Genetic study of resistance to beta-lactam, quinolones and aminoglycosides in bacteria isolated from patients with suspected meningitis in the State of São Paulo

Author(s)

Anelise Stella Ballaben

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2019-08-13

Examining board members

Ana Lucia da Costa Darini; Ilana Lopes Baratella da Cunha Camargo; Juliana Pfrimer Falcão; Cristiano Gallina Moreira

• Advisor: Ana Lucia da Costa Darini; Leonardo Neves de Andrade

Abstract

In Brazil, there are few reports of members of the order Enterobacterales-causing meningitis. Over the last 20 years, beta-lactamase-producing gram-negative bacilli (BGN) have become a major challenge in the treatment of infections caused by CTX-M-and KPC-producing isolates. This work aimed to investigate the genetic and molecular epidemiology of resistance to antibiotics in members of the order Enterobacterales and non-fermentative gram-negative bacilli (BGN-NF) isolated from patients with meningitis in São Paulo State. Sixty-six members of the order Enterobacterales and BGN-NF resistant to broad-spectrum beta-lactam antibiotics were studied. The microbiological investigations were previously performed at the Adolfo Lutz Institute in Ribeirão Preto and São Paulo. These bacteria were isolated from blood and CSF from patients with suspected meningitis from 2007 to 2014. The genes coding for beta-lactamases, plasmid mediated quinolone resistance (PMQR) and 16S ribosomal RNA methyltransferase (16S-RMTase) as well as plasmids typing were screened by PCR and confirmed by sequencing. In addition, molecular typing was performed by multilocus sequencing (MLST). The chromosomal or plasmid localization of the resistance genes was evaluated by I-Ceu-I-PFGE or S1-PFGE, respectively. Cloning experiments, transformation, conjugation and inhibition of efflux pumps as well as molecular investigation of virulence determinants and whole genome sequencing were also performed. Among the 66 isolates, 39 were identified as BGN-NF being 21 Acinetobacter baumannii, 11 Pseudomonas aeruginosa, 1 Pseudomonas putida, 3 Stenotrophomonas maltophilia and 3 Ochrobactrum antrophi; besides 27 members of the order Enterobacterales, being 17 Klebsiella pneumoniae, 5 Klebsiella aerogenes, 2 Klebsiella oxytoca, 2 Enterobacter cloacae and 1 Serratia marcescens. Among 21 A. baumannii isolates, blaOXA-23-like upstream to ISAba1 was detected in 17 isolates. Plasmids AB-GR2, AB-GR4, AB-GR6 and AB-GR8 were detected in 9 isolates while 4 isolates showed IncFrepB. However, in all isolates, chromosomal location of blaOXA-23-like was confirmed. Four different sequence typing (ST) were detected: two ST407 and ST690 singletons, three clonal complexes (CC), ST225 and ST438 (CC103), ST231 and ST442 (CC109), ST227, ST233 and ST783 (CC113). Among the 11 P. aeruginosa, iv P. aeruginosa 1206/13 (ST1602) and P. aeruginosa 463/12 (ST235) presented bla genes, blaCTX-M-2 was detected in both isolates and blaGES-1 only in isolate 1206/13. Sequencing data revealed blaGES-1 organized as a cassette gene associated with the new class I integron, In1600 in which blaCTX-M-2 downstream to ISCR1 which was also associated with the same In1600, resulting in a ~ 11,680 bp structure. S1-PFGE experiments determined that both bla genes were inserted into ~340kb IncP2 plasmid. Regarding P. aeruginosa 463/12, blaCTX-M-2 was also found downstream to ISCR1 inserted into the chromosome of the isolate. IMP-16-producing P. putida was detected among the isolates. The blaTEM, blaCTX-M-2, blaCTX-M-8, blaCTX-M-15 and blaKPC genes were the most detected among Klebsiella sp. Among the PMQRs, only qnrB-1 was found. Still, these isolates presented a great diversity of replicons, being colE and IncL/M the most detected ones. Several virulence determinants were found among K. pneumoniae isolates. Among the 5 K. aerogenes isolates, only 1 was CTX-M-15-producing isolate as well as qnrB-1 and aac(6\')-Ib. In addition, one E. cloacae isolate showed AmpC overexpression. Different Inc plasmids were detected among K. aerogenes and E. cloacae isolates. Those isolates that presented a phenotype of high-level aminoglycoside resistance (HLAR) had their genomes sequenced and resistance mechanisms were investigated in detail. In some isolates, the combination of the production of different aminoglycoside-modifying enzymes (AMEs) resulted in the HLAR phenotype presented, whereas in others, 16S-RMTase production was detected. In addition, presence of hyperexpression of efflux pumps was also found among the isolates. (AU)

• FAPESP's process: 15/23484-9 - Molecular study of gram-negative beta-lactamase producing bacteria isolated from patients with suspected meningitis in the Ribeirão Preto region
• Grantee: Anelise Stella Ballaben
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)