Genetics of resistance to trimethoprim in cotrimoxazole resistant uropathogenic Escherichia coli: integrons, transposons, and single gene cassettes.

Cotrimoxazole, the combined formulation of sulfamethoxazole and trimethoprim, is one of the treatments of choice for several infectious diseases, particularly urinary tract infections. Both components of cotrimoxazole are synthetic antimicrobial drugs, and their combination was introduced into medical therapeutics about half a century ago. In Gram-negative bacteria, resistance to cotrimoxazole is widespread, being based on the acquisition of genes from the auxiliary genome that confer resistance to each of its antibacterial components. Starting from previous knowledge on the genotype of resistance to sulfamethoxazole in a collection of cotrimoxazole resistant uropathogenic Escherichia coli strains, this work focused on the identification of the genetic bases of the trimethoprim resistance of these same strains. Molecular techniques employed included PCR and Sanger sequencing of specific amplicons, conjugation experiments and NGS sequencing of the transferred plasmids. Mobile genetic elements conferring the trimethoprim resistance phenotype were identified and included integrons, transposons and single gene cassettes. Therefore, strains exhibited several ways to jointly resist both antibiotics, implying different levels of genetic linkage between genes conferring resistance to sulfamethoxazole (sul) and trimethoprim (dfrA). Two structures were particularly interesting because they represented a highly cohesive arrangements ensuring cotrimoxazole resistance. They both carried a single gene cassette, dfrA14 or dfrA1, integrated in two different points of a conserved cluster sul2-strA-strB, carried on transferable plasmids. The results suggest that the pressure exerted by cotrimoxazole on bacteria of our environment is still promoting the evolution toward increasingly compact gene arrangements, carried by mobile genetic elements that move them in the genome and also transfer them horizontally among bacteria.

Strict relationship between class 1 integrons and resistance to sulfamethoxazole in Escherichia coli.

Antibiotic resistance is a health concern. Class 1 integrons (Int1) are genetic elements that contribute to the problem, as they carry different antibiotic resistance genes in their variable region, frequently dfrA (resistance to trimethoprim) and, in their conserved region, the sul1 gene (resistance to sulfonamides, e.g. sulfamethoxazole). These are synthetic antibiotics that work by blocking two enzymes in the folic acid synthesis pathway. In the clinic, the combination of trimethoprim (TMP) and sulfamethoxazole (SMX), called co-trimoxazole (SXT), is widely used. A collection of 230 uropathogenic Escherichia coli strains was studied with three objectives: i) to analyze their phenotype of susceptibility to antifolate antibiotics, ii) to determine the genetic basis of their resistance to SMX, and iii) to correlate the phenotypic and genotypic data with the presence of Int1. The prevalence of resistance to SMX, TMP, and SXT was 54%, 45%, and 41%, respectively. The mobile genes sul1, sul2 and sul3, which confer resistance to sulfonamides, were PCR-surveyed: all sulfa-resistant strains were found to contain at least one of these genes, with the exception of three strains. For these latter, the possibility of being target folP mutants could be ruled out, pointing to the existence of a still unknown mechanism of resistance to SMX in E. coli. All 50 strains previously cataloged as positive for Int1 (because they had an intI1 gene for the integrase) were resistant to SMX: most had sul1, alone or with sul2 or sul3, others only had sul2, and one lacked every sul gene. In addition, 16 sul1+intI1- strains were found to contain other typical integron sequences. That is, in no case was the sul1 gene detected independently of other Int1 sequences. Therefore, we propose that the sul1 gene would be a good marker for the presence of Int1, as well as the intI1 gene. Following this criterion, the prevalence of Int1 in our collection increased from 22% (50 intI1+) to 29% (66 intI1+ and/or sul1+). Of these 66 Int1+ strains, 63 were resistant to TMP. The main conclusion in this work is that the presence of a class 1 integron would always require a sulfamethoxazole resistant cellular context. In more general terms, these integrons appear to be closely related to resistance to antifolate compounds.

On sulfonamide resistance, sul genes, class 1 integrons and their horizontal transfer in Escherichia coli.

Class 1 integrons (Int1) contribute to antibiotic multiresistance in Gram-negative bacteria. Being frequently carried by conjugative plasmids, their spread would depend to some extent on their horizontal transfer to other bacteria. This was the main issue that was addressed in this work: the analysis of Int1 lateral transfer in the presence of different antibiotic pressures. Strains from a previously obtained collection of Escherichia coli K12 carrying natural Int1+ conjugative plasmids were employed as Int1 donors in conjugation experiments. Two recipient strains were used: an E. coli K12 and an uropathogenic E. coli isolate. The four antibiotics employed to select transconjugants in LB solid medium were ampicillin, trimethoprim, sulfamethoxazole, and co-trimoxazole. For this purpose, adequate final concentrations of the three last antibiotics had to be determined. Abundant transconjugants resulted from the mating experiments and appeared in most -but not all-selective plates. In those supplemented with sulfamethoxazole or co-trimoxazole, transconjugants grew or not depending on the genetic context of the recipient strain and on the type of gene conferring sulfonamide resistance (sul1 or sul2) carried by the Int1+ plasmid. The horizontal transfer of a recombinant plasmid bearing an Int1 was also assayed by transformation and these experiments provided further information on the viability of the Int1+ clones. Overall, results point to the existence of constraints for the lateral transfer of Int1 among E. coli bacteria, which are particularly evidenced under the antibiotic pressure of sulfamethoxazole or of its combined formula co-trimoxazole.

Horizontal transfer of class 1 integrons from uropathogenic Escherichia coli to E. coli K12.

Class 1 integrons are genetic elements that carry a variable set of antibiotic resistance genes, being frequently found in clinical Gram-negative isolates. It is generally assumed that they easily spread horizontally among bacteria, thus contributing to the appearance of multidrug resistant clones. However, there are few experimental studies on the lateral transfer of these elements performed with bacterial collections that had been gathered following an epidemiological design. In this work, a collection with these characteristics, comprising uropathogenic Escherichia coli (UPEC) isolates bearing class 1 integrons, was employed to study the horizontal transfer of the integron to an E. coli K12 strain by means of conjugation and transduction experiments. Donor and resultant strains were characterized for their antibiotic resistances, presence of sul1, sul2 and sul3 genes, integron cassette arrays, plasmid replicons and tra region. Conjugation assays were carried out using 45 UPEC isolates as integron donors and transconjugants were obtained in 18 cases (40%). P1-transduction experiments only added the integron transfer from a single donor isolate. Thus, a collection of E. coli K12 strains carrying the class 1 integron from 19 UPEC isolates was generated. In all cases, the integron was co-transferred with at least one low-copy-number plasmid, generally of the F replicon type. Several variables were searched for that could be related to the ability to horizontally transfer the integron. Although no strict correlation was observed, the phylogenetic background of the donor strain and the presence of the sul2 gene appeared as candidates to influence the process. Therefore, there appears that besides being carried by mobile genetic elements, class 1 integrons may be influenced by other factors to accomplish their horizontal transfer, a topic that requires further studies.

Analysis of RecA-independent recombination events between short direct repeats related to a genomic island and to a plasmid in Escherichia coli K12.

RecA-independent recombination events between short direct repeats, leading to deletion of the intervening sequences, were found to occur in two genetic models in the Escherichia coli K12 background. The first model was a small E. coli genomic island which had been shown to be mobile in its strain of origin and, when cloned, also in the E. coli K12 context. However, it did not encode a site-specific recombinase as mobile genomic islands usually do. It was then deduced that the host cells should provide the recombination function. This latter was searched for by means of a PCR approach to detect the island excision in E. coli K12 mutants affected in a number of recombination functions, including the 16 E. coli K12 site-specific recombinases, the RecET system, and multiple proteins that participate in the RecA-dependent pathways of homologous recombination. None of these appeared to be involved in the island excision. The second model, analyzed in a RecA deficient context, was a plasmid construction containing a short direct repeat proceeding from Saccharomyces cerevisiae, which flanked the cat gene. The excision of this gene by recombination of the DNA repeats was confirmed by PCR and through the detection, recovery and characterization of the plasmid deleted form. In sum, we present new evidence on the occurrence of RecA-independent recombination events in E. coli K12. Although the mechanism underlying these processes is still unknown, their existence suggests that RecA-independent recombination may confer mobility to other genetic elements, thus contributing to genome plasticity.

Integrons in uropathogenic Escherichia coli and their relationship with phylogeny and virulence.

Uropathogenic Escherichia coli (UPEC) comprise a heterogeneous group of strains. In a previous epidemiological survey performed on 230 UPEC isolates, five virulence profiles were described, each one defined by the presence of some virulence determinants and by the absence of others. Phylogenetic groups and antibiotic resistances distributed non-randomly among the isolates with different profiles. Based on these results, the presence of class 1 and 2 integrons was now investigated in these UPEC isolates in order to analyze the distribution of integrons among the phylogenetic groups and virulence profiles. As detected by PCR reactions targeted to the corresponding integrase genes, the class 1 integrons prevailed (22%) followed by those of class 2 (8%). Integrons distributed unevenly among the four main E. coli phylogenetic groups: class 1 integrons predominated in the isolates belonging to group D while class 2 were almost absent in this group. In relation to virulence, integrons frequently appeared in some virulence profiles and were particularly scarce in others. Concerning the class 1 integrons, the most notable findings were that they highly concentrated in isolates presenting one of the virulence profiles (profile V) and were absent in isolates bearing the K1 capsule. The analysis of the Pc promoter variants of the class 1 integrons revealed that all isolates with virulence profile V contained the same Pc version; PcH1. Findings in this work support the idea that, among UPEC strains, integrons would encounter constraints for their installation in some genetic backgrounds while other backgrounds would be propitious for their permanence.

Virulence profiles in uropathogenic Escherichia coli isolated from pregnant women and children with urinary tract abnormalities.

Uropathogenic Escherichia coli is the leading etiologic agent of urinary tract infections, encompassing a highly heterogeneous group of strains. Although many putative urovirulence factors have been described, none of them appear in all uropathogenic E. coli strains, a fact that suggests that this group would be composed of different pathogenic subgroups. In this work, a study was performed on two collections of E. coli isolates proceeding from urine cultures from two groups of patients with urinary tract infection: pregnant women and children with urinary tract abnormalities. The isolates were analyzed for their virulence content and for their phylogeny by means of PCR determinations and of phenotypic assays. Associations among the virulence traits analyzed were searched for and this approach led to the identification of five urovirulence profiles. From a total of 230 isolates, 123 (53%) could be assigned to one of these profiles. A few loci appeared as markers of these profiles so that their presence allowed predicting the general virulence content of the strains. It is presumed that these conserved associations among the virulence functions would be devoted to ensure the coherence of the bacterial pathogenic strategy. In addition, three profiles appeared with significantly different frequencies depending on the host of origin of the isolates, indicating the existence of a correlation between the virulence content of the strains and their host specificity.

Microcin H47 system: an Escherichia coli small genomic island with novel features.

Genomic islands are DNA regions containing variable genetic information related to secondary metabolism. Frequently, they have the ability to excise from and integrate into replicons through site-specific recombination. Thus, they are usually flanked by short direct repeats that act as attachment sites, and contain genes for an integrase and an excisionase which carry out the genetic exchange. These mobility events would be at the basis of the horizontal transfer of genomic islands among bacteria.Microcin H47 is a ribosomally-synthesized antibacterial peptide that belongs to the group of chromosome-encoded microcins. The 13 kb-genetic system responsible for its production resides in the chromosome of the Escherichia coli H47 strain and is flanked by extensive and imperfect direct repeats. In this work, both excision and integration of the microcin H47 system were experimentally detected. The analyses were mainly performed in E. coli K12 cells carrying the microcin system cloned in a multicopy plasmid. As expected of a site-specific recombination event, the genetic exchange also occurred in a context deficient for homologous recombination. The DNA sequence of the attachment sites resulting from excision were hybrid between the sequences of the direct repeats. Unexpectedly, different hybrid attachment sites appeared which resulted from recombination in four segments of identity between the direct repeats. Genes encoding the trans-acting proteins responsible for the site-specific recombination were shown to be absent in the microcin H47 system. Therefore, they should be provided by the remaining genetic context, not only in the H47 strain but also in E. coli K12 cells, where both excision and integration occurred. Moreover, a survey of the attachment sites in data banks revealed that they are widely spread among E. coli strains. It is concluded that the microcin system is a small island -H47 genomic island- that would employ a parasitic strategy for its mobility.

Microcins and urovirulence in Escherichia coli.

Urinary tract infections are among the most common infectious diseases encountered in humans and Escherichia coli is their leading etiologic agent. Uropathogenic E. coli encompasses a group of bacteria possessing a variable virulence gene assortment. It is generally agreed that many urovirulence factors remain to be discovered and that this information is required to gain knowledge on the pathogenic processes underlying the different clinical presentations of urinary tract infections. The production of higher-molecular-mass microcins, a group of ribosomally-synthesized peptide antibiotics comprising microcins H47, I47, E492, M and ColV, has been proposed as a virulence trait of some uropathogenic E. coli. To study this possibility, clones producing any of these microcins were selected from a collection of 160 Gram-negative clinical isolates from urine cultures and their virulence profile was analyzed. The study consisted in surveying genetic loci known to be relevant to urinary tract infection caused by E. coli. Depending on the type of microcin produced, different virulence patterns were observed which seemed to be determined by the degree of compatibility between virulence and microcin loci. In conclusion, results pointed to a relationship between higher-molecular-mass microcins and urovirulence.

Modular structure of microcin H47 and colicin V.

Microcins are gene-encoded peptide antibiotics produced by enterobacteria that act on strains of gram-negative bacteria. In this work, we concentrated on higher-molecular-mass microcins, i.e., those possessing 60 or more amino acids. They can be subdivided into unmodified and posttranslationally modified peptides. In both cases, they exhibit conserved C-terminal sequences that appear to be characteristic of each subgroup. In the hypothesis that these sequences could correspond to domains, gene fusions between the activity genes for the unmodified microcin colicin V and the modified microcin H47 were constructed. These two microcins differ in their mode of synthesis, uptake, target, and specific immunity. Through this experimental approach, chimeric peptides with exchanged C-terminal sequences were encoded. Cells carrying the fusions in different genetic contexts were then assayed for antibiotic production. Many of them produced antibiotic activities with recombinant properties: the toxicity of one microcin and the mode of uptake of the other. The results led to the identification of a modular structure of colicin V and microcin H47, with the recognition of two domains in their peptide chains: a toxic N-terminal domain and an uptake C-terminal domain. This modular design would be shared by other microcins from each subgroup.

Comparative analysis of chromosome-encoded microcins.

Microcins are ribosomally synthesized peptide antibiotics that are produced by enterobacterial strains. Although the first studies concentrated on plasmid-encoded activities, in the last years three chromosome-encoded microcins have been described: H47, E492, and M. Here, a new microcin, I47, is presented as a fourth member of this group. Common features exhibited by chromosome-encoded microcins were searched for. The comparison of the genetic clusters responsible for microcin production revealed a preserved general scheme. The clusters essentially comprise a pair of activity-immunity genes which determine antibiotic specificity and a set of microcin maturation and secretion genes which are invariably present and whose protein products are highly homologous among the different producing strains. A strict functional relationship between the maturation and secretion pathways of microcins H47, I47, and E492 was demonstrated through genetic analyses, which included heterologous complementation assays. The peptide precursors of these microcins share a maturation process which implies the addition of a catecholate siderophore of the salmochelin type. Microcins thus acquire the ability to enter gram-negative cells through the catechol receptors. In addition, they employ a common mode of secretion to reach the external milieu by means of a type I export apparatus. The results presented herein lead us to propose that chromosome-encoded microcins constitute a defined subgroup of peptide antibiotics which are strictly related by their modes of synthesis, secretion, and uptake.

Involvement of enterobactin synthesis pathway in production of microcin H47.

Microcin H47 (MccH47) is a gene-encoded peptide antibiotic produced by an Escherichia coli clinical isolate which is active on strains of gram-negative bacteria. Its uptake by E. coli K-12-susceptible cells depends on the presence of any of the outer membrane proteins Cir, Fiu, and FepA, the three catechol receptors of this organism. The nucleotide sequence of a portion of the MccH47 genetic system that had not yet been studied was elucidated. Five open reading frames were identified, three of which corresponded to genes encoding functions related to catechol-type siderophores: mchA and mchS1 are iroB and iroD homologues, respectively, and mchS4 was found to promote the production of the catecholate siderophore enterobactin. The possible relationship between enterobactin synthesis and MccH47 production was studied. Enterobactin-deficient strains failed to produce MccH47 when transformed with the antibiotic genetic determinants and upon introduction of the ent genetic cluster, the production of both the siderophore and MccH47 was restored. Further studies demonstrated that at least the enterobactin nonribosomal peptide synthase EntF is necessary for MccH47 synthesis. The relationship found between MccH47 and catecholate siderophore production is discussed, and a model outlining MccH47 synthesis is proposed.

The proton channel is the minimal structure of ATP synthase necessary and sufficient for microcin h47 antibiotic action.

It had been previously determined that the presence of F(o)F(1) ATP synthase was required for microcin H47 antibiotic action. In this work, microcin-resistant atp mutants were genetically analyzed. Their mutations, originated by Tn5 insertion, in all cases were found to affect determinants for the F(o) portion of ATP synthase. To discern if microcin action required the presence of the entire complex or if the F(o) proton channel would suffice, recombinant plasmids carrying different segments of the atp operon were constructed and introduced into an atp deletion strain. The phenotypic analysis of the strains thus obtained clearly indicated that the presence of the F(o) proton channel was absolutely required for microcin H47 action, while the F(1) catalytic portion was found to be dispensable. Furthermore, when any of the three components of the proton channel was missing, total resistance to the antibiotic ensued. Complementation analysis between atp::Tn5 chromosomal mutations and recombinant atp plasmid constructions further supported the idea that the proton channel would be the minimal structure of the ATP synthase complex needed for microcin H47 antibiotic action.