Isabelle Podglajen

Modes and Modulations of Antibiotic Resistance Gene Expression

SUMMARY Since antibiotic resistance usually affords a gain of function, there is an associated biological cost resulting in a loss of fitness of the bacterial host. Considering that antibiotic resistance is most often only transiently advantageous to bacteria, an efficient and elegant way for them to escape the lethal action of drugs is the alteration of resistance gene expression. It appears that expression of bacterial resistance to antibiotics is frequently regulated, which indicates that modulation of gene expression probably reflects a good compromise between energy saving and adjustment to a rapidly evolving environment. Modulation of gene expression can occur at the transcriptional or translational level following mutations or the movement of mobile genetic elements and may involve induction by the antibiotic. In the latter case, the antibiotic can have a triple activity: as an antibacterial agent, as an inducer of resistance to itself, and as an inducer of the dissemination of resistance determinants. We will review certain mechanisms, all reversible, that bacteria have elaborated to achieve antibiotic resistance by the fine-tuning of the expression of genetic information.

Insertion of a novel DNA sequence, IS 1186, upstream of the silent carbapenemase gene cfiA, promotes expression of carbapenem resistance in clinical isolates of Bacteroides fragilis

A small number of isolates of Bacteroides fragilis, an anaerobic pathogen of the human intestinal flora, carries a copy (or copies) of the carbapenem‐resistance gene, cfiA, which may be silent or expressed. We have studied the mechanism of activation of the frequently silent gene in in vitro‐selected mutants and in clinical isolates. In both types of strains, activation was observed as the consequence of the insertion, at several possible sites, of a novel 1.3 kb insertion sequence, IS1186, immediately upstream of the carbapenemase gene. IS1186 has two open reading frames, on opposite strands, with coding capacities for a 41.2kDa (ORF1) and a 22.5 kDa (ORF2) protein. The 41.2kDa protein has homology with some proteins predicted from open reading frames of IS elements or DNA direct repeats of aerobic, but not anaerobic, Gram‐negative bacteria. Upon insertion, transcription of cfiA was found to be driven from a promoter identified on the right end of IS1186. In one instance, insertion occurred into the putative ribosome‐binding site of cfiA, leaving intact the tetranucleotide AGAA which is concluded to be a fully functional ribosome‐binding site. Between 3 and 14 copies of IS1186 were detected per genome and the element was found, within the species B. fragilis, almost exclusively in the subgroup carrying the cfiA gene.

Impact of Antimicrobial Therapy on Nasopharyngeal Carriage of Streptococcus pneumoniae, Haemophilus influenzae, and Branhamella catarrhalis in Children with Respiratory Tract Infections

We conducted a multicenter prospective study to document changes in nasopharyngeal carriage of Streptococcus pneumoniae, Haemophilus influenzae, and Branhamella catarrhalis during antibiotic therapy. A cohort of 629 children with respiratory tract infections underwent nasopharyngeal sampling before and after antibiotic treatment. Susceptibility testing, serotyping, arbitrarily primed polymerase chain reaction, and pulsed-field gel electrophoresis were used to compare pretreatment and posttreatment strains of S. pneumoniae. A significant decrease in carriage of all 3 species (especially S. pneumoniae and B. catarrhalis) was recorded. The increase in the proportion of penicillin-resistant pneumococci (PRP; 66% vs. 44%) was due to the decreased carriage of penicillin-susceptible pneumococci (71 of 629 vs. 176 of 629). The risk of PRP carriage in a given child did not increase. None of the children was found to harbor genetically related strains with increased minimum inhibitory concentrations. Given the multiple resistance of PRP, beta-lactam antibiotic therapy also increased the incidence of macrolide-resistant strains, whereas macrolides selected both macrolide- and penicillin-resistant strains.

Comparative molecular and microbiologic diagnosis of bacterial endocarditis.

Sequencing of 16S rDNA, and of sodAint and rpoBint in some cases, was applied to DNA from heart valves of 46 patients (36 with definite and 10 with possible endocarditis). Sequence-based identifications were compared with those obtained with conventional methods. Among the 36 definite cases, 30 had positive blood cultures and 6 had negative cultures. Among the 30 positive cases, sequencing of 16S rDNA permitted identification of species (18), genus (8), or neither (4); sodAint and rpoBint sequencing was necessary for species identification in 8 cases. Species identifications were identical in only 61.5%, when conventional techniques and DNA sequencing were used. In five of the six blood culture–negative endocarditis cases, sequencing identified Bartonella quintana (3), B. henselae (1), and Streptococcus gallolyticus (1). Our results demonstrate a clear benefit of molecular identification, particularly in cases of blood culture–negative endocarditis and of possible endocarditis, to confirm or invalidate the diagnosis. Moreover, in 19.4% of the definite cases, the improvement in species identification by sequencing led to improved patient management.

In Vitro Exchange of Fluoroquinolone Resistance Determinants between Streptococcus pneumoniae and Viridans Streptococci and Genomic Organization of the parE-parC Region in S. mitis

Transfer of fluoroquinolone (FQ) resistance determinants between Streptococcus pneumoniae and viridans streptococci was explored by transformation in vitro. One-step FQ-resistant parC mutants were selected, and resistance could be transferred from DNA from S. oralis, S. mitis, S. sanguis, and S. constellatus to S. pneumoniae, with frequencies of 10(-3) to <10(-7) in correlation with the homologies of their quinolone resistance determining region sequences (95%, 91%, 85%, and 81%, respectively). Reciprocal transfers of mutated parC from DNA from S. pneumoniae to S. mitis and S. oralis were also observed. Simultaneous transfer of mutated parC and gyrA genes from S. mitis to S. pneumoniae yielded high-level-resistant pneumococcal transformants in one step at low frequencies. The parE-parC region of the type strain S. mitis 103335T had >90% homology with that of S. pneumoniae. The efficient interspecific transfer of quinolone resistance determinants in vitro leads us to anticipate their dissemination in the clinical setting.

Salmonella enterica Serovar Typhimurium blaPER-1-Carrying Plasmid pSTI1 Encodes an Extended-Spectrum Aminoglycoside 6′-N-Acetyltransferase of Type Ib

ABSTRACT We have studied the aminoglycoside resistance gene, which confers high levels of resistance to both amikacin and gentamicin, that is carried by plasmid pSTI1 in the PER-1 β-lactamase-producing strain of Salmonella enterica serovar Typhimurium previously isolated in Turkey. This gene, called aac(6′)-Ib11, was found in a class 1 integron and codes for a protein of 188 amino acids, a fusion product between the N-terminal moiety (8 amino acids) of the signal peptide of the β-lactamase OXA-1 and the acetyltransferase. The gene lacked a plausible Shine-Dalgarno (SD) sequence and was located 45 nucleotides downstream from a small open reading frame, ORF-18, with a coding capacity of 18 amino acids and a properly spaced SD sequence likely to direct the initiation of aac(6′)-Ib11 translation. AAC(6′)-Ib11 had Leu118 and Ser119 as opposed to Gln and Leu or Gln and Ser, respectively, which were observed in all previously described enzymes of this type. We have evaluated the effect of Leu or Gln at position 118 by site-directed mutagenesis of aac(6′)-Ib11 and two other acetyltransferase gene variants, aac(6′)-Ib7 and -Ib8, which naturally encode Gln118. Our results show that the combination of Leu118 and Ser119 confers an extended-spectrum aminoglycoside resistance, with the MICs of all aminoglycosides in clinical use, including gentamicin, being two to eight times higher for strains with Leu118 and Ser119 than for those with Gln118 and Ser119.

Evaluation of ultraviolet C for disinfection of endocavitary ultrasound transducers persistently contaminated despite probe covers.

OBJECTIVE To determine the rate of bacterial and viral contamination of endocavitary ultrasound probes after endorectal or endovaginal examination with the use of probe covers and to evaluate the antimicrobial efficacy of a disinfection procedure consisting of cleaning with a disinfectant-impregnated towel followed by disinfection with ultraviolet C (UVC) light. METHODS Endovaginal or endorectal ultrasound examinations were performed for 440 patients in 3 institutions. All probes were covered by a condom or sheath during the examination. For bacterial analysis, 1 swab was applied lengthwise across one-half the surface of the probe just after removal of the probe cover. The second swab was similarly applied over the probe immediately after the end of a 2-step process consisting of cleaning with a towel impregnated with a disinfectant spray and a 5-minute UVC disinfection cycle. Swabs were applied onto plates and incubated for 48 hours. The number of colony-forming units was counted, and organisms were identified. A similar protocol was used for viral detection of Epstein-Barr virus, human cytomegalovirus, and human papillomavirus, except that an additional swab was applied along the entire external surface of the probe cover before its removal. Viruses were detected by means of a polymerase chain reaction-based protocol. RESULTS After removal of probe covers, contamination by pathogenic bacteria was found for 15 (3.4% [95% confidence interval, 2.0%-5.6%]) of 440 probes, and viral genome was detected on 5 (1.5% [95% confidence interval, 0.5%-3.5%]) of 336 probes. After cleaning with a towel impregnated with a disinfectant spray and disinfecting with UVC light, neither bacterial pathogenic flora nor viral genome was recovered from the probe. CONCLUSIONS Endocavitary ultrasound probes may carry pathogens after removal of covers under routine conditions. A disinfection procedure consisting of cleaning with a disinfectant-impregnated towel followed by disinfection with UVC may provide a useful method for disinfecting endocavitary ultrasound probes.

Enzyme structural plasticity and the emergence of broad-spectrum antibiotic resistance.

The emergence of multi‐resistant pathogenic bacteria is a worldwide health issue. Recently, clinical variants of a single antibiotic‐modifying acetyltransferase, AAC(6′)‐Ib—a variant of aminoglycoside 6′‐N‐acetyltransferase—have been identified that confer extended resistance to most aminoglycosides and, more surprisingly, to structurally unrelated fluoroquinolones. The corresponding gene is carried by mobile genetic elements and is present in most multi‐resistant pathogenic strains, hence making it a serious threat to current therapies. Here, we report the crystal structures of both narrow‐ and broad‐spectrum resistance variants of this enzyme, which reveal the structural basis for the emergence of extended resistance. The active site shows an important plasticity and has adapted to new substrates by a large‐scale gaping process. We have also obtained co‐crystals with both substrates, and with a simple transition state analogue, which provides new clues for the design of inhibitors of this resistance mechanism.

Emergence in Klebsiella pneumoniae of a Chromosome-Encoded SHV β-Lactamase That Compromises the Efficacy of Imipenem

ABSTRACT A Klebsiella pneumoniae isolate was identified that had reduced susceptibility to several expanded-spectrum cephalosporins and imipenem. That isolate produced a chromosome-encoded SHV-type β-lactamase, SHV-38, that had an alanine to valine substitution in position Ambler 146 compared to β-lactamase SHV-1. The kinetic parameters for purified β-lactamases SHV-38 and SHV-1 showed that the hydrolytic spectrum of SHV-38 included only ceftazidime and imipenem. This report is the first example of an SHV-type β-lactamase capable of hydrolyzing imipenem.

An intrinsic control element for translational initiation in class 1 integrons

Integrons are genetic elements able to capture anti‐biotic resistance and other genes and to promote their transcription. Here, we have investigated integron‐dependent translation of an aminoglycoside 6′‐N‐acetyltransferase gene (aac(6′)‐Ib7) inserted at the attI1 site. N‐terminal sequencing revealed that translation of this gene was initiated at a GTG codon, which is not part of a plausible translation initiation region (TIR). A short open reading frame (called ORF‐11) overlapping the attI1 site was probed by site‐directed mutagenesis for its contribution to aac(6′)‐Ib7 translation. When ORF‐11 and its TIR were deleted en bloc, translational efficiency dropped by over 80%, as determined with an acetyltransferase– luciferase fusion product. Invalidation of the ATG start codon of ORF‐11 or its putative Shine–Dalgarno sequence resulted in a decrease of over 60%, whereas the decrease was much less pronounced when the amino acid sequence of the putative ORF‐11‐encoded peptide was altered or when the distance between ORF‐11 and aac(6′)‐Ib7 was doubled. This demonstrates that aac(6′)‐Ib7 translation is dependent upon the translation of ORF‐11, but almost certainly not upon the corresponding peptide. These results lead us to conclude that an intrinsic short ORF present in the 5′‐conserved segment of many class 1 integrons may substantially enhance expression at the translational level of captured TIR‐deficient anti‐biotic resistance genes.