Phenotypic, Antibiotyping, and Molecular Detection of Klebsiella Pneumoniae Isolates from Clinical Specimens in Kirkuk, Iraq

Document Type : Original Articles

Authors

1 Department of Biology, College of Education of Pure Science Kirkuk University, Iraq

2 Basic Science Department, Faculty of Dentistry, Al. Kitab University, Iraq

Abstract

Klebsiella pneumoniae is globally responsible for hospital- and community-acquired infections. This study aimed to determine the prevalence of K. pneumoniae and investigate the antibiotic resistance profile among clinical specimens at Azadi Teaching Hospital in Kirkuk, Iraq, and detect the rpoB gene for molecular identification of  K. pneumoniae in comparison with phenotypic and biochemical methods. In total, 250 clinical specimens were collected from patients in Azadi Teaching Hospital in Kirkuk, Iraq, between January 2018 and May 2018. The isolates were identified by morphologic and biochemical testing. Kirby-Bauer disk diffusion method was used in the antibiotics susceptibility test. Following that, 19 (7.6%) K. pneumoniae isolates were isolated from 250 clinical specimens (5 [5.61%] and 14 [8.69%] from males and females, respectively), and most of them (n=12; 11.76%) were isolated from the age group of 10-35 years old. The isolates were reported high resistance towards various types of antibiotics, especially penicillins and cephalosporins. In contrast, K. pneumoniae showed very low resistance to imipenem and amikacin (5.26% and 10.52%, respectively). The range of multidrug-resistant K. pneumoniae isolates in this study was estimated at 100%. In gene detection, all isolates in this study showed PCR product with 108 bp by K. pneumonia specific primer (rpoB). Developed antibiotic policies and regular surveillance of antibiotic susceptibility patterns may help to overcome the indiscriminate use of antibiotics that is a major cause of the emergence of drug resistance among pathogens.

Keywords

Main Subjects


1. Introduction

Klebsiella pneumoniae is an important opportunistic pathogen that causes a variety of infectious diseases in humans, including septicaemia, liver abscesses, diarrhea, and pneumonia. The development of antibacterial resistance is considered a serious challenge in hospitals and health care centers over the world. K. pneumoniae strains that are rapidly developing multidrug-resistant (MDR) are considered a critical threat to the patients causing a high fatality rate due to low therapy options. The World Health Organization announced antimicrobial resistance (AMR) as one of the main global problems ( 1 ). K. pneumoniae is globally known as one of the major causes of hospital- and community-acquired infections and plays a significant role in the propagation of antibacterial resistance genes from environmental bacteria to pathogenic bacteria ( 2 ).

This bacterium has developed resistance to antibacterial agents more readily than most bacteria by the production of Carbapenemase and Extended-Spectrum β- Lactamase enzymes ( 1 , 3 ). The most significant risk factor of AMR is exposure to antibiotics, and the main cause which contributes to expanding the spreading of resistant bacteria strains is the prolonged and intensive use of antimicrobial agents in health care settings ( 4 ). The pathogenic bacteria cause many infections, such as hospital-acquired pneumonia, urinary tract infection, bacteremia, surgical site infection, ventilator-associated pneumonia, and septicemia, in addition to the opportunistic infections that occur among immunocompromised patients ( 5 , 6 ).

β-subunit of RNA polymerase is encoded by the rpoB gene which is considered a core gene candidate for the identification of bacteria and phylogenetic analyses, particularly when studying closely related isolates ( 7 , 8 ).

The emergence of AMR in Klebsiella spp. isolates is of great concern worldwide in human medicine. Therefore, this study aimed to determine the prevalence of K. pneumoniae and investigate the antibiotic resistance profile among clinical specimens at Azadi Teaching Hospital in Kirkuk, Iraq, and detect the rpoB gene for the molecular identification of K. pneumoniae in comparison with phenotypic and biochemical methods.

2. Material and Methods

2.1. Samples

In total, 250 clinical specimens were collected from patients (89 and 161 males and females, respectively; age range: 10-70 years), including urine (n=100), vaginal swabs (n=50), wound (n=50), and throat (n=50) from Azadi Teaching Hospital in Kirkuk, Iraq, between January 2018 and May 2018.

2.2. Culturing and Identification

The samples were streaked on blood and MacConkey agar and then incubated at 37°C for 24 h. Gram stain, capsule stain, and many biochemical tests, such as oxidase, IMViC, urea hydrolysis, H2S production, lactose fermentation, lysine decarboxylase, coagulase, gas production, and catalyzes were used for K. pneumoniae identification ( 1 , 9 ).

2.3. Antibiotic Sensitivity Test

Kirby-Bauer disc diffusion method was utilized to detect the sensitivity of isolates to Gentamicin (10 µg), Cefotaxime (30 µg), Meropenem (10 µg), Ciprofloxacin(5µg), Tobramycin (10 µg), Amikacin (30 µg), Ampicillin (10 µg), Ceftazidime (30 µg), Nitrofurination (30 µg), Rifampin (5 µg), Amoxicillin (25 µg), Cefixime (5 µg), Doxycycline (10 µg), Imipenem (10 µg), Aztreonam (30 µg), Nalidixic (30 µg), Chloramphenicol (30 µg), Cephalothin (30 µg), and Trimethoprim/Sulphamthoxazol (1.25/23.75µg). K. pneumoniae ATCC 1290 was employed as control strains ( 10 ).

Resistance to three or more various classes of antibiotics was considered MDR K. pneumoniae ( 11 ).

2.4. Polymerase Chain Reaction

DNA of bacterial isolates was extracted by using the Bioneer kit. Polymerase chain reaction (PCR) test was performed with species-specific primers forward primer 5'-CAACGGTGTGGTTACTGACG-3', and reverse primer 5'-TCTACGAAGTGGCCGTTTTC-3' was used for the amplification of the K. pneumoniae target genes (rpoB). The reaction was performed in a 20 μl volume, 3 μl of a ready Master Mix, 2 µL of each primer, and 5 µL of DNA, while nuclease-free water was used to complete the volume. The PCR program included initial denaturation in one cycle for 5 min at 95°C, amplification in 35 cycles each of 30 sec. at 94°C, 30 sec. at 55°C, and 30 sec. at 72°C, followed by a final extension cycle for 7 min. at 72°C ( 1 , 4 ).

2.5. Data Analysis

The data were analyzed in SPSS software (version 16.0), and a p-value less than 0.05 was considered statistically significant.

3. Results

The prevalence of K. pneumoniae among clinical specimens was 19 (7.6%) isolates, and the highest percentage of the isolates from throat swabs was obtained at 12% (n=6) (Table 1).

Source of specimens No. of samples No .of K. pneumoniae (%)
Urine 100 7(7%)
Vagina 50 4 (8%)
Wound 50 2 (4%)
Throat 50 6 (12%)
Total 250 19 (7.6%)
Table 1. Prevalence of K. pneumoniae depending on the source of specimens

The isolates were distributed among 5 male (5.61%) and 14 female (8.69%) patients, and most of them (n=12; 11.76%) were isolated from patients within the age range of 10-35 years old (Table 2).

Age Groups (yrs.) No. of patients (n=250) K. pneumoniae (%) (n=19)
10-35 (11.76%) 102 12
36-55 98 4 (4.08%)
<56 50 3 (6%)
Gender of total patients
Female 161 14 (8.69%)
Male 89 5 (5.61%)
Table 2. Prevalence of K. pneumoniae depending on patients' gender and age

3.1. Antibiotic Susceptibility Pattern

K. pneumoniae isolates were reported high resistance towards various types of antibiotics, especially penicillins and cephalosporins. In contrast, K. pneumoniae showed very low resistance to imipenem and amikacin (5.26% and 10.52%, respectively) (Figure 1).

Figure 1. Resistance pattern of isolates

Table 3 tabulates the antibiotyping of the K. pneumoniae isolates that are classified under different groups (Types 1-16) relying on antimicrobial-resistant patterns. Antibiotyping of Type 1 shows resistance towards 20 antibiotics representing 100% of antibiotics used in this study, while the last type (Type 16) shows resistance toward 4 antibiotics.

Type No. of resistant antibiotic No. of isolates (%) Resistance of antibiotics
1 20 1 5.26% AM,RA,AX,CEP,CTX,CAZ,CFM,ATM,SXT,C,DO,MEM,NA,TOB,CIP,NT,AK, IMP.
2 19 1 5.26% AM,RA,AX,CEP,CTX,CAZ,CFM,ATM,SXT,C,DO,MEM,NA,TOB,CIP,NT,AK.
3 16 1 5.26% AM,RA,AX,CEP,CTX,CAZ,CFM,ATM,SXT,C,DO,MEM,NA,TOB,CIP,NT.
4 15 1 5.26% AM,RA,AX,CEP,CTX,CAZ,CFM,ATM,SXT,NT,NA,TOB,CIP,MEM,DO.
5 15 1 5.26% AM,RA,AX,CEP,CTX,CAZ,CFM,ATM,SXT,GN,NA,NT,CIP,TOB.DO.
6 14 1 5.26% AM,RA,AX,CEP,CTX,CAZ,CFM,ATM,SXT,GN,NA,NT,CIP,TOB.
7 13 1 5.26% AM,RA,AX,CEP,CTX,CAZ,CFM,ATM,SXT,C,DO,MEM,GN.
8 11 1 5.26% AM,RA,AX,CEP,CTX,CAZ,CFM,ATM, SXT ,NA,NT.
9 11 1 5.26% AM,RA,AX,CEP,CTX,CAZ,CFM,ATM, SXT ,C,GN.
10 11 1 5.26% AM,RA,AX,CEP,CTX,CAZ,CFM,ATM, SXT,GN,TOB.
11 9 2 10.52% AM,RA,AX,CEP,CTX,CAZ,CFM,ATM,SXT.
12 8 1 5.26% AM,RA,AX,CEP,CTX,CAZ,CFM,ATM.
13 6 1 5.26% AM,RA,AX,CEP,CAZ,CFM.
14 6 1 5.26% AM,RA,AX,CEP,CAZ,MEM.
15 5 1 5.26% AM,RA,AX,CEP,DO.
16 4 3 15.8% AM,RA,AX,CEP.
Table 3. Antibiotyping of K. pneumoniae isolates

3.2. DNA Extraction and Identification of K. pneumoniae by PCR

The DNA extraction from 19 K. pneumoniae was made by the Bioneer kit. The purity and the concentration of DNA specimens were ranged from (1.6- 2) to (60-130) ng/ul, respectively. All isolates in this study showed PCR product with 108 bp by K. pneumonia specific primer (rpoB) that performed K. pneumoniae (Figure 2).

Figure 2. PCR product of (rpoB) gene for K. pneumonia by gel electrophoresis. Lane L: 100bp DNA ladder; Lane N: negative; Lane Lanes 1-10: Clinical isolates

4. Discussion

The prevalence of K. pneumoniae was 7.6% in this study, while other studies reported such percentages as 4.03%, 17.36%, and 32.48% ( 12 - 14 ). These differences in the mean prevalence rates among various studies could be related to differences in geographical location and hygienic practices of the population ( 15 , 16 ). The highest percentage of K. pneumoniae isolates were reported among female patients since the highest number of collected samples in this study were from females; moreover, the age group of 10-35 years represented the highest percentage of K. pneumoniae (Table 2) because the young age group participated in outdoor activities, and they are the most activated group. This result is in line with the findings of the studies conducted by Al-Rubaye, Hamza ( 17 ), as well as Kadum ( 12 ) in Iraq. Ampicillin, amoxicillin, and cephalothin showed the lowest effect towards K. pneumoniae isolates, while amikacin and imipenem revealed the highest effect (Figure 1). These results were supported by studies performed by Namratha, Sreeshma ( 13 ), as well as Nirwati, Sinanjung ( 14 ).

The range of population exposure to antimicrobial agents with the hygienic culture of them and the type of clinical samples that examined were considered major reasons in the variation of prevalence rate of bacterial resistance among many studies ( 15 , 16 ). Several factors act in the growth of antibiotic resistance, such as the use of antibiotics in the community, hospital, environment, agriculture, and animal production. Furthermore, since there is a possibility to buy antimicrobial agents without prescription, this made the antimicrobial agents be used extremely. Prolonged and intensive use of antibiotics in health care settings are considered major factors in the wide spreading of severe dangerous nosocomial infections ( 4 ). The rate of MDR K. pneumoniae isolates in this study was estimated at 100% (Table 3), and the results of a study conducted by Manjula, CM ( 18 ) supported this finding. They showed that out of 41 isolates, 37 (90.2%) of them were MDR. Many studies have used a combination of antibiotics in their treatments to avoid emerging new resistant strains.

The MDR bacterial isolates are causing a global challenge in curing infections; as a result, the use of antibiotic stewardship programs is of critical importance in the optimization and monitoring of antibiotics use. Moreover, the Rational Use of Medicine Program is urged on the importance of the collaboration between microbiologists and clinicians to get effective management of infections ( 19 ). K. pneumoniae is the most prevalent cause of nosocomial infections and is considered an opportunistic pathogen due to the difficulty and misclassification in the detection of this bacterium in the laboratory ( 20 ). Therefore, molecular identification is highly necessary for accurate detection. All isolates in this study demonstrated PCR product with 108 bp by K. pneumonia specific primer (rpoB) that performed K. pneumoniae (Figure 2).

This finding is consistent with the results of a study performed by Hadi ( 21 ) in Kufa University that showed 100% PCR product representing K. pneumonia; however, it was not in line with the results of a study conducted by Al- Rubaye, who showed 87.93% PCR product that represented K. pneumonia ( 17 ). This could have been related to the type of clinical specimens and the type of laboratory identification methods used in various studies.

5. Conclusion

Careful selection of antimicrobial agents is suggested in this study for proper and accurate management. Due to the high prevalence of MDR K. pneumoniae infections, this will aid in decreasing the rate of mortality and morbidity. The development of antibiotic policies and regular surveillance of antimicrobial sensitivity patterns may aid to overcome the overuse of antibiotics that is the main cause of drug resistance development among pathogens.

Authors' Contribution

Study concept and design: S. A. H.

Acquisition of data: T. F. R.

Analysis and interpretation of data: H. M. A.

Drafting of the manuscript: H. M. A.

Critical revision of the manuscript for important intellectual content: S. A. H.

Statistical analysis: T. F. R.

Administrative, technical, and material support: S. A. H.

Ethics

All procedures performed in this study involving human participants were in accordance with the ethical standards of the Kirkuk University, Iraq.

Conflict of Interest

The authors declare that they have no conflict of interest.

References

  1. Munita JM, Arias CA. Mechanisms of antibiotic resistance. Microbiol Spectr. 2016; 4(2)
  2. Wyres KL, Holt KE. Klebsiella pneumoniae as a key trafficker of drug resistance genes from environmental to clinically important bacteria. Curr Opin Microbiol. 2018; 45:131-9.
  3. Bengoechea JA, Sa Pessoa J. Klebsiella pneumoniae infection biology: living to counteract host defences. FEMS Microbiol Rev. 2019; 43(2):123-44.
  4. Prestinaci F, Pezzotti P, Pantosti A. Antimicrobial resistance: a global multifaceted phenomenon. Pathog Glob Health. 2015; 109(7):309-18.
  5. Ashurst JV, Dawson A. Klebsiella Pneumonia: StatPearls Publishing, Treasure Island (FL); 2020.
  6. Seifi K, Kazemian H, Heidari H, Rezagholizadeh F, Saee Y, Shirvani F, et al. Evaluation of biofilm formation among Klebsiella pneumoniae isolates and molecular characterization by ERIC-PCR. Jundishapur J Microbiol. 2016; 9(1)
  7. Börner T, Aleynikova AY, Zubo YO, Kusnetsov VV. Chloroplast RNA polymerases: Role in chloroplast biogenesis. Biochim Biophys Acta Bioenerg. 2015; 1847(9):761-9.
  8. Goldstein BP. Resistance to rifampicin: a review. J Antibiot. 2014; 67(9):625-30.
  9. Beyene G, Tsegaye W. Bacterial uropathogens in urinary tract infection and antibiotic susceptibility pattern in jimma university specialized hospital, southwest ethiopia. Ethiop J Health Sci. 2011; 21(2):141-6.
  10. Zamani A, Mashouf RY, Namvar AME, Alikhani MY. Detection of magA Gene in Klebsiella spp. Isolated from clinical samplesdetection of magA. Iran J Basic Med Sci. 2013; 16(2):173.
  11. Magiorakos A-P, Srinivasan A, Carey R, Carmeli Y, Falagas M, Giske C, et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect. 2012; 18(3):268-81.
  12. Kadum SM. Colistin Susceptibility in Carbapenem Resistant Klebsiella Pneumoniae and their Ability of Biofilm Formation. Iraqi J Sci. 2020;517-27.
  13. Namratha K, Sreeshma P, Subbannayya K, Dinesh P, Champa H. Characterization and antibiogram of Klebsiella spp. isolated from clinical specimen in a rural teaching hospital. Sch J App Med Sci. 2015; 3(2E):878-83.
  14. Nirwati H, Sinanjung K, Fahrunissa F, Wijaya F, Napitupulu S, Hati VP, et al. Biofilm formation and antibiotic resistance of Klebsiella pneumoniae isolated from clinical samples in a tertiary care hospital, Klaten, Indonesia. BMC Proc. 2019; 13(11):20.
  15. Hasan SA, Abass KS. Prevalence of Gram Negative Bacteria Isolated from Patients with Burn Infection and their Antimicrobial Susceptibility Patterns in Kirkuk City, Iraq. Indian J Public Health Res Dev. 2019; 10(8)
  16. Hasan SA, Najati AM, Abass KS. Prevalence and antibiotic resistance of “pseudomonas aeruginosa” isolated from clinical samples in Kirkuk City, Iraq. Eurasia J Biosci. 2020; 14(1):1821-5.
  17. Al-Rubaye DS, Hamza HM, Abdulrahman TR. Genotyping of Klebsiella spp. isolated from different clinical sources. Iraqi J Sci. 2016; 57(3B):1937-51.
  18. Manjula G, N, CM GNK, Patil S, Gaddad S, Shivannavar C. antibiotic susceptibility pattern of ESbetaL producing Klebsiella pneumoniae isolated from urine samples of pregnant women in Karnataka. J Clin Diagn Res. 2014; 8(10)
  19. Wu H, Moser C, Wang H-Z, Høiby N, Song Z-J. Strategies for combating bacterial biofilm infections. Int J Oral Sci. 2015; 7(1):1-7.
  20. Alves MS, Dias RCdS, de Castro ACD, Riley LW, Moreira BM. Identification of clinical isolates of indole-positive and indole-negative Klebsiella spp. J Clin Microbiol. 2006; 44(10):3640-6.
  21. Hadi Z. Detection of extended-spectrum beta-lactamases of Escherichia coli and Klebsiella spp. isolated from patients with significant bacteriuria in Najaf: M. Sc Thesis College of Medicine Kufa University. 2008.