Extended spectrum β-lactamase producing uropathogenic Escherichia coli and the correlation of biofilm with antibiotics resistance in Nepal

Background Urinary tract infection (UTI) is one of the frequently diagnosed infectious diseases which is caused mainly by Escherichia coli. E. coli confers resistance against the two major classes of antibiotics due to the production of extended spectrum β-lactamase enzymes (ESBL), biofilm, etc. Biofilm produced by uropathogenic E. coli (UPEC) protects from host immune system and prevent entry of antimicrobial compounds. The main objective of this cross-sectional study was to determine the correlation of biofilm production and antibiotic resistance as well as to characterize the pgaA and pgaC genes responsible for biofilm formation among uropathogenic ESBL producing E. coli. Methods A total of 1977 mid-stream urine samples were examined and cultured for bacterial strain identification. ESBL was detected by combined disc method following CLSI whereas biofilm formation was analyzed by semi-quantitative method. Furthermore, the pgaA and pgaC genes responsible for biofilm formation in UPEC were detected by multiplex PCR. All the statistical analyses were done via IBM SPSS Statistics 21 where Pearson’s correlation test were used to determine correlation (−1 ≥ r ≤ 1). Results E. coli was the predominant causative agent, which accounted 159 (59.3%) of the Gram-negative bacteria, where 81 (50.9%) E. coli strains were found to be ESBL producers. In addition, 86 (54.1%) E. coli strains were found to be biofilm producers. Both the pgaA and pgaC genes were detected in 45 (93.7%) the UPEC isolates, which were both biofilm and ESBL producers. Moreover, there was a positive correlation between biofilm and ESBL production. Conclusion The analyses presented weak positive correlation between biofilm and ESBL production in which biofilm producing UPEC harbors both pgaA and pgaC genes responsible for biofilm formation.

polysaccharides in E. coli was recently discovered and acts as an adhesive in biofilms [4]. Biofilms help not only in the transfer of plasmid encoding resistance genes i.e., ESBL to other organisms via conjugation but also resist immune clearance [9][10][11][12][13]. The dissemination of ESBLs has emerged to a high proportion of CTX-M enzymes, notably E. coli, which is the major carriers of ESBLencoding genes i.e. bla CTX-M [11,14,15] so, the incidence of ESBL producing E. coli is now elevating in urinary tract infections [16].
The uropathogenic E. coli is now developing new trends of antimicrobial resistance as well as their biofilm is supporting to gain the resistance against numerous antibiotics [6,8,13]. To our knowledge, this study would be first in Nepal to determine the correlation of biofilm formation and antibiotic resistance as well as to characterize the biofilm producing genes located in pgaABCD locus among uropathogenic ESBL producing E. coli.

Methods
The cross-sectional study was carried out in the Department of Microbiology, Grande International Hospital, Tokha, Kathmandu, and Department of Microbiology, National College, Kathmandu from June to November, 2017. A clinical and socio-demographic study of patients was performed. A total of 1977 mid-stream urine were cultured semi-quantitatively on Cysteine Lactose Electrolyte Deficient Agar plates and incubated at 37 °C for 24 h [6,18,19]. The antibiotic susceptibility test was performed by modified Kirby-Bauer method of disk diffusion within the guidelines of Clinical and Laboratory Standard Institute (CLSI), 2015 [18][19][20].

Detection of ESBL producing uropathogenic E. coli
The resistance of cefotaxime (30 µg) in E. coli was used as the screening method for detection of ESBL which were then confirmed by combined disc method following CLSI, 2015 [20].

Detection of biofilm production in E. coli
The uropathogenic E.coli were cultured in 5 ml of Luria-Bertani (LB) broth at 37 °C for 24 h. The turbidity of cultured LB broth was compared with the 0.5 McFarland standard to maintain 10 8 CFU/ml followed by addition of LB broth supplemented with 1% glucose in the ratio 1:100 to maintain the concentration of approximately 10 6 CFU/ ml. It was then vortexed and 200 μl of diluted cultured LB broth was transferred per well in a microtiter plate in triplicate. A positive control i.e. 200 μl of E. coli ATCC 25922 cultured LB broth and a negative control i.e. 200 μl of LB broth were transferred into well of a microtiter plate in triplicate. The microtiter plates were covered with a tape and incubated at 37 °C for overnight. The plates were washed 3 times with 300 μl of sterile phosphate buffered saline (PBS, pH 7.2). Subsequently, plates were heat fixed by incubating at 60 °C for 1 h. Then, the plates were stained with 150 μl of 2% crystal violet for 15 min at room temperature. The plates were washed with distilled water until the stain was free. It was then air dried at room temperature. Afterward, 150 μl of 95% ethanol (v/v) was transferred per well in microtiter plates. The covered microtiter plates were left at room temperature for half an hour without shaking. The absorbance was measured at 570 nm using a spectrophotometer. The uropathogenic E. coli was classified as a non-biofilm producer, weak biofilm producer, moderate biofilm producer, or strong biofilm producer on the basis of findings evaluated [21,22].

Detection of biofilm genes i.e. pgaA and pgaC in E. coli
The genomic DNA was extracted from the ESBL and biofilm producing uropathogenic E. coli via a standard phenol-chloroform protocol [23]. Multiplex PCR was done to detect pgaA and pgaC genes in which the pgaA and pgaC primers were used for the amplification of 209 and 540 bp, respectively (Table 1) [24,25].
At first, 12.5 µl of Master Mix (Biolabs, New England) was added followed by 8.5 μl nuclease-free water, 0.5 µl of each primer (Macrogen, Inc., South Korea) of both genes and 2 µl of DNA from the bacterial strains to maintain 25 μl PCR mixture (TAKARA PCR Thermal Cycler Dice Gradient TP600, Takara bio, Tokyo, Japan). PCR conditions i.e., initial 5 min denaturation step at 94 °C was maintained followed by 32 cycles of 30 s at 94 °C, 30 s at 50 °C, and 45 s at 72 °C, and a final extension step of 5 min at 72 °C [24].

Data analysis
All the data collected were analyzed via IBM SPSS Statistics 21. Pearson's correlation test were used to determine correlation (−1 ≥ r ≤ 1) [1,6].

Clinical and socio-demographic study
Community-acquired infections was found to be higher which accounts 218 (70.1%) and female were affected by 173 (55.3%). Moreover, the higher number of cases was observed within the age group 60+ which accounts 93 (29.91%) ( Table 2).

Correlation between biofilm production by the semi-quantitative method and ESBL production in E. coli.
Amongst ESBL producing UPEC, 18.5%, 17.3%, and 23.5% showed strong, moderate and weak production of biofilm, respectively. There was a weak positive correlation between biofilm formation and ESBL production (r = 0.157) which is illustrated in Table 4.

Discussion
Urinary tract infections are frequently occurred infections in hospital where 93 (29.9%) were hospital acquired infections. The prevalence rate of urinary tract infections in female was found to be predominant (55.3%) than male (44.7%) because of the close proximity between vagina and anus [1], cystitis, sexual behavior, vaginal infections, pregnancy, diabetes mellitus, obesity and genetic sensitivity in female [2,36]. In addition, the prevalence rate of infection was found to be higher in age groups 60+ years.  From the above evidences, it was clear that urinary tract infections were found to be more prone to older ages rather than younger ages. It is due to the fact that with the ageing, immune response tends to decline gradually and also hormonal changes takes place which leads to infections of urinary tract [36]. E. coli was found to be a predominant causative agent of UTI which was highly resistant towards norfloxacin 99 (62.3%), cotrimoxazole 90 (56.6%) and cefotaxime 82 (51.6%) and their resistance patterns were found to be similar with the earlier study conducted [8,10,11].
There was a weak positive correlation (r = 0.157) relationship between biofilm production and ESBL productions. Within strong, moderate and weak biofilm producing E. coli, 65.2%, 50% and 54.3% were ESBL producer, respectively. There was positive correlation between biofilm and ESBL producing E. coli which was stated by Tabasi et al. and Neupane et al. [6,8]. This revealed that biofilm favors the ESBL gene transferred between the E. coli and other microorganisms because of matrix which stabilizes and enhances the transferability of genetic elements horizontally as well as resist the immune clearance [6,21,[30][31][32].
The development of resistance in E. coli may be due to haphazard use of antibiotics, plasmid-mediated genes, i.e. bla CTX-M , bla SHV , bla OXA , etc., quorum sensing, etc. [26][27][28][29]. The rise of multidrug-resistant UPEC poses a serious threat to manage UTI along with increment in treatment cost. The biofilm producing pathogens are sensitive towards co-therapy with macrolides i.e. erythromycin, clarithromycin and azithromycin, and other effective antibiotics as macrolides are considered as reliable antibiofilm agents [6,33].

Conclusion
In conclusion, tigecycline were found to be pragmatic approach for treatment as the result indicates in this research. There was found to be weak positive correlation between biofilm and ESBL production. In addition,   biofilm producing UPEC harbors both pgaA and pgaC genes responsible for biofilm production.

Limitations of study
The study of all genes responsible for biofilm production other than pgaA and pgaC genes and the genes ESBL productions could not be carried out. Genes like mcr-1 and NDM-1 for colistin and meropenem resistant strains were not performed respectively to confirm the resistivity.