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Intraspecific and Intergeneric Mobilization of Non-conjugative Resistance Plasmids by a 24-5 Megadalton Conjugative Plasmid of Neisseria gonorrhoeae
Abstract
pLE2451, a 24.5 megadalton conjugative plasmid from Neisseria gonorrhoeae, was capable of efficiently mobilizing gonococcal beta-lactamase plasmids between gonococci and from gonococci to Haemophilus influenzae and restriction-deficient Escherichia coli. Donor strains of N. gonorrhoeae carrying pLE2451 were also found to be capable of mobilizing a variety of non-conjugative plasmids originally derived from enteric bacteria or Haemophilus species when such plasmids were resident in E. coli. Nevertheless, pLE2451 was not detected physically in E. coli or H. influenzae transconjugants. This suggests that the plasmid is unstable in these hosts but survives transiently to provide transfer functions for mobilization. The proficiency of pLE2451 in promoting intraspecific and intergeneric mobilization was not paralleled by pUB701, pRI234 and pFR16017, a series of conjugative plasmids derived originally from Haemophilus species. These plasmids were incapable of mobilizing even Haemophilus beta-lactamase plasmids, such as RSF0885, between Haemophilus species.
... Since the 41 kb tet(M) conjugative plasmid cannot be maintained in E. coli (Flett et al., 1981;Morse et al., 1986;Sox et al., 1978) intergeneric triparental matings among N. gonorrhoeae and two strains of E. coli were performed as previously described (Flett et al., 1981) with minor modifications. Overnight cultures of donor N. gonorrhoeae strain 15315, intermediate E. coli DH10B with pSJ5.2 and recipient rifampicin-resistant E. coli HMS174 were grown in LB broth without antibiotics and adjusted to a concentration of 10 8 cells/ml. ...
... Since the 41 kb tet(M) conjugative plasmid cannot be maintained in E. coli (Flett et al., 1981;Morse et al., 1986;Sox et al., 1978) intergeneric triparental matings among N. gonorrhoeae and two strains of E. coli were performed as previously described (Flett et al., 1981) with minor modifications. Overnight cultures of donor N. gonorrhoeae strain 15315, intermediate E. coli DH10B with pSJ5.2 and recipient rifampicin-resistant E. coli HMS174 were grown in LB broth without antibiotics and adjusted to a concentration of 10 8 cells/ml. ...
We report the mobilization by cointegration of the gonococcal 5.2 kb beta-lactamase plasmid pSJ5.2 in an Escherichia coli background. Transfer of pSJ5.2 was measured by filter mating assays with five different conjugative plasmids from Enterobacteriaceae and the gonococcal 41 kb tet(M). Plasmid pSJ5.2 was mobilized to E. coli at frequencies of 1.7x10(-6), 9.3x10(-8) and 2.7x10(-5) by the tet(M), R64 drd-33 and N3 conjugative plasmids, respectively. Mobilization of pSJ5.2 by the 41 kb tet(M) conjugative plasmid resulted in stable Amp(R) E. coli transconjugants consisting of pSJ5.2 plasmid with an insertion located in the 2.4 kb BamHI-BamHI fragment. Mobilization of pSJ5.2 by R64drd-33 and N3 conjugative plasmids involved stable cointegrates as detected by Southern Blot with a DIG-labelled PstI-digested pSJ5.2 probe. Restriction analysis of the R64::pSJ5.2 and N3::pSJ5.2 cointegrates and Southern Blot with the pSJ5.2 probe showed that cointegrates formed by deletion of DNA regions within the 1.8 kb BamHI-HindIII fragment of pSJ5.2. The plasmid thus appears to use multiple recombination mechanisms for cointegration with different conjugative plasmids. The complete nucleotide sequence of pSJ5.2 was determined, and will be a useful tool to further investigate the molecular mechanisms leading to its cointegrative transfer.
... d.f. = 1, P < 0.0001), possibly reflecting the mobilization of pBlaTEM by the pConjugative plasmid 25 , and were completely absent from isolates resistant to extended-spectrum cephalosporins (ESCs) (Fig. 3). The gonococcal genomic island was found in 277 (67%) isolates ( Supplementary Fig. 6), but showed no clear association with AMR. ...
The sexually transmitted pathogen Neisseria gonorrhoeae is regarded as being on the way to becoming an untreatable superbug. Despite its clinical importance, little is known about its emergence and evolution, and how this corresponds with the introduction of antimicrobials. We present a genome-based phylogeographical analysis of 419 gonococcal isolates from across the globe. Results indicate that modern gonococci originated in Europe or Africa, possibly as late as the sixteenth century and subsequently disseminated globally. We provide evidence that the modern gonococcal population has been shaped by antimicrobial treatment of sexually transmitted infections as well as other infections, leading to the emergence of two major lineages with different evolutionary strategies. The well-described multidrug-resistant lineage is associated with high rates of homologous recombination and infection in high-risk sexual networks. A second, multisusceptible lineage is more associated with heterosexual networks, with potential implications for infection control.
... Previous studies on antimicrobial resistance have mainly focused on pathogenic organisms; however, nonpathogenic organisms within the Neisseria genus are particularly apt in horizontal gene transfer, which can occur within their genus, as well as with other bacteria such as Haemophilus influenzae and E. coli [10][11][12][13][14]. Genomic analyses have shown widespread genetic exchange of multiple virulence factors between pathogenic and nonpathogenic Neisseria [15]. ...
BACKGROUND:
Neisseria gonorrhoeae (NG) infections are a global health burden. Increasing reports of NG resistance to cephalosporins are an imminent threat to public health. Many hypothesize that commensal Neisseria species are an important reservoir for genetic material conferring antimicrobial resistance in NG, however clinical data are lacking.
METHODS:
Men-who-have-sex-with-men (MSM) of Hanoi, Vietnam completed a questionnaire regarding antibiotic use. We collected pharyngeal specimens, cultured Neisseria species, and measured minimum inhibitory concentrations (MIC) to ciprofloxacin, cefixime, ceftriaxone, and cefpodoxime. Using MIC criteria for antimicrobial susceptibility in NG, we categorized the Neisseria species and compared mean MIC levels between different antibiotic user groups.
RESULTS:
Of 207 participants, 38% used at least one antibiotic in the past 6 months; 52% without a prescription. A median of one Neisseria species was cultured from each participant (range 1-4) with 10 different Neisseria species identified overall. The proportion of Neisseria with reduced susceptibility to ciprofloxacin was 93%, cefpodoxime 84%, cefixime, 31% and ceftriaxone, 28%. Antibiotic use within the past month was strongly associated with Neisseria species having increased MICs to cefixime, ceftriaxone, and cefpodoxime; with mean MIC ratios of 6.27, 4.11, and 7.70, respectively, when compared to those who used antibiotics between 1 and 6 months prior (p< 0.05, all comparisons).
CONCLUSIONS:
MSM in our study often used antibiotics without a prescription. At least one commensal Neisseria species colonized all men. Recent use of any antibiotics may select for oropharyngeal Neisseria species with antimicrobial resistance. The normal flora of the oropharynx may be an important source of antimicrobial resistance in Neisseria gonorrhoeae.
... d.f. = 1, P < 0.0001), possibly reflecting the mobilization of pBlaTEM by the pConjugative plasmid 25 , and were completely absent from isolates resistant to extended-spectrum cephalosporins (ESCs) (Fig. 3). The gonococcal genomic island was found in 277 (67%) isolates ( Supplementary Fig. 6), but showed no clear association with AMR. ...
The sexually transmitted pathogen Neisseria gonorrhoeae is regarded as being on the way to becoming an untreatable superbug. Despite its clinical importance, little is known about its emergence and evolution, and how this corresponds with the introduction of antimicrobials. We present a genome-based phylogeographic analysis of 419 gonococcal isolates from across the globe. Results indicate that modern gonococci originated in Europe or Africa as late as the 16th century and subsequently disseminated globally. We provide evidence that the modern gonococcal population has been shaped by antimicrobial treatment of sexually transmitted and other infections, leading to the emergence of two major lineages with different evolutionary strategies. The well-described multi-resistant lineage is associated with high rates of homologous recombination and infection in high-risk sexual networks where antimicrobial treatment is frequent. A second, multi-susceptible lineage associated with heterosexual networks, where asymptomatic infection is more common, was also identified, with potential implications for infection control.
... The broad host-range pJD4 plasmid can be mobilised by a number of conjugative plasmids, including IncP, IncFIV, IncFII and IncIα plasmids from E. coli (Dillon and Yeung 1989). In the gonococcus, mobilisation of smaller plasmids is promoted by the large conjugative plasmids (Fig. 2C) (Flett, Humphreys and Saunders 1981;Roberts and Knapp 1988). It has been shown that mobilisation of pJD4 requires the mobilisation protein MobA, enabling the transfer of DNA during conjugation, and origin of transfer oriT, which MobA binds to (Rodriguez-Bonano and Torres-Bauza 2004). ...
Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhoeae, possesses several mobile genetic elements (MGEs). The MGEs such as transposable elements mediate intra-chromosomal rearrangements, while plasmids and the gonococcal genetic island are involved in inter-chromosomal gene transfer. Additionally, gonococcal MGEs serve as hotspots for recombination and integration of other genetic elements such as bacteriophages, contribute to gene regulation or spread genes through gonococcal populations by horizontal gene transfer. In this review we summarize the literature on the structure and biology of MGEs and discuss how these genetic elements may play a role in the pathogenesis and spread of antimicrobial resistance in N. gonorrhoeae. Although an abundance of information about gonococcal MGEs exists (mainly from whole genome sequencing and bioinformatic analysis), there are still many open questions on how MGEs influence the biology of N. gonorrhoeae.
... The further twist to the story added by the second type of cheating is not hypothetical alone. It is well known that nonconjugal plasmids are common and they can undergo conjugal transfer in presence of conjugal plasmids [9,[31][32][33]. Two mechanisms can be employed by the non-conjugal plasmids for hitch-hacking. ...
... Once established, it was stably maintained and could be transferred to other gonococci by conjugation. The first N. gonorrhoeae conjugative plasmid was identified in 1974 (210), and this plasmid can also transfer -lactamase-producing plasmids between different gonococcal strains, and to Neisseria meningitidis (211)(212)(213), Haemophilus influenzae, and Escherichia coli (214). The tetM-possessing conjugative plasmid was first described in 1985 in the United States and was designated the American tetM plasmid (107). ...
Neisseria gonorrhoeae is evolving into a superbug with resistance to previously and currently recommended antimicrobials for treatment of gonorrhea, which is a major public health concern globally. Given the global nature of gonorrhea, the high rate of usage of antimicrobials, suboptimal control and monitoring of antimicrobial resistance (AMR) and treatment failures, slow update of treatment guidelines in most geographical settings, and the extraordinary capacity of the gonococci to develop and retain AMR, it is likely that the global problem of gonococcal AMR will worsen in the foreseeable future and that the severe complications of gonorrhea will emerge as a silent epidemic. By understanding the evolution, emergence, and spread of AMR in N. gonorrhoeae, including its molecular and phenotypic mechanisms, resistance to antimicrobials used clinically can be anticipated, future methods for genetic testing for AMR might permit region-specific and tailor-made antimicrobial therapy, and the design of novel antimicrobials to circumvent the resistance problems can be undertaken more rationally. This review focuses on the history and evolution of gonorrhea treatment regimens and emerging resistance to them, on genetic and phenotypic determinants of gonococcal resistance to previously and currently recommended antimicrobials, including biological costs or benefits; and on crucial actions and future advances necessary to detect and treat resistant gonococcal strains and, ultimately, retain gonorrhea as a treatable infection.
... It is very important in the onset and development of multiple resistance to the bacteria as well as it enables fast spreading of resistant genera in the human populations.Attempts to visualize sex pili by electron microscopy were unsuccessful.44 The 24 MDa conjugal plasmid apparently replicates only transiently in Escherichia coi.47 A physical map of the plasmid has been prepared and restriction maps of 24 MDa plasmids isolated from different gonococcal strains are very similar, although they differ significantly in their efficiencies of mobilization of a PPNG plasmid. ...
... A number of related PPNG carrying plasmids of different sizes have since been described (27). There is evidence that the plasmid was initially acquired from Haemophilus species (43)(44)(45)(46)(47)(48). ...
The genus Neisseria includes both pathogenic and commensal species. N. meningitidis and N. gonorrhoeae are obligate human pathogens with no reservoir outside of the human host. N. lactamica, N. sicca, N. subfl ava (biovars subfl ava, fl ava, and perfl ava), N. mucosa, N. fl avescens, N. cinerea, N. polysaccharea, and N. elongata subspecies elongata, glycolytica, and nitroreducens are human commensal organisms that are rarely associated with disease. Commensal organisms found in animal respiratory tract
or oral fl ora include N. canis and N. weaveri in dogs, N. dentrifi cans in guinea pigs, N. macacae in rhesus monkeys, N. dentiae in cows, and N. iguanae in iguanid lizards.
... Once established, it was stably maintained and could be transferred to other gonococci by conjugation. The first N. gonorrhoeae conjugative plasmid was identified in 1974 (210), and this plasmid can also transfer -lactamase-producing plasmids between different gonococcal strains, and to Neisseria meningitidis (211)(212)(213), Haemophilus influenzae, and Escherichia coli (214). The tetM-possessing conjugative plasmid was first described in 1985 in the United States and was designated the American tetM plasmid (107). ...
The strict human pathogen Neisseria gonorrhoeae has caused gonorrhea for thousands of years, and currently gonorrhea is the second most prevalent bacterial sexually transmitted infection worldwide. Given the ancient nature of N. gonorrhoeae and its unique obligate relationship with humankind over the millennia, its remarkable ability to adapt to the host immune system and cause repeated infections, and its propensity to develop resistance to all clinically useful antibiotics, the gonococcus is an ideal pathogen on which to study the evolution of bacterial pathogenesis, including antimicrobial resistance, over the long term and within the host during infection. Recently, the first gonococcus displaying high-level resistance to ceftriaxone, identified in Japan, was characterized in detail. Ceftriaxone is the last remaining option for empirical first-line treatment, and N. gonorrhoeae now seems to be evolving into a true "superbug." In the near future, gonorrhea may become untreatable in certain circumstances. Herein, the history of antibiotics used for treatment of gonorrhea, the evolution of resistance emergence in N. gonorrhoeae, the linkage between resistance and biological fitness of N. gonorrhoeae, lessons learned, and future perspectives are reviewed and discussed.
... The conjugative plasmids were also shown to be involved in the mobilization of small non selftransmissible β-lactamase gonococcal plasmids [26]. Mobilization by the conjugative plasmids was observed after short mating to other Neisseria species, to Haemophilus influenzae and to restriction-deficient Escherichia coli [27]. Mobilization occurs either via the oriT-binding MobA mobilization protein [28] or with lower efficiency via co-integration with the conjugative plasmid [25]. ...
Many clinical isolates of the human pathogen Neisseria gonorrhoeae contain conjugative plasmids. The host range of these plasmids is limited to Neisseria species, but presence of a tetracycline (tetM) determinant inserted in several of these plasmids is an important cause of the rapid spread of tetracycline resistance. Previously plasmids with different backbones (Dutch and American type backbones) and with and without different tetM determinants (Dutch and American type tetM determinants) have been identified. Within the isolates tested, all plasmids with American or Dutch type tetM determinants contained a Dutch type plasmid backbone. This demonstrated that tetM determinants should not be used to differentiate between conjugal plasmid backbones. The nucleotide sequences of conjugative plasmids with Dutch type plasmid backbones either not containing the tetM determinant (pEP5233) or containing Dutch (pEP5289) or American (pEP5050) type tetM determinants were determined. Analysis of the backbone sequences showed that they belong to a novel IncP1 subfamily divergent from the IncP1alpha, beta, gamma, delta and epsilon subfamilies. The tetM determinants were inserted in a genetic load region found in all these plasmids. Insertion was accompanied by the insertion of a gene with an unknown function, and rearrangement of a toxin/antitoxin gene cluster. The genetic load region contains two toxin/antitoxins of the Zeta/Epsilon toxin/antitoxin family previously only found in Gram positive organisms and the virulence associated protein D of the VapD/VapX toxin/antitoxin family. Remarkably, presence of VapX of pJD1, a small cryptic neisserial plasmid, in the acceptor strain strongly increased the conjugation efficiency, suggesting that it functions as an antitoxin for the conjugative plasmid. The presence of the toxin and antitoxin on different plasmids might explain why the host range of this IncP1 plasmid is limited to Neisseria species. The isolated plasmids conjugated efficiently between N. gonorrhoeae strains, but did not enhance transfer of a genetic marker.
... The further twist to the story added by the second type of cheating is not hypothetical alone. It is well known that nonconjugal plasmids are common and they can undergo conjugal transfer in presence of conjugal plasmids [9,313233. Two mechanisms can be employed by the non-conjugal plasmids for hitch-hacking. First method is called mobilization, a process by which plasmids achieve transfer by 'borrowing' the gene products of a conjugal plasmid, as in the case of ColE1 and RSF1010 plasmids of Escherichia coli. ...
All genes critical for plasmid replication regulation are located on the plasmid rather than on the host chromosome. It is possible therefore that there can be copy-up "cheater" mutants. In spite of this possibility, low copy number plasmids appear to exist stably in host populations. We examined this paradox using a multilevel selection model. Simulations showed that, a slightly higher copy number mutant could out-compete the wild type. Consequently, another mutant with still higher copy number could invade the first invader. However, the realized benefit of increasing intra-host fitness was saturating whereas that of inter-host fitness was exponential. As a result, above a threshold, intra-host selection was overcompensated by inter-host selection and the low copy number wild type plasmid could back invade a very high copy number plasmid. This led to a rock-paper-scissor (RPS) like situation that allowed the coexistence of plasmids with varied copy numbers. Furthermore, another type of cheater that had lost the genes required for conjugation but could hitchhike on a conjugal plasmid, could further reduce the advantage of copy-up mutants. These sociobiological interactions may compliment molecular mechanisms of replication regulation in stabilizing the copy numbers.
... In N. gonorrhoeae Steinberg & Goldberg (1980) found that the larger plasmid was not essential for chromosomal gene transfer, and suggested that DNA might leak from some cells and be taken up by others. Flett, Humphreys & Saunders (1981) reported that the larger plasmid could mobilize penicillinase plasmids between gonococci and from gonococci to H. influenzae and to a restriction-deficient strain of E. coli, yet it could not be detected physically in the accepting H. influenzae or E. coli cells. This might imply that it does not itself transfer together with the smaller resistance plasmid or is unstable in the latter two genera; an alternative possibility is that it transfers but then becomes integrated into the accepting cell's chromosome. ...
... These R-plasmids in the pathogenic gonococci have the potential to be transferred by conjugation to commensal Neisseria spp. and other endogenous flora, creating a pool of resistance genes in healthy hosts (Kirvin and Thornsberry, 1977;Sox et al., 1978;Flett et al., 1981;Guinney and Ito, 1982;Mc Nichol et al., 1983;Piffareti et al., 1988;Roberts and Knapp, 1988;Van Passel et al. 2006). ...
We identified and characterized four different recombination mechanisms involved in the cointegrative transfer of the Neisseria gonorrhoeae beta-lactamase plasmid pSJ5.2 by the gonococcal 41 kb tet(M) and the Gram negative self-transmissible plasmids N3 and R64 drd-33 using an Escherichia colirecA-background. Mobilization of pSJ5.2 by the tet(M) plasmid occurred by cointegration through a replicative transposition of two IS1 elements inserted upstream from the beta-lactamase gene of pSJ5.2 and creating a IS1::beta-lactamase hybrid promoter. Two types of recombinational events occurred within the 1.8 kb BamH1-HindIII fragment of pSJ5.2 with the N3 and R64 plasmids. A non-homologous recombination was found at coordinates 1817 and 2849 of pSJ5.2 with sequences from R64. A non-homologous recombination combined with an IS26-mediated one-ended transposition was found at coordinates 1817 and 3010 of pSJ5.2 with N3. In both recombinational events, a deletion of over 1 kb of pSJ5.2 occurred. The fourth recombination event was detected in the 1.0 kb BamH1-HindIII fragment of pSJ5.2 by homologous recombination between DNA from the truncated Tn3 resolvase gene of pSJ5.2 and the resolvase sequences from R64 and N3.
... gonorrhoeae is able to mobilize gonococcal P-lactamaseencoding R factors intraspecifically as well as interspecifically (7,18). However, this plasmid is unable to mobilize these plasmids efficiently from other bacterial species into N. gonorrhoeae, except for Neisseria cinerea (8). ...
... The gonococcal beta-lactamase-producing plasmid is not self-transmissible, but is mobilized by a 24.5-Mdal conjugative plasmid present in some strains of gonococci (1,4,17,18,21,23). Transfer of penicillin resistance to other gonococci (2,7,13,19,22), N. flava (7), E. coli (7,13,19) and Haemophilus influenzae (9) has been demonstrated. The possibility of gonococcal beta-lactamase-producing plasmids reaching N. meningitidis seemed very feasible because these two pathogens are very closely related according to the DNA-DNA hybridization technique (12), and this appears to have been confirmed in a recent report which describes a strain of N. meningitidis which harbors a 4.5-Mdal beta-lactamase-producing plasmid identical to other plasmids isolated from gonococci (5). ...
Twenty clinical isolates of beta-lactamase-producing Neisseria gonorrhoeae from Japanese sources were studied to define their ability to serve as donors for their plasmids in conjugation with Neisseria meningitidis. These twenty strains of N. gonorrhoeae harbored the 4.5-megadalton (Mdal) beta-lactamase-producing plasmids and the 24.5-Mdal conjugative plasmids. We found that only three of twenty N. gonorrhoeae strains showed a detectable conjugation frequency (greater than 10(-5)) with N. meningitidis as the recipient although all strains were capable of mobilizing beta-lactamase-producing plasmids to N. gonorrhoeae and to Escherichia coli. The 4.5-Mdal beta-lactamase-producing plasmid was maintained in N. meningitidis, but the large 24.5-Mdal conjugative plasmid has not been found in N. meningitidis transconjugants.
The emergence of Neisseria gonorrhoeae isolates displaying resistance to antimicrobial agents is a major public health concern also considering the occurrence of untreatable gonorrhoea infections.
Antibiotic resistance represents a substantial threat in the treatment and control of Neisseria gonorrhoeae. Exhibiting high transformability, resistance mechanisms have spread rapidly throughout the species, resulting in the development of clinically significant resistance to every class of antibiotics used in the treatment of gonorrheal disease. Resistance to third-generation cephalosporins, the only remaining class suitable as a single-dose single-agent therapy, has developed, and effective treatment regimens vary depending on the geographic origin of infection. Ongoing surveillance programs are essential for identifying and limiting the emergence of cephalosporin- and multidrug-resistant gonococci in the future, a problem which to a lesser extent threatens the closely related pathogen, N. meningitidis.
High-level resistance to tetracycline (MIC 16 to 64 µg/ml) in Neisseria gonorrhoeae was due to acquisition of the tetM determinant, a transposon-borne determinant intially described in the genus Streptococcus. In all strains of N. gonorrhoeae tested to date, the tetM determinant was located on a 25.2 Mdal plasmid which arose from the insertion of tetM into the 24.5 Mdal gonococcal conjugative plasmid. The tetM determinant was transferred to suitable recipient strains of N. gonorrhoeae by both genetic transformation and conjugation. The 25.2 Mdal plasmid represent the first description of a self-transmissible R-factor in N. gonorrhoeae.
In Neisseria gonorrhoeae, all penicillinase-specifying plasmids isolated so far share homology with each other and carry a 60% deleted sequence of TnA. Plasmid pHD131, an element isolated from Haemophilus ducreyi and carrying an intact copy of the ampicillin resistance transposable element, was introduced from Escherichia coli into N. gonorrhoeae by both transformation and conjugative mobilization. Plasmids were recovered with no detectable deletion. After their transfer back into E. coli, transposition assays onto phage-lambda DNA demonstrated that the TnA elements were still functional. Plasmid pHD131 could be stably maintained in N. gonorrhoeae with or without the presence of penicillin. These results support the hypothesis that the absence in N. gonorrhoeae of plasmids carrying entire and functional TnA transposons cannot be ascribed to incompatibility between the genetic element and the host, but rather to a barrier to introduction of foreign DNA into gonococcus.
Regulation of capsular biosynthesis (rcs) genes, encoding the ability to induce the production of a colanic acid polysaccharide capsule, were transferred to Escherichia coli by conjugation with Klebsiella pneumoniae (aerogenes) of capsular serotype K36. Transfer was mediated by a 58.4-MDa conjugative plasmid of incompatibility group IncM, which carried a copy of Tn7 (specifying resistance to trimethoprim and streptomycin) together with determinants for several further resistances. This plasmid did not carry the rcs genes itself, but mediated the conjugative recA-dependent transfer of part of the Klebsiella chromosome to E. coli. Once resident in E. coli, the rcs gene(s) could not be mobilised to other strains of E. coli, and the mobilising plasmid could be cured from capsulate transconjugants without loss of the ability to produce colanic acid. All such cured transconjugants contained an insertion of Tn7 in the chromosome, suggesting that the transposon might be involved in mobilisation of the rcs genes from Klebsiella sp. to E. coli. These findings explain previous observations that the ability to manufacture capsular polysaccharide could be transferred by plasmids between Klebsiella sp. and E. coli.
Haemophilus species have been implicated as the source of plasmid-mediated ampicillin resistance in Neisseria gonorrhoeae. Previous attempts to transfer conjugally the resistance plasmids from Haemophilus species to N. gonorrhoeae have met with limited success. Using both biparental and triparental mating systems, it was found that transfer will occur if the commensal Neisseria species, Neisseria cinerea, is used as a transfer intermediate. This organism stably maintains resistance plasmids of Haemophilus and facilitates transfer of these plasmids to N. gonorrhoeae, in a triparental mating system, at a transfer frequency of 10(-8). Both Haemophilus ducreyi and N. gonorrhoeae carry mobilizing plasmids capable of mediating conjugal transfer of the same resistance plasmids. However, restriction endonuclease mapping and DNA hybridization studies indicate that the mobilizing plasmids are distinctly different molecules. Limited homology is present within the transfer region of these plasmids.
Recently, strains of Neisseria gonorrhoeae have been isolated which are highly resistant to tetracycline (MICs of 16 to 64
micrograms/ml). This resistance was due to the acquisition of the resistance determinant tetM, a transposon-borne determinant
initially found in the genus Streptococcus and more recently in Mycoplasma hominis, Ureaplasma urealyticum, and Gardnerella
vaginalis. In N. gonorrhoeae, the tetM determinant was located on a 25.2-megadalton plasmid. This plasmid arose from the insertion
of tetM into the 24.5-megadalton gonococcal conjugative plasmid. The tetM determinant could be transferred to suitable recipient
strains of N. gonorrhoeae by both genetic transformation and conjugation.
High-level tetracycline resistance in strains of Neisseria gonorrhoeae, Neisseria meningitidis, Kingella denitrificans, and
Eikenella corrodens has recently been described. The resistance in each species is due to the acquisition of 25.2-megadalton
conjugative plasmids that carry the tetracycline resistance determinant TetM. We examined the ability of commensal Neisseria
species to serve as recipients in conjugation for these new plasmids. Most of the recipients (n = 21) tested had detectable
conjugation frequencies (greater than 10(-9] with one or more of the donor strains. Transfer was not detected in Branhamella
catarrhalis. Transconjugants were able to maintain the plasmids and act as donors in subsequent matings.
The plasmid pUB307, a derivative of RP1, is a conjugative, broad-host-range plasmid. We have shown that this element mobilizes gonococcal resistance plasmids from Escherichia coli to Neisseria gonorrhoeae, thus providing evidence that extrachromosomal elements can efficiently enter gonococci by conjugation. Furthermore, pUB307 can also be used as a helper element to mobilize the cloning vector pLES2 into N. gonorrhoeae. This finding significantly increases the usefulness of pLES2 as a shuttle vector between E. coli and gonococcus.
Restriction endonuclease analysis and heteroduplex studies indicate that the only difference between the 5.3-kilobase (kb) and 7.4-kb plasmids from beta-lactamase-producing Neisseria gonorrhoeae is that the latter is the 5.3-kb plasmid with a 2.1-kb insertion. The insertion is bounded by inverted repeats of approximately 300 base pairs. Several plasmids from beta-lactamase-producing N. gonorrhoeae isolated at different times and in different countries were compared. Nine 5.3-kb plasmids were examined and found to be indistinguishable, as were sixteen 7.4-kb plasmids.
We have studied the genetic basis of beta-lactamase production in eight strains of Haemophilus ducreyi isolated in diverse
areas of the world. Beta-lactamase production in all strains was mediated by plasmids having a molecular mass of either 5.7
or 7.0 megadaltons. Plasmids of 5.7 megadaltons were shown to carry the entire sequence of pFA7, the beta-lactamase specifying
plasmid found in isolates of Neisseria gonorrhoeae epidemiologically linked to West Africa. Plasmids of 7.0 megadaltons were
shown to carry the entire sequence of pFA3, the beta-lactamase specifying plasmid found in Far Eastern isolates of N. gonorrhoeae.
Both groups of H. ducreyi plasmids were shown to carry physically complete and functional TnA sequences. Thus we have identified
two types of H. ducreyi beta-lactamase plasmid which are identical to the two types of N. gonorrhoeae beta-lactamase plasmid,
except that they carry complete TnA sequences.
Haemophilus influenzae plasmid pVe445 (4.6 MDa) has been compared with Neisseria gonorrhoeae plasmids pNG18 (3.3 MDa) and pNG10 (4.6 MDa) by heteroduplex and restriction enzyme analysis. This study demonstrates that pVe445 differs from pNG18 only that it carries an additional 1.3-MDa DNA fragment between coordinates 0.9 and 2.2. Heteroduplex studies demonstrated that pNG10 and pVe445 are indistinguishable whereas restriction endonuclease analysis indicated minor differences in the distribution of AluI sites.
The human pathogenic Neisseria species N. gonorrhoeae and N. meningitidis are closely related. In contrast to N. meningitidis, however, almost all clinical isolates of N. gonorrhoeae harbour a phenotypically cryptic plasmid. In some gonococcal strains regions of the cryptic plasmid have been found in the gonococcal genome and it has been suggested that large segments of the cryptic plasmid can be integrated into the gonococcal chromosome of both plasmid-bearing and plasmid-free strains. Here we report homology between parts of the cryptic gonococcal plasmid and genomic DNA from four different N. meningitidis strains from systemic disease isolates in which no plasmids have been found with the applied methods. Serogroup B strains, causing many of the cases of meningococcal disease in Norway, hybridized strongly to the cryptic plasmid probe, in contrast to serogroup A and C strains. Clones hybridizing to the cryptic plasmid were isolated from a meningococcal genomic lambda EMBL3 DNA library and characterized by restriction mapping. When using one such clone as a probe the parts of the cryptic plasmid showing homology to the genomic meningococcal DNA were confined to two small separate regions of 420 and 88 bp.
EndoR . NgoII, a class II restriction endonuclease isolated from Neisseria gonorrhoeae, was purified to electrophoretic homogeneity. We were able to separate it from another restriction endonuclease of N. gonorrhoeae, NgoI, by phosphocellulose chromatography. NgoII is an isoschizomer of HaeIII, a restriction endonuclease of Haemophilus aegyptius, and was found to recognize the deoxyribonucleic acid nucleotide base sequence GGCC. NgoII was able to digest phage lambda deoxyribonucleic acid over a wide pH range, with optimal activity at pH 8.5. The enzyme has an absolute requirement for Mg2+; maximal enzyme activity was observed at 1 mM Mg2+. The active enzyme has a molecular weight of 65,000 and appears to be composed of six subunits of identical molecular weight (11,000). No methylase activity could be detected in the purified enzyme preparation.
Tetracycline resistance in a strain of Haemophilus influenzae isolated in the United Kingdom was found to be determined by an apparently non-selftransmissible plasmid of 31 X 10(6) daltons (31 MDal), designated pUB701. Deoxyribonucleic acid hybridization studies indicated that pUB701 shares about 70% base sequence homology with the 30-MDal ampicillin resistance R plasmid RSF007 isolated in the United States from H. influenzae, and 64% sequence homology with the 38-MDal tetracycline and chloramphenicol resistance R plasmid pRI234, isolated in the Netherlands. Heteroduplex studies between RSF007 and pUB701 confirmed the fact that these plasmids were largely homologous, except that pUB701 contained the tetracycline resistance transposon TnD, whereas RSF007 contained the ampicillin resistance transposon TnA. A strain of H. parainfluenzae resistant to both chloramphenicol and tetracycline carried two species of plasmid deoxyribonucleic acid of 2.7 and 0.75 MDal. We were unable to prove that either resistance was plasmid-borne in this strain. Hybridization studies with a [3H]thymine-labeled tetracycline resistance enteric plasmid suggested that the tetracycline transposon was integrated into the chromosome of H. parainfluenzae UB2832. We conclude either that the strains we studied received R factors of the same incompatibility group bearing different resistance genes, or that different resistance genes were translocated to a commom resident plasmid of H. influenzae.
Several β-lactamase-producing, penicillin-resistant strains of Neisseria gonorrhoeae were examined for R plasmids. Penicillin-resistant strains isolated from men returning from the Far East and their contacts
contained a 4.4 × 106-dalton plasmid in common. Transformation studies and the isolation of a spontaneous penicillin-susceptible segregant showed
that the structural gene for β-lactamase was part of the 4.4 × 106-dalton plasmid. An additional penicillin-resistant gonococcal strain isolated in London was found to harbor a 3.2 × 106-dalton R plasmid. Deoxyribonucleic acid (DNA)-DNA duplex studies revealed that the penicillin-resistant gonococcal isolates
contained a significant portion (about 40%) of the transposable DNA sequence, TnA, which includes the β-lactamase gene commonly
found on R plasmids of the Enterobacteriaceae and Haemophilus influenzae.
RP1, a group of genes specifying resistance to carbenicillin, neomycin, kanamycin, and tetracycline and originating in a strain of Pseudomonas aeruginosa, was freely transmissible between strains of P. aeruginosa, Escherichia coli, and Proteus mirabilis. Acquisition of the multiple drug resistance specified by RP1 by these strains was accompanied by acquisition of an extrachromosomal satellite of covalently closed circular deoxyribonucleic acid of molecular weight about 40 million daltons and of buoyant density 1.719 g/cm(3) (60% guanine plus cytosine).
A conjugation system initially discovered in beta-lactamase-producing gonococci mobilized small non-selftransmissible R plasmids encoding beta-lactamase (penicillinase) production into other gonococci, Neisseria, and Escherichia coli. This conjugation system was mediated by a separate selftransmissible plasmid of 23.9 X 10(6) daltons, pFA2. Conjugative plasmids capable of mobilizing R plasmids were also found in nearly 8% of the non-penicillinase-producing gonococci. These were similar to pFA2 in size, buoyant density, and restriction endonuclease digest patterns but were less efficient than pFA2 in mobilization of the penicillinase plasmid pFA3. The presence of conjugative plasmids in gonococci isolated before the appearance of penicillinase-producing strains indicates that a conjugation system for plasmid transfer predated the appearance of R plasmids in gonococci.
Agarose gel electrophoresis may be employed effectively for the detection and preliminary characterization of plasmid deoxyribonucleic acid (DNA) present in clinical isolates and laboratory strains of gram-negative microorganisms. The method is sensitive and does not require radioisotopes or ultracentrifugation. The estimation of plasmid mass from the extent of DNA migration in gels compares favorably with results obtained by electron microscopy of plasmid DNA purified by equilibrium density centrifugation. The method has proved to be a useful tool for survey work and the epidemiological investigation of plasmid dissemination, as well as an important adjunct to the genetic analysis of plasmids.
Compatibility and molecular studies were performed on a number of non auto-transferring plasmids for drug resistance and colicinogeny. The ampicillin (A) and streptomycin-sulphonamide (SSu) resistance determinants of Salmonella typhimurium type 29 are compatible with each other, and thus represent different compatibility groups. The laboratory-made resistance determinant ASu is incompatible with SSu and was used for compatibility studies with other determinants. Nineteen of 26 wild streptomycin-sulphonamide resistant strains of salmonellae and Escherichia coli carried non-transferring SSu determinants incompatible with ASu, and therefore probably phylogenetically related to the SSu determinant of type 29. A wild tetracycline resistance determinant (T) and the non-transferring colicinogeny determinants E1, E2 and E3 were compatible with each other and with A and SSu. A tentative classification can thus be suggested for these non-transferring plasmids which places A, SSu and its homologues, T, ColE1, ColE2 and ColE3 in separate compatibility groups. Molecular studies of ten of the plasmids showed that they consisted of covalentlyclosed circular DNA molecules with mean contour lengths between 2.22 and 4.53 µm. All were present in multiple copies per chromosome in E. coli K12.
The 24.5-megadalton plasmid of Neisseria gonorrhoeae is required for transfer of R-factors and possibly chromosomal markers during conjugal matings between gonococcal strains. We constructed a physical map of one such plasmid, pLE2451, using EcoRI, BglII, and HincII site-specific restriction endonucleases. The patterns of deoxyribonucleic acid digestion obtained with this plasmid were identical to those obtained with three other plasmids of similar size.
Plasmid-mediated chloramphenicol resistance in Haemophilus inj7uen- zae Host specificity of DNA produced by Escherichia coli: bacterial mutations affecting the restriction and modification of DNA
- Van Klingeren
- B Van Embden
- J Dessens-Kroon
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KROON, M. (1977). Plasmid-mediated chloramphenicol resistance in Haemophilus inj7uen-
zae. Antimicrobial Agents and Chemotherapy 11,
WOOD, W. B. (1966). Host specificity of DNA
produced by Escherichia coli: bacterial mutations
affecting the restriction and modification of DNA.
Journal ofMolecular Biology 16, 118-133.