Abstract
Resistance to carbapenems due to metallo-beta-lactamase NDM-1 was first described in Brazil in 2013. To date, only a few scattered reports of the prevalence of NDM-1 in the country have been reported, and most of them indicated a very low prevalence of this metalloenzyme. In the present study, we report a steady increase in the frequency of NDM among Enterobacterales resistant to carbapenems in a tertiary care hospital in southern Brazil. Carbapenemase genes were evaluated by multiplex real-time polymerase chain using high-resolution melting analysis among 3501 isolates of 8 different species of Enterobacterales recovered from January 2015 to May 2020. The blaKPC-like was identified in 3003 isolates (85.8%) and the blaNDM-like was the second most common gene (351 isolates—10%). There was a steady increase in frequency of blaNDM-like, from 4.2% in 2015 to 24% in 2020. The increase of blaNDM frequency raises an important matter as novel therapeutic options are currently very limited for the treatment of patients infected by bacteria carrying the blaNDM.
Keywords: Carbapenemase, Dissemination, NDM-1, New Delhi metallo-beta-lactamase, Resistance
In Brazil, resistance to carbapenems due to NDM-1 was first described in 2013 in a Providencia rettgeri in the city of Porto Alegre [1]. Since then, NDM-producing bacteria was heralded as the next public health threat in Brazil. However, this metallo-beta-lactamase (MBL) has been reported only sporadically in different regions of the country and in very low prevalence [2]. Nevertheless, Klebsiella pneumoniae carbapenemase type 2 (KPC-2) has been reported as the main carbapenemase in Brazil reaching the prevalence of more than 76% among carbapenem-resistant Enterobacterales [3, 4]. Similar to KPC, NDM has been associated with multidrug resistance and has been reported from various Brazilian states and in different gram-negative species, including Enterobacter cloacae, E. hormaechei, P. rettgeri, Escherichia coli, Klebsiella pneumoniae, Acinetobacter pittii, K. oxytoca, Citrobacter freundii, A. bereziniae, Proteus mirabilis, and A. baumannii [2, 3]. In 2013, we described the prevalence of NDM as only 0.97% among Enterobacterales with reduced susceptibility to carbapenems from several institutions in southern Brazil [4].
In order to monitor the frequency of the carbapenemase-encoding genes among carbapenem-resistant Enterobacterales in a tertiary teaching hospital located in the city of Porto Alegre, Rio Grande do Sul state, Brazil, we have evaluated carbapenem-resistant isolates since 2015. Isolates exhibiting resistance to at least meropenem were considered carbapenem-resistant isolates. Bacterial identification was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Biomérieux, France) and antimicrobial susceptibility testing was performed by disk diffusion method as described by the Clinical and Laboratory Standards Institute guidelines [5]. Carbapenem-resistant isolates were selected and carbapenemase genes were identified by multiplex real-time polymerase chain reaction (RT-PCR) using a high-resolution melting (HRM) for blaKPC-like, blaNDM-like, blaOXA-48-like, blaIMP-like, blaVIM-like, and blaGES-like as described previously [6]. Statistical analysis was performed using the Cochran-Armitage test.
A total of 3501 isolates of Enterobacterales including Klebsiella pneumoniae complex, K. oxytoca, Enterobacter cloacae complex, E. aerogenes, E. gergoviae, E. hormaechei, Escherichia coli, Citrobacter freundii complex, C. braakii, C. koseri, C. werkmanii, Serratia marcescens, Morganella morganii, and Providencia rettgeri were evaluated from January 2015 to May 2020. The blaNDM-like was the second most common gene (351 isolates—10%), whereas the blaKPC-like was identified in the majority of the isolates (3003 isolates—85.8%). The blaOXA-48-like, blaGES-like, blaIMP-like, and blaVIM-like were also found among carbapenem-resistant Enterobacterales collection (6, 5, 2, and 2 isolates, respectively). A total of 48 isolates presented both blaKPC-like and blaNDM-like by HRM-RT-PCR (42 K. pneumoniae, 2 Enterobacter hormaechei, 2 Enterobacter cloacae complex, 1 E. coli, and 1 S. marcescens).
Relatively few studies have reported Enterobacterales isolates harboring more than one carbapenemase gene. Rozales et al. described the characteristics of 10 Enterobacterales co-harboring carbapenemase genes: 5 E. cloacae complex with blaNDM-1 and blaOXA-370 genes, 3 K. pneumoniae and 1 E. cloacae complex with blaNDM-1 and blaKPC-2 genes, and 1 K. pneumoniae with blaKPC-2 and blaOXA-370 genes [7]. Balm et al. and Both et al. reported a K. pneumoniae isolate co-harboring blaNDM and blaOXA-181 genes and a E.coli coproducing blaNDM-1 and blaOXA-232, respectively [8, 9].
In this report, we observed a significant increase in frequency of blaNDM-like, from 4.2% in 2015 to 24% in 2020 (p < 0.001; Fig. 1). The frequency of blaNDM-like per species was as follows: Klebsiella pneumoniae complex (224/3122 isolates), Klebsiella oxytoca (15/39 isolates), Enterobacter spp. (75/195 isolates), Escherichia coli (14/38 isolates), Citrobacter spp. (15/26 isolates), Serratia marcescens (5/31 isolates), Morganella morganii (1/4 isolates), Providencia rettgeri (1/1 isolate), and Providencia spp. (1/1 isolate). In fact, to the best of our knowledge, this is the first report of the blaNDM gene in a S. marcescens.
Fig. 1.
blaKPC-like (a) and blaNDM-like (b) producing Enterobacterales distribution from January 2015 to May 2020
Conversely to the increase of blaNDM-like, it was possible to note a trend of decrease of blaKPC-like frequency during the same period (from 90% in 2015 to 77% in 2020), which did not present a statistical significance (p = 0.668). It is worth to mention that only carbapenem-resistant isolates were included in this study, i.e., isolates with “intermediate” result of meropenem were not evaluated; thus, the frequency of blaNDM-like in our hospital may be even higher.
The increase of blaNDM-like frequency raises an important matter as therapeutic options are currently very limited for the treatment of patients infected by bacteria carrying blaNDM-like compared to blaKPC-like bacteria. MBLs are considered to increase the risk to public health because they hydrolyze all classes of beta-lactams except monobactams (aztreonam) and are not inhibited by the new beta-lactam/beta-lactamase inhibitor combinations, such as ceftazidime/avibactam; therefore, the predominance of NDM will affect the use of new therapeutic alternatives and treatment with new beta-lactam/beta-lactamase inhibitors targeting serine carbapenemases would need to be guided by the susceptibility test.
In this study, it was not possible to determine what led to an increase in the frequency of NDM-producing Enterobacterales. Since ceftazidime/avibactam has been introduced in our institution in early 2019 and it has been prescribed only to 6 patients in 2019 and 23 patients in 2020 (data not shown), we believe that selective pressure over KPC-producing isolates caused by a more extensive use of ceftazidime-avibactam does not explain the rise of isolates with this MBL noted since early 2017.
Another aspect to be considered is that NDM-positive Enterobacterales may co-harbor additional genes that can confer resistance by other routes. Unfortunately, aztreonam is readily inactivated by Ambler class A beta-lactamases, including extended-spectrum beta-lactamases and KPC, impairing the activity of aztreonam against these isolates [10].
A limitation of our study was that molecular typing of the blaNDM-positive isolates was not performed. However, as the blaNDM gene was identified in several different species of Enterobacterales, one could consider that the increased frequency of this MBL is not due to (at least not solely) the spread of a specific clone.
Our finds regarding the increase frequency of blaNDM-like in our institution highlight the importance of early detection of the molecular mechanism underlying carbapenem resistance to avoid the empirical treatment with ceftazidime/avibactam. Hence, there is an urgent need to establish recommendations for tackling NDM carbapenem-resistant infections in southern Brazil.
Funding
This work has been funded by INPRA - Instituto Nacional de Pesquisa em Resistência Antimicrobiana - Brazil (INCT/CNPq: 465718/2014-0 and INCT/FAPERGS: 17/2551-0000514-7) and by Fundo de Incentivo a Pesquisa e Eventos do Hospital de Clínicas de Porto Alegre (FIPE/HCPA) (Project no. 19-0203).
Compliance with ethical standards
Conflict of interest
APZ received a research grant from Pfizer.
Footnotes
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