The Open Microbiology Journal




ISSN: 1874-2858 ― Volume 13, 2019
RESEARCH ARTICLE

Study of Various Virulence Genes, Biofilm Formation and Extended-Spectrum β-lactamase Resistance in Klebsiella pneumoniae Isolated from Urinary Tract Infections



Sanaa S. Hamam2, Reem M. El Kholy3, Maysaa E. S. Zaki1, *
1 Clinical Pathology, Faculty of Medicine, Mansoura University, Mansoura, Egypt
2 Medical Microbiology & Immunology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
3 Clinical Pathology, Faculty of Medicine, Menoufia University- Menoufia, Egypt

Abstract

Objective:

The aims of the current study were to evaluate the capacity of K. pneumoniae isolated from hospital-acquired urinary tract infection to form biofilm, the relation of this capacity to various virulence genes and the prevalence of Extended Spectrum β-lactamases (ESBL) among these isolates by phenotypic and genotypic methods.

Material and Methods:

The study included 100 non-duplicate strains of K. pneumoniae isolated from 100 different urine samples from patients with hospital-acquired urinary tract infection. The isolated strains were studied for biofilm formation, ESBL production by phenotypic methods. Molecular studies were applied for the detection of ESβLs genes blaTEM, blaSHV, blaCTX-M and for detection of virulence genes fimH, uge, rmpA, mag A, wzy, kfa and aerobactin genes.

Result:

The majority of the isolates had the capacity to form a biofilm (81%), with ESBL prevalence rate 41%. The most prevalent gene among ESBL producing K. pneumoniae was blaCTX-M (73.2%) followed by blaSHV (53.6%) and blaTEM (51.2%). Among the virulence genes studied in K. pneumoniae isolates, the most prevalent gene was fimH (76%), uge (70%). There was significant association between ESBL production, and resistance to amikacin, cefepime, ceftazidime, gentamicin, imipenem and meropenem and biofilm production in K. pneumoniae isolates. There was significant association between blaCTX-M, blaSHV, fimH, mag, kfa, wzy, rmpA and aerobactin and biofilm production in K. pneumoniae.

Conclusion:

The present study highlights the prevalence of virulence genes among biofilm-forming strains of K. pneumoniae isolated from hospital-acquired urinary tract infection. Moreover, there was association between biofilm formation and ESBL production. Further studies are required to elucidate the clinical impact of the association of these different mechanisms.

Keywords: ESBL, Biofilm, K. pneumoniae, PCR, Virulence genes, Phenotypic methods.


Article Information


Identifiers and Pagination:

Year: 2019
Volume: 13
First Page: 249
Last Page: 255
Publisher Id: TOMICROJ-13-249
DOI: 10.2174/1874285801913010249

Article History:

Received Date: 26/04/2019
Revision Received Date: 02/09/2019
Acceptance Date: 05/09/2019
Electronic publication date: 30/09/2019
Collection year: 2019

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© 2019 Hamam et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: (https://creativecommons.org/licenses/by/4.0/legalcode). This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


* Address correspondence to this author at the Clinical Pathology, Faculty of Medicine, Mansoura University, Mansoura, Egypt; Tel: 0020502258877;
E-mail: May_s65@hotmail.com






1. INTRODUCTION

Klebsiella pneumoniae (K. pneumoniae), as a gram-negative bacillus, is known to be associated with various infections such as Urinary Tract Infection (UTI) especially hospital-acquired Catheter-Associated Urinary Tract Infection (CAUTI), sepsis and pneumonia [1Maria SA, Paolo O, Donatella P, Fernanda P, Luisa CM. An overview of carbapenem-resistant Klebsiella pneumoniae: Epidemiology and control measures. Rev Med Microbiol 2014; 25: 7-14.
[http://dx.doi.org/10.1097/MRM.0b013e328365c51e]
, 2Murphy CN, Clegg S. Klebsiella pneumoniae and type 3 fimbriae: Nosocomial infection, regulation and biofilm formation. Future Microbiol 2012; 7(8): 991-1002.
[http://dx.doi.org/10.2217/fmb.12.74] [PMID: 22913357]
].

There are many virulence factors in K. pneumoniae associated with its pathogenicity. Among these factors is the capacity of K. pneumoniae to form a biofilm that protects it from the host immune response as well as from the antibiotics [3Bandeira M, Carvalho PA, Duarte A, Jordao L. Exploring dangerous connections between Klebsiella pneumoniae biofilms and healthcare-associated infections. Pathogens 2014; 3(3): 720-31.
[http://dx.doi.org/10.3390/pathogens3030720] [PMID: 25438020]
, 4Chung PY. The emerging problems of Klebsiella pneumoniae infections: Carbapenem resistance and biofilm formation. FEMS Microbiol Lett 2016; 363(20)fnw219
[http://dx.doi.org/10.1093/femsle/fnw219] [PMID: 27664057]
]. The formation of biofilm leads to the prolonged persistence of K. pneumoniae on the epithelium and on the medical devices with difficulty in its eradication. There are genes related to biofilm formation in K. pneumoniae such as capsular genes like rmpA gene associated with K1/K2 virulent type, fimbriae genes such as fimH and mrkD that regulate fimbrial types I and 3, respectively and genes encoding the enzymes used for the biosynthesis of lipopolysaccharide such as uge gene that encodes for the uridine diphosphate galacturonate 4-epimerase [5Balestrino D, Ghigo JM, Charbonnel N, Haagensen JA, Forestier C. The characterization of functions involved in the establishment and maturation of Klebsiella pneumoniae in vitro biofilm reveals dual roles for surface exopolysaccharides. Environ Microbiol 2008; 10(3): 685-701.
[http://dx.doi.org/10.1111/j.1462-2920.2007.01491.x] [PMID: 1823 7304]
, 6Alcántar-Curiel MD, Blackburn D, Saldaña Z, et al. Multi-functional analysis of Klebsiella pneumoniae fimbrial types in adherence and biofilm formation. Virulence 2013; 4(2): 129-38.
[http://dx.doi.org/10.4161/viru.22974] [PMID: 23302788]
] and genes that regulate iron uptake by K. peumoniae including aerobactin and kfu genes [7Ma LC, Fang CT, Lee CZ, Shun CT, Wang JT. Genomic heterogeneity in Klebsiella pneumoniae strains is associated with primary pyogenic liver abscess and metastatic infection. J Infect Dis 2005; 192(1): 117-28.
[http://dx.doi.org/10.1086/430619] [PMID: 15942901]
]. Moreover, quorum sensing system especially type 2 and pga ABCD operon that regulates the synthesis and translocation of poly-b-1,6-N-acetyl-D-glucosamine adhesin are involved in the biofilm formation through enhancement of cell to cell communication and intercellular adhesion process [8Chen KM, Chiang MK, Wang M, Ho HC, Lu MC, Lai YC. The role of pgaC in Klebsiella pneumoniae virulence and biofilm formation. Microb Pathog 2014; 77: 89-99.].

Beside biofilm formation, clinical isolates of K. pneumoniae are known to possess resistance to third generation of cephalosporines through the production of Extended Spectrum β-Lactamase (ESBL).

Extended-spectrum beta-lactamase is associated with the mutation of genes that regulate β-lactamases mainly blaSHV, blaTEM, and blaCTX-M genes. More than 300 different ESBLs variants have been described [9Paterson DL, Bonomo RA. Extended-spectrum beta-lactamases: A clinical update. Clin Microbiol Rev 2005; 18(4): 657-86.
[http://dx.doi.org/10.1128/CMR.18.4.657-686.2005] [PMID: 1622 3952]
].

There are few reports about the association of biofilm and ESBL within isolates of K. pneumoniae from hospital-acquired urinary tract infection.

Therefore, the present study aimed to study the capacity of K. pneumoniae isolated from hospital-acquired urinary tract infection to form biofilm and to correlate this capacity to various virulence genes and finally detect the prevalence of ESBL among these isolates by phenotypic and genotypic methods.

2. MATERIALS AND METHODS

The study included 100 non-duplicate strains of K. pneumoniae isolated from 100 patients with hospital-acquired urinary tract infections from Mansoura University Hospital, Egypt as defined by CDC in a period from December 2016 till January 2018. The study was approved by Mansoura Faculty of Medicine ethical committee and approval was obtained from each participant.

2.1. Microbiological Culture

Urine samples from catheterized patients were collected as part of the routine microbiological investigation, after disinfection of the catheter collection part using 70% ethyl alcohol solution discarding the first few drops of urine and collecting 5-10 ml urine using a sterile syringe. Urine samples were immediately sent to the laboratory to be processed and examined within 2 hours. Quantitative urine culture was performed by the use of sterile calibrated loop with a culture of I micron of the urine onto MacConkey agar plate and incubation was performed at 37°C for 24-48 hours. The colonies were identified by gram staining and biochemical identification was performed by VITEK 2 system according to the instructions of the manufacturers. The isolated K. pneumoniae were subjected to antibiotics susceptibility testing by disc diffusion method, determination of ESBL done by combined disc diffusion method and study of biofilm formation by Microtiter plate method. Molecular studies were carried out for the detection of virulence genes and bla genes.

2.2. Antibiotic Susceptibility Testing

Antibiotic susceptibility was performed by discs diffusion method according to Clinical and Laboratory Standards Institute (CLSI) recommendations (CLSI 2018) [10Clinical Laboratory Standards Institute (CLSI). Performance standards for antimicrobial susceptibility testing: Twentieth informational supplement 2015. http://shop.clsi.org/site/Sample_pdf/M100S25_ sample.pdf]. The used discs were: amikacin, cefotaxime, ceftazidime, cefepime, ciprofloxacin, levofloxacin, gentamicin, piperacillin-tazobactam, trimethoprim/sulfamethoxazole, imipenem, and meropenem (Oxoid-UK). The interpretation of the results was done according to CLSI 2018 [10Clinical Laboratory Standards Institute (CLSI). Performance standards for antimicrobial susceptibility testing: Twentieth informational supplement 2015. http://shop.clsi.org/site/Sample_pdf/M100S25_ sample.pdf].

2.3. ESBL Production

Determination of ESBL by Combined disk diffusion confirmatory test.

Klebsiella pneumoniae strains resistant to ceftazidime (30 μg) and/ or cefotaxime (30 μg) were subjected to the screening of ESBL enzymes production by combined disk diffusion confirmatory test. The principle of the test depends upon the use of ceftazidime and cefotaxime discs alone and conjugated with clavulanic acid. The increase of the inhibition zone diameter around discs conjugated with clavulanic acid ≥5mm was interpreted as positive ESBL production.

The suspension of overnight growth of K. pneumoniae in Muller-Hinton broth was prepared with adjusted turbidity to 0.5 McFarland and was subculture on Muller-Hinton agar medium and antibiotic discs containing ceftazidime, ceftazidime (30 μg)+clavulanic acid (10 μg), cefotaxime, and cefotaxime (30 μg + clavulanic (10 μg) pairs were placed with 20 mm space between them. Plates were incubated at 37ºC for 24 hours. The increase of growth inhibition zone ≥ 5 mm around ceftazidime/clavulanic acid and cefotaxime/clavulanic compared to ceftazidime and cefotaxime respectively was regarded as an ESBL producing isolate [11Thomson KS. Extended-spectrum-beta-lactamase, AmpC, and Carbapenemase issues. J Clin Microbiol 2010; 48(4): 1019-25.
[http://dx.doi.org/10.1128/JCM.00219-10] [PMID: 20181902]
]. The strain of Klebsiella pneumoniae (ATCC 700603) was used as a positive quality control strain for ESBL production.

2.4. Biofilm Assay

Biofilm formation by K. pneumoniae isolates was assessed by the semi-quantitative method [12Wu MC, Lin TL, Hsieh PF, Yang HC, Wang JT. Isolation of genes involved in biofilm formation of a Klebsiella pneumoniae strain causing pyogenic liver abscess. PLoS One 2011; 6(8)e23500
[http://dx.doi.org/10.1371/journal.pone.0023500] [PMID: 21858144]
].

Briefly, 0.5 McFarland suspensions of K. pneumoniae isolates were prepared using brain heart infusion broth (Oxoid-UK) and 100 microliters of the suspensions were inoculated onto wells of 96-well polystyrene plate and incubated at 37°C for 18 hours. Negative control well was inoculated with sterile brain heart infusion broth. Then, wells were stained with 200 microliters of crystal violet with concentration 0.5% for 20 minutes and the excess stain was washed by 200 microns sterile distilled water for three times. Then the stain was eluted with 200 microliters of 95% of ethanol and the optical density was measured at 570 nm by enzyme-linked immunoassay reader. The positive Optical Density (OD) was defined as three standard deviations above the mean OD of the negative control. The K. pneumoniae NTUH-K2044 strain was used as positive control.

2.5. Molecular Studies of K. pneumoniae

2.5.1. Extraction of DNA

DNA of the isolated K. pneumoniae was prepared by boiling method. Briefly, colonies were suspended in 100 microliters of sterile distilled water and boiled at 100ºC in the water bath for 15 minutes then rapidly cooled at -20°C for one hour, then centrifugation and the supernatant were preserved for the use in the amplification processes [13Shah RK, Ni ZH, Sun XY, Wang GQ, Li F. The determination and correlation of various virulence genes, ESBL, serum bactericidal effect and biofilm formation of clinical isolated classical klebsiella pneumoniae and hypervirulent klebsiella pneumoniae from respiratory tract infected patients. Pol J Microbiol 2017; 66(4): 501-8.
[http://dx.doi.org/10.5604/01.3001.0010.7042] [PMID: 29319515]
].

2.5.2. PCR for Virulence Genes of K. pneumoniae

PCR was performed for the detection of virulence genes for K. pneumoniae. The genes detected and the sequences of the used primers were summarized in Table 1. The conditions of PCR were performed according to Shah et al., 2017 [13Shah RK, Ni ZH, Sun XY, Wang GQ, Li F. The determination and correlation of various virulence genes, ESBL, serum bactericidal effect and biofilm formation of clinical isolated classical klebsiella pneumoniae and hypervirulent klebsiella pneumoniae from respiratory tract infected patients. Pol J Microbiol 2017; 66(4): 501-8.
[http://dx.doi.org/10.5604/01.3001.0010.7042] [PMID: 29319515]
] for each gene. Negative controls were included as sterile distilled water and K. pneumoniae ATCC13883 was used as positive control. The products of the amplifications were analyzed by the use of gel electrophoresis 1.5% stained with ethidium bromide and the bands were visualized by UV.

2.5.3. Multiplex PCR for ESBL Genes

The extracted DNA was used for multiplex PCR for the detection of ESBL genes blaTEM, blaSHV and blaCTX-M. The sequences of the used primers were shown in Table 1. The amplification steps were primary denaturation at 94°C for 4 minutes, then 35 cycles of amplification (94°C for 45 seconds, 55°C for 45 seconds and 72°C for 1 minute) with final extension at 72°C for 7 minutes. K. pneumoniae ATCC 7881 was used as a positive control. The products of the amplifications were analyzed by the use of gel electrophoresis 1.5% stained with ethidium bromide and the bands were visualized by UV [13Shah RK, Ni ZH, Sun XY, Wang GQ, Li F. The determination and correlation of various virulence genes, ESBL, serum bactericidal effect and biofilm formation of clinical isolated classical klebsiella pneumoniae and hypervirulent klebsiella pneumoniae from respiratory tract infected patients. Pol J Microbiol 2017; 66(4): 501-8.
[http://dx.doi.org/10.5604/01.3001.0010.7042] [PMID: 29319515]
].

2.5.4. Statistical Analysis

Data were collected, revised, coded and entered to the Statistical Package for Social Science (SPSS) version 24. The quantitative data were presented as mean, standard deviations and ranges and qualitative data was presented by number and percentages. The comparison between qualitative data was done by the use of chi-square. P-value was considered significant if it was <0.05.

3. RESULTS

The study included non-duplicate 100 isolates of K. pneumoniae. The majority of the isolates had the capacity to form biofilm (81 isolates, 81%) with ESBL prevalence rate 41% (41 isolates). Forty-one isolates had the combined capacity to form biofilm and were ESBL, 40 isolates had the capacity to form biofilm and were non ESBL producers and 19 isolates were non ESBL and lack the capacity to form biofilm (Fig. 1).


Table 1
Genes, sequences of the used primers and the bp of the used products.


Fig (1)
The frequency of ESBLs production and biofilm formation among isolated K. pneumoniae.


Fig (2). shows the optical density of biofilm formed by each isolate. The genes detected among ESBL producing K. pneumoniae was blaCTX-M in 30 isolates followed by blaSHV 22 isolates and blaTEM in 21 isolates. Multiple genes were present in 25 of the isolates. Among the virulent genes' studies in K. pneumoniae isolates, the most prevalent gene was fimH (76%), uge (70%), rmpA (62%), mag (59%), wzy (59%) and aerobactin gene (51%). Lower prevalence was detected for and kfa gene (33%).

There was significant association between ESBL production, and resistance to amikacin, cefepime, ceftazidime, gentamicin, imipenem and meropenem and biofilm-producing K. pneumoniae data not shown. There was a significant association between blaCTX-M, blaSHV, fimH,magA, kfa, wzy, rmpA and aerobact and biofilm-producing K .pneumoniae (Table 2).

Table 2
Comparison between biofilm producing and non-biofilm producing K. pneumoniae species regarding genes associated with ESBLs production and other virulence genes.


Fig. (2)
Distribution of OD of biofilm formation among isolated K. pneumoniae.


4. DISCUSSION

Klebsiella pneumoniae has emerged as an important pathogen causing around 20% of infections in the hospitals worldwide [14De Rosa FG, Corcione S, Cavallo R, Di Perri G, Bassetti M. Critical issues for Klebsiella pneumoniae KPC-carbapenemase producing K. pneumoniae infections: A critical agenda. Future Microbiol 2015; 10(2): 283-94.
[http://dx.doi.org/10.2217/fmb.14.121] [PMID: 25689539]
].

There are several factors that increase the virulence of K. pneumoniae, of which the capacity to form biofilm is prevalent. The majority of the isolates in the present study had the capacity to form biofilm (81%). This finding was similar to the previous report concerning isolates from urinary tract infections [15Vuotto C, Longo F, Pascolini C, et al. Biofilm formation and antibiotic resistance in Klebsiella pneumoniae urinary strains. J Appl Microbiol 2017; 123(4): 1003-18.
[http://dx.doi.org/10.1111/jam.13533] [PMID: 28731269]
]. The formation of biofilm protects K. pneumoniae species against the host immune responses, the action of antibiotics and enhance its persistence [16Vuotto C, Longo F, Balice MP, Donelli G, Varaldo PE. Antibiotic resistance related to biofilm formation in Klebsiella pneumoniae. Pathogens 2014; 3(3): 743-58.
[http://dx.doi.org/10.3390/pathogens3030743] [PMID: 25438022]
, 17Hawkey PM. Prevalence and clonality of extended-spectrum beta-lactamases in Asia. Clin Microbiol Infect 2008; 14(Suppl. 1): 159-65.
[http://dx.doi.org/10.1111/j.1469-0691.2007.01855.x] [PMID: 1815 4540]
].

One of the other factors associated with infection by K. pneumoniae is the emergence of resistance to antibiotics by different mechanisms. The common mechanism is ESBL production. In the present study, the prevalence of ESBL production was 41%. The prevalence of ESBL production among K. pneumoniae species varied from 30% up to 65% in different studies [18Patzer JA, Dzierzanowska D, Pawińska A, Turner PJ. High activity of meropenem against Gram-negative bacteria from a paediatric Intensive Care Unit, 2001-2005. Int J Antimicrob Agents 2007; 29(3): 285-8.
[http://dx.doi.org/10.1016/j.ijantimicag.2006.09.012] [PMID: 17257 814]
, 19Patzer JA, Dzierzanowska D. Increase of imipenem resistance among Pseudomonas aeruginosa isolates from a Polish paediatric hospital (1993-2002). Int J Antimicrob Agents 2007; 29(2): 153-8.
[http://dx.doi.org/10.1016/j.ijantimicag.2006.08.044] [PMID: 1715 7481]
]. The variations of the prevalence of ESBL depend upon the empirical use of antibiotics in local regions with positive pressure on K. pneumoniae leading to the selection of resistant strains.

The most prevalent genes among ESBL producing K. pneumoniae were blaCTX-M followed by blaSHV and blaTEM. Several reports had a similar finding with blaCTX-M was the most prevalent gene followed by blaTEM and blaSHV [20Mahdi Yahya Mohsen S, Hamzah HA, Muhammad Imad Al-Deen M, Baharudin R. Antimicrobial Susceptibility of Klebsiella pneumoniae and Escherichia coli with Extended-Spectrum β-lactamase associated Genes in Hospital Tengku Ampuan Afzan, Kuantan, Pahang. Malays J Med Sci 2016; 23(2): 14-20.
[PMID: 27547110]
, 21Al-Marzooq F, Mohd Yusof MY, Tay ST. Molecular analysis of antibiotic resistance determinants and Plasmids in malaysian isolates of multidrug resistant Klebsiella pneumoniae. PLoS One 2015; 10(7)e0133654
[http://dx.doi.org/10.1371/journal.pone.0133654] [PMID: 26203651]
]. The high prevalence of blaCTX-M among K. pneumoniae may represent selective pressure due to the wide prescriptions of cephalosporins, particularly cefotaxime and ceftriaxone in many geographical regions including Egypt [22Ahmed O, El-Hady SA, Ahmed TM, Ahmed IZ. Detection of blaSHV and blaCTX-M genes in ESβL producing Klebsiella pneumoniae isolated from Egyptian patients with suspected nosocomial infections. Egyp. J of Med Human Gen 2013; 14(3): 277-83.]. However, the prevalence rates vary not only between different countries but even between different hospitals in the same country [23Guo Y, Wang S, Zhan L, et al. Microbiological and clinical characteristics of hypermucoviscous Klebsiella pneumoniae isolates associated with invasive infections in china. Front Cell Infect Microbiol 2017; 7: 24-9.
[http://dx.doi.org/10.3389/fcimb.2017.00024] [PMID: 28203549]
].

The ESBL enzymes are encoded by plasmid genes, therefore, it is expected for one isolate to carry different genes as the mixed genes were present in 65.8% of the isolates in our study. Similar findings were reported by [20Mahdi Yahya Mohsen S, Hamzah HA, Muhammad Imad Al-Deen M, Baharudin R. Antimicrobial Susceptibility of Klebsiella pneumoniae and Escherichia coli with Extended-Spectrum β-lactamase associated Genes in Hospital Tengku Ampuan Afzan, Kuantan, Pahang. Malays J Med Sci 2016; 23(2): 14-20.
[PMID: 27547110]
]. Further studies are required to understand the mechanisms mediated by multiple bla genes on the same isolate.

The present study revealed that the most prevalent virulence genes among K. pneumoniae species were fimH (76%), uge (70%), rmpA (62%), mag (59%) andwzy (59%) and the lowest prevalence was detected for aerobactin gene (50%) and kfa gene (33%). Similar prevalence rates were reported for fimH, uge and rmpA genes [23Guo Y, Wang S, Zhan L, et al. Microbiological and clinical characteristics of hypermucoviscous Klebsiella pneumoniae isolates associated with invasive infections in china. Front Cell Infect Microbiol 2017; 7: 24-9.
[http://dx.doi.org/10.3389/fcimb.2017.00024] [PMID: 28203549]
, 24Candan ED, Aksöz N. Klebsiella pneumoniae: Characteristics of carbapenem resistance and virulence factors. Acta Biochim Pol 2015; 62(4): 867-74.
[http://dx.doi.org/10.18388/abp.2015_1148] [PMID: 26637376]
]. The prevalence of aerobactin gene varies in different studies from 3% up to 92.8% [25Alizade H, Jajarmi M, Aflatoonian M, Neyestanaki DK, Shoja S, Ghanbarpour R. Comparative prevalence of blaCTX-M gene with virulence genes and serotypes in Klebsiella pneumonia. Jund J Microb 2018; 11(4)e61285, 26Russo TA, Shon AS, Beanan JM, 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
[http://dx.doi.org/10.1371/journal.pone.0026734] [PMID: 22039542]
]. The difference in the prevalence of virulence genes appears to be associated with the difference of the clinical sources of the isolated K. pneumoniae species.

Several studies demonstrated an association between the aerobactin gene and the regulatory genes of mucous in K. pneumoniae [26Russo TA, Shon AS, Beanan JM, 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
[http://dx.doi.org/10.1371/journal.pone.0026734] [PMID: 22039542]
, 27Jung SW, Chae HJ, Park YJ, 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.
[http://dx.doi.org/10.1017/S0950268812000933] [PMID: 22578630]
]. There are cumulative data that the virulence of K. pneumoniae is enhanced by the presence of aerobactin due to the presence of other multiple virulence genes on the same plasmid carrying it [27Jung SW, Chae HJ, Park YJ, 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.
[http://dx.doi.org/10.1017/S0950268812000933] [PMID: 22578630]
].

There was a significant association between resistance to amikacin, cefepime, ceftazidime, gentamicin, imipenem and meropenem and biofilm production by K. pneumoniae. The finding was explained by several mechanisms such as the adherent bacterial cells are resistant to the actions of different antibiotics due to the limitation of the penetration of the antibiotics, beside the slow growth of the bacterial cells inside the biofilm [16Vuotto C, Longo F, Balice MP, Donelli G, Varaldo PE. Antibiotic resistance related to biofilm formation in Klebsiella pneumoniae. Pathogens 2014; 3(3): 743-58.
[http://dx.doi.org/10.3390/pathogens3030743] [PMID: 25438022]
]. Moreover, the exchange of plasmids that carry antibiotics resistance genes between contact microbial cells may play role [28Angel Díaz M, Ramón Hernández J, Martínez-Martínez L, Rodríguez-Baño J, Pascual A. Grupo de Estudio de Infección Hospitalaria (GEIH). [Extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae in Spanish hospitals: 2nd multicenter study (GEIH-BLEE project, 2006)]. Enferm Infecc Microbiol Clin 2009; 27(9): 503-10.
[http://dx.doi.org/10.1016/j.eimc.2008.09.006] [PMID: 19329230]
, 29Juhas M. Horizontal gene transfer in human pathogens. Crit Rev Microbiol 2015; 41(1): 101-8.
[http://dx.doi.org/10.3109/1040841X.2013.804031] [PMID: 2386 2575]
]. ESBL production by isolated K. pneumoniae species was significantly associated with biofilm formation. This finding was similar to previous reports [29Juhas M. Horizontal gene transfer in human pathogens. Crit Rev Microbiol 2015; 41(1): 101-8.
[http://dx.doi.org/10.3109/1040841X.2013.804031] [PMID: 2386 2575]
, 30Singhai M, Malik A, Shahid M, Malik MA, Goyal R. A study on device-related infections with special reference to biofilm production and antibiotic resistance. J Glob Infect Dis 2012; 4(4): 193-8.
[http://dx.doi.org/10.4103/0974-777X.103896] [PMID: 23326076]
]. Again, this finding can be explained by the formation of protective environment to K. pneumoniae away from the antibiotics and the transmission of resistant genes. This was confirmed in the present study by significant association of blaSHV and blaCTX-M genes with biofilm formation.

Virulence genes of K. pneumoniae, fimH, magA, kfa, rmpA and aerobactin had a significant association with biofilm formation. Several studies correlated the association of genes of fimbriae, capsule and lipopolysaccharides with the biofilm formation by K. pneumoniae through initial adhesion by fimbriae and lipopolysaccharide followed by coverage of substratum and construction of mature biofilm architecture by the capsule [31Nepal HP, Neopane P, Shrestha R, et al. Biofilm formation and antimicrobial resistance in Klebsiella pneumoniaeisolated from patients visiting a tertiary care center of Nepal. Asian Pac J Trop Dis 2017; 7(6): 347-50.
[http://dx.doi.org/10.12980/apjtd.7.2017D7-15]
, 32Johnson JG, Clegg S. Role of MrkJ, a phosphodiesterase, in type 3 fimbrial expression and biofilm formation in Klebsiella pneumoniae. J Bacteriol 2010; 192(15): 3944-50.
[http://dx.doi.org/10.1128/JB.00304-10] [PMID: 20511505]
].

CONCLUSION

The present study highlights the prevalence of virulence genes among biofilm-forming strains of K. pneumoniae isolated from hospital-acquired urinary tract infections. Moreover, there was an association between biofilm formation and ESBL production. Further studies are required to elucidate the clinical impact of the association of these different mechanisms.

ETHICAL APPROVAL AND CONSENT TO PARTICIPATE

The study was approved by Mansoura Faculty of Medicine ethical committee, Egypt.

HUMAN AND ANIMAL RIGHTS

No Animals were used in this research. All human research procedures followed were in accordance with the ethical standards of the committee responsible for human experimentation (institutional and national), and with the Helsinki Declaration of 1975, as revised in 2013.

CONSENT FOR PUBLICATION

Informed consent was obtained from all the participants prior to publication.

AVAILABILITY OF DATA AND MATERIALS

Not applicable.

FUNDING

UGC-BSR Start-up grant by University Grant Commission, New Delhi, India.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.

ACKNOWLEDGEMENTS

Declared none

REFERENCES

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