Abstract: Background and Objective: Klebsiella pneumoniae is an important multi-drug-resistant Gram-negative pathogen, associated with nosocomially acquired infections. This study aimed to determine the genotypic and phenotypic characterization of Klebsiella pneumoniae isolates and to correlate the antibiotic resistance with the presence of virulence genes revealed by molecular genotypic testing in Riyadh, Saudi Arabia. Materials and Methods: About 23 Klebsiella pneumoniae isolates were collected from various specimen types. Identification of the organisms was carried out. Antimicrobial susceptibility performed against 12 antibiotics. The DNA was isolated and purified then genotypic confirmation was done through polymerase chain reactions (PCR) to detect TEM, SHV, CTX-M, IMP and KPC genes. PCR products were sequenced and aligned with GenBank sequences. Results: Out of 23 isolates of K. pneumoniae, the majority (43.5%) was from tracheal aspirate. The percentage of females (65.2%) was more than males (34.8%). The highest isolates prevalence was found in the age group of >58 (39.1%). About 100% of isolates were resistant to cefotaxime, ceftriaxone, ceftazidime, cefepime and ampicillin and 91.3% were sensitive to amikacin and Imipenem. Most isolates were SHV-9 gene positive (52.2%). It was found that tested isolates had 99-100% similarity when compared to GenBank sequences. Conclusion: There was a preponderance of SHV-9 gene which suggests dissemination of the gene in the tested isolates.
INTRODUCTION
Anti-biotic resistance is a natural phenomenon of bacteria found naturally in the surrounding environment or pathogens of humans and animals. As newly produced antibiotics push them to develop biochemical mechanisms for resistance through a large number of responsible genes. As a result of which, worldwide problems are being arrived with serious consequences on the treatment of infectious diseases1. These pathogenic bacteria are frequently reported as multi-drug-resistant bacteria (MDR) and may produce extended-spectrum β-lactamases (ESBL). The incidence and type of ESBL producer strains vary according to different geographical locations2. Infections with MDR organisms often lead to increased mortality, longer hospital stays and inflated healthcare costs. Hence, MDR bacterial pathogens are important health challenge worldwide3.
The highest incidence of bacterial resistance usually observed at intensive care units (ICU) due to excessive use of antibiotic agents. And so, understanding the resistance profile of hospital micro-biota against antibiotics is fundamental for the prevention and control of nosocomial infections4. Another risk factor is the international travel which forms a major mode of spread of MDR Gram-negative bacteria. So Gulf Cooperation Council countries (GCC) (Saudi Arabia, United Arab Emirates [UAE], Oman, Kuwait, Qatar and Bahrain) suffers this significant issue as large numbers of expats seek medical care in specialized centers hand in hand with millions visit the region annually for the Hajj and other religious events5.
Out of particular concern is the spread of MDR strains of Klebsiella pneumoniae, a Gram-negative bacterium that causes nosocomial infections. Klebsiella accounts for 3-7% of all nosocomial bacterial infections in the United States, placing them among the eight most important infectious pathogens in hospitals. The K. pneumoniae can harbor both extended-spectrum-lactamases (ESBL) and carbapenemases capable of hydrolyzing newer carbapenem drugs. Frequently associated with ESBL-producing K. pneumoniae is resistance to other antibiotics, including fluoroquinolones, amino glycosides, trimethoprim and sulfamethoxazoles6,7. The prevalence of ESBL in Klebsiella pneumoniae is increasing worldwide. Global studies showed that the frequency of ESBL-producing K. pneumoniae was 44% in South America, 33% in Europe, 22% in Asia and 12% in the United States4. The ESBL are enzymes produced by many bacterial species as a means for defense against β-lactam drugs with the genes encoding for those enzymes being mainly located on mobile genetic elements8.
The K. pneumoniae has recently gained popularity as an infectious agent due to a rise in the number of severe infections. These pathogens showed much resistant in response to treatment of urinary tract, intra-abdominal, skin infections and pneumonia, among neonates, elderly and immuno-suppressed individuals within the healthcare-associated settings9,10. Treatment of these infections depends heavily on effective antimicrobial therapy and delaying treatment may lead to a higher mortality rate. Therefore, the presence of MDR in the infecting pathogen would adversely affect the treatment outcome11.
In Gram-negative bacteria, MDR-ESBL mechanism of resistance may include a production of β-lactamases and these enzymes are usually acquired by gene transfer12. These strains have been isolated in several countries since the 1980s. Many outbreaks caused by such bacteria, especially Klebsiella pneumoniae. The predominant ESBLs/genes reported in Western and Asian countries are TEM- and SHV-derived ESBLs. These β-lactamases possess one or more amino acid substitutions that correspond to the narrow-spectrum prototype, TEM-1 and SHV-1 and these amino acid substitutions confer resistance to oxyimino β-lactam antibiotics13. About more than 390 different kinds of ESBLs are recognized around the world, among them blaSHV, blaTEM and blaCTX-M are more common14.
Although few studies have been conducted on carbapenem-resistant Klebsiella pneumoniae isolates in the Arabian Peninsula, almost all countries in the Gulf Cooperation Council (GCC) share the same ESBL and carbapenemase-producing Enterobacteriaceae, most of which were recovered from nosocomial infections. Moreover, a recent review of β-lactamase producing Gram-negative bacilli from GCC states showed that β-lactamases genes such as OXA-48, CTX-M-15 and NDM-1 are the most common and widespread β-lactamases15.
The developing regional surveillance of antibiotic resistance is urgent to detect multidrug-resistant (MDR) isolates. Clinical laboratories have to accurately screen isolates suspected of harboring virulence genes that are responsible for antibiotic resistance. For this reason, this study was conducted to determine the phenotypic and genotypic characterization of Klebsiella pneumoniae isolates and to correlate the antibiotic resistance with the presence of virulence genes revealed by molecular genotypic testing in Riyadh, Saudi Arabia.
MATERIALS AND METHODS
Bacterial Isolates: This study was carried out at King Khalid University Hospital, Riyadh, Saudi Arabia. The study included 23 non-duplicate, consecutive, phenotypically confirmed K. pneumoniae ESBL-producing isolates identified between June-December, 2016. These isolates were from various specimen types including blood, urine, wound swabs, sputum, tracheal aspirate, eye and groin, which were routinely cultured in the bacteriology laboratory. Identification of the organisms and susceptibility testing were carried out according to the laboratory policy by Vitek 2 (Biomerieux, Marcy l’ Etoile). The isolates flagged as ESBL by Vitek 2 were confirmed using the E-test method as recommended by CLSI16.
Anti-biotic susceptibility testing against Klebsiella Pneumoniae isolates: Anti-microbial susceptibility test by the disk diffusion method was performed to determine the resistance patterns of the isolates to 12 antibiotics: cefotaxime (CTX, 30 μg), ceftriaxone (CT, 30 μg), ceftazidime (CAZ, 30 μg), cefepime (Cefep 30 μg), gentamicin (GEN, 10 g), amikacin (AK, 30 μg), piperacillin/tazobactam (PIP/TAZ 100/10 μg), ciprofloxacin (CIP , 5 μg), tetracycline (TET, 30 μg), ampicillin (AMP, 10 μg), amoxicillin/clavulanic (AX/CLA, 30 μg), Imipenem (IMI, 10 g) [Oxoid, England, disc contents according to Clinical and Laboratory Standards Institute (CLSI) guidelines]. All antimicrobial testing was performed on Mueller-Hinton agar by the flooding technique and data interpreted according to the CLSI guidelines16.
Extended-spectrum beta-lactamase E-tests (AB Biodisk, Solna, Sweden) were performed as confirmatory tests according to the CLSI guidelines16.
Genotyping using PCR and sequence data analysis: DNA was isolated and purified using a Nucleospin kit (Macherey-Nagel), according to the manufacturer’s instructions. Genotypic confirmation was done through polymerase chain reactions (PCR) to detect TEM, SHV, CTX-M, IMP and KPC genes17.
The PCR products were sequenced by sending the products to GATC BIOTECH or by using Sanger sequencing in the lab. The DNA sequences were analyzed using Codon Code Aligner software and were assembled using Geneious 5.0.4. The nucleotide sequences of the genes of interest were aligned to the local database using a BLAST CLC Genomics Workbench 9.0 software package. Sequences obtained from the samples were aligned with GenBank sequences18.
Statistical analysis: All data were stored in Microsoft Excel, Version 2016. Data management and statistical analyses were also performed in Excel. Descriptive statistics of the data and variables were presented in the form of frequencies and percentages.
Ethical statement: The institutional review board, college of medicine, King Khalid University Hospital, Riyadh, Saudi Arabia, has granted permission to conduct this study.
RESULTS
Bacterial isolates: The study included a total number of 23 non-duplicate, consecutive, phenotypically confirmed Klebsiella pneumoniae ESBL-producing isolates from various body sites.
The majority of the isolates (n = 10, 43.5%) were isolated from tracheal aspirate samples and others as shown in Table 1. Male to female was 1:2 (Table 2). Table 3 shows that the highest isolates prevalence was mostly found in the age group of >58 (39.1%).
Anti-biotic susceptibility testing against Klebsiella Pneumoniae isolates: Antibiotic susceptibility patterns are shown in Table 4, 100% of the isolates were resistant to cefotaxime, ceftriaxone, ceftazidime, cefepime and ampicillin, while 91.3% of them were sensitive to amikacin and Imipenem.
Genotyping using PCR and sequence data analysis: Of the 23 clinical isolates of Klebsiella pneumoniae 19 ESBL-producing isolates comprising SHV, CTX-M and TEM genes and 4 carbapenemase isolates comprising KPC and IMP genes.
| Table 1: | Prevalence of Klebsiella pneumoniae in various specimen types (n = 23) |
| Table 2: | Prevalence of Klebsiella pneumoniae in different genders (n = 23) |
| Table 3: | Prevalence of Klebsiella pneumoniae in different age groups (n = 23) |
| Table 4: | Antibiotic susceptibility testing against Klebsiella pneumoniae isolates (n = 23) |
| Table 5: | Distribution of antibiotic resistance genes in Klebsiella pneumoniae isolates (n = 23) |
| Table 6: | Distribution of single gene and gene combinations in Klebsiella pneumoniae isolates (n = 23) |
| Table 7: | Analysis of antibiotic resistance genes in tested Klebsiella pneumoniae isolates (n = 23) |
For ESBL genes, distributions of SHV, CTX-M, TEM were demonstrated as 12 (52.2%), 8 (34.8%) and 6 (26.1%), respectively. On the other hand, carbapenemase genes were observed as 3 (13%) KPC and 2 (8.7%) IMP (Table 5). The distribution of single gene and gene combinations in Klebsiella pneumoniae isolates are shown in Table 6.
The SHV-9 gene was harbored by the majority of the isolates 12 (52.2%). However, about 8 isolates (34.8%) harbored the CTX-M-15 gene. Also, the TEM-1 gene was detected in 6 (26.1%) and 2 (8.7%) isolates were positive for IMP4. And for KPC gene 3(13%), it comprised of two groups, KPC2 (n = 1) and KPC3 (n = 2) (Table 7). On sequence similarity analysis, it was found that tested isolates had 99-100% similarity when compared to GenBank sequences (Table 7).
DISCUSSION
This result revealed that the majority of K. pneumoniae isolates were sensitive to amikacin and Imipenem. There was a preponderance of SHV-9 gene which suggested dissemination of the gene in the isolates. The majorities of the isolates were from tracheal aspirate samples and were mostly from females. The highest isolates prevalence was mostly found in the age group of 58, which is in agreement with Somily et al.19, who studied 77 isolates from various body sites. They stated that isolates were mostly from females with predominance of adult patients19 and in contrast to Podschun and Ullmann20, who reported that nosocomial Klebsiella infections most commonly involve the urinary tract20.
Klebsiella infections target immune-compromised patients, so serve as a paradigm of hospital-acquired infections21 especially for patients who had underlying diseases and the predominance of females was noticed which might be due to previous higher antibiotic intake22. As risk factors associated with women are previous surgeries (cesarean, normal delivery) and diabetes23. High frequency in (>58) age group was observed which might be because of low immunity2. The current study confirmed by the results of previous studies that carbapenems and amikacin remain the most active agents against K. pneumoniae, while cefotaxime, ceftriaxone, ceftazidime, cefepime and ampicillin were the most resistant agents21,24-27.
As noticed, many of the isolates were Multidrug-Resistant (MDR) strains and extracted from admitted immune compromised patients which confirm Klebsiella pneumoniae infection to be a serious nosocomial Hospital Acquired Infections (HAI)21,23,28. Major outbreaks were reported worldwide thus making them emerging pathogens. And Increasing drug resistance induce a real problem in therapeutic choices, especially with the rise of carbapenem resistance among isolates, which has emerged and been reported in many countries such as Saudi Arabia, Algeria, India, Ireland, Switzerland and Russia26. This multi-drug-resistant might be due to using antibiotics without restriction. In several studies, it has been shown that the high prescribing habits of the physicians and empirical use are the driving factors for the antibiotic resistance for this group of antibiotics25,29. Thus, it is highly recommended that practicing physicians should become aware of the antibiotic resistance problem and help in fighting this deadly threat by rational prescribing29,30.
The majority of the isolates harbored SHV-9 gene followed by CTX-M-15 gene and then comes the TEM-1 gene. The fewer percentages of isolates for IMP4 gene and KPC gene were noticed which comprised of two groups (KPC2 and KPC3). On sequence similarity analysis, it was found that tested isolates had 99-100% similarity when compared to GenBank sequences, which were in agreement with Tawfik et al.30, that characterized ESBL-producing K. pneumoniae in Al-Qassim area, Saudi Arabia. Their molecular study revealed that 89.1% of the isolates produced SHV, 70.9% produced TEM and 36.4% were CTX-M-producing strains. The prevalence of ESBL SHV-12 and SHV-5 was of 60% and 18.2%, respectively and various non-ESBL SHV, including SHV-1 (5.5%), -11 (3.6%) and -85 (1.8%) was detected. However, the prevalence of CTX-M-15 and CTX-M-14 was 34.5% and 1.8%, respectively30.
Therefore, implementing genetic analysis is recommended as routine work up in future research and diagnostic modalities for MDR infections and this can open fields for researches in the production of target-specific antibiotics.
CONCLUSION
Molecular genotyping using PCR and sequence data analysis results revealed the preponderance of the SHV-9 gene which could be the virulence gene disseminate in those strains in our region. And the correlation between the presence of the virulence gene and antibiotic resistance in Klebsiella pneumoniae may help diagnose these resistant isolates and pick up the appropriate antibiotic. Imipenem and amikacin are still recommended as good choices for treating Klebsiella pneumoniae infections.
SIGNIFICANCE STATEMENT
This study may add a new theory in the diagnosis and treatment of multidrug-resistance Klebsiella pneumoniae strain as the predominance of the SHV-9 gene was discovered that can be beneficial for choosing the suitable antibiotic and for increasing the researcher’s capabilities to uncover the critical areas of molecular genotyping aberration of Klebsiella pneumonia strain which extended chances for diagnosis and treatment of multi-drug-resistance Klebsiella pneumoniae in our region and worldwide.
ACKNOWLEDGMENT
This research project was funded by the Deanship of Scientific Research, Princess Nourah Bint Abdulrahman University, Group Funding Program, grant No