Resistance to third-generation cephalosporins and quinolones in Escherichia coli isolated from broiler chicken feces and carcasses at slaughter in Rio de Janeiro state, BrazilABSTRACT:
Escherichia coli is a significant component of the poultry intestinal microbiota and a critical indicator of antimicrobial resistance. This study investigated the antimicrobial susceptibility and the presence of cephalosporin and quinolone resistance genes in E. coli strains isolated from broiler chicken feces and carcasses in Rio de Janeiro, Brazil. Phenotypic resistance characterization was performed using Disk Diffusion and Double Disk Synergy Tests (DDST) for cephalosporin and quinolone antimicrobials, while resistance genes were detected by PCR. Statistical analyses were conducted using Chi-square and Fisher’s Exact tests. Results indicated that 44.4% of the strains were resistant to at least one cephalosporin, with a higher resistance frequency observed in carcass isolates. Furthermore, 61.4% of strains were identified as Extended-spectrum β-lactamases (ESBL) producers through DDST, also with a higher prevalence in carcass samples. Resistance to at least one quinolone was detected in 96% of the strains, with no significant difference between feces and carcass isolates, except for nalidixic acid, which showed higher resistance in fecal isolates. The bla TEM gene was most frequently detected in both sources, followed by bla CTX-M, which was more prevalent in carcasses. CTX-M groups 1, 2, 8, and 9 were identified, with CTX-M-2 being the most frequent. Among quinolones, qnrA, qnrB, qnrD, and qnrS genes were detected, with qnrB predominating. The bla SHV, bla CTX-M-25, and qnrC genes were not found. This study highlighted the detection of ESBL or genes encoding this characteristic and the high prevalence of quinolone-resistant E. coli carrying various qnr genes in poultry farms in Rio de Janeiro, Brazil.
Key words:
poultry; third-generation cephalosporins; quinolones; ESBL;
qnr
RESUMO:
Escherichia coli é um componente importante da microbiota intestinal das aves e um indicador crítico de resistência antimicrobiana. Este estudo investiga a suscetibilidade antimicrobiana e a presença de genes de resistência às cefalosporinas e quinolonas em cepas de E. coli isoladas de fezes e carcaças de frangos de corte no Rio de Janeiro, Brasil. A caracterização fenotípica da resistência foi realizada utilizando os testes Disco Difusão e Sinergismo de Duplo Disco (DDST) para antimicrobianos de cefalosporinas e quinolonas, enquanto os genes de resistência foram detectados por PCR. As análises estatísticas foram conduzidas utilizando os testes Qui-quadrado e Exato de Fisher. Os resultados indicaram que 44,4% das cepas eram resistentes a pelo menos uma das cefalosporinas, com uma maior frequência de resistência observada nos isolados de carcaça. Além disso, 61,4% das cepas foram identificadas como produtoras de β-lactamases de espectro estendido (ESBL) através do DDST, também com maior prevalência em amostras de carcaça. Em 96% das cepas foram detectadas resistência a pelo menos uma das quinolonas, sem diferença significativa entre isolados de fezes e carcaças, exceto para o ácido nalidíxico, que mostrou maior resistência em isolados fecais. O gene bla TEM foi o mais frequentemente detectado em ambas as fontes, seguido por bla CTX-M, que foi mais prevalente nas carcaças. Os grupos CTX-M 1, 2, 8 e 9 foram identificados, sendo o CTX-M-2 o mais frequente. Entre as quinolonas, os genes qnrA, qnrB, qnrD e qnrS foram detectados, com predominância do gene qnrB. Os genes bla SHV, bla CTX-M-25 e qnrC não foram encontrados. Este estudo destaca a detecção de E. coli produtora de ESBL ou de genes que codificam tal característica e a alta prevalência de E. coli resistente a quinolonas, portadora de vários genes qnr em granjas avícolas no Rio de Janeiro, Brasil.
Palavras-chave:
aves; cefalosporinas de terceira geração; quinolonas; ESBL;
qnr
INTRODUCTION
Escherichia coli is a bacteria naturally reported in the intestinal microbiota of humans and animals, including birds, where strains with commensal or pathogenic properties can coexist (ABUSHAHEEN et al., 2020). This microorganism is commonly recognized as an indicator of antimicrobial resistance in Gram-negative bacterial populations and serves as a model for antimicrobial resistance surveillance studies (CASTANHEIRA et al., 2021; FURMANEK-BLASZK et al., 2023). In humans, pathogenic E. coli is one of the main microorganisms responsible for causing diarrheal or systemic diseases ranging from moderate to acute severity (FURMANEK-BLASZK et al., 2023). One of the measures taken to control bacterial infections caused by E. coli, both in birds and humans, is the use of antimicrobials. Beta-lactams and quinolones stand out among the antimicrobials of choice for treating these diseases (CORREIA et al., 2017).
Beta-lactams, which include cephalosporins, act by inhibiting the synthesis of the bacterial cell wall, while quinolones hinder bacterial DNA replication by inhibiting the activity of DNA gyrase type II and topoisomerase type IV enzymes (ABUSHAHEEN et al., 2020). The primary mechanism of bacterial resistance to cephalosporins is the production of extended-spectrum beta-lactamases (ESBLs), enzymes capable of hydrolyzing 3rd and 4th generation cephalosporins. The most frequently encountered ESBLs belong to the bla TEM, bla SHV, and bla CTX-M groups, whose encoding genes are located on plasmids (CASTANHEIRA et al., 2021), which may also carry resistance genes for other antimicrobials, such as quinolones (FURMANEK-BLASZK et al., 2023).
Quinolone resistance can also be associated with the acquisition of genes located on plasmids or through chromosomal mutations (CORREIA et al., 2017). In recent years, the dissemination of Plasmid-Mediated Quinolone Resistance (PMQR) genes has been observed, mainly in Latin American countries, where the prescription of fluoroquinolones is high, as described in several studies (VIEIRA et al., 2020). These genes include those of the qnr family (qnrA, qnrB, qnrC, qnrD, and qnrS), which encode a homologous protein that hinders the binding of quinolones to DNA gyrase, rendering the interaction of this kind of drug with the target less viable (CUNHA et al., 2017). Over the past decades, ESBL and PMQR genes have increasingly been found in Enterobacteriaceae due to the horizontal transfer of resistance plasmids (BRIALES et al., 2012; SUN PARK et al., 2012)
Considering the increasing concern regarding antimicrobial resistance (AMR) in both human and veterinary medicine, it is essential to understand the prevalence and mechanisms of resistance in pathogens such as E. coli. The use of antimicrobials, particularly beta-lactams and quinolones, in livestock farming, especially in poultry production, may contribute to the emergence and dissemination of resistant strains that pose a significant threat to public health (RACEWICZ et al., 2022). Monitoring resistance patterns in animal-derived pathogens, such as E. coli in poultry, is crucial for developing more effective strategies for antimicrobial stewardship and resistance management. This paper sets out to investigate the antimicrobial susceptibility to third-generation cephalosporins and quinolones and to detect resistance genes to these antimicrobials in E. coli isolated from broiler chicken feces and carcasses from slaughterhouses in the state of Rio de Janeiro (RJ), Brazil, in 2016 and 2022. Thereby contributing to a better understanding of AMR dynamics in the country.
MATERIALS AND METHODS
Sample collection and processing
A total of 198 strains of E. coli from the stock of the Avian Health Laboratory at Universidade Federal Fluminense (UFF) stored on refrigerated nutrient agar (Himedia®) were used in this study, consisting of 90 strains obtained from fecal swabs of broiler chicken and 108 strains from carcass washes collected in abattoirs in the years 2016 and 2022 in the state of Rio de Janeiro, Brazil. The isolates were obtained from the largest poultry production center in Rio de Janeiro, located in the mountainous region of Rio de Janeiro, as well as from other slaughterhouses situated in various regions of the state. A single passage was made from the nutrient agar to the Brain Heart Infusion Broth (Kasvi®), which was subsequently used for antibiogram analysis and DNA extraction.
Characterization of the phenotypic profile of antimicrobial susceptibility
Antimicrobial susceptibility testing was performed using the disk diffusion method, following the guidelines of the Clinical and Laboratory Standards Institute (CLSI, 2024). The following cephalosporin antimicrobials were used: ceftazidime (30 μg), ceftriaxone (30 μg), cefotaxime (30 μg), and ceftiofur (30 μg). From the quinolone class, the following were used: enrofloxacin (5 μg), nalidixic acid (5 μg), and ciprofloxacin (5 μg). The diameter of inhibition zones was measured to classify strains as sensitive, intermediate, or resistant. Strains classified as intermediate were considered resistant.
Tests for detection of ESBL-producing strains
The antimicrobials ceftazidime, ceftriaxone, and cefotaxime were included in the antimicrobial susceptibility testing as markers of beta-lactamase production. Strains that produced inhibition zones smaller than 22 mm, 25 mm, and 27 mm; respectively, were considered possible ESBL producers (CLSI, 2024). Strains considered ESBL producers were subjected to the Double Disk Synergy Test (DDST) to confirm ESBL production. Disks containing cefotaxime, ceftazidime, and ceftriaxone were placed next to a beta-lactamase inhibitor disk (amoxicillin-clavulanic acid), and when the inhibition zones around any of the cephalosporin disks increased, or there was distortion of the inhibition zone toward the amoxicillin-clavulanic acid disk, the test was considered positive (CLSI, 2024).
Detection of bla and qnr genes by PCR
DNA from the strains was extracted using the Wizard® Genomic DNA Purification Kit (Promega, Brazil), following the manufacturer’s protocol. The detection of bla TEM, bla SHV, and bla CTX-M genes, which encode ESBL production, was performed by Multiplex Polymerase Chain Reaction (PCR) according to MONSTEIN et al. (2007). When positive for the bla CTX-M gene, CTX-M-1, 2, 8, 9, and 25 group genes were characterized by another Multiplex PCR (WOODFORD et al., 2006). The search for qnr A, B, C, D, and S genes, which encode quinolone resistance, was performed by Multiplex PCR, as described by KRAYCHETE et al. (2016). The samples were considered ESBL-positive in the presence of at least one of the tested bla genes.
Statistical analysis
Chi-square and Fisher’s Exact tests were used to compare whether there was statistical significance between fecal and carcass sources of isolates. A significance level of 0.05 was used in the calculations.
RESULTS
Phenotypic profile of antimicrobial susceptibility
Out of 198 strains of E. coli analyzed, 44.4% (88/198) were resistant to at least one of the tested cephalosporins, with a higher frequency of resistance among strains from carcasses (56.5% - 61/108) compared to fecal isolates (32.2% - 29/90) (P < 0.05). Among fecal strains, the highest frequency of resistance was for ceftiofur (16.7% -15/90) and cefotaxime (13.3% - 12/90), and in carcasses, for ceftriaxone (52.8% - 57/108), followed by ceftiofur (47.2% - 51/90) (Table 1).
Regarding quinolones, resistance to at least one of the antimicrobials in the class was observed in 96% (190/198) of the strains, with no statistical difference between fecal and carcass sources (P > 0.05), except for nalidixic acid antimicrobial where 97.8% (88/90) of resistant strains were isolated from feces and 90.7% (98/108) from carcasses (Table 1).
Tests for detection of ESBL-producing strains
In the screening assay, 22.2% (44/198) of strains tested positive, and in the confirmatory test, 61.4% (27/44) of strains were deemed ESBL producers. A higher frequency of ESBL was observed in strains sourced from carcasses (81.5% - 22/27) compared to those of fecal origin (18.52% - 5/27) (P < 0.05).
Detection of bla and qnr genes by PCR
The bla TEM gene was the most frequent. The qnr and bla CTX-M genes were more frequent in carcasses than in feces (P < 0.05) (Table 2). The bla SHV gene was not detected in any of the strains studied with no statistical difference between the sources (P > 0.05). Among the 61 strains positive for the bla CTX-M gene, seven distinct profiles were detected in feces and eight in carcasses regarding the CTX-M group genes.
Frequency of resistance genes in Escherichia coli isolated from fecal material and broiler chicken carcasses in slaughterhouses in the state of Rio de Janeiro (2016 and 2022).
The CTX-M-2 type was the most frequent, and none of the isolated strains belonged to the CTX-M-25 group. The coexistence of different groups of bla CTX-M occurred in 23.5% (4/17) and 25.0% (11/44) of fecal and carcass isolates, respectively (P > 0.05) (Table 3).
Distribution of blaCTX-M group patterns in E. coli isolates from broiler chicken carcasses and feces in abattoirs in the state of Rio de Janeiro (2016 and 2022).
Among the qnr group genes, the qnrB gene was the most frequent, being isolated in 71.6% (53/74) of the strains. The frequency of the qnrB gene in carcasses was 78.4% (40/51) and in feces was 56.5% (13/23), with no statistical difference between the sources (P > 0.05). However, when analyzing the pattern of strains that possessed only qnrB, carcass strains had a higher frequency, with a statistically significant difference compared to fecal strains (P < 0.05) (Table 4).
Distribution of qnr gene patterns in E. coli isolated from feces and broiler chicken carcasses in abattoirs in the state of Rio de Janeiro (2016 and 2022).
The qnrS gene alone was more frequent in fecal isolates (30.4% - 7/24) (P < 0.05) (Table 4), and qnrC was not detected in any of the isolated strains. Six distinct profiles were detected in feces and seven profiles in carcasses. The coexistence of different qnr genes occurred in 9.46% (7/74) of the isolates (Table 4).
When analyzed together, the bla and qnr genes produced six distinct genotypic profiles in feces and seven in carcasses. In feces, the most frequent profile presented the bla TEM gene alone (36.7% - 33/90), followed by the profile containing the bla TEM and associated qnr genes (15.6% -14/90). In carcass strains, the most frequent profile was also that which presented the bla TEM gene alone (18.5% - 20/108), followed by the profile containing the qnr gene alone (14.8% - 16/108) (Table 5).
Distribution of genotypic profiles in E. coli isolated from fecal material and broiler chicken carcasses in abattoirs in the state of Rio de Janeiro (2016 and 2022).
There was concomitant presence of bla CTX-M and bla TEM genes in 8.89% (8/90) and 8.3% (9/108) of fecal and carcass strains, respectively. In carcass-isolated strains, there was a higher frequency of the profile containing bla CTX-M, bla TEM, and qnr genes (10.2% - 11/108) (P < 0.05) when compared to fecal isolates (1.1% - 1/90). Among the strains studied, 20.0% (18/90) and 12.0% (13/108) of fecal and carcass strains, respectively, did not present any resistance genes (Table 5).
Relationships between phenotype and presence of resistance genes
Among ESBL-producing strains detected in the DDST, 77.8% (21/27) harbored at least one bla gene. In 100% of ESBL-producing fecal strains in the phenotypic test, at least one bla gene was detected. In carcass strains, among ESBL-producing strains, 72.7% (16/22) carried one of the bla genes. The complete profile can be observed in table 6.
A total of 70.6% (48/68) and 63.8% (30/47) of fecal and carcass strains, respectively, showed sensitivity to all tested cephalosporins, despite the presence of ESBL genes in these isolates. The percentage of resistant strains and presence of bla genes was 72.7% (16/22) in feces and 80.33% (49/61) in carcasses. Likewise, in 27.3% (6/22) and 19.7% (12/61) of strains, resistance was observed, and no bla genes were detected (Table 7). Additionally, 29.4% (20/68) of fecal strains and 36.2% (17/47) of carcass strains were sensitive to cephalosporins and did not present any ESBL genes. Thus, in fecal isolates, 40% (36/90) of phenotypes and genotypes corresponded, while in carcass isolates, their frequency was 61.1% (66/108) (Table 7).
Regarding quinolones, no fecal strains were sensitive nor had the qnr gene, whereas two carcass-isolated strains showed sensitivity to all tested quinolones and had the qnr gene. In 25.8% (23/89) and 48.5% (49/101) of quinolone-resistant strains, some type of qnr gene was detected in feces and carcasses, respectively. In 74.2% (66/89) and 51.5% (52/101) of resistant strains, no associated qnr genes were detected in feces and carcasses. Additionally, in 100% (1/1) and 71.4% (5/7) of strains, they were sensitive and did not present any qnr genes. Thus, in fecal isolates, 26.7% (24/90) of phenotypes and genotypes corresponded, while in carcass isolates, this frequency was 50% (54/108) (Table 7).
DISCUSSION
The present study verified the occurrence of resistance to third-generation cephalosporins and quinolones in E. coli strains from broiler chicken feces and carcasses at slaughter in Rio de Janeiro. Similarly, KORB et al. (2015) and CERUTTI et al. (2020) also detected a high frequency of E. coli strains resistant to these antimicrobials in poultry and in their carcasses. CERUTTI et al. (2020) reported 100% resistance to cephalosporins and quinolones in enteropathogenic E. coli strains isolated from frozen broiler chicken carcasses available for commercialization in the southern region of Brazil.
The presence of resistant bacteria in broiler chicken feces and carcasses at slaughter may allow their dissemination to the environment and to products at any point during the processing if control measures fail to sufficiently reduce bacterial load by the end of the production process. KORB et al. (2015) detected genotypic similarity using pulsed-field gel electrophoresis (PFGE) technique among E. coli strains from broiler chicken and caretakers from different poultry houses in the metropolitan region of Curitiba, demonstrating the potential diffusion of bacterial clones among different environments. The frequency of resistance to cephalosporins was higher in carcasses. Among quinolones there was no statistical difference between the sources, when compared to the results reported by other authors, who detected 86.2% (CASELLA et al., 2018) and 74.8% (AL-TAMIMI et al., 2022) of positive strains in this test.
In recent decades, the dissemination of CTX-M-producing enzymes has been mainly associated with the spread of E. coli. Furthermore, E. coli isolates with enzymes belonging to the bla CTX-M family in ESBL-positive bacteria are predominant worldwide (SALIU et al., 2017). Despite this, in our analysis, there was a predominance of the bla TEM gene (56.06%) over the bla CTX-M gene (30.80%) in the isolates. The predominance of the bla TEM gene over the blaCTX-M gene in E. coli isolates was also observed by other authors in carcass and fecal swabs in the United Kingdom (RACEWICZ et al., 2022), in broiler chicken fecal isolates in Nigeria (AKINBAMI et al., 2018), and in broiler chicken meat available for commercialization in South Korea (SEO et al., 2018). This result suggested that the bla TEM gene may be predominant in the state of Rio de Janeiro compared to the bla CTX-M and bla SHV genes.
The predominance of the bla TEM gene in the state of Rio de Janeiro highlights the need to investigate regional factors, including the use of beta-lactam antimicrobials in poultry farming and the circulation of plasmids in environmental and animal reservoirs. The implementation of temporal studies could provide crucial insights into the evolution of these genes, including the emergence of new resistant variants and the dissemination of bacterial clones associated with specific resistance factors. Furthermore, local management practices, including antimicrobial use, may directly influence the composition of resistance genes over time. The relationship between these genes and mobile genetic elements, including plasmids and integrons, should be explored, as they are essential for the horizontal spread of resistance, facilitating its propagation throughout the production chain and into other reservoirs, such as humans and the environment (WOODFORD et al., 2009). VON TIPPELSKIRCH et al. (2018) reported that E. coli strains isolated from carcasses may be more significant sources of ESBL resistance genes since it has been demonstrated that during broiler chicken processing steps, the product can be contaminated with strains carrying these genes (BLAAK et al., 2015).
Among the bla CTX-M groups investigated, CTX-M-2 was the most frequent group in accordance with other studies conducted in the country, since, in Brazil, this group is more frequent in ESBL-producing E. coli isolated from humans and broiler chicken meat (IARK et al., 2018). Furthermore, genes from groups CTX-M-1, 8, and 9 were isolated, and this result reinforces what has been described by other authors who highlight that these groups and CTX-M-2 are the most prevalent in Brazil (RABELLO et al., 2020; SILVA & LINCOPAN, 2012). The qnr A, B, D, and S genes were identified in E. coli strains. In the present study, we found a higher frequency of qnrB and qnrS genes, the most prevalent qnr alleles detected in Brazil (RABELLO et al., 2020).
These genes are typically found on mobile genetic elements, facilitating horizontal dissemination (WOODFORD et al., 2009). Additionally, these mobile genetic elements can carry a series of resistance genes to different antimicrobials, making the use of substances potentially exert a selective pressure, in which case elicits a co-selection and ensures, in the bacterial population, the maintenance of resistance to different classes of antimicrobials. The presence of genes encoding the production of ESBL has been frequently associated with qnr genes (FARAJZADEHSHEIKH et al., 2019).
In this paper, we used both DDST and PCR to investigate ESBL-producing isolates. PCR showed that a small percentage of ESBL-producing strains (11.12% - 3/27) did not carry any bla genes, and this may be because resistance to cephalosporins was determined by the presence of other ESBLs such as OXA, PER, VEB, GES (ANDRADE et al., 2017). However, as recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST, 2017), these strains should be characterized as ESBL producers because they may produce the enzyme in vivo after treatment initiation.
One limitation of this study is the prolonged storage of bacterial strains on nutrient agar under refrigeration. While a single passage to Brain Heart Infusion Broth (Kasvi®) was made prior to antibiogram analysis and DNA extraction to minimize genetic variation during experimental procedures, the long-term storage conditions may have influenced the genetic stability of the strains. Prolonged storage on nutrient agar, as opposed to cryopreservation techniques (e.g., -80 °C storage in glycerol or lyophilization), could potentially lead to genetic drift, plasmid loss, or recombination over time. These factors may affect the interpretation of our results, particularly concerning the stability of resistance genes and phenotypic resistance profiles. Future studies should consider employing more stable storage methods to preserve the genetic integrity of bacterial isolates over extended periods.
The inappropriate use of antimicrobials in human and animal medicine can generate selective pressure, resulting in multidrug-resistant strains. These strains contribute to the spread of resistance genes in the environment and in the food production chain, thereby affecting individuals and animals. Thus, studies like this are important to assess the impact of antimicrobial use in animals and their phenotypic and genotypic resistance to avian microbiota.
CONCLUSION
Our study highlighted a significant occurrence of resistance to third-generation cephalosporins and quinolones in E. coli strains from broiler chicken feces and carcasses at slaughter. The research also observed a higher occurrence of the bla TEM gene over bla CTX-M in poultry farms in the state of Rio de Janeiro, Brazil (in samples from 2016 and 2022), contrasting with global trends. The high frequency of qnrB genes suggested a potential for broad dissemination of resistance. The detection of ESBL-producing strains without bla genes indicates other mechanisms of resistance, such as OXA, PER, VEB, GES. The presence of these resistant bacteria poses a risk of environmental dissemination and contamination throughout the production process. These findings underscore the need for effective antimicrobial stewardship in both human and veterinary medicine to mitigate the spread of multidrug-resistant strains.
ACKNOWLEDGMENTS
This research was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001, and by the Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) Process No. E-26/211.662/2021. The authors would like to thank Centro Integrado de Tradução e Escrita (CITE/UFF) for assistance with English language translation and developmental editing.
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BIOETHICS AND BIOSECURITY COMMITTEE APPROVAL
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CR-2024-0443.R3
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We authors of the article entitled “Characterization of cephalosporin and quinolone resistance in Escherichia coli isolated from broiler chicken feces and carcasses at slaughter in Rio de Janeiro state, Brazil” declared, for all due purposes, the project that gave rise to the present data of the same has not been submitted for evaluation to the Ethics Committee of the University Federal Fluminense, but we are aware of the content of the Brazilian resolutions of the National Council for Control of Animal Experimentation - CONCEA <http://www.mct.gov.br/index.php/content/view/310553.html> if it involves animals. Thus, the authors assume full responsibility for the presented data and are available for possible questions, should they be required by the competent authorities.
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DATA AVAILABILITY STATEMENT
Not applicable.
Edited by
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ASSOCIATE EDITOR:
Rudi Weiblen (0000-0002-1737-9817)
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SCIENTIFIC EDITOR:
Juliana Felipetto Cargnelutti (0000-0002-3160-3643)
Not applicable.
