Comparative Prevalence of blaCTX-M-15 Gene with Virulence Genes and Serotypes in Klebsiella pneumoniae

Hesam Alizade; Maziar Jajarmi; Mohammad Reza Aflatoonian; Davood Kalantar-Neyestanaki; Saeed Shoja; Reza Ghanbarpour

doi:10.5812/jjm.61285

Comparative Prevalence of bla_CTX-M-15Gene with Virulence Genes and Serotypes in Klebsiella pneumoniae

Hesam Alizade ^{1
, 2} , Maziar Jajarmi ³ , Mohammad Reza Aflatoonian ⁴ , Davood Kalantar-Neyestanaki ^{5
, 6} , Saeed Shoja ⁷ and Reza Ghanbarpour ^{3
, *}

Authors Information

¹ Research Center for Tropical and Infectious Diseases, Kerman University of Medical Sciences, Kerman, Iran

² Sirjan Faculty of Medical Sciences, Sirjan, Iran

³ Molecular Microbiology Research Group, Faculty of Veterinary Medicine, Shahid Bahonar University of Kerman, Kerman, Iran

⁴ Bam University of Medical Sciences, Bam, Iran

⁵ Department of Microbiology and Virology, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran

⁶ Student Research Committee, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran

⁷ Infectious and Tropical Disease Research Center, Hormozgan Health Institute, Hormozgan University of Medical Sciences, Bandar Abbas, Iran

Article information

Jundishapur Journal of Microbiology: April 2018, 11 (4); e61285
Published Online: April 20, 2018
Article Type: Research Article
Received: October 23, 2017
Revised: January 18, 2018
Accepted: February 26, 2018
DOI: 10.5812/jjm.61285

To Cite: Alizade H, Jajarmi M, Aflatoonian M R, Kalantar-Neyestanaki D, Shoja S, et al. Comparative Prevalence of bla_CTX-M-15Gene with Virulence Genes and Serotypes in Klebsiella pneumoniae, Jundishapur J Microbiol. 2018 ; 11(4):e61285. doi: 10.5812/jjm.61285.

Abstract

Methods: One hundred and three non-duplicate K. pneumoniae strains were isolated from clinical samples. The isolates were identified by standard bacteriological tests. Confirmed isolates were examined to detect a selection of virulence genes (wabG, rmpA and iucB) and serotypes (K1, K2, K5 and K20) by PCR. The isolates were studied foe the presence of beta-lactamase (bla_CTX-M-15)gene. SPSS software (version 19.0) was used for data analysis.

Results: Among the 103 K. pneumoniae isolates, 61 (59.2%) isolates were positive for wabG, 4 (3.9%) for iucB and 3 (2.9%) for rmpA genes. The presence of K20 in 3.9% (4/103) of the isolates represented the most prevalent. Only 3 (2.9%) isolates possessed the K1 serotype, while K2 and K5 serotypes were not detected in any isolate. The bla_CTX-M-15gene was detected in 47 (45.6%) isolates. bla_CTX-M-15-positive isolates showed a higher prevalence of wabG among the studied isolates (P < 0.05).

Copyright © 2018, Jundishapur Journal of Microbiology. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited

1. Background

Classic Klebsiella pneumoniae are multidrug-resistant strains associated with nosocomial infections outbreak and its hypervirulent strains are related to severe community-acquired infections (1, 2). During the past decade, CTX-M enzymes have become the most prevalent extended spectrum beta-lactamase (ESBL) enzymes in nosocomial infections caused by K. pneumoniae (3, 4). Klebsiella pneumoniae strains with plasmids harbouring bla_CTX-M-15have been detected in clinical isolates worldwide (5).

Bacteria can acquire antimicrobial resistance by the spread of transmissible plasmids, which may also carry virulent traits. The acquisition of antibiotic resistance and virulence determinants may represent a survival benefit to the bacteria (6). Several virulence genes such as the regulator of mucoid phenotype A gene (rmpA), iron acquisition system aerobactin (iucB) and lipopolysaccharide synthesis (wabG) have been detected and determined to be associated with the hypervirulent variant of K. pneumoniae. Both aerobactin system and regulator of mucoid phenotype A gene are found almost in all reported hypervirulent variants of K. pneumoniae strains located on a large plasmid. The important roles of rmpA and wabG genes are in capsular polysaccharide and lipopolysaccharide syntheses, respectively (7, 8).

Capsular polysaccharide (K antigen) is a major virulence factor responsible phagocytosis and serum killing resistance. At least 78 capsular serotypes have been identified while, serotype-related variation in infection severity have been observed. Strains with capsular serotypes K1 and, to a lesser extent, K2, K5, K20 and K54, have been recognized as the predominant virulent strains (8, 9).

2. Objectives

3. Methods

3.1. Ethics Statement

This study has been approved by the ethics committee of Kerman University of Medical Sciences (Code Number: IR.KMU.REC.1394.496), and written consent has been obtained from all patients.

3.2. Bacteria and DNA Extract

From October 2014 to May 2015, 103 non-duplicate non-consecutive K. pneumoniae were collected from patients referred to different clinical laboratories and hospitals in Kerman city (southeastern Iran). Clinical isolates were mostly from urine, wound, tracheal secretions and blood. The isolates were identified by standard biochemical and bacteriological methods. Isolates were then preserved at -70°C in brain-heart infusion (BHI) (HiMedia, India) broth with 30% glycerol until further tests. Bacterial genomic DNA was extracted from isolates using a kit (Topaz gene research, Iran) according to the manufacturer’s instructions.

3.3. Virulence Genes

Specific PCR primers for the detection of rmpA, wabG and iucB genes were previously described by Chen et al. (8).

3.4. Serotypes

K1, K2, K5 and K20 serotypes were identified by the detection of capsular polysaccharide (K serotype-specific) wzx and wzy alleles, using the multiplex PCR method as previously described (8).

3.5. Detection of bla_CTX-M-15 Gene

All isolates were screened by PCR for the bla_CTX-M-15gene, as described by Messai et al. (10).

3.6. String Test

The presence of hypermucoviscosity was evaluated using the string test, and a positive result was defined as formation of a viscous capsular string > 5 mm in length by stretching bacterial colonies on an agar plate (11).

3.7. Statistical Analysis

Descriptive and inferential statics were used for data analyse. Differences in virulence genes and serotypes with antimicrobial resistance gene were compared using, SPSS version 19.0 (SPSS, Inc), with a P value of P < 0.05 indicating statistical significance.

4. Results

The K. pneumoniae isolates were screened for the presence of virulence genes. In total, 62 out of 103 (60.2%) K. pneumoniae isolates harbored at least one of the virulence genes chosen for analysis. The most prevalent virulence gene was wabG (59.2%; 61/103), followed by iucB (3.9%; 4/103). The regulator of mucoid phenotype encoding gene rmpA was present in 2.9% (3/103) of the isolates (Figures 1, 2) (Table 1).

Table 1. Number and Percent of Serotypes, Virulence and bla_CTX-M-15Genes Present in K. pneumoniae Isolates

Variables	Gene	Total, No. (%)
Serotypes	K1	3 (2.9)
	K2	-
	K5	-
	K20	4 (3.9)
Virulence trait	wabG	61 (59.2)
	iucB	4 (3.9)
	rmpA	3 (2.9)
ESBL	bla_CTX-M-15	47 (45.6)

Figure 1. Lane 1, 100 bp DNA Marker; Lane 2, 3, an Amplification of wabG Gene (683 bp); Lane 4, Negative Control

Figure 2. Lane 1, 100 bp DNA Marker; Lane 2, Negative Control; Lane 4, Amplification of rmpA Gene (322 bp) and iucB Gene (948 bp)

Overall, 7 of 103 (6.8%) K. pneumoniae isolates were positive for at least one of the examined serotype genes. The presence of K20 in 3.9% (4/103) of the isolates was the most prevalent represented. The K1 serotype was the least prevalent in three isolates (Figure 3). None of the K. pneumoniae isolates were positive for K2 and K5 serotypes. Among the isolates positive for K1 serotype, 0.9% (1/103) was in combination with wabG, iucB and rmpA genes (hypervirulent), 0.9% (1/103) of isolates in combination with bla_CTX-M-15, wabG, iucB and rmpA genes and 0.9% (1/103) was accompanied by bla_CTX-M-15, wabG and iucB genes. Also, among the isolates that possessed the K20 serotype, 2.9% (3/103) were in combination with wabG gene.

Figure 3. A, Lane 1, 100 bp DNA Marker; Lane 2, Negative Control; Lane 4, Amplification of K1 (1283 bp); B, Lane 1, 5, Amplification of K20 (741 bp); Lane 2, Negative Control

Based on the results of the string test, hypervirulent phenotypes were identified in 3 (2.9%) of the isolates, whit only one (0.9%) isolate having the characteristic hypervirulent phenotype, was positive for K1, rmpA, wabG and iucB genes. The ESBL encoding gene bla_CTX-M-15was observed in 45.6% (47/103) of isolates (Figure 4 and Table 1). Of the 47 K. pneumoniae isolates that possessed bla_CTX-M-15gene, 28 (59.6%) isolates were positive for wabG gene. Bivariate analysis confirmed that this difference between the number of isolates was significant (P = 0.000). Among 47 isolates that possessed bla_CTX-M-15gene, three isolates were positive for rmpA gene (P > 0.05). There was no significant difference in bla_CTX-M-15gene to iucB gene (3/47 isolates) (P > 0.05). In addition, there was no association between the presence of bla_CTX-M-15 gene and the K1 (2/47 isolates) and K20 (2/47 isolates) serotypes (P > 0.05).

Figure 4. Lane 1, 100 bp DNA Marker; Lane 2, 4, Amplification of bla_CTX-M-15 Gene (550 bp); Lane 3, Negative Control

Table 2. Prevalence of Virulence Genes and Serotypes According to Isolates Possessed bla_CTX-M-15Gene in Klebsiella pneumoniae Isolates

Variables	Virulence Genes and Serotypes Pattern	No.
bla_CTX-M-15^a	wabG, rmpA, iucB, K1	1
	wabG, iucB, K1	1
	wabG, rmpA, iucB	1
	wabG, K20	2
	wabG	23
	-	19
Total		47

^aIsolates possessed bla_CTX-M-15gene.

5. Discussion

Klebsiella pneumoniae is known as an important reservoir for various types of ESBL enzymes, and has spread worldwide (12). The high rate of CTX-M producing K. pneumoniae prevalence and its widespread dissemination in Iran is causing concern (13). Virulence genes thought to be associated with invasive community-acquired infections include lipopolysaccharide synthesis, iron acquisition system, hypermucoidy and specific polysaccharide capsule serotypes (2). The present study showed a correlation between the epidemiology of specific virulence genetic traits and serotypes with bla_CTX-M-15gene; a first step toward understanding whether there is a link between virulence and resistance. The results from previous researches have shown that there is a link between resistance and virulence in Escherichia coli isolates (6). Lavigne et al. demonstrated that 14 virulence factors were less prevalent in ESBL isolates than in susceptible E. coli isolates (14). In another study by Lee et al. showed that CTX-M producers of E. coli isolates had fewer virulence factors than TEM-producing isolates (15).

The results showed a high (45.6%) prevalence of bla_CTX-M-15gene among the K. pneumoniae isolates. The existence of lipopolysaccharide synthesis encoding gene (wabG gene) was one of the most common virulence genes identified among the studied isolates. The findings by Chen et al. showed that there was high rate of wabG gene among 327 K. pneumoniae clinical isolates in China (8). According to the results of the current study, K1 serotype was detected in ~3% of the isolates. Lipopolysaccharide and capsular polysaccharide components are important pathogenic traits in K. pneumonia-caused bacteremia and pneumonia (16). The K1 strains were wabG, iucB, rmpA and bla_CTX-M-15-positive genes, which indicate that most of the K1 isolates tested in this study, were closely related to hyper-virulence and ESBL. Previous studies have shown that the K1 serotype K. pneumoniae isolated from liver abscesses is less resistant to antibiotics when compared with other serotypes (17, 18), whereas previously published data indicated that the K1 serotype is associated with antibiotic resistance (19, 20).

Of the virulence genes, wabG was significantly more common in strains producing the bla_CTX-M-15group ESBLs. Derakhshan et al. compared the occurrence of rmpA and wcaG genes with the production of class 1 integron (intl1) among K. pneumoniae strains isolated from clinical specimens. The prevalence of wcaG was more frequent in the intl1 producing K. pneumoniae (21). The findings of this study indicated that the numbers of wabG-carrying isolates were more common in strains producing the bla_CTX-M-15 gene (P < 0.05). The results of the current study showed that the rmpA gene was detected in quite a low frequency compared to other isolates. The rmpA gene encodes a transcriptional activator of capsular polysaccharide which is one of the major contributing factors of virulence in K. pneumoniae, promoting biofilm formation and is closely associated with the hypermucoviscous phenotype (8, 22). Yu et al., showed that rmpA-positive isolates were related to the clinical syndrome caused by invasive strains and with the hypermucoviscous phenotype (23). Another study that demonstrated the relationship between rmpA, virulence and specific polysaccharide capsule serotypes is yet to be elucidated (9).

In the present study, the aerobactin encoding gene iucB was present in 3.9% of isolates. Several molecular epidemiologic studies were suggestive that hypervirulent K. pneumonia strains produced more aerobactin than classical K. pneumoniae strains and it may be that this factor enhanced the virulence of hypervirulent K. pneumoniae (24, 25). In this regard, Li et al. suggested that hypervirulent K. pneumoniae has the potential to acquire significant antibiotic resistance (26).

6. Conclusions

Acknowledgements

Footnotes

Authors’ Contribution: Hesam Alizade and Mohammad Reza Aflatoonian, study concept and design, the development of the study; Hesam Alizade, Maziar Jajarmi and Saeed Shoja, performing experimental procedures; Hesam Alizade, Reza Ghanbarpour and Davood Kalantar-Neyestanaki, data interpretation, manuscript drafting and manuscript revision.

Conflict of Interest: The authors declare that they have no conflict of interest.

Role of the Sponsor: The funding organization had no role in the design and conduct of the study; collection, management, and analysis of data; and/or preparation, review, and approval of the manuscript. The research center for tropical and infectious diseases, Kerman University of Medical Sciences, Iran is acknowledged for funding assistance on this project.

Financial Disclosure: There was no financial disclosure to report.

References

1. Fazeli H, Norouzi-Barough M, Ahadi AM, Shokri D, Solgi H. Detection of New Delhi Metallo-Beta-Lactamase-1 (NDM-1) in carbapenem- resistant Klebsiella pneumoniae isolated from a university hospital in Iran. Hippokratia. 2015;19(3):205-9. [PubMed: 27418777].
2. Holt KE, Wertheim H, Zadoks RN, Baker S, Whitehouse CA, Dance D, et al. Genomic analysis of diversity, population structure, virulence, and antimicrobial resistance in Klebsiella pneumoniae, an urgent threat to public health. Proc Natl Acad Sci U S A. 2015;112(27):E3574-81. doi: 10.1073/pnas.1501049112. [PubMed: 26100894].
3. Afzali H, Firoozeh F, Amiri A, Moniri R, Zibaei M. Characterization of CTX-M-Type Extend-Spectrum beta-Lactamase Producing Klebsiella spp. in Kashan, Iran. Jundishapur J Microbiol. 2015;8(10). e27967. doi: 10.5812/jjm.27967. [PubMed: 26587221].
4. Wang G, Huang T, Surendraiah PK, Wang K, Komal R, Zhuge J, et al. CTX-M beta-lactamase-producing Klebsiella pneumoniae in suburban New York City, New York, USA. Emerg Infect Dis. 2013;19(11):1803-10. doi: 10.3201/eid1911.121470. [PubMed: 24188126].
5. Mshana SE, Fritzenwanker M, Falgenhauer L, Domann E, Hain T, Chakraborty T, et al. Molecular epidemiology and characterization of an outbreak causing Klebsiella pneumoniae clone carrying chromosomally located bla CTX-M-15 at a German University-Hospital. BMC Microbiol. 2015;15(22). doi: 10.1186/s12866-015-0460-2.
6. Da Silva GJ, Mendonca N. Association between antimicrobial resistance and virulence in Escherichia coli. Virulence. 2012;3(1):18-28. doi: 10.4161/viru.3.1.18382. [PubMed: 22286707].
7. Ye M, Tu J, Jiang J, Bi Y, You W, Zhang Y, et al. Clinical and Genomic Analysis of Liver Abscess-Causing Klebsiella pneumoniae Identifies New Liver Abscess-Associated Virulence Genes. Front Cell Infect Microbiol. 2016;6:165. doi: 10.3389/fcimb.2016.00165. [PubMed: 27965935].
8. Chen Z, Liu M, Cui Y, Wang L, Zhang Y, Qiu J, et al. A novel PCR-based genotyping scheme for clinical Klebsiella pneumoniae. Future Microbiol. 2014;9(1):21-32. doi: 10.2217/fmb.13.137. [PubMed: 24328378].
9. Hsu CR, Lin TL, Chen YC, Chou HC, Wang JT. The role of Klebsiella pneumoniae rmpA in capsular polysaccharide synthesis and virulence revisited. Microbiology. 2011;157(Pt 12):3446-57. doi: 10.1099/mic.0.050336-0. [PubMed: 21964731].
10. Messai Y, Benhassine T, Naim M, Paul G, Bakour R. Prevalence of beta-lactams resistance among Escherichia coli clinical isolates from a hospital in Algiers. Rev Esp Quimioter. 2006;19(2):144-51. [PubMed: 16964332].
11. Shon AS, Bajwa RP, Russo TA. Hypervirulent (hypermucoviscous) Klebsiella pneumoniae: a new and dangerous breed. Virulence. 2013;4(2):107-18. doi: 10.4161/viru.22718. [PubMed: 23302790].
12. Meletis G, Tzampaz E, Protonotariou E, Sofianou D. Emergence of Klebsiella pneumoniae carrying bla(VIM) and bla(KPC) genes. Hippokratia. 2010;14(2):139-40. [PubMed: 20596274].
13. Taherpour A, Hashemi A. Detection of OqxAB efflux pumps, OmpK35 and OmpK36 porins in extended-spectrum-beta-lactamase-producing Klebsiella pneumoniae isolates from Iran. Hippokratia. 2013;17(4):355-8. [PubMed: 25031516].
14. Lavigne JP, Blanc-Potard AB, Bourg G, Moreau J, Chanal C, Bouziges N, et al. Virulence genotype and nematode-killing properties of extra-intestinal Escherichia coli producing CTX-M beta-lactamases. Clin Microbiol Infect. 2006;12(12):1199-206. doi: 10.1111/j.1469-0691.2006.01536.x. [PubMed: 17121626].
15. Lee S, Yu JK, Park K, Oh EJ, Kim SY, Park YJ. Phylogenetic groups and virulence factors in pathogenic and commensal strains of Escherichia coli and their association with blaCTX-M. Ann Clin Lab Sci. 2010;40(4):361-7. doi: 10.1111/j.1469-0691.2006.01536.x. [PubMed: 20947811].
16. Hsieh PF, Lin TL, Yang FL, Wu MC, Pan YJ, Wu SH, et al. Lipopolysaccharide O1 antigen contributes to the virulence in Klebsiella pneumoniae causing pyogenic liver abscess. PLoS One. 2012;7(3). e33155. doi: 10.1371/journal.pone.0033155. [PubMed: 22427976].
17. Fang CT, Lai SY, Yi WC, Hsueh PR, Liu KL, Chang SC. Klebsiella pneumoniae genotype K1: an emerging pathogen that causes septic ocular or central nervous system complications from pyogenic liver abscess. Clin Infect Dis. 2007;45(3):284-93. doi: 10.1086/519262. [PubMed: 17599305].
18. Lin YT, Jeng YY, Chen TL, Fung CP. Bacteremic community-acquired pneumonia due to Klebsiella pneumoniae: clinical and microbiological characteristics in Taiwan, 2001-2008. BMC Infect Dis. 2010;10:307. doi: 10.1186/1471-2334-10-307. [PubMed: 20973971].
19. Cheng NC, Yu YC, Tai HC, Hsueh PR, Chang SC, Lai SY, et al. Recent trend of necrotizing fasciitis in Taiwan: focus on monomicrobial Klebsiella pneumoniae necrotizing fasciitis. Clin Infect Dis. 2012;55(7):930-9. doi: 10.1093/cid/cis565. [PubMed: 22715175].
20. Su SC, Siu LK, Ma L, Yeh KM, Fung CP, Lin JC, et al. Community-acquired liver abscess caused by serotype K1 Klebsiella pneumoniae with CTX-M-15-type extended-spectrum beta-lactamase. Antimicrob Agents Chemother. 2008;52(2):804-5. doi: 10.1128/AAC.01269-07. [PubMed: 18056273].
21. Derakhshan S, Najar Peerayeh S, Bakhshi B. Association Between Presence of Virulence Genes and Antibiotic Resistance in Clinical Klebsiella Pneumoniae Isolates. Lab Med. 2016;47(4):306-11. doi: 10.1093/labmed/lmw030. [PubMed: 27498999].
22. Huang Y, Li J, Gu D, Fang Y, Chan EW, Chen S, et al. Rapid Detection of K1 Hypervirulent Klebsiella pneumoniae by MALDI-TOF MS. Front Microbiol. 2015;6:1435. doi: 10.3389/fmicb.2015.01435. [PubMed: 26733976].
23. Yu WL, Ko WC, Cheng KC, Lee HC, Ke DS, Lee CC, et al. Association between rmpA and magA genes and clinical syndromes caused by Klebsiella pneumoniae in Taiwan. Clin Infect Dis. 2006;42(10):1351-8. doi: 10.1086/503420. [PubMed: 16619144].
24. Russo TA, Shon AS, Beanan JM, Olson R, MacDonald U, Pomakov AO, et al. Hypervirulent K. pneumoniae secretes more and more active iron-acquisition molecules than "classical" K. pneumoniae thereby enhancing its virulence. PLoS One. 2011;6(10). e26734. doi: 10.1371/journal.pone.0026734. [PubMed: 22039542].
25. Jung SW, Chae HJ, Park YJ, Yu JK, Kim SY, Lee HK, et al. Microbiological and clinical characteristics of bacteraemia caused by the hypermucoviscosity phenotype of Klebsiella pneumoniae in Korea. Epidemiol Infect. 2013;141(2):334-40. doi: 10.1017/S0950268812000933. [PubMed: 22578630].
26. Li W, Sun G, Yu Y, Li N, Chen M, Jin R, et al. Increasing occurrence of antimicrobial-resistant hypervirulent (hypermucoviscous) Klebsiella pneumoniae isolates in China. Clin Infect Dis. 2014;58(2):225-32. doi: 10.1093/cid/cit675. [PubMed: 24099919].