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MOLECULAR IDENTIFICATION OF CTX GENE OF EXTENDED SPECTRUM BETA- LACTAMASES (ESBL) PRODUCING ESCHERICHIA COLI ON LAYER CHICKEN IN BLITAR, INDONESIA

Authors:

Abstract

Escherichia coli is one of the ESBL-producing bacteria responsible for the rise of antibiotic resistance. The most common type of ESBL-encoding gene is cefotaximase (CTX). The aim of study was to identify CTX-encoding gene among Escherichia coli on Layer Chicken in Blitar, Indonesia by using DNA-based techniques. To do this, a total of 130 swab cloacal samples were randomly collected from layer chicken in 4 districts of Blitar. Samples was initially cultured on MacConkey agar and Eosin Methyllen blue agar. Subsequently, the suspected isolates were identified in TSIA test and IMVIC biochemistry test. The method of Confirmation test ESBL-producing Escherichia coli was performed by Double Disc Synergy Test (DDST), and the final characterization of the isolates were conducted using VITEK® 2 Compact. Following that, the identified isolates were exposed to PCR amplification for the presence of CTX gene in ESBL-producing Escherichia coli, followed by the visualization of the amplicons in the electrophoresis. Overall, the results was showed that 80% of the ESBL-positive isolates contained CTX gene. In conclusion, this report the high frequency of CTX gene in ESBL producing Escherichia coli, and thereby posing a significant threat for the animal and human health.
Wibisono et al., The J. Anim. Plant Sci., 31 (4) 2021
954
MOLECULAR IDENTIFICATION OF CTX GENE OF EXTENDED SPECTRUM BETA-
LACTAMASES (ESBL) PRODUCING ESCHERICHIA COLI ON LAYER CHICKEN IN
BLITAR, INDONESIA
F. J. Wibisono1, B. Sumiarto2, T. Untari3, M. H. Effendi4*, D. A. Permatasari4 and A. M. Witaningrum4
1Doctoral Program in Veterinary Science, 2Department of Veterinary Public Health, Faculty of Veterinary Medicine,
3Department of Microbiology, Faculty of Veterinary Medicine, Gadjah Mada University, Yogyakarta, Indonesia;
4Department of Veterinary Public Health, Faculty of Veterinary Medicine, Airlangga University, Surabaya, Indonesia
Corresponding author’s email: mheffendi@yahoo.com
ABSTRACT
Escherichia coli is one of the ESBL-producing bacteria responsible for the rise of antibiotic resistance. The most common
type of ESBL-encoding gene is cefotaximase (CTX). The aim of study was to identify CTX-encoding gene among
Escherichia coli on Layer Chicken in Blitar, Indonesia by using DNA-based techniques. To do this, a total of 130 swab
cloacal samples were randomly collected from layer chicken in 4 districts of Blitar. Samples was initially cultured on
MacConkey agar and Eosin Methyllen blue agar. Subsequently, the suspected isolates were identified in TSIA test and
IMVIC biochemistry test. The method of Confirmation test ESBL-producing Escherichia coli was performed by Double
Disc Synergy Test (DDST), and the final characterization of the isolates were conducted using VITEK® 2 Compact.
Following that, the identified isolates were exposed to PCR amplification for the presence of CTX gene in ESBL-producing
Escherichia coli, followed by the visualization of the amplicons in the electrophoresis. Overall, the results was showed
that 80% of the ESBL-positive isolates contained CTX gene. In conclusion, this report the high frequency of CTX gene in
ESBL producing Escherichia coli, and thereby posing a significant threat for the animal and human health.
Key words: CTX gene; ESBL; Escherichia coli; Human health; PCR; VITEK®
https://doi.org/10.36899/JAPS.2021.4.0289
Published online December 15, 2020
INTRODUCTION
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 β- 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.
The Journal of Animal & Plant Sciences, 31(4): 2021, Page: 954-959
ISSN (print): 1018-7081; ISSN (online): 2309-8694
Wibisono et al., The J. Anim. Plant Sci., 31 (4) 2021
955
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 ºC, 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 VITEK® 2
compact using a VITEK® 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 VITEK® 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 30μg (Oxoid, England),
Cefotaxime 30μg (Oxoid, England), Ceftazidime 30μg
(Becton Dickinson, USA), and Aztreonam 30μg (Oxoid,
England).
Culture was incubated at 35-37 ºC 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 VITEK® 2 compact. The bacterial
suspension was homogenized and a bacterial turbidity of
0.50 to 0.63 Mc Farland was made using VITEK® 2
DensiCHEK (Biomerieux, 2017). Antimicrobial
susceptibility and phenotypic detection of ESBL
producers using AST N280 cards (bioMérieux, Marcy-
L'Étoile, 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 QIAamp® DNA kit (QIAGEN, Germany).
Escherichia coli ATCC35218 was used as positive
control standard for strains of ESBL-producing bacteria,
and Escherichia coli ATCC25922 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 94oC, 2 minutes; extended
denaturation 94oC, 1 minute; annealing 54 oC, 30 seconds;
extension 72oC), 45 seconds; extended extension 72 oC, 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).
RESULTS
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
VITEK® 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.
Subdistric
Location
Sample
size
No of ESBL positive strain
PCR
DDST
VITEK®2 compact
No of CTX gene positive
strain
Ponggok
40
-
-
-
Srengat
45
6
6
6
Talun
20
1
1
-
Kademanga
n
25
3
3
2
Total in
Blitar
130
10
(10/115;
8.69%)
10
(10/10; 100%)
8
(8/10; 80%)
Wibisono et al., The J. Anim. Plant Sci., 31 (4) 2021
956
Figure 1. Escherichia coli on MacConkey Agar (A) and on Eosin Methylen Blue Agar (B)
Figure 2. ESBL-producing Escherichia coli confirmation test with Double Disc Synergy Test (DDST) (A) positive
result (B) negative result. Note: ATM: Aztreonam, CAZ: Ceftasidime, AMC: Amoxycillin clavulanic, and
CTX: Cefotaxime.
Figure 3. Molecular identification CTX gene of ESBL-producing Escherichia coli by PCR on 550 bp.
Legend: Code sample 140 and 142 there were no CTX gene.
DISCUSSION
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,
A
B
A
B
CTX
CTX
CAZ
CAZ
AMC
AMC
ATM
ATM
Wibisono et al., The J. Anim. Plant Sci., 31 (4) 2021
957
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 ≥ 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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... Escherichia coli was isolated using selective eosin methylene blue agar (EMBA) media (Oxoid, England) and incubated at 37°C for 18-24 h (Putra et al. 2020). Identification of E. coli using Gram staining and biochemical tests IMVIC (Indol-motility, methyl red, Voges Proskauer, citrate) and TSIA (Triple sugar iron agar) Wibisono et al. 2021). ...
... The cultures were incubated at 35°C±2°C for 16-18 hours. The test was considered positive when there was synergy and an increase in zone diameter of 5 mm for any of the antimicrobial agents tested in combination with clavulanate (Guo et al. 2019;CLSI 2020;Wibisono et al. 2021). ...
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Yanestria SM, Dameanti FNAEP, Musayannah BG, Pratama JWA, Witaningrum AM, Effendi MH, Ugbo EN. 2022. Antibiotic resistance pattern of Extended-Spectrum β-Lactamase (ESBL) producing Escherichia coli isolated from broiler farm environment in Pasuruan district, Indonesia. Biodiversitas 23: 4460-4465. Escherichia coli is one of the bacteria that can be used as an indicator of environmental pollution. This bacterium has the ability to become an antimicrobial-resistant bacterium that impacts public health. The antimicrobial ability can develop into a bacterium that produces extended-spectrum β-lactamase (ESBL). The purpose of this study was to reveal the occurrence of extended-spectrum β-lactamase (ESBL) producing E. coli from a broiler farm environment in Pasuruan district and to describe the phenotypic pattern of E. coli producing ESBL that has been detected. A total of 175 samples were used in this study consisting of 115 samples of coop swabs and 65 samples of wastewater around the farm. The samples were isolated and identified to find E. coli by using different culture media viz. McConkey agar (MCA), eosin methylene blue agar (EMBA), Gram staining, indole test, methyl red Voges Proskauer (MR-VP), citrate, and triple sugar iron agar (TSIA). Detection of ESBL using the double disc synergy test (DDST) according to standard Clinical and Laboratory Standards Institute procedures and the VITEK®2 compact apparatus. The results of ESBL confirmation with DDST and VITEK®2 showed that 16 (9.14%) of the 175 environmental samples confirmed E. coli produced ESBL. The results of the VITEK®2 test also produced a phenotypic pattern of resistance properties of ESBL-producing E. coli and found 12 types of resistance patterns. The combination of "AM AMP ATM KZ CTX CRO CIP" and "AM AMP ATM KZ CTX CRO GM CIP SXT" are the 2 most common resistance patterns (18.75%), while the other 10 resistance patterns occur at the same level (6.25%). The data presented here confirmed the presence of ESBL-producing E. coli in the farm environment, which can contribute to the dissemination of MDR bacteria in the environment if not monitored. Therefore, the presence of ESBL-producing E. coli in Pasuruan is worrisome since it can lead to an impact on human health.
... Control and prevention of infectious diseases in animals can be done by giving antibiotics to infected birds (Schwarz et al. 2004). However, inappropriate administration of antibiotics can also cause bacteria to become resistant (Enne et al. 2014;Wibisono et al. 2021). ...
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Efendi MH, Faridah HD, Wibisono FM, Wibisono FJ, Nisa N, Fatimah F, Ugbo EN. 2022. Detection of virulence factor encoding genes on Escherichia coli isolated from broiler chicken in Blitar District, Indonesia. Biodiversitas 23: 3437-3442. Broiler chicken is a source of protein that is widely consumed by the public. However, broiler chicken production sometimes decreases due to infectious diseases such as colibacillosis caused by pathogenic Escherichia coli possessing virulence genes. Virulence factors function to facilitate colonization and invasion of host cells to cause disease. The presence of these virulence factors is encoded by various genes such as the increased serum survival gene and P fimbriae gene which plays a role in surface adhesion. The present study aims to detect the presence of virulence genes from extended-spectrum beta-lactamase (ESBL) producing E. coli isolated from broiler chickens in the Blitar District. A total of 110 cloacal swabs collected by systematic random sampling from broiler poultry farms in four different sub-districts were screened for ESBL-producing E. coli and virulence genes by phenotypic and molecular methods, respectively. Out of 110 E. coli recovered, 95 (86.4%) were observed to show a high level of resistance to the tested antibiotics, and 34 (35.7%) were ESBL-producers. Among ESBL producing E. coli isolates, 22 (73.5%) and 1 (2.9%) were found to have the iss and papC gene virulence factors, respectively using the polymerase chain reaction (PCR) method. The results of this study indicate that virulence genes can be found in E. coli from poultry farms. The iss gene is the most predominant virulence gene. The report of these virulence factors in E. coli isolated from broiler could impose a serious potential public health problem.
... After the emergence of CTX-M-type beta-lactamases which were reported as Kluyvera species, members of the Enterobacteraceae family intrinsically possessed unique genes on their chromosomes to encode CTX-M-like beta-lactamases such as KLUA-1, KLUA-2, KLUC-1 and KLUG-1. Kluyvera georgiana encoded an enzyme very similar (99%) to CTX-M-8 in the amino acid sequence (Ishii et al, 1995), which was first identified in human-isolated Enterobacteriaceae in Brazil (Poirel et al, 2002) and later found in poultry and chicken meat samples worldwide (Bonnet et al, 2000;Ferreira et al, 2014;Wibisono et al, 2021). Because the chromosome-like beta-lactamasemediated CTX-M genes of the Kluyvera species have little or no promoter activity at the top of the gene, they tend to be silent. ...
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The study was isolated Escherichia coli from cloacal swab of broiler chicken farms in Blitar area to investigate cases of Multidrug Resistance (MDR) Escherichia coli and their Extended Spectrum Beta-Lactamase (ESBL) producing Escherichia coli. This research was conducted on broiler chicken farms in Blitar district in June until August 2019. Samples using cloaca swabs on broiler chickens were 160 animals from 6 districts in Blitar district. Samples were taken at random and brought to the laboratory for isolation of Escherichia coli bacteria. Positive isolates of Escherichia coli were tested for antibiotic sensitivity and Escherichia coli resistant to beta lactam groups were then confirmed using the Double Disc Synergy Test (DDST) confirmation test, to confirm as ESBL-producing bacteria. The results showed the percentage of antibiotic resistance to Escherichia coli bacteria in broilers in Blitar District was 88.75% (Ampicillin), 78.75% (Streptomycin), 76.87% (Erithromycin), 50.63% (Tetracyclin) and 75% (sulphamethoxazole-trimethoprim). The incidence of Multi-Drug Resistant (MDR) Escherichia coli bacteria in Broiler chickens in Blitar District was 85.63%, and around 28.75% were Escherichia coli bacteria that produce Extended Spectrum Beta-Lactamase (ESBL). In conclusion, the high level of ESBL-producing Escherichia coli in broiler chicken cloaca swabs is a threat to public health and the environment and is an important concern to reduce the rate of its spread.
... In addition, Escherichia coli also has the potential to be the biggest cause of AMR and plays an important role in the acquisition and dissemination of the AMR mechanism because it produces the Extended Spectrum Beta Lactamase (ESBL) enzyme (Inderbinen et al 2020;Rahmahani et al 2020;. Escherichia coli causes antimicrobial resistance and often identified in veal calves (Jarrige et al. 2020;Widodo et al 2020;Putra et al 2020) and poultry Effendi et al 2021;Wibisono et al 2021). ...
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Escherichia coli is a commensal bacteria in the digestive tract of animals that is very influential on health. There has been a lot of research on Escherichia coli, but no studies have addressed the idea of publishing Escherichia coli in Veterinary Medicine which shows the big overview using data from all countries. This study aims to map the status of Escherichia coli publications in Veterinary Medicine which are published internationally and indexed by Scopus using bibliometric visualization. Bibliometric methods and research data analysis were implemented using analysis search results services from Scopus and VOSviewer applications. In this study, the details collected apply to 5836 documents published from 2011 to 2020. Research reveals that Kim, I. H., Universiteit Gent, the United States and Veterinary Microbiology are individual scientists, affiliated, country and sources of dissemination most active in Escherichia coli publications in Veterinary Medicine. There are six maps of collaborative research groups in Escherichia coli publications in Veterinary Medicine around the world. To identify a body of knowledge created from ten years of publication, this study builds a convergence axis grouping consisting of Escherichia coli studies: Immunology, Virulence Factors, Microbiology and Molecular Examination, and Animal abbreviated with the theme IVMA.
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Fanissa F, Effendi MH, Tyasningsih W, Ugbo EN. 2022. Multidrug-resistant Salmonella species from chicken meat sold at Surabaya Traditional Markets, Indonesia. Biodiversitas 23: 2823-2829. Salmonella species is one of the major infectious pathogens associated with poultry birds, which also has an impact on public health. Antimicrobials are used as growth promoters or treatment of diseases thus; this encourages the persistent spread of antimicrobial resistance (AMR). This study aimed to identify multidrug-resistant Salmonella species from traditional chicken meat sold at Surabaya markets. A total of 150 chicken meat swab samples were collected from five different traditional markets and identified using microbiological standard methods. The Kirby-Bauer disk diffusion method identified multidrug-resistant Salmonella species on five different antibiotics discs. A low prevalence of Salmonella species was detected with a frequency of 11.3% (17/150). Some of the Salmonella isolates showed strong resistance to trimethoprim-sulfamethoxazole, tetracycline, aztreonam, and chloramphenicol. Five (29.4%) out of seventeen were multidrug-resistant Salmonella species. The presence of multidrug-resistant Salmonella species in these traditional markets is worrisome, since, it can lead to an outbreak of Salmonellosis as a result of the consumption of contaminated chicken meat. Therefore, the detection of multidrug-resistant Salmonella species is significant in understanding their prevalence and development of AMR. More strong awareness education and monitoring programs are recommended to mitigate the persistent spread of antimicrobial resistance.
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Occurence of extended spectrum beta-lacta-mases (ESBL) in Escherichia coli (E. coli) from animal origin is a growing health concern of global signi cance. The objective of this study was to determine the occurrence of ESBL producing E. coli and the characteristics of their encoding genes from 115 rectal swab samples of dairy cows from Tululungagung and Surabaya farms. All samples were positive for Escherica coli as per indole test. To con rm the ESBL the Double Disc Synergy Test (DDST). Betalactam antibiotic disk namely Amoxicylyn-clavulanate, Ceftazidime and Cefotaxime were used for DDST. Molecular identi cation for bla CTX-M and bla TEM ESBL encoding genes was done by used Polymerase Chain Reaction. The Escherichia coli bacteria isolated from rectal swabs of dairy cows was 5.21% (6/115). PCR results showed that bla CTX-M gene was 6 ESBL isolates and bla TEM gene was 2 ESBL isolates. It can be concluded that dairy cows can be potential as reservoir for spreading ESBL isolates to human health.
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17 and Identifi cation of Aerobic Bacteria Detected From Sheep Infected With Pneumonia. Adv. Environ. Biol. 10(5): 214-219. Munoz MA, Welcome FL, Schukken YH and Zadoks RN (2007) Molecular epidemiology of two Klebsiella pneumoniae mastitis outbreaks on a dairy farm in New York State. J Clin Microbiol. 45: 3964-3971 Ohnishi M, Okatani AT, Harada K, Sawada T and Marumo K (2013) Genetic Characteristics of CTX-M-Type Extended-Spectrum-b-Lactamase (ESBL)-Producing Enterobacteriace-ae Involved in Mastitis Cases on Japanese Dairy Farms, 2007 to 2011. J Clin Microbiol. 51: 3117-3122. Santos MV and Fonseca, LFL. (2007) (Eds.) Estratégias para controle de mastite e melhoria da qualidade do leite. São Paulo: Manole, pp 314. Schukken YH, Bennett GJ, Zurakowski MJ, Sharkey HL and Rauch BJ (2011) Randomized clinical trial to evaluate the efficacy of a 5-day ceftiofur hydrochloride intramammary treatment on nonsevere gram-negative clinical mastitis. J Dairy Sci. 94: 6203-6215. Zadoks RN, Middleton JR, McDougall S, Katholm J and Schukken YH (2011) Molecular epidemiology of mastitis pathogens of dairy cattle and comparative relevance to humans. Abstract The purpose of this study was to identify the ESBL producing E. coli and the characteristics of the encoding genes. 84 rectal dog swabs from several animal clinics in Surabaya, Indonesia were taken for the study. All the 84 positive E. coli samples were con rmed by the Double Disc Synergy Test (DDST) method. Molecular identi cation of bla CTX-M and bla TEM ESBL encoding genes using mutiplex Polymerase Chain Reaction (PCR). ESBL-producing E. coli bacteria from rectal swabs were found to be 9.52% (8/84). The PCR results showed that the bla CTX-M gene consisted of 8 ESBL isolates and bla TEM gene as many as 1 ESBL isolate which have the potential as a reservoir in spreading antibiotic resistance to humans and animals.
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p>Antibiotika telah banyak digunakan sekarang ini. Pemakaian antibiotika yang berlebihan dan tidak sesuai dengan klinis dapat menyebabkan terjadinya resistensi terhadap antibiotika tersebut Salah satu antibiotika yang dipakai adalah antibiotika golongan beta-lactam yang bekerja menghambat dinding sel. Pemakaian antibiotika beta-lactam yang tidak sesuai dapat menyebabkan terjadi resistensi terhadap antibiotika tersebut. Resistensi terhadap beta-lactam dapar terjadi di berbagai tingkatan. Salah satu resistensi dapat terjadi adalah resistensi terhadap extendedspectrum broad lactamase (ESBL) Extended spectrum beta-lactamase adalah enzim yang mempunyai kemampuan dalam menghidrolisis antibiotika golongan penicillin, cephalosporin generasi satu, dua, dan tiga serta golongan monobactam dan menyebabkan resistensi ke seluruh antibiotika tersebut. ESBL banyak dihasilkan oleh Enterobactericeae (terutama Escherichia coli ) dan Klebsiella pneumoniae. Enterobacteriacea e mempunyai 3 pola resistensi yang disebabkan b road spectrum beta-lactamase,inhibitor resistant beta-lactamase (derivat TEM) , Cephalosporinase yang berlebihan. ESBL dapat sulit terdeteksi karena ESBL mempunyai perbedaan tingkatan aktifitas terhadap bermacam-macam cephalosporin ESBL dapat dideteksi secara clinical microbiology (phenotypic ) dan molecular detection (genotypic). Keyword s : Antiobiotika, resistensi, ESBL</p
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Aim The study aimed to detect the invA gene in Salmonella isolated from milkfish in the Sidoarjo wet fish market. Materials and Methods A total of 84 samples were prepared in enrichment media and isolated on the surface of Salmonella Shigella Agar. Salmonella growth produces transparent colonies with blackish color in the middle due to H2S gas formation. Samples were identified as Salmonella based on macroscopic colony morphology. Presumptive Salmonella sp. was put on Bismuth Sulfite Agar media. Salmonella was determined based on the results of the biochemical test that has been carried out using Microbact identification kits from negative gram staining. Results The results of this study indicate that 32 of 84 samples (38.09%) were Salmonella bacteria. Furthermore, the invA gene detection was carried out using the polymerase chain reaction technique. Electrophoresis results showed four positive samples contained invA gene with a length of 284 bp. Conclusion Results in this study indicate that contamination of milkfish with Salmonella needs strict hygienic measures to prevent their transmission to human.
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This study aims to calculate the estimated economic losses in national poultry farming in Indonesia that are infected with pathogenic Escherichia coli. Poor management of chicken preservation is a major predisposing factor. Escherichia coli is a normal flora found in the gastrointestinal tract of chicken, but when the chicken stress or decrease immune system, Escherichia coli develops into a pathogenic agent. Pathogenic Escherichia coli appears as secondary infections that aggravate other disease infection. Cost of illness approach was divided into two main categories namely direct losses from disease and indirect losses from other related costs. Direct losses in broiler farms that were infected with pathogenic Escherichia coli through calculation of weight loss of harvest and total mortality, while in layer farms that were infected with pathogenic Escherichia coli, direct loss calculations included decreased chicken egg production and total mortality. Indirect losses on broiler and layer farms included calculation of other expenditure costs at the time of the occurrence of pathogenic Escherichia coli infections such as cleaning, disinfection and labor compensation costs. Based on the total calculation results obtained that the estimated economic losses incurred on national scale broiler farms reached IDR 14,167,792,041,150, - per harvest period of broiler, while estimated total loss of layer farms on national scale based on the calculation results reached IDR 13,391,996,617,850, - per month. The overall total loss due to this colibacillosis reached 13.10% of total poultry assets in Indonesia. The large proportion of losses incurred to total livestock assets can indicate how important the disease is to be controlled or overcome.
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Objective: To genetically characterize clusters of virulence factors (VFs) among extended spectrum β-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae and assess whether these clusters are associated with genetic determinants or clinical outcomes. Methods: One hundred forty-eight E. coli and 82 K. pneumoniae clinical isolates were obtained from 213 patients in Paris, France. Isolates underwent ESBL characterization, MultiLocus Sequence Typing (MLST) typing and phylogenetic group identification. Detection of ten E. coli and seven K. pneumoniae VF-encoding genes were assessed, from which a k-medians partition algorithm with Jaccard similarity measure was used to construct clusters. Results: CTX-M was the predominant ESBL and susceptibility to trimethoprim-sulfamethoxazole (32%), ciprofloxacin (22%) and aminoglycosides (32%) was low. In E. coli, there were five identified clusters, with significantly different distributions of ESBL-sequence type (P<0.001), ST131 (P<0.001) and phylogenetic group (P=0.001) between clusters. "Siderophore exclusive", "siderophore exclusive with iroN " and "adhesin sfa/papGIII-rich" clusters had higher 12-month mortality rates compared to others (49% vs 22%, respectively, P=0.02). In K. pneumoniae, three different clusters, with significantly different distributions of aminoglycoside-sensitivity (P<0.004), MLST-type (P<0.001) and relaxase plasmids (P=0.001) were described. Conclusion: Distinct clusters of E. coli and K. pneumoniae VFs are observed within ESBL-producing isolates and are strongly associated with several genetic determinants. Their association with overall morbidity and mortality requires further evidence.
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Klebsiella pneumoniae is one of 9 bacteria resistance to antibiotics in concern. This research aimed to detect any gene of blaTEM in bacteria of the K. pneumoniae isolated from swab of food-producing animals. In this study, 195 swab samples were taken from 17 sampling locations. Samples obtained were cultivated on selective medium and had several tests including identification, antibiotic sensitivity test using Kirby-Bauer method against antibiotics of ampicillin, cefotaxime, amoxicillin, meropenem, and trimetrophrim-sulfamethoxazole, and followed by PCR test for detecting the gene that was responsible for the antibiotic resistances. The results showed that 10 out of 195 samples were found to be K. pneumonia, those were 4 samples originated from dairy cows (SP-S1, SP-S3, SP-B2, SP-G4), 2 samples originated from beef (SPT-K1, SPT-K2), 1 sample originated from chickens (A-W5), and 3 samples originated from fish (IN-P2, IN-P3, IN-S3). Most of isolates (9/10) were found to be resistant toward amoxicillin. These isolates were SP-S3, SP-B2, SP-G4, SPT-KI, SPT-K2, A-W5, IN-P2, IN-P3, and the IN-S3 and all of them also showed to be positive of blaTEM gene. It could be concluded that most of K. pneumoniae isolates from food animals harbour had Extended Spectrum Beta-Lactamase (ESBL) encoding gene.
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The purpose of this research was to identify the presence of shiga toxin-producing Escherichia coli (STEC) in raw milk samples in Surabaya dairy cows using Multiplex Polymerase Chain Reaction (MPCR) assay. Approximately 10 ml milk samples from 75 apparently healthy Holstein Friesian cows from Surabaya, Indonesia were analyzed. Milk samples were inoculated with brilliant green bile broth (BGBB), subcultured in eosin methylene blue agar (EMBA) and were confirmed biochemically using Indol test. Multiplex PCR using primer flicH7 and primer stx2 (gene coding shiga toxin) were then performed. Results showed that 26 out of 75 samples were E. coli in bacterial isolation and MPCR. Moreover, two samples (7.7%) were positive for stx2 gene. The MPCR assay described in the present study can be employed to identify and screen for E. coli harboring stx2 gene in raw milk from dairy cows in Indonesia. © University of the Philippines at Los Banos. All rights reserved.
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Objective: To determine the multidrug resistance extended spectrum β-lactamase and AmpC (ESBL/AmpC producing) Escherichia coli (E. coli) isolated from the environment of Bogor slaughterhouse, Indonesia. Methods: A total of 35 samples from 7 locations in slaughterhouse i.e., source of water, slaughtering floor, swab of carcass area floor, swab of evisceration area floor, untreated waste water, treated waste water, drinking water for cattle were collected from March to April 2016. Presence of ESBL/AmpC producing E. coli and susceptibility testing against 8 antimicrobial agents (penicillin G, streptomycin, gentamycin, ciprofloxacin, enrofloxacin, tetracycline, trimethoprim-sulfamethoxazole, and polymyxin B) were detected by disk diffusion test according to Clinical and Laboratory Standards Institute. Results: ESBL/AmpC producing E. coli were identified in 14.3% (5/35) of the collected samples from the environment of Bogor slaughterhouse. ESBL/AmpC-producing E. coli isolates were detected in untreated waste water (n = 3), slaughtering floor (n = 1), and carcass area floor (n = 1). Most of ESBL/AmpC-producing E. coli isolates (80%) showed multidrug resistance phenotypes against at least three classes of antibiotics. The highest incidence of antibiotics resistance was against penicillin G (100.0%) and streptomycin (100.0%), followed by gentamicin (60.0%), trimethoprim-sulfamethoxazole (60.0%), tetracycline (40.0%), ciprofloxacin (40.0%), enrofloxacin (20.0%), and polymyxin B (0.0%). Conclusions: The transmission of antimicrobial resistant bacteria into the environment may be a potential risk for human health.
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Background Agricultural use of antimicrobials in subtherapeutic concentrations is increasing in response to the rising demand for food animal products worldwide. In India, the use of antimicrobials in food animal production is unregulated. Research suggests that many clinically important antimicrobials are used indiscriminately. This is the largest study to date in India that surveys poultry production to test for antimicrobial resistance and the occurrence of extended-spectrum β-lactamases (ESBLs) modulated by farming and managerial practices. Objectives Our goal was to survey poultry production for resistance to eleven clinically relevant antimicrobials and phenotypic occurrence of ESBLs as modulated by farming and managerial practices. Methods Eighteen poultry farms from Punjab were surveyed, and 1,556 Escherichia coli isolates from 530 birds were tested for susceptibility to 11 antimicrobials using the disk diffusion method and validated using VITEK 2 (bioMérieux, Marcy-L’Étoile, France). Samples from 510 of these birds were phenotypically tested for ESBL production using the combination disk method and confirmed using VITEK 2. Generalized linear mixed models were used to infer differences in resistance profiles associated with different farming practices and facility types. Results Resistance profiles were significantly different between broiler and layer farms. Broiler farms were 2.2 [ampicillin (AMP), p=0.017] to 23 [nalidixic acid (NX), p<0.001] times more likely to harbor resistant E. coli strains than layer farms. Adjusting for farm type (broiler vs. layer), the odds of resistance (although not statistically significant) to all antimicrobials except nitrofurantoin (NIT) were higher in independent facilities (IUs) as compared to contracted facilities (CFs). Increased prevalence of multidrug resistance (MDR; 94% compared to 60% in layers), including prevalence of ESBL-producing strains (87% compared to 42% in layers), was observed in broiler farms. Conclusions Our findings suggest that unregulated use of clinically relevant antimicrobials in Indian broiler and layer farms may contribute to the emergence of resistance and support the need to curb the nontherapeutic use of medically important antimicrobials in food animal production. https://doi.org/10.1289/EHP292