Antimicrobial resistance and virulence studies in broiler chicken production: a one health approach

[Estudos de resistência antimicrobiana e virulência na produção de frangos de corte: uma abordagem de saúde única]

ABSTRACT

The aim of this study was to characterize bacterial species and their resistance and virulence profiles in a poultry production located in Rio de Janeiro, Brazil. Samples from 1-day-old chicks and broilers were evaluated using dependent and independent cultivation techniques. The 25 strains of Staphylococcus spp. presented the mecA gene. Of the 51 strains of Enterococcus spp., a strain of E. faecium presented the vanB gene, and one strain of E. faecalis presented vanA and vanB genes simultaneously. Analysis of scraped litter from wood used for poultry animals revealed the presence of a carbapenemase resistant gene, blaVIM, in one of the samples evaluated. Of the 44 Enterobacterales isolates, 45% (20/44) were extended-spectrum beta-lactamase (ESBL) producers. Of these, 9 (45%) tested positive for the blaSHV gene, 4 (20%) for the blaCTX gene, 3 (15%) for the blaTEM gene, 2 (10%) for the blaSHV and blaCTX genes and 2 (10%) for the blaSHV and blaTEM genes. Some virulence factors related to avian pathogenic Escherichia coli (APEC) were detected in the E. coli strains. The role of animal production in the emergence and spread of resistance genes is a matter of One Health, requiring studies to be carried out in animal environments.

Keywords:
Escherichia coli; broiler chickens; resistance genes; coagulase negative Stapylococcus; chicks

RESUMO

O objetivo deste estudo foi caracterizar as espécies bacterianas e seus perfis de resistência e virulência em uma produção avícola localizada no Rio de Janeiro, Brasil. Amostras de pintinhos de um dia e frangos de corte foram avaliadas usando-se técnicas de cultivo dependentes e independentes. As 25 cepas de Staphylococcus spp. apresentaram o gene mecA. As 51 cepas de Enterococcus spp., uma cepa de E. faecium, apresentaram o gene vanB, e uma cepa de E. faecalis apresentou os genes vanA e vanB simultaneamente. A análise da cama raspada de madeira usada para aves domésticas revelou a presença de um gene resistente à carbapenemase, blaVIM, em uma das amostras avaliadas. Dos 44 isolados de Enterobacterales, 45% (20/44) eram produtores de beta-lactamase de espectro estendido (ESBL). Desses, nove (45%) testaram positivo para o gene blaSHV, quatro (20%) para o gene blaCTX, três (15%) para o gene blaTEM, dois (10%) para os genes blaSHV e blaCTX e dois (10%) para os genes blaSHV e blaTEM. Alguns fatores de virulência relacionados à Escherichia coli patogênica aviária (APEC) foram detectados nas cepas de E. coli. O papel da produção animal no surgimento e na disseminação de genes de resistência é uma questão de saúde única, exigindo a realização de estudos em ambientes animais.

Palavras-chave:
Escherichia coli; frangos de corte; genes de resistência; Stapylococcus coagulase negativa; pintos

INTRODUCTION

Brazilian agricultural activities, particularly their exports, stand out at the world stage. The Brazilian poultry industry has shown high growth rates over the past 30 years. At the beginning of 2021, 14.1 million tons of broiler chickens were produced, topping the global ranking of product exports (Statistics, 2021).

To increase poultry production, the industry regularly uses antimicrobials and anticoccidials as zootechnical additives for growth promoters at a dosage lower than the minimum inhibitory concentrations during the rearing period (up to 42 days); these are removed before slaughter, depending on the half-life of the product used (Savin et al., 2021). However, this practice favors the dissemination of resistance genes in the environment, with the possibility of these resistant bacteria infiltrating the human microbiome (Moraes et al., 2022; Bezerra et al., 2017) thus reducing the antimicrobial effectiveness in humans.

A report for the British government (O’Neill, 2015) estimated that by 2050, the current 700,000 annual deaths caused by superbugs, which are microorganisms that are resistant to all classes of antimicrobials available, could reach 10 million. The global cost of antimicrobial resistance can reach $100 trillion if preventive measures are not systematically adopted. This number is higher than the total number of deaths currently caused by cancer, which is 8.2 million per year.

In 2017, the World Health Organization (Statistics, 2020) published a list of 12 superbugs of concern to human health ranked by risk level, considering the resistance mechanisms presented and therapeutic alternatives available. Since 2018, our research group started the analysis of antimicrobial resistance using the One Health approach, where we extrapolated the research on superbug strains in animal environments. Given the contribution of some species to the etiology of infectious processes in several animals, we decided to investigate the following groups: carbapenemase-resistant and ESBL-producing Enterobacteriaceae (a critical priority strain) and vancomycin-resistant Enterococcus faecium, methicillin-resistant Staphylococcus aureus, and intermediate and resistant vancomycinin (high priority strains).

Among the animal environments studied, those related to animal production, especially poultry, are the most important, given their significant use of zootechnical additives to accelerate animal growth.

The integration of human, veterinary, and environmental clinical studies is a key aspect of the One Health approach and is of paramount importance in antimicrobial resistance surveillance and development of possible effective control measures (Diallo et al., 2020). The objective of this study was to identify and evaluate the diversity of bacterial species and characterize their antimicrobial resistance profiles in poultry.

MATERIALS AND METHODS

Stuart swabs were used to collect sixty samples from animals located in the Poultry Sector of UFRRJ (Seropédica, Rio de Janeiro, Brazil). Of these sixty samples, 35 from the cloaca and 30 from trachea of adult chickens, and 25 from the cloaca and trachea of 1-day-old chicks. In addition to the animal samples, litter samples were randomly collected at three different times (first batch, windrowing, and second batch) from 2021 to 2022. This study was approved by the Animal Experimentation and Use Committee (CEUA) protocol number 6239180418/001020.

Phenotypic identification of the different bacterial species was performed according to previous research (Koneman et al., 2008) and confirmed via proteomic identification using MALDI-TOF mass spectrometry (microbiology laboratory at the Federal University of Rio de Janeiro).

Isolates were cultured in 1.5mL of Brain Heart Infusion broth at 35°C for 24 h. The microtubes were centrifuged for 2 min at 8000g, the supernatant was discarded, and this procedure was repeated thrice. Cells were resuspended in 200µL of ultrapure water, vortexed, and incubated at 100°C for 10 min. The microtubes were then cooled to 28°C and centrifuged for 2 min at 8000g. Approximately 180µL of the supernatant was transferred to a new microtube (600µL) and stored at -20°C (Buyukcangaz et al., 2013).

Briefly, 0.5 g of litter was collected, resuspended in 1000µL of extraction buffer (1% CTAB, 1.5 M NaCl, 0.1 M EDTA, 0.1 M NaH₂PO₄, 0.1 M Tris-HCl, pH 8.0) with 0.4 g of glass beads, and vortexed for 5 min. After that, 500 µL of 20% sodium dodecyl sulfate were added and gently mixed, incubated at 65°C for 1 h with gentle agitation every 15 min, and then centrifuged for 10 min at 8,349 rpm. The supernatant was collected, and 500 µL of extraction buffer was added. The mixture was incubated again at 65°C for 10 min and centrifuged at 6000g for 10 min. The supernatant was transferred to another tube and mixed with 330µL of PEG8000 (30% polyethylene glycol) and NaCl to a final concentration of 1.6 M. Subsequently, it was centrifuged at 10,000g for 20 min. The precipitate was resuspended in 400µL of TE (10 mM Tris HCl, 1 mM EDTA, pH 8.0), and ammonium acetate was added to a final concentration of 2.5 M. It was then placed in ice for 5 min and centrifuged at 16,000 g for 30 min. The supernatant was transferred and resuspended in an equal volume of chloroform:isoamyl alcohol (24:1). The samples were then centrifuged at 16,000g for 5 min. Further, the upper phase was removed, transferred to a new tube, mixed with one volume of 100% isopropanol, incubated overnight at room temperature, and centrifuged at 16,000 g for 30 min. The resulting supernatant was discarded, and the mixture was washed twice with 500µL of ice-cold 70% ethanol. The pellet was resuspended in 50µL of ultrapure water and lightly vortexed to thoroughly homogenize (Yeates et al., 1998).

β-lactamases production, resistance to penicillin (10µg), and the Edge-Zone test were phenotypically evaluated, and the blaZ gene was detected according to previous research (Rosato et al., 2003; CLSI…, 2020a). To analyze methicillin resistance, cefoxitin (30 µg) and oxacillin (1µg) resistance were evaluated according to CLSI… (2020a), followed by the mecA gene search (Melo et al., 2020).

A disk diffusion test using gentamicin and streptomycin was performed to detect HLAR. Vancomycin resistance (30µg) was determined by amplifying the vanA and vanB genes (Clark et al., 1993).

Antibiograms were obtained using ampicillin (AMP 10µg), ceftazidime (CAZ 30µg), cefoxitin (CFO 30µg), cefotaxime (CTX 30µg), aztreonam (ATM 30µg), imipenem (IMP 10µg), cefepime (CPM 30µg), and amoxicillin + clavulanic acid (AMC 30µg). Confirmatory assay was performed by evaluating a difference of 5 mm when using ceftazifime with clavulanate (CCA 30µg) and cefotaxime with clavulanate (CCT 30µg) to disks without clavulanate (Disc…, 2020; CLSI, 2020b). Genotypic characterization was performed by detecting the bla _TEM (Minarini et al., 2007), bla _CTX (Geser et al., 2012), and bla _SHV (Shahid, 2010) genes.

Antibiograms were obtained using meropenem (MPM 10µg), which has high specificity (CSLI…, 2020a). Genotypic detection of carbapenemases was performed through multiplex detection of the following genes: blaIMP, blaVIM (Fallah et al., 2014), blaKPC, blaNDM, and blaoxa-48 (Monteiro et al., 2012).

The multiplex PCR technique was used to identify APEC strains by detecting genes such as siderophil receptor (iroN), episomal outer membrane protein (ompT), E. coli putative hemolysin encoding avian (hlyF), coding for complement system resistance (iss) proteins, and aerobactin siderophil receptor (iutA) (Johnson et al., 2008).

The sequences of the primers and amplification conditions for the genes used in the search for virulence factors are set out in Annex 3. The multiplex PCR technique was used for presumptive identification of APECs strains (pathogenic avian E. coli) searching for the genes: siderophyll (iroN) (Johnson et al., 2008), coding for episomal outer membrane protein (ompT) (Johnson et al., 2008), coding for putative avian E. coli hemolysin (hlyF) (Morales et al., 2004), encoding resistance proteins of the complement system (iss) (Johnson et al., 2008), aerobactin siderophil receptor (iutA) (Johnson and Stella., 2000).

RESULTS

Cloacal swabs from 30 broiler chickens and 25 chicks were analyzed. Phenotypically, 25 strains were identified as coagulase-negative Staphylococcus, 51 as Enterococcus spp., and 44 as Gram-negative bacteria. Among the coagulase-negative Staphylococcus strains, 48% (12/25) were identified as Staphylococcus gallinarum, 40% (10/25) were Staphylococcus sciuri, 8% (2/25) were Staphylococcus cohnii, and 4% (1/25) were Staphylococcus slow (Table 1). MALDI-TOF mass spectrometry showed high confidence scores for all the identified strains. None of the 25 coagulase-negative Staphylococcus strains presented phenotypic resistance to cefoxitin, but one strain (4%) harbored the mecA gene. In addition, none of the isolates showed phenotypic resistance to penicillin or had the blaZ gene.

Table 1:
SCN identification, sample site and resistance detection in poultry strains

Number of Strains	Origin	Resistance gene
9 Staphylococcus gallinarum	cloaca
9 Staphylococcus sciuri	cloaca
1 Staphylococcus cohnii	cloaca
1 Staphylococcus slow	cloaca
3 Staphylococcus gallinarum	trachea
1 Staphylococcus sciuri	trachea
1 Staphylococcus cohnii	trachea	mecA

Of the 51 Enterococcus spp. strains, 47.06% (24/51) were identified as Enterococcus gallinarum, 31.37% (16/51) were Enterococcus faecium, 19.61% (10/51) were Enterococcus faecalis, and 1.96% (1/51) were Enterococcus avium (Table 2). One Enterococcus faecalis strain exhibited phenotypic resistance to vancomycin. One strain of Enterococcus faecium from chick cloaca presented the vanB gene, and one strain of Enterococcus faecalis from adult chicken cloaca presented vanA and vanB genes simultaneously.

The 51 isolates of Enterococcus spp. were also evaluated for HLAR. Twelve strains (23.53%) presented streptomycin resistance, and two (8%) were inconclusive, according to the CLSI standard [16]. In addition, six strains (11.74%) exhibited gentamicin phenotypic resistance. Thus, 35.30% (18/51) of the isolates had high resistance to glycosides.

Table 2
Enterococcus identification, sample site and resistance detection in poultry strains

Number of Strain	Origin	Resistance gene
24 Enterococcus gallinarum	cloaca
16 Enterococcus faecium	cloaca
10 Enterococcus faecalis	cloaca	vanB
1 Enterococcus avium	cloaca

Among the 44 identified Gram-negative strains, proteomic analysis confirmed 36 E. coli (81.82%), 4 Enterobacter bugandensis (9.09%), 3 Klebsiella pneumoniae (6.82%), and 1 Pseudomonas aeruginosa (2.27%) strain. Further, β-lactamases production was investigated; the disk diffusion method revealed that 26 strains (59.09%) possessed phenotypic antimicrobial resistance for ESBL, and one strain was positive for ESBL and carbapenemase production, simultaneously.

Of the 26 beta-lactamases-producing strains, 15 were ampicillin-resistant E. coli strains (57.69%), 9 were cefotaxime-resistant strains (34.61%) (3 E. coli, 3 Enterobacter bugandensis, and 3 Klebsiella pneumoniae); 7 were ceftazidime-resistant strains (26.92%) (3 E. coli, 2 Enterobacter bugandensis, and 2 Klebsiella pneumoniae); 6 were cefepime-resistant strains (23.07%) (3 Enterobacter bugandensis, 2 Klebsiella pneumoniae, and 1 E. coli); 4 were aztreonam-resistant strains (15.38%) (2 E. coli and 2 Enterobacter bugandensis); 4 were ampicillin + clavulanic acid-resistant strains (15.38%) (2 Enterobacter bugandensis, 1 E. coli, and 1 Pseudomonas aeruginosa); and 3 were cefoxitin-resistant strains (11.54%) (2 Enterobacter bugandensis and 1 Pseudomonas aeruginosa). An Enterobacter bugandensis strain exhibited phenotypic resistance to meropenem; thus, it was simultaneously selected as an ESBL- and carbapenemase-producer. Furthermore, 18 strains (69.23%) tested positive for resistance genes; These included 7 bla _SHV (38.89%), 4 bla _CTX (22.22%), 3 bla _TEM (16.67%), 2 bla _SHV and bla _CTX (11.11%), and 2 bla _SHV and bla _TEM genes (11.11%). Notably, among the 18 ESBL-resistant isolates, 22.22% (4/18) came from 1-day-old chicks, which were not treated with antimicrobials. No carbapenemase-producing genes were detected.

Thirty-five E. coli strains were evaluated for virulence genes related to Avium Pathogenic Escherichia coli (APEC). Of these, five strains (14.28%) harbored at least one virulence gene; further, they contained the gene encoding episomal outer membrane protease (ompT). Two strains (40%) harbored the gene encoding the putative enzyme of avian E. coli (hlyF), and one strain (20%) harbored a siderophil receptor, samochelin (iroN). Notably, those that expressed hlyF and iroN also expressed ompT (Table 3). Strain 4 presented ompT, hlyF, and bla _TEM, simultaneously.

Table 3
Characteristics of Escherichia coli isolates according to their origin and virulence factor

Strain	Origin	Virulence factor
1	Poultry trachea	ompT e hlyF
2	Poultry cloaca	ompT e hlyF
3	Poultry cloaca	ompT
4	Poultry cloaca	ompT e iroN
5	Chick cloaca	ompT

• The genotypic analysis revealed the bla _VIM gene, linked to carbapenemase production, from a DNA sample of the litter.

DISCUSSION

The broiler production cycle in the Poultry Sector at UFRRJ comprises obtaining 1-day-old chicks, followed by a 40-day term in the sheds, and subsequent sale. Therefore, sampling included three steps: the first was carried out in adults (final stage/sale), the second in 1-day-old chicks, and the third after 30 d of the same chicks at broiler age to evaluate the relationship between the animals and their environment. Random samples of the shaved bedding were collected simultaneously with the animal samples and after the windrowing time to observe whether the fermentation process was effective.

Coagulase-negative Staphylococci were the prevalent species in the present study, especially Staphylococcus gallinarum. Sorour et al. (2023) found the biochemical analysis of the suspected CoNS colonies revealed that the identifed isolates were 8 (29,63%) S. gallinarum, 5 (18,52%) S. saprophyticus, 5 (18,52%) S. chromogens, 3 (11,11%) S. warneri, 2 (7,40%) S. hominis, S. caprae, and S. epidermidis wich was different from our samples. All isolated CoNS species did not ferment mannitol and grew as small red colonies except eight isolates of S. gallinarum that fermented mannitol and produced yellow colonies (Sorouor et al., 2023). The presence of resistance genes in the isolates from this work was not necessarily related to their phenotypic expression, once that were no found phenotypic resistance in all strains who were detected the gene. Methicilin resistance CoNS in livestock was first reported in healthy chickens in Japan in 1996. Despite the increasing interest in CoNS in recent years, there is very limited information on their prevalence and resistance profiles in poultry production, and information is even more limited regarding methicillin resistance in CoNS. Silva et al. (2022) investigated the presence of methicillin resistance CoNS in healthy quails and commercial and homebred chickens (Silva et al., 2022). This study was performed on chickens, with no quails production. This finding points to the importance of PCR as a gold standard test to prevent the spread of resistance genes through the incorrect usage of some antimicrobial classes, especially beta-lactams, which exert selective pressure on bacteria carrying genetic markers and choose the better antibiotic therapy. Methicillin resistance mediated by mecA is acquired by the horizontal transfer of a mobile genetic element called the staphylococcal chromosome cassette mec (SCCmec) (Turlej and Hryniewicz, 2011). This is concerning as methicillin-resistant Staphylococcus aureus is a high-priority superbug according to WHO, raising an alert for other animal staphylococci species (Holmström et al., 2020).

Bacterial identification is even more challenging for strains with diverse animal origins. In this study on bacterial diversity and antimicrobial resistance patterns in poultry production, it was observed that some Enterococcus spp. strains were incongruent in phenotypic identification, presenting some differences in the preliminary analysis. However, it is difficult to find reports of this misidentification in specialized literature (Spinali et al., 2015), which emphasizes the importance of data interpretation for accurately identifying clinically relevant species and establishing guidelines for microbial typing. In the last decade, MALDI-TOF has ensured reliable genus and species characterization when the score exceeds 2.0.

Because Enterococcus spp. can affect the respiratory system, gastrointestinal tract, and integument, particularly in poultry production, it is crucial to establish a reliable identification procedure (Song et al., 2019). In addition to these issues, it is also considered important by the WHO, once these bacteria have been identified, they may be resistant to vancomycin, which generates an alert in the lack of treatment for these strains. Vancomycin-resistant Enterococcus faecium is ranked as a high-priority superbug by WHO and because of this was the focus of this work. It is noteworthy that the resistant strain detected via phenotypic assay was not the same as the one detected via the genotypic assay. Intrinsic resistance to vancomycin was not detected in Enterococcus gallinarum or Enterococcus casseliflavus, as reported by the CLSI. However, it is important to note that the reference values are based on human CLSI/EuCast tables. Vancomycin is an antimicrobial used as the last resort for multidrug-resistant bacteria that are not responsive to treatment. Thus, the natural circulation of these resistance genes in the bird microbiomes facilitates their dispersion in the environment, thereby posing a risk to human health.

Injectable gentamicin has been used in newborn chicks and turkeys to prevent or control the horizontal transmission of various bacteria (Ito et al., 2005), and advertisements from agricultural companies have confirmed this use. Gentamicin and streptomycin are two important antimicrobials belonging to the aminoglycoside group used in clinical practice. Therefore, the propagation of HLAR in bacteria leads to severe difficulties in treating bacterial infections (Özdemir and Tuncer, 2020).

According to Albornoz (Albornoz et al., 2014), the intestinal microbiota of birds is mainly composed of Lactobacillus spp., Streptococcus spp., Bacteroides spp., Enterococcus spp., Clostridium spp., and E. spp. Sanz et al. (2021) reported a predominance of Enterococcus spp., followed by E. coli, Staphylococcus saprophyticus, and Pantoea agglomerans, quite different from this work that it was SCN, Enterococcus spp., E. coli, 4 Enterobacter bugandensis, Klebsiella pneumoniae and Pseudomonas aeruginosa.

The intestinal environment contains commensal microorganisms that live harmoniously with their hosts, especially healthy animals, maintaining environmental homeostasis. This symbiosis is relevant to animal health and welfare and directly affects production in a win-loss relationship (Feitosa et al., 2020). Microbiotas regulate nutrient absorption and use and favor intestinal maturation, integrity, and immunomodulation (Alexandrino et al., 2020). Therefore, identifying the isolates belonging to animal microbiota is essential. These animals are destined for food; therefore, if they experience microbial dysbiosis, this may cause heavy financial losses in agrobusiness with the disposal of animal products and contamination of humans and the environment an One Health vision. Several studies have used sequencing techniques to analyze the intestinal microbiota of birds to identify the most prevalent species in their microbiome, establish their functionality, and monitor their dynamics (Christofoli et al., 2020). Therefore, future studies using these techniques are necessary for a better understanding of the bird microbiota.

The management used in breeding is concerned with feeding and the use of food additives to maximize the animals, this directly interferes with the intestinal microbiota. Commercial antimicrobials are used to prevent respiratory diseases and diarrhea in chicks as additives. Generally, these antimicrobials are poorly absorbed, and large proportions are excreted in feces and urine as unmetabolized compounds (Heuer et al., 2011), could be one of the reasons to finding resistance genes. However, in the UFRRJ farms, antimicrobials are not used for poultry health. Therefore, it is plausible that the animals were born with genes that were passed on through horizontal contamination by the matrix (Lee et al, 2019). These data raise concerns about the presence of antimicrobial residues in animal products as they can spread throughout the body of treated animals, such as the ovaries and oviducts of birds (Ospina-Barrero et al., 2021), and similarly transfer resistance genes, generating an alert in One Health. Likewise, Saliu (Saliu et al., 2017) reported a high level of heterogeneity in ESBL genes and plasmids in poultry production.

Several studies suggest that the broiler production industry acts as a reservoir for bacteria resistant to third-generation cephalosporins, such as ESBL or plasmid-encoded AmpC β-lactamase-producing E. coli (pAmpC) (ESBL/pAmpC-EC); the poultry industry presents a higher prevalence of these bacteria compared to other animal sectors (Saliu et al., 2017). Apostolakos et al. (2020) used next-generation sequencing to investigate 100 E. coli isolates from broiler chickens, including 1-day-old chicks, and detected 31 sequences, including a sequence found in human blood and another from a urinary tract infection. All isolates carried ESBL genes, such as bla _CTX, bla _TEM, and bla _SHV, corroborating that next-generation sequencing could be used to screen genes other than those investigated in this study. Bacteria’s ESBL Enterobacteriaceae producers are challenging in human and veterinary medicine because of their limited therapeutic options (Projahn et al., 2018). Although, in this study, no carbapenemase-producing genes were detected in Enterobacteriaceae, this does not imply that the analyzed strains would not have genes other than those studied, given the vast diversity of genes related to the production of these enzymes, these genes were research in front of the WHO (2024) call Enterobacteriaceae ESBL producer and carbapenemase resistance as a superbug.

The findings regarding Gram-negative strains are alarming because many genes were related to ESBL production, possessing enzymes capable of hydrolyzing third- and fourth-generation cephalosporins and aztreonam (monobactams), which are inactivated by clavulanate, sulbactam, and tazobactam (Disc…, 2021; Bush et al., 1995). The WHO considers ESBL-producing bacteria of the order Enterobacterales to be critical pathogens, which poses a serious global problem regarding the consumption of these foods. A strong correlation was observed between the phenotypic and genotypic tests for ESBL-producing genes (Table 4). Several genes encode ESBL, and enzymes with such characteristics belong to the 2be group (TEM, SHV, and CTX types) and are known for being more dispersed (Iovleva and Bonomo, 2017). In the present study, 69.23% (18/26) samples had one or more resistance genes. Of these, 22.22% (4/18) of 1-day-old chicks that arrived in the flock already harbored resistance genes and could act as a contamination source for other animals. In this study, E. coli harbored the ompT and iroN genes as well as bla _TEM, a resistance gene linked to ESBL production.

One of the reasons of the use Antimicrobials are used on the farm to circumvent challenges arising from APEC strains; therefore, E. coli strains were also evaluated for virulence factors as a parameter for their classification as commensal, intestinal pathogenic, or extraintestinal pathogenic. In this study, no strain was characterized as APEC, considering the evaluated genes; however, there were other genes related to APEC that were not evaluated. Classification was based on the genetic criteria for pathogenicity, in which isolates containing at least five virulence genes were considered APEC. Conversely, isolates containing fewer than five virulence genes were considered non-pathogenic (non-APEC) avian E. coli. However, another study used the criterion that APEC harbors at least four genes, while Johnson et al. (2008) considered a strain APEC if it simultaneously presented three virulence genes.

Table 4
Bacterial identification, sample site, characterization of the phenotypic profile of resistance, and frequency of ESBL in poultry strains

Bacteria	Sample	Phenotypic resistance	N
Escherichia coli	Trachea	AMP	3
Escherichia coli	Trachea	AMP, ATM	1
Escherichia coli	Cloaca	AMP	7
Escherichia coli	Cloaca	AMP, CPM, CTX, CAZ, ATM	1
Escherichia coli	Cloaca	AMP, CPM, CTX, CAZ	1
Escherichia coli	Cloaca	AMP, CTX	1
Escherichia coli	Cloaca	AMP, CAZ, AMC	1
Klebsiella pneumoniae	Cloaca	AMP	1
Klebsiella pneumoniae	Cloaca	AMP, CPM, CTX, CAZ	2
Klebsiella pneumoniae	Cloaca	AMP, CTX	1
Enterobacter bugandensis	Cloaca	AMP, CTX, CAZ, ATM	1
Enterobacter bugandensis	Cloaca	AMP, CPM, CTX, CAZ, CFO, AMC, MPM	1
Enterobacter bugandensis	Cloaca	AMP, CAZ, CTX	1
Pseudomonas aeruginosa	Cloaca	AMC, CTX	1

• *ampicilin (AMP 10µg), ceftazidime (CAZ 30µg), cefoxitin (CFO 30µg), cefotaxime (CTX 30µg), aztreonam (ATM 30µg), meropenem (MPM 10µg), cefepime (CPM 30µg), amoxicilina + clavulanic acid (AMC 30µg).

The use of poultry litter is limited to each production cycle. Its reuse demands the absence of sanitary episodes and treatment for the inactivation or reduction of pathogens, while respecting sanitary voids. The windrowing process, in which litter is placed in a corner to allow fermentation, is widely used. In this study, eight poultry litter samples were evaluated to identify the resistance genes of importance, according to One Health. This analysis allowed the detection of bla _VIM, a gene linked to carbapenemase production, in a DNA litter sample at the end of the production cycle. Therefore, ensuring the importance of litter treatment for reuse is a valid alternative for reducing the spread of bacterial agents and resistance genes. However, in some cases, management is insufficient to prevent this dispersion.

As Brazil is one of the largest chicken producers in the world, it is necessary for their poultry industry to establish guidelines on the use of antimicrobial additives in a One Health context. In addition, the presence of carbapenemase-producing bacteria in wood-shaved bedding before composting and toilet vacuum raises questions about bedding reuse and suggests the need for in-depth studies to gain a better understanding of the protocols for bedding management and reuse in multiple cycles. In addition, the phenotypic and genotypic detection of resistance elucidated the selection pressure exerted by antimicrobials and zootechnical additives in disseminating resistance genes to the environment, other animals, and humans.

CONCLUSIONS

This work made clear the importance of evaluating bacterial populations in healthy animals and their relationship with One Health, especially since they are birds raised for human consumption. Other studies on poultry microbiota and their correlation should be carried out in the future. Another interesting point to be highlighted is the detection of resistance gene in different bacteria and animals, such as mecA gene in the non-coagulasis Staphylococcus of the trachea of adult chickens and vanA gene in the cloaca of adult chickens and 1-day-old chicks. Other important data were APEC-related virulence genes in E. coli strains were detected in five strains.

The interconnection of management within the production system favors the dissemination of these genes through excreta into the environment, through direct consumption of meat, through the reuse of bedding wood shavings, failures in sanitary management, among others. Production gene detection of carbapenemase in the wood shavings bed before the composting process and sanitary emptying, raises questions about its reuse.

ACKNOWLEDGEMENTS

This study received support from the Carlos Chagas Foundation for Research Support of the State of Rio de Janeiro (FAPERJ, E-26/210.085/2020 and E-26/202.604/2019), the Coordination for the Improvement of Higher Education Personnel (CAPES), Technological Development (CNPq) and the Foundation for Scientific and Technological Research Support of UFRRJ (FAPUR). We would like to thank Editage (www.editage.com) for the English language edition.

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Publication Dates

• Publication in this collection
  21 Feb 2025
• Date of issue
  Mar-Apr 2025

History

• Received
  14 Mar 2024
• Accepted
  25 Aug 2024