First report of a blaNDM-resistant gene in a Klebsiella aerogenes clinical isolate from Brazil

Cynthia Regina Pedrosa Soares Jorge Belém Oliveira-Júnior Elza Ferreira Firmo About the authors

Abstract

INTRODUCTION:

Carbapenemase-resistant enterobacteria that produce the bla NDM gene are found worldwide. However, this is the first report of blaNDM in Klebsiella aerogenes in Brazil.

METHODS:

The identification of bacterial species was performed using anautomated system and confirmed by biochemical tests, 16S rRNA gene sequencing, and detection of resistance genes.

RESULTS:

The clinical isolate showed minimum inhibitory concentration resistance to meropenem and polymyxin B at 8mg/L and 4mg/L, respectively. Only the blaNDM gene was detected.

CONCLUSIONS:

The current report of the blaNDM gene in isolated MDR enterobacteria indicates that this gene can spread silently in a hospital setting.

Keywords:
Klebsiella aerogenes; blaNDM gene; metallo-betalactamase; Carbapenemase; Multidrug-resistant

New Delhi metallo-beta-lactamase (NDM), which codes for the bla NDM gene, is found in carbapenemase-producing Enterobacteriaceae, and the highest distributions of bla NDM variants have been detected in Klebsiella pneumoniae and Escherichia coli11. Khan AU, Maryam L, Zarrilli R. Structure, Genetics and Worldwide Spread of New Delhi Metallo-β-lactamase (NDM): a threat to public health. BMC Microbiol. 2017; 17(1):101.,22. Pérez-Vázquez M, Sola Campoy PJ, Ortega A, Bautista V, Monzón S, Ruiz-Carrascoso G, et al. Emergence of NDM-producing Klebsiella pneumoniae and Escherichia coli in Spain: phylogeny, resistome, virulence and plasmids encoding blaNDM-like genes as determined by WGS. J Antimicrob Chemother. 2019;74(12):3489-96.. The occurrence of the bla NDM gene in Klebsiella aerogenes has also been documented in countries such as China33. Shen Y, Xiao WQ, Gong JM, Pan J, Xu QX. Detection of New Delhi Metallo-Beta-Lactamase (Encoded by blaNDM-1) inKlebsiella aerogenesin China. J Clin Lab Anal. 2017;31(2):1-5., South Korea44. Yoon EJ, Kang DY, Yang JW, Kim D, Lee H, Lee KJ, et al. New Delhi Metallo-Beta-Lactamase-Producing Enterobacteriaceae in South Korea Between 2010 and 2015. Front Microbiol. 2018; 29;9:571., Tunisia55. Ben Helal R, Dziri R, Chedly M, Klibi N, Barguellil F, El MS Asli, et al. Occurrence and Characterization of Carbapenemase-Producing Enterobacteriaceae in a Tunisian Hospital. Microb Drug Resist. 2018;24(9):1361-7., Japan66. An J, Guo L, Zhou L, Ma Y, Luo Y, Tao C, et al. NDM-producing Enterobacteriaceae in a Chinese hospital, 2014-2015: identification of NDM-producing Citrobacter werkmanii and acquisition of blaNDM-1-carrying plasmid in vivo in a clinical Escherichia coli isolate. J Med Microbiol. 2016;65(11):1253-9., and India77. Khalid S, Ahmad N, Ali SM, Khan AU. Outbreak of Efficiently Transferred Carbapenem-Resistantbla NDM-Producing Gram-Negative Bacilli Isolated from Neonatal Intensive Care Unit of an Indian Hospital. Microb Drug Resist. 2020;26(3):284-9.,88. Ahmad N, Khalid S, Ali SM, Khan AU. Occurrence ofbla NDMVariants Among Enterobacteriaceae From a Neonatal Intensive Care Unit in a Northern India Hospital. Front Microbiol . 2018;9:407.. In South America, the bla NDM gene was first reported in 2012 in Uruguay99. Carvalho-Assef AP, Pereira PS, Albano RM, Berião GC, Chagas TP, Timm LN, et al. Isolation of NDM -producing Providencia rettgeri in Brazil. J Antimicrob Chemother. 2013;68(12):2956-7.. In Brazil, this gene has been detected in only 11 isolates (0.97%) from Porto Alegre, with nine from Enterobacter cloacae and two from Morganella morganii complexes. Moreover, other isolates showed high resistance to carbapenems1010. Rozales FP, Ribeiro VB, Magagnin CM, Pagano M, Lutz L, Falci DR, et al. Emergence of NDM‐1‐producing Enterobacteriaceae in Porto Alegre, Brazil. Int J Infect Dis. 2014;(25):79-81.. The spread of the bla NDM gene in various species of gram-negative bacilli has occurred in different Brazilian regions1111. da Silva IR, Aires CAM, Conceição-Neto OC, de Oliveira Santos IC, Ferreira Pereira N, Moreno Senna JP, et al. Distribution of Clinical NDM-1-Producing Gram-Negative Bacteria in Brazil. Microbial Drug Resistance. 2019;25(3):394-9.. In 2019, the first report of Proteus mirabilis and Serratia marcescens carrying bla KPC-2 and bla NDM-1 genes in Brazil was published1212. Firmo EF, Beltrão EMB, Silva FRF, Alves LC, Brayner FA, Veras DL, et al. Association of blaNDM-1 with blaKPC-2 and aminoglycoside-modifying enzymes genes among Klebsiella pneumoniae, Proteus mirabilis and Serratia marcescens clinical isolates in Brazil. J Glob Antimicrob Resist. 2019;21:255-61.. The bla NDM-1 gene product is NDM-1, which hydrolyzes a wide range of β-lactam antimicrobials, including carbapenems, which are the last-resort antimicrobials in the treatment of infections caused by antimicrobial-resistant bacteria. The emergence of bacteria carrying these genes represents a challenge in the treatment of infections11. Khan AU, Maryam L, Zarrilli R. Structure, Genetics and Worldwide Spread of New Delhi Metallo-β-lactamase (NDM): a threat to public health. BMC Microbiol. 2017; 17(1):101..The objective of this study was to describe the occurrence of the bla NDM gene in a clinical isolate of K. aerogenes resistant to aminopenicillin, beta-lactam + beta-lactamase inhibitors, and 1st, 2nd, 3rd, and 4th generation beta-lactams, as well as carbapenems.

Identification of bacterial species was performed usinga Vitek-2 automated system (bioMérieux, Marcy I’Etoile, France), and confirmed by biochemical tests and 16S rRNA gene sequencing. PCR and DNA sequencing were performed to track the presence of the bla NDM gene in two duplicates, collected at a referral hospital in Recife (Pernambuco, Brazil) in 2019. A universal primer pair, NDM-F (5-CGGAATGGCTCATCACGATC-3) and NDM-R (5-GGTTTGGCGATCTGGTTTTC-3)1313. Nithia N, Remitha R, Jayasree PR, Faisal M, Manish Kumar PR. Analysis of beta-lactamases, blaNDM-1 phylogeny & plasmid replicons in multidrug-resistant Klebsiella spp. from a tertiary care centre in south India. Indian J Med Res. 2017;146(1):38-45.were used for screening, as previously described. Furthermore, the entire bla NDM region was amplified and sequenced based on consensus sequences flanking the gene. The analysis showed that the bla NDM gene was present in a K. aerogenes clinical isolate from a patient with bacteremia hospitalized in an intensive care unit (ICU). The minimum inhibitory concentrations (MIC) of meropenem and polymyxin B for this isolate were 8mg/L and 4mg/L, respectively. In addition, this isolate presented a susceptibility of 8mg/L to amikacin and 1mg/L to colistin, according to the Brazilian Committee on Antimicrobial Susceptibility Testing (BrCAST)1414. BrCAST - Brazilian Committee on Antimicrobial Susceptibility Testing. Tabelas de pontos de corte clínicos 2020-01-05-2020 [updated 2020 January 05; cited 2020 Fev 14]. Available in: Available in: http://brcast.org.br/documentos/ .
http://brcast.org.br/documentos/...
. These data were confirmed by colorimetric assays with resazurin1515. Palomino JC, Martin A, Camacho M, Guerra H, Swings J, Portaels F. Resazurinmicrotiter assay plate: simple and inexpensive method for detection of drug resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother. 2002;46(8):2720-2..

The K. aerogenes isolate showed an antimicrobial resistance profile (R) to cefepime, ceftazidime, cefoxitin, ceftriaxone, ciprofloxacin, ampicillin, ampicillin/sulbactam, cefotaxime, and carbapenems (imepenem, meropenem, ertapenem), which differs from its resistance profile in China33. Shen Y, Xiao WQ, Gong JM, Pan J, Xu QX. Detection of New Delhi Metallo-Beta-Lactamase (Encoded by blaNDM-1) inKlebsiella aerogenesin China. J Clin Lab Anal. 2017;31(2):1-5.. High resistance to carbapenems has also been observed in other Enterobacteriaceae isolates1010. Rozales FP, Ribeiro VB, Magagnin CM, Pagano M, Lutz L, Falci DR, et al. Emergence of NDM‐1‐producing Enterobacteriaceae in Porto Alegre, Brazil. Int J Infect Dis. 2014;(25):79-81.. However, it was susceptible (S) to amikacin, gentamicin, tobramycin, tigecycline, colistin, and polymyxin B, and sensitive, with increasing exposure (I), to ciprofloxacin, according to BrCAST1414. BrCAST - Brazilian Committee on Antimicrobial Susceptibility Testing. Tabelas de pontos de corte clínicos 2020-01-05-2020 [updated 2020 January 05; cited 2020 Fev 14]. Available in: Available in: http://brcast.org.br/documentos/ .
http://brcast.org.br/documentos/...
. The present study detected the presence of the broad-spectrum beta-lactamase and beta-lactamase class C beta-lactamase phenotypes in K. aerogenes isolates. The presence of bla NDM was confirmed by polymerase chain reaction (PCR). To our knowledge, this is the first report of the bla NDM gene in clinical isolates of K. aerogenes from Brazil. In addition, other carbapenemase genes, such as bla IMP, bla VIM, bla GES, bla OXA-48, and bla KPC were investigated, as well as the mcr-1 gene. Despite being susceptible to polymyxin B by automated testing, the MIC showed resistance, but the mcr-1 gene was not detected. However, none of the other genes were detected in the clinical isolates used. NDM-producing strains, including carbapenems, cephamycin, extended-spectrum cephalosporins, aminoglycosides, monobactamines, tetracyclines, fluoroquinolones, and polymyxin and colistin88. Ahmad N, Khalid S, Ali SM, Khan AU. Occurrence ofbla NDMVariants Among Enterobacteriaceae From a Neonatal Intensive Care Unit in a Northern India Hospital. Front Microbiol . 2018;9:407., were detected in neonates in the ICU with high antibiotic resistance.

NDM-1-producing Enterobacteriaceae has spread rapidly in hospitalized patients in several countries. Thus, the spread of carbapenemase-resistant genes has been a major concern worldwide, as it poses a threat to the antimicrobial treatment of infections caused by multidrug-resistant isolates. Although strains that produce bla NDM appear less frequently in Brazil, it is nonetheless necessary to control epidemiological surveillance to control infections.

Nucleotide sequence accession number: The nucleotide sequence of the blaNDM gene in the K. aerogenes isolate was GenBank/EMBL/DDBJ, accession Nº. MN735678.1.

This study was approved by the ethics committee of our institution (CAAE: 20195119.3.0000.5586), opinion number 3,787,364, and (CAAE: 20195119.3.3001.5198) opinion number 3,838,892, following Resolution 466/12 of the National Health Council.

ACKNOWLEDGMENTS

We thank all the staff and patients of the referral public hospital for their collaboration.

REFERENCES

  • 1
    Khan AU, Maryam L, Zarrilli R. Structure, Genetics and Worldwide Spread of New Delhi Metallo-β-lactamase (NDM): a threat to public health. BMC Microbiol. 2017; 17(1):101.
  • 2
    Pérez-Vázquez M, Sola Campoy PJ, Ortega A, Bautista V, Monzón S, Ruiz-Carrascoso G, et al. Emergence of NDM-producing Klebsiella pneumoniae and Escherichia coli in Spain: phylogeny, resistome, virulence and plasmids encoding blaNDM-like genes as determined by WGS. J Antimicrob Chemother. 2019;74(12):3489-96.
  • 3
    Shen Y, Xiao WQ, Gong JM, Pan J, Xu QX. Detection of New Delhi Metallo-Beta-Lactamase (Encoded by blaNDM-1) inKlebsiella aerogenesin China. J Clin Lab Anal. 2017;31(2):1-5.
  • 4
    Yoon EJ, Kang DY, Yang JW, Kim D, Lee H, Lee KJ, et al. New Delhi Metallo-Beta-Lactamase-Producing Enterobacteriaceae in South Korea Between 2010 and 2015. Front Microbiol. 2018; 29;9:571.
  • 5
    Ben Helal R, Dziri R, Chedly M, Klibi N, Barguellil F, El MS Asli, et al. Occurrence and Characterization of Carbapenemase-Producing Enterobacteriaceae in a Tunisian Hospital. Microb Drug Resist. 2018;24(9):1361-7.
  • 6
    An J, Guo L, Zhou L, Ma Y, Luo Y, Tao C, et al. NDM-producing Enterobacteriaceae in a Chinese hospital, 2014-2015: identification of NDM-producing Citrobacter werkmanii and acquisition of blaNDM-1-carrying plasmid in vivo in a clinical Escherichia coli isolate. J Med Microbiol. 2016;65(11):1253-9.
  • 7
    Khalid S, Ahmad N, Ali SM, Khan AU. Outbreak of Efficiently Transferred Carbapenem-Resistantbla NDM-Producing Gram-Negative Bacilli Isolated from Neonatal Intensive Care Unit of an Indian Hospital. Microb Drug Resist. 2020;26(3):284-9.
  • 8
    Ahmad N, Khalid S, Ali SM, Khan AU. Occurrence ofbla NDMVariants Among Enterobacteriaceae From a Neonatal Intensive Care Unit in a Northern India Hospital. Front Microbiol . 2018;9:407.
  • 9
    Carvalho-Assef AP, Pereira PS, Albano RM, Berião GC, Chagas TP, Timm LN, et al. Isolation of NDM -producing Providencia rettgeri in Brazil. J Antimicrob Chemother. 2013;68(12):2956-7.
  • 10
    Rozales FP, Ribeiro VB, Magagnin CM, Pagano M, Lutz L, Falci DR, et al. Emergence of NDM‐1‐producing Enterobacteriaceae in Porto Alegre, Brazil. Int J Infect Dis. 2014;(25):79-81.
  • 11
    da Silva IR, Aires CAM, Conceição-Neto OC, de Oliveira Santos IC, Ferreira Pereira N, Moreno Senna JP, et al. Distribution of Clinical NDM-1-Producing Gram-Negative Bacteria in Brazil. Microbial Drug Resistance. 2019;25(3):394-9.
  • 12
    Firmo EF, Beltrão EMB, Silva FRF, Alves LC, Brayner FA, Veras DL, et al. Association of blaNDM-1 with blaKPC-2 and aminoglycoside-modifying enzymes genes among Klebsiella pneumoniae, Proteus mirabilis and Serratia marcescens clinical isolates in Brazil. J Glob Antimicrob Resist. 2019;21:255-61.
  • 13
    Nithia N, Remitha R, Jayasree PR, Faisal M, Manish Kumar PR. Analysis of beta-lactamases, blaNDM-1 phylogeny & plasmid replicons in multidrug-resistant Klebsiella spp. from a tertiary care centre in south India. Indian J Med Res. 2017;146(1):38-45.
  • 14
    BrCAST - Brazilian Committee on Antimicrobial Susceptibility Testing. Tabelas de pontos de corte clínicos 2020-01-05-2020 [updated 2020 January 05; cited 2020 Fev 14]. Available in: Available in: http://brcast.org.br/documentos/
    » http://brcast.org.br/documentos/
  • 15
    Palomino JC, Martin A, Camacho M, Guerra H, Swings J, Portaels F. Resazurinmicrotiter assay plate: simple and inexpensive method for detection of drug resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother. 2002;46(8):2720-2.

  • Financial Support: This research did not receive any grant from funding agencies in the public, commercial, or not-for-profit sectors.

Publication Dates

  • Publication in this collection
    21 Dec 2020
  • Date of issue
    2021

History

  • Received
    11 May 2020
  • Accepted
    02 Sept 2020
Sociedade Brasileira de Medicina Tropical - SBMT Caixa Postal 118, 38001-970 Uberaba MG Brazil, Tel.: +55 34 3318-5255 / +55 34 3318-5636/ +55 34 3318-5287, http://rsbmt.org.br/ - Uberaba - MG - Brazil
E-mail: rsbmt@uftm.edu.br