococcus epidermidis and Staphylococcus aureus are important causes of infections associated with catheters and other medical devices. It has recently been shown that not only S. epidermidis but also S. aureus can produce slime and carries the ica operon responsible for slime production. In the operon, coexpression of icaA and icaD is required for full slime synthesis. In this study, the presence of icaA and icaD was determined in a collection of 91 staphylococcal (68 S. epidermidis and 23 S. aureus) strains from intravenous catheter-associated infections, in 10 strains from the skin and mucosa of healthy volunteers, and in two reference strains by a PCR method. Slime-forming ability was tested on Congo red agar plates; 49% of S. epidermidis strains from catheters and, surprisingly, 61% of S. aureus strains were icaA and icaD positive and slime forming. All the saprophytic strains turned out to be negative for both icaA and icaD and also non-slime forming. Two S. aureus and one S. epidermidis strain from catheters, detected as icaA and icaD positive by PCR analysis and as slime forming (black colonies) at 24 h on Congo red agar, at 48 h exhibited tiny red spikes at the center of black colonies. The onset of these variants could not be ascribed to a mutagenic potential of Congo red, which, in the Ames test, was devoid of mutagenicity. PCR analysis showed that these red variants were negative for both icaA and icaD and even lacking the entire icaADBC operon. The data reported indicate an important role of ica genes as a virulence marker in staphylococcal infections from intravenous catheters.