Polymyxin resistance is a public health concern -present in humans, animals and the environment -caused by chromosomal-encoding or plasmid-encoding mechanisms. Chromosomal alterations in MgrB are frequently detected in Klebsiella spp., but not yet reported and characterised in Klebsiella variicola ( K. variicola ). This study performed microbiological and genomic characterisation of three polymyxin- resistant K. variicola isolates (M14, M15 and M50) recovered from the microbiota of migratory birds in Brazil. The isolates were submitted to SpeI-PFGE, broth microdilution and whole genome sequencing us- ing Illumina MiSeq for analysis of genetic relatedness, sequence typing and detection of antimicrobial- resistance genes. K. variicola isolates belonged to two clones, and susceptibility tests showed resistance only for polymyxins. Sequences of chromosomal two-component systems (PmrAB, PhoPQ, RstAB, CrrAB) and MgrB were evaluated by blastN and blastP against a polymyxin-susceptible K. variicola (A58243), and mutations with biological effect were checked by the PROVEAN tool. K. variicola isolates belonged to two clones, and susceptibility tests showed resistance for polymyxins. In M14 and M15, phoQ deleterious mutations (D90N, I122S and G385S) were identified, while an mgrB variant containing a single deletion (C deletion on position 93) leading to the production of a non-functional protein was detected in M50. mgrB complementation studies showed restoration of polymyxin susceptibility (64 to ≤ 0.25 mg/L) as a wild-type mgrB was inserted into the mgrB-deficient M50. This study confirmed the role of a non-functional mgrB variant in conferring polymyxin resistance, stressing the role of this regulator in K. variicola and drawing attention to novel polymyxin resistance mechanisms emerging in wildlife.
SPM-1-producing Pseudomonas aeruginosa ST277 clone recovered from microbiota of migratory birds
The production of Sao Paulo metallo-ß-lactamase (SPM-1) is the most common carbapenem resistance mechanism detected among multidrug-resistant Pseudomonas aeruginosa clinical isolates in Brazil. Dissemination of SPM-1-producing P. aeruginosa has been restricted to the nosocomial settings, with sporadic reports of environmental isolates due to contamination by hospital sewage. Herein, we described the detection and molecular characterization of SPM-1-producing P. aeruginosa recovered from the microbiota of migratory birds in Brazil. Three hundred gram-negative bacilli were recovered from cloacal and choanal swabs of Dendrocygna viduata during a surveillance study for detection of carbapenem-resistant isolates. All isolates were identified by matrix-assisted laser desorption ionization?time of flight mass spectrometry. Molecular typing was performed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. MICs were determined by agar dilution, except for polymyxin B. Antibiotic resistance genes were detected by polymerase chain reaction (PCR) followed by DNA sequencing. Transcriptional levels of oprD and efflux system encoding genes were also carried out by quantitative real-time PCR. Nine imipenem resistant P. aeruginosa isolates were recovered with 7 of them carrying blaSPM-1. Additional resistance genes (rmtD-1, blaOXA-56, aacA4, and aac(6′)-Ib-cr) were also detected in all 9 isolates. The SPM-1-producing isolates showed high MICs for all ß-lactams, fluoroquinolones, and aminoglycosides, being susceptible only to polymyxin B. Interestingly, all isolates showed the same PFGE pattern and belonged to ST277. Overexpression of MexXY-OprM and MexABOprM was observed in those isolates that did not harbor blaSPM-1. Our results suggest that migratory birds might have played a role in the dissemination of SPM-1 producing P. aeruginosa within the Brazilian territory.