Introduction

Klebsiella pneumoniae is an opportunistic bacterium that can cause various infections [1, 2]. Carbapenem-resistant K. pneumoniae (CR-Kp) isolates are considered “critical concern” by the World Health Organization (WHO) [1, 2]. Carbapenemases are beta-lactamases capable of hydrolyzing the oxy-amine side chains of carbapenem antibiotics. K. pneumoniae carbapenemase (KPC), oxacillinase (OXA), Verona integron-encoded metallo-β-lactamase (VIM), imipenamas (IMP), and New Delhi metallo-β-lactamase (NDM) are among the most common carbapenemase genes in K. pneumonia [3, 4].

Since there are scare data on the prevalence of carbapenemase genes in CR-Kp from the northwestern region of Iran, this study aimed to evaluate the antibiotic resistance patterns and prevalence of carbapenemase genes in K. pneumoniae isolates from different clinical samples in Tabriz city, northwestern Iran. The results of this study may provide a better background for local antimicrobial prescribing protocols, local empirical treatments, and infection control programs.

Main text

Materials and methods

Ethics approval and consent to participate

This study was approved by the IRB of the Kazerun Branch of the Islamic Azad University, Kazerun, Iran in accordance with the World Medical Association Declaration of Helsinki (no registered code). Clinical samples were collected as the routine laboratory analysis and to check any potential infection for referred and admitted patients and not as a part of this study. Therefore, written informed consent was waived by the IRB of the Kazerun Branch of the Islamic Azad University, Kazerun, Iran.

Clinical isolates collection

This cross-sectional study was performed on 100 K. pneumoniae isolates over a six-month period (from June to November 2020). The isolates were collected from different clinical samples of inpatients and outpatients referred to hospitals in Tabriz, northwestern Iran. None of the patients had taken antibiotics three days prior to sample collection. Also, none of the patients had any underlying disease. Clinical samples included blood, wounds, sputum, and urine. The 100 K. pneumoniae strains were isolated from 49 female and 51 male patients, respectively. The mean age of the patients was 47.4 ± 23 years and ranged from a minimum of 10 months to a maximum of 70 years.

Identification of K. pneumoniae isolates

Clinical samples were cultured on blood agar and MacConkey agar (Quelab, Canada) plates. The plates were incubated at 37 °C for 24 h. The grown colonies were confirmed by standard bacteriological tests, which included Gram stain, lysine iron agar (LIA), triple sugar iron agar (TSI), SIM (sulfide-indole- motility), Simon citrate, MR-VP (Methyl Red-Voges Proskauer), and urea broth [5]. The confirmed K. pneumoniae isolates were suspended in trypticase soy broth (TSB, Merck, Germany) with 20% (v/v) glycerol and stored at -80 °C for further investigations. K. pneumoniae ATCC® 13,883™ was used as quality control strain. All media were purchased from Merck Co, Germany.

Antimicrobial susceptibility testing (AST)

The disc diffusion method was performed on Mueller-Hinton (MH) agar (Merck, Germany) for antibiotic susceptibility testing of K. pneumoniae based on the Clinical and Laboratory Standards Institute (CLSI) guidelines [6]. Antibiotics used included cephalothin (30 µg), imipenem (10 µg), meropenem (10 µg), ceftazidime (30 µg), ciprofloxacin (5 µg), cefoxitin (30 µg), gentamicin (10 µg), amikacin (30 µg), nalidixic acid (30 µg), ampicillin (10 µg), cotrimoxazole (23.75 µg), cefotaxime (30 µg), ceftriaxone (30 µg), tetracycline (30 µg), and azteronam (30 µg) (Himedia Co. India). Isolates that showed resistance to 3 or more antibiotic categories were classified as multidrug resistant (MDR) [7]. Escherichia coli ATCC® 25,922™ and K. pneumoniae ATCC® 13,883™ were used as quality control strains.

Identification of carbapenemase genes

DNA was extracted from carbapenem-resistant isolates using the Invitek Strateg Business kit (Invitek-Molecular, Germany) according to the manufacturer instruction. Then uniplex conventional polymerase chain reaction (PCR) was performed using previously described specific oligonucleotide primers of blaVIM, blaNDM, blaKPC, blaOXA, and blaIMP genes (Table 1) [8, 9]. The final reaction mixture was 25 µl including 12.5 µl of Buffer 10X PCR, 1 mg/µl of MgCl2, 0.75 µM of deoxynucleotide triphosphates (dNTPs) mix, 1 µl of each primer, 0.2 units of Taq Polymerase, 3 µl of DNA, and the DNA/RNA free water. The PCR program was performed in a thermocycler (Eppendorf Master Cylinder, Germany) as follows: initial denaturation for 10 min at 94 °C, 35 cycle of denaturation stage for 20 s at 94 °C, annealing stage for 40 s at 52 °C, extension stage for 30 s at 72 °C, and the final elongation step was performed for 5 min at 72 °C. Following loading the PCR products on a 1% agarose gel with 0.5 µg/ml safe stain (Sinaclon, Iran), the gel was subjected to electrophoresis at 85 V for 60 min and the bands were then seen under UV light with the help of a gel documentation device. Positive and negative controls in this reaction include K. pneumoniae ATCC® BAA-1705™ and K. pneumoniae ATCC® BAA-1706™, respectively.

Table 1 Primers used in this study for the detection of the different carbapenemase genes
Full size table

Statistical analysis

The twentieth version of SPSS software (IBM Corporation, Armonk, NY, USA) was used for statistical analysis of the data. Results were presented as descriptive statistics in the form of relative frequencies. Values were expressed as percentages of the variables. An analysis of possible correlations between variables was conducted using Fisher’s exact test. The correlation was considered statistically significant if the P-value was less than 0.05.

Results

Distribution of the K. pneumoniae isolates in clinical samples

In our investigation, 100 K. pneumoniae isolates were collected from 57 urine samples, 27 blood samples, 13 wound samples, and 3 sputum samples. In total, 70.0% of the samples were collected from inpatients, while 30.0% were collected from outpatients.

Antimicrobial susceptibility

The results of AST of the 100 K. pneumoniae isolates against 15 antibiotics are shown in Fig. 1. Overall, the most resistance rates of K. pneumoniae isolates were related to ampicillin (94.0%), cefotaxime (67.0%) and ceftazidime (62.0%), and the lowest resistance rates were related to imipenem (18.0%) and meropenem (20.0%) antibiotics. In total, 25.0% of isolates were carbapenem-resistant, of which 13.0% were resistant to both imipenem and meropenem antibiotics. The carbapenem-resistant isolates were related to 18 (72.0%) males and 7 (28.0%) females. In total, 49.0% of K. pneumoniae isolates were MDR.

Fig. 1
figure 1

Antibiotic resistance percent rates in Klebsiella pneumoniae isolates collected from clinical samples in Tabriz, northwestern Iran

Full size image

Distribution of the carbapenemase genes

The distribution of different carbapenemase genes is presented in Table 2. In this study, among the 25 carbapenem-resistant isolates, the blaVIM, blaKPC, blaOXA−48−like, and blaIMP genes were detected by PCR. Eighteen isolates (72.0%) had the blaKPC gene, 3 (12.0%) isolates had the blaVIM gene, 1 (4.0%) isolate had the blaIMP gene, and 1 (4.0%) isolate had the blaOXA−48−like gene. Two carbapenem-resistant isolates did not carry any gene. The blaNDM gene was not detected in any isolate. Coexistence of carbapenemase genes was also not detected in any isolate. The 18 blaKPC positive K. pneumoniae were isolated from 10 urine, 5 blood, and 3 wound samples. Of 3 blaVIM positive isolates, 2 were from urine and one from blood. The blaIMP and blaOXA-4− like positive isolates were from urine and blood, respectively.

Table 2 Distribution of blaVIM, blaNDM, blaKPC, blaOXA, and blaIMP carbapenemase genes in the carbapenem-resistant Klebsiella pneumoniae isolates based on gender, referral type, and sample type
Full size table

Association of carbapenemase genes with antibiotic resistance patterns

The results of Fisher’s exact test showed that there was a significant association between the presence of carbapenemase genes with the resistance to meropenem (P-value = 0.000), imipenem (P-value = 0.000), cephalothin (P-value = 0.007), ceftazidime (P-value = 0.000), cefotaxime (P-value = 0.006), and ceftriaxone (P-value = 0.006).

Discussion

In this study, the lowest resistance rates were found with the antibiotics imipenem (18.0%) and meropenem (20.0%). Also, 25.0% of the isolates were resistant to carbapenems. In contrast to our study, researchers found an unusual increase in carbapenem resistance in a retrospective cross-sectional study conducted from 2014 to 2018 in Saudi Arabia, with 38.4% for imipenem and 46.1% for meropenem [10]. However, the resistance rate to amikacin (36.3%) was lower, and the ciprofloxacin resistance was the same as in this study [10]. In another study conducted in Baluchistan in 2021, the lowest resistance was related to imipenem (0%), which was lower than in our study, and the highest resistance was related to cefotaxime (100.0%), which was higher than in our result [11]. In another study from Russia, the rate of carbapenem-resistance phenotypes (45.7%) was higher than in our study [12]. In contrast to this study, higher prevalence rates of CR-Kp isolates were found in the Iranian cities of Tehran (more than 60.0%) and Isfahan (more than 50.0%) [13, 14]. By comparing these studies, we concluded that the rate of CR-Kp in northwestern Iran was relatively lower than in other regions of the country. However, further studies with a larger sample size are needed to support these results. These findings seem to be useful for epidemiologists and physicians to improve their understanding of hospital-acquired infections and to implement antibiotic resistance control programs. In this study, K. pneumoniae isolates showed a high resistance rate (more than 50.0%) against third-generation cephalosporins that may be due to the presence of extended spectrum β-lactamase (ESBL) enzyme in the plasmids harbored by K. pneumoniae isolates [10, 11]. Also, this study revealed that aminoglycosides including amikacin and gentamicin were among the highly effective antibiotics against CR-Kp that was in line with a previous report from Saudi Arabia [10]. Also, Ferreira et al. [15] and Awoke et al. [16] reported the amikacin as one of the most effective antibiotics against K. pneumoniae isolates. Hence, aminoglycosides may be effective in the empirical treatment of infections caused by CR-Kp in northwestern Iran.

Another finding of this study was the frequency rate of 49.0% of MDR K. pneumoniae, which was lower than in previous studies from Brazil (84.0%) [15] and Ethiopia (98.5%) [16]. Compared with our results, Farhadi et al. [17] from Mazandaran province in northern Iran reported a higher prevalence (58.0%) of MDR K. pneumoniae. Antibiotic resistance patterns of K. pneumoniae isolates vary in different areas and studies due to differences in antibiotic prescribing, lack of drug resistance control programs, overuse of antibiotics in food and agricultural industries, and differences in epidemiology among regions. Data on K. pneumoniae and its antimicrobial resistance profiles in different geographic areas can help clinicians choose the best empiric antibiotic therapy.

In this study, the prevalence of blaVIM, blaNDM, blaKPC, blaOXA, and blaIMP genes was investigated in CR-Kp isolates. The results showed that a total of 23.0% of K. pneumoniae isolates carried various carbapenemase genes. Also, blaKPC (72.0%) was the most prevalent carbapenemase gene among CR-Kp isolates followed by blaVIM (12.0%), blaIMP (4.0%), and blaOXA−48−like (4.0%) genes. However, the blaNDM was not detected in any isolate. Coexistence of carbapenemase genes was also not detected in any isolate. In contrast to the current study, in a previous report from Busher in southern Iran, the rate of carbapenemase-producing K. pneumoniae (CP-Kp) was lower (7.9%), the blaNDM−1 (91.6%) and blaOXA−48−like (33.3%) genes were the most prevalent carbapenemase genes among CP-Kp isolates, and the coexistence of blaNDM and blaOXA−48−like genes was present in 25.0% of the isolates [18]. In previous studies from Iran by Gheitani et al. [14] and Khorvash et al. [19], no CR-Kp isolate carried blaKPC, which was in contrast to the current study. However, similar to this study, Khorvash et al. [19] did not find blaNDM gene in their isolates. Although the blaNDM gene was not detected in this study, it has been speculated that Middle Eastern countries may serve as reservoirs for the spread of this type of carbapenemase [20]. Shahcheraghi et al. [21] have identified blaNDM−1 containing K. pneumoniae in Iran for the first time.

Pourgholi et al. [20] from Tehran, Iran (67.6%) and Ssekatawa et al. [8] from Uganda (36.4%) reported the blaOXA−48−like as the most frequent carbapenemase in K. pneumoniae isolates, which was much higher than the current study (4.0%). There is ample evidence that the blaOXA−48−like gene is present in several other countries, including Nepal [22] and South Africa [23]. However, this gene was not detected in the previous studies from Brazil [15] and China [24]. In this study, the blaVIM and blaIMP genes were detected in 3 (12.0%) and 1 (4.0%) isolates, respectively. In a previous study by Ragheb et al. [25] from Egypt, the blaVIM gene was detected as the most frequent carbapenemase (84.62%) in K. pneumoniae isolates, which was higher than in this study. Likewise, they reported the blaIMP gene in a higher proportion (58.97%) of isolates than in the current research [25]. In line with the current study, Bilal et al. [26] from Pakistan, reported low frequency of blaIMP (7.2%) and blaVIM (3.2%) genes in K. pneumoniae isolates. In contrast to this study, in a previous report from Turkey, the blaKPC and blaVIM genes were not detected in Enterobacterales isolates [27]. Likewise, no blaIMP, blaVIM, and blaKPC genes were detected among clinical isolates of K. pneumoniae by Yasbolaghi Sharahi et al. [13] from Tehran, Iran that was in contrast to this study. In another study by Hosseinzadeh et al. [28] from Shiraz, southwestern Iran, blaOXA−48−like and blaNDM−1 genes were found in 0.9% and 10.9% of K. pneumoniae isolates, respectively. These results confirmed the discrepancies in the distribution of various carbapenemase genes in different geographical areas. Reasons for discrepancies in the results include differences in the study population, the source of the isolates, and the technique used to identify the carbapenemases. In our study, no carbapenemase genes were found in 2 carbapenem-resistant isolates, suggesting that their resistance may be due to other mechanisms, such as production of AmpC beta-lactamases, ESBLs, efflux pumps, decreased permeability of outer membranes, absence of the CRISPR/Cas system, or possibly other carbapenemase genes that were not studied [20, 29, 30]. In this study, a total of 36.0% (n = 9/25) of CR-Kp isolates were detected in the outpatient department. In line with our results, a high rate of carbapenem-resistant Enterobacterales (57.5%) collected from the outpatient department has been previously reported by Abdelaziz [31] from Egypt. This can be a warning sign to implement surveillance programs to hinder any further spread of this strains. Presently, the only three nations that have an orderly organized national surveillance system are Thailand, the European Union, and the United States [32]. Gathering national information is necessary for establishing appropriate policy, revising lists of vital drugs for infection treatment, and assessing the effects of intervention strategies [33].

The WHO highlighted 12 antibiotic-resistant pathogens in 2017, including K. pneumoniae, to be the most threatening to human health [34]. Considering the increasing resistance of bacteria to many antibiotics, including carbapenems, more studies are necessary to achieve new treatment methods using plant metabolites or vaccine design [34, 35].

Limitations

This study had several limitations, including the lack of sequencing of the carbapenemase gene and the failure to evaluate the clonal association of the CR-Kp isolates by a PCR-based method. Also, the other carbapenem resistance mechanisms, including efflux pumps expression and porin profiles of the isolates, were not investigated.

Conclusion

This study showed that the prevalence of CP-Kp with carbapenemase genes was lower in northwestern Iran compared to other regions of the country. Moreover, blaKPC was the most frequent carbapenemase gene. To prevent the further spread of CR-Kp in northwestern Iran, infection control measures, better screening methods, and optimal use of existing antibiotics should be emphasized.