A macrolide antimicrobial drug was administered to a newborn with cough. On day 23 of hospitalization, macrolide-resistant Bordetella pertussis was isolated from nasopharyngeal aspirates. DNA sequencing and PCR–restriction fragment length polymorphism showed a 2047 A-to-G mutation in the 3 copies of the 23S rRNA gene. Monitoring for macrolide resistance is essential in infants <6 months of age.

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L egionella spp. are ubiquitous bacteria present in natural and artificial water systems. Inhalation of Legionella spp. in aerosolized water droplets from contaminated water sources is known to cause a type of pneumonia called Legionnaires' disease (LD). In the event of an LD outbreak, the successful outcome of an epidemiological investigation can help prevent further cases by rapidly identifying and containing the source of contamination.Legionella pneumophila is responsible for more than 90% of the cases of LD, and serogroup 1 alone accounts for almost 85% of cases (9, 23). Diagnosis of LD can be made by serology, direct immunofluorescence, PCR, urinary antigen detection, or culturing of clinical specimens; almost 20% of confirmed LD cases are detected by culture (2). Epidemiological analyses based on pulsedfield gel electrophoresis (PFGE) and/or sequence-based typing (SBT) of clinical isolates of L. pneumophila serogroup 1 have been used to classify isolates as sporadic, epidemic, or endemic (1). A strain is considered endemic when several isolates of an identical genotype are responsible for several epidemiologically unrelated cases of LD. Among the endemic strains of L. pneumophila serogroup 1, sequence type 1 (ST1) strains are among the most prevalent, in particular the ST1/Paris pulsotype. This endemic type was responsible for 8.2% of French culture-proven cases of LD from 1995 through 2006 (1, 10, 15). ST1/Paris pulsotype isolates have also been detected in clinical and environmental samples from several other countries around the world, including Switzerland, Italy, Spain, Sweden, the United States, Japan, Senegal, and Canada (1, 4). The high isolation rate of this strain in clinical and environmental samples makes it difficult, and frequently impossible, to identify the environmental source of an infection during epidemiological investigations.Recent studies have demonstrated the value of using the diversity of CRISPR spacers as genotyping markers for several pathogenic agents, and spoligotyping tools have been successfully developed for this purpose (14,17,19,20). The aim of this study was to design the first spoligotyping tool for subtyping L. pneumophila ST1/Paris pulsotype isolates and to evaluate its performance and efficiency on a collection of clinical and environmental isolates from France.(These results were presented in part as an oral communication at the EWGLI Meeting in 2010 and as a poster at the FEMS Microbiology Congress in 2011.) MATERIALS AND METHODSStrains and growth conditions. Reference strains used in this study were L. pneumophila Paris CIP107629 (ST1/Paris pulsotype) and L. pneumophila 130b ATCC BAA-74 (non-ST1/non-Paris pulsotype). All other clinical (257) and environmental (149) L. pneumophila isolates were part of the collection from the French Centre National de Référence des Légion-elles and were selected based on their genotypes (PFGE and SBT). Among the 406 isolates, 46 belonged to the ST1/non-Paris pulsotype (11 unrelated and 35 isolates from the same water sample), 15 t...

Monitoring the emergence of antibiotic resistance is a recent issue in the treatment of Legionnaires' disease. Macrolides are recommended as first-line therapy, but resistance mechanisms have not been studied in Legionella species. Our aim was to determine the molecular basis of macrolide resistance in L. pneumophila. Twelve independent lineages from a common susceptible L. pneumophila ancestral strain were propagated under conditions of erythromycin or azithromycin pressure to produce high-level macrolide resistance. Whole-genome sequencing was performed on 12 selected clones, and we investigated mutations common to all lineages. We reconstructed the dynamics of mutation for each lineage and demonstrated their involvement in decreased susceptibility to macrolides. The resistant mutants were produced in a limited number of passages to obtain a 4,096-fold increase in erythromycin MICs. Mutations affected highly conserved 5-amino-acid regions of L4 and L22 ribosomal proteins and of domain V of 23S rRNA (G2057, A2058, A2059, and C2611 nucleotides). The early mechanisms mainly affected L4 and L22 proteins and induced a 32-fold increase in the MICs of the selector drug. Additional mutations related to 23S rRNA mostly occurred later and were responsible for a major increase of macrolide MICs, depending on the mutated nucleotide, the substitution, and the number of mutated genes among the three rrl copies. The major mechanisms of the decreased susceptibility to macrolides in L. pneumophila and their dynamics were determined. The results showed that macrolide resistance could be easily selected in L. pneumophila and warrant further investigations in both clinical and environmental settings.

Identification and antimicrobial susceptibility testing (AST) are critical steps in the management of bloodstream infections. Our objective was to evaluate the performance of the Accelerate Pheno™ System, CE v1.2 software, for identification and AST of Gram-negative pathogens from positive blood culture bottles. A total of 104 bottles positive for Gram-negative bacteria collected from inpatients throughout our institution were randomly selected after Gram staining. The time-to-identification and AST results, and the raw AST results obtained by the Accelerate Pheno™ system and routine techniques (MALDI-TOF MS and VITEK®2, EUCAST guidelines) were compared. Any discrepant AST result was tested by microdilution. The Pheno™ significantly improved turn-around times for identification (5.3 versus 23.7 h; p < 0.0001) and AST (10.7 versus 35.1 h; p < 0.0001). Complete agreement between the Accelerate Pheno™ system and the MALDI-TOF MS for identification was observed for 96.2% of samples; it was 99% (98/99) for monomicrobial samples versus 40% (3/5) for polymicrobial ones. The overall categorical agreement for AST was 93.7%; it was notably decreased for beta-lactams (cefepime 84.4%, piperacillin-tazobactam 86.5%, ceftazidime 87.6%) or Pseudomonas aeruginosa (71.9%; with cefepime 33.3%, piperacillin-tazobactam 77.8%, ceftazidime 0%). Analysis of discrepant results found impaired performance of the Accelerate Pheno™ system for beta-lactams (except cefepime) in Enterobacteriales (six very major errors) and poor performance in P. aeruginosa. The Accelerate Pheno™ system significantly improved the turn-around times for bloodstream infection diagnosis. Nonetheless, improvements in the analysis of polymicrobial samples and in AST algorithms, notably beta-lactam testing in both P. aeruginosa and Enterobacteriales, are required for implementation in routine workflow.

Currently, several methods are used for the detection of Legionella in clinical samples, and these methods constitute part of the criteria for defining legionellosis cases. Urinary antigen detection is the first-line diagnostic test, although this test is limited to L. pneumophila serogroup 1 (Lp1) (Helbig et al., J Clin Microbiol 41:838-840, 2003). The use of molecular techniques can improve Legionaire's disease (LD) diagnosis by detecting other serogroups and species (Diederen et al., J Clin Microbiol 46:671-677, 2008). The isolation of Legionella strains from pulmonary samples by axenic culture is still required to perform further epidemiological investigations (Blyth et al., N S W Public Health Bull 20:157-161, 2009; Fields et al., Clin Microbiol Rev 15:506-526, 2002) but demonstrates various sensitivities. Amoebic coculture has been described as a method to recover Legionella from clinical culture-negative specimens (La Scola et al., J Clin Microbiol 39:365-366, 2001; Rowbotham, J Clin Pathol 36:978-986, 1983) and can be proposed for optimizing Legionella strain isolation from samples contaminated by oropharyngeal flora. Identification of Legionella isolates is based on serological characterization, genotypic methods (with sequencing of the mip gene as the standard method) and, more recently, the Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method.This chapter is limited to the identification of Legionella in clinical samples; antibody detection in human serum will not be discussed.

LpeAB are components of an efflux pump, which is a macrolide resistance determinant in L. pneumophila Paris strain. Mutations observed in the upstream sequence of lpeAB operon in resistant lineages led to an overexpression of this efflux pump. Sub-inhibitory concentrations of macrolides themselves participated in upregulating this efflux and could constitute a first step in the acquisition of a high macrolide resistance level.

Background Rarely, Legionnaires’ disease (LD) can progress into a slowly or nonresolving form. Methods A nationwide retrospective study was conducted by the French National Reference Center for Legionella (2013–2017) including cases of slowly or nonresolving LD defined as persistent clinical symptoms, computed tomography (CT) scan abnormalities, and Legionella detection in lower respiratory tract specimens by culture and/or real-time (RT) polymerase chain reaction (PCR) &gt;30 days after symptom onset. Results Twelve cases of community-acquired slowly or nonresolving LD were identified among 1686 cases of culture-positive LD. Median (interquartile range [IQR]) age was 63 (29–82) years. Ten (83.3%) patients had ≥1 immunosuppressive factor. Clinically, 9 patients transiently recovered before further deterioration (median [IQR] symptom-free interval, 30 [18–55] days), 3 patients had uniformly persistent symptoms (median [IQR] time, 48 [41.5–54] days). Two patients had &gt;2 recurrences. CT scan imagery found lung abscess in 5 (41.6%) cases. Slowly or nonresolving LD was diagnosed on positive Legionella cultures (n = 10, 83.3%) at 49.5 (IQR, 33.7–79) days. Two cases were documented through positive Legionella RT PCR at 52 and 53 days (cycle threshold detection of 21.5 and 33.7, respectively). No genomic microevolution and no Legionella resistance to antibiotics were detected. The median (IQR) duration of treatment was 46.5 (21–92.5) days. Two empyema cases required thoracic surgery. At a median (IQR) follow-up of 26 (14–41.5) months, LD-attributable mortality was 16.6% (n = 2). Conclusions Slowly or nonresolving LD may occur in immunocompromised patients, possibly leading to lung abscess and empyema.