Plasmid-mediated sulfonamide resistance in Neisseria meningitidis.

An 8.5-megadalton plasmid coding for sulfonamide resistance was found in a clinical isolate of Neisseria meningitidis, as demonstrated by plasmid elimination and transformation experiments. The plasmid complemented a mutation which determines the production of a thermosensitive dihydropteroate synthetase in Escherichia coli, thus suggesting that the mechanism of resistance involved a plasmid-encoded dihydropteroate synthetase.     

Molecular and phenotypic characterization of penicillinase-producing Neisseria gonorrhoeae from Canadian sources.

The incidence of penicillinase-producing Neisseria gonorrhoeae (PPNG) infections has increased in Canada during the past 2 years. Most of these cases were imported from abroad. The PPNG strains from these cases were characterized with respect to susceptibility to 11 antibiotics, auxotype, and plasmid content. Rosaramicin and cefuroxime proved to be the most potent of the antibiotics tested. The molecular characterization of the isolates indicated that all carried a 2.6-megadalton cryptic plasmid. Most of the PPNG isolates (87%) harbored a 4.5-megadalton penicillinase-producing plasmid, whereas only 13% harbored the 3.2-megadalton penicillinase-producing plasmid. In those cases where contact tracing was possible, the correlation linking strains of Far Eastern etiology with carriage of the 4.5-megadalton plasmid was upheld. The penicillinase-producing strains were typed auxanographically in either the proline-requiring (57%) or prototrophic groups (42%). Substrate hydrolysis profiles and analytical isoelectric focusing of crude beta-lactamase extracts of several isolates has reconfirmed that these strains elaborate a type TEM-1 enzyme. Several of the penicillinase-producing plasmids were also examined for plasmid stability.     

Isolation and molecular characterization of beta-lactamase-producing Haemophilus parainfluenzae from the genital tract.

Three Haemophilus parainfluenzae strains isolated from the urogenital tract harbored a beta-lactamase-coding 3.2-megadalton plasmid identical, by restriction endonuclease digestion and hybridization with radioactive and biotin-labeled probes specific for the TEM-1 beta-lactamase and TnA sequences, to the 3.2-megadalton "African-type" plasmid found in Neisseria gonorrhoeae.     

Plasmid-determined resistance to fosfomycin in Serratia marcescens.

Multiple-antibiotic-resistant strains of Serratia marcescens isolated from hospitalized patients were examined for their ability to transfer antibiotic resistance to Escherichia coli by conjugation. Two different patterns of linked transferable resistance were found among the transconjugants. The first comprised resistance to carbenicillin, streptomycin, and fosfomycin; the second, and more common, pattern included resistance to carbenicillin, streptomycin, kanamycin, gentamicin, tetracycline, chloramphenicol, sulfonamide, and fosfomycin. The two types of transconjugant strains carried a single plasmid of either 57 or 97 megadaltons in size. Both of these plasmids are present in parental S. marcescens strains resistant to fosfomycin. The 57-megadalton plasmid was transformed into E. coli.     

Bactericidal activity of trimethoprim alone and in combination with sulfamethoxazole on susceptible and resistant Escherichia coli K-12.

The combined effects of trimethoprim and sulfamethoxazole on the viability of Escherichia coli K-12 and resistant strains possessing resistance plasmids were examined in minimal medium. When methionine, glycine, and adenine were present, sulfamethoxazole could enhance trimethoprim activity against E. coli K-12 so that the combination was bactericidal. However, this enhancement occurred over a narrow range of trimethoprim concentrations (0.04 to 0.2 mg liter-1) and only when the sulfamethoxazole concentration was more than 10 times that of trimethoprim. Under certain conditions, sulfamethoxazole enhanced trimethoprim bactericidal activity against E. coli K-12 carrying plasmid R1 at concentrations of sulfamethoxazole far below those required to inhibit the organism, but there was no such enhancement with the same host containing the SSu plasmid. Similar differences were found with strains possessing trimethoprim resistance plasmids R483 and R751. Sulfamethoxazole can promote a bactericidal response with trimethoprim in E. coli K-12 and some of its resistant derivatives, but only under a narrow range of concentrations.     

Novel plasmid-mediated beta-lactamase in clinical isolates of Klebsiella pneumoniae more resistant to ceftazidime than to other broad-spectrum cephalosporins.

Multiresistant Klebsiella pneumoniae strains isolated from three patients in the same intensive care unit were more resistant to ceftazidime than to cefotaxime and aztreonam but remained susceptible to moxalactam and imipenem. Resistance to beta-lactams, kanamycin, streptomycin, sulfonamides, and tetracyclines was transferable to Escherichia coli by conjugation and was lost en bloc after treatment with ethidium bromide. Agarose gel electrophoresis of wild types and transconjugants indicated that these resistances were mediated by a 150-kilobase plasmid, pCFF14. The strains constitutively produced a beta-lactamase with isoelectric point close to 5.6 and which had a higher Vmax for ceftazidime and cephalothin than for cefotaxime. The substrate profile and isoelectric point of this enzyme thus differ from those of other known plasmid-mediated beta-lactamases, including the broad-spectrum enzyme CTX-1. Hybridization studies support the derivation of the novel enzyme from a TEM-type beta-lactamase.     

Transfer of beta-lactamase plasmids from Neisseria gonorrhoeae to Neisseria meningitidis and commensal Neisseria species by the 25.2-megadalton conjugative plasmid.

Two highly tetracycline-resistant, beta-lactamase-producing Neisseria gonorrhoeae strains were used as donors for conjugation with Neisseria meningitidis and commensal Neisseria species. We found that both strains were able to transfer the 4.4- and 3.2-megadalton beta-lactamase plasmids to the recipients tested, with frequencies between 10(-1) and 10(-9).     

High-level resistance to gentamicin in clinical isolates of Streptococcus (Enterococcus) faecium.

During a 14-month period beginning in July 1986, three distinct clinical isolates of Streptococcus (Enterococcus) faecium demonstrating high-level resistance (MIC, greater than 2,000 micrograms/ml) to gentamicin, kanamycin, tobramycin, and streptomycin were recovered from individual patients at one institution. Combinations of ampicillin with any of these agents failed to show bactericidal synergism. By filter-mating techniques, high-level gentamicin resistance could be transferred into a susceptible recipient of the same species at frequencies as high as 1 x 10(-4); transfer into Streptococcus faecalis JH2-7 occurred at lower frequencies (less than 2 x 10(-7). Aminoglycoside substrate profile analysis of clinical isolates as well as of laboratory-derived cured strains and transconjugants revealed 2"-aminoglycoside phosphotransferase and 3'-aminoglycoside phosphotransferase (III) phosphorylating enzymes, AAC-6' acetylating activity above that attributable to the intrinsic activity characteristic of S. faecium, and a streptomycin adenylylating enzyme. All three isolates carried a 51-megadalton plasmid. Curing of this plasmid or conjugative transfer into susceptible recipients was associated with the loss or acquisition of high-level gentamicin resistance, respectively. Loss of high-level gentamicin resistance was also observed when curing techniques resulted in a decrease in the size of this plasmid equivalent to a 10-megadalton deletion. Transferable, high-level resistance to gentamicin and other aminoglycosides, which was previously recognized in S. faecalis, has now emerged in clinical isolates of S. faecium, with the attendant concerns for possible spread.     

Molecular nature of a streptomycin and sulfonamide resistance plasmid (pBP1) prevalent in clinical Escherichia coli strains and integration of an ampicillin resistance transposon (TnA).

A small, nonconjugative plasmid, designated pBP1, was originally found in different fecal Escherichia coli serotypes isolated from a healthy proband. Of a total number of 130 hospital strains of E. coli subsequently studied, 8.5% yielded plasmid of the pBP1 type. This R plasmid specifies resistance to streptomycin (Sm) and sulfonamides (Su) and has a mass of 4.0 megadaltons. Inactivation of streptomycin is due to the aminoglycoside phosphotransferase APH-(3 "). A physical map was constructed by analysis with restriction endonucleases. Another small plasmid, pBP1-1, was isolated from one of the hospital strains and characterized as an enlarged pBP1 replicon containing an additional deoxyribonucleic acid sequence identified as a transposable element for ampicillin resistance (TnA). Plasmid pBP1-1 was cleaved by restriction enzymes for identification of the transposon sequence which codes for a TEM 1 beta-lactamase. The sequence organizations in the Sm Su plasmids RSF1010 and pBP1 were shown to be identical for regions specifying streptomycin and sulfonamide resistance, but different for the region containing the origin of replication and genes for replicative functions. Thus, RSF1010, which has been considered as the prototype of Sm Su plasmids, and pBP1, which is at least as frequent in clinical isolates as RSF1010, do not have a single common ancestor.     

Comparative study of a novel plasmid-mediated beta-lactamase, CAZ-2, and the CTX-1 and CAZ-1 enzymes conferring resistance to broad-spectrum cephalosporins.

Infections caused by strains of Klebsiella pneumoniae resistant to broad-spectrum cephalosporins have been observed recently in hospitals in Clermont-Ferrand, France. beta-Lactam resistance resulted primarily from the plasmid-mediated, expanded-spectrum CTX-1 beta-lactamase. Furthermore, since 1987 some K. pneumoniae isolates more resistant to ceftazidime than to other cephalosporins have been observed. This new resistance phenotype was the result of the production of ceftazidimase CAZ-1 and, more recently, CAZ-2. As in CTX-1-producing strains, resistance to beta-lactams resulting from CAZ-2 was associated with resistance to aminoglycosides except gentamicin, sulfonamide, and tetracycline and was transferable to Escherichia coli by conjugation. Agarose gel electrophoresis of plasmid DNA from wild-type strains and transconjugants indicated that CAZ-2 production was mediated by a plasmid of 85 kilobases highly related to plasmid pCFF04 coding for CTX-1 beta-lactamase. The isoelectric point, close to 6.0, of this novel enzyme differed from those of CTX-1 and CAZ-1. Like CAZ-1, the CAZ-2 enzyme efficiently hydrolyzed ceftazidime and aztreonam, but as with CTX-1, cefotaxime gave the maximal reaction rate. For each expanded-spectrum beta-lactamase, the activity of broad-spectrum cephalosporins was restored by clavulanic acid or sulbactam.     

Antimicrobial susceptibility testing and phenotyping of Neisseria gonorrhoeae isolated from patients with ophthalmia neonatorum in Nairobi, Kenya.

Antimicrobial susceptibility testing, auxotyping-serotyping, and plasmid analysis were performed on 41 ocular isolates, 7 nasopharyngeal isolates, and 18 cervical isolates of Neisseria gonorrhoeae obtained during a recent treatment trial of gonococcal ophthalmia neonatorum in Nairobi, Kenya. Fourteen distinct serovar-auxotype patterns were observed with IB-1/Pro-strains which accounted for 59% of the isolates. Infection with multiple types of gonococci appeared to occur in 22% of the mothers since 4 of 18 paired maternal cervical and neonatal ocular isolates had mismatched serovar-auxotype patterns. Among 10 treatment failure isolates only 1 had a mismatched serovar-auxotype pattern. Six (15%) of the ocular isolates were penicillinase-producing N. gonorrhoeae (PPNG). Five had the 4.4-megadalton (Md) beta-lactamase plasmid and one had the 3.2-Md beta-lactamase plasmid. The 24.5-Md plasmid was found in 5 of 6 PPNG strains and in 8 of 35 non-PPNG strains (P less than 0.02). For most antimicrobial agents, PPNG and non-PPNG strains showed similar patterns of susceptibility. Ceftriaxone was the most active of the antibiotics tested, with all strains having an MIC less than or equal to 0.06 mg/liter. Among non-PPNG strains, 15 (43%) had a penicillin MIC greater than or equal to 2 mg/liter and were considered intrinsically resistant to penicillin. Overall, non-PPNG intrinsically resistant strains had greater resistance to other antibiotics than did non-intrinsically resistant strains (P less than or equal to 0.006). The Mtr phenotype was found in 53% of these strains.     

Occurrence of transposable trimethoprim resistance in clinical isolates of Escherichia coli devoid of self-transmissible resistance plasmids.

Fifty trimethoprim-resistant clinical isolates of Escherichia coli, devoid of self transmissible trimethoprim resistance plasmids, were examined for the presence of trimethoprim resistance transposons. Trimethoprim resistance was mobilized from 12 strains by transposition onto plasmid RP4. The trimethoprim resistance transposons isolated comprised two groups: those with and without linked streptomycin resistance.     

TEM-type beta-lactamase production in Haemophilus ducreyi.

A TnA-containing 6.0-megadalton plasmid (pJB1) isolated from Haemophilus ducreyi was shown to code for a beta-lactamase similar to the TEM-1-type beta-lactamase originating from the ampicillin transposon Tn2.     

Identification of antimicrobial resistance and class 1 integrons in Shiga toxin-producing Escherichia coli recovered from humans and food animals

OBJECTIVES: The objective of this study was to identify antimicrobial resistance and class 1 integrons among Shiga toxin-producing Escherichia coli (STEC). METHODS: Two-hundred and seventy-four STEC recovered from poultry, cattle, swine and humans were characterized by antimicrobial susceptibility testing, screened for the presence of class 1 integrons by PCR, and assayed for integron transfer by conjugation. RESULTS: Ninety-three (34%) of the isolates were resistant to streptomycin, followed by 89 (32%) to sulfamethoxazole, 83 (30%) to tetracycline, 48 (18%) to ampicillin, 29 (11%) to cefalothin, 22 (8%) to trimethoprim/sulfamethoxazole, 18 (7%) to gentamicin, 13 (5%) to chloramphenicol and 10 (4%) to cefoxitin. Class 1 integrons were detected in 43 (16%) of the 274 isolates. The adenyl acetyltransferase gene, aadA, which confers resistance to streptomycin, was identified in integrons from 41 (95%) of these 43 isolates, and the dfrA12 gene, which confers resistance to trimethoprim, was identified in integrons from eight (19%) of the isolates. The sat1 gene, which confers resistance to streptothricin, an antimicrobial that has never been approved for use in the United States, was identified in integrons from three (7%) of the isolates. Transfer of integrons by conjugation between strains of E. coli resulted in transfer of antimicrobial-resistant phenotypes for ampicillin, chloramphenicol, cefalothin, gentamicin, tetracycline, trimethoprim, sulfamethoxazole and streptomycin. CONCLUSIONS: Antimicrobial resistance is common in STEC. Class 1 integrons located on mobile plasmids have facilitated the emergence and dissemination of antimicrobial resistance among STEC in humans and food animals.     

Detection of PSE-2 beta-lactamase in enterobacteria

PSE-2, a plasmid-mediated beta-lactamase which was previously considered pseudomonas specific, was observed in clinical isolates of Escherichia coli, Klebsiella pneumoniae, Providencia stuartii, and Enterobacter cloacae. All four isolates transferred PSE-2 production, together with resistance to gentamicin, tobramycin, kanamycin, and sulfamethoxazole, into Escherichia coli J62-1 and Pseudomonas aeruginosa PU21. Transfer correlated with acquisition by the transconjugants of a 65-megadalton plasmid, and this element was considered to encode PSE-2 expression. The PSE-2 enzyme conferred high-level carbenicillin resistance on both J62-1 and PU21 transconjugants, although the PSE-2-producing Providencia stuartii isolate was susceptible to carbenicillin.     

Plasmid-coded ampicillin resistance in Haemophilus ducreyi.

Seven of the 96 ampicillin-resistant isolates of Haemophilus ducreyi reported in the preceding article (Bilgeri et al., Antimicrob. Agents Chemother. 22:686-688, 1982) were investigated and found to harbor plasmids of 3.95, 5.2, 5.8, and 6.4 megadaltons. All except the 5.8-megadalton plasmid have been shown to code for beta-lactamase. The 6.4- and 5.2-megadalton plasmids of three isolates were conjugally transferable to a streptomycin-resistant mutant of H. ducreyi at high frequencies, perhaps due to the presence in these strains of a high-molecular-weight plasmid.     

Properties of PSE-2 beta-lactamase and genetic basis for its production in Pseudomonas aeruginosa.

The properties of PSE-2 beta-lactamase have been examined by using two new PSE-2-producing plasmids, pMG33 and pMG74, as well as plasmid R151, found in Pseudomonas aeruginosa. PSE-2 beta-lactamase resembled other PSE enzymes in activity against carbenicillin, but it also resembled OXA enzymes, such as OXA-1, in rapid hydrolysis of oxacillin, cloxacillin, and methicillin and in inhibition by sodium chloride but not by cloxacillin. Antisera that inactivated TEM-1, TEM-2, OXA-1, or PSE-1 and PSE-4 beta-lactamase failed to cross-react with PSE-2, which thus appears to be immunologically distinct. The plasmids determining PSE-2 varied in geographical origin, size, transfer proficiency, and incompatibility specificity, but all determined resistance to carbenicillin, gentamicin, kanamycin, streptomycin, spectinomycin, sulfonamide, and tobramycin. From a pUZ8-R151 recombinant plasmid in Escherichia coli, the PSE-2 beta-lactamase gene could be transposed to a second plasmid in a 6.4-megadalton unit together with resistance to gentamicin, kanamycin, streptomycin, spectinomycin, sulfonamide, and tobramycin. Transposition was recA independent. We propose the designation Tn1404 for this unit, which, like transposons carrying OXA-1, PSE-1, PSE-4, and some transposons determining TEM-1, includes genes for beta-lactam, aminoglycoside, and sulfonamide resistance.     

Molecular cloning and mechanism of trimethoprim resistance in Haemophilus influenzae.

We studied 10 trimethoprim-resistant (Tmpr) Haemophilus influenzae isolates for which agar dilution MICs were 10 to greater than 200 micrograms/ml. Trimethoprim resistance was transferred from two Tmpr H. influenzae isolates to a Tmps strain by conjugation or transformation. Wild-type Tmpr strains and Tmpr transcipients did not contain detectable plasmid DNA. The trimethoprim resistance gene was cloned into a cosmid vector, and recombinant plasmids were transduced into Escherichia coli. A 0.50-kilobase intragenic probe derived from a 12.9-kilobase fragment which encoded trimethoprim resistance hybridized with whole-cell DNA from Tmps and Tmpr strains. Southern blot analysis of restricted DNA from isogenic Tmps and Tmpr H. influenzae indicated that acquisition of trimethoprim resistance involved a rearrangement or change in nucleotide sequence. Hybridization was not seen with DNA derived from Tmpr E. coli containing dihydrofolate reductase I, II, and III genes or with Tmpr Neisseria meningitidis, Neisseria gonorrhoeae, and Pseudomonas cepacia. Southern hybridization with 12 multiply resistant encapsulated H. influenzae strains confirmed that the trimethoprim resistance gene was chromosomally mediated. Dihydrofolate reductase activity was significantly greater in cell sonicate supernatants of Tmpr strains in comparison with isogenic Tmps recipients. Differences were not found in the trimethoprim inhibition profile of dihydrofolate reductase activity in Tmps and Tmpr strains. We conclude that the mechanism of trimethoprim resistance in H. influenzae is overproduction of chromosomally located dihydrofolate reductase.     

Quinolone, fluoroquinolone and trimethoprim/sulfamethoxazole resistance in relation to virulence determinants and phylogenetic background among uropathogenic Escherichia coli

INTRODUCTION: The goal of this study was to assess how resistance to quinolones, fluoroquinolones and trimethoprim/sulfamethoxazole relates to the virulence potential and phylogenetic background of clinical Escherichia coli isolates. METHODS: Among 150 uropathogens (21% resistant to quinolones, 12% resistant to fluoroquinolones and 29.3% resistant to trimethoprim/sulfamethoxazole), E. coli phylogenetic group, 15 virulence-associated genes and 7 O antigens were analysed. Clonal group A (CGA) and genomic PCR profiles were studied among trimethoprim/sulfamethoxazole-resistant isolates. RESULTS: Isolates susceptible to the three antimicrobial agents were significantly associated with phylogenetic group B2, whereas resistant isolates exhibited shifts to non-B2 groups (quinolone and fluoroquinolone-resistant isolates to group A; trimethoprim/sulfamethoxazole-resistant isolates to group D). Diverse virulence traits, including UTI-associated O antigens, were significantly less frequent among resistant isolates, particularly those resistant to fluoroquinolones (median score, 3.9 virulence factors/strain) and also to quinolones (5.2) or trimethoprim/sulfamethoxazole (6.4), as compared with the corresponding drug-susceptible isolates (median scores of 7.9, 8.6 and 7.9, respectively). Among 44 trimethoprim/sulfamethoxazole-resistant isolates, 3 (6.8%) belonged to CGA. All these 3 CGA strains caused pyelonephritis (P=0.02) and exhibited the consensus virulence profile of previously described CGA strains from abroad. CONCLUSIONS: E. coli isolates resistant to quinolones, trimethoprim/sulfamethoxazole and especially fluoroquinolones were associated with reductions in virulence traits and shifts to non-B2 phylogenetic groups. Moreover, fluoroquinolone resistance usually occurred in low-virulence E. coli group A isolates rather than in isolates from groups B2 and D which had lost virulence traits. CGA accounted for 23% of trimethoprim/sulfamethoxazole-resistant E. coli producing pyelonephritis.     

Transfer of Plasmid-Borne Beta-Lactamase in Neisseria gonorrhoeae

Neisseria gonorrhoeae strain GC82 contains a plasmid specifying a beta-lactamase (beta-Lam(+)). Mixed incubation of strain GC82 with a penicillin-susceptible (beta-Lam(-)), streptomycin-resistant mutant of strain GC9 results in the expression of beta-lactamase activity and streptomycin resistance in the transcipients. The frequency of transfer of the plasmid-specified resistance to penicillin seems to be proportional to the initial input ratio of the mating mixture of donor to recipient and to correlate positively with bacterial density. Cell-to-cell transmission of the deoxyribonucleic acid (DNA) appears to be by a conjugal mechanism or, alternatively, by an as yet undescribed transducing phage. Additionally, whole-cell DNA from a beta-lactamase-producing strain could be used to transform streptomycin-resistant recipients, resulting in the expression of both beta-lactamase activity and streptomycin resistance in the transformants, and purified gonococcal plasmid DNA transformed Escherichia coli but not the gonococcus. Circular DNA extracted from donor GC82 comprised three molecular species (approximately 2.7, 4.8, and 25 megadaltons [Mdal]), whereas the recipients GC9-S (Str(r)) contained only the 2.7-Mdal cryptic DNA species. DNA from the GC9-S82 (Str(r), beta-Lam(+)) transcipient contained a 4.8-Mdal species in addition to the cryptic molecular species (2.7 Mdal). The finding that the transcipient will not retransfer beta-lactamase is consistent with the hypothesis that the 25-Mdal plasmid promotes mobilization of the smaller 4.8-Mdal R plasmid.