Filter Mating Experiments Research Articles

First Occurrence of an Escherichia coli Clinical Isolate Producing the VIM-1/VIM-2 Hybrid Metallo-β-Lactamase VIM-12

Gram-negative bacteria producing VIM-type metallo-β-lactamases (MBLs) are increasingly isolated worldwide (8). The VIM group includes at least 13 variants, clustered into three evolutionary lineages, driven by VIM-1, VIM-2, and VIM-7 (www.lahey.org/studies/other.asp#table1). blaVIM-12 is a recently identified blaVIM-1/VIM-2 hybrid gene, originally found in 2005 in a Klebsiella pneumoniae clinical isolate from Greece and ranked as intermediate between blaVIM-1 and blaVIM-2 (6). Its gene cassette resides in a class 1 integron, designated In-h12 (6); it has not since been detected in other gram-negative species. Kinetic parameters of the purified VIM-12 enzyme have not been described. We document herein the identification of the blaVIM-12 gene in an Escherichia coli clinical isolate. E. coli strain 28 was recovered in February 2006 from a decubitus ulcer infection of an 85-year-old male hospitalized at Hippokration General Hospital, Thessaloniki, Greece, for lower respiratory tract infection. MICs of several β-lactams, aminoglycosides, ciprofloxacin, tetracycline, and cotrimoxazole were determined by using Etest (AB Biodisk, Solna, Sweden). The isolate was phenotypically screened for MBL production by using Etest MBL (AB Biodisk) and the imipenem-EDTA double disc synergy test (DDST) (5). Pseudomonas aeruginosa ATCC 27853 was used as a control in all susceptibility assays. PCR detection of various genes encoding MBLs, extended-spectrum beta-lactamases, and AmpC enzymes, including blaVIM, blaIMP, blaSPM, blaTEM, blaSHV, blaGES, blaCTX-M and blaCMY/LAT, and PCR integron mapping were performed as described previously (6), followed by DNA sequencing. Filter mating experiments were performed using E. coli 26R793 (Rifr) as the recipient. Selection of transconjugants was made on MacConkey agar plates containing rifampin (100 μg/ml) and ceftazidime (2 to 4 μg/ml). Plasmid isolation was performed using the ChargeSwitch Plasmid ER minikit (Invitrogen Corporation, Carlsbad, CA) and a standard alkaline lysis protocol, using E. coli 39R861 as the standard plasmid control. Restriction fragment length polymorphism (RFLP) of the plasmid extract of E. coli strain 28 and the transconjugant derived from Klebsiella pneumoniae 2873 carrying VIM-12 in plasmid p2873 (6) was performed using BamHI. The location of the blaVIM gene was determined as described previously (3) by Southern blotting of unsheared genomic DNA, followed by gene-specific hybridization using a digoxigenin-labeled blaVIM-1 probe (7). E. coli strain 28 was susceptible to imipenem and meropenem (MICs, 1 and 0.25 μg/ml, respectively), aztreonam, ciprofloxacin, and cotrimoxazole but resistant to all other antimicrobials tested (Table ​(Table1).1). The presence of MBL was indicated by positive DDST and Etest MBL. PCR for β-lactamase genes showed that E. coli strain 28 was positive for blaVIM and blaCMY/LAT genes but negative for the remaining genes. PCR assays using primers 5′CS and 3′CS in various combinations with primers blaVIM and aacA and nucleotide sequencing revealed an integron structure identical with that of integron In-h12, originally found in the blaVIM-12-producing K. pneumoniae (GenBank accession number {type:entrez-nucleotide,attrs:{text:DQ143913,term_id:72536051}}DQ143913) (6). TABLE 1. Susceptibilities of strain EC28 and its respective transconjugant carrying pEC28 Repeated mating experiments yielded, at a median frequency of 1.8 × 10−2 per donor cell, transconjugant colonies that had elevated MICs of penicillins, cephalosporins, and aminoglycosides but not of carbapenems and tetracycline (Table ​(Table1).1). Plasmid analysis of both clinical and transconjugant colonies showed a single plasmid of approximately 70 kb. PCR specific for blaVIM and blaCMY/LAT in several transconjugant colonies was positive only for the latter gene. RFLP analysis with BamHI showed that the conjugative plasmid of E. coli strain 28 had a different restriction pattern from that of p2873 (6), tested in parallel. The location of In-h12 in the chromosome was indicated by hybridization of the unsheared genomic DNA with the blaVIM-1 probe. Production of VIM-12, a VIM-1/VIM-2 hybrid MBL, is documented here for the first time for an E. coli isolate after its original detection in K. pneumoniae. It is noteworthy that several blaVIM-12-producing K. pneumoniae strains have been identified in the same hospital since the original detection in 2005 (unpublished data), indicating a rather wide dissemination of this gene among our K. pneumoniae isolates. E. coli strain 28 carried an integron identical to In-h12, although not lying in the transferable plasmid p2873, which harbored blaVIM-12 in K. pneumoniae (6). It could be speculated here that the integron In-h12 containing the blaVIM-1/VIM-2 hybrid gene blaVIM-12 has arisen within our hospital settings, where blaVIM-1- and blaVIM-2-carrying gram-negative pathogens are common (1, 2, 4, 7). Alternatively, the blaVIM-12 gene might be independent and have a wide natural distribution in bacterial populations, as was observed previously for other blaVIM genes (8).

Antimicrobial susceptibilities of Lactobacillus, Pediococcus and Lactococcus human isolates and cultures intended for probiotic or nutritional use

To determine MICs of 16 antimicrobials representing all major classes for 473 taxonomically well-characterized isolates of lactic acid bacteria (LAB) encompassing the genera Lactobacillus, Pediococcus and Lactococcus. To propose tentative epidemiological cut-off (ECOFF) values for recognizing intrinsic and acquired antimicrobial resistances in numerically dominant species. On the basis of depositors' information, LAB were grouped in categories of probiotic, nutritional, probiotic or nutritional research, human and animal isolates and tested for their antibiotic susceptibilities by broth microdilution using LAB susceptibility test medium (LSM). Tentative ECOFFs were defined according to the recommendations of the European Committee on Antimicrobial Susceptibility Testing. Isolates showing acquired antimicrobial resistance(s) were selected for PCR-based detection of resistance gene(s) and in vitro conjugative transfer experiments. Tentative ECOFF values of 13 antibiotics were determined for up to 12 LAB species. Generally, LAB were susceptible to penicillin, ampicillin, ampicillin/sulbactam, quinupristin/dalfopristin, chloramphenicol and linezolid. LAB exhibited broad or partly species-dependent MIC profiles of trimethoprim, trimethoprim/sulfamethoxazole, vancomycin, teicoplanin and fusidic acid. Three probiotic Lactobacillus strains were highly resistant to streptomycin. Although erythromycin, clindamycin and oxytetracycline possessed high antimicrobial activities, 17 Lactobacillus isolates were resistant to one or more of these antibiotics. Eight of them, including six probiotic and nutritional cultures, possessed erm(B) and/or tet(W), tet(M) or unidentified members of the tet(M) group. In vitro intra- and interspecies filter-mating experiments failed to show transfer of resistance determinants. Finding of acquired resistance genes in isolates intended for probiotic or nutritional use highlights the importance of antimicrobial susceptibility testing in documenting the safety of commercial LAB.

DfrA25, a novel trimethoprim resistance gene from Salmonella Agona isolated from a human urine sample in Brazil

To describe a novel trimethoprim resistance gene, designated dfrA25, which was detected as a gene cassette within a class 1 integron in Salmonella Agona. The gene was cloned into Escherichia coli MT102 and resistance to 10 different antimicrobial drugs was measured. A phylogenetic tree was constructed based on representative trimethoprim-resistance-mediating DfrA proteins retrieved from GenBank. Filter-mating experiments and Southern blots of plasmid preparations were performed with the donor and selected transconjugants. dfrA25 encodes a dihydrofolate reductase of 157 amino acids with closest identity (85%) to dfrA5 dihydrofolate reductase. dfrA25 was located on a transferable plasmid (approximately 150 kb) that also harboured the tetracycline resistance gene tet(A).

Incidence and behaviour of Tn916‐like elements within tetracycline‐resistant bacteria isolated from root canals

Tetracycline resistance is commonly found in endodontic bacteria. One of the most common tetracycline-resistance genes is tet(M), which is often encoded on the broad-host-range conjugative transposon Tn916. This study aimed to determine whether tet(M) was present in bacteria isolated from endodontic patients at the Eastman Dental Institute and whether this gene was carried on the transferable conjugative transposon Tn916. The cultivable microflora isolated from 15 endodontic patients was screened for resistance to tetracycline. Polymerase chain reactions for tet(M) and for unique regions of Tn916 were carried out on the DNA of all tetracycline-resistant bacteria. Filter-mating experiments were used to see if transfer of any Tn916-like elements could occur. Eight out of 15 tetracycline-resistant bacteria isolated were shown to possess tet(M). Furthermore, four of these eight were shown to possess the Tn916-unique regions linked to the tet(M) gene. Transfer experiments demonstrated that a Neisseria sp. donor could transfer an extremely unstable Tn916-like element to Enterococcus faecalis. The tet(M) gene is present in the majority of tetracycline-resistant bacteria isolated in this study and the conjugative transposon Tn916 has been shown to be responsible for the support and transfer of this gene in some of the bacteria isolated.

Increased conjugation frequencies in clinical Enterococcus faecium strains harbouring the enterococcal surface protein gene esp

This study compared the in-vitro ability of Enterococcus faecium isolates of different origin to acquire vanA by conjugation in relation to the occurrence of the esp gene. In total, 29 clinical isolates (15/29 esp+), 30 normal intestinal microflora isolates (2/30 esp+) and one probiotic strain (esp-) were studied with a filter-mating assay. Conjugation events were confirmed by PCR and pulsed-field gel electrophoresis. Among the infection-derived isolates, the esp+ isolates had higher conjugation frequencies compared with esp- isolates (p < 0.001), with a median value of 6.4 x 10(-6) transconjugants/donor. The probiotic strain was shown to acquire vanA vancomycin resistance in in-vitro filter mating experiments.

Identification of Tn5397-like and Tn916-like transposons and diversity of the tetracycline resistance gene tet(M) in enterococci from humans, pigs and poultry

To analyse the sequence diversity of the tetracycline resistance gene tet(M) and its location on mobile elements in Enterococcus faecium and Enterococcus faecalis from humans, pigs and poultry in Denmark. A total of 76 isolates were screened for Tn916/Tn1545-like and Tn5397-like transposons using PCR. tet(M) was sequenced in 15 of the isolates and compared with tet(M) sequences submitted to GenBank (phylogenetic analysis and signs of recombination). Plasmids were extracted, filter-mating experiments were performed and Tn5397-like transposons were further characterized in selected isolates. In 8 of 13 isolates of E. faecium from broilers, tet(M) was present on Tn5397-like transposons, whereas tet(M) was predominantly associated with Tn916/Tn1545-like transposons in E. faecium from pigs and humans, as well as in E. faecalis from humans, pigs and broilers (50 of 63 isolates). The tet(M) genes were divided into three major subgroups according to the phylogenetic analysis. Subgroup I consisted of tet(M) from Clostridium difficile and E. faecium associated with Tn5397-like elements, subgroup II consisted of tet(M) located on Tn916/Tn1545 family transposons and subgroup III consisted of tet(M) associated with composite elements containing several resistance genes. We found evidence of recombination both within and between these groups. Moreover, we identified an E. faecium isolate with both Tn916/Tn1545-like and Tn5397-like elements. This study showed that enterococci contain diverse tet(M) genes present on different mobile elements, which may suggest that enterococci play an important role in the evolution and horizontal spread of mobile elements carrying tet(M). This is the first report of Tn5397-like elements in enterococci.

Characterisation and transfer of antibiotic resistance genes from enterococci isolated from food

The genetic determinants responsible for the resistances against the antibiotics tetracycline [ tet(M), tet(O), tet(S), tet(K) and tet(L)], erythromycin ( ermA ,B,C; mefA,E; msrA/B; and ereA,B) and chloramphenicol ( cat) of 38 antibiotic-resistant Enterococcus faecium and Enterococcus faecalis strains from food were characterised. In addition, the transferability of resistance genes was also assessed using filter mating assays. The tet(L) determinant was the most commonly detected among tetracycline-resistant enterococci (94% of the strains), followed by the tet(M) gene, which occurred in 63.0% of the strains. Tet(K) occurred in 56.0% of the resistant strains, while genes for tet(O) and tet(S) could not be detected. The integrase gene of the Tn 916- 1545 family of transposons was present in 81.3% of the tetracycline resistant strains, indicating that resistance genes might be transferable by transposons. All chloramphenicol-resistant strains carried a cat gene. 81.8% of the erythromycin-resistant strains carried the ermB gene. Two (9.5%) of the 21 erythromycin-resistant strains, which did not contain ermA,B,C, ereA,B and mphA genes harboured the msrC gene encoding an erythromycin efflux pump, which was confirmed by sequencing the PCR amplicon. In addition, all E. faecium strains contained the msrC gene, but none of the E. faecalis strains. Transfer of the genetic determinants for antibiotic resistance could only be demonstrated in one filter mating experiment, where both the tet(M) and tet(L) genes were transferred from E. faecalis FAIR-E 315 to the E. faecalis OG1X recipient strain. Our results show the presence of various types of resistance genes as well as transposon integrase genes associated with transferable resistances in enterococci, indicating a potential for gene transfer in the food environment.

Class 1 Integrons and Tetracycline Resistance Genes in Alcaligenes , Arthrobacter , and Pseudomonas spp. Isolated from Pigsties and Manured Soil

The presence of tetracycline resistance (Tc(r)) genes and class I integrons (in-1), and their ability to cotransfer were investigated in Tc(r) gram-negative (185 strains) and gram-positive (72 strains) bacteria from Danish farmland and pigsties. The isolates belonged to the groups or species Escherichia coli, Enterobacter spp., Arthrobacter spp., Alcaligenes spp., Pseudomonas spp., and Corynebacterium glutamicum. The 257 isolates were screened for in-1. Eighty-one of the gram-negative isolates were also screened for the Tc(r) genes tet(A), tet(B), and tet(C), and all (n = 72) gram-positive isolates were screened for tet(33). Fourteen (7%) of the soil isolates and eleven (25%) of the pigsty isolates contained in-1. All isolates that contained tet genes also contained in-1, except one gram-negative isolate from a pigsty that contained tet(B). All gram-positive isolates with in-1 also contained tet(33). No isolates contained more than one tet gene. The in-1-positive isolates were tested for resistance to selected antimicrobial agents and showed resistance to three to nine drugs. Filter-mating experiments showed cotransfer of Tc(r) and class I integrons from soil isolates to Escherichia coli and/or Pseudomonas putida. We conclude that soil bacteria in close contact to manure or pigsty environment may thus have an important role in horizontal spread of resistance. Use of tetracyclines in food animal production may increase not only Tc(r) but also multidrug resistance (caused by the presence tet genes and in-1) in bacteria.

Prevalence and molecular characterization of tetracycline resistance in Enterococcus isolates from food.

In the present study, a collection of 187 Enterococcus food isolates mainly originating from European cheeses were studied for the phenotypic and genotypic assessment of tetracycline (TC) resistance. A total of 45 isolates (24%) encompassing the species Enterococcus faecalis (n = 33), E. durans (n = 7), E. faecium (n = 3), E. casseliflavus (n = 1), and E. gallinarum (n = 1) displayed phenotypic resistance to TC with MIC ranges of 16 to 256 microg/ml. Eight of these strains exhibited multiresistance to TC, erythromycin, and chloramphenicol. By PCR detection, TC resistance could be linked to the presence of the tet(M) (n = 43), tet(L) (n = 16), and tet(S) (n = 1) genes. In 15 isolates, including all of those for which the MIC was 256 micro g/ml, both tet(M) and tet(L) were found. Furthermore, all tet(M)-containing enterococci also harbored a member of the Tn916-Tn1545 conjugative transposon family, of which 12 erythromycin-resistant isolates also contained the erm(B) gene. Filter mating experiments revealed that 10 E. faecalis isolates, 3 E. durans isolates, and 1 E. faecium isolate could transfer either tet(M), tet(L), or both of these genes to E. faecalis recipient strain JH2-2. In most cases in which only tet(M) was transferred, no detectable plasmids were acquired by JH2-2 but instead all transconjugants contained a member of the Tn916-Tn1545 family. Sequencing analysis of PCR amplicons and evolutionary modeling showed that a subset of the transferable tet(M) genes belonged to four sequence homology groups (SHGs) showing an internal homology of > or = 99.6%. Two of these SHGs contained tet(M) mosaic structures previously found in Tn916 elements and on Lactobacillus and Neisseria plasmids, respectively, whereas the other two SHGs probably represent new phylogenetic lineages of this gene.

In vitro conjugal transfer of tetracycline resistance from Lactobacillus isolates to other Gram-positive bacteria

The ability of 14 Lactobacillus strains, isolated from fermented dry sausages, to transfer tetracycline resistance encoded by tet(M) through conjugation was examined using filter mating experiments. Seven out of 14 tetracycline-resistant Lactobacillus isolates were able to transfer in vitro this resistance to Enterococcus faecalis at frequencies ranging from 10 −4 to 10 −6 transconjugants per recipient. Two of these strains could also transfer their resistance to Lactococcus lactis subsp. lactis, whereas no conjugal transfer to a Staphylococcus aureus recipient was found. These data suggest that meat lactobacilli might be reservoir organisms for acquired resistance genes that can be spread to other lactic acid bacteria. In order to assess the risk of this potential hazard, the magnitude of transfer along the food chain merits further research.

Analysis of the conjugal transfer system of the pheromone-independent highly transferable Enterococcus plasmid pMG1: identification of a tra gene (traA) up-regulated during conjugation.

pMG1 (65.1 kbp) is a pheromone-independent Enterococcus faecium conjugative plasmid conferring gentamicin resistance. pMG1 is able to transfer to enterococci at a high frequency in broth mating experiments, and it does not respond to the sex pheromones which are involved in the high-frequency transfer system of some conjugative plasmids in Enterococcus faecalis. To analyze regulation of tra gene expression in pMG1, transcripts of pMG1 were examined during conjugation. RNA samples were prepared from mating mixtures 20, 40, 80, and 160 min after initiation of mating and were subsequently analyzed by Northern hybridization by using a variety of pMG1 DNA fragments as probes. One transcript of gene 71ORF2 increased to the maximal level 20 min after the start of mating. The level of this transcript decreased after 40 min of mating to the same level as the level in the control donor culture. The increase was not observed in cultures of the donor cells or recipient cells. These findings suggested that the increase was a conjugation-specific event. The 71ORF2 gene of pMG1 was disrupted by using the suicide vector pMG226 carrying an erythromycin resistance gene that is expressed in enterococci. The transfer frequencies of mutant plasmid pMG229, which had a disrupted 71ORF2 gene in pMG1, and the parent plasmid pMG1 were 1.6 x 10(-7) and 1.1 x 10(-3) per donor cell, respectively, in broth mating experiments, and the transfer frequencies of pMG229 and pMG1 were 2.7 x 10(-3) and 8.5 x 10(-2) per donor cell, respectively, in filter mating experiments. This indicated that the transfer frequency of plasmid pMG229 was reduced during broth mating and was not altered during filter mating. 71ORF2, which is designated traA, is a gene involved in the tra gene system for conjugation, and the product of this gene is associated with the formation or stabilization of mating aggregates during broth mating.

Transfer of TN916-like elements in microcosm dental plaques.

Microcosm dental plaques were grown from an inoculum of human saliva in a constant-depth film fermentor. The inoculum contained four tetracycline-resistant streptococcal species, each of which contained a Tn916-like element. This element was shown to transfer to other streptococci both in filter-mating experiments and within the biofilms in the fermentor.

Contribution of the Earthworm Lumbricus rubellus (Annelida, Oligochaeta) to the Establishment of Plasmids in Soil Bacterial Communities.

The contribution of the earthworm Lumbricus rubellus in spreading plasmids from a nonindigenous bacterial species to the soil microbial community was studied with Escherichia coli strains as donor organisms. The selected donor strains harbored marker-gene tagged plasmids with different transfer properties and host ranges. Prototrophic benzoate degrading indigenous bacteria were analyzed as potential recipients. In filter-mating experiments, donor strains were mixed with bacterial cell consortia extracted from earthworm casts (feces) and incubated on nutrient agar at 28 degrees C. Transfer was detected with the broad host range IncP plasmid pRP4luc; with the IncQ plasmid, pSUP104luc, but only when it was present in a mobilizing donor strain; and with the transposon delivery vector pUTlux. No transfer was detected with the nonmobilizable pUCluc and the mobilizable pSUP202luc, both of narrow host range. In microcosm studies with E. coli inoculated soil incubated at 12 degrees C, transconjugants were only detected in casts of L. rubellus but not in bulk soil, indicating that the gut passage was a precondition for plasmid transfer. Plasmid pRP4luc was transferred at higher frequencies than detected in filter mating. Results of the filter matings were confirmed except that transfer of pUTlux could not be detected. The majority of transconjugants isolated in this study lost their acquired plasmid upon further cultivation. Stable transconjugants, however, were obtained and identified at the 16S rRNA gene level as members of the b- and g-subgroups of Proteobacteria. Incubation of E. coli and selected transconjugants in soil microcosms with L. rubellus demonstrated that the gut passage resulted in a slight but significant reduction of ingested cells. In contrast to the donor strains, however, the population sizes of transconjugants in bulk soil and in casts did not decrease over time. This demonstrated that the transferred plasmids had established themselves in the soil microbial community.

Role of transposon Tn5482 in the epidemiology of vancomycin-resistant Enterococcus faecium in the pediatric oncology unit of a New York City Hospital.

During a 36-month period between 1993 and 1995 in the Pediatric Oncology Unit of Memorial Sloan Kettering Cancer Center, 74 patients experienced episodes of infection or colonization caused by vancomycin-resistant enterococci (VRE). Characterization of the 74 bacterial isolates by microbiological and molecular techniques (pulsed-field gel electrophoresis and hybridization with DNA probes specific for the vanA and vanB genes and for IS1251) identified 73 Enterococcusfaecium and one Enterococcusfaecalis (vanB) among the primary VRE isolates. Most (69/73) of the E. faecium isolates carried vanA and four isolates, the vanB gene complex. The overwhelming majority (67/69) of the vanA -positive isolates also gave hybridization signal for IS1251, indicating the presence of the newly described conjugative transposon Tn5482. No hybridization with IS1251 was obtained with the four vanB-carrying isolates. About 30% of the vanA-positive strains (23/69) were represented by PFGE subtype variants of a single clone, most isolates of which were recovered during a 4-month period between April to June of 1994. The larger portion of the vanA-carrying VRE represented by close to 70% of the isolates (46/69) belonged to as many as 37 different clonal types, indicating tremendous genetic diversity. Among 67 of the 69 vanA-carrying isolates, the localization of the Tn5482-associated vanA gene complex could be unequivocally identified either on the chromosome (40/69) or in plasmids (27/69). Transconjugants recovered from filter mating experiments using either a chromosomally located or plasmid-borne vanA donor strain and a single vancomycin-susceptible strain of either E. faecium or E. faecalis were analyzed by molecular typing techniques. Seven out of 10 independent transconjugants recovered from the same cross showed extensive differences in PFGE pattern and also in the localization of the vanA hybridizing DNA fragment transferred from the common VRE donor with chromosomally located vanA. The observations suggest that the extensive genetic diversity observed among the clinical isolates of VRE may be generated during conjugation between vancomycin-resistant and -susceptible enterococcal isolates. The observations also suggest that the epidemic spread of VRE in the United States may be linked to the frequent presence of Tn5482 among the American isolates.

Genetic analysis of the bacteriocin-encoding plasmids pRJ6 and pRJ9 of Staphylococcus aureus by transposon mutagenesis and cloning of genes involved in bacteriocin production.

pRJ6 and pRJ9, small Staphylococcus aureus plasmids which code for bacteriocins, exhibited a bactericidal activity against several lactic acid bacteria and strains of Listeria monocytogenes, an important food-borne pathogen. Filter-mating experiments using plasmid derivatives tagged with either Tn551 or Tn917-lac showed that pRJ6, but not pRJ9, could be mobilized by staphylococcal conjugative plasmids. Transposon mutagenesis of both plasmids was also performed. The bacteriocin and immunity structural genes of pRJ6 are part of the same operon, which is located around co-ordinate 4.0, being transcribed from right to left. However, gene cloning experiments using a staphylococcal vector showed some evidence for the involvement of additional functions of pRJ6 in bacteriocin expression. One function involved in pRJ6 mobilization mapped around co-ordinate 5.2, and it appears to be transcribed from left to right. The bactericidal action exerted by strains harbouring pRJ9 appears to reflect the activity of at least two bacteriocins, whose combined action results in a broader spectrum of activity and in a higher antagonistic activity. Gene cloning experiments also supported these assumptions.

Characterization of Mupirocin-Resistant Staphylococcus aureus from Different Geographic Areas

With the increasing use of mupirocin, resistance among staphylococci has emerged (1). High-level resistance (MICs, >500 μg/ml) is usually mediated by a plasmid-associated mupA gene. Low-level resistance (MICs, <100 μg/ml) is more common, can be selected in vitro with increasing concentrations of mupirocin, and, except for one report (7), has not been associated with mupA. We sought to define the characteristics and relatedness of Mupr strains from different geographic regions in the United States. A total of 18 Staphylococcus aureus strains with high-level resistance (Hi-Mupr strains) and 19 S. aureus strains with low-level resistance (Lo-Mupr strains) were obtained from Ann Arbor, Mich.; Johnson City, Tenn.; and New Haven, Conn. (4–6, 8). Contour-clamped homogeneous electric field electrophoresis (CHEF) of genomic DNA was performed as described previously (7). Plasmid profile analysis was done following extraction of DNA by a rapid mini-prep procedure, treatment with HindIII, and separation of fragments by conventional agarose electrophoresis. Southern analysis of chromosomal and plasmid DNA was performed according to standard procedures with a PCR-amplified product of an intragenic 1.65-kb NcoI fragment of mupA for the probe (7). Previously described oligonucleotide primers Mup1 and Mup2 were used for PCR amplifications (2). Filter mating experiments were carried out as described previously (3). There were 11 Michigan strains (2 Hi-Mupr methicillin-resistant S. aureus [MRSA] and 9 Lo-Mupr [6 MRSA and 3 methicillin-susceptible S. aureus] strains), 14 Tennessee MRSA strains (6 Hi-Mupr and 8 Lo-Mupr strains), and 12 Connecticut strains (10 Hi-Mupr and 2 Lo-Mupr strains). Eight different CHEF patterns were noted among the 37 strains. All 14 Tennessee strains were closely related and were similar to the 2 Hi-Mupr and to 5 of the 9 Lo-Mupr strains from Michigan; the 4 other Michigan strains showed three different patterns. Of the 12 Connecticut strains, 8 showed one predominant pattern and the other 4 showed three different patterns; all were unrelated to the Michigan and Tennessee strains. All strains contained plasmids. Analysis of plasmid profiles showed differences between the Michigan and Tennessee strains that had appeared similar by CHEF typing; there were three different patterns among the 11 Michigan strains, and four entirely different patterns among the 14 Tennessee strains. The 12 strains from Connecticut showed five unique plasmid profiles. All 18 Hi-Mupr strains transferred mupirocin resistance and contained mupA on plasmid DNA. The 2 Michigan strains carried mupA on a 9.1-kb fragment, the 6 Tennessee strains carried it on a 4.6- or a 5.5-kb fragment, and all 10 Connecticut strains carried it on a 4.6-kb fragment. No Lo-Mupr strains transferred mupirocin resistance and, based on hybridization results, none had plasmid-associated mupA. In nine Michigan Lo-Mupr strains and two of eight Tennessee Lo-Mupr strains, the mupA probe hybridized to genomic DNA, indicating mupA is on the chromosome. No Connecticut Lo-Mupr strain showed hybridization to the mupA probe. Both chromosomal and plasmid typing indicated that Connecticut strains were not related to Michigan and Tennessee strains. Although most of the strains from Michigan and Tennessee were closely related by CHEF typing, their plasmid profiles differed. Hi-Mupr S. aureus strains from the three geographic areas transferred resistance, but the plasmid location of the mupA gene varied. Low-level mupirocin resistance was associated with chromosomal mupA in some but not all strains. Both high-level and low-level mupirocin resistance appeared to have arisen from multiple different clones within an individual institution and in different geographic areas of the United States.

Retrotransfer kinetics of R300B by pQKH6, a conjugative plasmid from river epilithon

The kinetics of conjugation, retrotransfer and mobilisation were studied at 5–15 min intervals between strains of Pseudomonas putida using plasmid pQKH6, isolated from river epilithon, and R300B. Transconjugants from the direct conjugation of pQKH6 and mobilisation of R300B by pQKH6 appeared rapidly, reaching maximum densities within 30–60 min of the start of both filter and liquid mating experiments. However, retrotransconjugants only appeared after a delay. This delay was short (approx. 45–60 min) in filter mating and much longer (2–5 h) for liquid mating experiments. Attempts at predicting the time course of retrotransconjugant development from (1) numbers of transconjugants from the conjugation and mobilisation experiments and (ii) mathematical models based on a mass action approach, both failed to reproduce the observed delay. It was concluded that retrotransfer did not proceed by either a one-step mechanism occuring early in conjugation or two separate conjugation and mobilisation steps. The clear demonstration of a delay in retrotransconjugant formation implies that a new mechanism must be sought. The likely importance of retrotransfer in the environment is discussed.

Identification and nucleotide sequence analysis of a transfer-related region in the streptococcal conjugative transposon Tn5252

To obtain a functional map of Tn5252, a 47.5-kb streptococcal conjugative transposon, a series of defined deletion and insertion mutations were introduced within the transposon. Interruptions at several regions were found to affect the conjugal transposition functions of the element in filter-mating experiments. The nucleotide sequence of the left terminus of Tn5252 showed two open reading frames, ORF1 and ORF2, adjoining the att site. The organization of this region and the structure of the predicted integrase encoded by ORF1 were found to be similar to those of other site-specific recombination systems.

Characterization of pGA1, a new plasmid from Corynebacterium glutamicum LP-6

A new plasmid, pGA1, has been isolated from Corynebacterium glutamicum LP-6, and its detailed restriction map has been prepared. The 4.9-kb plasmid has a G + C content of 57%. It replicates in C. glutamicum ATCC13032 and is compatible with the three other plasmids, pCC1, pBL1 and pHM1519, commonly used for vector construction for amino acid-producing corynebacteria. Fusions of pGA1 with different Escherichia coli replicons (transferred from E. coli to Corynebacterium via transformation of spheroplasts or by filter mating experiments with intact cells) are shown to be suitable as shuttle plasmids; some of them are highly stable in C. glutamicum, even when propagated without any selection pressure.

Transmissible mupirocin resistance inStaphylococcus aureus

The spread of two strains of Staphylococcus aureus with high level resistance to mupirocin is described. The resistance proved to be easily transferred to other S. aureus strains by filter mating experiments and on the skin of mice. No plasmid band corresponding to the resistance could be demonstrated by agarose gel electrophoresis or by caesium chloride gradient centrifugation but cleavage of 'chromosomal' DNA from resistant recipients showed bright bands of DNA absent from sensitive controls.