Detection of ESBLs was tested by the combination disk diffusion test (CDDT) for the K. pneumoniae isolates. The CDDT was performed by ceftazidime (30 μg), cefotaxime (30 μg), cefepime (30 μg), cefpodoxime (30 μg), cefotaxime-clavulanic acid (30/10μg), cefotaxime-clavulanic acid (30/10 μg), cefepime-clavulanic acid (30/10 μg) and cefpodoxime-clavulanic acid (30/10 μg) (Roscoe, Denmark) (Ho et al., 1998). Escherichia coli ATCC 25922 and K. pneumoniae ATCC 7881 were utilized for negative and positive controls, respectively.

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 hypermucoviscosity of K. pneumoniae (hv-KP) phenotype was determined by forming a viscous string more than 5 in standard bacteriological loops 3.

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.

The association between the ESBL production, clinical specimen type, sex ,and hvKP phenotype and presence of ybtS, entB, mrkD, magA, kfu, iutA, rmpA and K2 genes among the ESBL and non-ESBL K.pneumoniae isolates was analyzed by Fisher tests with STATA software program v12.

Out of the 70 K. pneumoniae isolates, 37 (52.9%), 32 (45.7 %) and 1 isolates (1.4%) were collected from women, men and the hospital environment. Moreover, 50 isolates (71.4%) were obtained from urine, 8 isolates (11.4%) from blood, 10 isolates (14.3%) from tracheal aspirates, 1 isolate (1.4%) from wound, and 1 isolate (1.4%) from the environment (Table 2).

Table 2

Sample No	Origin	Sex	ESBL production	Virulence genes
Kp6	urine	male	positive	ybt S,entB, mrkD
Kp12	urine	female	positive	ybt S,entB, mrkD
Kp16	urine	male	positive	entB, mrkD, kfu
Kp32	urine	female	positive	ybt S,entB, mrkD, magA
Kp39	tracheal	male	positive	entB, mrkD
Kp43	urine	female	positive	ybt S,entB, mrkD, rmpA
Kp46	urine	female	positive	ybt S,entB, mrkD, rmpA
Kp53	urine	female	positive	ybt S,entB, mrkD
Kp58	urine	female	positive	entB, iutA
Kp60	urine	female	positive	entB

The results of screening for the ESBL showed that 62 isolates (88.6%) were ESBL-producing K. pneumoniae isolates. Of the 70 clinical isolates, 10 isolates (14.3%) were positive and 60 isolates (85.7%) were negative for the hv-KP test. Table 2 shows characteristics of hypermucoviscosity clinical isolates.

According to the results of screening virulence genes, entB (n= 57, 81.4%) was the most prevalent among all the clinical isolates, followed by the mrkD (n=46, 65.7 %%), ybtS (n=42, 60%), iutA (n=8, 11.4%), kfu (n=8, 11.4%), rmpA (n=4, 5.7%), magA (n=1, 1.43%), and K2 was not detected in any of the isolates. The presence of the ybtS, entB, mrkD, magA, kfu, iutA, rmpA and K2 genes coding virulence factors was detected among the ESBL and non-ESBL K. pneumoniae isolates (Table 3).

Figure 1

Table 3

	Total isolates	ESBL	Non-ESBL	P-Value
ybt S	42(60%)	38(90.5%)	4(9.5%)	0.705
entB	57(81.4%)	52(91.2%)	5(8.8%)	0.161
mrkD	46(65.7%)	43(93.5%)	3(6.5%)	0.113
magA	1(1.4%)	1(100%)	0(0%)	1.000
iutA	8(11.4%)	6(75%)	2(25%)	0.225
Kfu	8(11.4%)	7(87.5%)	1(12.5 %)	1.000
rmpA	4(5.7%)	4(100%)	0(0%)	1.000
K2	0(0 %)	0(0%)	0(0 %)	0.231

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 ESBL-producing 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 virulence genes in K. pneumoniae isolates in Kurdistan Province, Iran. So far, there has been no report of virulence genes 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 were the 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 28. 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 2826. 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).

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.

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.

CDDT: Combination disk diffusion test; ESBL: Extended-spectrum betalactamase; LPS: Lipopolysaccharide; MDR: Multi-drug resistant; PCR: Polymerase Chain Reaction; UTI: Urinary tract infection

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.

The authors declare no conflict of interest.

None

PS: Study design, doing experiments, data collection, writing; MKT: Study design, writing, critical review; RR: Supervision, study design, writing, critical review; AA: Data collection, data analysis, critical review; SR: Doing experiments, data collection, data analysis, critical review.

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