This study evaluated 21 K. pneumoniae strains isolated from different patients admitted to four hospitals in Manaus, located in the state of Amazonas, northern Brazil. All strains were subjected to antimicrobial susceptibility test, with a total of forty-one antibiotics of different classes (Figure 1), most strains were non-susceptible to the class folate pathway antagonists, sulphonamide (SUL) (95.2 % n = 20); trimethoprim-sulfamethoxazole (SUT) (85.7 %) and trimethoprim (TRI) (85.7 %); as well nitrofurantoin (NIT) and cefuroxime (CRX) (both 90.5 %); a high rate of non-susceptibility was also demonstrated for β-lactam combination agents, ticarcillin-clavulanate (TAC) and ampicillin-sulbactam (APS) (both 80.9 %). Non-susceptibility rates above 70 % were also observed for the antibiotics: streptomycin (EST), tobramycin (TOB), cefaclor (CFC) and ceftaroline (CTL). The results of non-susceptibility of each antibiotic are shown in Figure 1.
The majority of strains (61.9 % n = 13) were classified as MDR, according to the classification suggested by Magiorakos et al. (2012). Moreover, five strains (23.8 %) were classified as XDR (Figure 1). Ferreira et al. (2019) investigated the resistance profile of 25 K. pneumoniae from the Tocantins, northern Brazil, finding a high incidence of MDR among them (84 %). Gonçalves et al. (2017) studied 26 K. pneumoniae strains from patients admitted in a tertiary hospital in Londrina, southern Brazil, being 53.8 % classified as MDR and 26.9 % as XDR and 11.5 % PDR. A study conducted by Pereira et al. (2019) evaluated the frequency of different extended-spectrum β-lactamases (ESBL), associating it with antimicrobial resistance in E. coli (362 strains) and Klebsiella spp. (73 strains), demonstrate a relevant frequency of ESBL (11 %) and a significantly higher percentage of resistance in 91.3 % (n = 21/23) antimicrobials analysed: ampicillin (AMP), ampicillin-sulbactam (APS), piperacillin-tazobactam (PIT), amoxicillin-clavulanate (AMC), cefazolin (CFZ), cefotaxime (CTX), ceftazidime (CAZ), ceftriaxone (CRO), cefuroxime (CRX), cefepime (CPM), cefoxitin (CFO), aztreonam (ATM), ertapenem (ERT), meropenem (MPM), imipenem (IPM), amikacin (AMI), tobramycin (TOB), nitrofurantoin (NIT), sulfamethoxazole (SUT), tetracycline (TET), ciprofloxacin (CIP), levofloxacin (LEV). The exceptions are APS and IPM. Therefore, these works, corroborate with the results of the present study.
PCR analyses detected in eleven strains (52.4 %) presence of the blaOXA−1−like genes, ten with blaCTX−M−Gp1 genes (47.6 %) and seven strains harboring blaKPC (33.3 %). Accession numbers MT330306, MT330308 and MT330310. The XDR KpAm06 and KpAm08 encoded all the three of these β-lactamases genes, another ten strains (KpAm01, KpAm02, KpAm03, KpAm07, KpAm09, KpAm13, KpAm14, KpAm17, KpAm19 and KpAm21) harboured two of these three β-lactamases. The same genes have been reported in K. pneumoniae from similar studies in southern, southeastern and northern Brazil (Gonçalves et al. 2017; Azevedo et al. 2019; Ferreira et al. 2019).
Among the virulence genes, the fimH gene was detected in majority strains (95.2 % n = 20), the second most prevalent gene was ycfM (90.5 % n = 19), followed by entB (85.7 % n = 18) and mrkD (80.9% n = 17) (Figure 1). Accession numbers of all detected virulence genes MT330312, MT330313, MT330314, MT330316, MT330317, MT330319, MT330320, MT330322 and MT330324. Remya et al. (2019) studied virulence factors in 370 K. pneumoniae clinical strains and showed that entB was present in 90 % of the strains, fimH 89.1 %, ybtS 44.3 %, kfu 27.8 %, rmpA 5.1 %, K2 2 %, allS 1 % and magA 0.2 %. The large detection of entB and fimH; reasonable ybtS and; low rmpA and magA are similar to our results. Fimbrial adhesins (fimH, mrkD), siderophores (entB, mainly) and lipopolysaccharides (ycfM) were the most common genes in this study and similar results were found by Candan et al. (2015), with 74 % ycfM and 65 % of mrkD, fimH and entB, which studied 15 K. pneumoniae clinical strains isolated from different sources. The high prevalence of fimH, mrkD and entB genes found in this study was already expected, other studies have shown a high correlation of these genes in K. pneumoniae strains (Azevedo et al. 2019; Ferreira et al. 2019; Kus et al. 2017). The strain KpAm11 presented nine of the virulence genes studied, only K2 was not found in this strain (Figure 1). Eight strains (38.1 %) presented the HMV phenotype (Figure 1), the rmpA2 (mucoid phenotype A2 regulator) and magA genes (mucoviscosity-associated A gene) were associated with HMV phenotype, however, subsequent studies showed that the magA is associated to specific capsular serotype K1 (wzy K1) (Catalán-Nájera et al. 2017). Among the eight strains positive for this phenotypic in our study, only KpAm11and KpAm24 presented magA and rmpA genes. Similar results were found by Lee et al. (2010) which found three (8 %) among 35 strains that presented the phenotype without the presence of at least one of the three genes, magA, rmpA and rmpA2. Other studies also showed equivalents results, 10.3 % (n = 6/58) HMV K. pneumoniae from Taiwan did not have the magA and rmpA genes (Yu et al. 2006) and among the results of Mohammed et al. (2018), one out of six clinical strains demonstrated the characteristic in question. Moreover, our findings corroborate the results recently described by our research group (Nakamura-Silva et al. 2021) and also by Garza-Ramos (2015) who did not find these genes in K. variicola HMV strains. These results indicate that other genes may be involved in this phenotype in different species of Klebsiella which need further investigation, Walker et al. (2020) suggest that the HMV phenotype is probably due to factors not exclusive to the capsule. These authors suggest that it is necessary to investigate and clarify the link between the HMV phenotype and the capsule.
MLST analysis demonstrated a great diversity of STs among the strains, totaling 12 different STs (ST11, ST23, ST198, ST277, ST307, ST340, ST378, ST462, ST502, ST3991, ST3993 and ST5209). Four of these strains (ST11, ST23, ST307 and ST340) belong to the clonal group (CG) 258, which has been globally described as a KPC spreader and is often related to carbapenemase production. Therefore, strains with STs grouped into this CG are classified as international high-risk clones (Munoz-Price et al. 2015; Gonçalves et al. 2017; Azevedo et al. 2019). In fact, among the eight strains harbouring blaKPC gene in this study, seven belong to CG258. K. pneumoniae strains belonging to CG258 harbouring virulence and resistance genes have been described causing both hospital and community infections (Azevedo et al. 2019). ST11 was found in three strains in the study (KpAm01, KpAm02 and KpAm13), all classified as XDR, this ST is widespread in Brazil and is internationally considered a high-risk clone. ST340 was found in three strains (KpAm06, KpAm08 and KpAm09) with XDR profile in the first two and MDR in the last, and is also known to be widespread in Brazil (Gonçalves et al. 2017). In a study of the genomic population of K. pneumoniae, Wyres et al. (2020) describes the six hypervirulent global problem clones, the ST23 found in our strains belong to this group.
The KpAm11 and KpAm24 (ST23), both isolated from tracheal secretion, have the HMV phenotype harboring several virulence genes and wzi K1, indicating that these strains are hypervirulent, several other studies describe ST23 worldwide as a hypervirulent clone (Cheng et al. 2015; Mukherjee et al. 2020; Pereira et al. 2017). Moreover, the ST23 was previously described in Brazil in a colistin-resistant K. pneumoniae harboring blaKPC and several other resistance genes (Boszczowski et al. 2019), Coutinho et al. (2014) reported an invasive liver abscess syndrome caused by K. pneumoniae clone ST23 and also in non-human primates (Alouatta clamitans) with hypervirulent and hypermucoid characteristics (Anzai et al. 2017). KpAm16 presented ST307 and Wyres et al. (2019) emphasizes the need for more attentive epidemiological surveillance of this clone, due to its rapid and emerging global spread. This ST was also reported in other studies in southeastern Brazil as described by Dropa et al. (2016), that found K. pneumoniae harboring CTX-M-15 from wastewater, and Sartori et al. (2019) related MDR CTX-M-15 causing urinary tract infection in a dog. ST277 is a double locus variant ST258, and was previously reported by Chmelnitsky et al. (2013) in two K. pneumoniae ST277 carrying blaKPC gene and the isolate KpAm03 presented the same characteristic. The other STs belonging to CG258 (ST378 and ST462) appear in few studies, composing the bacterial collection among several STs (Lin et al. 2016; Zhong et al. 2014; Yan et al. 2015; Zhan et al. 2021; Saxenborn et al. 2021). The ST198 and ST502 grouped into smaller CGs, the first one was found in the strains KpAm14, KpAm17 and KpAm21 and was previously described in Brazil in K. pneumoniae producing CTX-M-15 isolated from commercial lettuce (Lopes et al. 2017).
The ICEKp is a mobile genetic element associated with invasive infection in K. pneumoniae (Lam et al. 2018), where ICEKp12 with ybt lineage 60 were identified in the WGS analyses in the three ST198 strains. The ST502 was found in three MDR strains (KpAm12, KpAm18 and KpAm19), this ST has been described to cause infections in humans, including community infections in Brazil (Azevedo et al. 2019). Three new STs were determined in this study, ST3991 (KpAm22) due to a new allele number combination, ST3993 (KpAm10), due to the new infB (177), mdh (298) and phoE (417) alleles and ST5209 (KpAm05), due the new tonB (705) allele. Through data analysis by the curators of the MLST database, the KpAm10 strain had its species re-identified as Klebsiella quasipneumoniae subsp. quasipneumoniae and was deposited in the database with this identification. Accession numbers of all WGS strains are listed in Supplementary Table 1.