Chromosome- and plasmid-mediated gentamicin resistance in Staphylococcus aureus encoded by Tn4001

DNA sequences corresponding to the 4.7-kb gentamicin, tobramycin and kanamycin resistance (GmrTmrKmr) transposon Tn4001 have been detected on a series of nine structurally-related plasmids that mediate this phenotype in Australian isolates of Staphylococcus aureus. Tn4001 sequences have also been demonstrated on the chromosomes of GmrTmrKmr isolates that do not possess these plasmids, and the exhibited diversity of chromosomal sites occupied by this element implies that Tn4001 has transposed to the chromosome on numerous occasions in vivo. These results suggest that the rapid emergence of nosocomial GmrTmrKmr S. aureus in the early 1980s may have been the result of the transposition of Tn4001 from a chromosomal site to a readily disseminated plasmid.     

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.     

Identification of Shigella sonnei form I plasmid genes necessary for cell invasion and their conservation among Shigella species and enteroinvasive Escherichia coli.

A series of Tn1 insertions in pSS120, the 120-megadalton form I plasmid of Shigella sonnei, were constructed by a Tn1-mediated conduction system previously described (H. Watanabe and A. Nakamura, Infect. Immun. 48:260-262, 1985, and screened for cell invasion in a tissue culture assay. The analysis of Tn1 insertion sites of seven noninvasive mutants suggested that four separate HindIII fragments were necessary for cell invasion. HindIII fragments including Tn1 of mutant plasmids were cloned into a vector plasmid, pACYC184. The DNA was used as a DNA probe to identify the corresponding, parental HindIII fragments. We identified one contiguous molecule of 2.6- and 4.1-kilobase pair (kb) HindIII fragments as being responsible for restoring cell invasiveness to the three mutant plasmids, pHW505, pHW510, and pHW511. Polypeptide analysis in minicells demonstrated that the contiguous HindIII fragments of 2.6 and 4.1 kb coded for at least four polypeptides, of 38, 41, 47, and 80 kilodaltons (kDa). A comparison of polypeptides synthesized by parental and mutant plasmids strongly suggested that the 38-kDa protein was essential for cell invasion. The 4.1-kb DNA which encoded the 38-kDa protein was conserved among plasmids of Shigella species and enteroinvasive Escherichia coli.     

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.     

The virulence plasmids of Salmonella serovars typhimurium, choleraesuis, dublin, and enteritidis, and the cryptic plasmids of Salmonella serovars copenhagen and sendai belong to the same incompatibility group, but not those of Salmonella serovars durban, gallinarum, give, infantis and pullorum

We examined the compatibility of the Salmonella virulence plasmids of serovars choleraesuis, dublin, enteritidis, gallinarum and pullorum and the cryptic Salmonella plasmids of serovars copenhagen, durban, give, infantis and sendai, with the 90 kilobase pair (kb) virulence plasmid of S. typhimurium. The 90 kb virulence plasmid of S. typhimurium in the form of pWR33, a cointegrate of F'::Tn10lac+ts and the 90 kb virulence plasmid, was transferred by bacterial conjugation into the Salmonella strains (except for S. sendai). The compatibility of their plasmids with the 90 kb virulence plasmid of S. typhimurium was then tested. Separately, a 90 kb virulence plasmid tagged with Tn5 was transformed into the S. sendai strain. The 90 kb virulence plasmid of S. typhimurium was found to be incompatible with the plasmids of serovars choleraesuis, copenhagen, dublin, enteritidis, and sendai, but compatible with the plasmids of serovars durban, gallinarum, give, infantis, and pullorum.     

Typing of Staphylococcus aureus and Staphylococcus epidermidis strains by PCR analysis of inter-IS256 spacer length polymorphisms.

IS256 elements are present in multiple copies in the staphylococcal genome, either flanking the transposon Tn4001 or independent of it. PCR-based analysis of inter-IS256 spacer polymorphisms was developed for typing of methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis strains. Using SmaI macrorestriction analysis resolved by pulsed-field gel electrophoresis (PFGE) as the reference method for MRSA typing, excellent reproducibility (100%), discriminatory power (97%), and in vivo stability were observed. Good concordance of the results with those of other molecular typing methods was found for two MRSA collections. Inter-IS256 PCR analysis of a U.S. collection of MRSA strains (n = 36), previously characterized by 15 typing methods, showed more limited discrimination. Agreement was 78% with PFGE analysis and 83% with ribotyping (HindIII). Analysis of a second set of Belgian MRSA strains (n = 17), categorized into two widespread epidemic clones by PFGE analysis, showed 65% agreement. For typing of S. epidermidis strains (n = 26), inter-IS256 PCR showed complete typeability (100%) and good discriminatory power (85%). Inter-IS256 PCR analysis is proposed as an efficient molecular typing assay for epidemiological studies of MRSA or S. epidermidis isolates.     

Possible relationship of a 36-megadalton Salmonella enteritidis plasmid to virulence in mice.

All of the Salmonella enteritidis strains isolated from diseased animals (61 strains) and from beef (2 strains) in Japan and in West Germany (1 strain), except for 2 strains isolated from ducks, harbored either a 36-megadalton (Md) plasmid alone or in combination with several other plasmids of different sizes. It is likely that these 36-Md plasmids from various S. enteritidis strains were derived from the same origin because their plasmid DNAs showed the same cleavage patterns obtained with EcoRI, HindIII, and BamHI. We also suggested that this plasmid is native to S. enteritidis. Tests carried out on two strains isolated from ducks which naturally lacked this plasmid and one strain whose plasmid was artificially cured showed that the strains without the 36-Md plasmid showed less virulence compared to a wild-type strain harboring the 36-Md plasmid, suggesting that this 36-Md plasmid might be associated with virulence for mice.     

DNA fingerprinting by pulsed-field gel electrophoresis is more effective than ribotyping in distinguishing among methicillin-resistant Staphylococcus aureus isolates.

Pulsed-field gel electrophoresis (PFGE) after SmaI restriction of DNA from 239 methicillin-resistant Staphylococcus aureus isolates (from 142 patients) produced 26 different fingerprints. The deduced chromosome sizes ranged from 2,200 to 3,100 kb (+/- 100 kb). A total of 81 isolates taken from 65 patients were then typed by PFGE and ribotyping with ClaI, EcoRI, and HindIII. Ribotypes were less discriminating than PFGE. Ribotyping did not discriminate isolates from a given PFGE fingerprint into different subsets. PFGE may be a more effective epidemiological tool than ribotyping for the typing of methicillin-resistant S. aureus strains.     

Isolation and characterization of transposon mutants of Staphylococcus epidermidis deficient in capsular polysaccharide/adhesin and slime.

We used transposon (Tn) mutagenesis to study the role of capsular polysaccharide/adhesin (PS/A) and slime in adherence of Staphylococcus epidermidis to catheters. pLTV1, containing Tn917-LTV1, was transformed into S. epidermidis M187 by protoplast fusion with S. aureus RN4220(pLTV1), creating M187(pLTV1). Tn mutants were isolated following growth at 42 degrees C; mutants deficient in PS/A and slime production were selected. PS/A- and slime-deficient Tn mutants had a 10-fold decrease in vitro in the initial phase of adherence to catheters, comparable to levels of strains that do not produce PS/A. Introduction of Tn917-LTV1-interrupted DNA from PS/A-deficient mutant M187sn3 into the parental strain via transformation of protoplasts yielded recipients with inserts identical to those of the Tn mutant that were PS/A and slime deficient. Chromosomal DNA flanking the Tn in mutant M187sn3 was cloned into Escherichia coli. The cloned DNA was found to hybridize to approximately 5-kb EcoRI fragments from the parental strain and from control Tn mutants that express parental levels of PS/A and to either approximately 9- or approximately 14-kb EcoRI fragments from other highly adherent, PS/A-producing strains. Mapping studies demonstrated that in the eight PS/A-deficient mutants that have been isolated, the Tn insertions all occur within a region of approximately 11.6 kb that is defined by three EcoRI sites. These results support previous findings indicating that in S. epidermidis PS/A is involved with in vitro adherence to plastic biomaterials and elaboration of PS/A is closely associated with slime production.     

Tn5 mutagenesis of the Salmonella typhimurium 100 kb plasmid: definition of new virulence regions.

In this study, the 100 kb plasmid of Salmonella typhimurium, which is known to contribute to the pathogenicity of the organism, was tagged with the transposon Tn5 to define regions of the plasmid contributing to overall virulence. Eleven randomly selected vir::Tn5 plasmids carried by the plasmid-free S. typhimurium strain WS1321 were physically mapped and then examined in mice for subcutaneous LD50 value, ability to induce splenomegaly, and ability to grow to high numbers in the spleens of infected mice. Nine strains were found to be virulence-attenuated and showed varied levels of growth in the spleens of subcutaneously infected BALB/c mice. Eight of these nine strains carried Tn5 insertions which lie outside the previously defined virulence region. These studies corroborate the findings of other investigators as well as defining novel regions of the 100 kb virulence plasmid involved in the pathogenicity of this organism.     

Comparison of genes involved in penicillin resistance in staphylococci of bovine origin

Ten penicillin-resistant and -susceptible staphylococci, isolated from bovine mastitis milk, were studied for the presence of genes that are, or may be, involved in resistance against penicillin. The repressor (blaI), antirepressor (blaR1), and structural (blaZ) genes of the beta-lactamase-operon were found to be closely linked in all penicillin-resistant strains. The beta-lactamase gene cluster was more commonly located on chromosomal rather than plasmid DNA in the strains studied. The transposase (p480) gene, which has been identified in the Staphylococcus aureus beta-lactamase transposon Tn552, was found in only one single penicillin-resistant S. aureus strain. The other penicillin-resistant S. aureus isolates contained IS1181 in close location with the beta-lactamase gene cluster. In only one S. haemolyticus isolate was the beta-lactamase gene cluster found in close association with IS257. Penicillin-resistant S. aureus strains, which were additionally resistant to tetracycline, contained IS257 in close association with the tetracycline resistance gene (tetK). Sequence analysis of blaI, blaR1, and blaZ in two penicillin-resistant S. aureus strains revealed 94-96% sequence homology with bla in staphylococci of human origin. The results indicate a predominance of class I bla transposons rather than Tn3 family class II transposons in the isolates used in this study.     

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.     

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 unrelated strains of Staphylococcus schleiferi by using ribosomal DNA fingerprinting, DNA restriction patterns, and plasmid profiles.

The molecular characteristics of 31 unrelated strains of Staphylococcus schleiferi isolated from 13 hospitals between 1973 and 1991 were determined by ribosomal DNA fingerprinting by using a digoxigenin-labeled DNA probe, genomic DNA restriction patterns, and plasmid profiles. Only six strains harbored one or two plasmids. DNA restriction analysis, which was carried out with five endonucleases (EcoRI, HindIII, PstI, PvuII, and ClaI), did not allow us to discriminate between isolates. Ribotyping with HindIII, ClaI, or EcoRI enzymes generated six, seven, and nine distinct patterns, respectively. With the combination ClaI-EcoRI, 13 ribotypes were obtained among the 31 strains, suggesting a relative heterogeneity within the species. Moreover, all strains shared two or three common bands, according to the endonuclease used, which were relatively specific for S. schleiferi in comparison with the ribosomal banding patterns described for other coagulase-negative staphylococci. These results illustrate that ribotyping can be used for the epidemiological investigation of S. schleiferi isolates and possibly for taxonomic analysis in this species.     

Genomic fingerprinting of bacteriocin-producer strains of Staphylococcus aureus

Among 363 strains of Staphylococcus aureus, 21 were shown to produce bacteriocins (Bac), antimicrobial peptides with potential biotechnological applications. This collection includes strains which are either isolated from food, patients and healthy cattle, or are involved in subclinical bovine mastitis. From these 21 strains, 17 were shown to carry closely-related 8.0-kb Bac plasmids encoding bacteriocins either identical to or similar to aureocin A70, a bacteriocin able to inhibit strains of Listeria monocytogenes, a food-borne pathogen. Such findings prompted us to investigate the genetic relationships among these Bac+ strains. To obtain more discriminatory results, a combined analysis of AP-PCR, rep-PCR, and a modified PCR technique that we designated SD-PCR was employed. The 17 Bac+ strains harboring 8.0-kb Bac plasmids exhibited seven fingerprint patterns. One such genotype was composed of 8 out of the 11 strains associated with bovine mastitis, which suggests the prevalence of a clone of Bac+ strains involved in this animal infection carrying 8.0-kb Bac plasmids. Our data support the assumption that Bac+ strains of S. aureus carrying genetically related 8.0-kb Bac plasmids do not belong to a single clone. It seems, therefore, that 8.0-kb Bac plasmids have spread horizontally among different S. aureus strains. There also seems to be genetic diversity among the remaining Bac+ strains analyzed.     

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.     

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.     

Cloning and transposon insertion mutagenesis of virulence genes of the 100-kilobase plasmid of Salmonella typhimurium.

We have cloned regions of the 100-kilobase (kb) plasmid, pStSR100, of Salmonella typhimurium SR-11 that confer virulence to plasmid-cured S. typhimurium. Cells carrying recombinant plasmids that conferred virulence were selected by inoculating mice orally with recombinant libraries in virulence plasmid-cured S. typhimurium and harvesting isolates that infected spleens. Three plasmids, pYA401, pYA402, and pYA403, constructed with the cosmid vector pCVD305 conferred wild-type levels of virulence to plasmid-cured S. typhimurium and had a common 14-kb DNA insert sequence. Another recombinant plasmid, pYA422, constructed with the vector pACYC184, conferred to plasmid-cured S. typhimurium a wild-type 50% lethal dose (LD50) level, but mice died more slowly than when infected with wild-type S. typhimurium. Furthermore, pYA422 conferred the ability to cause a higher, but not a wild-type, level of splenic infection on plasmid-cured S. typhimurium. pYA422 had a 3.2-kb insert sequence which mapped to the center of the 14-kb common sequence of the cosmid clones. Transposon Tn5 insertion mutations in pYA403 inhibited virulence to various degrees, and when transduced into the native virulence plasmid of S. typhimurium, these Tn5 insertions decreased virulence to degrees similar to those observed when the Tn5 insertions were present in pYA403. vir-22::Tn5 in pStSR100 greatly lowered infection of spleens relative to unmutagenized virulence plasmid, while vir-26::Tn5 and vir-27::Tn5 lowered splenic infection to lesser degrees. At least three proteins were encoded by pYA403 containing 23 kb of insert sequence and subclone pYA420, containing the 14-kb common insert sequence present in all of the cosmid clones. One of these proteins, with an apparent molecular weight of 28,000, was also encoded by pYA422. The Tn5 insertion that most attenuated virulence, vir-22::Tn5, inhibited synthesis of the 28,000-molecular-weight protein. The vir-22::Tn5 insertion was complemented by recombinant plasmids encoding only the 28,000-molecular-weight protein, suggesting a role of this protein in virulence. However, recombinant plasmids, exemplified by pYA422, that encoded only the 28,000-molecular-weight protein did not confer full virulence.     

Genetic and molecular studies of plasmids coding for colonization factor antigen I and heat-stable enterotoxin in several escherichia coli serotypes.

Plasmids coding for colonization factor antigen I (CFA/I) and heat-stable enterotoxin (ST) were identified in 10 strains of human enterotoxigenic Escherichia coli. The strains, which belonged to serogroups O63, O114, O128, and O153, were isolated in Bangladesh, Latin America, Spain, and South Africa. Two strains produced heat-labile enterotoxin in addition to ST. CFA/I-ST plasmids were mobilized from two O128 strains into E. coli K-12 with the R factor R1-19K-. Like the prototype CFA/I-ST plasmid NTP113, mobilized previously from an E. coli O78 strain into K-12, these two plasmids were non-autotransferring. All 10 CFA/I-ST plasmids were incompatible with NTP113 and had molecular weights ranging from 59 X 10(6) to 72 X 10(6). The molecular properties of seven of these plasmids were compared with those of six CFA/I-ST plasmids previously mobilized from O78 strains from Ethiopia, South Africa, and Bangladesh and with those of one plasmid coding for CFA/I, ST and heat-labile enterotoxin from a South African strain of serogroup O63. Digestion with the restriction endonuclease HindIII showed that several plasmids had very similar fragment patterns and two were identical. Generally, a larger proportion of HindIII fragments were of common size in digests of plasmids identified in strains from related geographical areas, regardless of serogroup. However, all except one plasmid shared five or six HindIII fragments of the same size, one of which had been shown previously to be involved in CFA/I production. There was at least 90% DNA homology between CFA/I-ST plasmids with a molecular weight of about 58 X 10(6) from O78 strains from different sources. Most of the DNA sequences of these plasmids were present in a larger CFA/I-ST plasmid (72 X 10(6) from an O128 strain. The results of genetic and molecular studies suggest that CFA/I and ST production is determined by very similar plasmids in different serogroups of human enterotoxigenic E. coli from several sources.