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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 dynamics of L. pneumophila DNA load and gyrA83 mutations in respiratory samples from patient #4.(a) The results of qPCRmip, qPCRgyrALp and NGS assays are shown, together with the antibiotic therapy for patients #4 over his hospitalization stay (see legend of Fig. 2).(b) Dynamics of L. pneumophila DNA load, expressed as log GU/mL clinical sample, in respiratory samples of patient # 4 over time after admission in ICU. The total, reference, and mutant gyrA QRDR DNA loads are shown by circles, triangles and squares, respectively. Data were extracted from the qPCRmip assay and NGS experiments.(c) Relative proportion of the reference (triangles) and mutant (squares) gyrA QRDR allele in respiratory samples of patient # 4 over time after admission in ICU. Data were extracted from the NGS experiments.
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f0015: Clinical history, and dynamics of L. pneumophila DNA load and gyrA83 mutations in respiratory samples from patient #4.(a) The results of qPCRmip, qPCRgyrALp and NGS assays are shown, together with the antibiotic therapy for patients #4 over his hospitalization stay (see legend of Fig. 2).(b) Dynamics of L. pneumophila DNA load, expressed as log GU/mL clinical sample, in respiratory samples of patient # 4 over time after admission in ICU. The total, reference, and mutant gyrA QRDR DNA loads are shown by circles, triangles and squares, respectively. Data were extracted from the qPCRmip assay and NGS experiments.(c) Relative proportion of the reference (triangles) and mutant (squares) gyrA QRDR allele in respiratory samples of patient # 4 over time after admission in ICU. Data were extracted from the NGS experiments.

Mentions: For patients # 4, a reference gyrA QRDR was detected in the D0 sample, but a C248T mutation resulting in the T83I GyrA change was found in both D3 and D5 samples, suggesting in vivo selection of fluoroquinolone-resistant mutants. Using NGS, the C248T gyrA83 mutation was detected in 1.05% of 8063 reads at D0, increasing to 75% of 15,824 reads and 85% of 16,050 reads at D3 and D5, respectively, while the patient was receiving ciprofloxacin and erythromycin in the intensive care unit (ICU) (Table 1). The high proportion of gyrA83 mutants in samples D3 and D5 confirmed the melting curve profiles of the qPCRgyrALp assay. For this patient, we investigated the dynamics of both the DNA load and gyrA83 allele by combining the qPCRmip and NGS approaches. While the global DNA load remained stable in respiratory samples from D0 to D5, the gyrA83 mutated alleles progressively replaced the wild-type alleles, suggesting an increase in the fluoroquinolone-resistant population of L. pneumophila, parallel to the reduction of the susceptible population (Fig. 3). Patient #4 was first admitted to a hematologic ward for severe pneumonia and aplasia, and transferred after 11 days at ICU because of health worsening despite a three-day course of ciprofloxacin, ceftazidime and teicoplanin (Fig. 3). He was cured under ciprofloxacin and erythromycin therapy, with a 32-day hospital stay including 7 days at ICU.


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 dynamics of L. pneumophila DNA load and gyrA83 mutations in respiratory samples from patient #4.(a) The results of qPCRmip, qPCRgyrALp and NGS assays are shown, together with the antibiotic therapy for patients #4 over his hospitalization stay (see legend of Fig. 2).(b) Dynamics of L. pneumophila DNA load, expressed as log GU/mL clinical sample, in respiratory samples of patient # 4 over time after admission in ICU. The total, reference, and mutant gyrA QRDR DNA loads are shown by circles, triangles and squares, respectively. Data were extracted from the qPCRmip assay and NGS experiments.(c) Relative proportion of the reference (triangles) and mutant (squares) gyrA QRDR allele in respiratory samples of patient # 4 over time after admission in ICU. Data were extracted from the NGS experiments.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0015: Clinical history, and dynamics of L. pneumophila DNA load and gyrA83 mutations in respiratory samples from patient #4.(a) The results of qPCRmip, qPCRgyrALp and NGS assays are shown, together with the antibiotic therapy for patients #4 over his hospitalization stay (see legend of Fig. 2).(b) Dynamics of L. pneumophila DNA load, expressed as log GU/mL clinical sample, in respiratory samples of patient # 4 over time after admission in ICU. The total, reference, and mutant gyrA QRDR DNA loads are shown by circles, triangles and squares, respectively. Data were extracted from the qPCRmip assay and NGS experiments.(c) Relative proportion of the reference (triangles) and mutant (squares) gyrA QRDR allele in respiratory samples of patient # 4 over time after admission in ICU. Data were extracted from the NGS experiments.
Mentions: For patients # 4, a reference gyrA QRDR was detected in the D0 sample, but a C248T mutation resulting in the T83I GyrA change was found in both D3 and D5 samples, suggesting in vivo selection of fluoroquinolone-resistant mutants. Using NGS, the C248T gyrA83 mutation was detected in 1.05% of 8063 reads at D0, increasing to 75% of 15,824 reads and 85% of 16,050 reads at D3 and D5, respectively, while the patient was receiving ciprofloxacin and erythromycin in the intensive care unit (ICU) (Table 1). The high proportion of gyrA83 mutants in samples D3 and D5 confirmed the melting curve profiles of the qPCRgyrALp assay. For this patient, we investigated the dynamics of both the DNA load and gyrA83 allele by combining the qPCRmip and NGS approaches. While the global DNA load remained stable in respiratory samples from D0 to D5, the gyrA83 mutated alleles progressively replaced the wild-type alleles, suggesting an increase in the fluoroquinolone-resistant population of L. pneumophila, parallel to the reduction of the susceptible population (Fig. 3). Patient #4 was first admitted to a hematologic ward for severe pneumonia and aplasia, and transferred after 11 days at ICU because of health worsening despite a three-day course of ciprofloxacin, ceftazidime and teicoplanin (Fig. 3). He was cured under ciprofloxacin and erythromycin therapy, with a 32-day hospital stay including 7 days at ICU.

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