MOLECULAR IDENTIFICATION OF CTX GENE OF EXTENDED SPECTRUM BETA-LACTAMASES (ESBL) PRODUCING ESCHERICHIA COLI ON LAYER CHICKEN IN BLITAR, INDONESIA.
Key words: CTX gene; ESBL; Escherichia coli; Human health; PCR; VITEKA(r)
Antibiotic resistance is a serious problem worldwide, especially center of layer poultry in Blitar, Indonesia (Wibisono et al., 2020a). Escherichia coli is one of the causes responsible for infection and antibiotic resistance in humans and animals through ESBL-mechanism (Paterson and Bonomo, 2005; Amelia et al., 2016). The presence of pathogenic Escherichia coli infections in poultry can adversely affect economy (Wibisono et al., 2018). The usage antibiotics can pose a risk of antibiotic resistance in poultry (Santos et al., 2013). There is restricted data related to the prevalence of antibiotic resistance in developing countries, in particular, Indonesia. Surveillance of antibiotic resistance is needed to monitor the emergence of antibiotic resistance (Kurniawati et al., 2015).
Antibiotic resistance which produced from Escherichia coli is Extended Spectrum [beta]-lactamase (ESBL) (Santos et al., 2013; Hammerum et al., 2014). Extended-spectrum beta lactamase is an enzyme that is characterized by the ability to hydrolyze third generation cephalosporins and aztreonam but are inhibited by clavulanic acid (Paterson and Bonomo, 2005; Public Health England, 2014). Exposure large amounts of beta-lactam antibiotics can induces production and mutation of beta-lactamase type enzyme.
This mutation causes an increase in the enzymatic activity of beta-lactamase so that this enzyme can hydrolyze third generation cephalosporins and aztreonam (Paterson and Bonomo, 2005; Lim et al., 2013). ESBL-producing bacteria can also be resistant to antibiotics from aminoglycoside, fluoroquinolone, tetracycline, chloramphenicol, and sulfamethoxazole-trimethoprim (Brower et al., 2017; Sudarwanto et al., 2017). Extended Spectrum Beta-Lactamase genes are often found in mutated genes, namely cefotaximase (CTX-M), temoneira (TEM) and variable sulfhydryl (SHV). The CTX-M gene in ESBL-producing Escherichia coli is a gene that codes and produces enzymes that can hydrolyze beta lactam rings from third-generation beta lactam antibiotics and cephalosporins (Biutifasari, 2018). The molecular detection is a genotypic confirmatory test to screen the ESBL encoding gene in Escherichia coli bacteria using PCR (Bradford, 2001).
The highest prevalence of ESBL-producing bacteria with CTX-M-1 is the most common type of ESBL in poultry (Saliu et al., 2017; Upadhyay et al., 2015; Rao et al., 2014).
ESBL-producing Escherichia coli in India was about 42% in layer chicken (Brower et al., 2017). In this study, we aimed to identify CTX gene for encoding ESBL producing Escherichia coli from cloacal swabs of layer chicken in Blitar, Indonesia.
MATERIALS AND METHODS
Isolation and Identification of Escherichia coli: A total of 130 cloacal swab samples was randomly taken from 4 districts in Blitar. The samples were kept in Amies Swab Viscosa (deltalab, Spain) transport medium at 4 AdegC, and immediately taken to the laboratory for further analyses (Seni et al., 2016). Isolation of Escherichia coli bacteria using selective media Mac Conkey Agar no. 3 CM0115 (Oxoid, England) and differential media Eosin Methylene Blue Agar CM0069 (Oxoid, England), incubated at of Escherichia coli were identified by biochemical testing of IMVIC (Indol-Motility, Methyl Red, Voges Proskauer, Citrate) and TSIA (Triple Sugar Iron Agar) (Effendi et al., 2018a; Effendi et al., 2019). The identification of Escherichia coli bacteria was confirmed by VITEKA(r) 2 compact using a VITEKA(r) 2 GN card (Biomerieux, 2017).
Confirmation for ESBL producing Escherichia coli: ESBL-producing Escherichia coli bacteria isolated from cloacal swabs of layer chicken were confirmed by the Double Disc Synergy Test (DDST) and VITEKA(r) 2 compact. This confirmation test with DDST was conducted to evaluate the presence of inhibitory zones of ESBL activity with clavulanic acid using the Kirby-Bauer disk diffusion method on Mueller-Hinton agar (Merck, Germany). Double Disc Synergy Test uses the antibiotic disc Amoxycillin-clavulanic 30ug (Oxoid, England), Cefotaxime 30ug (Oxoid, England), Ceftazidime 30ug (Becton Dickinson, USA), and Aztreonam 30ug (Oxoid, England).
Culture was incubated at 35-37 AdegC for 18-24 hours (CLSI, 2017; Effendi et al., 2018b). The results of the evaluation after incubation showed that the inhibition zone that appeared in the plate was measured based on CLSI 2017 guidelines (CLSI, 2017) as shown on Figure 2. The antibiotic sensitivity 102 test of ESBL-producing Escherichia coli bacteria by the DDST was then confirmed by the VITEKA(r) 2 compact. The bacterial suspension was homogenized and a bacterial turbidity of 0.50 to 0.63 Mc Farland was made using VITEKA(r) 2 DensiCHEK (Biomerieux, 2017). Antimicrobial susceptibility and phenotypic detection of ESBL producers using AST N280 cards (bioMerieux, Marcy-L'Etoile, France). These results are analyzed automatically by the system and interpreted as sensitive, intermediate, and resistant (Biomerieux, 2017; Brower et al., 2017).
Characterization of CTX gene by Polymerase Chain Reaction (PCR): The ESBL-positive strains were then subjected to molecular screening of CTX-gene. To do this, DNAs were extracted according to the instructions by a mini QIAampA(r) DNA kit (QIAGEN, Germany). Escherichia coli ATCC(tm) 35218 was used as positive control standard for strains of ESBL-producing bacteria, and Escherichia coli ATCC(tm) 25922 is used as negative control or non-ESBL-producing bacteria. The primers designed for screening CTX-M gene were used to encode CTX encoding genes using CTX-MA primers (CGCTTTGCGATGTGCAG), CTX-MB (ACCGCGATATCGTTGGT), respectively and the amplicon is 550-bp.
The PCR conditions were given as with denaturation temperatures 94AdegC, 2 minutes; extended denaturation 94AdegC, 1 minute; annealing 54 AdegC, 30 seconds; extension 72AdegC), 45 seconds; extended extension 72 AdegC, 5 minutes, this reaction is carried out for 30 cycles (Ali et al., 2016), and the amplification was carried out by PCR (Blue-Ray Biotech Turbo Cycler, TST-9620). After that, the amplicons were visualized by electrophoresis using 2% agarose gel (Invitrogen, USA) (Yanestria et al., 2019).
This study was conducted for the characterization of CTX gene among ESBL-positive E. oli strains isolated from 130 cloacal swab samples in layer poultry. The results were 8.69% (10) confirmation positive of ESBL-producing Escherichia coli on layer chicken cloacal swab by the Double Disc Synergy Test (DDST), shown on Figure 2. The presence of ESBLs-producing bacteria by double discs synergy test (DDST) to detect ESBL producing bacteria and then confirmed by the automatic VITEKA(r) 2 compact and indicated 100% ESBL producing Escherichia coli as shown in Table 1. For the identification of CTX-encoding gene present in ESBL producing E. coli PCR was used (Putra et al, 2019), as shown on Figure 3.
Tabel 1. Data ESBL producing Escherichia coli from cloacal swabs layer chicken in Blitar.
###No of ESBL positive strain###PCR
Subdistric###Sample###No of Escherichia###DDST###VITEKA(r)2 compact###No of CTX gene positive
###Blitar###(115/130; 88,5%)###(10/115;###(10/10; 100%)###(8/10; 80%)
Several other studies have examined the number of E. coli isolates that isolated from animal and animal products, showing concordance results between studies as shown on Table 1 (Saliu et al., 2017; Upadhyay et al., 2015; Rao et al., 2014). The relative abundance of the ESBL producing E. coli in samples from cattle and dogs has been shown to vary with geographic location (Putra et al., 2019; Kristianingtyas et al., 2020). In this study, isolates including ESBL producing E. coli were dominated by encoding CTX gene.
Table 1 showed the spread of ESBL-producing Escherichia coli in 3 districts from 4 districts. Srengat District has 6 samples, one sample from Talun district and Kademangan district was 3 samples from 10 ESBL-producing Escherichia coli samples, while Ponggok district was not found any ESBL producing Escherichia coli.
Cefotaxime synergy with the combination of amoxilin-clavulanate in the form of an expansion inhibition zone between the two discs represent that the bacteria is positive ESBL, synergy of third generation cephalosporins with combination of cephalosporin-clavulanic acid in the form of an expansion inhibition zone between the two discs. Positive results on ESBL-producing bacteria confirmed that there was increase in inhibition zone [greater than or equal to] 5 mm between diameter of cephalosporin disc and cephalosporin-clavulanate disc combination revealed an ESBL positive (CLSI, 2017).
The incidence of ESBL producing Escherichia coli from cloaca swabs on layer chicken was consistent with the incidence of Escherichia coli on slaughterhouses in Bogor by 8.6% (Sudarwanto et al., 2016), but smaller compared to the incidence of Escherichia coli as ESBL producing Escherichia coli from feces of broiler chickens in Bogor ESBL by 25% (Masruroh et al., 2016) and the incidence of ESBL producing Escherichia coli in India on layer chicken was around 42% (Brower et al., 2017).
Molecular identification as shown in Table 1 that 80% (8/10) samples of ESBL producing Escherichia coli encoding CTX gene. The CTX encoding gene is most commonly found in Escherichia coli. CTX enzymes have hydrophilic ability against cephalosporins, especially cefotaxime, so called CTX (Bradford, 2001). Molecular identification shown in Figure 3 that visualization of the CTX gene fragment band. Electrophoresis results of CTX gene represent samples describing the same fragments as positive controls with a gene length of 550 bp (Ali et al., 2016) as shown on Figure 3.
ESBL bacteria can be identified by detecting the presence of ESBL encoding genes (Bhoomika et al., 2016, Surgers et al., 2019). This research showed that the CTX gene was found in 80% ESBL samples. Other studies have been carried out mainly ESBL producing Kleibsiella pneumoniae (Hayati et al., 2019). Some investigations show that the dominant genotype found was CTX gene (Zarfel et al., 2014; Ibrahim and Hameed, 2015). This type of ESBL is often seen as single or combination. In this study, the ESBL encoding CTX gene was detected dominance of ESBL producing Escherichia coli samples from layer chicken. The CTX beta lactamase is the most prevalent ESBL type among chicken (96%) samples (Valentin et al, 2014). In many countries CTX gene is one of the most frequent ESBL types in ESBL-producing bacteria, causing human infections (Alonso et al., 2017), therefore the evidence of CTX gene in this study should be used as reference in controlling the spread of ESBL encoding gene in poultry farms (Wibisono et al., 2020b).
The spreading of genetic elements such as transposons, insertion and integrons in the bacteria cause ESBL genes move quickly from animals to humans or vice versa. Genetic factors can also spread the virus nature of resistance to other bacteria in animals digestive tract. The bacteria then spread from cage to the surrounding environment through facilitated waste by poor hygiene and sanitation, which pollutes land and water around agriculture. ESBL bacteria are also detected in vegetables, soil and surrounding water agriculture and markets (Wu et al., 2016).
The presence of ESBL producing Escherichia coli is threat to the public health and animal health (Kristianingtyas et al., 2020). This condition can occur in limited maintenance options. The steps that can be done is to build supervision program, supervising feed and poultry. Farmers also need to improve biosecurity practice. Garbage and laying chicken manure must be correct managed in an intensive production system, to prevent air, soil and water contamination, as well negative consequences for human health (Thyagarajan et al., 2014).
Conclusion: One hundred and fifteen Escherichia coli samples were isolated from cloacal swabs layer chicken in layer farms Blitar, East Java, Indonesia. Ten E. coli were classified as ESBL bacteria. Through PCR testing, ESBL encoding gene of CTX gene was identified in eight samples. The presence of ESBL encoding gene in bacteria has potential to spread its resistance to the other bacteria in the gastrointestinal tract of layer chickens as well as in the poultry environment.
Conflict of interest: We certify that there is no conflict of interest with any financial, personal, or other relationships with other people or organization related to the material discussed in the manuscript.
Acknowledgements: The authors would like to thank the Rector of Airlangga University for providing HIBAH MANDAT research funds with grant numbers; 371/UN3.14/LT/2019 on the research title: PENANGGULANGAN VIRULENCE FACTOR ISOLAT GRAM NEGATIVE BACTERIA DARI SUMBER PANGAN ASAL HEWAN DENGAN PENGGUNAAN POLYMERASE CHAIN REACTION. This article is part of the research.
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|Author:||F. J. Wibisono, B. Sumiarto, T. Untari, M. H. Effendi, D. A. Permatasari and A. M. Witaningrum|
|Publication:||Journal of Animal and Plant Sciences|
|Date:||Jun 22, 2021|
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