Whole genome sequencing analysis on antibiotic-resistant Escherichia coli isolated from pig farms in Banten Province, Indonesia.

IMPORTANCE: The emergence and rapid increase in the incidence of multidrug-resistant (MDR) bacteria in pig farms has become a serious concern and reduced the choice of effective antibiotics. OBJECTIVE: This study analyzed the phylogenetics and diversity of antibiotic resistance genes (ARGs) and molecularly identified the source of ARGs in antibiotic-resistant Escherichia coli isolated from pig farms in Banten Province, Indonesia. METHODS: Forty-four antibiotic-resistant E. coli isolates from fecal samples from 44 pig farms in Banten Province, Indonesia, were used as samples. The samples were categorized into 14 clusters. Sequencing was performed using the Oxford Nanopore Technologies MinION platform, with barcoding before sequencing with Nanopore Rapid sequencing gDNA-barcoding (SQK-RBK110.96) according to manufacturing procedures. ARG detection was conducted using ResFinder, and the plasmid replicon was determined using PlasmidFinder. RESULTS: Three phylogenetic leaves of E. coli were identified in the pig farming cluster in Banten Province. The E. coli isolates exhibited potential resistance to nine classes of antibiotics. Fifty-one ARGs were identified across all isolates, with each cluster carrying a minimum of 10 ARGs. The ant(3'')-Ia and qnrS1 genes were present in all isolates. ARGs in the E. coli pig farming cluster originated mainly from plasmids, accounting for an average of 89.4%. CONCLUSIONS AND RELEVANCE: The elevated potential for MDR events, coupled with the dominance of ARGs originating from plasmids, increases the risk of ARG spread among bacterial populations in animals, humans, and the environment.

Detection of tetracycline resistance genes and their diversity in Escherichia coli isolated from pig farm waste in Banten province, Indonesia.

Background and Aim: Livestock waste in the form of feces and liquid represents an important reservoir of antibiotic resistance genes (ARGs). Because many ARGs can be horizontally transferred to other pathogens, livestock waste plays an essential role in the emergence and transmission of various ARGs in the environment. Therefore, this study aimed to detect and assess the diversity of tet genes in Escherichia coli isolated from pig farm waste in Banten province, Indonesia. Materials and Methods: Solid waste (feces) and wastewater were collected from 44 pig farms in Banten province. The isolation and identification of E. coli referred to the Global Tricycle Surveillance extended-spectrum beta-lactamase E. coli World Health Organization (2021) guidelines. tet genes were detected using quantitative real-time polymerase chain reaction after dividing pig farms in the province into four clusters based on their adjacent areas and characteristics. Results: tetA, tetB, tetC, tetM, tetO, and tetX were detected in solid waste and wastewater from pig farms, whereas tetE was not detected in either sample type. tetX (100%) and tetO (75%) were the most dominant genes in solid waste, whereas wastewater samples were dominated by tetA, tetM, tetO, and tetX (prevalence of 50% each). Furthermore, eight tet gene patterns were found in pig farm waste (prevalence of 12.5% each). Conclusion: The results showed a high prevalence of tetO and tetX in solid waste and wastewater from pig farms in Banten province. This significant prevalence and diversity indicated the transmission of tet genes from pigs to the environment, posing a serious threat to public health.

Distribution analysis of tetracycline resistance genes in Escherichia coli isolated from floor surface and effluent of pig slaughterhouses in Banten Province, Indonesia.

Background and Aim: Slaughterhouses and their effluents could serve as a "hotspot" for the occurrence and distribution of antibiotic-resistant bacteria in the environment. This study aimed to understand the distribution of tetracycline resistance genes in Escherichia coli isolated from the floor surface and effluent samples of pig slaughterhouses in Banten Province, Indonesia. Materials and Methods: Ten samples, each from floor surface swabs and effluents, were collected from 10 pig slaughterhouses in Banten Province. Escherichia coli strains were isolated and identified by referring to the protocol of the Global Tricycle Surveillance extended-spectrum beta-lactamase E. coli from the WHO (2021). Quantitative real-time polymerase chain reaction (qPCR) was used to detect the tet genes. Results: The tetA, tetB, tetC, tetM, tetO, and tetX genes were distributed in the isolates from the floor surface samples, and the tetA, tetC, tetE, tetM, tetO, and tetX genes were distributed in the isolates from the effluent samples. The tetO gene (60%) was the most dominant gene in the isolates from floor surface samples, while the tetA gene was the dominant one in the isolates from the effluent samples (50%). The tetA + tetO gene combination was the dominant pattern (15%) in the E. coli isolates. Conclusion: The high prevalence and diversity of the tet genes in floor surface and effluent samples from pig slaughterhouses in Banten Province indicated that the transmission of the tet genes had occurred from pigs to the environment; thus, this situation should be considered a serious threat to public health.

A preliminary metagenomics study of bacteria present in the dirt of Swiftlet farmhouses based on nitrite levels in edible bird's nest on Sumatera Island, Indonesia.

Background and Aim: Since the past decade, metagenomics has been used to evaluate sequenced deoxyribonucleic acid of all microorganisms in several types of research. Nitrite contamination originates from the natural environment in Swiftlet farmhouses (SFHs) and can influence nitrite levels in edible bird's nest (EBN). It is strongly speculated that the conversion process into nitrite is influenced by the bacteria present in SFHs. Nitrite can cause adverse effects on human health. The previous research has focused on the characteristics of bacteria that may influence the nitrite conversion process in SFHs. This study aimed to a metagenomics analysis of bacteria present in the dirt of SFHs and evaluated nitrite levels in EBN on Sumatera Island. Materials and Methods: In total, 18 SFHs on Sumatera Island were selected, and EBN and dirt samples were collected from each SFH, resulting in 18 EBN and 18 dirt SFH samples. Raw uncleaned white EBN and dirt from three areas of SFH were collected. The samples were analyzed for nitrite levels using a spectrophotometer, and the metagenomics sequencing of SFH dirt samples was performed using the MinIon nanopore method. The sequenced data were analyzed using the EPI2ME software. Results: Of the 18 raw uncleaned white EBN samples, 9 (50%) had <30 ppm nitrite levels. The top five bacterial genera in SFH dirt samples in Group A (nitrite levels >30 ppm) were Aeromonas, Escherichia, Acinetobacter, Arcobacter, and Acetoanaerobium. Those in Group B (nitrite levels <30 ppm) were Aeromonas, Pseudomonas, Shewanella, Escherichia, and Acinetobacter. There were 12 genera of nitrifying bacteria in Group A and 8 in Group B. The total cumulative read of nitrifying bacteria in Groups A and B were 87 and 38 reads, respectively. Conclusion: This is the first study to show that characteristic bacteria present in the dirt of SFHs might significantly influence the conversion from nitrogen to nitrite. Approximately 50% of raw uncleaned EBN samples had <30 ppm nitrite levels. Aeromonas was the most dominant bacterial genus found in Groups A and B. The variations in genus and cumulative reads nitrifying bacteria in group A were greater than those in Group B. This study provides information on the characteristics of bacteria that may influence the nitrite conversion process in SFHs. Metagenomics data were obtained from the reading using the software EPI2ME. Further research is needed on the bacterial target species that can convert nitrite in SFHs.

An investigation of heavy metals in edible bird's nest from Indonesia using inductively coupled plasma mass spectrometry.

Background and Aim: In 2020, Indonesia, which has the highest global production of edible bird's nest (EBNs), exported up to 1312.5 tons of this product at a value of USD 540.4 million. Recently, food safety aspects related to EBNs, including contamination with heavy metals, have become a serious concern. However, data on the presence and concentration of heavy metals in EBNs in Indonesia are not yet available. This study aimed to determine and compare the presence and concentrations of arsenic (As), mercury (Hg), lead (Pb), cadmium (Cd), and tin (Sn) in EBNs originating from several primary Indonesian islands. The study also analyzed the effect of washing on the heavy metal content in EBNs. Materials and Methods: A study on 44 swiftlet farmhouses (SFHs) was conducted to determine the concentrations of heavy metals in EBNs. The number of samples from the SFHs was allocated proportionally to the main EBN-producing islands in Indonesia, that is, Kalimantan, Sumatera, Sulawesi, and Java (22, 13, 7, and 2, respectively). The concentrations of the above five elements in the samples before washing (raw-unclean EBNs) and after washing (raw-clean EBNs) were determined by inductively coupled plasma mass spectrometry. Washing was conducted according to the general procedures at an EBN processing plant. Results: The raw-unclean EBNs from the four islands contained As, Pb, Cd, and Sn at varying concentrations. However, Hg was not detected in the raw-unclean EBN samples from Sulawesi. The raw-unclean EBNs from Kalimantan had lower concentrations of Pb and Cd compared with the other islands. The concentrations of As, Pb, Cd, and Sn in the EBNs decreased significantly after washing with clean water. Conclusion: Heavy metals (As, Hg, Pb, Cd, and Sn) were detected at a low level in most of the raw-unclean EBNs originating from the main Indonesian island where they were produced. The concentrations of all the heavy metals reviewed in the raw-unclean EBNs samples decreased significantly after washing.

Identification and differentiation of Campylobacter isolated from chicken meat using real-time polymerase chain reaction and high resolution melting analysis of hipO and glyA genes.

BACKGROUND AND AIM: Campylobacter species have been recognized as the most frequently identified bacterial cause of human gastroenteritis. The aims of this study were to identify Campylobacter jejuni and Campylobacter coli species isolated from chicken meat and to analyze the differences in the melting curve patterns of both species. MATERIALS AND METHODS: A total of 105 chicken meat samples collected from slaughterhouses and retailers in six provinces in Indonesia were examined for the isolation and identification of Campylobacter spp. A total of 56 positive isolates of Campylobacter spp. were analyzed using the quantitative real-time polymerase chain reaction and high resolution melting method. RESULTS: The prevalence of Campylobacter spp. in chicken meat was found to be 61.9%. Regarding the identification, 23 isolates (41.07%) were C. jejuni, 22 (39.29%) were C. coli, six (10.71%) were a mix between C. jejuni and C. coli, and five isolates (8.93%) were Campylobacter spp. All the C. jejuni and C. coli isolates produced varied melting curve patterns. CONCLUSION: The high prevalence of C. jejuni and C. coli in chicken meat in Indonesia indicates a high risk of the incidence of campylobacteriosis in humans.

Sumateran wild boar (Sus scrofa vittatus) meat antibody production as immunodiagnostic reagent candidate.

AIM: Meat authentication gives significance values in view of religious, food safety, public health, quality assurance, and legal concern. Most of the meat authentication is based on molecular assay; a simpler method to authenticate meat is needed to develop. An immunoassays technique may offer a solution for simpler test. The aim of our current study was to develop a polyclonal antibody of Sus scrofa vittatus (Sumateran wild boar) as an immunodiagnostic reagent candidate. MATERIALS AND METHODS: Three male New Zealand white rabbits were used in this study for antibody production. Antigen used was meat extract of Sumateran wild boar, each rabbit was immunized with meat extract antigen (0.5 mg/ml) emulsified in Freund's complete adjuvant at a 1:1 (v/v) ratio as much as 1 ml at subcutaneous route. Booster was carried out 3 times with interval time of 14 days, using meat extract antigen emulsified in Freund's incomplete adjuvant at a 1:1 (v/v) ratio. Serum samples were taken every week, start from 1 week after the first immunization up to 1 week after the third booster. Antibody purification was performed using ammonium sulfate precipitation and Protein A. The presence of specific antibody was determined using agar gel precipitation test and enzyme-linked immunosorbent assay, while purified specific IgG was characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis method. RESULTS: Specific antibody was detected at 14 days after the first immunization and still detected until 2 weeks after the third booster. Highest absorbance of specific antibody was detected 1 week after the third booster. CONCLUSION: The present study demonstrated that specific antibody of Sumateran wild boar is favorable to be produced in rabbit and showed that antibody produced is applicable to detect Sumateran wild boar meat antigen in immunodiffusion assay, indicating that it is promising as a reagent candidate in immunodiagnostic assay/kit.

Multidrug resistance extended spectrum β-lactamase and AmpC producing Escherichia coli isolated from the environment of Bogor Slaughterhouse, Indonesia

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, enro-floxacin, 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.

CTX-M producing Escherichia coli isolated from cattle feces in Bogor slaughterhouse, Indonesia

Objective: To determine the occurrence of CTX-M producing Escherichia coli (E. coli) from cattle feces in Bogor slaughterhouse, Indonesia. Methods: A total of 220 cattle feces samples were collected from Bogor slaughterhouse from March to April 2015. Presence of extended-spectrum beta-lactamase (ESBL) producing E. coli was detected by disc diffusion test based on the recommendation from Clinical and Laboratory Standards Institute (2014). Bacterial strains which were confirmed as producing ESBLs were further analyzed for the presence of bla genes of the ESBL by PCR. Results: The results showed that CTX-M producing E. coli isolates were detected in 19 samples from 220 samples (8.6%). The b-lactamase genes detected were CTX-M-1 (n = 10) and CTX-M-9 (n = 9). All of the CTX-M producing E. coli isolates showed multidrug resistance phenotypes to at least four antibiotics. The highest incidence of an-tibiotics resistance was showed to ampicillin (100.0%), cefotaxime (100.0%), and cef-podoxime (100.0%), followed by streptomycin (84.3%), trimethoprim-sulfamethoxazole (73.7%), erythromycin (52.6%), kanamycin (26.3%), doxycycline (10.5%), and ceftazi-dime (0.0%). Conclusions: Detection of CTX-M-producing E. coli in cattle feces raises important questions as they can represent a potential risk factor to public health.

Immunochemical analysis of fumigaclavine mycotoxins in respiratory tissues and in blood serum of birds with confirmed aspergillosis.

The ergoline alkaloid fumigaclavine A (FuA) is one of the major mycotoxins produced by Aspergillus fumigatus, the main causative fungal agent of avian aspergillosis. To study in situ production of FuA, post-mortem respiratory tissues of various avian species, as well as blood samples of falcons (Falco sp.), were analysed by enzyme immunoassay (EIA). At a detection limit of 1.5 ng/ml, FuA EIA positive results were obtained for tissue samples from seven (64%) out of 11 birds with confirmed aspergillosis, with a maximum concentration of 38 ng/g, while all controls (n = 7) were negative. No FuA could be detected in blood serum (detection limit 0.7 ng/ml) of 15 falcons, experimentally inoculated with A. fumigatus conidia. Fungal mycelium material from tissue of clinical aspergillosis cases, cultured on malt extract agar, was highly positive in the FuA EIA in milligrams per gram range. Chromatographic analysis of mycelium extracts revealed the co-presence of FuA and the structurally related fumigaclavine C (FuC). Alkaline hydrolysis of FuA and FuC yielded the corresponding deacetylation products, FuB and FuE. This is the first report showing that fumigaclavine alkaloids are produced by A. fumigatus in situ during the course of clinical aspergillosis in birds; however, the role of these compounds in the pathogenesis of this disease is still unknown.

Enzyme immunoassay for tenuazonic acid in apple and tomato products.

The Alternaria mycotoxin tenuazonic acid was derivatized with succinic anhydride and conjugated to keyhole limpet hemocyanin (KLH) and to horseradish peroxidase (HRP), respectively. The KLH conjugate was used to produce polyclonal antibodies in rabbits. A competitive direct enzyme immunoassay (EIA) for tenuazonic acid was established, which was moderately sensitive for tenuazonic acid [50% inhibition concentration (IC(50)): 320 ± 130 ng/mL] but strongly reacted with tenuazonic acid acetate (IC(50): 23.3 ± 7.5 ng/mL). Therefore, an optimized EIA protocol was established, which employed acetylation of standard and sample extract solutions. The mean standard curve detection limit (IC(30)) for tenuazonic acid acetate was 5.4 ± 2.0 ng/mL, enabling detection limits for tenuazonic acid in apple and tomato products of 25-50 ng/g (150 ng/g in tomato paste). Recoveries in a concentration range of 50-2000 ng/g were 60-130% in apple juice and tomato juice and 40-150% in other tomato products. Tenuazonic acid was detected in apple juice and tomato products from German retail shops at levels of 50-200 ng/g. In conclusion, this novel EIA for tenuazonic acid could be useful within a screening program for Alternaria mycotoxins in food.

Widespread occurrence of low levels of alternariol in apple and tomato products, as determined by comparative immunochemical assessment using monoclonal and polyclonal antibodies.

This study investigated the production of polyclonal (pAB) antibodies and the first time production of monoclonal (mAB) antibodies against the mycotoxin alternariol, and their implementation in enzyme immunoassay (EIA) for the rapid determination of alternariol in foods. Both EIAs were highly sensitive, with detection limits (IC20) of 35 ± 6.9 pg/mL (mAb EIA) and 59 ± 16 pg/mL (pAb EIA). Food products (n = 109; apple and tomato products, white wine) from German retail shops were analyzed. At a detection limit of 1-2 µg/kg, alternariol at 1-13 µg/kg was found with high frequency in apple (67%) and tomato (93%) products. Tomatoes with visible signs of Alternaria infection, stored at room temperature for up to 4 weeks, contained alternariol at levels up to 50 mg/kg, as determined by EIA and HPLC-FLD. It is concluded that the alternariol immunoassays present a versatile screening tool which could facilitate food control for Alternaria toxins.

Production and characterization of antibodies against fumigaclavine A.

Polyclonal antibodies against fumigaclavine A (FuA) were obtained from rabbits after immunization with a FuA-keyhole limpet hemocyanine conjugate prepared by formaldehyde condensation. Using these antibodies and a FuA-bovine serum albumine conjugate, a competitive indirect enzyme immunoassay (EIA) was established. The antiserum obtained from one rabbit enabled highly sensitive detection of FuA, with an IC50 level and detection limit of the standard curve of 3.3 ng/ml and approx. 1 ng/ml, respectively. The EIA was very specific for FuA, with 1.3% cross-reactivity with FuB. Several other lysergic acid derivatives (ergonovine, ergotamine, alpha-ergocryptine) were tested but did not cross-react in the FuA EIA. This is the first description of antibodies against FuA and the first development of an EIA for FuA.