INVESTIGATION ON ANTIMICROBIAL RESISTANCE RATES IN COMMENSAL ESCHERICHIA COLI ISOLATES FROM BROILERS ORIGINATING FROM BULGARIA (2020-2024)

Prevalence and genetic basis of tetracycline resistance in Vibrioparahaemolyticus isolates recovered from food products in Shenzhen, China during 2013 to 2021

Tetracycline resistance is one of the most frequently encountered resistance properties in bacteria of animal origin. The aim of the present study was to investigate the prevalence and diversity of tetracycline resistance (tet) genes among Escherichia coli clinical isolates from diseased ducks in China and to report the identification and sequencing of the tet(M) gene. The susceptibility of 85 Escherichia coli strains to tetracyclines was determined by broth microdilution, and the presence of tet genes was investigated by multiplex PCR. All of the 85 isolates were fully resistant to both oxytetracycline and tetracycline, and 76.5 % were resistant to doxycycline. Seventy-seven of the isolates (90.6 %) encoded multiple tet genes, with 17.6, 38.8 and 34.1 % encoding two, three and four tet genes, respectively, and only 7.1 % encoded a single tet(A) gene. The MICs of oxytetracycline and tetracycline for all isolates ranged from 16 to ≥128 µg ml(-1) with a MIC90 of >128 µg ml(-1), regardless of the type or number of tet genes encoded. Isolates containing tet(M) commonly had more than one tet gene per strain. The doxycycline resistance rate in the tet(M)-positive isolates was significantly higher than in the tet(M)-negative isolates (P<0.05). A full-length tet(M) gene, including the promoter region, was obtained by PCR in seven of the 41 tet(M)-positive isolates and was sequenced and cloned. The cloned tet(M) gene conferred resistance to tetracyclines in the recombinant Escherichia coli host strain. These results revealed that, in these isolates, the prevalence of multiple tet genes was strikingly high and that tet(M) played a role in doxycycline resistance.

Simple SummaryThe extensive use of tetracyclines in clinical practice and livestock has subjected bacterial populations to selection pressure and increased the prevalence of tetracycline resistance, one of the most abundant antibiotic resistances among pathogenic and commensal microorganisms. In the present survey, DNA extracted from cloacal swabs from 195 broiler chickens in Tunisia were molecularly tested for 14 tetracycline resistance genes. A high frequency and diversity of tetracycline resistance genes in the chickens sampled were detected. The results confirm the antimicrobial resistance urgency in Tunisia’s poultry sector and suggest that the investigation of antibiotic resistance genes directly in biological samples could be a useful means for epidemiological studies on the spread of the antimicrobial resistance.Tetracycline resistance is still considered one of the most abundant antibiotic resistances among pathogenic and commensal microorganisms. The aim of this study was to evaluate the prevalence of tetracycline resistance (tet) genes in broiler chickens in Tunisia, and this was done by PCR. Individual cloacal swabs from 195 broiler chickens were collected at two slaughterhouses in the governorate of Ben Arous (Grand Tunis, Tunisia). Chickens were from 7 farms and belonged to 13 lots consisting of 15 animals randomly selected. DNA was extracted and tested for 14 tet genes. All the lots examined were positive for at least 9 tet genes, with an average number of 11 tet genes per lot. Of the 195 animals tested, 194 (99%) were positive for one or more tet genes. Tet(L), tet(M) and tet(O) genes were found in 98% of the samples, followed by tet(A) in 90.2%, tet(K) in 88.7% and tet(Q) in 80%. These results confirm the antimicrobial resistance impact in the Tunisian poultry sector and suggest the urgent need to establish a robust national antimicrobial resistance monitoring plan. Furthermore, the molecular detection of antibiotic resistance genes directly in biological samples seems to be a useful means for epidemiological investigations of the spread of resistance determinants.

Escherichia coli affects human health through intestinal and extraintestinal infections. Avian pathogenic E. coli (APEC) contributes to colibacillosis in poultry and can develop public health risks. Antibiotic resistance and biofilm-producer strains are challenges in infection control options. This study aimed to characterize phenotypic and genotypic antibiotic resistance profiles as well as biofilm formation assay in E. coli isolates from clinical and poultry samples. In the study, 42 E. coli isolates were collected and confirmed from clinical and poultry sources. The isolates were evaluated for pathotypes using polymerase chain reaction (PCR). Antibiotic resistance was evaluated using the disk diffusion technique and minimum inhibitory concentration (MIC) tests. PCR was utilized to identify antimicrobial resistance genes associated with fluoroquinolones, sulphonamides, tetracyclines and beta-lactams. Biofilm formation was evaluated using a 96-well microtiter plate. Three clinical isolates, including enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC) and enterotoxigenic E. coli (ETEC), were identified as pathogenic strains. The highest rates of resistance were recorded against tylosin (100%), neomycin (92.85%), tetracycline (85.7%), ampicillin (73.8%), doxycycline (71.4%), ciprofloxacin (64.28%), trimethoprim/sulfamethoxazole (64.28%) and enrofloxacin (57.1%). The most prevalent resistance genes detected as blaTEM and gyrA/B (97.6% and 76.1%, respectively). The overall prevalence of blaCTX, sul1, sul2, tetA and tetB genes were 21.4%, 45.2%, 11.9%, 33.3% and 7.1%, respectively. The qnrB, qnrB4 and qnrS genes were absent in the clinical samples, whereas present in poultry isolates. All isolates were biofilm producers, and 96.4% of poultry isolates had strong biofilm formation capacity. The alarming levels of resistance genes and biofilm formation of isolates in the present study emphasize the need for antibiotic management practices and further research on resistance transmission dynamics in the food industry.

Anaerobic digestion is used for the treatment of animal manure by generating biogas. Heavy metals cause environmental pollutions and co-select for antimicrobial resistance. We evaluated the impact of mesophilic anaerobic digestion of cattle manure (CM), swine manure (SM) and poultry litter (PL) on the concentrations of seven tetracycline [tet(A), tet(B), tet(G), tet(M), tet(O), tet(Q), and tet(W)], macrolide [erm(B)], methicillin (mecA and mecC), copper (copB, pcoA, pcoD, and tcrB) and zinc (czrC) resistance genes, and three bacterial species (E. coli, Enterococcus spp. and Staphylococcus aureus). The total bacterial population and total abundance of the seven tet genes significantly increased in the three manure types after digestion. Concentration of tet(M) was strongly correlated with that of erm(B) and enterococci. As concentration of tetracyclines declined during anaerobic digestion, that of four tet genes (A, B, Q, and W) and 16S rRNA increased, that of tet(M) decreased, and that of tet(G) and tet(O) did not change. Concentrations of copB and pcoA did not change; while that of pcoD did not change in the PL, it increased in the SM and CM. While the concentration of enterococci remained unchanged in CM, it significantly increased in the PL and SM. Concentrations of tcrB significantly increased in the three manure types. While concentrations of S. aureus significantly increased in the CM and PL, that of SM was not affected. Concentrations of mecC significantly increased in all manure types after digestion; while mecA concentrations did not change in the SM, they significantly increased in CM and PL. While concentration of czrC remained low in the CM, it increased in the PL but declined in the SM. In conclusion, while mesophilic anaerobic digestion of animal manure decreased concentration of tetracyclines, it increased the concentrations of total bacteria, tet genes, E. coli, enterococci and S. aureus and methicillin resistance genes. It did not have any effect on concentrations of heavy metals; concentrations of heavy metal resistance genes either increased or remained unaffected depending on the animal species. This study showed the need for post-digestion treatments of animal manure to remove bacteria, antibiotic resistance genes, heavy metals and their resistance genes.

Simple SummaryCampylobacter spp. has been the leading cause of human diarrhea in EU since 2005. Although poultry and poultry meat are considered as the primary source of transmission of campylobacteriosis to humans, pigs can be a significant reservoir of the pathogen, as well. Moreover, the increase of antibiotic resistance in the specific pathogen, especially against fluroquinolones and macrolides is considered a significant threat for public health. The purpose of the current study was to evaluate and molecularly characterize the antimicrobial resistance of Campylobacter infection in pig farms in Greece at both phenotypic and molecular level.The purpose of this research was to characterize the antibiotic resistance patterns of Campylobacter spp. isolated from commercial farrow to finish farms in Greece, and analyze the relevant molecular resistance mechanisms among the resistant Campylobacter isolates. Susceptibility testing to five different classes of antibiotics was performed in 100 C. coli and 100 C. jejuni, previously isolated and identified. All isolates were found susceptible to meropenem. Very high rates of resistance were recorded for tetracyclines (84.5%), medium rates of resistance were recorded regarding quinolones (23%), and low and very low rates of resistance were identified for macrolides such as erythromycin and aminoglycosides (12% and 4%, respectively). Only 12.5% of the Campylobacter isolates displayed MDR. Regarding the molecular mechanisms of resistance, all ciprofloxacin resistant isolates hosted the mutant type Thr-86-Ile region of the quinolone resistance-determining region (QRDR) of the gyrA gene. In all erythromycin resistant isolates, the transitional mutations A2075G and A2074C in the 23S rRNA gene were only amplified. Molecular screening of tetracycline resistance genes indicated that the vast majority of Campylobacter isolates (92.3%) were positive for the tet(O) gene. In summary, these findings and especially the very high and medium rates of resistance for tetracyclines and fluroquinolones, respectively recommend that a continuous monitoring of Campylobacter isolates susceptibility in combination with the proper use of antimicrobials in livestock production is of great importance for public health.

Introduction: Multidrug resistance among Enterobacteriaceae is in increasing trend these days. The objective of this study was to determine the antibiogram of clinical isolates of Enterobacteriaceae with special reference to multidrug resistance and extended spectrum beta-lactamases production.Materials and Methods: A descriptive cross sectional study was conducted over a period of six months (February -July, 2017) in the microbiology laboratory of Nepal Medical College Teaching Hospital, Kathmandu, Nepal. A total of 936 bacterial isolates of Enterobacteriaceae from clinical specimens were processed for antimicrobial susceptibility testing and screened for multidrug resistance. ESBL production was detected among potential isolates by combination disk diffusion test.Results: The rate of multidrug resistance and extended spectrum beta-lactamases production was 54.2% and 23.8% respectively. Of the total ESBL producers 92.4% were multidrug resistance. The rate of multidrug resistance and extended spectrum beta-lactamases production were higher in organisms isolated from clinical samples collected from inpatients. High rate of multidrug resistance and extended spectrum beta-lactamases production was seen in E. coli (54.4% &amp; 27.7%), Klebsiella spp. (67.1% &amp; 28.2%) and Citrobacter spp. (70.3% &amp; 10.9%). The antimicrobial resistance rate was highest against ampicillin (76.7%) followed by cefixime (54. 0%), ceftazidime (51.5%), ceftriaxone (51.0%), cotrimoxazole (48.7%), ciprofloxacin (43.9%) and ofloxacin (41.1%).Conclusions: Multidrug resistance is common among Enterobacteriaceae. These bacteria have high rate of resistance against commonly used groups of antibiotics like cephalosporins and quinolones. Continuous monitoring, surveillance of antimicrobial resistance, proper infection control and practices are important to combat with these issues.

Research Note: Longitudinal monitoring of chicken houses in a commercial layer farm for antimicrobial resistance in Escherichia coli with special reference to plasmid-mediated quinolone resistance

Campylobacter species, especially C. coli and C. jejuni, have been associated with a range of human gastrointestinal diseases. During the last two decades, due to the irrational use of antibiotics in poultry farms, high rates of antimicrobial resistance have been globally reported in C. coli and C. jejuni isolates. Recently, acquired linezolid-resistance mechanisms have been reported in Campylobacter spp. isolates, which is a cause of concern to human health. In this study, we performed a retrospective analysis of 139 C. coli isolates previously collected from broilers (n = 41), laying hens (n = 53), eggs (n = 4), and environment (n = 41) to detect acquired genes implicated in linezolid resistance. Isolates were tested for their susceptibility to antimicrobial agents using the Kirby-Bauer disk diffusion assay. Chloramphenicol- and linezolid-resistant isolates were subjected to PCR screening for the following genes: fexA, fexB, floR, RE-cmeABC, cfrA, and optrA. The genetic relatedness of eight multidrug-resistant isolates was determined by multilocus sequence typing (MLST). Among the 139 C. coli isolates, high rates of resistance (57.55%-100%) were detected toward nalidixic acid, ciprofloxacin, erythromycin, azithromycin, ampicillin, chloramphenicol, linezolid, and kanamycin. Among 135 chloramphenicol-resistant isolates, the optrA, cfr, fexA floR, RE-cmeABC, and fexB genes were detected in 124 (124/135, 91.85%), 108 (80%), 105 (77.7%), 64 (47.4%), 56 (41, 48%), and 27 (20%) isolates, respectively. In addition, the majority of isolates harbored more than one of these genes. The selected eight isolates belonged to the same sequence type ST13450, which is a new sequence type (ST), not belonging to ST828 and ST1150 complexes. In conclusion, the emergence of optrA gene in Campylobacter spp. isolates makes this genus an optrA reservoir and vector to other pathogens such as Staphylococcus aureus and Enterococcus spp., which is a cause of concern for human and animal health.

Anaerobic bacteria insensitive to chlortetracycline (64 to 256 microg/ml) were isolated from cecal contents and cecal tissues of swine fed or not fed chlortetracycline. A nutritionally complex, rumen fluid-based medium was used for culturing the bacteria. Eight of 84 isolates from seven different animals were identified as Megasphaera elsdenii strains based on their large-coccus morphology, rapid growth on lactate, and 16S ribosomal DNA sequence similarities with M. elsdenii LC-1(T). All eight strains had tetracycline MICs of between 128 and 256 microg/ml. Based on PCR assays differentiating 14 tet classes, the strains gave a positive reaction for the tet(O) gene. By contrast, three ruminant M. elsdenii strains recovered from 30-year-old culture stocks had tetracycline MICs of 4 microg/ml and did not contain tet genes. The tet genes of two tetracycline-resistant M. elsdenii strains were amplified and cloned. Both genes bestowed tetracycline resistance (MIC = 32 to 64 microg/ml) on recombinant Escherichia coli strains. Sequence analysis revealed that the M. elsdenii genes represent two different mosaic genes formed by interclass (double-crossover) recombination events involving tet(O) and tet(W). One or the other genotype was present in each of the eight tetracycline-resistant M. elsdenii strains isolated in these studies. These findings suggest a role for commensal bacteria not only in the preservation and dissemination of antibiotic resistance in the intestinal tract but also in the evolution of resistance.

Antibiotic resistance in Escherichia coli is a global health concern. We studied all possible routes of cross contamination of broiler meat with resistant E. coli from broiler feces at poultry shops. Various sample categories namely poultry feces, meat (n=225 for each), slaughterer hands, consumer hands, slaughterer knife, canister, tap water, carcass, feed and drinking water (n=50 for each) were collected from local poultry processing market. Samples were screened for prevalence of E. coli, resistance of isolates against ten antibiotics and presence of tetracycline- resistance genes in the isolates. Fecal samples had greatest colony count (4.1×104 CFU/g) as compared to meat (1.9×104 CFU/g) samples. Samples of consumer hands (6%) and tap water (12%) had less prevalence percentages of E. coli as compared to slaughterer hands (92%) and drinking water of broiler (86%). Isolates of eight sample categories had high resistant rate (≥90%) against oxytetracycline. On average, about 94% of the isolates from various sample categories possessed multidrug-resistance (MDR). Tetracycline-resistance genes (tetA and tetB) were identified in all sample categories except isolates of consumer hands and tap water. The distribution of tetracycline-resistance genes was significantly greater in fecal isolates (42%) than meat isolates (25%). The study depicted the spread of resistant E. coli in broiler meat through all studied routes of contamination of slaughtering periphery. This problem can be mitigated by strict monitoring of antibiotics use at poultry farms, prevention of cross contamination by adopting hygienic slaughter and vigorously screening the market meat for resistant E. coli.

Antimicrobial resistance in pathogenic bacteria is a serious problem in public health. Antibiotic-resistant pathogens are the cause of many deaths. Escherichia coli (E. coli) is one of the bacteria that experienced multi-drug resistance (MDR). Infection of Escherichia coli in humans occurs through transmission of fecal-oral. This study, conducted at the Veterinary Public Health Laboratory of Gadjah Mada University, aimed to assess MDR E. coli prevalence in 200 chicken egg samples sourced from poultry farms and supermarkets, alongside 63 cloacal swab samples from broiler poultry in Sleman, Yogyakarta. The study focused on detecting resistance genes including tetA, aadA1, aph(3)IIa, and blaTEM1, also analyzing polymorphisms in the blaTEM1 gene associated with antibiotic resistance. Identification technique of E. coli positivity refers to the Indonesian National Standard (SNI) 2897:2008, then E. coli identification was performed using the Analytical Profile Index (API) Test 20E Kit. Antibiotic sensitivity was determined by the Kirby Bauer method. Detection of antibiotic resistance genes in E. coli were determine using Polymerase Chain Reaction (PCR) method. Sequencing and analysis of polymorphism and phylogenetic were performed only in blaTEM1. There were 12 samples identified as having E. coli (1 from chicken eggs and 11 from cloacal swabs), resistance percentages were highest for erythromycin (100%), ampicillin (91.7%), ciprofloxacin (91.7%), sulfamethoxazole (83.3%), streptomycin (83.3%) gentamicin (75%), tetracycline (41.7%), and chloramphenicol (25%). respectively. All of 12 E. coli samples were bacteria with MDR. Resistant genes were prevalent, notably blaTEM1 and aadA1 (100% each), with aph(3)IIa and tetA genes also detected in 58.3% of samples each. Sequencing of the blaTEM1 gene revealed polymorphisms in isolate A8. However, these did not alter its antibiotic resistance phenotype. Sequences of E. coli isolates showed similarities to strains from Vietnam, China, and India, countries with high antibiotic consumption, particularly ampicillin.

Antibiotic susceptibility and resistance of Streptococcus iniae and Streptococcus parauberis isolated from olive flounder ( Paralichthys olivaceus)

Background: In this study, we aimed to estimate the prevalence, tetracycline resistance and presence of Tet(O) in Campylobacter strains isolated from chicken in markets of Lima, Peru. Methods: A total of 250 chicken samples were obtained from traditional markets (skin, n = 120) and supermarkets (meat, n = 130). Samples were subjected to microbiological assays for identification of Campylobacter spp. according to ISO 10272-2017, and the isolates were then submitted to species identification by PCR. Phenotypic resistance to tetracyclines was assessed by the Kirby–Bauer test, and the presence of the Tet(O) gene was determined by PCR. Results: A significantly higher prevalence (p < 0.0001) of Campylobacter coli in skin samples from traditional markets (97.5%) than in meat samples from supermarkets (36.2%) was observed. On the other hand, Campylobacter jejuni was confirmed only in 3.1% of meat samples. All Campylobacter species isolated from skin and meat samples were phenotypically resistant to tetracyclines; however, the presence of the Tet(O) gene in C. coli was identified in 76.9% and 66.0% of skin and meat samples, no significant statistical difference (p = 0.1488) was found between these prevalence. All C. jejuni isolated from chicken meat samples from supermarkets were positive for Tet(O) gene. Conclusions: This study confirms the high prevalence of C. coli isolated from chicken sold in traditional markets and supermarkets in Lima, Peru, and in more than 70% of these strains, phenotypic resistance to tetracyclines could be linked with expression of the Tet(O) gene. It is necessary to evaluate other genes involved in resistance to tetracyclines and other groups of antibiotics in campylobacter strains isolated from chicken meat.

In order to meet consumer demand, the global poultry business is constantly expanding; hence it is crucial to ensure the bio-safety of the poultry farms. In poultry farms, the main sources of bacterial development, increased antibiotic resistance, and environmental dispersion may be poultry feed, litter, and water. The focus of this research is on the identification and characterization of bacterial pathogens found in commercial poultry farms, drinking water samples, feed samples, and litter samples. The 100 samples were gathered from chicken farms in different districts of Tamil Nadu and transported to the lab for additional examination. Serial dilution was used to analyse the samples, which were then examined for colony morphology, microscopic features, and the biochemical traits of the bacteria Escherichia coli, Pseudomonas spp., Proteus spp., Salmonella spp., Shigella spp., Bacillus spp., staphylococcus spp., and Streptococcus spp. The disc diffusion method was used to evaluate bacterial isolates against various antibiotic drugs. The results of this research indicate that the main sources of bacterial infections are chicken feed, drinking water samples and poultry litter. Salmonella, Pseudomonas, and E. coli prevalence rates were 94%, 80%, and 75%, respectively. Chloramphenicol and Tetracycline both had overall antibiotic resistance rates of 65.65% and 59.23%, respectively. Public health is alarmingly threatened by the presence of these harmful microorganisms in poultry habitats. To reduce the chance of transmitting the bacterial diseases linked to poultry, strict hygiene procedures must be put in place. The use of antibiotics in chicken for promoting growth and illness prevention needs to be properly regulated and executed.