Common origin and fixation of Plasmodium falciparum dhfr and dhps mutations associated with sulfadoxine-pyrimethamine resistance in a low-transmission area in South America

Recent studies indicated that sensitive parasites could increase in frequency in a population when drugs are removed, suggesting that the life span of affordable antimalarial drugs could be expanded. We studied 97 samples from Bolivar State, Venezuela, an area where sulfadoxine-pyrimethamine (SP) has not been used for 8 years due to its ineffectiveness. We characterized point mutations in two genes that have been implicated in resistance to SP, dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps). We also assayed neutral microsatellite markers around the dhfr (chromosome 4) and dhps (chromosome 8) genes and on chromosomes 2 and 3 to track the origin and spread of resistant alleles. We found that drug-resistant SP mutants are fixed in the population. Two genotypes were present in the samples, dhfr(50R/51I/108N) dhps(437G/540E/581G) (90.7%) and dhfr(51I/108N) dhps(437G/581G) (9.3%). We show a single microsatellite haplotype for all of the dhfr and dhps alleles, and the alleles at the microsatellite loci are different from those present in Africa. Thus, in these samples from Venezuela, there is a single origin for both dhfr and dhps SP-resistant alleles, and these alleles originated independently of those characterized from Africa. Furthermore, this is the first report of a "hitchhiking effect" on the genetic variation around dhps due to selection by SP using an extensive set of microsatellite markers. Our results indicate that, in areas where there is limited gene flow, the fixation of drug-resistant parasites in the population is stable, even after drug selection is relaxed.     

Multiple origins of Plasmodium falciparum dihydropteroate synthetase mutant alleles associated with sulfadoxine resistance in India

With the spread of chloroquine (CQ)-resistant malaria in India, sulfadoxine-pyrimethamine (SP) alone or in combination with artesunate is used as an alternative antimalarial drug. Due to continuous drug pressure, the Plasmodium falciparum parasite is exhibiting resistance to antifolates because of mutations in candidate genes dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps). Our earlier study on flanking microsatellite markers of dhfr mutant alleles from India had shown a single origin of the pyrimethamine resistance and some minor haplotypes which shared haplotypes with Southeast Asian (Thailand) strains. In the present study, we have analyzed 193 of these Indian P. falciparum isolates for 15 microsatellite loci around dhps to investigate the genetic lineages of the mutant dhps alleles in different parts of the country. Eighty-one of these samples had mutant dhps alleles, of which 62 were from Andaman and Nicobar Islands and the remaining 19 were from mainland India. Of 112 isolates with a wild-type dhps allele, 109 were from mainland India and only 3 were from Andaman and Nicobar Islands. Consistent with the model of selection, the mean expected heterozygosity (H(e)) around mutant dhps alleles (H(e) = 0.55; n = 81) associated with sulfadoxine resistance was lower (P ≤ 0.05) than the mean H(e) around the wild-type dhps allele (H(e) = 0.80; n = 112). There was more genetic diversity in flanking microsatellites of dhps than dhfr among these isolates, which confirms the assertion that dhps mutations are at a very early stage of fixation in the parasite population. Microsatellite haplotypes around various mutant dhps alleles suggest that the resistant dhps alleles have multiple independent origins in India, especially in Andaman and Nicobar Islands. Determining the genetic lineages of the resistant dhps alleles on Andaman and Nicobar Islands and mainland India is significant, given the role of Asia in the intercontinental spread of chloroquine- and pyrimethamine-resistant parasites in the past.     

Novel Plasmodium vivax dhfr alleles from the Indonesian Archipelago and Papua New Guinea: association with pyrimethamine resistance determined by a Saccharomyces cerevisiae expression system

In plasmodia, the dihydrofolate reductase (DHFR) enzyme is the target of the pyrimethamine component of sulfadoxine-pyrimethamine (S/P). Plasmodium vivax infections are not treated intentionally with antifolates. However, outside Africa, coinfections with Plasmodium falciparum and P. vivax are common, and P. vivax infections are often exposed to S/P. Cloning of the P. vivax dhfr gene has allowed molecular comparisons of dhfr alleles from different regions. Examination of the dhfr locus from a few locations has identified a very diverse set of alleles and showed that mutant alleles of the vivax dhfr gene are prevalent in Southeast Asia where S/P has been used extensively. We have surveyed patient isolates from six locations in Indonesia and two locations in Papua New Guinea. We sequenced P. vivax dhfr alleles from 114 patient samples and identified 24 different alleles that differed from the wild type by synonymous and nonsynonymous point mutations, insertions, or deletions. Most importantly, five alleles that carried four or more nonsynonymous mutations were identified. Only one of these highly mutant alleles had been previously observed, and all carried the 57L and 117T mutations. P. vivax cannot be cultured continuously, so we used a yeast assay system to determine in vitro sensitivity to pyrimethamine for a subset of the alleles. Alleles with four nonsynonymous mutations conferred very high levels of resistance to pyrimethamine. This study expands significantly the total number of novel dhfr alleles now identified from P. vivax and provides a foundation for understanding how antifolate resistance arises and spreads in natural P. vivax populations.     

In vitro generation of novel pyrimethamine resistance mutations in the Toxoplasma gondii dihydrofolate reductase

Pyrimethamine is a potent inhibitor of dihydrofolate reductase and is widely used in the treatment of opportunistic infections caused by the protozoan parasite Toxoplasma gondii. In order to assess the potential role of dhfr sequence polymorphisms in drug treatment failures, we examined the dhfr-ts genes of representative isolates for T. gondii virulence types I, II, and III. These strains exhibit differences in their sensitivities to pyrimethamine but no differences in predicted dhfr-ts protein sequences. To assess the potential for pyrimethamine-resistant dhfr mutants to emerge, three drug-sensitive variants of the T. gondii dhfr-ts gene (the wild-type T. gondii sequence and two mutants engineered to reflect polymorphisms observed in drug-sensitive Plasmodium falciparum) were subjected to random mutagenesis and transfected into either wild-type T. gondii parasites or dhfr-deficient Saccharomyces cerevisiae under pyrimethamine selection. Three resistance mutations were identified, at amino acid residues 25 (Trp-->Arg), 98 (Leu-->Ser), and 134 (Leu-->His).     

Distinctive Origin and Spread Route of Pyrimethamine-Resistant Plasmodium falciparum in Southern China

Southeast Asia (the Thailand-Cambodia border) has been considered the primal epicenter for most antimalarial drug resistance; however, numerous molecular epidemiological studies have successively reported multiple independent origins of sulfadoxine-pyrimethamine (SP) resistance-associated Plasmodium falciparum dhfr (pfdhfr) and pfdhps alleles in other areas. To better understand the origin and evolutionary pathway of the SP resistance in Southeast Asia, a total of 374 P. falciparum field isolates from the Yunnan-Burma border and Hainan Island in southern China have been collected for comprehensive investigations on the mutation patterns of the pfdhfr/pfdhps genes as well as their microsatellite haplotypes. By comparative analysis of single-nucleotide polymorphism (SNP) genotyping and flanking microsatellite haplotypes, we reveal a unique origin of pyrimethamine-resistant mutations in Pfdhfr gene in Hainan Island and an oriented spread route of the pyrimethamine resistance from the Thailand-Cambodia border into the Hainan area, which reflects the geographical traits and SP administration histories in the two geographically independent areas. Moreover, genetic linkages between the high-level SP resistance-conferring pfdhfr/pfdhps alleles have been established in the isolates from the Yunnan-Burma border, raising the concern of a genetic basis in adopting combination chemotherapies against falciparum malaria.     

Novel point mutations in the dihydrofolate reductase gene of Plasmodium vivax: evidence for sequential selection by drug pressure

Mutations in the dihydrofolate reductase (dhfr) genes of Plasmodium falciparum and P. vivax are associated with resistance to the antifolate antimalarial drugs. P. vivax dhfr sequences were obtained from 55 P. vivax isolates (isolates Belem and Sal 1, which are established lines originating from Latin America, and isolates from patient samples from Thailand [n = 44], India [n = 5], Iran [n = 2], and Madagascar [n = 2]) by direct sequencing of both strands of the purified PCR product and were compared to the P. vivax dhfr sequence from a P. vivax parasite isolated in Pakistan (isolate ARI/Pakistan), considered to represent the wild-type sequence. In total, 144 P. vivax dhfr mutations were found at only 12 positions, of which 4 have not been described previously. An F-->L mutation at residue 57 had been observed previously, but a novel codon (TTA) resulted in a mutation in seven of the nine mutated variant sequences. A new mutation at residue 117 resulted in S-->T (S-->N has been described previously). These two variants are the same as those observed in the P. falciparum dhfr gene at residue 108, where they are associated with different levels of antifolate resistance. Two novel mutations, I-->L at residue 13 and T-->M at residue 61, appear to be unique to P. vivax. The clinical, epidemiological, and sequence data suggest a sequential pathway for the acquisition of the P. vivax dhfr mutations. Mutations at residues 117 and 58 arise first when drug pressure is applied. Highly mutated genes carry the S-->T rather than the S-->N mutation at residue 117. Mutations at residues 57 and 61 then occur, followed by a fifth mutation at residue 13.     

Genetic analysis of the dihydrofolate reductase-thymidylate synthase gene from geographically diverse isolates of Plasmodium malariae

Plasmodium malariae, the parasite responsible for quartan malaria, is transmitted in most areas of malaria endemicity and is associated with significant morbidity. The sequence of the gene coding for the enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS) was obtained from field isolates of P. malariae and from the closely related simian parasite Plasmodium brasilianum. The two sequences were nearly 100% homologous, adding weight to the notion that they represent genetically distinct lines of the same species. A survey of polymorphisms of the dhfr sequences in 35 isolates of P. malariae collected from five countries in Asia and Africa revealed a low number of nonsynonymous mutations in five codons. In five of the isolates collected from southeast Asia, a nonsynonymous mutation was found at one of the three positions known to be associated with antifolate resistance in other Plasmodium species. Five isolates with the wild-type DHFR could be assayed for drug susceptibility in vitro and were found to be sensitive to pyrimethamine (mean 50% inhibitory concentration, 2.24 ng/ml [95% confidence interval, 0.4 to 3.1]).     

Use of a semi-nested PCR diagnosis test to evaluate antifolate resistance ofPlasmodium falciparumisolates

The antifols, inhibitors of the dihydrofolate reductase (DHFR), such as pyrimethamine and proguanil, have been used againstPlasmodium falciparumin the areas where chloroquine resistance is widespread. This use has selected resistant strains in Southeast Asia and South America. The resistance is correlated with point mutations in precise positions of the DHFR gene. A reliable semi-nested PCR diagnosis test was established and used to determine the genotypic features of 29 isolates ofP. falciparumoriginating from Africa. The results obtained by the PCR technique were compared with those of thein vitrodrug sensitivity test. Some isolates were found to be polyclonal. Among the mutations tested, only mutations on codon 108 which generate an asparagine induce a decrease in sensitivity to pyrimethamine and cycloguanil, whereas mutation on codon 59 strengthens resistance to both antifols. No mutation on codon 16 or codon 164 was found.     

Plasmodium falciparum isolates in India exhibit a progressive increase in mutations associated with sulfadoxine-pyrimethamine resistance

The combination of sulfadoxine-pyrimethamine (SP) is used as a second line of therapy for the treatment of uncomplicated chloroquine-resistant Plasmodium falciparum malaria. Resistance to SP arises due to certain point mutations in the genes for the dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) enzymes of the parasite. We have analyzed these mutations in 312 field isolates of P. falciparum collected from different parts of India to assess the effects of drug pressure. The rate of mutation in the gene for DHFR was found to be higher than that in the gene for DHPS, although the latter had mutations in more alleles. There was a temporal rise in the number of isolates with double dhfr mutations and single dhps mutations, resulting in an increased total number of mutations in the loci for DHFR and DHPS combined over a 5-year period. During these 5 years, the number of isolates with drug-sensitive genotypes decreased and the number of isolates with drug-resistant genotypes (double DHFR mutations and a single DHPS mutation) increased significantly. The number of isolates with the triple mutations in each of the genes for the two enzymes (for a total of six mutations), however, remained very low, coinciding with the very low rate of SP treatment failure in the country. There was a regional bias in the mutation rate, as isolates from the northeastern region (the state of Assam) showed higher rates of mutation and more complex genotypes than isolates from the other regions. It was concluded that even though SP is prescribed as a second line of treatment in India, the mutations associated with SP resistance continue to be progressively increasing.     

Characterization of class 1 integrons associated with R-plasmids in clinical Aeromonas salmonicida isolates from various geographical areas

Class 1 integrons were found in 26 of 40 antibiotic-resistant isolates of the fish pathogen Aeromonas salmonicida from Northern Europe and North America. Three different dhfr genes, conferring trimethoprim resistance, and one ant(3")1a aminoglycoside resistance gene were identified as gene inserts. The gene cassettes tended to be conserved among isolates from a particular geographical area. Nineteen isolates transferred R-plasmids carrying different tet determinants to Escherichia coli in filter mating assays, and in 15 cases, the class 1 integrons were co-transferred. Transferable sulphadiazine, trimethoprim and streptomycin resistances were invariably encoded by integrons. It thus appears that integron-encoded antibiotic resistance genes contribute substantially to the horizontal spread of antimicrobial resistance within this species, being associated with conjugative plasmids.     

Sulfadoxine-pyrimethamine resistance in the rodent malaria parasite Plasmodium chabaudi

We have studied resistance to sulfadoxine-pyrimethamine (S/P) in the rodent malaria parasite Plasmodium chabaudi. A stable S/P-resistant mutant, AS(50S/P), was selected by drug treatment of a clone, AS(PYR), already resistant to pyrimethamine. The sequences of the P. chabaudi dhfr and dhps genes were obtained and found to be identical in AS(50S/P) and AS(PYR), showing that resistance to S/P in AS(50S/P) was not due to additional mutations in either gene. AS(50S/P) was crossed with a drug-sensitive clone, AJ, and 16 independent recombinant progeny were obtained. These clones were phenotyped for their susceptibility to S/P and to sulfadoxine and pyrimethamine separately. Pyrimethamine resistance was invariably associated with S/P resistance, but no correlation was found between resistance to S/P and resistance to sulfadoxine. Quantitative trait locus analysis of the progeny with 31 chromosome-specific markers showed that mutant P. chabaudi dhfr, or one or more genes closely linked to it, was a major determinant of S/P resistance. In addition, the inheritance of genes on chromosomes 5 and 13 from the sensitive parent appeared to contribute to the level of resistance observed. These results demonstrate that the S/P resistance of the AS(50S/P) mutant of P. chabaudi does not involve mutation in dhps and is not due simply to a combination of two genes determining resistance to pyrimethamine and sulfadoxine separately.     

Association of genetic mutations in Plasmodium vivax dhfr with resistance to sulfadoxine-pyrimethamine: geographical and clinical correlates

Mutations in the Plasmodium falciparum gene (dhfr) encoding dihydrofolate reductase are associated with resistance to antifols. Plasmodium vivax, the more prevalent malaria parasite in Asia and the Americas, is considered antifol resistant. Functional polymorphisms in the dhfr gene of P. vivax (pvdhfr) were assessed by PCR-restriction fragment length polymorphism using blood samples taken from 125 patients with acute vivax malaria from three widely separated locations, Thailand (n = 100), India (n = 16), and Madagascar and the Comoros Islands (n = 9). Upon evaluation of the three important codons (encoding residues 57, 58, and 117) of P. vivax dhfr (pvdhfr), double- or triple-mutation genotypes were found in all but one case from Thailand (99%), in only three cases from India (19%) and in no cases from Madagascar or the Comoros Islands (P < 0.0001). The dhfr PCR products of P. vivax from 32 Thai patients treated with the antifolate sulfadoxine-pyrimethamine (S-P) were investigated. All samples showed either double (53%) or triple (47%) mutations. Following treatment, 34% of the patients had early treatment failures and only 10 (31%) of the patients cleared their parasitemias for 28 days. There were no significant differences in cure rates, but parasite reduction ratios at 48 h were significantly lower for patients whose samples showed triple mutations than for those whose samples showed double mutations (P = 0.01). The three mutations at the pvdhfr codons for residues 57, 58, and 117 are associated with high levels of S-P resistance in P. vivax. These mutations presumably arose from selection pressure.     

Hitchhiking and selective sweeps of Plasmodium falciparum sulfadoxine and pyrimethamine resistance alleles in a population from central Africa

Sulfadoxine-pyrimethamine (SP) resistance in Plasmodium falciparum is encoded by a number of mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes. Here, we have characterized point mutations in dhfr and dhps and microsatellite loci around dhfr on chromosome 4 and dhps on chromosome 8 as well as neutral markers on chromosomes 2 and 3 in 332 samples from Yaoundé, Cameroon. The triple mutant dhfr haplotype that originated in Southeast Asia is the most predominant in this sample set, but we also find additional independent haplotypes at low frequency and an incipient process of genetic differentiation among alleles of Southeast Asian origin. As reported for other African populations, we find evidence of a selective sweep for resistant dhfr mutants in this Cameroonian population due to drug selection. Although we find evidence for a selective sweep in dhps mutants associated with SP resistance, the dynamics of dhps mutants appear different than those observed for dhfr mutants. Overall, our results yield support for the use of microsatellite markers to track resistant parasites; however, the detection of resistant dhfr alleles in low frequency, the evidence of divergence among dhfr alleles that share a common evolutionary origin, and the distinct dynamics of resistant dhps alleles emphasize the importance of comprehensive, population-based investigations to evaluate the effects of drug selection on parasite populations.     

Characterization of streptomycin resistance mechanisms among Mycobacterium tuberculosis isolates from patients in New York City.

From a collection of 367 isolates of Mycobacterium tuberculosis from patients in New York City in 1994, 45 isolates (12.3%) were resistant in vitro to 2 micrograms or more of streptomycin (SM) per ml. We further evaluated these isolates for levels of SM resistance and for mutations previously associated with resistance in the rpsL (S12 ribosomal protein) gene and the rrs (16S rRNA)-coding region. Twenty-four isolates, representing nine distinct patterns of susceptibility to antituberculosis drugs, were resistant to 500 micrograms of SM per ml and shared a common point mutation at nucleotide 128 in the rpsL gene. This mutation, which substitutes lysine for arginine in the S12 ribosomal binding protein, was not present in isolates with low-level SM resistance or in SM-susceptible control isolates. Among 20 isolates with low-level SM resistance, one possessed a substitution (C-->G865) in the 912 loop of the rrs gene. No mutations in the 530 loop of the rrs coding region were detected, suggesting the presence of an alternative SM resistance mechanism in 19 isolates. Single-strand conformation polymorphisms of mutants were readily detected by a nonradioactive gel screen.     

Quinolone resistance mutations in topoisomerase IV: relationship to the flqA locus and genetic evidence that topoisomerase IV is the primary target and DNA gyrase is the secondary target of fluoroquinolones in Staphylococcus aureus.

Mutations in the flqA (formerly ofx/cfx) resistance locus of Staphylococcus aureus were previously shown to be common after first-step selections for resistance to ciprofloxacin and ofloxacin and to map on the S. aureus chromosome distinctly from gyrA, gyrB, and norA.grlA and grlB, the genes for the topoisomerase IV of S. aureus, were identified from a genomic lambda library on a common KpnI fragment, and grlB hybridized specifically with the chromosomal SmaI A fragment, which contains the flqA locus. Amplification of grlA sequences (codons 1 to 251) by PCRs from nine independent single-step flqA mutants, one multistep mutant, and the parent strain identified mutations encoding a change from Ser to Phe at position 80 in four mutants, a novel change from Ala to either Glu or Pro at position 116 in three mutants, and no change in three mutants. In the multistep mutant, another resistance locus, flqC, was mapped by transformation to the chromosomal SmaI G fragment by linkage to omega(ch::Tn551)1051 (58%) and nov (97.9%), which encodes resistance to novobiocin. This fragment contains the gyrA gene, and flqC mutants had a mutation in gyrA encoding a change from Ser to Leu at position 84, a change previously found in resistant clinical isolates. In genetic outcrosses, the flqC (gyrA) mutation expressed resistance only in flqA mutants, including those with both types of grla mutations. The silent mutant allele of gyrA was present in a flqA background and expressed resistance only upon introduction of a grlA mutation. At fourfold the MIC of ciprofloxacin, the bactericidal activity of ciprofloxacin was reduced in a grlA mutant and was abolished in gyrA grlA double mutants. These findings provide direct genetic evidence that topoisomerase IV is the primary target of current fluoroquinolones in S. aureus and that this effect may result from the greater sensitivity of topoisomerase IV relative to that of DNA gyrase to these agents. Furthermore, resistance from an altered DNA gyrase requires resistant topoisomerase IV for its expression.     

Reduced impact of pyrimethamine drug pressure on Plasmodium malariae dihydrofolate reductase gene

Molecular investigations performed following the emergence of sulfadoxine-pyrimethamine (SP) resistance in Plasmodium falciparum have allowed the identification of the dihydrofolate reductase (DHFR) enzyme as the target of pyrimethamine. Although clinical cases of Plasmodium malariae are not usually treated with antifolate therapy, incorrect diagnosis and the high frequency of undetected mixed infections has probably exposed non-P. falciparum parasites to antifolate therapy in many areas. In this context, we aimed to assess the worldwide genetic diversity of the P. malariae dhfr gene in 123 samples collected in Africa and Asia, areas with different histories of SP use. Among the 10 polymorphic sites found, we have observed 7 new mutations (K55E, S58R, S59A, F168S, N194S, D207G, and T221A), which led us to describe 6 new DHFR proteins. All isolates from African countries were classified as wild type, while new mutations and haplotypes were recognized as exclusive to Madagascar (except for the double mutations at nucleotides 341 and 342 [S114N] found in one Cambodian isolate). Among these nonsynonymous mutations, two were likely related to pyrimethamine resistance: S58R (corresponding to C59R in P. falciparum and S58R in Plasmodium vivax; observed in one Malagasy sample) and S114N (corresponding to S108N in P. falciparum and S117N in P. vivax; observed in three Cambodian samples).     

Molecular epidemiology and variants of the multidrug-resistant Streptococcus pneumoniae Spain14-5 international clone among Spanish clinical isolates

OBJECTIVE: To analyse the molecular structure of several antimicrobial resistance determinants in isolates of the Spain(14)-5 clone to better understand its emergence and spread. METHODS: The distinct genes and mechanism of resistance to penicillin, erythromycin, clindamycin, tetracycline, chloramphenicol and trimethoprim were studied in an apparently homogeneous group of 117 isolates of the multidrug-resistant Spain(14)-5 major clone isolated in Spain between 1981 and 2004. RESULTS: Several genotyping techniques such as PFGE, BOX-PCR and multilocus sequence typing (MLST) revealed a high degree of homogeneity among these isolates over time. Nevertheless, distinct variants of the clone could be established according to the restriction fragment length polymorphism (RFLP) patterns of the penicillin-binding protein (pbp) genes and the sequences of the dihydrofolate reductase (dhfr) gene. In addition, an association between the pbp2b RFLP patterns, the ddl alleles identified by MLST and the dhfr alleles was found. The emergence of variants of the Spain(14)-5 clone, which had lost macrolide and tetracycline resistance, while harbouring the ins and xis genes of the Tn916-Tn1545 family of conjugative transposons, was documented. Two different tet(M) alleles were detected in isolates of the clone, one of them with a mosaic structure. CONCLUSIONS: The finding of different patterns or alleles of the genes responsible for antibiotic resistance among isolates of the Spain(14)-5 clone from different Spanish cities indicates different evolutionary events within isolates of a unique Streptococcus pneumoniae clone.     

Characteristic resistance mechanism of Mycobacterium tuberculosis to DC-159a, a new respiratory quinolone

A G88C mutation in GyrA is one of the key alterations by which Mycobacterium tuberculosis mutants acquire DC-159a resistance in vitro. A novel double mutation in GyrA, G88C D94H, conferred high DC-159a resistance. Different mutation patterns in GyrA were demonstrated for DC-159a-resistant mutants and quinolone-resistant multidrug-resistant (QR-MDR) M. tuberculosis isolates, with a mutation either at position 90 or 94 and double mutations at 90 and 91 or at 90 and 94. DC-159a might be promising for QR M. tuberculosis treatment.     

Amdinocillin (Mecillinam) Resistance Mutations in Clinical Isolates and Laboratory-Selected Mutants of Escherichia coli

Amdinocillin (mecillinam) is a β-lactam antibiotic that is used mainly for the treatment of uncomplicated urinary tract infections. The objectives of this study were to identify mutations that confer amdinocillin resistance on laboratory-isolated mutants and clinical isolates of Escherichia coli and to determine why amdinocillin resistance remains rare clinically even though resistance is easily selected in the laboratory. Under laboratory selection, frequencies of mutation to amdinocillin resistance varied from 8 × 10⁻⁸ to 2 × 10⁻⁵ per cell, depending on the concentration of amdinocillin used during selection. Several genes have been demonstrated to give amdinocillin resistance, but here eight novel genes previously unknown to be involved in amdinocillin resistance were identified. These genes encode functions involved in the respiratory chain, the ribosome, cysteine biosynthesis, tRNA synthesis, and pyrophosphate metabolism. The clinical isolates exhibited significantly greater fitness than the laboratory-isolated mutants and a different mutation spectrum. The cysB gene was mutated (inactivated) in all of the clinical isolates, in contrast to the laboratory-isolated mutants, where mainly other types of more costly mutations were found. Our results suggest that the frequency of mutation to amdinocillin resistance is high because of the large mutational target (at least 38 genes). However, the majority of these resistant mutants have a low growth rate, reducing the probability that they are stably maintained in the bladder. Inactivation of the cysB gene and a resulting loss of cysteine biosynthesis are the major mechanism of amdinocillin resistance in clinical isolates of E. coli.     

Early Strains of Multidrug-Resistant Salmonella enterica Serovar Kentucky Sequence Type 198 from Southeast Asia Harbor Salmonella Genomic Island 1-J Variants with a Novel Insertion Sequence

Salmonella genomic island 1 (SGI1) is a 43-kb integrative mobilizable element that harbors a great diversity of multidrug resistance gene clusters described in numerous Salmonella enterica serovars and also in Proteus mirabilis. The majority of SGI1 variants contain an In104-derivative complex class 1 integron inserted between resolvase gene res and open reading frame (ORF) S044 in SGI1. Recently, the international spread of ciprofloxacin-resistant S. enterica serovar Kentucky sequence type 198 (ST198) containing SGI1-K variants has been reported. A retrospective study was undertaken to characterize ST198 S. Kentucky strains isolated before the spread of the epidemic ST198-SGI1-K population in Africa and the Middle East. Here, we characterized 12 ST198 S. Kentucky strains isolated between 1969 and 1999, mainly from humans returning from Southeast Asia (n = 10 strains) or Israel (n = 1 strain) or from meat in Egypt (n = 1 strain). All these ST198 S. Kentucky strains did not belong to the XbaI pulsotype X1 associated with the African epidemic clone but to pulsotype X2. SGI1-J subgroup variants containing different complex integrons with a partial transposition module and inserted within ORF S023 of SGI1 were detected in six strains. The SGI1-J4 variant containing a partially deleted class 1 integron and thus showing a narrow resistance phenotype to sulfonamides was identified in two epidemiologically unrelated strains from Indonesia. The four remaining strains harbored a novel SGI1-J variant, named SGI1-J6, which contained aadA2, floR2, tetR(G)-tetA(G), and sul1 resistance genes within its complex integron. Moreover, in all these S. Kentucky isolates, a novel insertion sequence related to the IS630 family and named ISSen5 was found inserted upstream of the SGI1 complex integron in ORF S023. Thus, two subpopulations of S. Kentucky ST198 independently and exclusively acquired the SGI1 during the 1980s and 1990s. Unlike the ST198-X1 African epidemic subpopulation, the ST198-X2 subpopulation mainly from Asia harbors variants of the SGI1-J subgroup that are encountered mainly in the Far East, as previously described for S. enterica serovars Emek and Virchow.