Enterobacter cloacae Complex and CTX-M Extended-Spectrum ß-Lactamases in Algeria.

We evaluated the ß-lactam resistance phenotypes of clinical and environmental strains of the Enterobacter cloacae complex (ECC) isolated from three Algerian hospitals. The first combination of API 20E, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and hsp60 genetic clustering methodologies were carried out for the identification of ECC strains. Our research showed that API 20E and MALDI TOF MS are satisfactory in genus identification of ECC strains, but sequence-based methods are then necessary to discriminate the species and subspecies levels. Among 36 ECC strains, 94.44% belonged to Enterobacter hormaechei species. Twenty-five isolates clustered with the reference strain of E. hormaechei subsp. xiangfangensis, making it the most frequently isolated subspecies. Enterobacter kobei was found only once (2.77%). All ECC isolates were phenotypically extended-spectrum ß-lactamase (ESBL) producers and were resistant to ticarcillin, piperacillin, cefoxitin, cefotaxime, ceftazidime, ceftriaxone, and aztreonam, but susceptible to ertapenem and imipenem. The genetic analyses only allowed the detection of resistance genes of the CTX-M-1 group (32 strains, 88.9%), including CTX-M-15 (30 strains), CTX-M-3 (1 strain), and CTX-M-22 (1 strain). We report for the first time the detection of CTX-M-22 among ECC strains in an Algerian hospital (Tlemcen hospital). None of the isolated strains harbored CTX-M-2, CTX-M-9, or CTX-M-8/25 group genes. In this review, we address recent comparison in the identification methods of multidrug-resistant E. cloacae complex in Algeria, focusing also on the CTX-M ESBLs. This represents a serious public health challenge, which requires the clarification of the current situation and warrants the reinforcement of hygiene measures in the Algerian hospitals.

Evaluation of Microbiological Performance and the Potential Clinical Impact of the ePlex® Blood Culture Identification Panels for the Rapid Diagnosis of Bacteremia and Fungemia.

Molecular rapid diagnostic assays associated with antimicrobial stewardship have proven effective for the early adaptation of empiric therapy in bloodstream infections. The ePlex® BCID (GenMark Diagnostics) Panels allow identification of 56 bacteria and fungi and 10 resistance genes in 90 min directly from positive blood cultures. We prospectively evaluated 187 sepsis episodes at Grenoble University Hospital and retrospectively analyzed the cases to measure the potential clinical impact of the ePlex BCID results. Identification of all pathogens was obtained for 164/187 (88%) bloodstream infections with 100% detection of antimicrobial resistance genes (17 blaCTX-M , 1 vanA, and 17 mecA genes). Only 15/209 (7%) strains were not covered by the panels. Sensitivity for detection of micro-organisms targeted by the RUO BCID-GP, BCID-GN, and BCID-FP Panels was respectively 84/84 (100%), 103/107 (96%), and 14/14 (100%). Interestingly, accurate identification of all pathogens was achieved in 15/17 (88%) polymicrobial samples. Retrospective analysis of medical records showed that a modification of antimicrobial treatment would have been done in 45% of the patients. Treatment modifications would have been an optimization of empiric therapy, a de-escalation or an escalation in respectively 16, 17, and 11% of the patients. Moreover, 11% of the samples were classified as contaminants or not clinically relevant and would have led to early de-escalation or withdrawal of any antibiotic. Detection of resistance genes in addition to identification alone increased escalation rate from 4 to 11% of the patients. Absence of the ePlex result was considered a lost opportunity for therapy modification in 28% of patients.

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

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.

Mutational paths towards increased fluoroquinolone resistance in Legionella pneumophila.

OBJECTIVES: Fluoroquinolone resistance has been poorly studied in Legionella pneumophila, an intracellular pathogen responsible for legionellosis. Our goal was to further characterize molecular mechanisms involved in fluoroquinolone resistance in this species. METHODS: Eight independent lineages were founded from a common fluoroquinolone-susceptible L. pneumophila ancestor and propagated by serial passages in moxifloxacin-containing culture medium. We identified the substituted mutations that affected the DNA topoisomerase II-encoding genes, determined the order of substitution of the mutations leading to the stepwise MIC increases of moxifloxacin over evolutionary time and demonstrated their direct involvement in the resistance process. RESULTS: Adaptation occurred through parallel stepwise increases in the moxifloxacin MICs up to 512-fold the MIC for the parental strain. Mutations affected the topoisomerase II-encoding genes gyrA, parC and gyrB, reflecting a high degree of genetic parallelism across the independent lineages. During evolution, the T83I change in GyrA occurred first, followed by G78D or S80R in ParC and D87N in GyrA, or S464Y or D426N in GyrB. By constructing isogenic strains, we showed that the progressive increase in resistance was linked to a precise order of mutation substitution, but also to the co-existence of several subpopulations of bacteria bearing different mutations. CONCLUSIONS: Specific mutational trajectories were identified, strongly suggesting that intermolecular epistatic interactions between DNA topoisomerases underlie the mechanism of fluoroquinolone resistance in L. pneumophila. Our results suggest that L. pneumophila has strong potential to become resistant to fluoroquinolone compounds and warrant further investigation of resistance in clinical and environmental strains of this pathogen.