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Hidden Selection of Bacterial Resistance to Fluoroquinolones In Vivo: The Case of Legionella pneumophila and Humans.

Shadoud L, Almahmoud I, Jarraud S, Etienne J, Larrat S, Schwebel C, Timsit JF, Schneider D, Maurin M - EBioMedicine (2015)

Bottom Line: By investigating the mutational dynamics in patients, we showed that antibiotic resistance occurred during hospitalization most likely after fluoroquinolone treatment.In vivo selection of antibiotic resistances in L. pneumophila may be associated with treatment failures and poor prognosis.This hidden resistance must be carefully considered in the therapeutic management of legionellosis patients and in the control of the gradual loss of effectiveness of antibiotics.

View Article: PubMed Central - PubMed

Affiliation: Univ. Grenoble Alpes, Laboratoire Adaptation et Pathogénie des Microorganismes (LAPM), F-38000 Grenoble, France ; Centre National de la Recherche Scientifique (CNRS), LAPM, F-38000 Grenoble, France ; Centre Hospitalier Universitaire (CHU) Grenoble, Institut de Biologie et de Pathologie, Grenoble, France.

ABSTRACT

Background: Infectious diseases are the leading cause of human morbidity and mortality worldwide. One dramatic issue is the emergence of microbial resistance to antibiotics which is a major public health concern. Surprisingly however, such in vivo adaptive ability has not been reported yet for many intracellular human bacterial pathogens such as Legionella pneumophila.

Methods: We examined 82 unrelated patients with Legionnaire's disease from which 139 respiratory specimens were sampled during hospitalization and antibiotic therapy. We both developed a real time PCR assay and used deep-sequencing approaches to detect antibiotic resistance mutations in L. pneumophila and follow their selection and fate in these samples.

Findings: We identified the in vivo selection of fluoroquinolone resistance mutations in L. pneumophila in two infected patients treated with these antibiotics. By investigating the mutational dynamics in patients, we showed that antibiotic resistance occurred during hospitalization most likely after fluoroquinolone treatment.

Interpretation: In vivo selection of antibiotic resistances in L. pneumophila may be associated with treatment failures and poor prognosis. This hidden resistance must be carefully considered in the therapeutic management of legionellosis patients and in the control of the gradual loss of effectiveness of antibiotics.

No MeSH data available.


Related in: MedlinePlus

Clinical history and gyrA83 mutational dynamics for patients #2.The results of qPCRmip, qPCRgyrALp and NGS assays are shown, together with the antibiotic therapy for patients #2 over his hospitalization stay. The number of days for relevant steps during hospitalization is given inside circles according to D0 which corresponds to the day of legionellosis diagnosis as determined by a positive urinary antigen test (UAT +). qPCR-mip + indicates a positive qPCRmip assay in respiratory samples. Results of the qPCRgyrALp assay are indicated as “neg” (no amplification of the gyrA QRDR), “wt” and “mut” (a positive PCR assay with a reference and mutant-type melting peak, respectively). The proportion of the reads corresponding to the T83I gyrA83 mutation, determined by next-generation sequencing, is shown (NGS-mut). Small arrows indicate the periods of antibiotic therapy. Lev: levofloxacin; Cp: ciprofloxacin; Azi: azithromycin; Ery: erythromycin; Amc: amoxicillin-clavulanate; Ca: ceftazidime; Tc: teicoplanin.
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f0010: Clinical history and gyrA83 mutational dynamics for patients #2.The results of qPCRmip, qPCRgyrALp and NGS assays are shown, together with the antibiotic therapy for patients #2 over his hospitalization stay. The number of days for relevant steps during hospitalization is given inside circles according to D0 which corresponds to the day of legionellosis diagnosis as determined by a positive urinary antigen test (UAT +). qPCR-mip + indicates a positive qPCRmip assay in respiratory samples. Results of the qPCRgyrALp assay are indicated as “neg” (no amplification of the gyrA QRDR), “wt” and “mut” (a positive PCR assay with a reference and mutant-type melting peak, respectively). The proportion of the reads corresponding to the T83I gyrA83 mutation, determined by next-generation sequencing, is shown (NGS-mut). Small arrows indicate the periods of antibiotic therapy. Lev: levofloxacin; Cp: ciprofloxacin; Azi: azithromycin; Ery: erythromycin; Amc: amoxicillin-clavulanate; Ca: ceftazidime; Tc: teicoplanin.

Mentions: For patients #2, a reference gyrA QRDR was detected in the D0 sample, but a C248T mutation resulting in the T83I GyrA change was found in the analyzed D4 sample, suggesting in vivo selection of fluoroquinolone-resistant mutants. Using NGS, we confirmed the presence of the C248T gyrA83 mutation in respiratory samples. It occurred at a frequency of 2.9% of 7205 reads at D0, increasing to 94% of 16,794 reads in the D4 sample after 4 days of treatment with levofloxacin and azithromycin (Table 1). The high proportion of gyrA83 mutants in sample D4 of patients #2 confirmed the melting curve profiles of the qPCRgyrALp assay. Patient #2 recovered from legionellosis under levofloxacin and azithromycin therapy, after 31 days at ICU and a total of 79 days at hospital (Fig. 2).


Hidden Selection of Bacterial Resistance to Fluoroquinolones In Vivo: The Case of Legionella pneumophila and Humans.

Shadoud L, Almahmoud I, Jarraud S, Etienne J, Larrat S, Schwebel C, Timsit JF, Schneider D, Maurin M - EBioMedicine (2015)

Clinical history and gyrA83 mutational dynamics for patients #2.The results of qPCRmip, qPCRgyrALp and NGS assays are shown, together with the antibiotic therapy for patients #2 over his hospitalization stay. The number of days for relevant steps during hospitalization is given inside circles according to D0 which corresponds to the day of legionellosis diagnosis as determined by a positive urinary antigen test (UAT +). qPCR-mip + indicates a positive qPCRmip assay in respiratory samples. Results of the qPCRgyrALp assay are indicated as “neg” (no amplification of the gyrA QRDR), “wt” and “mut” (a positive PCR assay with a reference and mutant-type melting peak, respectively). The proportion of the reads corresponding to the T83I gyrA83 mutation, determined by next-generation sequencing, is shown (NGS-mut). Small arrows indicate the periods of antibiotic therapy. Lev: levofloxacin; Cp: ciprofloxacin; Azi: azithromycin; Ery: erythromycin; Amc: amoxicillin-clavulanate; Ca: ceftazidime; Tc: teicoplanin.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4588375&req=5

f0010: Clinical history and gyrA83 mutational dynamics for patients #2.The results of qPCRmip, qPCRgyrALp and NGS assays are shown, together with the antibiotic therapy for patients #2 over his hospitalization stay. The number of days for relevant steps during hospitalization is given inside circles according to D0 which corresponds to the day of legionellosis diagnosis as determined by a positive urinary antigen test (UAT +). qPCR-mip + indicates a positive qPCRmip assay in respiratory samples. Results of the qPCRgyrALp assay are indicated as “neg” (no amplification of the gyrA QRDR), “wt” and “mut” (a positive PCR assay with a reference and mutant-type melting peak, respectively). The proportion of the reads corresponding to the T83I gyrA83 mutation, determined by next-generation sequencing, is shown (NGS-mut). Small arrows indicate the periods of antibiotic therapy. Lev: levofloxacin; Cp: ciprofloxacin; Azi: azithromycin; Ery: erythromycin; Amc: amoxicillin-clavulanate; Ca: ceftazidime; Tc: teicoplanin.
Mentions: For patients #2, a reference gyrA QRDR was detected in the D0 sample, but a C248T mutation resulting in the T83I GyrA change was found in the analyzed D4 sample, suggesting in vivo selection of fluoroquinolone-resistant mutants. Using NGS, we confirmed the presence of the C248T gyrA83 mutation in respiratory samples. It occurred at a frequency of 2.9% of 7205 reads at D0, increasing to 94% of 16,794 reads in the D4 sample after 4 days of treatment with levofloxacin and azithromycin (Table 1). The high proportion of gyrA83 mutants in sample D4 of patients #2 confirmed the melting curve profiles of the qPCRgyrALp assay. Patient #2 recovered from legionellosis under levofloxacin and azithromycin therapy, after 31 days at ICU and a total of 79 days at hospital (Fig. 2).

Bottom Line: By investigating the mutational dynamics in patients, we showed that antibiotic resistance occurred during hospitalization most likely after fluoroquinolone treatment.In vivo selection of antibiotic resistances in L. pneumophila may be associated with treatment failures and poor prognosis.This hidden resistance must be carefully considered in the therapeutic management of legionellosis patients and in the control of the gradual loss of effectiveness of antibiotics.

View Article: PubMed Central - PubMed

Affiliation: Univ. Grenoble Alpes, Laboratoire Adaptation et Pathogénie des Microorganismes (LAPM), F-38000 Grenoble, France ; Centre National de la Recherche Scientifique (CNRS), LAPM, F-38000 Grenoble, France ; Centre Hospitalier Universitaire (CHU) Grenoble, Institut de Biologie et de Pathologie, Grenoble, France.

ABSTRACT

Background: Infectious diseases are the leading cause of human morbidity and mortality worldwide. One dramatic issue is the emergence of microbial resistance to antibiotics which is a major public health concern. Surprisingly however, such in vivo adaptive ability has not been reported yet for many intracellular human bacterial pathogens such as Legionella pneumophila.

Methods: We examined 82 unrelated patients with Legionnaire's disease from which 139 respiratory specimens were sampled during hospitalization and antibiotic therapy. We both developed a real time PCR assay and used deep-sequencing approaches to detect antibiotic resistance mutations in L. pneumophila and follow their selection and fate in these samples.

Findings: We identified the in vivo selection of fluoroquinolone resistance mutations in L. pneumophila in two infected patients treated with these antibiotics. By investigating the mutational dynamics in patients, we showed that antibiotic resistance occurred during hospitalization most likely after fluoroquinolone treatment.

Interpretation: In vivo selection of antibiotic resistances in L. pneumophila may be associated with treatment failures and poor prognosis. This hidden resistance must be carefully considered in the therapeutic management of legionellosis patients and in the control of the gradual loss of effectiveness of antibiotics.

No MeSH data available.


Related in: MedlinePlus