Prevalence of pathogenic Klebsiella pneumoniae based on PCR capsular typing harbouring carbapenemases encoding genes in Uganda tertiary hospitals

Klebsiella pneumoniae is an opportunistic pathogen that has been implicated as one of commonest cause of hospital and community acquired infections. The K. pneumoniae infections have considerably contributed to morbidity and mortality in patients with protracted ailments. The capacity of K. pneumoniae to cause diseases depends on the presence of an array virulence factors. Coexistence and expression of virulence factors and genetic determinants of antibiotic resistance complicates treatment outcomes. Thus, emergence of pathogenic MDR K. pneumoniae poses a great threat to the healthcare system. However, the carriage of antibiotic resistance among pathogenic K. pneumoniae is yet to be investigated in Uganda. We sought to investigate the carbapenem resistance profiles and pathogenic potential based on capsular serotypes of K. pneumoniae clinical isolates. This was a cross sectional study involving use of archived Klebsiella pneumoniae isolates collected between January and December, 2019 at four tertiary hospitals in Uganda. All isolates were subject to antimicrobial susceptibility assays to determine phenotypic antibiotic resistance, pentaplex PCR to detect carbapenemases encoding genes and heptaplex PCR to identify capsular serotypes K1, K2, K3, K5, K20, K54 and K57. The study found an overall phenotypic carbapenem resistance of 23.3% (53/227) and significantly higher genotypic resistance prevalence of 43.1% (98/227). Over all, the most prevalent gene was blaOXA-48-like (36.4%), followed by blaIMP-type (19.4%), blaVIM-type (17.1%), blaKPC-type (14.0%) and blaNDM-type (13.2%). blaVIM-type and blaOXA-48-like conferred phenotypic resistance in all isolates and 38.3% of isolates that harbored them respectively. Capsular multiplex PCR revealed that 46.7% (106/227) isolates were pathogenic and the predominantly prevalent pathotype was K5 (18.5%) followed by K20 (15.1%), K3 (7.1%), K2 (3.1%) and K1 (2.2%). Of the 106 capsular serotypes, 37 expressed phenotypic resistance; thus, 37 of the 53 carbapenem resistant K. pneumoniae were pathogenic. The high prevalence of virulent and antibiotic resistant K. pneumoniae among clinical isolates obtained from the four tertiary hospital as revealed by this study pose a great threat to healthcare. Our findings underline the epidemiological and public health risks and implications of this pathogen.


Background
The growing prevalence of antibiotic-resistant clinical bacterial isolates is one of the main burdens to the Open Access *Correspondence: wamps@covab.mak.ac.ug 1 College of Veterinary Medicine, Animal Resources and Biosecurity, Makerere University, P. O. Box 7062, Kampala, Uganda Full list of author information is available at the end of the article Page 2 of 10 Ssekatawa et al. Antimicrob Resist Infect Control (2021) 10:57 healthcare systems worldwide [1,2]. Knowledge of antibiotic resistance genetic determinants is critical in thwarting the emergence and spread of multidrug-resistant (MDR) bacteria. Of great concern, is the spread of MDR strains of pathogenic Klebsiella pneumoniae, the Gram-negative bacteria that cause healthcare associated infections (HAI), community acquired infections, urinary tract infections (UTI) and wound infections. K. pneumoniae can harbor and express beta lactamases, most importantly carbapenemases capable of hydrolyzing newer carbapenem drugs used in the treatment of MDR bacterial infections [3][4][5]. An array of virulent factors responsible for pathogenesis such as endotoxins, capsules, iron-scavenging systems, siderophores and adhesions can be expressed by K. pneumoniae. A capsule is a vital virulence factor, because it confers two pathogenic mechanisms; shielding the invading bacteria from phagocytosis, and neutralizing the host immune response [6]. Klebsiella capsular serotyping (K typing) differentiates K. pneumoniae into approximately 77 K types [7]. Several capsular (K) types, predominantly K1, K2, K54, K57, K20, and K5, are frequently linked to community-acquired invasive septicemia, pyogenic liver abscess syndrome and pneumonia [8,9]. K3 is the usual cause of rhinoscleroma [10]. Pathogen survival requires the acquisition of drug resistance and virulent factors [11] and the acquired traits have been postulated to play an important part in the pathogenesis of K. pneumoniae infections [12].
Molecular capsular typing is presently the main technique employed in characterization of K. pneumoniae isolates and demonstrates excellent reproducibility in distinguishing clinical isolates [13]. Multiplex PCRs for detecting of the capsule repeat unit polymerase Wzy genes can be utilized for capsule typing of K. pneumoniae [14,15]. Therefore, virulence factors encoding genes can be used to characterize the different pathotypes of K. pneumonia. In spite of this, the carbapenem resistance profiles of pathogenic K. pneumoniae in Uganda are yet to be documented. Thus, to obtain insights into this, we characterized the carbapenem genetic resistance determinants among the pathogenic Klebsiella pneumoniae clinical isolates collected from four main referral hospitals in Uganda.

Bacterial strains
The study used 227 out of 284 archived MDR Klebsiella pneumoniae isolated between January and December, 2019 from clinical specimens in the Microbiology Laboratories of Mulago National Referral Hospital (MNRH) located in the central region 03381°N, 32.5761°E, Mbale Regional Referral Hospital (MRRH) in the Eastern region 1.0766°N, 34.1768°E, Mbarara Regional Referral Hospital (MBRRH),Western region 0.6171° S, 30.6577° E and Kampala International University Teaching Hospital (KIU-TH), Western region, 0.5468° S, 30.1387° E, Table 1. The isolates were initially assayed and confirmed to be resistant to several antibiotics.

Molecular characterization of K. pneumoniae
Klebsiella pneumoniae capsular molecular typing and characterization of carbapenem resistant genes was done by multiplex PCR employing adjusted methods used in the typing of E. coli by Toma et al. [17]. The first Multiplex PCR typing was based on primers targeting the K1, K2, K5, K20, K54, K57, and K3 capsular antigen genes to detect the major seven serovars [18],  [20] were used as positive controls.

Statistical analysis
Data analysis was performed using SPSS Version 25 statistical software. Chi square tests and Spearman's correlation were used to compare the frequencies of carbapenem resistant isolates, carbapenem resistance genes, capsular serotypes and correlation of resistance genes to phenotypic resistance. A P-value of ≤ 0.05 signified substantial statistical variation.

Results
Klebsiella pneumoniae isolates were obtained from different clinical samples of patients who were referred to the microbiology laboratories of the respective hospitals. A total of 284 isolates were obtained. However, 57 isolates were excluded because 24 isolates failed grow and 33 isolates were not Klebsiella pneumoniae. Of the 227 isolates used in this study, 128 were obtained from urine, 48 from pus swabs, 23 from blood, 16 rectal swabs, seven were from vaginal swabs, three from tracheal aspirate and two from sputum, Table 2.

Phenotypic carbapenem resistance profiles
Isolates used in this study were MDR as they exhibited resistance to different types of antibiotics, Table 3. Out of the 227 K. pneumoniae clinical isolates collected from different hospitals, 53 displayed phenotypic resistance to ertapenem; thus, this study established an overall phenotypic carbapenem resistance prevalence of 23.4%. Among  Fig. 1.

Correlation of phenotypic resistance with genotypic resistance
Variation between phenotypic and genotypic was registered. The prevalence of VIM gene was 9.7% and conferred phenotypic resistance to 100% of the isolates that harbored it. This was followed by KPC-like which exhibited phenotypic resistance in 72.2% of the isolates, then IMP at 60%, NDM at 41.2% and then OXA-48 protected only 38.3% of isolates that housed it. All the carbapenemase encoding genes significantly correlated to phenotypic resistance with chi square P value < 0.05, Table 5 (3), rectal swab (7) WzyK54  (21), rectal swabs (19), blood (11), and other clinical specimens (04) Table 6.

Discussion
Klebsiella pneumoniae has been implicated as one of the main human pathogens causing nosocomial and community acquired infections over a long period of time. Due to antimicrobial resistance, treatment of K. pneumoniae infections has become exceedingly complicated (Moradigaravand et al. 2017 [21,22]. The study screened 227 MDR K. pneumoniae isolates obtained from four tertiary hospitals located in different regions for carbapenem resistance. High overall phenotypic carbapenem resistance prevalence of 23.3% was detected. This is in agreement with other studies in Uganda and Tanzania that reported phenotypic carbapenem resistance prevalence among Enterobacteriaceae of 22.4% [23] and 24% [24] respectively. However, a similar study at MBRRH detected lower phenotypic prevalence of 12.6% [25]. Contrary to this, studies in North Africa and West Africa reported remarkably higher phenotypic resistance of > 50% and K. pneumoniae were the most prevalent isolates [26][27][28][29]. Furthermore, a larger study which covered Gauteng, KwaZulu-Natal, Western Cape and Free State provinces in South Africa documented overwhelming phenotypic resistance of between 47 and 50% to imipenem, meropenem and doripenem, 84% and 89% to ertapenem [30,31]. In comparison with previous studies at MNRH [23] and MBRRH [25] this study shows that the prevalence of carbapenem resistance in Uganda is on the rise and this is terrifying as recent meta-analyses revealed a substantial correlation between carbapenem resistant infections and increased risk of death [32]. Through molecular characterization, we detected carbapenem genotypic resistance frequency ranging from 32.4% at KIU-TH to 70.8% at MRRH and overall genotypic resistance prevalence of 43.2% in Uganda. In contrast, the overall genotypic prevalence was lower than that reported in Tunisia (86.3%) [33], Egypt (56%) [27], South Africa (86.0%), [31], India (76.3%) [34]. Among the five genes which were detected by multiplex PCR, the most encountered gene was OXA-48-like at a genotypic frequency of 36.4%. This corroborates well with recent studies which documented OXA-48-like gene and its variants as the most prevalent gene [27,31,33,35]. OXA-48 was first detected in K. pneumoniae isolate in Turkey 2003. OXA-48 producers spread sporadically to the neighboring countries located in the southern and eastern part of the Mediterranean Sea, and north Africa [36]. This provides an insight why the occurrence of OXA-48 is predominantly high in Egypt and Tunisia [27,33]. Previous studies reported NDM as the most dominant gene in South Africa [30,37], VIM and IMP as the most prevalent genes in East Africa [23][24][25]38] in contrast with the results of this study. This trend of events clearly shows that OXA-48 like producing E. coli and K. pneumoniae have invaded sub-Saharan Africa through immigration of individuals from the endemic region.
The overall phenotypic resistance registered by this study was lower than the genotypic resistance. For example, all isolates which harbored VIM expressed phenotypic resistance to ertapenem. Whereas OXA-48 like provided protection in only 38.3% of the isolates that sheltered it in disc diffusion assays. Oxacillinases encoded for by OXA-48 and its variant genes have been reported to possess low carbapenems hydrolyzing activity [36,39,40]. This enlighten why 61.7% of the isolates that housed OXA-48-like genes were sensitive to carbapenems. Furthermore, results of this study outlines that not all isolates that harbored carbapenemase genes were carbapenem insusceptible. This agrees with [40] findings who reported that modification and down regulation of outer membrane proteins through which drugs diffuse to reach their targets complements gene products and among the carbapenems, ertapenem is affected most by this scenario. This elucidates why resistance to ertapenem was significantly high. Thus, presence of a carbapenemase encoding genes alone does not guarantee resistance.
The capsule is one of the major factors that influence virulence in K. pneumoniae. Several studies have documented how capsular types influence pathogenicity of K. pneumoniae associated infections [9,41]. Previous studies unraveled the structures of the gene cluster in Klebsiella spp responsible for capsular polysaccharide synthesis (CPS) in some types [42,43]. The genetic structure is composed of a cluster of six highly conserved genes among different capsule types namely galF, cpsACP, wzi, wza, wzb and wzc that encodes for proteins that play a role in CPS translocation and processing at the bacterial surface and are located at the 5′ end of the cps regions and genes encoding glucose-6-phosphate dehydrogenase (gnd) and UDP-glucose dehydrogenase (ugd) found at the 3′ end. In the middle of the CPS loci lies a variable region that contains certain genes (Wzy and Wzx) that transcribe proteins accountable for polymerization and putting together of the specific CPS subunits. Thus, the great sequence variation of the wzy gene among the different capsular types is the basis of PCR capsular typing assays [43][44][45]. In light of this, we exploited the Wzy gene to characterize the most clinically important K. pneumoniae capsular serotypes isolated from different tertiary hospitals in Uganda. Capsular typing by heptaplex PCR revealed that K1, K2, K3, K5 and K20 accounted 46.7% (106/227) of the K. pneumoniae clinical isolates. K54 and K57 were not detected in any of the isolates. Klebsiella pneumoniae K1 and K2 have been reported as the most virulent capsular types causing septicemia and liver abscess [41,43]. However, other capsular serotypes are equally important as K5 and K20 are also associated with community acquired ailments whereas K3 causes chronic granulomatous infection of the nasal cavity and in some patients, the infection advance and lead to severe respiratory impairment [10,13]. Thus, the high prevalence of pathogenic capsular serotypes isolated from clinical specimens is a great threat to the healthcare system. There is no data about incidence of K. pneumoniae K types within the sub-Saharan region for comparison. However, results of this study are in line with Lin et al. [46], who reported K1, K2, K3, K5 and K20 as the most prevalent capsular types in Taiwan. Furthermore, out of the 106 klebsiella pneumoniae capsular types, 37 exhibited resistance to carbapenems yet carbapenems are regarded as the drugs of choice for treatment of MDR Gram-Negative HAI when the first line drugs have failed [47]. Acquisition of carbapenem resistance in pathogenic bacteria correlates with treatment failure in addition to increased morbidity and mortality [21]. Investigations elsewhere which looked clinical samples, associated coexistence of capsular and other virulent factors such as rmpA and aerobactin genes with hypervirulent or hypermucoviscous K. pneumoniae variant (hvKP) [48,49]. Despite the fact that this study did not attempt to detect other virulence factors, high occurrence of carbapenem resistance in capsular serotypes detected in study suggests possible existence carbapenem resistant hypervirulent K. pneumoniae (CR-HvKP) in Uganda clinical settings. Indeed, this has been case in clinical settings with substantial carbapenem resistance [50][51][52].

Conclusion
Findings of this study show that clinical K. pneumoniae isolates obtained from representative tertiary hospitals in Uganda exhibit high diversity of the main virulent capsular types and antibiotic resistance profiles to the frontline and last resort antibiotics. Based antimicrobial susceptibility assay, PCR capsular and carbapenemase gene typing, substantial prevalence of highly virulent MDR K. pneumoniae isolates were present in clinical specimens. High incidence of such isolates poses great health risks within healthcare and community settings; thus, should be treated with urgent attention. To the best of our knowledge, this is the first study to unravel the carriage of carbapenem resistance in pathogenic K. pneumoniae clinical isolates in Uganda. Thus, our data informs the need for regular surveillance of antibiotic resistance in pathogenic bacteria in clinical settings for meaningful control of emergence and spreading of AMR pathogens. Furthermore, this study only investigated carbapenem resistance carriage in capsular serotypes. However, to provide an insight into carbapenem resistance carriage in all K. pneumoniae pathotypes, additional virulence genes such as the rmpA gene expressing regulator of mucoid phenotype A; allS gene which is translated into the activator of the allantoin regulon, connected to allantoin metabolism; endotoxin encoding genes wabG, uge, and wcaG; iron acquisition system codifying genes iucB, iroNB, ybtA, and kfuBC; adhesin gene fimH (type I fimbriae); and ureA gene coding for a-subunit of the urease should be characterized.