The tracking resistance patterns, changing the prescribing habits, and increasing the infection control are the best way in reducing the development of bacterium antibiotic resistance, improving and saving in treatment to patients and health care facilities. The nosocomial infections by K.pneumoniae are still prevalent, and may be more dangerous due to the rapid development and spread of antimicrobial resistance in hospital [31]. In this study, most of the XDR K.pneumoniae isolates were collected from male patients. This result was similarly with a survey data from health care centers in Nigeria, here K.pneumoniae infection was higher in males than females [32]. Akter et al. also reported that male patients had a higher risk to get Klebsiella infection than females J Akter, AMMA Chowdhury and MAI Forkan [33]. There may be a association in poor lifestyle choices, smoking and alcoholism between male and female [32]. In addition, most of K.pneumoniae in this study were isolated from patients aged 23 to 92 years old. In the previous studies revealed a greater number of K.pneumoniae isolates were obtained from patients aged among 40 to 65 years or over 70 years old [34, 35]. The differences in age distribution of patients may be related to the response of the immune system, such as under 40 years have stronger immune systems, and more pressure to fight to K.pneumoniae [36]. Besides, an increasing age leads to increase the comorbid illness cause of a higher risk of K.pneumoniae infection [37]. These results also revealed that K.pneumoniae isolates were mainly isolated from ICU and respiratory specimens (namely is sputum samples). Ashurst and Dawson [38] emphasized that K.pneumoniae typically colonizes human mucosal surfaces of the oropharynx. For this reason, K.pneumoniae is considered to be the most common cause of hospital acquired pneumonia in the Vietnam [39]. The finding is in line to a study by Wang et al., which reported that the respiratory tract was the main infection site of K.pneumoniae in China [40].
Antimicrobial resistance bacteria are commonly associated with nosocomial infections cause of the overuse of antibiotics, and without monitoring or control [41]. In this study, most of K.pneumoniae was resisted to various antibiotics, fully resistant to ampicillin, piperacillin/tazobactam, cefotaxime, ceftazidime, cefepime, ciprofloxacin, and norfloxacin, whereas colistin, amikacin, nitrofurantoin, and fosfomycin were being the most effective for K.pneumoniae. The finding was higher than other results, Cepas et al. reported that 40% of K.pneumoniae strains were resisted to ciprofloxacin and amoxicillin-clavulanic acid [42]. Manjula et al. showed that 90.2% of K.pneumonia isolates were MDR, and high resistance to penicillin, cephalosporin, fluoroquinolone, aminoglycoside, and sulfonamide [43]. These MDR microbes are causing a great challenge in controlling infections, thus it is very important to monitor and optimize antibiotic use through antibiotic management programs [44, 45].
In the resistant phenotype of K.pneumoniae, the XDR phenotype showed resistance to most antibiotics, especially strong resistance to carbapenem, evidencing that they hold a variety of resistance mechanisms. They are not only secreting the enzyme that destroys carbapenem but also showing the resistance mechanisms such as the porin channel closes and the pump expels antibiotics out. Previous studies have shown that antibiotic consumption leads to selective pressure increasing β-lactam resistance in the genus Enterobacteriaceae bacteria [46]. However, XDR may also a result of gene circulation in the bacterial population. Currently in Vietnam, research on carbapenem resistant genotype of K.pneumoniae has not been conducted much. In this study, whole genome sequencing of XDR K.pneumoniae strains, analysis and confirmation of carbapenem resistance genes, also the relationship between carbapenem resistance and multi-drug resistance of the strains of K.pneumoniae were performed. This will provide a clearer and more indepth look at about the XDR capacity of these strains. Curently, millions of MDR genes have been sequenced that includes mostly bla genes (TEM, SHV, KPC, CTX-M, OXA, VIM, CMY, GIM, and SPM etc), drug modifying genes like catB, aadA, aacA, aph, as well as sul, arr3, dhfr, mcr-1 and vanA genes [47–49]. Antimicrobial resistance genes can be transferred to other bacterial species and thereafter can lead to the production of various enzymes in order to inactivate antimicrobial activities. The several members of Enterobacteriaceae group also can against carbapenems when combined mutations in chromosomal porin to prevent accumulation of β-lactam agents in the bacteria [50]. This study was also determined the antimicrobial susceptibility gene lists (Supplementary Table 1), the presence and mutation of five relevant carbapenem genes such as KPC-2, NDM-1 and 4, and OXA-48 and 181. The K.pneumoniae isolates had 93 difference resistance genenotyles separating in 10 antibiotic groups (e.g., aminocyclitol, aminoglycoside, beta-lactam, fluoroquinolone, folate pathway antagonist, fosfomycin, macrolide, rifamycin, tetracycline, and under development groups). Among the resistance against carbapenems K.pneumoniae isolates, the results have shown a high percentage of harboring the KPC-2 gene (41.2%), NDM-4 gene (29.4%), NDM-1 gene (23.5%), and a low percentage of both OXA-48 and OXA-181 genes (only 1/17 strain, accounting 5.9%). These findings were in accordance with the other reports that showed a low percentage of the OXA-48 gene in K.pneumoniae isolates [51, 52], whereas a previous study in Romania was revealed that OXA-48 was the most predominant genotype, followed by NDM-1 and KPC-2 in the carbapenem resistance K.pneumoniae clinical isolates [53]. There are a global epidemiology of the spread of carbapenem resistant K.pneumoniae strains with mainly carbapenemase genes as KPC-2 in Bulgaria [54], KPC-3 in Portugal [55], OXA-48 in Spain [56] and Vietnam [57], OXA-232 in China [58] and NDM-1 in Nepal [59]. Hoang et al. also reported similar distributions of NDM and KPC-producing Klebsiella spp. in hospital and aquatic isolated in Vietnam [60].
In particularlly, the mutations in KPC-2 gene, a main gene is causing the carbapenem resistance of K.pneumoniae, has been identified. This mutation is located in position acid amin 25 Glycine changed to a Serine. Another mutation on NDM-4 gene also indicated as amino acid 187 Proline changed to an Alanine. These mutations have not been mentioned in previous studies in carbapenem resistant K.pneumoniae strains. Mutations in DNA can have a significant impact on the resistance phenotype and hence the amino acid substitutions detected in two K.pneumoniae isolates may result in high levels of carbarpenem resistance (Table 3). Although, the antibiotic resistance mechanism of these related genes has not been studied in this experiment, this helps researchers to localize and be more interested in these gene groups in other related studies. An interesting note in our study, of the two isolates containing mutations (MH16-335M and MH17-011M) were belongs to the multilocus sequence type ST15. The most frequent STs of carbapenem resistant K.pneumoniae are characterized by different strains that distributed geographically, namely, ST258 being predominant in Europe and USA, ST11 being most common in East Asia, whereas ST15 is a less frequently occurring strain but has been indicated in clinical cases within outbreaks worldwide [45]. In the specific outbreaks, ST15 isolates can be highly homogenic, a high variability in antibiotic susceptibility and potential horizontal gene acquisition (Björn Berglund, 2019). K.pneumoniae ST15 strains had few reports from Asia or Vietnam previously, however, near time, Jiansheng Huang et al. was the first description of nosocomial outbreaks caused by K.pneumoniae ST15 strains in China [61]. This may indicate the spread of ST15 strains between geographically close regions. These findings may contribute to understanding the existence and spread of resistant bacteria such as strain ST15. Although ST15 strains are low in carbapenem resistance and virulence, it is important to note the importance of careful assessment of phenotypic and genetic characteristics for increasing anti infection treatment.