Epidemic Klebsiella pneumoniae ST258 incidence in ICU patients admitted to a university hospital in Istanbul

Introduction: Klebsiella pneumoniae sequence type 258 (ST258) strains are globally distributed multi-drug resistant pathogens and can spread rapidly throughout the world, causing severe healthcare-associated invasive infections with limited antimicrobial treatment options. The aim of this study was to reveal the incidence of Klebsiella pneumoniae ST258 strains among the intensive care unit patients in a university hospital in Istanbul. Methodology: Consecutive nonreplicated 83 K. pneumoniae strains were isolated from various clinical samples of intensive care unit patients admitted to a university hospital in Istanbul, between November 2016 to December 2018. Bacterial identifications were performed via VITEK2. Antimicrobial susceptibility tests were conducted with Kirby Bauer’s disc diffusion test except for colistin which was performed with broth microdilution. Real-time PCR method was utilized in order to reveal ST258 positivity among the strains. Results: Antimicrobial susceptibility results revealed that 56 (67%) K. pneumoniae strains were carbapenem-resistant. Real-time PCR results demonstrated that 15 out of 83 (18%) K.pneumoniae strain were ST258. According to antimicrobial susceptibility test results of ST258 strains, 8 were found as carbapenem-resistant whereas 7 were found as carbapenem susceptible. 3 out of 8 (37.5%) carbapenem-resistant ST258 strains were found as resistant against all antibiotics tested. Conclusions: Our study revealed that K. pneumoniae ST258 which caused severe infections worldwide so far has also spread to Istanbul. We believe that rapid molecular methods for monitorization of these clones are useful. our results showed that ST258 is not linked to a multiresistant strain and suggested that it does not contribute to multi-resistance formation alone.


Introduction
Klebsiella pneumoniae are Gram-negative, encapsulated, nonmotile bacteria that are colonized on human mucosal surfaces [1]. Due to their ability to invade and disseminate to other tissues, it causes several types of life-threatening infections in humans [2,3]. K. pneumoniae strains have been known as the most frequent cause of multidrug-resistant gramnegative bacterial infections [4]. Moreover, in the intensive care unit (ICU), in case of any infection with multidrug-resistant bacteria, broad-spectrum antibiotics are needed for treatment and these infections associated with worse outcomes compared to infections due to susceptible strain [5]. The majority of the K. pneumoniae strains express carbapenemases that break down most of the beta-lactams [6]. Genes that code for carbapenemases often spread worldwide through clonal expansion and K. pneumoniae sequence type 258 (ST258) strains are considered as a "high-risk international clone" [6,7].
K. pneumoniae ST258 clones are globally distributed multi-drug resistant pathogens and have capacity to spread rapidly throughout the world, causing severe healthcare-associated invasive infections with limited antimicrobial treatment options [8,9]. These strains may share genetic features that predispose them to pathogenicity or increased transmissibility. Carbapenem resistance is frequent among the strains, thus infections with ST258 strains are treated with regimens containing colistin. However, colistin-resistant strains have been reported from different parts of the world which is one of the major concerns of public health [10,11]. Although there are studies revealing the prevalence of ST258 clones in different parts of the world, there is only one case report the presence of ST258 clone in Turkey [12]. However, there is not any study investigating the prevalence of the clone in our country. In light of this information, we aimed to reveal the incidence and antimicrobial susceptibility of Klebsiella pneumoniae ST258 strains among the intensive care unit patients in a university hospital in Istanbul for the first time.

Methodology
Consecutive nonreplicated 83 K. pneumoniae strains were isolated from various clinical samples of intensive care unit patients admitted to a university hospital in Istanbul, between November 2016 to December 2018. Bacterial identifications were conducted with VITEK2 automated system, using VITEK 2 Identification cards, which provide rapid, accurate species-level identification of clinically relevant bacteria (BioMerieux, Marcy L'Etoile, France). Mucoid and lactose positive colony forming, gram negative bacilli are suspected as K. pneumonie, and further identification performed with automated system using VITEK2 Gram-negative bacilli Identification test card (VITEK2 GN ID card). Except for colistin, all other antimicrobial susceptibility tests were performed with Kirby Bauers disc diffusion tests on Mueller-Hinton agar (BioMerieux, Marcy L'Etoile, France). Briefly, a density of 0.5 McFarland bacterial suspension was inoculated in Mueller-Hinton broth and streaked on Mueller-Hinton agar plates, then antibiotic discs were placed on plates and incubated at 37 o C overnight to detect antimicrobial susceptibility tests except colistin. Colistin susceptibility performed by the broth microdilution method and the results were interpreted according to the EUCAST clinical breakpoints [13,14]. The following antibiotic discs were used, amoxicillin-clavulanic acid (20/10 μg, Cat: CT0223B), piperacillin-tazobactam (30/10 μg, Cat: All K. pneumoniae strains was collected and stored at -80 o C using specific Microbank cryovials containing 20% glycerol (Pro-Lab, Texas, USA). A cryovial bead was inoculated 10 mL Tryptone Soya Broth (TSB, Cat: CM1016B Oxoid Basingstoke, UK) and incubated at 37 o C overnight. One hundred μL inoculum from TSB, streaked on eosin methylene blue (EMB) agar incubated at 37 o C overnight to obtain a single colony for DNA extraction. Real-time PCR method was performed in order to reveal ST258 positivity among the strains. In order to extract bacterial DNA, in a single colony of each strain's fresh overnight culture on eosin methylene blue (EMB) agar was suspended in 50 μL of ultrapure water. The suspension was heated at 95 o C for 10 minutes and centrifuged at 14.000 rpm for 10 minutes. Thirty microliters of the supernatant were used as a DNA template for real-time PCR [15]. All DNA was stored at -80 o C until processing. Previously designed primers targeting the pilv-l region in the following sequences 5′-TTGGAGCTGATCCTTGCTCT and 5′-TCGATCCATGCTGATGATGT were used to detect ST258 clones among the strains [8]. Real-time PCR amplification and melting curve analysis were performed using a LightCycler 480 II system with software version 1.5 (Roche Diagnostics, Mannheim, Germany). The real-time PCR mixture was prepared using the LightCycler 480 SYBR Green I Mastermix kit (Roche Diagnostics, Mannheim, Germany) following the manufacturer's instructions. Cycling conditions for the ST258 assays were: initial denaturation for 5min at 95 °C and 40 cycles of 5 seconds at 95 °C and 10 seconds at 58 °C [8]. The fluorescence signal was measured at the end of each annealing step. Following amplification, a melting curve was generated by heating the PCR product to 95 °C with a ramp rate of 0.05 °C/s. Statistical analysis was performed with chi-square test on SPSS vers. 20 software (IBM, USA).

Results
Antimicrobial susceptibility results revealed that 56 out of 83 (67%) K. pneumoniae strains were carbapenem-resistant. It has been observed that all strains were resistant to ampicillin (100%). Also, 67 out of 83 (81%) K. pneumoniae strains are resistant to the third generation cephalosporins, which are usually used to treat Enterobacteriaceae infections. In addition to this, resistance rates against trimethoprimsulfamethoxazole, tigecycline and colistin, which are therapeutic options used instead of beta-lactam antibiotics, were found as 63.86%, 26.67% and 20.0%, respectively. Also, 3 out of 8 (37.5%) carbapenemresistant ST258 strains were found as resistant against all antibiotics tested. According to susceptibility results, colistin was found to be the best therapeutic choice for all patients which was followed by tigecycline, amikacin and gentamicin. Antimicrobial susceptibility results of all strains were given in Table 1.
Real-time PCR results demonstrated that 15 out of 83 (18%) K. pneumoniae strain were ST258. According to antimicrobial susceptibility test results of ST258 strains, 8 were found as carbapenem-resistant whereas 7 were found as carbapenem susceptible. There was no statistically significant difference between ST258 incidence and carbapenem resistance (p > 0.05). Moreover, in three out of eight (37.5%) ST258 positive Carbapenem-resistant K. pneumoniae (CR-Kp) strains were colistin-resistant whereas there was not encountered any colistin-resistant strains among ST258 Carbapenem sensitive K. pneumoniae (CS-Kp). In other words, in three out of 15 (20%) ST258 strains carbapenem and colistin resistance co-existed. Table 2 and Figure 1 demonstrates the antimicrobial susceptibility results of ST258 positive stains.
In addition to this, when the data of patients infected with Klebsiella pneumoniae ST258 strains were   analyzed, it was noticed that the first treatment that some patients received contradicted with the in-vitro antimicrobial susceptibility results of the clone (Table  3). Six patients infected with ST258 received piperacillin and tazobactam combination therapy, however, in-vitro antimicrobial susceptibility tests revealed that only two strains were susceptible to the antibiotic in interest.

Discussion
Antimicrobial resistance is an important worldwide problem in the treatment of diseases caused by resistant bacteria. Klebsiella pneumoniae is defined as a member of the ESKAPE group microorganisms which comes from their ability to "escape" from the effects of antimicrobial drugs [16]. Therefore, K. pneumoniae is known to be an important cause of morbidity and mortality among hospital-acquired and long-term carerelated infections [17].
Over the last few decades, there has been a concerning increase in the resistance of K. pneumoniae strains to a wide range of antibiotics. Expression of extended spectrum beta-lactamases (ESBLs) and carbapenemases are two major types of antimicrobial resistance mechanisms mainly observed in these strains [1,2]. The prevalence of multiple antibiotic-resistant strains such as CR-Kp has increased in recent years and CR-Kp has become a major public health problem worldwide, as carbapenems are the first-line therapy for infections caused by K. pneumoniae, especially ESBL producers [16,18]. Although not all the CR-Kp strains belonging to ST258 clone, previous studies revealed that there is a strong relationship between carbapenem resistance and ST258 clone. In addition to carbapenem resistance, ST258 positive K. pneumoniae strains are known as resistant to all β-lactam antibiotics, also typically have plasmid-derived genes that encode aminoglycoside modifying enzymes and chromosomal mutations that confer fluoroquinolone-resistance, that make them multi-drug resistant strains [16][17][18][19]. Furthermore, treating CR-Kp infections is a major clinical challenge, due in part to limited antibiotic options. In spite of being a major health concern worldwide, the data related to the molecular epidemiology and molecular characteristics of these strains are insufficient in our country.  [22]. Moreover, Bogdanovich et al. reported that all five colistin-resistant CR-Kp strains were ST258 positive in their study conducted in 2011 [11]. Lomonaco et al. did not detect ST258 clone in any of the 10 multi-drug resistant K. pneumoniae strains and reported that these strains were belonging to different sequence types in the study conducted in Pakistan in 2018 [23]. In our study, we found colistin resistance rate as 37.5% (three out of eight) ST258 positive CR-Kp strains, which was not as high as it was found in Greece [22]. However, there was no statistically significant difference between ST258 incidence and carbapenem resistance, also ST258 was not linked to a multiresistant strain.
In addition to this, there is only one case report on the ST258 clone, however, there is not any study revealing the prevalence of ST258 clone in our country [12]. Becker et al. . [25]. Villa et al. reported that firstly they detected ST258 positive CR-Kp in intraabdominal abscess of a kidney transplant patient but during treatment with tigecycline they isolated ST258 positive CS-Kp in 2013. After examination of these two strains with the next generation sequence system, they reported that plasmid loss and carbapenem susceptibility could be seen after treatment with noncarbapenem antibiotics [26]. Diago-Navarro et al. reported 19% ST258 positivity among 300 K. pneumoniae strains in 2016 [27]. Moreover, they reported that 13% of ST258 positive strains were carbapenem susceptible due to the loss of carbapenemase gene which was attributed to ICU residence time and antibiotic use of ST258 positive patients, similarly to the study of Villa et al. [25][26][27]. The primers used in our study for detecting ST258 clones were targeting the pilv-1 region [8]. Adler et al. [8]. reported ST258 positivity in 9 CS-Kp strains when they used these primers in 2014 [8]. Similarly, the studies of Becker et al. [24], Diago-Navarro et al. [25], Diago-Navarro et al. [27] and Adler et al. [26], showed that ST258 clones were detected in carbapenem sensitive K. pneumoniae strains as well as CR-Kp. This may be a result of the loss of carbapenemase plasmids due to different antibiotic therapy regimens applied to the patients. However, although the previous studies reported the loss of carbapenemase plasmid, it is seen that majority of the studies on the ST258 clone have been tried to associate the clone only with carbapenemresistant strains and CS-Kp strains were ignored [19][20][21]. In the study of Kontopidou et al. [28], conducted in Greece in 2014, it has been reported that they used betalactam inhibitors for the first 10 days as the first treatment option in ICU patients infected with CR-Kp which were strongly associated with bacteremia [28]. Clancy et al. [29]. reported that piperacillin-tazobactam was the first therapeutic option given to transplant recipients in the first 30 days after the detection of CR-Kp-induced bacteremia [29]. Moreover, in 2016, Ocampo et al. [19] reported that piperacillintazobactam was the treatment for patients with CR-Kp, which was similar to other studies in 2016 [19]. Similarly, in our study, CR-Kp and CS-Kp strains were mostly associated with bacteremias and it was found that beta-lactam/beta-lactamase inhibitor combinations such as piperacillin-tazobactam were frequently given during the treatment of patients. However, it was noticed that the treatment was started empirically, without obtaining in-vitro susceptibility results. Patient survival rates can be increased by rapid detection and control of infection source and infectious agent-specific combination therapy, whereas inappropriate empirical treatments result in poor clinical outcomes in patients [30].

Conclusions
This molecular epidemiological study is the first study conducted in Turkey in order to reveal the prevalence of ST258 clone, which was reported in only one case study in 2014 [12], among K. pneumoniae strains isolated from intensive care unit patients from a university hospital in Istanbul. In addition to the presence of ST258 clone in CR-Kp strains, ST258 clone positivity was also found in CS-Kp strains. Our results showed that ST258 is not linked to a multi-resistant strain and suggested that it does not contribute to multiresistance formation alone. We believe that in order to be successful with empirical treatment in intensive care unit patients who are infected with MDR strains, the epidemiological data on K. pneumoniae strains with different clones that can rapidly disseminate multi-drug resistance should be developed with new multicenter and extensive studies.