Relationships among some R plasmids found in Haemophilus influenzae.

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.     

Molecular properties of transmissible R factors of Haemophilus influenzae determing tetracycline resistance.

The tetracycline-resistant Haemophilus influenzae strains LU121 and FR16017, recently isolated in West Germany, each harbour a plasmid; that of the former (pLU12U) has a mol. wt of 31.5 X 10(6) and that of the latter (pFR16017) has a mol. wt of 33 X 10(6). Conjugation and DNA-DNA hybridization studies have shown that both plasmids are self-transmissible and carry tetracycline-resistance genes. The purified plasmid DNA of H. influenzae strain LU121 transformed a sensitive Escherichia coli strain to tetracycline resistance. The two R factors are closely related to the H. influenzae plasmid specifying ampicillin resistance (pKRE5367). Electron microscope DNA heteroduplex analysis indicated that pLU121 and pFR15017 probably carry the tetracycline-resistance transposon TnD and that pKRE5367 probably carries the ampicillin-resistance transposon TnA. There is more than one integration site for the insertion which probably represents TnD in pFR15017. All three plasmids have a similar plasmid core and could have a common evolutionary origin.     

Intraspecific and intergeneric mobilization of non-conjugative resistance plasmids by a 24.5 megadalton conjugative plasmid of Neisseria gonorrhoeae

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.     

Origin of Haemophilus influenzae R factors.

The Haemophilus influenzae R plasmids specifying resistance against one, two, or three antibiotics which have emerged in different parts of the world were shown to have closely related but not identical plasmid cores. The gene for ampicillin resistance in the H. influenzae plasmid pKRE5367 is part of a transposon similar to Tn3, which was transposed from pKRE5367 onto RSF1010 in Escherichia coli. An indigenous H. influenzae plasmid (pW266) was isolated. Its properties correspond to those of the H. influenzae R plasmids, except for the presence of a drug resistance transposon. The in vitro-generated H. influenzae R plasmids carrying an ampicillin resistance transposon, a tetracycline resistance transposon, and a transposon for combined tetracycline-chloramphenicol resistance resembled the natural isolates. The findings support the hypothesis that the R plasmids of H. influenzae are of multiclonal evolutionary origin.     

A series of Tn5 variants with various drug-resistance markers and suicide vector for transposon mutagenesis

A series of variants of transposon Tn5 were constructed by replacement of the 2.7-kb central segment which encodes kanamycin resistance with various other resistance-coding genes: tetracycline, chloramphenicol, gentamicin, trimethoprim, streptomycin or ampicillin. A thermosensitive replication mutant of the broad-host-range transmissible plasmid R388 was also constructed for use as a suicide vector for the delivery of transposable elements.     

Antibiotic resistance in Streptococcus pneumoniae and Haemophilus influenzae. Report of a study group on bacterial resistance.

Twenty laboratories in England and Scotland took part in 1977 in a survey of antibiotic resistance in Streptococcus pneumoniae and Haemophilus influenzae. In Str pneumoniae 59 (6.8%) of the 866 strains studied were resistant to tetracycline and three to chloramphenicol, and one strain showed a decreased susceptibility to penicillin. The prevalence of resistance to tetracycline was lower than that found in a similar study performed in 1975. Nine hundred and fifty-two strains of H influenzae were examined: 15 (1.6%) were resistant to ampicillin (all were beta-lactamase producers) and 26 (2.7%) to tetracycline. Only two strains were resistant to chloramphenicol and two to trimethoprim. Sixty-three H influenzae strains were capsulated. Thirty-four of these were of Pittman type b, and antibiotic resistance, particularly to ampicillin, was more common in these than in other serotypes or non-typable strains. Some variation was seen in the resistance rate of both H influenzae and Str pneumoniae to tetracycline in strains from different centres, but too few were isolated to assess whether this represented a true geographical difference.     

Increase in antibiotic resistance in Haemophilus influenzae in the United Kingdom since 1977: report of study group.

A survey of antibiotic resistance in Haemophilus influenzae was carried out in the United Kingdom with 25 laboratories participating. The incidence of resistance in the 1841 strains examined was: tetracycline 3.1%, ampicillin 6.2%, chloramphenicol 1.03%, trimethoprim 1.4%, and sulphamethoxazole 1.5%. Of the 115 strains resistant to ampicillin, 106 produced beta-lactamase. Seventy-nine strains were capsulate, none of which was chloramphenicol resistant, but nine produced beta-lactamase (11.4%). Comparison of these figures of antibiotic resistance with those from a similar survey performed in 1977 showed a significant increase in resistance of H influenzae to ampicillin, chloramphenicol, and trimethoprim.     

Molecular characterization of a small Haemophilus influenzae plasmid specifying beta-lactamase and its relationship to R factors from Neisseria gonorrhoeae.

The ampicillin-resistant Haemophilus influenzae strain Ve445 which caused purulent meningitis and septicaemia in a newborn child in Germany contained a 4.4 megadalton (Mdal) plasmid (pVe445) and produced a TEM type beta-lactamase. The transformation to ampicillin resistance of a sensitive Escherichia coli strain with isolated pVe445 DNA proved that the structural gene for the beta-lactamase resided on this plasmid genome. Molecular DNA-DNA hybridization studies and electron microscope DNA heteroduplex analysis indicated that pVe445 probably contained 38 to 41% of the ampicillin translocation DNA segment (TnA) found on R factors of enteric origin. The TnA fragment present in pVe445 most likely does not contain both of the inverted repeat sequences of TnA. DNA-DNA polynucleotide sequence studies indicated that the 4.4 Mdal plasmid pVe445 was unrelated to the 30 to 38 Mdal H. influenzae R plasmids but was closely related to the 4.1 Mdal ampicillin resistance specifying H. influenzae plasmid RSF0885 isolated in the U.S.A. The H. influenzae plasmid pVe445 shared 91% of its base sequences with the beta-lactamase specifying Neisseria gonorrhoeae plasmid pMR0360 (4.4 Mdal) and had 85% of its base sequences in common with the beta-lactamase specifying N. gonorrhoeae plasmid pMR0200 (3.2 Mdal). All of the four 3.2 to 4.4 Mdal beta-lactamase specifying R plasmids of H. influenzae and N. gonorrhoeae investigated probably have a common evolutionary origin.     

Sensitivity of Haemophilus influenzae to Antibiotics

The use of many different antibiotics to treat chest infection has led us to test the sensitivity of 68 strains of Haemophilus influenzae to 15 different compounds. These included established compounds such as ampicillin and tetracycline and newer agents such as cephalosporins and clindamycin. The minimum inhibitory concentrations of the compounds for H. influenzae were then compared with blood levels attained after the usual dose regimens. There has been a significant increase in tetracycline resistance in the last few years, but all strains were sensitive to ampicillin, chloramphenicol, sulphamethoxazole, and trimethoprim, Several antibiotics were found to be microbiologically unsuitable for treating H. influenzae infections.     

Frequency of R Factor-mediated Multiple Drug Resistance in Klebsiella and Aerobacter

A comparative study was done on the transfer frequency of R factors from 90 strains of multiple drug-resistant Aerobacter and 81 strains of Klebsiella to Escherichia coli CSH-2 (F(-), met(-), pro(-), Nal-r). The most common resistance patterns for the Aerobacter isolants were ampicillin streptomycin chloramphenicol tetracycline and ampicillin streptomycin chloramphenicol tetracycline kanamycin neomycin; for the Klebsiella isolants, the most common resistance pattern was ampicillin kanamycin streptomycin tetracycline chloramphenicol neomycin. R factors were isolated from 14.1% of the Aerobacter strains; 61.5% of these R factors harbored R determinants for ampicillin streptomycin tetracycline. R factors were isolated from 79.1% of the Klebsiella strains; four R factors were isolated with significant frequency; streptomycin chloramphenicol kanamycin neomycin, 37.5%; ampicillin streptomycin tetracycline kanamycin neomycin, 14.1%; ampicillin streptomycin tetracycline, 12.5%; and streptomycin chloramphenicol tetracycline, 12.5%.Chloramphenicol, kanamycin, and neomycin resistance was rarely transferred from the Aerobacter strains, although over 50% of the clinical isolants possessed resistance to these antibiotics. In contrast, over 75% of the Klebsiella strains transferred resistance to chloramphenicol, kanamycin, neomycin. Highest frequency of transferred resistance to individual drugs in the Aerobacter strains was to streptomycin (14.8%), whereas in the Klebsiella group resistance to four drugs was transferred at a very high frequency: streptomycin (80.8%), chloramphenicol (78.5%), kanamycin (76.4%), and neomycin (75.9%).     

Construction of a hybrid plasmid capable of replication in the bacterium Escherichia coli and the cyanobacterium Anacystis nidulans.

A hybrid plasmid was constructed between the 5.3-megadalton plasmid (pUH24) of Anacystis nidulans R2 and the Escherichia coli plasmid pBR322. This was accomplished by adding a transposon to pBR322 and transforming this DNA into A. nidulans. One resultant hybrid, pLS103, had a molecular weight of 6.8 x 10(6), replicated in both organisms, had unique sites for two restriction endonucleases, conferred ampicillin resistance on both organisms, and could be used as a cloning vector in A. nidulans.     

Molecular nature of two beta-lactamase-specifying plasmids isolated from Haemophilus influenzae type b.

The molecular nature of two beta-lactamase-specifying plasmids isolated from two separate ampicillin-resistant Haemophilus influenzae type b strains was examined. A 30 X 10(6)-dalton (30-Mdal) plasmid (RSF007) had a copy number of approximately 3 per chromosomal equivalent and a mole fraction guanine plus cytosine content of 0.39. By heteroduplex analysis the 30-Mdal plasmid was found to contain the entire ampicillin translocation DNA segment (TnA) found on R factors of enteric origin. A 3.0-Mdal plasmid (RSF0885) was found as a multicopy pool of approximately 28 copies per chromosomal equivalent, had a mole fraction guanine plus cytosine content of 0.40, and contained only about one-third of the transposable TnA sequence. RSF007 and RSF0885 appeared to be unrelated plasmids in that they share base sequence homology only within the confines of the TnA segment. The 3.0-Mdal Haemophilus plasmid was used to transform E. coli to ampicillin resistance but was found to be unstable in this host in the absence of antibiotic. The possibility that R-plasmids arose in Haemophilus by the translocation of TnA from a donor R-factor onto an indigenous H. influenzae plasmid is discussed.     

Deletion mutants from R plasmids with increased copy number.

Rms 201-12 and Rms 201-46 are R mutants with increased copy number, and are derived from a conjugative plasmid Rms 201 that encodes resistance to five drugs, ampicillin (Apc), tetracycline (Tc), chloramphenicol (Cm), streptomycin (Sm), and sulfonamides (Sa). The mutants expressed increased levels of resistance to Apc, Sm, and Cm, and a decreased level of resistance to Tc than those of the parent Rms 201 plasmid. When the Rms 201-12+ or Rms 201-46+ cells were inoculated onto plates containing a high concentration of Tc, colonies developed on the plate at a frequency of 10-3 to 10-4 after overnight incubation. The cells grown on the Tc plate carried a tet (Tc gene)-deleted R mutant besides tet-possessing Rms 201-12 or Rms 201-46, and we isolated the tet-deleted R mutant by purifying the R+ cells on drug-free plates. On the other hand, various deletion mutants possessing tet were isolated by prolonged culture of the cells. We have presented a circular gene order of Rms 201 by comparing the genetic markers of all deletion mutants derived from Rms 201, Rms 201-46, and Rms 201-12. The gene(s) regulating the copy number was closely linked to the rep gene. The gene(s) specifying entry exclusion was jointly lost with the tra region.     

Tn10 mediated integration of the plasmid R100.1 into the bacterial chromosome: Inverse transposition

Summary Upon integration into the bacterial chromosome the drug resistance plasmid R100.1 often loses its tetracycline resistance character. We have analyzed an Hfr strain formed by such an integration and an R-prime plasmid derived from it. We find that integration took place within the Tn10 transposon, that the two IS10 sequences were retained, but that at least 80% of the transposon segment located between them, and carrying the tetracycline resistance genes, had been lost. We suggest that integration of R100.1 was mediated by an inverse transposition using the IS10 sequences.     

Mobilization of nonconjugative antibiotic resistance plasmids in Haemophilus ducreyi.

A clinical isolate of Haemophilus ducreyi was found to harbor three plasmids: a 23.5-megadalton (Mdal) phenotypically cryptic plasmid, a 7.0-Mdal ampicillin resistance plasmid, and a 4.0-Mdal sulfonamide resistance plasmid. The two smaller plasmids were transferable by conjugation to Haemophilus recipients, but only if the donor cell harbored the 23.5-Mdal plasmid as well, indicating that this large plasmid had mobilizing capabilities. Transfer was also possible to Escherichia coli recipients. Haemophilus influenzae transconjugants which had acquired both the 23.5-Mdal plasmid and one of the R-plasmids could subsequently retransfer the R-plasmid to other Haemophilus recipients at higher frequencies. A derivative of the 23.5 Mdal plasmid was isolated which was shown by restriction endonuclease analysis to contain an ampicillin resistance transposon and to have retained its conjugative ability.     

Resistance of clinical isolates of Haemophilus influenzae in United Kingdom 1986.

Between 1 January and 31 March 1986, 2434 strains of Haemophilus influenzae collected from 23 laboratories in the United Kingdom were examined. With the same criteria as previous studies in 1977 and 1981 the prevalence of resistance was: ampicillin 7.8% (6.2% beta-lactamase producers and 1.6% non-producers), tetracycline 2.7%, chloramphenicol 1.7%, trimethoprim 4.2%, and sulphamethoxazole 3.5%. of the 87 capsulated strains, 15 produced beta-lactamase, nine were resistant to ampicillin but did not produce beta-lactamase, and two strains, one of which produced beta-lactamase, were resistant to chloramphenicol and tetracycline. Since 1977 the prevalence of resistance to ampicillin, chloramphenicol, and trimethoprim has increased significantly. During 1981-6 strains resistant to ampicillin but not producing beta-lactamase and strains resistant to trimethoprim have significantly increased.     

Beta-lactamase-specifying plasmids isolated from Neisseria gonorrhoeae have retained an intact right part of a Tn3-like transposon.

In three beta-lactamase-producing strains of Neisseria gonorrhoeae, ampicillin resistance is due to the presence of a 7.4-kilobase plasmid. Heteroduplex analysis has shown that the R plasmid contains a 1.6-kilobase segment homologous to the right part (the region coding for the beta-lactamase) of the Tn3-like transposon Tn2301 the 1.6-kilobase DNA segment is not transposable, but it can give rise to a functional transposon, when linked to the left part of TN2301. This provides strong evidence that the R plasmids of N. gonorrhoeae are deletion derivatives of a plasmid that contained an entire TN3-like transposon.     

Serotypes and biotypes and antibiotic susceptibility of Haemophilus influenzae encountered in a clinical laboratory in Taiwan.

Forty-four serologically and biochemically typable Haemophilus influenzae isolates from clinical specimens in Taiwan were subjected to analysis in their relationship with source of isolation and age distribution. It was found that all isolates from blood and cerebrospinal fluid were serotype b, biotype I, and all were in children less than 4 years of age. Serotypes b and e, biotypes I and III were encountered to have the highest incidence of infection caused by H. influenzae in this area. All H. influenzae isolates were further tested for susceptibility to several selected antibiotics. All strains of this organism were susceptible to erythromycin and chloramphenicol. All but two strains were susceptible to tetracycline, whereas more strains were resistant to carbenicillin, gentamycin, keflin, and penicillin. Thirty-four percent strains were found to be resistant to ampicillin and all were beta-lactamase producer. No direct correlation between ampicillin resistance and serotypes or biotypes was recognized.     

Tetracycline resistance transposon Tn1721: recA-dependent gene amplification and expression of tetracycline resistance

The 7.1-megadalton transposon Tn1721 codes for inducible tetracycline resistance (Tcr). The transposable element consists of a "minor transposon" (3.6 megadaltons) encoding functions required for transposition and a "tet region" (3.5 megadaltons) encoding resistance. Multiple tandem repeats of the tet region can be generated by recA-dependent gene amplification. This feature of Tn1721 has been used to analyze the relationship between gene dosage and Tcr. Derivatives of plasmid R388:Tn1721 containing from one to nine copies of the tet region were isolated and separately transformed into recA host cells, where they are stably maintained. The results of the study of Tcr in these strains were as follows: (i) the uninduced, "basal" level of Tcr was linearly related to gene dosage between 4 and 36 copies of tet per chromosome equivalent; (ii) the underlying mechanism could not be attributed to reduced accumulation of the drug; and (iii) induction with tetracycline elicited a four- to fivefold reduction in drug accumulation, independent of the gene dosage.     

Construction and characterization of new cloning vehicles. IV. Deletion derivatives of pBR322 and pBR325

In vitro recombinant DNA experiments involving restriction endonuclease fragments derived from the plasmids pBR322 and pBR325 resulted in the construction of two new cloning vehicles. One of these plasmids, designated pBR327, was obtained after an EcoRII partial digestion of pBR322. The plasmid pBR327 confers resistance to tetracycline and ampicillin, contains 3273 base pairs (bp) and therefore is 1089 bp smaller than pBR322. The other newly constructed vector, which has been designated pBR328, confers resistance to chloramphenicol as well as the two former antibiotics. This plasmid contains unique HindIII, BamHI and SalI sites in the tetracycline resistance gene, unique PvuI and PstI sites in the ampicillin resistance gene and unique EcoRI, PvuII and BalI sites in the chloramphenicol resistance gene. The pBR328 plasmid contains approx. 4900 bp.