ORIGINAL ARTICLE Year : 2022  |  Volume : 9  |  Issue : 3  |  Page : 129-134

Prevalence of multidrug-resistant, extensively drug-resistant, and pandrug-resistant phenotypes among Klebsiella pneumoniae collected from referral therapeutic centers in Sari, North Iran

Mojgan Farhadi1, Mohammad Ahanjan2, Hamid Reza Goli1, Mohammad Reza Haghshenas1, Mehrdad Gholami3
1 Department of Microbiology and Virology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran
2 Antimicrobial Resistance Research Center, Communicable Diseases Institute, Mazandaran University of Medical Sciences, Sari, Iran
3 Department of Microbiology and Virology, Faculty of Medicine, Mazandaran University of Medical Sciences; Antimicrobial Resistance Research Center, Communicable Diseases Institute, Mazandaran University of Medical Sciences, Sari, Iran

Date of Submission10-Sep-2021Date of Decision07-Sep-2022Date of Acceptance09-Sep-2022Date of Web Publication11-Apr-2023

Correspondence Address:
Dr. Mehrdad Gholami
Antimicrobial Resistance Research Center, Communicable Diseases Institute, Mazandaran University of Medical Sciences, Sari
Iran

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/iahs.iahs_173_21

Aims: Klebsiella pneumoniae is an important cause of nosocomial infections. The present study sought to detect resistance status and the frequency of multidrug-resistant (MDR), extensively drug-resistant (XDR), and pandrug-resistant (PDR) K. pneumoniae strains isolated from hospitalized patients. Materials and Methods: Confirmation of K. pneumoniae isolates was performed by polymerase chain reaction (PCR) for rpoB fragment. Drug susceptibility was done by disk diffusion method based on the Clinical and Laboratory Standards Institute. According to the susceptibility pattern, the strains were categorized as MDR, XDR, and PDR. Results: On the basis of PCR results, rpoB gene was identified in all 100 K. pneumoniae isolates. Most K. pneumoniae strains showed a high percentage of resistance against ampicillin-sulbactam (93%). On the other hand, tigecycline and fosfomycin were active against 100% and 90% isolates, respectively. The multiple resistance analysis of the strains showed that 58% and 13% of isolates were identified as MDR and XDR, respectively. Overall, no PDR isolate was detected in any tested strains. Conclusion: Our results demonstrated that the vast majority of K. pneumoniae strains indicated the MDR phenotype including a high resistance rate to common antibiotics. Therefore, it is suggested to implement antimicrobial susceptibility testing before prescribing, to assist in selecting the most effective agents within the antimicrobial categories for the treatment of infections with multiple antibiotic-resistant K. pneumoniae strains in hospitalized patients.

Keywords: Extensively drug-resistant, fosfomycin, Klebsiella pneumoniae, multidrug-resistant, nosocomial infections

How to cite this article:
Farhadi M, Ahanjan M, Goli HR, Haghshenas MR, Gholami M. Prevalence of multidrug-resistant, extensively drug-resistant, and pandrug-resistant phenotypes among Klebsiella pneumoniae collected from referral therapeutic centers in Sari, North Iran. Int Arch Health Sci 2022;9:129-34
How to cite this URL:
Farhadi M, Ahanjan M, Goli HR, Haghshenas MR, Gholami M. Prevalence of multidrug-resistant, extensively drug-resistant, and pandrug-resistant phenotypes among Klebsiella pneumoniae collected from referral therapeutic centers in Sari, North Iran. Int Arch Health Sci [serial online] 2022 [cited 2023 Apr 15];9:129-34. Available from: http://www.iahs.kaums.ac.ir/text.asp?2022/9/3/129/374081   Introduction 

The word “ESKAPE” covers six groups of bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) with developing rate of resistance to available antimicrobial agents and pathogenicity. ESKAPE pathogens are responsible for most health-care-associated infections.[1],[2] The K. pneumoniae belongs to the Enterobacterales family and is a Gram-negative opportunistic pathogen that is encapsulated and nonmotile.[3] The K. pneumoniae can cause pneumonia, urinary tract infections, bloodstream infections, and liver abscesses in hospitalized and/or immunocompromised patients.[4]K. pneumoniae uses diverse virulence factors during infection to avoid immune-mediated clearance.[5] A variety of virulence factors are found in this bacterium, including capsular antigen, lipopolysaccharide, adhesions, siderophores, biofilms, fibrils, and toxins. Increasing antibiotic resistance and the emergence of infection are closely linked to these factors.[1],[4],[5] Currently, K. pneumoniae displays a considerable resistance to a broad spectrum of antimicrobial agents, including beta-lactam, fluoroquinolones, and aminoglycosides.[6],[7] Meanwhile, infections caused by multidrug-resistant K. pneumoniae (MDRKp) strains are getting increasingly difficult to treat worldwide as they become increasingly resistant to antibiotics.[7],[8] Although several studies were performed in different parts of Iran on the drug resistance of this bacterium and the rate of MDR, extensively drug-resistant, (XDR), and pandrug-resistant (PDR) strains, there is a limited study on the frequency of this resistance phenotypes among K. pneumoniae strains, especially in northern Iran, Mazandaran.[9],[10],[11] Increasing the emergence of multiple drug resistance among K. pneumoniae strains has been a big challenge to clinicians. Moreover, limited treatment options are also associated with high morbidity and mortality for hospitalized patients. The characterization of drug susceptibility patterns will help clinicians make the best management decisions and help to prevent and control infections caused by resistant strains of K. pneumoniae. Considering this evidence, this research was designed to investigate antibiotic susceptibility pattern and the rate of MDR, XDR, and PDR K. pneumoniae isolated from clinical specimens of teaching hospitals of Mazandaran University of Medical Sciences (MAZUMS) located in Sari, Iran.

  Materials and Methods 

This project was approved by the Ethics Committee of MAZUMS (Approved Number: IR.MAZUMS.REC.1398.628). Although, we did not have a direct connection with the patients. We only obtained the clinical samples of the patients without their names from the hospital laboratories, and the data were kept secret by the authors. In this cross-sectional study, a total of 100 nonduplicate K. pneumoniae isolates from clinical samples were collected from inpatients, who were referred to three teaching hospitals of MAZUMS during November 2018 to October 2019. Clinical samples were obtained as portion of the common diagnostics from hospitalized patients and included samples from sterile and/or nonsterile body sites. The samples were cultured on blood agar and MacConkey agar (Merck, Darmstadt, Germany) and incubated at 37°C for 24 h. In order to identification of isolates, standard microbiological tests were conducted, including oxidase; citrate; triple sugar iron; urease; lysine decarboxylase; sulfide production, indole production, and motility; methyl red; and Voges–Proskauer tests.[12] Furthermore, the sex and age of the patients and the source of samples were documented. The K. pneumoniae isolates were stored at −70°C in trypticase soy broth (Merck, Darmstadt, Germany) plus 20% glycerol.

Confirmatory assessment of K. pneumoniae at species level was accomplished by polymerase chain reaction (PCR) of rpoB gene.[13] Briefly, a single colony of each strain was grown at 37°C in Mueller–Hinton agar plate. The total genomic DNA of tested isolates was extracted as previously described.[14] After extraction, NanoDrop spectrophotometer (Thermo Fisher Scientific, USA) was used for assessment of the extracted DNA yield/purity. Amplification of rpoB gene among the tested strains was achieved with the following species/specific primers: (F: 5′-CAACGGTGTGGTTACTGACG-3′/R: 5′-TCTACGAAGTGGCCGTTTTC-3′) (BIONEER company).[15] In order to make sure of the primer specificity, its sequence is rechecked and confirmed in BLAST-NCBI primer program. K. pneumoniae American Type Culture Collection (ATCC) 700603 were used as positive control. Negative controls (all essential components of the amplification reaction except the template) were considered for every run of PCRs. Reactions were done in a thermal cycler system (Bio-Rad, USA). PCRs were carried out in 25-μl reaction volumes with 14 μL of ready-to-use 2X Master Mix (Amplicon), 6 μL of chromosomal DNA (50 ng), 1 μL (20 pM/μL) of each primer (forward/reverse), and 3 μl of sterile distilled water. PCR reactions were achieved by thermal cycler (Eppendorf, Germany) in the following steps: step 1 (initial denaturation): 95°C 10 min, step 2 (denature): 95°C 30 s, step 3 (anneal primers): 52°C 40 s, step 4 (extend DNA): 72°C 50 s, and step 5 (final extension): 72°C 5 min. Step 2/3/4 was repeated in 35 cycles. PCR products were electrophoresed in a 1% (w/v) agarose gel alongside a DNA size marker 100 bp (Cat No. DM003-R500).

According to the Clinical and Laboratory Standards Institute documents, disk diffusion was used to determine susceptibility and/or resistance to 18 antimicrobial agents.[16] A panel of 18 antimicrobial agents from different classes were tested, including trimethoprim-sulfamethoxazole (SXT, 1.25/23.75 μg), cefepime (FEP, 30 μg), amikacin (AN, 30 μg), imipenem (IPM, 10 μg), meropenem (MEM, 10 μg), cefotaxime (CTX, 30 μg), gentamicin (GM, 10 μg), ceftriaxone (CRO, 30 μg), ceftazidime (CAZ, 30 μg), ciprofloxacin (CIP, 5 μg), levofloxacin (LEV, 5 μg), nitrofurantoin (NIT, 300 μg), ampicillin/sulbactam (SAM, 20 μg), ertapenem (ETP, 10 μg), cefoperazone (CEP, 75 μg), tetracycline (TE 30 μg), and fosfomycin (FOS, 200 μg) (MAST UK). In summary, a bacterial suspension equal to #0.5 McFarland standard (1.5 × 108 CFU/mL) was prepared from overnight cultures and then cultured on Mueller–Hinton agar (Merck, Darmstadt, Germany). Then, antibiotic disks were put in plate. After 24 h of incubation at 37°C, the zone of the inhibition was measured and the data were reported as susceptible (S), intermediate (I), and resistant. Escherichia coli ATCC 25922 was used as a quality control. In all analyzed strains, tigecycline (TGC, 15 μg) (HiMedia) disk was done to investigate the trend of TGC susceptibility.[17] In this study, MDR and XDR phenotypes among the strains were defined as an instruction previously described by Basak et al.[18]

The data derived from this study were analyzed by SPSS (Statistical Package for the Social Sciences, Chicago, IL, USA) statistical program (ver. 16) using Chi-square and Fisher's exact statistical tests at the confidence level of 95%.

  Results 

In this study, among the entire samples, 58% of the subjects were female and 42% were male. Based on Chi-square test, there was no significant relationship between relative antibiotic resistance and gender of patients (P > 0.05). In our study, rpoB gene was detected in all 100 K. pneumoniae isolates. Therefore, rpoB can be a good marker for identification of K. pneumoniae at the species level [Figure 1]. Of the 100 K. pneumoniae isolates, 64 (64%) were isolated from urine, 15 (15%) from tissue, 10 (10%) from blood, 7 (7%) from wound and 4 (4%) from sputum samples. The age range of the patients was 5–90 years. The isolates were obtained from patients in different age groups: 1–19 years (n = 5), 20–39 (n = 22), 40–59 years (n = 45), 60–79 years (n = 19), and 80–100 (n = 9). The overall susceptibility, intermediate, and resistance were determined, and the outcomes are displayed in [Figure 2]. Most K. pneumoniae isolates indicated a high ratio of resistance against studied antibiotics: SAM (93%), NIT (57%), CEP (52%), SXT (50%), CAZ (49%), CTX (48%), MEM (42%), CRO (42%), FEP (41%), CIP (34%), IPM (33%), LEV (29%), GM (23%), ETP (23%), TE (22%), AN (8%), FOS (2%), and TGC (0%). [Table 1] shows the details of the antibiotic resistance profile of the strains based on antimicrobial class. Furthermore, in our study 13% of the strains were XDR and 58% were MDR. The antimicrobial resistance among MDR strains was found meaningfully higher than that of non-MDR strains (P < 0.05). It should be noted that no PDR isolate was detected in this study.

Figure 1: PCR amplification fragments for the detection of rpoB gene (108 bp) among Klebsiella pneumoniae isolates. Lane: M: 100 base-pairs (bp)-3K DNA size marker, Lane P: Positive control, lane N: Negative control, lanes 1–14: Tested strains, PCR: Polymerase chain reaction

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Figure 2: Antimicrobial resistance profiles of 100 Klebsiella pneumoniae isolates against 18 antibiotics. R: Resistance, S: Sensitive, and I: Intermediate. SXT: Trimethoprim-sulfamethoxazole, FEP: Cefepime, AN: Amikacin, IPM: Imipenem, MEM: Meropenem, CTX: Cefotaxime, GM: Gentamicin, CRO: Ceftriaxone, CAZ: Ceftazidime, CIP: Ciprofloxacin, LEV: Levofloxacin, NIT: Nitrofurantoin, SAM: Ampicillin/sulbactam, ETP: Ertapenem, TGC: Tigecycline, CEP: Cefoperazone, TE: tetracycline, FOS: Fosfomycin

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Table 1: Susceptibility testing profile of 100 Klebsiella pneumoniae isolates based on antimicrobial class

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  Discussion 

K. pneumoniae is one of the major causes of community-and hospital-acquiring infections. It is thought that this bacterium is one of the most common pathogens that cause high rates of mortality in hospitals.[19],[20],[21] It is unfortunate that the increase in antibiotic resistance is becoming a serious threat to controlling infectious diseases caused by this pathogen.[22] Recent years have seen an increase in infections caused by MDR/XDR and PDR K. pneumoniae strains among critically ill patients, which are often associated with limited treatment options. The main subject of managing these infections is to find an effective antibiotic regimen or alternative treatment strategies.[18],[23] Based on the results of the current study the highest rate of resistance was observed for SAM (93% resistant and 6% intermediate) and the lowest for FOS (2%). In addition, no resistant isolates against TGC were found. In our study, a small number of K. pneumoniae isolates (only 1%) were susceptible to SAM. Therefore, we concluded that SAM could not be a good choice for the initial empiric antimicrobial therapy in hospitalized patients infected with MDR K. pneumoniae strains. This result is in agreement with the outcomes stated by prior work conducted by Qadeer et al. in Pakistan.[24] Carbapenems were commonly used for treating infections caused by K. pneumoniae.[25] Three members of the carbapenem antibiotics were investigated in our study. The isolates' susceptibility rates to ETP (73%), IPM (65%), and MEM (56%) were consistent with those reported in another study in Tehran, Iran.[26] Gheitani et al., in their work which was directed in Isfahan, Iran, reported that 28%, 24%, and 44% of the studied K. pneumoniae isolates were susceptible against MEM, IPM, and ETP, respectively.[27] These inconsistent outcomes may be related to the variable sample sizes and the geographical areas. In this study, the resistance to LEV and CIP antibiotics has been achieved 34% and 29%, respectively. According to Akya et al.,[28] study in Kermanshah, Iran, CIP and LEV resistance rates are 28%, similar to our findings for quinolone antibiotics. These data are similar to Yedekci et al.[29] Another study showed that of the 142 K. pneumoniae isolates analyzed, 76% were resistant to CAZ, and 73% were resistant to CTX and FEP.[30] In our study, the rate of third-generation cephalosporin resistance was 48%, 41%, 42%, and 49% against CTX, FEP, CRO, and CAZ, respectively. Various studies have shown controversial results with regard to the effectiveness of AN.[31],[32] In a study from India, among 73 K. pneumoniae isolates, 74% were resistant to AN.[33] Furthermore, other studies from Iran such as Bayati et al.[34] and Azimi et al.,[35] the rate of susceptibility to AN was 48% and 79%, respectively. In the study conducted by Asadpour and Nahavandinejhad,[36] from northern of Iran in 2015, GM and AN were the most appropriate antimicrobial agents against K. pneumoniae isolates. The difference between the results is probably due to the difference in source of clinical samples, the quality of antimicrobial disks and/or culture medium, and geographical regions. The rates of MDR and XDR isolates detected in this study are worrying. The prevalence of MDR and XDR phenotypes among our isolates was found to be 58% and 13%, respectively. The rate of MDR and XDR strains observed in this study was similar to previously reported rates from Iran and China,[37],[38] and it was higher than reported rates from a prospective surveillance study in 10 Asian countries.[39] This could be due to the difference on the study region population, geographical site, and type of antibiotics used in the treatment regimens. According to the results of the current study, all of collected K. pneumoniae were susceptible to TGC. Bokaeian et al. in 2018 determined that TGC is the most effective antibiotic for the treatment of K. pneumoniae infections.[40] Shokri from Iran stated that the resistance rate of the K. pneumoniae isolates to TGC in their study was 3.1%.[41] Fortunately, TGC also can be used in antibiotic therapy if MDR K. pneumoniae was isolated.[42],[43] However, the use of it should be the main focus in hospital settings. This study has some limitations: results of this cross-sectional study were derived from the patients referred from three teaching health facilities, which do not represent the actual view of the Sari city in Mazandaran Province. There was a lack of adequate demographic information and underlying diseases about the studied patients. Furthermore, in order to reach better outcomes, detection of common antibiotic-resistance genes is also necessary.

  Conclusion 

In summary, the high prevalence of MDR strains of K. pneumoniae is alarming in our study. Therefore, it is suggested to implement antimicrobial susceptibility testing before prescribing, to assist in selecting the most effective antimicrobial agents within the antimicrobial categories for the treatment of infections caused by K. pneumoniae in hospitalized patients. In our work, TGC was the only antibacterial agent that inhibited 100% K. pneumoniae strains. Hence, the use of TGC and FOS in combination with other antibiotics are useful to treatment of infection caused by MDR K. pneumoniae isolates. Furthermore, timely identification and systematic monitoring and surveillance of highly resistant strains in hospital settings will help to prevent the spread of these strains. Multicenter research is essential to gaining a comprehensive understanding of antibiotic resistance in other Iranian regions.

Acknowledgments

We are grateful to the laboratory staffs of the hospitals studied for their assistance in collecting the clinical isolates, as well as Mazandaran University of Medical Sciences for their financial support.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 

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  [Figure 1], [Figure 2]
 
 
  [Table 1]