Emergence of sulfamethoxazole-trimethoprim resistant Shigella flexneri in Northeastern Brazil.

In contrast to prior experience in this setting, three of four Shigella flexneri strains recently isolated from patients in Northeastern Brazil with acute inflammatory diarrhea were found to be resistant to sulfamethoxazole, trimethoprim and the combination in vitro. We performed mating studies to determine if the resistance was transferable, and then isolated and characterized plasmid DNA from the resistant Shigella isolates, other resistant Enterobacteriaceae isolated simultaneously from the stools of these individuals, and transconjugant strains. Each of the resistant Shigella strains contained a large plasmid. These plasmids were of different molecular weights ranging from 30 to 50 Mdal in size. Two of these plasmids were transferred with sulfamethoxazole-trimethoprim resistance to E. coli K-12 recipient strains. These findings of transferable resistance to sulfamethoxazole-trimethoprim associated with plasmids in Shigella and in other Enterobacteriaceae raises concerns about the potential limitations of this widely used antimicrobial combination.

Sulfamethoxazole-trimethoprim-resistant Shigella flexneri in northeastern Brazil.

In contrast to prior experience in northeastern Brazil, three of four Shigella flexneri strains recently isolated from patients with acute inflammatory diarrhea in this setting were found to be resistant to sulfamethoxazole-trimethoprim. The resistant strains contained large, different plasmids, two of which were transferred with sulfamethoxazole-trimethoprim resistance to Escherichia coli K-12 recipient strains.

Identification and cloning of the genetic determinant that encodes for the K88ac adherence antigen.

Strains of Escherichia coli capable of causing diarrhea in young pigs are often able to proliferate in the upper small intestine of the infected animal due to the presence of a specific surface antigen, K88. The genetic determinants for K88 antigen production and the ability to utilize the trisaccharide raffinose (Raf) are carried on a 50-megadalton plasmid. Recombinant deoxyribonucleic acid techniques were used to insert an 8.2-megadalton HindIII fragment carrying the K88ac gene(s) from the K88/Raf plasmid pPS100 into the vector pBR322. At lease six polypeptides encoded by this fragment were expressed in minicells. These polypeptides ranged in size from 18,000 to 70,000 daltons. The K88ac antigenic subunit, which has an apparent molecular weight of 23,500, was identified by immunoprecipitation with staphylococcal protein A as the coprecipitant.

Genetic and molecular characteristics of Vir plasmids of bovine septicemic Escherichia coli.

Three wild strains of bovine septicemic Escherichia coli were selected on the basis of their production of a toxin lethal for mice and chickens and their characteristic surface antigen. The transfer of these virulence (Vir) properties from two of the three to recipient E. coli was detected after mating. One Vir plasmid (pJL1) was derepressed for transfer and associated with mobilization of chromosomal markers. The other, pJL2, was repressed. Both plasmids were tagged with transposon Tn5 (kanamycin resistance), and transfer parameters of the tagged plasmids were studied. The Tn5 insertion in pJL2 usually increased transfer efficiency 100-fold. Plasmid pJL1 was classified as a member of the FIV incompatibility group. A pJL1::Tn5 derivative plasmid was incompatible with ColV1. Plasmid pJL2 behaved as an fi+ plasmid. Both plasmids pJL1 and pJL2 had a molecular weight of 92 x 10(6) and were present at about four copies per chromosome; their deoxyribonucleic acid (DNA) structures were not identical on the basis of restriction enzyme analysis. DNA-DNA hybridization revealed a polynucleotide sequence homology of at least 58% between the two plasmids. No plasmids could be detected in one wild or certain laboratory-derived Vir+ E. coli strains.

Characterization of plasmids that encode for the K88 colonization antigen.

K88 antigen, and important virulence factor in porcine enteropathogenic Escherichia coli (EEC), can be transferred along with the ability to ferment the trisaccharide raffinose (Raf). The plasmids from a number of EEC strains that encode these two properties were isolated and characterized. In most strains the K88 and Raf genes were found on a single nonconjugative plasmid approximately 50 x 10(6) daltons in size. This plasmid core was conserved with only slight variation among the strains tested. In some transconjugants, larger conjugative plasmids were observed that were apparently recombinants between the Raf/K88 plasmid and a transfer fa(tor. Occasionally plasmids carrying only the raffinose fermentation genes arose by deletion of a deoxyribonucleic acid segment of about 20 x 10(6) daltons that included the K88 antigen gene(s).

Isolation of a nontransmissible antibiotic resistance plasmid by transductional shortening of R factor RP1.

A plasmid segregant carrying tetracycline and carbenicillin resistance markers has been isolated from R factor RP1 by transductional shortening with phage P22. The new plasmid RP1-S2, which has a molecular weight of 23 times 10-6, has lost the transfer, phage sensitivity, and neomycin resistance functions of RP1. It combines readily with a W group plasmid, R388, to form a transmissible carbenicillin and trimethoprim resistance plasmid, RWP1.

RP1 properties and fertility inhibition among P, N, W, and X incompatibility group plasmids.

Incompatibility group P plasmids demonstrate strong entry exclusion properties. Stringent incompatibility is also observed in the absence of entry exclusion. These observations have been facilitated by the study of a nontransmissible plasmid, RP1-S2, derived from RP1 by transductional shortening. RP1-S2 retains carbenicillin and tetracycline resistances as well as loci that cause either the loss of P plasmids (incp) or a locus specifying susceptibility to curing (sinp) in the presence of a P plasmid. RP1-S2 can be mobilized by an incompatibility group W plasmid, R388, and also freely forms recombinants with R388. P, N, and W incompatibility group plasmids all encode information for the receptor of the cell wall-adsorbing phage PRD1. Based on the premise that the location of this receptor is analogous to entry exclusion factors for F-like plasmids and hence a regulated transfer region determinant, we tested fertility inhibition relationships among these plasmid groups. We detected both reciprocal and nonreciprocal fertility inhibition relationships for bacteria containing various combinations of W, N, and P group plasmids. The nonreciprocal nature of some combinations, we believe, reflects the identity of the point mutation reading to derepression of the plasmid in question. Reciprocal fertility inhibition, on the other hand, may reflect the reconstruction of a fertility inhibition system through complementation. An X incompatibility group plasmid, known to affect the fertility of an N group plasmid, was also shown to inhibit P plasmid fertility. These observations may indicate a possible evolutionary relationship(s) of plasmids unrelated by the criteria of incompatibility, pilus phage specificity, or plasmid host range.

Characteristics and expression of tetracycline resistance in gram-negative bacteria carrying the Pseudomonas R factor RP1.

The Pseudomonas R factor RP1 determines an inducible tetracycline resistance similar to that described for Escherichia coli R factors. The level of RP1-determined resistance measured by minimal inhibitory concentration testing is dependent on the host bacteria and corresponds to the magnitude of decrease in tetracycline accumulation by RP1-containing organisms. The tetracycline resistance mechanism is inactivated at low temperatures. The effect of metabolic inhibitors on tetracycline accumulation by susceptible organisms under various conditions indicates a possible nonspecific effect of cellular energy metabolism on tetracycline uptake.