Multidrug-resistant Klebsiella pneumoniae harboring extended spectrum β-lactamase encoding genes isolated from human septicemias

Klebsiella pneumoniae is a major pathogen implicated in nosocomial infections. Extended-spectrum β-lactamase (ESBL)-producing K. pneumoniae isolates are a public health concern. We aim to characterize the type of β-lactamases and the associated resistance mechanisms in ESBL-producing K. pneumoniae isolates obtained from blood cultures in a Portuguese hospital, as well as to determine the circulating clones. Twenty-two cefotaxime/ceftazidime-resistant (CTX/CAZR) K. pneumoniae isolates were included in the study. Identification was performed by MALDI-TOF MS and the antimicrobial susceptibility testing by disk-diffusion. The screening test for ESBL-production was performed and ESBL-producer isolates were further characterized. The presence of different beta-lactamase genes (blaCTX-M, blaSHV, blaTEM, blaKPC, blaNDM, blaVIM, blaOXA-48, blaCMY-2, blaDHA-1, blaFOX, blaMOX, and blaACC) was analyzed by PCR/sequencing in ESBL-producer isolates, as well as the presence of other resistance genes (aac(6’)-Ib-cr, tetA/B, dfrA, qnrA/B/S, sul1/2/3) or integron-related genes (int1/2/3). Multilocus-sequence-typing (MLST) was performed for selected isolates. ESBL activity was detected in 12 of the 22 CTX/CAZR K. pneumoniae isolates and 11 of them carried the blaCTX-M-15 gene (together with blaTEM), and the remaining isolate carried the blaSHV-106 gene. All the blaCTX-M-15 harboring isolates also contained a blaSHV gene (blaSHV-1, blaSHV-11 or blaSHV-27 variants). Both blaSHV-27 and blaSHV-106 genes correspond to ESBL-variants. Two of the CTX-M-15 producing isolates carried a carbapenemase gene (blaKPC2/3 and blaOXA-48) and showed imipenem resistance. The majority of the ESBL-producing isolates carried the int1 gene, as well as sulphonamide-resistance genes (sul2 and/or sul3); the tetA gene was detected in all eight tetracycline-resistant isolates. Three different genetic lineages were found in selected isolates: ST348 (one CTX-M-15/TEM/SHV-27/KPC-2/3-producer isolate), ST11 (two CTX-M-15/TEM/SHV-1- and CTX-M-15-TEM-SHV-11-OXA-48-producer isolates) and ST15 (one SHV-106/TEM-producer isolate). ESBL enzymes of CTX-M-15 or SHV-type are detected among blood K. pneumoniae isolates, in some cases in association with carbapenemases of KPC or OXA-48 type.


Introduction
During the last decades, the selective pressure exerted by antibiotics has given rise to bacterial species which are increasingly resistant to these agents, and this increase in multi-resistant pathogenic strains has been extremely high [1,2].
Klebsiella pneumoniae is a major pathogen implicated in nosocomial infections that is known to spread easily, and it is frequently associated with resistance to the highest-priority critically important antimicrobial agents [3,4]. During the last years, the diffusion of broadspectrum cephalosporin-, carbapenem-and colistin-resistant K. pneumoniae isolates is now reducing treatment options and the containment of infections. Recently, the World Health Organization (WHO) [5] published a global priority list of antibiotic resistant bacteria, where third-generation cephalosporin-and/or carbapenem-resistant Enterobacteriaceae (K. pneumoniae and others), were included in the Priority 1 group. According to Amit, Mishali [6], carbapenem-resistant K. pneumoniae isolates implicated in bloodstream infections are associated with a high mortality rate of 40% to 70%.
K. pneumoniae isolates can acquire different mechanisms that confer antibiotic resistance to commonly used antibiotics. Among the most common mechanisms, the Extended-spectrum β-lactamases (ESBLs) and Acquired AmpC enzymes (qAmpCs) are widely reported [6][7][8]. One of the main concerns is that resistance caused by these enzymes may result in efficacy reduction of antimicrobial therapy, or in failed treatment [9]. Carbapenems are considered a last-resort antibiotic group for the treatment of infections caused by multidrug-resistant (MDR) Enterobacteriaceae [10]. ESBL-and carbapenemase-producing K. pneumoniae isolates are usually found after prolonged hospital stay and tends to affect debilitated patients with poor functional status [11]. Antimicrobial resistance is commonly related to the spread of plasmids, and the acquisition of resistance genes that normally occur by horizontal gene transfer (HGT) [12,13]. International high-risk clones of K. pneumoniae are frequently detected not only among humans' infections but also in those of companion animals [14][15][16][17][18][19][20][21].
Previous studies have been performed in Portugal analyzing the diversity of ESBLs in clinical K. pneumoniae isolates [4,[22][23][24][25][26], but none of them have been performed in our geographical region among invasive infections. The aim of this study was to characterize the type of ESBLs and the associated resistance mechanisms in broad-spectrum cephalosporin-resistant K. pneumoniae isolates recovered from blood cultures in a Portuguese hospital, as well as to determine the genetic lineages of these isolates.

Bacterial isolates
A collection of 22 cefotaxime/ceftazidime-resistant (CTX/CAZ R ) K. pneumoniae isolates obtained from blood cultures of hospitalized patients (one isolate/patient) in a Portuguese

DNA extraction and quantification
Genomic DNA from ESBL-producing K. pneumoniae isolates was extracted using the Insta-Gene Matrix (Bio-Rad), according to the manufacturer's instructions.

Antibiotic resistance genes
PCR (polymerase chain reaction) was the selected methodology to analyze the presence of resistance genes. K. pneumoniae isolates were screened by PCR and sequencing for the presence of genes encoding beta-lactamases: bla CTX-M , bla SHV , bla TEM , bla CMY-2 , bla DHA-1, bla FOX, bla MOX, bla ACC, bla KPC , bla OXA-48 , bla VIM and bla NDM [28,29]. The isolates were also screened by PCR (and sequencing when required) for the presence of the genes encoding for resistance to tetracycline (tetA, tetB), fluoroquinolones (aac(6´)-Ib-cr, qnrA, qnrB, and qnrS), sulfamethoxazole (sul1, sul2 and sul3), and trimethoprim (dfrA genes) [28]. The presence of the integrase gene of class 1, class 2 and class 3 integrons (int1, int2 and int3, respectively) were analyzed by PCR [30]. Furthermore, the mcr-1 colistin resistance gene was tested in all K. pneumoniae isolates [31]. Analysis of DNA sequences was performed with the BLAST program, available at the National Center for Biotechnology Information. Positive controls of the University of La Rioja were used in all PCR assays.

Multilocus sequence typing of K. pneumoniae isolates
The multilocus-sequence-typing (MLST) with seven housekeeping genes (gapA, phoE, infB, pgi, rpoB, tonB and mdh) was performed by PCR and sequencing in selected K. pneumoniae isolates (https://bigsdb.pasteur.fr/klebsiella/klebsiella.html); the allelic combination of the seven genes allowed the determination of the sequence type (ST).

Antimicrobial resistance phenotype in CTX/CAZR K. pneumoniae isolates
Amongst the 22 CTX/CAZ R K. pneumoniae isolates, twelve of them showed a positive ESBL screening test (54.5%) and these isolates were considered for further genetic resistance analysis.

Genetic determinants in ESBL-producing K. pneumoniae isolates
As shown in Table 1, most of the ESBL-producing K. pneumoniae isolates (11 out of 12) carried the bla CTX-M-15 gene and co-harbored a β-lactamase gene of SHV-type, with the following variants (number of isolates): bla SHV-1 (9 isolates), bla SHV-11 (1 isolate) and bla  (1 isolate, ESBL-variant). Moreover, the remaining ESBL-producing isolate carried the bla SHV-106 gene, a genetic variant that confers an ESBL phenotype. A bla TEM gene was found among most of the ESBL-producing isolates (all except one). Three of the ESBL-positive isolates showed resistance to cefoxitin and amoxicillin-clavulanic acid, but all were negative by PCR for the genes encoding qAmpC beta-lactamases (bla CMY-2 , bla DHA-1, bla FOX, bla MOX, and bla ACC ). Moreover, four of the ESBL-positive isolates also were IMP R ; a carbapenemase gene was detected in two of these isolates: 1) one of them carried the bla KPC2/3 gene (it was not possible to distinguish between both variants after sequencing the PCR amplicon), together with bla CTXM-15 , bla SHV-27 and bla TEM genes; 2) the other one carried the bla OXA-48 gene, together with bla CTXM-15 , bla SHV-11 and bla TEM genes ( Table 1); the two remaining IMP R isolates were negative for all carbapenemase genes tested.
Tetracycline resistance was mediated in all eight resistant isolates by the tetA gene and the int1 gene, encoding the integrase of class 1 integrons, was present in 11 out of 12 ESBL-producing isolates ( Table 1). The int2 and int3 genes (encoding the integrase of class 2 and 3 integrons, respectively) were not detected in this study. Furthermore, sulfamethoxazole-resistance was mediated by sul2 (n = 10) and sul3 genes (n = 5) in ESBL-producers. The aac(6¨)-Ib-cr Table 1. Resistance phenotype and genotype present in K. pneumoniae isolates from human septicemias in Portugal. gene was detected in four ciprofloxacin-resistant isolates and qnrS was identified among 10 isolates ( Table 1).

Discussion
The mechanisms of resistance implicated in a collection of ESBL-producing K. pneumoniae isolates obtained from invasive infections (blood cultures) in a Portuguese hospital have been analyzed in this study. In agreement with the current global epidemiology based on the bla CTX-M , we detected the CTX-M-15 β-lactamase in most of ESBL-producer isolates (11 out of 12). This enzyme is widely disseminated among human isolates, particularly in Portugal [8,[23][24][25][26]32]. It is worth noting that the first report of the CTX-M-15 enzyme isolated from blood culture in Portuguese hospitals goes back to 2005 [33]. Since then, the occurrence of this genotype was announced from K. pneumoniae isolates of various environments in Portugal, more recently in sick and healthy dogs [20], which can be explained by the close contact between humans and pets. Actually, it has been claimed that the bla CTX-M-15 among humans has, outstandingly, increased over time in most countries. Our finding seems to match completely with surveys conducted on hospitals located in different parts of Europe [19,[34][35][36]. Likewise, it was shown that this genotype has been disseminated in Asia [37] and Africa [38]. This study constitutes additional evidence that the CTX-M-15 remains the most important CTX-M enzyme in K. pneumoniae due to its large diffusion and relation to infections in human settings. Accordingly, this global spread could be, mostly, explained by the HGT between bacteria, mediated by conjugative plasmids [24]. Other ESBL variants of SHV-type were detected, either associated (SHV-27) or not associated to CTX-M-15 (SHV-106). Similarly, other authors detected SHV-106 producing K. pneumoniae isolates in Portuguese health institutions [24,25,39]. Moreover, SHV-27-producing K. pneumoniae isolates have been reported among human clinical infections [40,41] and also in companion animals in Japan and Germany [42,43]. This ESBL gene is frequently associated to other ESBL genes of the CTX-M-type, both in human and in animal settings.
Interestingly, two of the ESBL-producing isolates (with the bla CTX-M-15 gene, associated or not to bla SHV-27 ) also carried a carbapenemase encoding gene (bla KPC2/3 or bla OXA-48 ) as well as other beta-lactamase genes (bla TEM and bla SHV-11 ). In this respect, the bla OXA-48 gene was found in one ESBL-producer isolate (recovered in 2017), in association with bla CTX-M-15, bla TEM and bla SHV-11 genes. Moreover, another isolate carried the bla KPC2/3 gene, together with two ESBL encoding genes (bla CTX-M-15 and bla . Similarly, some international reports showed the presence of bla OXA-48 gene among hospitalized patients [44]. This gene had been reported in human isolates, mainly in Iberian Peninsula [45][46][47][48]. Particularly in Portugal, the OXA-181 carbapenemase was detected among K. pneumoniae isolates of hospitalized patients [8].
Moreover, carbapenemases of the KPC-2, KPC-3 and OXA-48 type have been recently reported among carbapenem resistant K. pneumoniae isolates of different origins from the same hospital analyzed in our study, few of them of blood origin [46,49]. Carbapenems are generally considered the most effective antibacterial agents and the first-choice treatment for infections caused by ESBL-producing Enterobacteriaceae. The current study emphasizes the relevance of co-occurrence of ESBL and carbapenemase encoding genes in K. pneumoniae isolates implicated in invasive infections with the difficulties that could have for effective therapeutic options.
Three different sequence types belonging to major international high-risk K. pneumoniae clones were identified in this study among four selected K. pneumoniae producer isolates (ST11, ST15 and ST348), revealing clonal diversity, in line with previous reports [3,14]. The ST11 lineage has been frequently detected worldwide among CTX-M-15- [50] and KPC-producing K. pneumoniae isolates [51]. In addition, isolates of ST348 or ST15 lineages producing CTX-M-15 and/or KPC-2/3 enzymes have been reported either in humans or animals in different studies performed in Portugal [21,32,46]. The ST15 lineage was identified in our study in a SHV-106-producing K. pneumoniae isolate, and similar isolates were previously circulating in a Portuguese hospital [24].
Rodrigues, et al. [24] considered that the dissemination of CTX-M-15 and the persistence of diverse ESBLs of SHV-type among K. pneumoniae isolates was mainly linked to a few epidemic and international clones (as ST15), in line with our study. It is important to note that this ST15 lineage has been disseminated in different settings. The ST15 lineage was found in an intensive care unit in Brazil [12] and among CAZ R clinical isolates in France, Poland and Portugal [52]. Moreover, ertapenem-resistance associated with ST11, ST15 and ST348 K. pneumoniae lineages have been previously found in another hospital in the same region [32]. K. pneumoniae ST15, which is a high-risk clonal lineage, seems to predominate among clinical CTX-M-15-producing isolates from companion animals [3,42,53]. All these findings indicate that these clonal lineages are frequently circulating, suggesting their important contribution to the expansion of β-lactamases in Portuguese hospitals.

Conclusions
Antimicrobial resistance can make infections difficult to treat, which represents a public health problem due to the negative consequences for human health.
Enterobacteriaceae isolated from septicemias in this human population study were frequently MDR and harbored clinically relevant antimicrobial resistance genes. The findings demonstrate that CTX-M-15-and ESBL-variants of SHV-type (SHV-106 and SHV-27) (associated in two cases with carbapenemases) are the most frequent mechanisms of resistance in ESBL-producing K. pneumoniae isolates implicated in bacteremia in the tested hospital. Additionally, our study demonstrates the presence of high-risk international clones (ST11, ST15 and ST348) among these ESBL-producing K. pneumoniae isolates. More studies should be carried out in the future to track the evolution of these type of β-lactamases in different environments.