Characterization of multidrug-resistant and virulent Klebsiella pneumoniae strains belonging to the high-risk CG258 isolated from inpatients in northeastern Brazil

Multidrug-resistant (MDR) and hypervirulent Klebsiella pneumoniae (hvKp) clones have become a major threat to global public health. The CG258 is considered a high-risk CG and the K. pneumoniae strains belonging to it are known to be often multi-resistant and to spread mainly in the hospital environment. This study aimed to characterize the antimicrobial resistance profile, virulence factors, and the clonal relationships among 13 K. pneumoniae strains belonging to CG258 from patients admitted to a tertiary hospital in Teresina, in the state of Piauí, northeastern Brazil. Ten strains were classified as MDR and three as extensively drug-resistant (XDR). Three different β-lactamase-encoding genes ( bla KPC , bla OXA-1- like , and bla CTX-M-Gp1) and six virulence genes ( fimH , ycfM , mrkD , entB , ybtS , and kfu ) were detected. Moreover, two hypermucoviscous K. pneumoniae strains and one capsular K-type 2 were found. Multilocus sequence typing analysis revealed 10 different sequence types (STs) (ST14, ST17, ST20, ST29, ST45, ST101, ST268, ST1800, ST3995, and ST3996) belonging to CG258, being two (ST3995 and ST3996) described for the first time in this study.


Introduction
Klebsiella pneumoniae is a Gram-negative bacillus, usually encapsulated, nonmotile bacterium and belongs to the Enterobacteriaceae family. It is an opportunistic pathogen that can cause several infections and is among the most common nosocomial pathogens worldwide (Wyres et al. 2020). K. pneumoniae strains can contain a wide range of virulence and antimicrobial resistance factors (Ashurst et al. 2020). Several virulence factors further increase the severity of infections by K. pneumoniae, such as siderophores that are systems of chelating molecules that can competitively eliminate iron from host proteins and other sources; capsules, including their overproduction characterized by the hypermucoviscous (HM) phenotype; among others (Paczosa et al. 2016).
This study aimed to determine the pathogenic potential, antimicrobial resistance profile, and the clonal relationships among K. pneumoniae strains belonging to CG258 from patients admitted to hospitals in northeastern Brazil.

Bacterial strains
This study included 13 K. pneumoniae strains isolated from different patients admitted to a tertiary hospital in Teresina, a Brazilian municipality located in the northeast region, being capital of the state of Piauí, with an estimated population of 868,075 4 inhabitants. The strains were randomly isolated between August to September 2013 from several sources (catheter tip, hemoculture, purulent secretion, tracheal secretion, and urine) ( Table 1). The strains were identified by the VITEK 2 system (bioMérieux, France) and confirmed by the 16S rRNA sequencing, after amplification and sequencing with the primers fD1 (5'-AGAGTTTGATCCTGGCTCAG-3') and rP2 (5'-ACGGCTACCTTGTTACGACTT-3'), according to Weisburg et al. (1991). The strains were maintained at -80 °C in 15 % glycerol for subsequent experiments.

Hypermucoviscosity test
The HM phenotype was investigated using the string test according to Wiskur et al. (2008). The strains were inoculated on Mueller-Hinton agar (Oxoid Ltd., United Kingdom) and incubated for 18 h at 37 ºC. After bacterial growth, using a bacteriological loop, an isolated colony was touched and raised vertically. The HM phenotype was considered positive when there was the formation of a viscous filament ≥ 5 mm.

Antimicrobial susceptibility testing
Antimicrobial susceptibility testing was realized by the disk diffusion method on  Magiorakos et al. (2012).

Detection of virulence genes and ARGs by Polymerase Chain Reaction (PCR)
The strains were subjected to conventional PCR assays to detect K1 (magA, K1 capsule-specific wzy gene) and K2  One amplicon from each gene found was randomly selected to confirm its identity by sequencing using an automated sequencer (ABI 3500xL Genetic Analyzer; Applied Biosystems, USA). The obtained sequences were compared with those available in

Determination of capsular type
The capsular type (K-type) of the strains was determined by amplification and sequencing of the wzi gene as described by Brisse et al. (2013). The wzi gene was amplified by PCR and the amplicons were sequenced using the automated sequencer (ABI 3500xL Genetic Analyzer; Applied Biosystems, USA). Subsequently, the obtained sequences were submitted to the K. pneumoniae MLST database (https://bigsdb.web.pasteur.fr/klebsiella/klebsiella.html) to determine the alleles corresponding to each K-type and K-locus of the strains.

Multilocus Sequence Typing (MLST)
The strains were evaluated by the MLST technique using protocol 2 of the K.

Antimicrobial resistance profiles
The majority of strains were non-susceptible to nitrofurantoin (NIT), ciprofloxacin (CIP), and trimethoprim-sulfamethoxazole (SUT) (n = 13, 92.8 %), the other members of the class of folate pathway antagonists also demonstrated high percentages, such as sulphonamide (SUL) and trimethoprim (TRI) (both 85.7 %). Similar results were obtained for the tetracyclines class, such as doxycycline (DOX), tetracycline (TET) (both 85.7 %) and minocycline (MIN) (78.6 %). All strains were susceptible to cefotetan (CTT). Almost all were susceptible to COL since the minimal inhibitory concentration (MIC) found was < 1 mg/L, except for the KpPi151 strain that showed resistance to COL (MIC 32 mg/L). However, the mcr-1 gene was not found in this strain, indicating that resistance to COL is due to other genes or mechanisms not investigated in this study, as described by El-Sayed et al. (2020). Figure 1 shows the percentage of nonsusceptibility of each antimicrobial.
A large and worrying level of multidrug resistance strains was found, being ten strains classified as MDR and three strains (KpPi150, KpPi156 and KpPi159) as XDR (  (Braun et al. 2018), and south (Nava et al. 2019).
In a study by Gonçalves et al. (2017), a total of 26 strains of K. pneumoniae, including 14 MDR, seven XDR and three PDR strains were found in a university hospital in southern Brazil, corroborating with our results.

Presence of ARGs and the relationship with antimicrobial resistance profiles
Three different ARGs were detected among the strains, including blaOXA-1-like, blaCTX-M-Gp1 and blaKPC (GenBank accession numbers MT330307, MT330309 and MT330311). The blaOXA-1-like was detected in four strains -KpPi148, KpPi152, KpPi153 8 and KpPi159 (Table 1). According to Sugumar et al. (2014), Three strains (KpPi150, KpPi153 and KpPi157) harbored the blaKPC gene (Table   1). KPC-producing bacteria are closely related to infections associated with a high-level of morbidity and mortality because carbapenem antimicrobials are generally not effective against these bacteria (Arnold et al. 2011). The KpPi153 strain presented resistance to all tested carbapenems (DOR, ERT, IPM, and MPM) and KpPi150 strain to IPM and MPM.
Interestingly, although the KpPi157 strain presented the blaKPC gene, it was susceptible to carbapenems. On the other hand, as described by Marschall et al. (2009) andVillegas et al. (2006), the presence of the blaKPC gene may not always result in resistance in vitro to carbapenems, resulting in a possible failure to detect this phenotype during routine workup. Therefore, an accurate investigation to detect KPC and the real resistance to 9 carbapenems is crucial to establish control of their silent dissemination in strains that remain susceptible in vitro (Marschall et al. 2009;Villegas et al. 2006).

Occurrence of virulence genes and HM phenotype
Several virulence genes were found among the strains. All strains presented the genes fimH and ycfM, followed by mrkD ( variicola without the genes rmpA and magA was described causing primary endodontic infection and none of these genes were detected (Nakamura-Silva et al. 2020). These studies corroborate our results indicating that other genes may be involved in the HM phenotype.

Molecular typing of K. pneumoniae strains
MLST analysis showed 10 different STs (ST14, ST17, ST20, ST29, ST45, ST101, ST268, ST1800, ST3995, and ST3996) ( Table 1). Two STs (ST3995 and ST3996) were described for the first time in this study, since the strains presented new alleles for tonB gene (allele 573 for ST3995 and allele 574 for ST3996). All STs were found to belong to CG258 (Figure 2), which is considered a high-risk CG and is well known for spreading ARGs, mainly blaKPC and virulence genes worldwide (Pitout et al., 2015). CG258 and ST11 have been described as widespread in Brazil (Gonçalves et al. 2017), including outpatient infections (Azevedo et al. 2019).
The new ST3996 was found in an XDR strain (KpPi156) which is a single-locus variant (SLV) of ST11 ( Figure 2). Two MDR strains (KpPi146 and KpPi153) presented ST101, which is considered an emerging clone that has been identified worldwide and has the potential to become a persistent threat to global public health (Roe et al. 2019).
Moreover, the Kpi153 strain presented the blaKPC gene (Table 1). These characteristics were also found in southern Brazil and Italy (Gonçalves et al. 2017;Roe et al. 2019).
Another new ST found in this study was ST3995 that was described in the strains Kpi152 and Kpi159, which is also an SLV of ST101. The appearance of strains with the same ST in different patients and sources suggests the spread of these STs, a characteristic observed in ST3995, ST101 and ST17 (Table 1).
The Kpi145 strain presented ST268/KL20, which corroborates with several other studies that demonstrate an association between ST268 and K20 in K. pneumoniae strains (Table 1)  Tanzania (Mshana et al. 2013), and K. pneumoniae co-producing blaOXA-48 and blaNDM carbapenemases in Dubai (Moubareck et al. 2018). Furthermore, the KpPi148 strain presented the capsular serotype K2 which is frequently associated with invasive infections when found in hv clones (Wyres et al. 2019); however, ST14 is a non-hv clone and K. pneumoniae ST14/K2 strains have been found in several other studies (Brisse, et al. 2009;Harada et al. 2018;Musicha et al. 2019).
These same characteristics were found in an outbreak caused by MCR-1-producing K.
pneumoniae strains (24 strains of ST45 and one of ST1112) isolated from patients in a hospital in Porto, Portugal (Mendes et al. 2018). Moreover, in a study of ESBL-producing Enterobacteriaceae causing sepsis in neonates at a tertiary hospital in Tanzania were found in our strains (Table 1).
The strains KpPi149 and KpPi150 presented ST17, both have wzi allele 141, similar virulence genes, and HM phenotype, but only Kpi150 presented the blaKPC gene and an XDR profile (Table 1). ST17 has also been shown to carry other ARGs in southern Brazil, such as blaOXA-370 and blaCTX-M-8 (Aires et al., 2016). In addition, ST17 was also found in a CTX-M-15-producing K. pneumoniae strain in Norway, which caused an outbreak in a neonatal intensive care unit and subsequent intestinal colonization of affected children for up to two years (Löhr et al. 2015). Some STs found in this study were described as causing neonatal outbreaks in other countries (Mshana et al. 2013;Marando et al. 2018;Jin et al. 2015;Löhr et al. 2015). However in the present study, none of the strains were isolated from neonates, with the average age of the patients being 57 years old and the youngest a child of 3 years old (KpPi159) ( Table 1).

Conclusions
The results presented in this study raise worrying data related to multidrug resistance and virulence in K. pneumoniae strains isolated from inpatients in the northeast  Table 1. General data related to 13 K. pneumoniae strains used in this study.  pneumoniae strains indicating clonal group 258 (CG258) (blue) and sequence types (STs) found (red).