Jesús Blázquez

Mutations in Putative Mutator Genes of Mycobacterium tuberculosis Strains of the W-Beijing Family

Alterations in genes involved in the repair of DNA mutations (mut genes) result in an increased mutation frequency and better adaptability of the bacterium to stressful conditions. W-Beijing genotype strains displayed unique missense alterations in three putative mut genes, including two of the mutT type (Rv3908 and mutT2) and ogt. These polymorphisms were found to be characteristic and unique to W-Beijing phylogenetic lineage. Analysis of the mut genes in 55 representative W-Beijing isolates suggests a sequential acquisition of the mutations, elucidating a plausible pathway of the molecular evolution of this clonal family. The acquisition of mut genes may explain in part the ability of the isolates of W-Beijing type to rapidly adapt to their environment.

The mismatch repair system (mutS, mutL and uvrD genes) in Pseudomonas aeruginosa: molecular characterization of naturally occurring mutants

We have recently described the presence of a high proportion of Pseudomonas aeruginosa isolates (20%) with an increased mutation frequency (mutators) in the lungs of cystic fibrosis (CF) patients. In four out of 11 independent P. aeruginosa strains, the high mutation frequency was found to be complemented with the wild‐type mutS gene from P. aeruginosa PAO1. Here, we report the cloning and sequencing of two additional P. aeruginosa mismatch repair genes and the characterization, by complementation of deficient strains, of these two putative P. aeruginosa mismatch repair genes (mutL and uvrD). We also describe the alterations in the mutS, mutL and uvrD genes responsible for the mutator phenotype of hypermutable P. aeruginosa strains isolated from CF patients. Seven out of the 11 mutator strains were found to be defective in the MMR system (four mutS, two mutL and one uvrD). In four cases (three mutS and one mutL), the genes contained frameshift mutations. The fourth mutS strain showed a 3.3 kb insertion after the 10th nucleotide of the mutS gene, and a 54 nucleotide deletion between two eight nucleotide direct repeats. This deletion, involving domain II of MutS, was found to be the main one responsible for mutS inactivation. The second mutL strain presented a K310M mutation, equivalent to K307 in Escherichia coli MutL, a residue known to be essential for its ATPase activity. Finally, the uvrD strain had three amino acid substitutions within the conserved ATP binding site of the deduced UvrD polypeptide, showing defective mismatch repair activity. Interestingly, cells carrying this mutant allele exhibited a fully active UvrABC‐mediated excision repair. The results shown here indicate that the putative P. aeruginosa mutS, mutL and uvrD genes are mutator genes and that their alteration results in a mutator phenotype.

Antibiotic-Selective Environments

The evolution and spread of antibiotic resistance depends on the antibiotic pressure exerted in the microbial environment. Selective effects occur in selective compartments, where particular antibiotic concentrations result in a differential growth rate of resistant bacterial variants. This may happen even at very low antibiotic concentrations able to select low-level-resistant bacteria. When more than one antibiotic is present in the environment, the multiple and fluctuating pressure produces the selection of bacterial variants that use multiple or multipurpose mechanisms or optimize a single mechanism of resistance to survive under the variable environmental conditions. Host factors such as immunity contribute to the selective process. Antibiotics themselves may promote bacterial diversity, either mediated by the random drift effect or triggering the increase of mutational events under bacterial stress. Analysis of selective environment-related antibiotic-host-bacteria interactions is essential to understanding the biology of antibiotic resistance.

β-Lactam Resistance Response Triggered by Inactivation of a Nonessential Penicillin-Binding Protein

It has long been recognized that the modification of penicillin-binding proteins (PBPs) to reduce their affinity for β-lactams is an important mechanism (target modification) by which Gram-positive cocci acquire antibiotic resistance. Among Gram-negative rods (GNR), however, this mechanism has been considered unusual, and restricted to clinically irrelevant laboratory mutants for most species. Using as a model Pseudomonas aeruginosa, high up on the list of pathogens causing life-threatening infections in hospitalized patients worldwide, we show that PBPs may also play a major role in β-lactam resistance in GNR, but through a totally distinct mechanism. Through a detailed genetic investigation, including whole-genome analysis approaches, we demonstrate that high-level (clinical) β-lactam resistance in vitro, in vivo, and in the clinical setting is driven by the inactivation of the dacB-encoded nonessential PBP4, which behaves as a trap target for β-lactams. The inactivation of this PBP is shown to determine a highly efficient and complex β-lactam resistance response, triggering overproduction of the chromosomal β-lactamase AmpC and the specific activation of the CreBC (BlrAB) two-component regulator, which in turn plays a major role in resistance. These findings are a major step forward in our understanding of β-lactam resistance biology, and, more importantly, they open up new perspectives on potential antibiotic targets for the treatment of infectious diseases.

Evolution of antibiotic resistance

Abstract World-wide spread of bacterial resistance to antimicrobial agents may limit the future progress of medicine. A huge environmental antibiotic pressure, resulting from industrial production and marketing of these drugs, has simultaneously contributed to the increase in the diversity of resistant phenotypes, to the selection of the fittest among them, and to the dispersal of resistance genes, which is expected to result in a significant acceleration of the rate of microbial evolution. Current research is focused on the mechanisms involved in the genesis, selection and dispersal of resistance genetic determinants; strategies based on molecular epidemiology and mathematical models may contribute to control or reverse the frightening trend towards a new pre-antibiotic era.

Mycobacterium tuberculosis subsp. caprae subsp. nov.: A taxonomic study of a new member of the Mycobacterium tuberculosis complex isolated from goats in Spain

Isolates from the Mycobacterium tuberculosis complex cultured from caprine pathological tissue samples were biochemically and genetically characterized. The isolates were negative for nitrate reduction and niacin accumulation, they weakly hydrolysed Tween 80, were sensitive to pyrazinamide (50 micrograms ml-1) and were resistant to 1 and 2 micrograms tiophene-2-carboxylic acid hydrazide ml-1 but not to 5 or 10 micrograms tiophene-2-carboxylic acid hydrazide ml-1. Sequencing of the pncA gene revealed a polymorphism characteristic of M. tuberculosis, whereas oxyR, katG and gyrA sequences were characteristic of Mycobacterium bovis. The fingerprinting patterns obtained with IS6110, direct repeats and polymorphic G+C-rich sequence-associated RFLP and direct variable repeat-spacer oligonucelotide typing (spoligotyping) segregated these isolates from the other members of the complex. The results of this testing, together with the repeated association of this micro-organism with goats, suggest that a new member of this taxonomic complex not matching any of the classical species had been identified. This unusual mycobacterium may play a role in the epidemiology of animal and human tuberculosis in Spain. The name Mycobacterium tuberculosis subsp. caprae subsp. nov. is proposed for these isolates. The type strain of Mycobacterium tuberculosis subsp. caprae subsp. nov. is gM-1T (= CIP 105776T).

β-lactam antibiotics promote bacterial mutagenesis via an RpoS-mediated reduction in replication fidelity

Regardless of their targets and modes of action, subinhibitory concentrations of antibiotics can have an impact on cell physiology and trigger a large variety of cellular responses in different bacterial species. Subinhibitory concentrations of β-lactam antibiotics cause reactive oxygen species production and induce PolIV-dependent mutagenesis in Escherichia coli. Here we show that subinhibitory concentrations of β-lactam antibiotics induce the RpoS regulon. RpoS-regulon induction is required for PolIV-dependent mutagenesis because it diminishes the control of DNA-replication fidelity by depleting MutS in E. coli, Vibrio cholerae and Pseudomonas aeruginosa. We also show that in E. coli, the reduction in mismatch-repair activity is mediated by SdsR, the RpoS-controlled small RNA. In summary, we show that mutagenesis induced by subinhibitory concentrations of antibiotics is a genetically controlled process. Because this mutagenesis can generate mutations conferring antibiotic resistance, it should be taken into consideration for the development of more efficient antimicrobial therapeutic strategies.

Hypermutation as a Factor Contributing to the Acquisition of Antimicrobial Resistance

Contrary to what was thought previously, bacteria seem to be, not merely spectators to their own evolution, but, through a variety of mechanisms, able to increase the rate at which mutations occur and, consequently, to increase their chances of becoming resistant to antibiotics. Laboratory studies and mathematical models suggest that, under stressful conditions, such as antibiotic challenge, selective pressure favors mutator strains of bacteria over nonmutator strains. These hypermutable strains have been found in natural bacterial populations at higher frequencies than expected. The presence of mutator strains in the clinical setting may indicate an enhanced risk of acquiring antibiotic resistance through mutational and recombinational events. In addition, some antibiotics are inducers of mechanisms that transiently increase the mutation rate, and thus probably act, not only as mere selectors of antibiotic resistant clones, but also as resistance-promoters.

Characterization of a new TEM-type beta-lactamase resistant to clavulanate, sulbactam, and tazobactam in a clinical isolate of Escherichia coli.

A clinical Escherichia coli strain highly resistant to the combinations of amoxicillin-clavulanate, ampicillin-sulbactam, and piperacillin-tazobactam was isolated from a patient with a community-acquired urinary tract infection who was previously treated with amoxicillin-clavulanate. These resistances were carried by a 45-kb conjugative plasmid encoding for a single beta-lactamase with a pI of 5.4. Cloning and sequencing of the new beta-lactamase, IRT-3, revealed identity with the blaT1 gene encoding the TEM-1 beta-lactamase except for a replacement of the methionine residue at position 67 by isoleucine and of the methionine residue at position 180 by threonine. Both mutations were segregated by the construction of hybrid genes, and only the mutation at methionine at position 67 was related to resistance to the suicide inhibitors. The inhibitory effects of clavulanate, sulbactam, and tazobactam on the TEM-1 enzyme were substantially decreased in comparison with those on IRT-3, as indicated by the 50% inhibitory concentrations.

Hypermutation and the Preexistence of Antibiotic-Resistant Pseudomonas aeruginosa Mutants: Implications for Susceptibility Testing and Treatment of Chronic Infections

ABSTRACT Whether or not resistant mutants will be present before the start of antibiotic treatment of an initially susceptible population of bacteria depends on the size of the infecting population, the rate of mutation to resistance, and the amount of time that the population has been maintained. In the present investigation, we argue that for the treatment of chronic infections caused by hypermutable Pseudomonas aeruginosa of the sort frequently found in cystic fibrosis patients, mutants resistant to all single antipseudomonal drugs will almost invariably be present in a high proportion at the onset of treatment, and consequently, these strains should be considered resistant to all agents when they are used as monotherapy. Using a construct of P. aeruginosa strain PAO1 with a mutS deletion (strain PAOΔmutS), we show that when in vitro populations of less than 5 × 104 seemingly susceptible hypermutable bacteria are confronted with any of 11 antipseudomonal agents, mutants for which the MICs and the minimum bactericidal concentrations are in the range of clinical resistance will almost invariably ascend to dominance within 24 to 36 h. This does not occur for PAO1 without the mutS deletion. The results of our detailed analysis of this evolution of acquired resistance to two of these antibiotics, imipenem and ciprofloxacin, indicate that although the rates of mutation to resistance in PAOΔmutS are on the order of 1 × 10−6 per generation, resistant mutants are very likely to either be present in cultures of between 2 × 104 and 4 × 104 bacteria or arise after the bacterial populations are confronted with antibiotics. We also demonstrate with in vitro experiments that the problem of acquired resistance to treatment with single antibiotics can be thwarted by combination therapy with pairs of antibiotics of different classes with synergistic activities. We discuss the clinical implications of our analysis of these observations.