| Resumo: | Background: Tigecycline is increasingly used to treat infections caused by Gram negative isolates, because these bacteria express multidrug-resistance (MDR) phenotypes in an escalating frequency. Thus, this study aimed: 1) to correlate mediated β-lactam and fluoroquinolone resistance mechanisms (RMs) among Gram-negative isolates causing untreatable bacterial infections nowadays; 2) to study the in vitro activity of tigecycline to identify the importance of this antibiotic; 3) and to characterize tigecycline resistance mechanisms (RM) in order to understand how to extend its activity. Material/methods: A total of 2560 unduplicated Gram-negative (2309 Enterobacteriaceae and 251 Acinetobacter baumannii) isolates, collected in healthcare facilities in Portugal (2009-2013), were studied. MICs of tigecycline were determined against all isolates, and interpreted according to EUCAST (Enterobacteriaceae) or CLSI (A. baumannii, Ab) guidelines. Antimicrobial susceptibility of other 17 antibiotics was determined to all clinical isolates by disk diffusion method and interpreted by EUCAST. Fluoroquinolone and β-lactam RMs were reached by PCR and sequencing of 623 Enterobacteriaceae and 133 Ab using specific primers targeting PMQR-, Class A- and D β-lactamase-, Class B/MBL- and PMAβ-encoding genes. Tigecycline resistance due to efflux pump production was studied by molecular methods: ramR gene of Klebsiella pneumoniae and marR gene of Escherichia coli isolates. Results: We identified 27.2%/35.8% and 96.4%/96.2% tigecycline non-susceptible/MDR Enterobacteriaceae and Ab isolates, respectively. The molecular analyses of tigecycline RMs revealed deletions/insertions/point mutations in the ramR gene that might contribute to the overexpression of AcrAB efflux pump in 63 out of 108 K. pneumoniae isolates showing reduced susceptibility to tigecycline. Point mutations observed in marR gene from E. coli isolates with or without tigecycline resistance, might contribute to MDR scenarios. A great diversity of β-lactamases was observed in Enterobacteriaceae isolates: penicillinases, ESBLs (CTX-M-1/-14/-15/-32/-G1-type/-G2-type, TEM-4/-10, SHV-12/-55, GES-7), carbapenemases (KPC-3, GES-5/-6, OXA-48, VIM-2/-34) and PMAβ (CMY-2, DHA-1, MIR-type, ACT-type); Enterobacteriaceae were fully susceptible to tigecycline for: 48.3% of β-lactamase producers; 65.6% carrying PMQR determinants; 69.1% presenting both RMs. Concerning Ab all 133 isolates tested (3.8% tigecycline susceptible) expressed an acquired carbapenemase (OXA-23 and/or OXA-24). Conclusions: This study showed that tigecycline remains a substrate of MDR Ab isolates. However, the in vitro susceptibility of Enterobacteriaceae isolates to tigecycline showed its decisive importance when these bacteria presented resistance to other antibiotic classes, specifically through plasmid-mediated β-lactam and fluoroquinolone RMs. Thus, tigecycline contribution against the most important MDR Gram-negative pathogens should be preserved principally to bacterial infections untreatable by other antibiotics (such as carbapenems and colistin).
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