Prevalence of antibiotic resistance and biofilm formation in Klebsiella pneumoniae carrying fimbrial genes in Egypt

 

Sara H. Mohamed¹, Mary S. Khalil², Mona I. Mabrouk¹, Mahmoud S.M. Mohamed²*

¹Department of Microbiology, National Organization for Drug Control and Research, Giza, Egypt

²Department of Botany and Microbiology, Faculty of Science, Cairo University, PO Box 12613, Giza, Egypt

 

ABSTRACT:

Biofilm formation is closely related to the pathogenic processes of Klebsiella pneumoniae, which frequently causes infections that are difficult to treat with antimicrobial agents. The aim of this study was to evaluate biofilm formation ability among clinical Klebsiella pneumoniae isolates from Egypt, and to study its antibiotic resistance, extended spectrum β-lactamases (ESBLs) production and fimbrial genes occurrence. A total of 90 clinical Klebsiella pneumoniae isolates were collected from different sources. Antimicrobial susceptibility, phenotypic and genotypic detection of ESBLs and biofilm assay were determined. SEM was applied to confirm K. pneumoniae biofilm formation. PCR assay was performed to investigate the distribution of fimbrial, as well as β-lactamases genes which were further confirmed by DNA sequencing. The results reveal high prevalence of multidrug resistance (86.66%) and biofilm formation ability (51%) among Klebsiella pneumoniae isolates. Furthermore, ESBL producing Klebsiella pneumoniae isolates had a higher ability to form a biofilm compared to non-ESBL forming ones. The occurrence of bla_CTX-M and bla_TEM among ESBLs-biofilm producers demonstrated high predominance of isolates harboring bla_CTX-M. The distribution of fimbrial (mrkD and fimH) among biofilm former isolates were 100% and 86.95% respectively. The present study revealed high prevalence of multi-drug resistance (MDR) among Klebsiella pneumoniae in Egypt, in addition to biofilm formation, and also conclude that ESBL producing Klebsiella pneumoniae isolates had a higher ability to form a biofilm in comparison with non-ESBL forming ones. In addition, our study strongly supports that type 3 fimbriae strongly promote biofilm formation in Klebsiella pneumoniae.

 

 

 

INTRODUCTION:

Klebsiella pneumoniae is one of the most common clinical Gram-negative pathogens causing severe infections, such as pneumonia, urinary tract infections, wound infections and others^[1,2]. Great attention has been specified to the antimicrobial level of resistance due to the elevated death rate from illnesses caused by K. pneumoniae specially to children and immune-compromised patients^[3]. This pathogens acquires resistance to antibiotic by multiple procedures resulting in developing multidrug-resistant (MDR) strains that leads to serious conditions in medical center configurations and wellness concern^[4].

 

The rise of extended spectrum β-lactamases (ESBL) producing K. pneumoniae represent one of the most frequent multidrug-resistant groups of Gram-negative bacilli bacteria globally ^[5,6].

 

Another important factor that contributes to the establishment and spread of infection is the biofilm formation. Biofilm production is a mechanism exhibited by bacteria to survive under unfavorable conditions at which the bacteria in this biofilm are highly resistant to antibiotic treatments. K. pneumoniae is a biofilm developing bacteria accountable for a number of attacks, putting it among the eight most essential nosocomial disease^[7]. The capability of the K. pneumoniae of forming biofilm facilitate their attachment on medical devices and biological surfaces such as epithelial cells [8]^. Therefore, formation of biofilm by harmful bacteria is a current subject of interest as it may represent a virulence aspect in individual attacks renders the necessity for continuous evaluation of pathogen development ^[9,10].

 

The most essential virulence aspects adding to K. pneumoniae pathogenesis pertaining to the intensity of its attacks are capsular polysaccharides, type 1 and type 3 pili, which can play a major role in the biofilm development ^[11]. Because cell attachment is an essential step in infection, the type 3 fimbriae may be indispensable for K. pneumoniae virulence ^[12,13].

 

Therefore, this study was aimed to study the ability of K. pneumoniae to form biofilm, as well as to detect ESBLs production and the occurrence of type 1 and type 3 adhesins among MDR biofilm-producing K. pneumoniae. In addition, study the occurrence of bla_CTX-M-I and bla_TEM among ESBLs-biofilm producers.

 

MATERIAL AND METHODS:

Collection and identification of bacterial isolates

The study was performed on 90 K. pneumoniae isolates (one isolate per patient) collected along the period of October 2015 until March 2016 from the main laboratory of Kasr Alaini Hospital, Cairo, Egypt. Isolates were identified biochemically as K. pneumoniae based on colony morphology on MacConkey's agar, Gram staining and laboratory biochemical tests, including catalase test, citrate test, motility indole ornithine test, methyl red test, oxidase test, triple sugar iron, urease test, Voges Proskauer test and lactose fermentation ability^[1]. Isolates which identified as K. pneumoniae, were stored on glycerol nutrient broth at -80 °C freezer. Ten K. pneumoniae isolates were previously identified and characterized for antibiotic susceptibility and biofilm formation ^[9].

 

Antibiotic susceptibility testing and phenotypic detection of ESBLs:

The susceptibility of K. pneumoniae to different antibiotics was performed by disk diffusion method according to CLSI^[14] recommendations using commercially available antibiotic discs (Oxoid, UK): imipenem (10µg), meropenem (10µg), cefuroxime sodium (30µg), ceftazidime (30µg), cefotaxime (30µg), cefepime (30µg), cefoxitin (30µg), gentamicin (10µg), amikacin (30µg), nalidixic acid (30µg), ciprofloxacin (5µg), levofloxacin (5µg), ampicillin (10µg), piperacillin-tazopactam (110µg) and co-trimoxazole (1.25/23.75μg).

 

ESBL confirmatory test was done to all isolates using combination disk method (Bio-Rad, France) as recommended by CLSI^[15] and include cefotaxime (30 μg), cefotaxime/clavulinic acid (30/10 μg), ceftazidime (30 μg), and ceftazidime/clavulinic acid (30/10 μg). Klebsiella pneumoniae ATCC 700603 (positive control) was kindly provided by the U.S. naval medical research unit no. 3 (NAMRU-3) in Egypt.

 

Biofilm formation assay:

Evaluation of biofilm formation by K. pneumoniae was performed according to the method described by O’Toole and Kolter, ^[16] with minor modifications. Wells originally containing uninoculated media were considered as negative control. The optical density (O.D) was measured at 630 nm using STAT FAX 2100 microplate reader. The cut-off value (ODc) is defined as three standard deviations (SD) above the mean OD of the negative control, that is, sample’s ODc = average OD of negative control + (3*SD of negative control). After comparing the O.D of biofilm to the control and according to the readings, the isolates were classified as follows: O.D ≤ O.Dc no biofilm producer, O.Dc <O.D ≤ 2×O.Dc weak biofilm, 2×O.Dc <O.D ≤ 4×O.Dc moderate and 4×O.Dc <O.D strong biofilm as described previously ^[17].

 

Scanning Electron Microscopy:

In order to confirm the K. pneumoniae biofilm formation random isolate was examined using scanning electron microscope (Philips XL30, Eindhoven, Netherlands) run at 20KV. Polystyrene tubes covered with K. pneumoniae biofilm formed after 24 hours were washed with 100 mM phosphate buffer pH 7, fixed for 12 hours in 2% glutaraldehyde (v/v) at 4°C. The biofilm samples were similarly processed with the method adopted by Mohamed et al., ^[18].

 

Genotypic detection of fimberial and ESBLs genes by Polymerase Chain Reaction (PCR):

DNA extraction was done using GeneJET™ DNA Purification Kit (Thermo scientific-USA). Fimbrial and ESBLs genes were detected by PCR using specific primers presented in Table 1. The reaction mixture composition was 1 μl of each primer (0.4 μM), 50 ng of bacterial genomic DNA, 12.5 μl of DreamTaq Green PCR Master Mix (2X), and nuclease-free water was added to complete a final volume to 25 μl; the mixture was mixed gently before starting the program. Cycling conditions for fimbrial genes were done as described elsewhere ^[19], while for bla_CTX-M and bla_TEM were done as described previously^[20]. PCR products were analyzed by gel electrophoresis with 1.6 % agarose gels in 1X TAE buffer.

 

Table 1. Primers used in this study

Gene	Primer Sequence (5–3)	Expected size (bp)	Annealing temperature (°C)	Refs.
fimH	5´- ATG AAC GCC TGG TCC TTT GC -3´	688	55	19
	5´- GCT GAA CGC CTA TCC CCT GC -3´
mrkD	5´- CCA CCA ACT ATT CCC TCG AA -3´	240	52	19
	5´- ATG GAA CCC ACA TCG ACA TT -3´
blaCTX-M	5´- GAC GAT GTC ACT GGC TGA GC -3´	499	55	20
	5´- AGC CG C CGA CGC TAA TAC A -3´
blaTEM	5´- AGA TCA GTT GGG TGC ACG AG -3´	750	55	20
	5´- TGC TTA ATC AGT GAG GCA CC -3´

 

Sequencing of PCR products:

Randomly selected products were sequenced after been purified on an Applied Biosystems 3500 Genetic Analyzer (Hitachi, Thermo Fisher) to identify the specific ESBL type. The primers used for sequencing were the same used for amplification. The PCR products were purified with high pure PCR product purification kits (QIAquick PCR Purification Kit, Qiagen). BigDye Terminator v3.1 Cycle Sequencing Kit and 5X Sequencing Buffer (Thermo Fisher) were used. Nucleotide sequences were compared to the known variants by BLAST at website search (http://www.ncbi.nlm.nih.gov/ blast).

 

Statistical analysis:

Chi-square test (χ2) was conducted to analyze the significance of the resistance level to various antibiotics in biofilm and non-biofilm producing isolates. A value of p < 0.05 was considered to be statistically significant.

 

RESULTS:

Out of 90 K. pneumoniae clinical isolates, 34 (37.78%) isolates were derived from urine cultures of hospitalized patients, 27 (30%) from wounds, 16 (17.78%) from sputum, 5 (5.55%) from blood, 4 (4.44%) from pus, 2 (2.22%) from bronchial lavage, and 1 (1.1%) sample from each of pleural fluid and periumbilial swab. The rates of isolates resistance to the antimicrobial agents were recorded and summarized in Table 2. The results shown that K. pneumoniae isolates were characterized by high rates of resistance to many antibiotics including ampicillin, ceftazidime, nalidixic acid, cefuroxime and cefotaxime.

 

 

 

Table 2. Antimicrobial resistance percentages of biofilm and non-biofilm producing K. pneumoniae isolates

Antimicrobial category	Antibiotic	Biofilm formers (N=46)		Non-biofilm formers (N=44)		p-value	Total (N=90)
		NO.	(%)	NO.	(%)		NO.	(%)
Carbapenems	Imipenem	10	21.73	4	9.09	˃0.05	14	15.55
	Meropenem	29	63.04	22	50.00	˃0.05	51	56.66
Cephalosporins	Cefuroxime	45	97.82	41	93.18	˃0.05	86	95.55
	Ceftazidime	45	97.82	38	86.36	0.042	83	92.22
	Cefotaxime	45	97.82	37	84.09	0.022	82	91.11
	Cefepime	43	93.47	31	70.45	0.004	74	82.22
Cephamycins	Cefoxitin	22	47.82	22	50.00	˃0.05	44	48.88
Aminoglycosides	Gentamicin	26	56.52	18	40.90	˃0.05	44	48.88
	Amikacin	21	45.65	15	34.09	˃0.05	36	40.00
Fluoroquinolones	Nalidixic acid	45	97.82	41	93.18	˃0.05	86	95.55
	Ciprofloxacin	35	76.08	22	50.00	0.01	57	63.33
	Levofloxacin	27	58.69	16	36.36	0.034	43	47.77
Penicillins	Ampicillin	46	100	44	100	-	90	100
Penicillins/ß-lactamase inhibitors	piperacillin-tazopactam	25	54.34	29	65.9	˃0.05	54	60.00
Sulfonamides	Co-trimoxazole	39	84.78	37	84.09	˃0.05	76	84.44

Difference in resistance level between biofilm and non-biofilm producers was significant at p<0.05 as tested by Chi-square test.

 

 

In contrast, high susceptibility rates were demonstrated for imipenem and amikacin. ESBL production was observed in 32 (35.55%) of the isolates, ESBLs production among biofilm producers was 22 (47.82%) and among non-biofilm producers were 10 (22.72%).

 

Biofilm formation assay showed that 51.11% of K. pneumoniae isolates formed biofilms. Herein, there is no significant difference between the groups of isolates in their ability to form biofilm (Figure 1). The biofilm analysis demonstrated that 15.55% (n=14) were categorized as strong biofilm-producing strains, 21.11% (n=19) as moderate biofilm-producing strains, 14.44% (n=13) as weak biofilm producing and 48.88% (n=44) of the strains were non-biofilm producers.

 

Figure 1. Number of biofilm and non-biofilm producing Klebsiella pneumoniae isolates from the major clinical sources in this study. Biofilm formation ability was not significantly associated with the bacterial source (p-value = 0.64208) at p<0.05 as tested by Chi-square test.

 

Indeed, K. pneumoniae biofilm former exhibited higher resistance to all antibiotics except piperacillin-tazopactam and cefoxitin (Table 2). High percentage of biofilm-producing isolates 93.478% (n=43) were MDR K. pneumoniae, MDR was defined according to Magiorakos et al. [21] categories as non-susceptibility to at least one agent in three or more antimicrobial categories (Table 3). The majority of biofilm producers were isolated from urine (36.95%) followed by wound (34.78%).

 

In order to confirm the formation of biofilm morphologically by K. pneumoniae the scanning electron microscopy was employed. The image of biofilm former isolate demonstrates dense matrix and several bacilli cells were collected together and embedded in it (Figure 2).

 

Figure 2. Scanning electron microscope image of Klebsiella pneumoniae biofilm formed in Brain Heart Infusion culture medium after 24 hours of incubation (magnification: 3,000×).

 

PCR was carried out on all biofilm producers (n=46) and 4 non-biofilm producers as a control to detect the occurrence of fimH and mrkD genes responsible for expression of type 1 and type 3 fimbrial adhesions, respectively. The results showed that fimH gene was detected in 40 isolates (90.9%) among biofilm producers and only one non-biofilm producer isolate. However, mrkD gene was detected in all biofilm producers and not detected in non-biofilm producers (Table 3).

 

The occurrence of bla_CTX-M and bla_TEM genes among the 22 biofilm-ESBLs producers presented in Table 3 showed the high predominance of isolates harboring bla_CTX-M-I which represent 68.18% (15 isolates), containing 3 isolates harboring bla_TEM in association. ESBLs genes were detected in all ESBL-biofilm producing isolates (Table 3).

 

Sequencing of two random amplicons from bla_CTX-M-I group was characterized as bla_{CTX-M-I -15} variant sharing 99% identity with the nucleotide sequences of reference GenBank accession no. KP455328.1.

 

Table 3. Antibiotic resistance patterns and occurrence of tested ESBL as well as fimbrial genes among biofilm producing isolates.

ID	Source	Resistance pattern	Biofilm formation ability	Fimbrial genes	ESBL Genes (bla)	ID	Source	Resistance pattern	Biofilm formation ability	Fimbrial genes	ESBL Genes (bla)
K3†	Urine	CAZ CXM CTX FEP NA CIP AMP SXT	Moderate biofilm	mrkD, fimH1	CTX	K47‡	Urine	CAZ CXM CTX FEP GM NA CIP LEV AMP SXT	No biofilm	-	_
K4†	Wound	CAZ CXM CTX NA CIP LEV AMP SXT	Moderate biofilm	mrkD, fimH1	CTX, TEM	K48†	Wound	IPM MEM CAZ CXM CTX FEP GM NA CIP LEV AMP TZP SXT	Strong biofilm	mrkD, fimH1	CTX
K6	Wound	IPM MEM CAZ CXM CTX FEP FOX GM AK NA CIP LEV AMP TZP SXT	Moderate biofilm	mrkD, fimH1	_	K49	Blood	MEM CAZ CXM CTX FEP FOX GM AK NA AMP TZP SXT	Weak biofilm	mrkD, fimH1	_
K7‡	Bronchial lavage	CAZ CXM CTX FOX NA AMP TZP	No biofilm	-	_	K50†	Sputum	CAZ CXM CTX FEP NA AMP	Moderate biofilm	mrkD, fimH1	CTX
K10†	Urine	CAZ CXM CTX FEP NA CIP AMP SXT	Moderate biofilm	mrkD, fimH1	_	K51	Sputum	MEM CAZ CXM CTX FEP FOX NA CIP LEV AMP SXT	Weak biofilm	mrkD, fimH1	_
K11	Urine	IPM MEM CAZ CXM CTX FEP FOX GM AK NA CIP LEV AMP TZP SXT	Weak biofilm	mrkD, fimH1	_	K56†	Wound	IPM MEM CAZ CXM CTX FEP GM AK NA CIP LEV AMP TZP SXT	Strong biofilm	mrkD, fimH1	CTX, TEM
K13	Urine	CAZ CXM CTX GM NA CIP LEV AMP SXT	Moderate biofilm	mrkD, fimH1	_	K62†	Wound	MEM CAZ CXM CTX FEP GM AK NA AMP TZP	Moderate biofilm	mrkD, fimH1	_
K15†	Wound	CAZ CXM CTX FEP NA AMP SXT	Moderate biofilm	mrkD		K63†	Wound	MEM CAZ CXM CTX FEP NA AMP SXT	Strong biofilm	mrkD, fimH1	CTX
K16†	Wound	CAZ CXM CTX FEP NA CIP LEV AMP SXT	Weak biofilm	mrkD, fimH1	CTX	K65†	Blood	CAZ CXM CTX FEP NA CIP LEV AMP SXT	Weak biofilm	mrkD, fimH1	CTX
K17	Sputum	CAZ CXM CTX FEP FOX GM AK NA AMP TZP SXT	Moderate biofilm	mrkD	_	K75	Sputum	MEM CAZ CXM CTX FEP FOX GM AK NA CIP LEV AMP TZP SXT	Weak biofilm	mrkD, fimH1	_
K18	Urine	IPM MEM CAZ CXM CTX FEP FOX GM AK NA CIP LEV AMP SXT	Moderate biofilm	mrkD, fimH1	_	K81†	Urine	MEM CAZ CXM CTX FEP AK NA CIP LEV AMP TZP SXT	Moderate biofilm	mrkD, fimH1	CTX
K19	Sputum	MEM CAZ CXM CTX FEP FOX AK NA CIP LEV AMP	Moderate biofilm	mrkD	_	K84†	Wound	MEM CAZ CXM CTX FEP GM NA CIP AMP SXT	Weak biofilm	mrkD, fimH1	_
K22	Urine	CAZ CXM CTX FEP FOX GM NA CIP LEV AMP TZP SXT	Weak biofilm	mrkD, fimH1	_	K85†	Wound	MEM CAZ CXM CTX FEP GM AK NA CIP LEV AMP TZP SXT	Moderate biofilm	mrkD, fimH1	_
K23†	Urine	CAZ CXM CTX FEP FOX AMP SXT	Weak biofilm	mrkD, fimH1	_	K86	Sputum	MEM CAZ CXM CTX FEP FOX GM NA CIP AMP TZP SXT	Moderate biofilm	mrkD, fimH1	_
K24	Urine	CAZ CXM CTX FEP FOX NA CIP AMP SXT	Moderate biofilm	mrkD, fimH1	_	K89‡	Urine	CAZ CXM CTX FEP FOX GM AMP TZP SXT	No biofilm	-	_
K25	Wound	MEM CAZ CXM CTX FEP FOX NA CIP AMP TZP SXT	Weak biofilm	mrkD	_	Kp1	-	-	Moderate biofilm	mrkD, fimH1	_
K26†	Wound	CAZ CXM CTX FEP NA AMP	Weak biofilm	mrkD, fimH1	_	Kp2	-	-	Strong biofilm	mrkD, fimH1	_
K27	Periumbilial swap	IPM MEM CAZ CXM CTX FEP FOX GM AK NA CIP LEV AMP TZP SXT	Moderate biofilm	mrkD, fimH1	_	Kp3	-	-	Strong biofilm	mrkD, fimH1	_
K29†	Sputum	MEM CAZ CXM CTX FEP GM NA CIP AMP SXT	Strong biofilm	mrkD, fimH1	CTX, TEM	Kp4†	-	-	Strong biofilm	mrkD, fimH1	CTX
K31	Urine	CAZ CXM CTX FEP FOX NA AMP SXT	Moderate biofilm	mrkD, fimH1	_	Kp5	-	-	Moderate biofilm	mrkD, fimH1	_
K34	Wound	IPM MEM CAZ CXM CTX FEP FOX GM AK NA CIP LEV AMP TZP SXT	Weak biofilm	mrkD	_	Kp6	-	-	Strong biofilm	mrkD, fimH1	_
K36†	Urine	CAZ CXM CTX FEP NA AMP	Weak biofilm	mrkD, fimH1	CTX	Kp7†	-	-	Strong biofilm	mrkD, fimH1	CTX
K39‡	Urine	MEM CAZ CXM CTX FEP FOX GM AK NA CIP LEV AMP TZP SXT	No biofilm	fimH1	_	Kp8	-	-	Strong biofilm	mrkD	_
K42†	Urine	MEM CAZ CXM CTX FEP AK NA CIP LEV AMP TZP	Strong biofilm	mrkD, fimH1	CTX	KP9	-	-	Strong biofilm	mrkD, fimH1	_
K46†	Wound	CAZ CXM CTX FEP NA AMP TZP	Strong biofilm	mrkD, fimH1	CTX	KP10	-	-	Strong biofilm	mrkD, fimH1	_

Imipenem IPM, meropenem MEM, cefuroxime CXM, ceftazidime CAZ, cefotaxime CTX, cefepime FEP, cefoxitin FOX, gentamicin GM, amikacin AK, nalidixic acid NA, ciprofloxacin CIP, levofloxacin LEV, ampicillin AMP, piperacillin-tazopactam TZP, co-trimoxazole SXT. Previously characterized isolates (KP1-KP10) are highlighted at the end of the table[9]^.

† ESBLs producing isolates.

‡ Non-biofilm producing isolates (control).

Sequenced isolates are underlined.

 

DISCUSSION:

Antibiotic resistance in K. pneumoniae has become a major concern and a threat to public health through the world. According to studies conducted in recent years, resistance has spread to most antibiotics ^[22,23]. In our study, it was recorded that the majority of K. pneumoniae were isolated from urine (37.78%). Urinary tract infection (UTI) is considered the most common bacterial infection worldwide ^[4,24] which accounts for an estimated 25% to 40% of nosocomial infections ^[25].

 

K. pneumoniae showed the highest resistance against commonly used antibiotics at which 86.66% of isolates were MDR. In this regard, different rates of MDR were reported in many studies from different countries such as 71.73% ^[26] and 67% ^[27] of MDR K. pneumoniae isolates. In this context, ESBL-producing Gram-negative bacilli especially K. pneumoniae have emerged as serious causative pathogens recently in both nosocomial and community-acquired infections worldwide ^[28,29].

 

In this study, the prevalence of ESBL-producing K. pneumoniae isolates was 35.55% of all clinical K. pneumoniae isolates. Other developed countries reported a prevalence of 76% in Serbia ^[30], 18% in Algeria ^[31], 25.5% in Morocco^[32] and 42.3 % in Ghana^[33]. Outbreaks of ESBL-producing K. pneumoniae infections had increased and the prevalence of ESBL producing isolates varies in different countries, depending upon various factors such as antibiotic policy and the type of disinfection used especially in the intensive care units ^[5].

 

Among the three phenotypic methods used to detect biofilm formation, tissue culture plate method (TCP) was found to be a method with good reproducibility and specificity which can be used routinely in the microbiology laboratory to detect biofilm formation ^[10]. Therefore, biofilm formation was evaluated in our study using TCP method and reported that 51.11% of K. pneumoniae isolates have the ability to form biofilm. Evaluation of K. pneumoniae biofilm varies among different studies at which the highest percent recorded was reported by Cruz-Córdova et al. ^[25] in Mexico, which found that all strains were able to form biofilms (100%). Other studies observed percentage of 93% ^[11], 71% ^[8] 48.13% ^[34] and 18% ^[24]. The bacterial ability to form biofilm was confirmed by morphological examination using scanning electron microscope (SEM) of representative biofilm former K. pneumoniae isolate and the results indicating reliability of the biofilm assay using TCP method ^[35–38].

 

By comparing the antibiotic sensitivity results of biofilm and non-biofilm producing isolates, we observed that the number of resistant isolates in biofilm-former was higher in most of tested antibiotics (Table 2), which supports that the increase in antibiotic resistance is a general trait associated with biofilm in bacteria reported formerly ^[19].

 

fimH gene was detected in 86.95% among biofilm-producers and only one urine non-biofilm isolate, this is in agreement with the result early reported ^[39]. This study concluded that type 1 fimbriae did not influence the ability of K. pneumoniae to colonize, but was determined to be a significant virulence factor in urinary tract infection.

 

Our study strongly agrees with the studies ^[40,41] which stated that type 3 fimbriae but not type 1 fimbriae strongly promote biofilm formation in K. pneumoniae, as all biofilm producers harboring mrkD gene (100%).

 

In our study, 68.75% of ESBLs producing isolates versus 41.38% of non-ESBLs producers had the ability to form a biofilm which supports the evidence that ESBL producing K. pneumoniae had a greater ability to form biofilm compared to non ESBL forming K. pneumoniae ^[25,34]. ESBLs genes were detected in all ESBL-biofilm producing isolates (Table 3). Isolates harboring blaCTX-M represent 68.18%, and that harboring bla_TEM represent only 13.64% of all ESBLs producers among K. pneumoniae. Randomly selected two isolates which have been chosen for sequencing showed that both isolates harboring bla_CTX-M-₁₅ variants. bla_CTX-M β-lactamases have become the most prevalent ESBL enzymes during the past decade, more specifically, recent reports have shown that the frequency of bla_CTX-M genes among Klebsiella isolates is a rising alarming rate ^[31,42]. Because of all that, recently researches has been focused on the antibacterial and antibiofilm activity using alternative strategies other than antibiotics to fight against pathogenic organisms ^{[9,18,23,36,43–45]}

 

This study showed high prevalence of MDR among K. pneumoniae (86.66%), high percentage (51.11%) of K. pneumoniae isolates have the ability to form a biofilm, and also concludes that ESBL producing K. pneumoniae isolates had a higher ability to form a biofilm compared to non ESBL forming ones. Furthermore, it strongly supports that type 3 fimbriae, but not type 1 fimbriae, strongly promote biofilm formation in K. pneumoniae.

 

CONFLICT OF INTEREST:

We declare that we do not have conflicts of interest.

 

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Received on 11.09.2019           Modified on 16.11.2019

Accepted on 21.12.2019         © RJPT All right reserved