Characterization of plasmids encoding cefotaximases group 1 enzymes in Escherichia coli recovered from cattle in England and Wales

In the study, we examined the molecular characteristics of cattle-associated Escherichia coli carrying CTX-M genes and their plasmids. Between July 2006 and July 2007, 18 E. coli were collected from cattle that were found to possess a bla(CTX-M) belonging to group 1. bla(CTX-M-15/28) was the predominant type, and it was associated with plasmids of several different inc/rep types. In addition, bla(CTX-M-1) and bla(CTX-M-3) were also detected. Plasmids encoding the bla(CTX-M) genes belonged to incompatibility groups I1, F, and A/C. Analysis of the non-beta-lactam resistance genes associated with each CTX-M-bearing plasmid demonstrated that F plasmids frequently carried a larger number of resistance genes than IncI1 plasmids, which rarely carried additional resistance genes. All bla(CTX-M) carrying plasmids were positive by polymerase chain reaction for an ISEcp1-like element.     

Restriction enzyme map of cryptic plasmid accompanying pR711b and pR409; identity of pR409 and pR388

A reference strain containing the IncW plasmid, pR409, was found to contain also a cryptic, conjugative plasmid, here named pJR15. After the separation of the two plasmids into different strains of bacteria, pR409 was found to have an identical restriction enzyme map to the IncW plasmid, pR388. Both pR409 and pR388 were isolated from the same hospital in London and confer sulphonamide and trimethoprim resistance. Originally pR409 was reported to confer resistance to tetracycline, but this was not confirmed by the Plasmid Reference Center. pR409 appears to represent another isolate of pR388 in a different host background. The restriction enzyme map of the cryptic plasmid, pJR15 (39 kb), has been determined. pJR15 was found compatible with plasmids from 15 different incompatibility groups and has been found also in the reference strain of the IncD plasmid, pR711b. The HindIII and BglII digests of pR711b are shown. The possible presence of the conjugative plasmid, pJR15, should be examined for studies on the sex pili or chromosomal mobilization properties of pR711b.     

Presence of F-like OriT base-pair sequence on the virulence plasmids of Salmonella serovars Gallinarum,Enteritidis, and Typhimurium,but absent in those of Choleraesuis and Dublin

The 90 kb virulence plasmids of Salmonella biosers Gallinarum and Pullorum (expressed as serovar Gallinarum) are non-conjugative. They were, however, found to be readily mobilized by IncFI and IncFIV plasmids, but not by conjugative IncA, IncB = C, IncH, IncL and IncM R plasmids. No virulence plasmids of serovars Choleraesuis, Dublin, Enteritidis, and Typhimurium were mobilized by any of these conjugative plasmids. The 90 kb virulence plasmid of Gallinarum was shown to hybridize to the Tra genes region of IncFI and IncFIV plasmids, suggesting it contains some F Tra genes region. It also hybridized with 75% homology to the virulence plasmid of Typhimurium, and, in decreasing order, to those of Dublin, Enteritidis, and Choleraesuis. A probe made of 319 basepairs of the F OriT region hybridized with approximately 45% homology to the virulence plasmid of Gallinarum, 63% to that of Typhimurium and 50% to that of Enteritidis. The probe, however, failed to hybridize to the plasmid DNAs of IncA, IncB = C, IncH, IncL and IncM, and to the virulence plasmids of Choleraesuis and Dublin.     

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.     

Construction of a conjugative plasmid with potential use in vaccines against heat-labile enterotoxin.

A conjugative plasmid with potential usefulness for vaccine strains was constructed. In the first step, a 5.9-kilobase DNA segment containing the two loci for the A and B subunits of heat-labile enterotoxin with a mutation in the gene for the A subunit was joined to the cloning vehicle pGA22, generating the nonconjugative plasmid pPMC4 with genes for resistance to tetracycline and chloramphenicol. In the second step, a segment of pPMC4 containing the genes for the A and B subunits, the gene for chloramphenicol resistance, and the replication genes of pGA22 was ligated to the genes for conjugal transfer of the F plasmid, generating the 54.9-kb plasmid pPMC5. Eleven porcine Escherichia coli isolates were tested as recipients for pPMC4 and pPMC5. For pPMC4, transformation and mobilization with a conjugative R plasmid were used to effect plasmid transfer. Only 1 of the 11 strains acted as a recipient in transformation. Mobilization with the R plasmid occurred with two strains, but the plasmids were altered during transfer. In contrast, pPMC5 was transferred with high frequency and unaltered to 9 of the 11 E. coli strains. Transconjugants from these nine matings produced high titers of the B subunit and no active heat-labile enterotoxin. Plasmid pPMC5 was stable in three porcine E. coli strains tested; plasmid pPMC4 was somewhat less stable in these strains. The method we describe for the construction of conjugative chimeric plasmids offers an opportunity for introducing genes with potential for immunization into bacterial strains that are suitable for colonizing the appropriate host sites.     

Molecular analysis and identification of virulence gene on pR(ST98) from multi-drug resistant Salmonella typhi

pR_(ST98) is a large and conjugative resistant plasmid(R plasmid)of 98.6 mega-dalton from multi-drug resistantSalmonella typhi(S.typhi),which was classified to incompatibility group C(Inc C).It has been found that pR_(ST98)made its host bacteria not only antibiotic resistant but also more virulent.In this study we explored the possibilityof plasmid pR_(ST98) in S.typhi carrying the Salmonella plasmid virulence gene-spv.The plasmid pR_(ST98) was isolated,purified and then digested by nine restriction endonucleases to make the plasmid enzyme profile.Spv-specific PCRand Southern blot were applied to identify the virulence gene on pR_(ST98).The amplified spv fragments spvR andspvB were cloned into pGEM-T EASY and then the DNA sequences were analysed.The fragments of pR_(ST98)digested by endonucleases Bgl Ⅱ,Pst Ⅰ and Sac Ⅱ were identified,which may be useful for molecular analysis andfurther epidemiological surveillance of pR_(ST98).The results of PCR and Southern blot showed that spv homologousgenetic sequence which had been found in all pathogenesis Salmonella spp.except S.typhi was also presented onpR_(ST98).The ORF of spvR and spvB of pR_(ST98) were 894 bp and 1,776 bp,respectively.They have more than 99%homology with that of spvR and spvB on virulence plasmid in S.typhmurium.The genotype research on pR_(ST98)revealed that there is a plasmid carrying genes responsible for drug resistance and virulence in S.typhi.This is thefirst report for such kind chimerical plasmid in S.typhi.Cellular & Molecular Immunology.2005;2(2):136-140.     

Organization of aerobactin, hemolysin, and antibacterial resistance genes in lactose-negative Escherichia coli strains of serotype O4 isolated from children with diarrhea.

Epidemiologically related, non-lactose-fermenting (NLF) Escherichia coli strains of serotype O4 have been isolated at a high frequency from children with diarrhea in Somalia (M. Nicoletti, F. Superti, C. Conti, A. Calconi, and C. Zagaglia, J. Clin. Microbiol. 26:524-529, 1988). In order to define the virulence potential of these strains, we characterized the replication properties of their high-molecular-weight plasmids and studied the genetic locations and organization of the aerobactin (aer) and hemolysin (hly) determinants encoded by 23 NLF O4 E. coli strains. Southern blot hybridizations, mobilization assays of nonconjugative plasmids, and incompatibility-exclusion experiments conducted with a conjugative incompatibility group FI (IncFI) plasmid showed that (i) 20 out of the 23 strains examined harbor a 160- to 180-kb IncFI plasmid that shares homology with the basic replicons RepFIA, RepFIB, and (except for the plasmid of one strain) RepFIC, and 22 strains also contain a 40- to 140-kb IncFII plasmid sharing homology with the RepFIIA replicon; (ii) the IncFI plasmid is nonconjugative and carries antibiotic resistance genes; (iii) the aer system is located on the IncFI plasmids and/or the chromosomes in the three strains not harboring IncFI, and it is found in an inverted orientation; (iv) the hly determinants are located on the chromosome, and their genetic organization is well conserved and closely resembles that of the reference hemolytic plasmid pHly152; and (v) Hly- mutants obtained by transposon insertion mutagenesis are not cytotoxic to HeLa cell monolayers, indicating that hemolysin is responsible for the high cytotoxic activity we have previously reported for these strains. The structural organization of the plasmid-encoded aer operon, together with the finding that those plasmids also carry antibiotic resistance genes, indicates that the IncFI plasmid of the NLF O4 E. coli strains studied more closely resembles aer-encoding virulence IncFI Salmonella R plasmids than E. coli ColV plasmids. The data presented here cannot rule out whether the strains examined are potentially intestinal or extraintestinal pathogens. Nevertheless, the genetic organization of the virulence genes, together with the epidemiological behavior and the wide spectrum of antibiotic resistance of the NLF O4 E. coli strains, indicates that these strains are structured as typical E. coli pathogenic isolates of human origin.     

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.     

耐药质粒pRST98在不同种属肠道杆菌间的接合及表达

目的 研究伤寒杆菌耐药质粒ｐＲＳＴ９８在不同种属肠道杆菌间（大肠杆菌、伤寒杆菌、鼠伤寒杆菌及痢疾杆菌）互相接合传递的规律及表达。方法 以３株携带不相容性Ｃ群（ＩｎｃＣ）ｐＲＳＴ９８的伤寒杆菌作供体,用传统及改良的两种接合转移试验方法,研究其在肠道杆菌间的传递及耐药标志的表达,并用核酸内切酶对供体菌及受体接合了的Ｒ质粒进行分析。结果 ｐＲＳＴ９８能在４种肠道杆菌间传递;但在不同促属中Ｒ质粒接合难易程     

Interaction of drug resistance plasmids and bacteriophages with diarrheagenic strains of Escherichia coli.

Seven transfer-derepressed plasmids from different incompatibility groups in Escherichia coli K-12 were tested for their ability to enter 43 strains of diarrheagenic E. coli (mostly enteropathogenic E. coli clinical isolates) representing 12 serogroups and including rough and semirough mutants (characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Strains in some serogroups were more competent as recipients of plasmids than were those in others. Five test plasmids in an E. coli K-12 (rough) donor transferred significantly less efficiently to two smooth strains than to their rough or semirough isogenic derivatives. When the same smooth and rough strains were used as donors, the plasmids transferred to E. coli K-12 equally well. These results suggested that the O-antigenic lipopolysaccharide side chains of diarrheagenic E. coli isolates shielded the outer membrane receptors for conjugative pili, thus preventing plasmid entry. The different receptors for eight bacteriophages were also covered by O side chains. In addition, a limited survey of clinical isolates for drug resistance markers and resident plasmids was carried out.     

Characterization of multidrug-resistant Escherichia coli by antimicrobial resistance profiles, plasmid replicon typing, and pulsed-field gel electrophoresis

The objective of this study was to examine the distribution of multidrug resistance in Escherichia coli in relation to plasmid replicon types, animal sources, and genotypes. E. coli isolates (n?=?35) from seven different animal sources were selected and tested for susceptibility to 15 antimicrobials; pulsed-field gel electrophoresis was used to determine genetic relationships among the E. coli isolates. Plasmid types based on their incompatibility (Inc) replicon types were determined, and linkage disequilibrium analysis was performed for antimicrobial resistance profiles, replicon types, and animal source. A high degree of genotypic diversity was observed: 34 different pulsed-field gel electrophoresis types among the 35 isolates examined. Twelve different plasmid Inc types were detected, and all isolates carried at least one replicon type. IncF (n?=?25; 71.4%) and IncFIB (n?=?19; 54.3%) were the most common replicon types identified. Chloramphenicol resistance was significantly linked with four Inc types (A/C, FIIA, F, and Y), and amoxicillin/clavulanic acid was linked with three Inc types (B/O, P and Y). Resistance to any other antimicrobial was linked to two or fewer replicon types. The isolate source was linked with resistance to seven antimicrobials and IncI1. We conclude that commensal E. coli from animal sources are highly variable genotypically and are reservoirs of a diverse array of plasmids carrying antimicrobial resistance.     

Identification and antigenic characterization of virulence-associated, plasmid-coded proteins of Shigella spp. and enteroinvasive Escherichia coli.

Seven plasmid-coded polypeptides, designated a through g, were identified by two-dimensional nonequilibrium pH gradient electrophoresis of radiolabeled extracts from minicells of virulent Shigella flexneri serotypes 2a and 5 and enteroinvasive Escherichia coli O143. These polypeptides were deemed to be products of 140-megadalton (MDa) virulence-associated plasmids because they were not synthesized in minicells which were not harboring a 140-MDa plasmid or in minicells which were carrying an F lac plasmid of the same incompatibility group. Synthesis of these polypeptides was repressed in minicells incubated at 30 degrees C and in minicells isolated from a noninvasive opaque colonial variant, even though these strains harbored a 140-MDa plasmid. Enriched fractions of polypeptides b, c, and d were obtained from S. flexneri serotype 5 by preparative isoelectric focusing, and polyclonal rabbit antisera recognizing each polypeptide were raised. These antisera were able to detect cross-reacting plasmid-coded polypeptide antigens in S. flexneri serotype 3, Shigella sonnei, and enteroinvasive E. coli O143. In addition, Western blots of minicell extracts from S. flexneri serotype 5 or E. coli O143 indicated that plasmid-coded polypeptides a through d were recognized by convalescent antiserum from a monkey infected with S. flexneri serotype 2a.     

Analysis of phenotype resistance of clinical strains of Escherichia coli

The multiplasmid clinical E. coli KL4 strain isolated from urine of a patient was examined for resistance to antibacterial substances and the number of plasmids. The distribution of resistance genes to antibiotics in different plasmids was assessed. All assessed resistances were transmitted by the mechanism of bacterial conjugation. By means of conjugation of the clinical E. coli strain KL4 and E. coli DH1, transformation of the DH1 strain by plasmid DNA and electrophoretic analysis two R plasmids were identified. The largest plasmid carries the gene for resistance against tetracycline and it is conjugative. Another three plasmids were mobilized by this plasmid during conjugation. The largest among them codes resistance against ampicillin, azlocillin and ticarcillin. From the total of five plasmids of the KL4 strain three were cryptic with regard to the resistance phenotype.     

Molecular evolution of large virulence plasmid in Shigella clones and enteroinvasive Escherichia coli.

Three genes, ipgD, mxiC, and mxiA, all in the invasion region of the Shigella virulence plasmid, were sequenced from strains representing a range of Shigella serotypes and from two enteroinvasive Escherichia coli (EIEC) isolates. The plasmids can be classified into two relatively homogeneous sequence forms which are quite distinct. pINV A plasmids are found in Shigella flexneri strains F6 and F6A, S. boydii strains B1, B4, B9, B10, B14, and B15, S. dysenteriae strains D3, D4, D6, D8, D9, D10, and D13, and the two EIEC strains (M519 and M520). pINV B plasmids are present in S. flexneri strains F1A, F2A, F3A, F3C, F4A, and FY, two S. boydii strains (B11 and B12), and S. sonnei. The D1 pINV plasmid is a recombinant with ipgD gene more closely related to those of pINV A but with mxiA and mxiC genes more closely related to those of pINV B. The phylogenetic relationships of the plasmid and those of the chromosomal genes of Shigella strains are largely consistent. The cluster 1 and cluster 3 strains tested (G.M. Pupo, R. Lan, and P. R. Reeves, Proc. Natl. Acad. Sci. USA 97:10567-10572, 2000) have pINV A and pINV B plasmids, respectively. However, of the three cluster 2 strains (B9, B11, and B15), B9 and B15 have pINV A while B11 has a pINV B plasmid. Those Shigella (D8 and D10 and S. sonnei) and EIEC strains which do not group with the main body of Shigella strains based on chromosomal genes were found to have plasmids belonging to one or the other of the two types and must have acquired these by lateral transfer.     

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.     

Transfer of a CFA/I-ST plasmid promoted by a conjugative plasmid in a strain of Escherichia coli of serotype O128ac:H12.

Escherichia coli strains belonging to serotype O128ac:H12 and producing heat-stable enterotoxin (ST) and colonization factor CFA/I were found in Sao Paulo in children with diarrhea, but not in normal children. Segregants occurred in such strains with a frequency of about 10%, which have lost the ability to produce ST and CFA/I at the same time. From one strain, both properties were transformed jointly in matings to an E. coli K-12 strain. All such ST+ CFA/I+ progeny had received two plasmids of length 97 and 64 kilobases in the matings. Insertion of a transposon, Tn5, carrying a gene for kanamycin resistance, into the two plasmids enabled us to select for kanamycin-resistant progeny in further matings. Analysis of such progeny strains in terms of plasmid content and production of ST and CFA/I revealed that the larger plasmid carries the genes for St and CFA/I and is not self-transmissible, whereas the smaller plasmid does not carry any recognizable phenotypic traits, but is conjugative and promotes cotransfer of the larger plasmid with a frequency of about 30%.     

Self-transmissible R plasmids encoding CS31A among human Escherichia coli strains isolated from diarrheal stools.

The CS31A antigen was first described for septicemic and enterotoxigenic bovine E. coli strains. In our study, of 597 human Escherichia coli strains isolated from diarrheagenic stools of hospitalized patients, 30 (5%) hybridized with the CS31A DNA probe. These CS31A-positive E. coli strains diffusely adhered to Caco-2 and/or HEp-2 cells and produced a major surface protein of either 30 or 30.5 kDa according to the strain. These proteins were antigenically related to the two forms of the CS31A antigen, namely, CS31A-L and CS31A-H. Genes encoding CS31A were located on 140-kb conjugative R plasmids. E. coli transconjugants expressed major surface proteins similar to those of the wild-type strains and adhered to Caco-2 and/or HEp-2 cells. An association of CS31A and another adhesive factor of the Dr family was found in 70% of wild-type strains, since 21 strains hybridized with the diffuse adhesion DNA probe corresponding to the accessory gene (daaC) of the F1845 adhesin. Comparison of the restriction patterns of the 140-kb R plasmids of the CS31A-positive E. coli strains showed these plasmids to be similar. Hybridization experiments indicated that the genes encoding CS31A and resistance to penicillin were located together on either of two 20- or 27-kb EcoRI restriction fragments in four E. coli strains. We reported a similar linkage between these genes in Klebsiella pneumoniae strains which produced CF29K, a CS31A-like antigen. These results suggest a horizontal transfer between E. coli and K. pneumoniae strains.     

The complete DNA sequence and analysis of the virulence plasmid and of five additional plasmids carried by Shiga toxin-producing Escherichia coli O26:H11 strain H30

Shiga toxin-producing Escherichia coli (STEC) strains belonging to serogroup O26 have been associated with sporadic cases and outbreaks of hemorrhagic colitis and hemolytic uremic syndrome. In addition to chromosomal virulence genes, STEC strains usually harbor a large plasmid that carries genes associated with pathogenicity. The complete nucleotide sequence and genetic organization of 6 plasmids carried by STEC O26:H11 strain H30 were determined. The large virulence plasmid (pO26-Vir) was approximately 168 kb in size and contained 196 open reading frames (ORFs). pO26-Vir possesses a mosaic structure and shows similarity to the virulence plasmids in locus of enterocyte effacement (LEE)-negative STEC O113:H21 EH41 (pO113), in E. coli clinical strain C1096 (pSERB1), and in E. coli O157:H7 RIMD 0509952 (pO157). Plasmid pO26-Vir shares several highly conserved regions with pO157 and carries important virulence genes, including toxB, katP, espP, and the hly gene cluster. In addition, pO26-Vir possesses genes encoding for type IV pili (pilL-V). The second largest plasmid, pO26-L (73 kb) contains 101 ORFs. pO26-L carries the tetracycline resistance gene and has regions that show similarity to the E. coli conjugative resistance plasmid NR1. The third largest plasmid, pO26-S4 (5.8 kb), is homologous to the ColE2 colicinogenic plasmid that encodes for colicin E2. The remaining 3 plasmids, pO26-S1 (1.5 kb), pO26-S2 (3.1 kb), and pO26-S3 (4.2 kb), carry very little genetic information except for putative proteins involved in plasmid replication and DNA maintenance. The data presented underscore the diversity among the STEC virulence plasmids and provide insights into the evolution of these plasmids in STEC strains that cause serious human illness.     

Distribution of conjugative-plasmid-mediated 16S rRNA methylase genes among amikacin-resistant Enterobacteriaceae isolates collected in 1995 to 1998 and 2001 to 2006 at a university hospital in South Korea and identification of conjugative plasmids mediating dissemination of 16S rRNA methylase

The distribution of conjugative-plasmid-mediated 16S rRNA methylase genes among amikacin-resistant Enterobacteriaceae collected between 1995 and 1998 and between 2001 and 2006 at a university hospital in South Korea was examined, and conjugative plasmids carrying the 16S rRNA methylase genes were characterized by PCR-based replicon typing and by determination of their antimicrobial resistance pattern. Among the 7,127 isolates, 463 isolates showed a high level of resistance to amikacin, and 218 of the 463 isolates transferred amikacin resistance by conjugation. Among the 218 isolates, armA was detected in 153 isolates (88 Klebsiella pneumoniae, 28 Escherichia coli, 19 Enterobacter cloacae, and 6 Serratia marcescens isolates and 12 isolates of other organisms), and rmtB was detected in 51 isolates (32 K. pneumoniae isolates, 18 E. coli isolates, and 1 Citrobacter freundii isolate). The first appearance of armA was in 1997. The armA gene was carried by conjugative plasmids of replicon groups IncL/M, IncFIIAs, IncF, IncA/C, IncHI2, and Inc(unidentified) in 38, 20, 7, 9, 4, and 75 strains, respectively. The rmtB gene was carried by conjugative plasmids of groups IncA/C, IncF, and IncI1-Iγ in 43 strains, 7 strains, and 1 strain, respectively. Transconjugants that received the IncL/M plasmid carrying armA or the IncA/C plasmid carrying rmtB showed an additional resistance to cefotaxime. Transconjugants that received the IncFIIA plasmid or Inc(unidentified) plasmid carrying the armA gene showed an additional resistance to cefoxitin and a high MIC₅₀ (0.25 mg/liter) of ciprofloxacin. In conclusion, this study demonstrated that the dissemination of 16S rRNA methylase genes among the Enterobacteriaceae is mediated by conjugative plasmids of various incompatibility groups that confer resistance to multiple drugs, including aminoglycosides, extended-spectrum β-lactams, and/or quinolones.     

A new class of conjugative plasmid in Staphylococcus aureus

Plasmid pWBG637, a Staphylococcus aureus conjugative plasmid having no known resistance phenotype, was compared with other conjugative plasmids in S. aureus by restriction endonuclease analysis, incompatibility testing and DNA-DNA hybridisation. It differed from the other conjugative plasmids on all three criteria and thus belongs to a new class of conjugative plasmids.