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. Author manuscript; available in PMC: 2024 Mar 27.
Published in final edited form as: Infect Genet Evol. 2020 Nov 30;87:104658. doi: 10.1016/j.meegid.2020.104658

Extensively drug-resistant IMP-16-producing Pseudomonas monteilii isolated from cerebrospinal fluid

Anelise Stella Ballaben a, Renata Galetti a, Leonardo Neves Andrade a, Joseane Cristina Ferreira a, Doroti de Oliveira Garcia b, Yohei Doi c,d, Ana Lucia Costa Darini a,*
PMCID: PMC10966726  NIHMSID: NIHMS1970128  PMID: 33271337

Abstract

IMP-1-producing Pseudomonas aeruginosa was first reported in Japan and since then, bacteria with this metallo-β-lactamase have been detected worldwide. Pseudomonas monteilii (part of P. putida group) were considered an environmental pathogen with low virulence potential; however, multidrug-resistant and carbapenem-resistant P. monteilii have emerged. The present study reports the draft sequence of an extensively drug-resistant IMP-16-producing P. monteilii 597/14 isolated from cerebrospinal fluid in 2014. The sequencing data revealed blaIMP-16 as a gene cassette on class 1 integron, In1738 characterized in this study. Furthermore, the resistome of Pm597/14 consisted of 7 resistance genes (aadA1b, strA, strB, aacA4, blaIMP-16, blaOXA-2, sul1) and diverse virulence determinants involved in the adherence, LPS, antiphagocytosis, iron uptake and mercuric resistance. Although different virulence determinants were found in this study, using Galleria mellonella infection model, Pm597/14 did not kill any larvae between 7 days post-infection. P. monteilii isolates have been reported from clinical and environmental sources, carrying different MBL genes showing its potential role as their reservoir.

Keywords: Metallo-β-lactamases, Brazil, Meningitis, Galleria mellonella

Production of metallo-β-lactamases (MBLs) is recognized as one of the leading carbapenem resistance mechanisms among Gram-negative bacilli. In 1988, IMP-1-producing Pseudomonas aeruginosa was first reported in Japan and subsequently the gene blaIMP-1 was found in a class 1 integron located on a conjugative plasmid. Since then, bacteria with IMP enzymes have been detected worldwide (Matsumura et al., 2017) and according to National Database of Antibiotic Resistant Organisms, the IMP family now comprises 85 variants. Pseudomonas monteilii (P. putida group) were previously considered an environmental pathogen with low virulence potential; however, multidrug-resistant and carbapenem-resistant P. monteilii have emerged, being responsible for opportunistic nosocomial infections (Bogaerts et al., 2011; Ocampo-Sosa et al., 2015).

P. monteilii 597/14 (hereafter called Pm597/14) was obtained from the cerebrospinal fluid of a patient admitted at a hospital of São Paulo State, Brazil, in 2014 and showed resistance to 3rd-4th generation cephalosporins, aztreonam and carbapenems.

Antimicrobial susceptibility testing by disk diffusion tests (DDTs) and minimal inhibitory concentrations (MICs) by broth microdilution were performed according to the CLSI recommendations (2017). Conventional PCR was conducted to detect 28 different resistance genes, including acquired β-lactamase genes (Woodford et al., 2006; Andrade et al., 2010; Nicoletti et al., 2015; Bogaerts et al., 2013), qnr, aac(6′)-Ib, qepA (Cattoir et al., 2007; Wang et al., 2009; Cavaco et al., 2009; Xia et al., 2010) and 16S ribosomal RNA methyltransferase genes (Doi & Arakawa, 2007; Corrêa et al., 2014; Bueno et al., 2013). Plasmid incompatibility groups were investigated by PCR-based replicon typing methods (PBRT and AB-PBRT) (Carattoli et al., 2005; Bertini et al., 2010). S1 and I-Ceu-I-nuclease digestion followed by pulsed-field gel electrophoresis, Southern blot and hybridization with specific probes were performed to determine location of the bla gene.

DDTs and MICs revealed an extensively drug-resistant phenotype of Pm597/14 (Table 1), which only remained susceptible to amikacin, tetracycline and colistin. Double disk testing showed the presence of synergy between imipenem (10 μg) and EDTA (10 μL of 0.125 M EDTA stock), which was highly suggestive of metallo-β-lactamase production. PCR sequencing confirmed Pm597/14 as an IMP-16-producing isolate. Based on S1-PFGE, Pm597/14 did not possess any plasmid. The Southern blot followed by hybridization with specific probes revealed that the blaIMP-16 gene was located on the chromosome. Hybridization with probes for Pseudomonas sp. 16S rRNA and bla genes after I-Ceu-I-PFGE further confirmed chromosomal location.

Table 1.

In vitro evaluation of antimicrobial drugs activity against P. monteilii 597/14.

Druga Susceptibility profileb MIC (μg/mL)c
CTX R ≥256 μg/mL
CAZ R ≥256 μg/mL
CPM R ≥256 μg/mL
ATM R 32 μg/mL
ETP R ≥32 μg/mL
IPM R ≥32 μg/mL
MER R ≥32 μg/mL
TZP I 24/4 μg/mL
TIM R ≥128/2 μg/mL
GEN R ≥256 μg/mL
TOB R 128 μg/mL
AMK S –
CIP R 8 μg/mL
LVX R ≥32 μg/mL
TET S –
CHL R 64 μg/mL
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a

AMC: SAM, TZP: piperacillin-tazobactam, TIM: ticarcillin-clavulanate, AMP: ampicillin, CTX: cefotaxime, CAZ: ceftazidime, CPM: cefepime, ATM: aztreonam, ETP: ertapenem, IPM: imipenem, MER: meropenem, GEN: gentamicin, TOB: tobramycin, AMK: amikacin, CIP: ciprofloxacin, LVX: levofloxacin, TET: tetracycline, CHL: chloramphenicol.

b

S: susceptible, I: intermediate, R: resistant.

c

MIC testing was performed by Etest® (bioMerieux). MIC breakpoints were evaluated according to Clinical and Laboratory Standards Institute (CLSI, 2018).

Whole genome sequencing (WGS) was performed in order to further investigate the isolate’s resistance and virulence genetic context. Briefly, the genomic DNA was extracted by the Qiagen Blood and Tissue Kit and sequenced using Illumina NextSeq 250-bp paired-end sequencing. De novo assembly was carried out using CLC Genomics Workbench 8.0 (CLC bio, Aarhus, Denmark) and generated 125 contigs, with contig N50 of 43,319 bp, an average coverage of 63×, and the assembled genome of approximately 5.9 Mb in size (draft sequence). Gene prediction was performed for the draft sequence using the RAST server (http://rast.nmpdr.org/).

Pm597/14 was initially identified as P. putida by Vitek®, however, this isolate was subsequently confirmed as P. monteilii by WGS using the average nucleotide identity (ANI) method available through the NCBI.

The sequencing data also revealed blaIMP-16 as a gene cassette on class 1 integron, In1738 (intI1-aacA4-blaIMP-16-aadA1-blaOXA-2-qacEΔ-sul1) (http://integrall.bio.ua.pt/), characterized in this study. Additional antimicrobial resistance genes were predicted using ResFinder 3.1 (https://cge.cbs.dtu.dk/services/ResFinder/), which showed a resistome consisting of 7 resistance genes (aadA1b, strA, strB, aacA4, blaIMP-16, blaOXA-2, sul1). In silico analysis of the draft sequence showed the presence of two insertion sequences (IS) associated with resistance genes, an IS1595-family (Siguier et al., 2009) and IS6 (Harmer & Hall, 2019) up-and-downstream, respectively, of In1738 described here. Besides, Pm597/14 genome was compared to P. monteilii strain B5 genome (Accession Number NZ_CP022562.1) and the reference strain did not present any resistance genes or any gene that could be involved in any resistance mechanism. In silico analysis also revealed diverse virulence determinants (http://www.mgc.ac.cn/cgi-bin/VFs/genus.cgi?Genus=Pseudomonas) involved in the adherence (flagella [fleN], type IV pili [fimV] and LPS [waaF and waaG]), antiphagocytosis (alginate [algA, algB, algD and algE]) and in the iron uptake such as pvdE and pvdS. Furthermore, genes involved in mercuric resistance such as merA, merP, merR and merT were also found. Although different virulence determinants were found in this study, using Galleria mellonella infection model, Pm597/14 did not kill any larvae between 7 days post-infection with an inoculum of 1 × 108 CFU/mL, consistent with the relatively low virulence of this species in the clinic (Shariff & Beri, 2017). E. coli ATCC25922 and P. aeruginosa ATCC27853 were used as negative control.

IMP-16 was first reported in P. aeruginosa isolate from Brazil (Mendes, 2004) and since then, this metallo-β-lactamase was only been reported in P. putida in 2010 (Carvalho-Assef et al., 2010). Regarding P. monteilii isolates, it has been reported from clinical and environmental sources, carrying different MBL genes, such as blaIMP-13 and blaVIM-2 (Bogaerts et al., 2011; Ocampo-Sosa et al., 2015), showing its potential role as their reservoir. As genome-based species identification becomes more widely available, MBL-producing Pseudomonas sp. strains previously considered to be P. putida may be identified as P. monteilii as an emerging drug-resistant pathogen.

Acknowledgements

We thank to São Paulo Research Foundation (FAPESP) and National Council for Scientific and Technological Development (CNPq) for the constant support for our research. Dr. Vaughn Cooper for his assistance with whole genome sequencing. Profª. Drª. Marcia Regina von Zeska Kress and Mario Henrique Paziani for the provision of Galleria mellonella experiments.

Funding

This work was supported by FAPESP [grant 2014/14494–8]. Y.D. was supported by research grants from the National Institutes of Health (R21AI135522, R01AI104895). ASB was supported by a PhD fellowship [grant 2015/23484–9]. RG was supported by a postdoctoral fellowship from FAPESP [grant 2015/11728–0]. Also, this study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.

Footnotes

GenBank accession numbers

This BioSample has been deposited at the DDBJ/ENA/GenBank database under accession number SAMN12983224.

Transparency declarations

None to declare.

Declaration of Competing Interest

We would like to declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.

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