The Spread of NDM-1 and NDM-7-Producing Klebsiella pneumoniae Is Driven by Multiclonal Expansion of High-Risk Clones in Healthcare Institutions in the State of Pará, Brazilian Amazon Region

Carbapenem resistance among Klebsiella pneumoniae isolates is often related to carbapenemase genes, located in genetic transmissible elements, particularly the blaKPC gene, which variants are spread in several countries. Recently, reports of K. pneumoniae isolates harboring the blaNDM gene have increased dramatically along with the dissemination of epidemic high-risk clones (HRCs). In the present study, we report the multiclonal spread of New Delhi metallo-beta-lactamase (NDM)-producing K. pneumoniae in different healthcare institutions in the state of Pará, Northern Brazil. A total of 23 NDM-producing isolates were tested regarding antimicrobial susceptibility testing features, screening of carbapenemase genes, and genotyping by multilocus sequencing typing (MLST). All K. pneumoniae isolates were determined as multidrug-resistant (MDR), being mainly resistant to carbapenems, cephalosporins, and fluoroquinolones. The blaNDM-7 (60.9%—14/23) and blaNDM-1 (34.8%—8/23) variants were detected. MLST genotyping revealed the predomination of HRCs, including ST11/CC258, ST340/CC258, ST15/CC15, ST392/CC147, among others. To conclude, the present study reveals the contribution of HRCs and non-HRCs in the spread of NDM-1 and NDM-7-producing K. pneumoniae isolates in Northern (Amazon region) Brazil, along with the first detection of NDM-7 variant in Latin America and Brazil, highlighting the need for surveillance and control of strains that may negatively impact healthcare and antimicrobial resistance.


Introduction
Klebsiella pneumoniae is among the major pathogens causing healthcare-related infections (HAIs) and outbreaks in several healthcare institutions. This is due to its antimicrobial resistance (AMR) and virulence traits, leading to the increasingly frequent reports of severe infections, poor prognosis outcomes, and limitation of antimicrobial therapy options [1,2].
MLST genotyping and genetic relationship analysis revealed a genetic background mostly composed by HRCs, including nine sequence types (STs) associated with four clonal complexes (CCs), including: HRC ST11/CC258 ( (Table 1 and Figure 1).

Discussion
The emergence and spread of MDR CR-Kp strains have been reported worldwide and in Brazil, causing a critical impact on the increasing levels of antimicrobial resistance and leading to a public health crisis due to the limitation of antimicrobial therapy options, infection severity, and challenging spread control. Such strains are usually related to epidemic HRCs harboring plasmid-borne carbapenemases, particularly the blaKPC, and recently blaNDM, emphasizing the importance of epidemiological vigilance and recognizing medically relevant resistant features strains. The present study reports the spread of NDM-producing K. pneumoniae strains associated with HRCs in different healthcare institutions in the state of Pará, Brazilian Amazon region.

Discussion
The emergence and spread of MDR CR-Kp strains have been reported worldwide and in Brazil, causing a critical impact on the increasing levels of antimicrobial resistance and leading to a public health crisis due to the limitation of antimicrobial therapy options, infection severity, and challenging spread control. Such strains are usually related to epidemic HRCs harboring plasmid-borne carbapenemases, particularly the bla KPC , and recently bla NDM , emphasizing the importance of epidemiological vigilance and recognizing medically relevant resistant features strains. The present study reports the spread of NDMproducing K. pneumoniae strains associated with HRCs in different healthcare institutions in the state of Pará, Brazilian Amazon region.
The resistance trend for 3rd/4th generation cephalosporins and carbapenems among K. pneumoniae isolates have been reported in several countries, and in Latin America, countries including Brazil, Argentina and Chile account for most of the cases [1,2,5]. Indeed, on recently reported data of national surveillance on antimicrobial resistance, K. pneumoniae isolated in adult and pediatric Brazilian ICUs presented resistance rates ranging 49.3% to 68.1% and 19.3% to 51.8% to 3rd/4th generation cephalosporins and carbapenems, respectively [19]. Previous reports suggest the increasing prevalence of MDR CR-Kp isolates in the Brazilian Amazon region [20][21][22]. As in line with the discussed data, the antimicrobial susceptibility features of K. pneumoniae isolates in our study demonstrated that all tested isolates were MDR (possible XDR), predominantly exhibiting combined resistance to 3rd/4th generation cephalosporins, carbapenems, and fluoroquinolones. Furthermore, even though the transmission of MDR K. pneumoniae between patients is an important mechanism for outbreaks occurrence, especially in ICUs [23,24], the presence of highly resistant isolates in non-ICU settings (13/23-56.5%) was observed. This points out the spread of these strains in different wards and/or different healthcare facilities, which may also be associated and contribute to disseminating antibiotic resistance markers, such as bla NDM and bla KPC .
CR-Kp has been typically related to transmissible genetic elements added to carbapenemases genes, such as bla NDM and bla KPC , which have been increasingly detected in different countries, emphasizing their worldwide dissemination [7,8,25,26]. In Brazil, KPC has become endemic and widely disseminated among K. pneumoniae from hospitals in all Brazilian regions [5,14,17,20,21,[27][28][29][30]. Since 2013, NDM-producing isolates have been reported in different Brazilian regions and associated with a wide variety of bacterial species, including Escherichia coli, Acinetobacter baumannii, A. nosocomialis, A. pittii, Enterobacter cloacae, Enterobacter hormaechei, and K. pneumoniae [12,15,16,[31][32][33][34][35]. Recently, Silva et al. [16], in a study evaluating 81 bacterial isolates from different regions, demonstrated that K. pneumoniae is responsible for the widespread of this carbapenemase in Brazilian hospitals, confirming more efficient dissemination compared with the bla KPC variant. The study, however, was limited by not including isolates from the Brazilian Amazon region, evidencing the absence of data on NDM-producing K. pneumoniae isolates in the region. Interestingly, the present study revealed 23 CR-Kp isolates were harboring bla NDM gene in nine different hospitals in the same region. This elevated frequency of NDM-producing CR-Kp has been only reported by Vivas et al. [15] in Sergipe (Northeast Brazil), where among the 147 investigated isolates, over 50.0% were bla NDM carriers.
The NDM-7 variant was firstly described in E. coli and presented in its structure two amino acid substitutions compared to NDM-1 [36,37]. This variant has been related to infection by MDR microorganisms, predominantly in Asian countries, such as India, Japan, and China [38][39][40]. Despite being mainly associated with E. coli and K. pneumoniae isolates, NDM-7 has already been described in other members of Enterobacterales, reinforcing its ability to spread among different bacterial genera [41]. Furthermore, some studies have suggested more enzymatic hydrolysis activity against carbapenem, comparing NDM-7 to NDM-1 [42]. This study describes the alarming dissemination of NDM -1 and NDM-7 in association with K. pneumoniae HRCs in different healthcare institutions in northern Brazil, being the first detection of NDM-7 circulating in Latin America and Brazil.
Worryingly, the co-production and accumulation of genetic resistance determinants have become typically reported among highly resistant K. pneumoniae isolates. Certainly, most of the NDM-producing isolates co-harbor a broad variety of resistance mechanisms, such as CTX-M, SHV, KPC, VIM, and OXA-48, highlighting the increasing incidence of bla NDM and bla KPC co-producing strains, as reported in China, USA, Greece, India, Pakistan and Turkey [7,25,[43][44][45][46][47][48]. In April 2021, Argentina's National Antimicrobial Reference Laboratory was alerted to the emergence and spread of Enterobacterales producing different combinations of carbapenemases, especially during the first wave of the COVID-19 pandemic. Approximately one-third of the isolates received at this center (27.0%-52/196) were co-producers, with a combination of serine and MβLs, of which 60% had a combination of KPC and NDM [49]. In Brazil, the presence of over 20 bla NDM and bla KPC K. pneumoniae co-producing isolates, distributed among the Northeast, Southeast and South regions, have been described [12,15,27,31,50]. In the present study, four CR-Kp isolates were detected co-harboring bla NDM and bla KPC . Three were recovered from patients hospitalized in the pediatric ICU at the same hospital (H3), suggesting that the persistence of high resistance levels strains in the environment and urgent strengthening of surveillance measures.
Investigations on the molecular epidemiology of K. pneumoniae isolates demonstrated that a small subset of successful HRCs is responsible for undermining antimicrobial therapy options, severe and poor prognosis infections, and nosocomial outbreaks globally. K. pneumoniae HCRs exhibit a high resistance degree, including resistance to 3rd/4th generation cephalosporins and carbapenems, and are remarkably effective reservoirs and vehicles for disseminating genetic resistance mechanisms, such as ESBLs and carbapenemases [7,10,51]. Molecular epidemiology analysis based on MLST revealed an absence of relationship among most of the evaluated K. pneumoniae strains, including the presence of nine distinct STs belonging to four CCs and predominance of MDR HRCs, which corroborates the hypothesis of epidemic multiclonal expansion of K. pneumoniae in Brazilian hospitals and globally.
Carbapenemase-producing clones are mostly associated with CC258, including STs 11, 101, 258, 340, 437, 512, 874, and 1264. ST11 and ST340/CC258 are globally distributed and have been detected harboring carbapenemases regardless of their type, and in Brazil, they are mostly associated with KPC-producing strains in almost all Brazilian regions [7,13,14,17,[52][53][54][55]. ST1264 has emerged (and probably highly restricted) in China, causing bloodstream infection, and recently associated with ESBL-producing isolates [56,57]. Recently, several reports indicate ST340/CC258 has a critical role in expanding NDM-producing strains in Brazil, being associated with monoclonal outbreaks, colistinresistant-isolates, and isolates co-harboring bla NDM and bla KPC genes [9,12,18,58]. Epidemic ST11 was the most prevalent in our study, found dispersed in five distinct hospitals in different periods, and interestingly present in pediatric settings at H3, co-harboring bla NDM, and bla KPC , indicating endemicity and spread these resistance markers in this environment. ST340 was related to a single isolate recovered from a patient in the adult ICU at H3. To the best of our knowledge, the present study first provides data on the dissemination of NDM and NDM/KPC-producing K. pneumoniae associated with ST11/CC258 among clinical isolates in Brazil and corroborates the dissemination of ST340 NDM-producing strains in Brazil. Finally, our results also contribute to a better comprehension of the epidemiology of clinically important carbapenem resistance markers in the Brazilian territory and globally.
The HRC ST15/CC15 is usually related to CR-Kp carrying plasmid-borne MβLs in several locations in Europe, India, Nepal, Pakistan, and China, indicating a high capacity for the horizontal acquisition of resistance genes [6,10,59,60]. In Latin America, ST15/CC15 has been less frequently described than ST11 and ST340. However, it has demonstrated a concerning spread, with strains particularly harboring the bla NDM gene and rarely associated with bla KPC [54,61]. Since its first report in Brazil by Gonçalves et al. [17], ST15/CC15 has been detected in clinical and environmental samples, but with few reported NDMproducing strains in Porto Alegre (South region), Rio de Janeiro (Southeast), and Brasilia (Midwest region) cities [9,50,54,62]. According to our data, NDM-producing ST15/CC15 strains have been found circulating in four different hospitals since 2018 and mostly present adult settings, suggesting an early and rapid spread across Brazil.
K. pneumoniae ST392/CC147 has been reported related to nosocomial infection in various countries and described as an emerging clinically important HRC. Its endemicity has been related to the clonal spread of KPC-3-producing strains in Italy, VIM-producing strains in Grecia, while in Colombia, Mexico, and Iran with NDM-producing strains [63][64][65][66][67].
In Brazil, few reports revealed strains co-harboring bla KPC /mcr-1 and bla KPC /bla OXA in Espírito Santo (Southern Brazil) and Tocantins (Northern Brazil), respectively [12,21]. In the present study, ST392 was detected in the pediatric clinic at H3, and, as far as we know, this is the first report of NDM-producing K. pneumoniae belonging to ST392/CC147 in Brazil.
Despite the non-HRCs being found related with the minority of NDM-producing K. pneumoniae isolates in our study, they were interestingly detected carrying the bla NDM-7 variant. ST1401 was first described in a human blood sample from Kuwait and has been reported in the USA and Mexico [68][69][70]. ST138/CC138 has been associated with KPC-2-producing and NDM-7-producing isolates in Brazil and Canada, respectively [14,71]. ST3449 was detected in China in an isolate harboring the bla NDM gene [72]. An NDM/KPC-Antibiotics 2021, 10, 1527 8 of 13 producing K. quasipneumoniae isolate belonging to ST3512 was to be causing bloodstream infection in Bahia State (Northeast Brazil) [11]. Wyres et al. [10] highlight that non-HCR may only cause localized problems; however, their emergence, spread, and persistence as HRC are influenced by several factors, which are mostly unknown. In this sense, our results draw attention to the importance of non-HRCs in the spread of NDM-7-producing strains at four hospitals (H1, H2, H7, and H8) in the Brazilian Amazon region, also been, to the best of our knowledge, the first report of ST1401, ST138, ST3449 and ST4398 carrying the bla NDM-7 gene in Latin America and Brazil.

Bacterial Isolates and Species Identification
The present cross-sectional study included 23 non-duplicated K. pneumoniae isolates stored at the Bacteriology and Mycology Section, Evandro Chagas Institute, a referral surveillance center located in the State of Pará, Brazilian Amazon region. From January 2018 to February 2021, K. pneumoniae isolates were obtained from several clinical sources of non-consecutive patients admitted at nine different hospitals in the region (H1-H9). Bacterial suspensions for each sample were prepared to match the 0.5 McFarland standard, followed by isolates identification on the automated VITEK-2 system (bioMérieux, Marcy l'Etoile, France) using the VITEK-2 card GN Test Kit Ref: 21341 for Gram-negative species identification.
Additionally, isolates were phenotypically categorized as MDR if resistant to ≥1 drug in ≥3 antimicrobial categories, according to criteria proposed by Magiorakos et al. [75]. Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as quality control strains. Finally, all isolates exhibiting non-susceptibility to carbapenems (IMP, MER and/or ETP) were phenotypically tested for the presence of carbapenemase using the test of inactivation of carbapenem (mCIM/eCIM), following the CLSI recommendations [73].

Molecular Screening of β-Lactamase-Encoding Genes
Bacterial genomic DNA was extracted from overnight cultures, where a single K. pneumoniae colony was suspended in 300µL of distilled water and boiled at 95 • C for 10 min, followed by incubation at −20 • C for 15 min, and final centrifugation at 12,000 rpm for 7 min. The obtained DNA samples were stored at −20 • C until testing and used for all molecular assays. The molecular detection of β-lactamase-related genes (bla KPC , bla NDM , bla IMP , bla VIM , and bla OXA-48 ) was performed by PCR using the previously described primers and methodology [76]. PCR products were analyzed under UV light after electrophoresis on 1% agarose gel stained with SYBR™ Safe DNA gel stain (Life Technologies, Carlsbad, CA, USA). Definition of bla NDM and bla KPC subtypes was performed by direct sequencing of purified PCR products using the BigDye™ Terminator v3.1 Cycle Sequencing Kit (Life Technologies, Carlsbad, CA, USA) on an ABI Prism 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). This was followed by the analysis of the obtained sequences at the BLAST search, available at the NCBI website (http://blast.ncbi.nih.gov/Blast.cgi (accessed on: 21 October 2021)) [76,77].

Genetic Diversity Assessment by MLST
Molecular typing by MLST was performed in accordance with a protocol previously described by Diancourt et al. [78], slightly modified by using universal sequencing primers. The seven housekeeping genes included in the scheme (gapA, infB, mdh, pgi, phoE, rpoB, and tonB) were amplified by PCR, followed by sequencing of reaction products using the BigDye™ Terminator v3.1 Cycle Sequencing Kit (Life Technologies, Carlsbad, CA, USA) on an ABI Prism 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Determination of allele profiles and sequence types (STs) was conducted by comparing the obtained sequences to the documented data at Klebsiella PasteurMLST database (https://bigsdb.web.pasteur.fr/Klebsiella/Klebsiella.html (accessed on 21 October 2021). PHYLOViZ 2.0 platform was used for data management and analysis of clonal complexes (CCs), which were defined by related ST clusters exhibiting variation in a single locus (single locus variants-SLV) [79].

Conclusions
In conclusion, the present study demonstrates the multiclonal dissemination of MDR K. pneumoniae HRCs producing NDM-1 and NDM-7 carbapenemases in different hospitals in Northern (Amazon region) Brazil. This highlights the need for reinforcement of surveillance and control measures for such strains. Epidemic HRCs ST11/CC258 and ST392/CC147 were firstly associated with NDM-producing strains in Brazil and the first detection of the NDM-7 variant in Latin America and Brazil. Finally, the concerning diversity of NDM variants associated with a diverse genetic background of K. pneumoniae strains suggests an early endemicity for this carbapenemase in the country, which may negatively impact healthcare and antimicrobial resistance scenarios locally and nationally.