An Alliance of Carbapenem-Resistant Klebsiella pneumoniae with Precise Capsular Serotypes and Clinical Determinants: A Disquietude in Hospital Setting

Carbapenemase-resistant Klebsiella pneumoniae (CRKP) is a genuine burden for physicians and researchers. We aimed at carbapenemase resistance and its relation with capsular serotyping in K. pneumoniae and studied some clinical determinants, which may influence the clinical infections. Initially, 61 K. pneumoniae isolates obtained from various clinical specimens were confirmed at the molecular level and then antimicrobial susceptibility test was performed followed by capsular serotyping performed by multiplex PCR. All isolates were subjected to the detection of carbapenemase genes including blaKPC, blaNDM-1, blaOXA-48, blaVIM, and blaIMP. Clinical and demographic data of all patients were reviewed including age, gender, underlying diseases, and the treatment obtained. Multidrug-resistance was a predominant feature in 77% K. pneumoniae strains. Presence of extended-spectrum beta-lactamase was detected phenotypically in 59% K. pneumoniae strains. Carbapenem resistance was noticed phenotypically in 24.6% isolates. blaOXA-48 and blaNDM-1 were the most frequent carbapenemase genes. blaNDM-1 positive isolates correlated with gentamicin, amikacin, imipenem, and meropenem resistance (p < 0.05). The nosocomial isolates mostly harbored blaOXA-48 gene (p < 0.02). Amongst all the K. pneumoniae isolates, 59% isolates could be typed and serotype K54 had the highest prevalence followed by K20 and K5. Correlation between the carbapenemase genes, serotype and type of infection showed that blaOXA-48 positive strains had a significant association with K20 serotype and urinary tract infections (p=0.2) while, K20 serotype and blaKPC positive strains were significantly associated with wound infections (K20, p=0.3 and blaKPC, and p=0.4). Mucoid phenotype was not found related to presence of specific carbapenemase genes or serotypes except serotype K20 (p < 0.001). Patients with monotherapy had treatment failure in comparison to the combination therapy for blaKPC-associated infections. In conclusion, the present investigation exhibited the significant association between K20 serotype with blaOXA-48. The predominance of K54 reveals the possibility of endemicity in our hospital setting. K. pneumoniae isolated from wound specimens significantly harbors K20 serotype and blaKPC gene. Comprehensive clinical information and the distribution of antibiotic resistance genes, and serotypes may play important roles in the treatment process.


Introduction
Klebsiella pneumoniae has become more afuent in antibiotic resistance mechanisms and virulence features such that no one ever thought would come on too strong turning opportunistic bacteria into a potent pathogen. Clinical infections caused by this organism have become resistant to the treatment and increasingly life-threatening [1]. Acquiring the antibiotic resistance mechanisms, specifcally, extended-spectrum β-lactamases (ESBLs) have rendered the bacteria resistant to cephalosporins and monobactams. Eventually, the emergence of carbapenemase [2] curbed the usage of imipenem and meropenem, thereby putting profound constraints on the therapeutic strategies [1]. In fact, the frst case of K. pneumoniae expressing a carbapenemase was identifed in 1996 and was named as KPC (Klebsiella pneumoniae carbapenemase) [3]. Later years witnessed an unprecedented increase in some additional enzymes inhibiting carbapenems, such as VIM (Verona integronencoded Metallo-β-lactamase), IMP (Imipenemase), NDM (New Delhi Metallo-beta-lactamase) [4]. Enzymes IMP and VIM hydrolyze cephalosporins, penicillins, monobactam, and carbapenem except aztreonam [5], while NDM is a plasmid-borne [6] and OXA-48, the class D β-lactamase contains the carbapenemase activity which hydrolyzes imipenem and penicillin [7].
K. pneumoniae possesses several virulence treasures including a capsule, described as K types, which gives mucoid phenotype to the organism [8]. Tough the organism has been discriminated into 79 capsular serotypes [9], nevertheless, the distribution of these types varies geographically and the type of infections.
To date, studies showing the correlation between antimicrobial resistance and serotypes of K. pneumoniae are limited worldwide, especially serotyping of carbapenemresistant K. pneumoniae. In the past few years, emerging carbapenem-resistant hypervirulent K. pneumoniae (CR-hvKp) has become a serious threat for the treatment [10][11][12]. Moreover, the high potential dissemination of carbapenemase genes through serotype K1 have been reported in research studies [13,14]. In this investigation, for the frst time the dissemination of carbapenemase genes encoding the OXA-48, KPC, NDM-1, VIM, and IMP types were determined among six diferent capsular serotypes of K. pneumoniae strains. Tis prospective investigation covered various clinical infections, serotypes involved, and the presence of carbapenem-resistant genes to understand any relation amongst them in the context of K. pneumoniae infections. Host factors were given the insight to perceive the infuence of them on the rise of antibiotic resistance.

Bacterial Isolates.
Te study was conducted on 61 K. pneumoniae clinical isolates obtained as a routine process in the Division of Microbiology, Sina Educational, Research and Treatment Center, Tabriz, Iran. Duplicate isolates from the same patient were not enrolled. In general, the inclusion criteria comprised of those K. pneumoniae isolates which were obtained as a pure isolate, the clinical manifestations of the patients matched with infectious conditions, and the infectious specialist suspected an infection. All clinical isolates were initially identifed by conventional biochemical tests as described previously [15] and were confrmed by K. pneumoniae 16S-23S ITS (internal transcribed spacer) gene at the molecular level [16]. Te isolates were defned phenotypically as mucoid when colonies were touched with a loop and a string-like growth was observed which adhered to the loop as it was lifted from the agar plate [17]. Te pertinent information on any underlying disease, other demographic data, and the treatment regimens were collected from records of each patient. Response to the treatment of infection was assessed by the infectious disease specialist using clinical, biochemical, and microbiological parameters [18]. Te identifed strains were stored in tryptic soy broth containing 20% glycerol at −70°C for further experiments.

DNA Isolation.
Te commercial DNA extraction kit (Stratec Biomedical systems, Birkenfeld, Germany) was used for the extraction of DNA from K. pneumoniae isolates. In brief, 1 mL of bacterial suspension matched equivalent to 0.5 McFarland was prepared from an overnight culture and then centrifuged. DNA was extracted as per the instructions provided in the kit from the pellet and fnally resolved in 100 µL TE bufer.

Statistical
Methods. Statistical analysis was performed using descriptive statistics done by the x 2 test and Fisher's exact test (if needed) to fnd the relationship between carbapenemase genes and other variables. Spearman's rank correlation was tested between carbapenemase genes and antibiotic resistance, mucoid phenotype, and hospitalacquired infection that were found to be statistically signifcantly correlation between values. Variables were analyzed using the SPSS statistics (version 20) program (IBM Corporation). All the tests were performed two sided and a p value ≤0.05 were considered statistically signifcant.

Prevalence of Carbapenemase Genes in Predominant
Serotypes. Figure 2 depicts the information on the distribution of carbapenemase genes and capsular serotypes among the various clinical specimens. Most of the isolates obtained from the wound specimens belonged to K20 serotype (p � 0.03) and harbored bla KPC gene (p � 0.04) in their genome. On the other hand, bla OXA-48 positive K. pneumoniae strains were mostly isolated from urine specimens (p � 0.02). In the present investigation, carbapenemase gene bla OXA-48 was observed in K. pneumoniae isolates obtained from patients admitted to internal and infectious wards; however, this gene was strongly associated with K20 serotype in K. pneumoniae-infected patients admitted to burn wards including burn ICU, especially in-patients who developed K. pneumoniae infections after the grafting procedure (Table 4).

Discussion
K. pneumoniae has become notorious for causing nosocomial and difcult to treat clinical infections. In this study, prevalence of hospital-acquired K. pneumoniae infections was 55.6%. Our record is higher than other nosocomial infections reported earlier from Iran, Turkey, and Southern Europe (23.5%) [24], which may be due to diverse infections analyzed in medical practices at diferent countries. Similar to other research studies [25,26], bla OXA-48 was the most prevalent (78.8%) carbapenemase factor in the present research followed by bla NDM-1 . Seven (11.47%) K. pneumoniae isolates produced both bla OXA-48 and bla NDM-1 . Coproduction of bla OXA-48 and bla NDM-1 carbapenemase genes have been reported in the investigations conducted in Turkey [22] and the United States [27]. Te coexistence of these two carbapenemase genes in a pathogen constrains the treatment options for the clinicians and potential for global dissemination by means of cross-border transfer [28]. It is apparent that carbapenem resistance is on move and has increased comparatively since last fve years. Te current study was in concordance with other investigations conducted on imipenem resistance in Brazil and New York [29,30]. Tere exist a variation in prevalence of imipenem resistance geographically as well as the usage of antibiotics. Investigations carried out recently in Iran and earlier in New York [5,29], respectively, reported higher resistance to imipenem while Indian research found much lower resistance [31].
Te present investigation found high prevalence of carbapenemase genes which is compatible with another US study where one-third of gross K. pneumoniae isolates carried the carbapenemase enzymes [29]. Prevalence of the bla VIM genes in our study was 11.4% which is though lower than the two studies conducted in Canada and the US [32,33] earlier. Tese diferences may be due to the plasmid-born bla VIM gene and the genetic diversity among strains. IMPtype enzymes are one of the major groups of MBLs [34], encoded on plasmid and integrons and thus, spread easily [35] but they have low prevalence comparatively [22,33]. Te current study observed bla IMP gene merely in 4.91% isolates.
K. pneumoniae carbapenemase (KPC) associated with K. pneumoniae infections are predominantly nosocomial and systemic. Tese type of infections are frequently encountered in patients possessing multiple risk factors [36]. Similar to a study conducted on the bacteremic patients for the outcome of the treatment [37], we observed that 55.5% of bla KPC positive isolates were associated with mortality. Both monotherapy and combination therapy regimens are used for the treatment of KPC infections in our hospital. Te present study observed that 60% of expired patients were infected with KPC-producing isolates and had received monotherapy. Lee and Burgess [18] concluded in their study that combination therapy should be considered for the treatment of KPC infections as monotherapy lead to higher rates of treatment failure.
Some capsular serotypes including K1, K2, K5, K16, K20, K54, K57, and KN1 are known as hyper virulent variants of K. pneumoniae [9]. Te results of our study did not fnd much variability in the presence of serotypes. In the current study, K54 was the most frequent (29.5%) serotype followed by K20 (21.3%) and K5 (8.2%). In total, 36 (59%) isolates were typeable. Diferences are observed when seroepidemiology is compared geographically thus, knowledge of the existing serotypes is mandatory. A study conducted on the frequencies of capsular serotypes among 703 Klebsiella isolated from the blood of hospitalized patients found more than 90% of the isolates typeable [38], while another research from Taiwan, despite the inclusion of the high number of isolates, could not type the isolates enormously [39]. A recent study reported from Iran found K54 as the most frequent (68%) capsular serotype while K1 (8%) had the lowest frequency [40]. Prevalence of K5, K20, and K54 serotypes is signifcantly lower in Europe and Taiwan in comparison to our study [38,41]. High prevalence of serotypes K54 and K20 in our K. pneumoniae isolates requires medical attention.
About 26.22% of K. pneumoniae isolates in this study showed a mucoid phenotype that was associated with capsular serotypes other than K1 and K2. In a study conducted by Victor et al., similar to our work, about 24% of isolates with serotypes other than K1 and K2 had a mucoid phenotype, while a higher percentage of mucoid phenotype was associated to serotypes K1 and K2 [17].
A study in Uganda accomplished by Ssekatawa et al., found that serotypes K1, K2, K5, and K20 were identifed among K. pneumoniae isolates and were not belonged to serotypes K54 and K57. In addition, they reported which carbapenemase resistance genes were identifed among 16/ 42 serotype K5 and in 11/35 serotype K20 [42]. Nevertheless, comparison of carbapenem resistance among the Kserotypes showed chi square p values >0.05 indicating insignifcant correlation between them. Similarly, in this survey, 4 of 5 serotype K5, all serotype K20, and 15 of 18 serotype K54 harboring carbapenemase genes. We assessed the relationship between capsular serotypes and the presence of carbapenemase genes. Ours is the frst of its kind research study which showed serotype K20 to be associated with the presence of bla OXA-48 -mediated carbapenem resistance (p < 0.05). It is noteworthy that fve K. pneumoniae isolates with the same antibiotic resistance profle (GM, CIP, AN, SXT, CAZ, CTX, PTZ, and NI) belonged to K20 serotype and all had acquired plasmid containing bla OXA-48 gene. According to our fndings, serotype K20 was associated with amikacin and gentamicin resistance while ciprofoxacinresistant isolates belonged to serotype K54. In addition, NDM-1 had a high prevalence in MDR isolates with resistance against aminoglycosides and carbapenems (p < 0.05 ). Our study is similar to Flores et al. research conducted in the year 2020. Tey characterized NDM-producing K. pneumoniae isolates and found that this gene was mostly detected in MDR, PDR, and XDR isolates and also described the coexistence of NDM-producing K. pneumoniae with other carbapenemase genes such as bla OXA-48 , bla VIM , and bla KPC [43]. Ciprofoxacin, which is the most common antibiotic prescribed for the treatment of K. pneumoniae infections in our hospitals, should also be given a second thought as the most frequent serotype K54 isolates were characterized belonging to ciprofoxacin-resistant.
Acquisition of bla OXA-48 -mediated carbapenem resistance with intimacy with serotype K20 was a general feature in K. pneumoniae-infected patients admitted to burn wards including burn ICU, especially in-patients who developed K. pneumoniae infections after the grafting procedure. However, bla OXA-48 positive K. pneumoniae were also isolated from in-patients admitted to the internal ward. Extension of bla OXA-48 gene coding epidemically signifcant carbapenemase among hospital pathogens is important for the regional and global epidemiology of antimicrobial resistance [44]. We also observed that urine and wound specimens had the largest number of bacteria carrying carbapenemase-encoding gene with bla OXA-48 gene associated with urinary tract infections while bla KPC was mostly positive in wound infections. Tese fndings are similar to those observed in African countries [45][46][47]. Earlier an association of KPC positivity and bacteremia has been witnessed with higher mortality [37].
In this investigation, a high percentage (19.6%) K. pneumoniae isolates carried bla NDM-1 gene. NDM-harboring Gram-negative strains are known as a serious public health concern [48]. Te bla NDM-1 positive K. pneumoniae isolates with the multidrug-resistant feature could quickly disseminate all around the world and create an alarming risk situation. Moreover, a large 180-kb plasmid specialized for K. pneumoniae which possesses bla NDM-1 gene has high potential for transfer to susceptible E. coli J53 at a high frequency. Also, multiple resistance genes are located on this plasmid which create resistance against almost all antibiotics. As bla NDM-1 gene has potential to spread rapidly among clinically relevant bacteria, it may lead to a severe threat in therapeutics [48]. A previous report suggested a high attention to colonization pressure and the infection prevention control strategies for minimizing the rapid dissemination of bla NDM-1 harboring plasmids in specifed geographical areas [49].

Conclusion
To the best of our knowledge, this is the frst study performed on K. pneumoniae strains isolated from Northwest Iran, aiming to investigate the correlations among the capsular serotypes, carbapenem resistance, and the clinical determinants involved. Tis study highlighted a high prevalence of carbapenem-resistant genes in K. pneumoniae isolates. Of the fve carbapenemase genes studied, the association of bla OXA-48 was observed in serotype K20 isolates. bla OXA-48 positivity was correlated with patients aficted with urinary tract infections with the hospital as a source. bla KPC positive strains and K20 serotype were signifcantly associated with wound infections. Appropriate information regarding the distribution of antibiotic resistance genes, serotypes, and other characteristic features in relation to the specifc clinical specimens and medical wards could help physicians to choose the appropriate treatment.

Data Availability
Te authors declare that the data used to support the fndings of this study are available from the corresponding author upon request.

Ethical Approval
Tis study was approved by the Research Ethical Committee (IR.TBZMEDE.REC.1397.058) at Tabriz University of Medical Sciences, Tabriz, Iran.

Disclosure
Tis is a report of a database from MSc thesis of the frst author registered in the Tabriz University of Medical Sciences.

Conflicts of Interest
Te authors declare that they have no conficts of interest.