Studies on molecular epidemiology of ESβL-producing Klebsiella pneumoniae isolated from patients hospitalized in a specialist hospital in southern Poland

The effective worldwide spread of extended-spectrum β-lactamase (ESβL)-producing Enterobacteriaceae, particularly Klebsiella pneumoniae, remains a critical concern for therapies against multidrug-resistant (MDR) bacteria [1]. K. pneumoniae is an opportunistic ESKAPE pathogen responsible for a wide spectrum of infections, including bacteremia, pneumonia, meningitis, purulent abscesses, urinary tract infections, and wound infections, at diverse sites [2]. Additionally, K. pneumoniae is known for its high prevalence and variety of antimicrobial resistance (AMR) genes [3]. These microorganisms are intrinsically resistant to ampicillin due to the presence of the SHV-1 penicillinase in their chromosomes. Resistance to additional drugs sporadically occurs through chromosomal mutations; however, most AMR in this species results from acquisition of these genes via HGT (horizontal gene transfer) mainly via large conjugative plasmids [3]. The most common mechanism of resistance to oxyimino-cephalosporins observed in K. pneumoniae is the production of ESβLs (extended-spectrum β-lactamases), followed by AmpC-like cephalosporinases, and KPC (Klebsiella pneumoniae carbapenemase) [4].

ESβLs are β-lactamases able of conferring bacterial resistance to the penicillins; first-, second-, third-generation cephalosporins; and aztreonam (but not the cephamycins or carbapenems) by hydrolysis of these antibiotics, which are inhibited by β-lactamase inhibitors such as clavulanic acid [5]. In recent years, most ESβL-producing organisms have also been found to be resistant to other major classes of antibiotics, such as aminoglycosides, fluoroquinolones, and sulfonamides, owing to the coexistence of associated resistant genes encoded in and transmitted by plasmids [6].

ESβL-producing bacteria were first detected in Europe in the 1980s. Although the initial reports were from Germany and England, the vast majority of reports in the first decade after the discovery of ESβLs were from France [5]. According to the SENTRY Antimicrobial Surveillance Program (1997 to 2016), the prevalence rate of ESβL-producing Enterobacteriaceae in 4 main geographic areas is high, although the rates vary substantially in different regions: Europe (19.5%), North America (12.3%), Latin America (11%), and Asia-Pacific (10.2%) [7]. The study conducted in 16 European countries over 20 years reported that ESβL screen-positive Enterobacteriaceae showed an increasing trend from 10.3% in 1997-2000 to 24% in 2013-2016, and the percentage of ESβL screen-positive K. pneumoniae isolates increased from 7.1% to 19.4% [7]. In this global survey, MDR K. pneumoniae rates decreased from 18.9% in 2013 to 15.8% in 2016; however, in many European countries these rates ranged from 16.9% to 55.7% in 2016 [7]. Meanwhile, the proportions of MDR K. pneumoniae in the study by Chong et al. in 2018 were 15%–60% [1]. By contrast, already in 2016, Morris et al. published data from the European Antimicrobial Surveillance Network which indicated that across Europe the percentages of invasive K. pneumoniae resistant to third-generation cephalosporins ranged from 0% to 70.1%, with ESβL producers accounting for between 85% and 100% of all strains reported [8].

The global problem of the presence of ESβL-producing K. pneumoniae is treated with great care; but still, the epidemiological situation in individual countries varies. In Poland, it is diverse, as it ranges from sporadic cases to dangerous epidemic outbreaks. Due to the small number of scientific reports on the spread of pathogenic K. pneumoniae strains in southern Poland, there is a need to learn more about the epidemiological situation in highly specialized hospitals in this area. This need served as a launching pad for carrying out this study.

Our work aimed to characterize a collection the clinical isolates of K. pneumoniae with an emphasis on the antibiotic resistance profile, identification of ESβL genes, virulence-associated gene detection, and the genetic relationship, thereby helping guide the monitoring of the status, prevalence, and intra-hospital spread of such pathogenic strains.

ESβL-producing Enterobacteriaceae are among the most common pathogens involved in nosocomial infections worldwide, with species other than Escherichia coli (especially K. pneumoniae) being increasingly reported in recent years [16].

In K. pneumoniae rods, ESβLs are still some of the most important mechanisms of drug resistance clinically and epidemiologically, due to their potential for rapid spread, as the genes encoding them are usually transferred on mobile genetic elements such as plasmids. Moreover, the widespread use of antibiotics has led to the emergence of MDR strains which accumulate mechanisms conditioning resistance to various groups of antibiotics. Currently, such strains are a global problem [3, 6, 7, 17]. Studies on molecular epidemiology of ESβL-producing K. pneumoniae isolates are needed, particularly under multi-level epidemiological monitoring, which is considered essential to understanding trans-hierarchical evolutionary changes in antibiotic resistant populations, and ultimately to establish appropriate control interventions [16].

This study investigates drug resistance profiles, the presence of genes encoding selected virulence factors, and genetic diversity of the ESβL-producing K. pneumoniae strain collection. Considering the latest research, this examination is part of a general trend of research on the molecular epidemiology of the species K. pneumoniae, and the obtained strain characterization results are similar to the results presented by other Polish authors [4, 18, 19, 20].

In their analysis of hospital-acquired infections caused by ESβL-producing K. pneumoniae, Castanheira et al. demonstrated that these rods were mainly the cause of wound infections (20.8%), respiratory tract infections (16.3%), urinary tract infections (13%), and generalized infections (11.9%) [7]. Therefore, respiratory secretions, urine, wound swabs, and blood are the main materials from which these pathogens were isolated [7]. In our study, the greatest number of K. pneumoniae isolates (40.3%) came from materials from the respiratory tract, urine (32.4%), wound swabs (19.4%), and blood (5%). Similarly, Wang et al. described most K. pneumoniae isolates that were cultured from urine (38.8%), materials from the respiratory tract (28.25%), followed by wound swabs (11.04%) and blood (7.22%) [6]. Correspondingly, most K. pneumoniae isolates characterized by Ranjbar et al. came from urine (63.75%), blood (37.5%), sputum (35.21%) and wounds (33.33%) [21].

The results of drug susceptibility testing as regards the K. pneumoniae collection under study demonstrated a resistance profile to β-lactam antibiotics that is typical of ESβL producers. All isolates were found to be resistant to several marker oxyimino-β-lactams of the ESβL mechanism: for instance, ceftazidime, cefotaxime, cefepime and aztreonam.

It should be underlined that our investigations revealed a high percentage (54.7%) of strains resistant to piperacillin/tazobactam. Similar results were obtained by other Polish researchers, Mrowiec et al., who recorded 53.5% of K. pneumoniae isolates resistant to this antibiotic in the adult population [18]. On the other hand, an investigation carried out by Korean researchers, Lee et al., found a lower percentage (20.9%) of K. pneumoniae strains which were resistant to piperacillin/tazobactam [22]. In his 1995 study, Livermoore proved that ESβL-producing strains frequently turn out to be resistant in vitro to combinations of β-lactams with inhibitors, despite the fact that ESβLs, by definition, are inhibited by β-lactamase inhibitors (clavulanic acid, sulbactam, or tazobactam) [23]. Furthermore, literature data indicate that the development of resistance to piperacillin/tazobactam in clinical K. pneumoniae isolates may be the result of several different resistance mechanisms, including the hyperproduction of constitutive chromosomal β-lactamases SHV-1 or the hyperproduction of TEM-1 β-lactamase due to the presence of either strong promoters or multiple bla_TEM-1 copies [22]. In our study, among all isolates resistant to piperacillin/tazobactam, as many as 93.4% of the strains demonstrated the presence of several (2–3) variants of ESβL enzymes. Hence, the observed growing resistance to piperacillin/tazobactam in K. pneumoniae is caused by the co-occurrence of different variants of ESβL enzymes in individual strains.

In this study, examinations conducted using PCR confirmed the presence of genes encoding ESβL in all the studied K. pneumoniae isolates; in as many as 72% of the strains, genes belonging to three different families were detected: CTX-M-type (85.6%), SHV (82%), and TEM (77.7%). Within the cefotaxime group, however, a clear dominance of the CTX-M-9 enzyme group was demonstrated at 75.6%, while the presence of CTX-M-1 was confirmed in only two (1.4%) strains. The study showed a high percentage of isolates (65.5%) that were characterized by the coexistence of three genes: bla_CTX-M-9/bla_SHV/bla_TEM. We have even observed that 5% of strains harbored bla_CTX-M-9 and bla_SHV, 2.9% of strains harbored bla_CTX-M-9 and bla_TEM, and 2.2% of strains harbored bla_SHV and bla_TEM,. Contrarily, in 2019, a similar Polish research into K. pneumoniae isolates carried out by Mrowiec et al. showed that 91.2% of strains carried bla_CTX-M-1, 63.5% carried bla_TEM, and 11.8% carried bla_SHV; 7% of strains had all 3 genes, 67.4% had bla_CTX-M-1 and bla_TEM, 1.2% had bla_TEM and bla_SHV, and 1.2% had bla_CTX-M-1 and bla_SHV [18]. However, the K. pneumoniae isolates coming from 2011 and described by Walter de Walthoffen et al. in Poland demonstrated the presence of bla_CTX-M at 94%, bla_TEM at 94%, and bla_SHV at 52.8%, while at the same time, 41.6% of strains had all 3 genes, 86.1% had bla_TEM and bla_CTX-M, 50% had bla_SHV and bla_CTX-M, and 44.4% had bla_TEM and bla_SHV [24]. The studies quoted above show that a spread of many different variants of ESβL enzymes is observed in Poland. In Portugal, studies carried out by Rodrigues et al. confirm the fact that, among K. pneumoniae strains, there is coexistence of β-lactamases belonging to CTX-M-15, SHV and TEM, detected at 47%, 45%, and 9%, respectively [16]. Yet, as early as in 2003 in North America, Paterson et al. documented the occurrence of K. pneumoniae with multiple ESβL types in different regions of the world, among others, Argentina, Australia, South Africa, Turkey, and United States [25].

A review of the literature indicates that in K. pneumoniae genes encoding plasmid-transmitted ESβL β-lactamases often coexist with other plasmid-encoded resistance mechanisms, including to fluoroquinolones, aminoglycosides, co-trimoxazole, tetracyclines, and chloramphenicol [26]. The characterization of the collection of ESβL-producing K. pneumoniae strains that was carried out confirmed the high percentage of isolates resistant to antibiotics from therapeutic groups other than penicillins, such as fluoroquinolones (ciprofloxacin, 90.6%), aminoglycosides (amikacin, 0.7%) and trimethoprim/sulfamethoxazole (88.5%). Studies carried out in Poland by Ojdana et al. found a high percentage of K. pneumoniae strains resistant to trimethoprim/sulfamethoxazole (100%), amikacin (58.3%), and 16.7% of isolates resistant to ciprofloxacin [19]. In Brazil, Wang et al. recorded 10.5%, 59.8%, and 80.6% of strains resistant to, respectively, amikacin, ciprofloxacin, and trimethoprim/sulfamethoxazole [6]. The differences observed in the abovementioned investigations prove the fact that drug resistance profiles in individual ESβL-producing K. pneumoniae strains vary and can be characteristic of a given region, hospital, or unit. In this study, 82.7% of the examined K. pneumoniae isolates were classified into the category of MDR strains, which is similar to the results of the Brazilian researchers, who recorded 84% of strains from this species with the MDR phenotype [26].

In the course of analysis of the most common genes encoding virulence factors in the studied pool of K. pneumoniae isolates, 55.4%, 10.1%, and 7.9% demonstrated the presence of genes iutA, mrkD, and rmpA, respectively. Contrarily, in the examination by Compain et al., the mrkD gene was detected in 100% of K. pneumoniae isolates, while the presence of the iutA gene was not found [2]. Also Ranjbar et al. demonstrated the presence of the mrkD gene in 84.34% and the rmpA gene in 13.91% of the studied K. pneumoniae isolates [21]. On the other hand, Ferreira et al. recorded iutA in 40% and mrkD in 96% of isolates, but they did not detect the presence of the rmpA gene [26]. Compain et al. showed the presence of the rmpA gene in 7.1% of isolates, which is similar to the proportion in this study (7.9%). The remaining virulence-associated genes found among the isolates were entB (91.4%), ybtS (55.4%), kfu (14.4%), K2 (11.5%), allS (5%). The French researchers also detected these virulence genes: entB (100%), kfu (35.7%), ybtS (21.4%), and allS (7.1%) [2], and the researchers from Brazil, respectively: entB (100%), and ybtS (60%) [26]. The magA gene was detected in 53.2% of the strains, contrary to Brisse et al. in France and Lascols et al. in Canada, who did not demonstrate the presence of the magA gene in any of the K. pneumoniae isolates studied [27, 28].

As for the K. pneumoniae strains under study, it was observed according to the data in the literature [8, 20], that the majority of the isolates (61%) were of clonal origin. The assessment of the genetic similarity of the studied K. pneumoniae rods was done with the use of PFGE. According to Paterson and Bonomo, PFGE is the most frequently used molecular reference method to genotype ESβL-producing Enterobacteriaceae in the world [5]. It is confirmed by numerous international, as well as Polish, publications concerning molecular analysis of the K. pneumoniae population: in France, Ireland, Brazil, China, and Poland [8, 14, 20, 26, 27]. Our work showed good typeability, reproducibility of SpeI for K. pneumoniae subtyping, and revealed high genetic diversity among the studied isolates. Analysis of the SpeI-PFGE genetic profiles of the strains demonstrated that their genetic similarity ranged from 66.6% to 100%. The highest degree of similarity (100%) was found between isolates classified into four PFGE clonal types (A–D) and seven minor clonal lineages (G-M). These isolates came from various clinical materials from patients hospitalized in different hospital wards. The obtained results of genotyping indicate that K. pneumoniae epidemic clones spread between different hospital departments. Similarly, Morris et al. demonstrated clonal organization of the K. pneumoniae strain collection: among 137 isolates, they identified 111 various PFGE profiles, which fell into 16 major clusters (A–P) in 112 isolates based on similarity of >85%, with each cluster comprising 2–43 isolates [8]. The clonal genetic structure in K. pneumoniae was also confirmed by Lascols et al. in their work published in 2013, which identified 3 major clonal groups among the isolates under study with more than 10 isolates per cluster, 10 minor clusters with less than 10 isolates per cluster, and 13 singletons [28]. In our study, most isolates (28%) were classified into clonal group A (cluster A), and in the research by Morris et al., the percentage of isolates classified into the largest clonal group (cluster A) was very similar and amounted to 31% (Morris et al. 2016). Yet, Lascols et al. described the largest clonal group (cluster C) comprising 53% of K. pneumoniae isolates [28].

The epidemiological situation of a hospital or a given ward may be influenced by many factors; among them, the specificity of the facility, the profile of patients that are treated there, the antibiotics employed, hygienic and sanitary measures undertaken or the persistence of certain pathogenic microorganisms.

To conclude, due to worldwide reports of a significant increase in resistance of K. pneumoniae to the antibacterial drugs used and the observed regional changes in the scale of this phenomenon, it is necessary to find the ESβL+ phenotype in all K. pneumoniae isolates as part of routine microbiological diagnostics. In addition, it should be remembered that when choosing empirical therapy for serious infections with an etiology of K. pneumoniae ESβL+, it is extremely important to take into account the local epidemiological characteristics. The application of molecular techniques to identify multidrug-resistant strains can contribute to improved surveillance and rapid detection of epidemic clones, and as a consequence, to a reduction in their spread in the hospital environment.