Molecular detection of virulence genes in Klebsiella Pneumoniae clinical isolates from Kurdistan Province, Iran

Shakib et al. Biomedical Research and Therapy 2018, 5(8): 2581-2589 Biomed. Res. Ther. 2018, 5(8): 2581-2589 2581


Background
Klebsiella pneumoniae is a prominent opportunistic pathogen which causes upper respiratory tract infection, diarrhea, pneumonia, urinary tract infection (UTI), and septicemia [1][2][3]. The prevalence of drug resistance in K. pneumoniae has increased, which is because of extended-spectrum betalactamase (ESBL) enzymes and appearance of multi-drug resistant (MDR) K. pneumoniae [4,5]. In addition, K. pneumoniae possesses different virulence factors that contribute to its pathogenicity including lipopolysaccharide (LPS) O-side chain (endotoxin), capsular polysaccharide, adhesions and sidrophores [4,6,7]. The LPS contains lipid A, core, and O-polysaccharide antigen [8]. Capsule polysaccharide (CPS) is a major factor for virulence of K. pneumoniae and classified into 77 serological types (K) [9]. Capsular layers engulf the surface of bacteria and prevent bacteria phagocytosis. K1 and K2 capsular antigens are the most important ones [10].
Genome of the K. pneumoniae capsule comprises gene clusters cps (capsular polysaccharide synthesis), magA (mucoviscosity associated gene A), rmpA and wb (O-specific polysaccharide directed by the wb gene cluster) [11]. MagA (35-Kbp) was identified as a K1-specific capsular polymerase gene which acts as a trans-acting activator for biosynthesis of cps. Moreover, magA is homologous with the genes involved in glycosylation, transfer and biosynthesis of the LPS [12]. In 2004, magA was determined as the major virulence factor of K. pneumoniae [2]. It has been reported that rmpA can magnify the colony mucoidy of different serotypes of K. pneumoniae and act as a plasmid-mediated regulator of extra capsular polysaccharide synthesis [13]. Adhesives include Pilli, the building of protein, and the attachment of bacteria to the host. MrkD gene mediates binding to the extracellular matrix, and also codes type 3 fimbria adhesion [14]. K. pneumoniae by different siderophores (iron-bound) including enterobactin, yersiniabactin and hydroxamate siderophore obtain iron from transferrin and lactoferrin in host transport proteins. EntB, ybtS, kfu and iutA genes encode enterobactin, yersiniabactin, iron-uptake system and hydroxamate siderophore [15]. The main purpose of the current study was to detect ybtS, entB, mrkD, magA, kfu, iutA, rmpA, and K2 genes in ESBL and non-ESBL producing K. pneumoniae isolated from clinical specimens in Kurdistan Province, Iran.

Methods
(a) Identification of bacterial strains Seventy K. pneumoniae isolates were taken from specimens including urine, blood, tracheal aspirates and wound from October 2015 to July 2016 from general hospitals of Kurdistan Province, Iran. All the isolates were cultured on blood and MacConkey agar (Merck, Germany). Colonies were identified by Gram stain and biochemical tests such as urea hydrolysis, H2S production, lysine decarboxylase, lactose fermentation, indole, methyl red, voges proskauer, citrate (IMViC) and oxidase tests [16].
(c) Determination of hypermucoviscosity K. pneumoniae (hv-KP) phenotype Seventy K. pneumoniae isolates were separated from the clinical samples and cultivated on blood agar medium (Merck, Germany); then, they were incubated at 37 • C for 24 h. Subsequently, the

(d) Virulence genes identification by PCR amplification
The K. pneumoniae isolates were cultured on Luria broth (LB) medium overnight. Then, DNA samples were extracted using Genomic DNA Extraction Kit (SinaClon, Iran). Gene coding virulence factors were detected by the PCR method. PCRs were carried out by using the thermo cycler system (Bio-Rad, Australia) and master mix PCR (YT1553, Iran) and primers were designed by Compain et al. (Table 1) [14]. Amplification was carried out as follows: initial denaturation at 95 • C for 15 min followed by 30 cycles of denaturation at 94 • C for 30 s, 60 • C for 90 s, and 72 • C for 60 s and elongation at 72 • C for 10 min. One multiplex PCR was not performed for detection of genes due to unavailability of one control isolate with multiple genes. Control positive isolates were obtained from the Lorestan University of Medical Sciences, Iran.

Discussion
Generally, one of the classes of antibiotics used for treating K. pneumoniae is beta-lactams such as cephalosporin [17]. However, the presence of ESBL enzymes impairs the performance of these antibiotics [18]. The difference in sensitivity and drug resistance in different geographic regions can be associated with different patterns of antibiotic use in different areas [19]. In this survey, 88.6% of the clinical isolates were the ESBL producers. Moreover, as shown in the study by Ghasemi, 60% of K. pneumoniae isolates were ESBL producers in Shiraz, Iran [20]. Jaskulski et  al. in Brazil reported that all K. pneumoniae isolates were ESBL-positive. The prevalence of ESBLproducing clinical isolates is related to different risk factors such as current antibiotic use, resent hospitalization [21]. K. pneumoniae has many virulence factors such as capsular polysaccharide, adhesions and siderophores which contribute to the pathogenicity of these bacteria. Presence of virulence factors in K. pneumoniae is important because they are the most prominent cause of death in patients before starting antibiotic therapy [15]. YbtS, entB, mrkD, magA, kfu, iutA, rmpA and K2 genes are among genes that code virulence factors [22]. Our study focused on detection of ybtS, entB, mrkD, magA, kfu, iutA, rmpA, and K2 genes in ESBL and non-ESBL producing K. pneumoniae isolates. The important point in this study is that it was the first study to report the presence of virulencegenes in K. pneumoniae isolates in Kurdistan Province, Iran. So far, there has been no report of virulencegenes in K. pneumoniae on Google Scholar and PubMed. In the present study, entB was determined in 81.43% of the isolates whereas no isolates carried K2 among all the K. pneumoniae isolates taken from the clinical specimens. Nevertheless, entB and K2 werethe highest and lowest prevalent virulence factors in the current study. In this investigation, among the 70 isolates collected from clinical specimens such as blood, tracheal, wound, and urine, 10 isolates (14.3%) were hv-KP isolates. Frequency of rmpA was (n=4, 5.7 %) that all the isolates were ESBL. According to table 2, this is while all the hvKP-isolates had the entB gene and ESBL phenotype. In contrast, in previous studies, such as Yu et al. in Taiwan, the prevalence of hv-KP, rmpA, and magA was reported to be 38%, 48% and 17%, respectively; the result of their study showed that strains carrying rmpA were significantly associated with hv-Kp [23]. On the other hand, Nahavandinejad et al. in northern Iran demonstrated that the hv-KP isolates were not restricted to magA [24]. MagA was only found in one ESBL isolate that contained the ybtS, entB, mrkD, and kfu genes. In contrast, MagA was much higher than the magA and K2 genes detected in Korea [25] and Taiwan [8]. These difference between the prevalence of MagA and K2 could be related to sample type of infection [26]. In the majority of those studies, K. pneumoniae was isolated from liver and meninges curtains infections whereas in our study, the isolates were collected from the clinical specimens [2,8]. In a study conducted by Feizabadi et al. in Iran on 89 isolates of K. pneumoniae, 10 (11.2%) isolates belonged to K1 and 13 (14.6%) isolates belonged to K2 serotypes, respectively [27]. Amraie et al. in Shahrekord, Iran, reported low frequency of MagA among clinical isolates, which is similar to our results [26]. Prior studies suggested that the magA gene can be infrequently seen in K. pneumoniae isolated clinical samples except liver abscesses [26,28]. Compain et al. in France designed a multiplex PCR for identifying seven virulence factors and K1/K2 capsular serotypes of K. pneumoniae. The multiplex PCR was used on 65 K. pneumoniae isolates between 2004 and 2014, which included 45 clinical isolates identified as hvKP; most isolates (64 /65) were found to possess mrkD [14] which is dissimilar to our results. Unfortunately, there has been no report of virulence genes in ESBL and non ESBL K. pneumoniae.
As a result of these investigations, the presence of virulence genes in ESBL-producing isolates more than clinical isolates of K. pneumoniae lacking ESBL. Our results indicated that there were no statistically significant differences between the ESBL productions and presence of the ybtS, entB, mrkD, magA, kfu, iutA, rmpA, and K2 genes. Moreover, the presence of these genes and variables such as presence of sex, clinical specimen type and hv-KP phenotype between ESBL and non -ESBL K. pneumoniae isolates (0.05< p).

Conclusions
In conclusion, frequency of ESBL-producing K. pneumoniae is increasing now. Detection of virulence factors that positively impact the pathogenicity of K. pneumoniae is of immense importance. The results of the current study showed that entB was the major virulence factor for K. pneumoniae (ESBL and non-ESBL) isolated from the clinical specimens in the hospitals of Kurdistan Province, Iran.

Open Access
This article is distributed under the terms of the Creative Commons Attribution License (CCBY4.0) which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

Ethics approval and consent to participate
The study was approved by Kurdistan University of medical science, Iran. All the members were fully informed of the purpose of the investigation, and were informed.

Competing interests
The authors declare no conflict of interest.

Acknowledgments
This work was retrieved from the thesis of PhD student, Pegah Shakib and, Kurdistan University of Medical Sciences supported this study.