Multidrug and vancomycin-resistant Enterococcus isolates in different productive stages of a poultry farm

Abstract

The poultry industry represents an important economic sector in Argentina. In this study we recovered 26 vancomycin-resistant Enterococcus (VRE) isolates from different productive stages of a poultry farm located in Tandil, Argentina. Ten isolates were Enterococcus faecium and seven, Enterococcus faecalis. Total resistance to vancomycin (96.2%), erythromycin (80.8%), levofloxacin (57.7%), chloramphenicol (26.7%), penicillin (23.1%), ampicillin (7.7%) was detected and 20 isolates (76.9%) were identified as multidrug-resistant (MDR). With respect to the distribution of glycopeptide resistance genes, 57.7% of the isolates harbored the vanC-1 gene, and 11.5%, carried the vanC-2/C-3 gene. The vanA and vanB variants were not detected. This study provides evidence that healthy chickens from the studied region can be a reservoir of MDR–VRE. Further surveillance should be conducted to control their dissemination through the food chain.

Resumen

La industria avícola representa un sector económico importante en Argentina. En el presente estudio se obtuvieron 26 aislamientos de Enterococcus resistentes a vancomicina (ERV) en diferentes etapas productivas de una granja avícola ubicada en Tandil, Argentina. De los aislamientos obtenidos, 10 correspondieron a Enterococcus faecium y 7 a Enterococcus faecalis. Se detectó resistencia a vancomicina (96,2%), eritromicina (80,8%), levofloxacina (57,7%), cloranfenicol (26,7%), penicilina (23,1%) y ampicilina (7,7%). Veinte aislamientos (76,9%) fueron multirresistentes (MDR). En cuanto a la distribución de los genes de resistencia a glicopéptidos, el 57,7% de los aislamientos presentó el gen vanC-1 y el 11,5% presentó vanC-2/C-3. No se detectaron las variantes vanA ni vanB. Este estudio aporta evidencia de que los pollos sanos de la región estudiada pueden ser reservorios de ERV MDR y destaca la necesidad de continuar con la vigilancia para controlar su diseminación a través de la cadena alimentaria.

Enterococcus, mainly E. faecium and E. faecalis, represents one of the main agents causing nosocomial infections. This genus exhibits intrinsic resistance to common antibiotics and a plastic genome which allows it to readily acquire resistance to further antibiotics, such as vancomycin, either through mutation or by horizontal transference of genetic elements⁵. Despite their low virulence, resistance to antimicrobials and adaptability have enabled enterococci to transition from intestinal commensals to major causes of healthcare-associated infections¹¹.

The WHO has identified the most critical antimicrobial-resistant bacteria worldwide, which must be monitored due to the urgent need for new treatments to combat them. Vancomycin-resistant Enterococcus (VRE) is classified as a high priority pathogen (Group 2)¹⁴. Vancomycin is considered a last-resort antibiotic for treating infections caused by Gram-positive bacteria. Resistance is mediated by van genes, with vanA and vanB located on transposons and capable of being horizontally transferred, while vanC and vanD are chromosomal and apparently not transferred in that way⁷.

Enterococcus contributes to the spread of antimicrobial resistance. Antibiotic resistance genes present in mobile genetic elements of Enterococcus isolates of animal origin can be transmitted to Enterococcus isolates of human origin¹⁰. The poultry industry is one of the most crucial food producers globally, and Argentina is among the top 10 poultry meat producers⁹. The emergence of VRE in food production systems has been attributed not only to the hospital use of glycopeptides but also to the use of avoparcin as a growth promoter in animals¹.

This study aimed to isolate VRE from a poultry farm located in Tandil, Pampean region, Argentina, to determine which isolates were E. faecium/E. faecalis and to investigate the antimicrobial susceptibility profiles and the glycopeptide resistance genotypes. Thirty samples obtained in 2022 in a previous study⁸ from a conventional farm raising broiler chickens, with a slaughter room, were selected. Originally, the samples were grown in LB medium and stored at −80 °C with the addition of glycerol. Samples from cloacal swabs of 40-day-old and 30-day-old broiler chickens, barn beds, poultry drinker surfaces, balanced feed, carcasses and slaughter room surfaces were incubated in glucose azide broth with shaking (120 rpm) at 37 °C for 18 h. An aliquot of bacterial cultures was inoculated into bile esculin azide (BEA) agar (Britania, Argentina) supplemented with vancomycin (6 μg/ml) at 37 °C for 48 h. Presumptive colonies that grew on the selective medium were confirmed as Enterococcus spp. by esculin hydrolysis and catalase tests, and Gram stain. Identification of E. faecium and E. faecalis and distribution of glycopeptide resistance genes were performed by PCR, according to Dutka-Malen et al.⁴, with modifications. Additionally, biochemical tests, such as PYR (l-pyrrolidonyl arylamidase) test, arabinose, mannitol, and sorbitol fermentation, were performed on isolates in which the species could not be defined by PCR. The isolates were tested for susceptibility to six antibiotics representing five antimicrobial classes, using the disk diffusion method, according to the CLSI² guidelines (Supplementary Table 1). The antibiotics were ampicillin (AMP, 10 μg), penicillin (PEN, 10 U), vancomycin (VAN, 30 μg), erythromycin (ERY, 15 μg), chloramphenicol (CHL, 30 μg), and levofloxacin (LVX, 5 μg). All samples analyzed showed growth on BEA agar supplemented with vancomycin and 30 isolates suspected of exhibiting antimicrobial resistance (AMR) to vancomycin were obtained.

Twenty-six isolates were confirmed as Enterococcus spp. (Catalase-negative/Gram-positive diplococci that hydrolyzed esculin), of which 10 were E. faecium, 7 E. faecalis, 7 E. avium, and 2 E. durans. The largest number of isolates and the greatest specific diversity (E. avium, E. durans, E. faecalis, E. faecium) were found in the slaughter room (Table 1). Total resistance to VAN (96.2%), ERY (80.8%), LVX (57.7%), CHL (26.9%), PEN (23.1%), AMP (7.7%) was detected among the 26 Enterococcus isolates (Table 2). Taking into account resistant and intermediate isolates, 15 AMR profiles were observed. VAN–ERY–LVX was the most predominant profile (19.2%). Twenty isolates (76.9%) were multidrug-resistant (MDR), showing resistance to three or more antimicrobial classes. One E. faecium isolate obtained from a slaughter room utensil (#12) was resistant to all tested antimicrobial agents (Table 1). With regard to the distribution of glycopeptide resistance genes, 57.7% of the isolates analyzed harbored the vanC-1 gene, and 11.5%, carried the vanC-2/C-3 (Table 1). VanA and vanB variants were not detected. Isolates from henhouse 1 showed the greatest diversity of van genotypes (Table 3). It can be hypothesized that the same strain may be distributed at different stages of production in some cases, for example, E. faecalis isolates #15 and #20 from henhouses 2 and 1, respectively (AMR profile: AMN-S/PEN-S/VAN-R/ERY-R/CHL-S/LVX-S, without van genes). However, this could only be confirmed after applying some subtyping method. Although future studies that include the identification of sequence types (ST) will be necessary to determine the lineages and study the epidemiology, the information provided here on antimicrobial resistance profiles and van genotypes show genetic diversity among VRE strains circulating in the analyzed farm.

Table 1.

Origin and antimicrobial susceptibility of vancomycin-resistant Enterococcus (VRE) isolates obtained from a poultry farm in Argentina.

Isolate	Identification	Source	Production phase	Antimicrobial susceptibility profile						van genotype
				AMN	PEN	VAN	ERY	CHL	LVX
1	E. faecalis	Equipment	Slaughter room	S	S	R	R	S	I	vanC-1
2	E. faecalis	Table		S	S	R	R	S	S	vanC-1
3	E. faecalis	Equipment		S	S	R	I	S	S	vanC-1
4	E. faecium	Utensil		S	S	R	S	S	S	vanC-1
5	E. faecium	Carcasses		S	S	R	S	S	S	vanC-1
6	E. avium	Carcasses		S	S	R	R	S	I	vanC-1
7	E. avium	Utensil		S	S	R	R	S	I	vanC-1
8	E. avium	Carcasses		S	S	R	S	S	I	–
9	E. faecium	Carcasses		S	S	R	R	S	I	vanC-1
10	E. durans	Carcasses		R	R	I	R	S	R	–
11	E. durans	Carcasses		S	S	R	S	S	R	–
12	E. faecium	Utensil		R	R	R	R	R	R	vanC-1
13	E. faecium	40-Day-old broiler chicken	Henhouse 2	S	S	R	R	S	R	vanC-1
14	E. faecium	40-Day-old broiler chicken		S	R	R	R	S	R	vanC-1
15	E. faecalis	40-Day-old broiler chicken		S	S	R	R	S	R	–
16	E. faecalis	40-Day-old broiler chicken		S	S	R	R	I	R	–
17	E. faecalis	Barn bed		S	S	R	R	R	R	vanC-2/C-3
18	E. faecium	Balanced feed		S	S	R	R	R	R	–
19	E. avium	30-Day-old broiler chicken	Henhouse 1	S	S	R	R	R	R	–
20	E. faecalis	30-Day-old broiler chicken		S	S	R	R	S	R	–
21	E. avium	30-Day-old broiler chicken		S	S	R	R	S	R	–
22	E. faecium	30-Day-old broiler chicken		S	R	R	R	R	R	vanC-1
23	E. faecium	30-Day-old broiler chicken		S	S	R	R	S	R	vanC-1, vanC-2/C-3
24	E. faecium	Poultry drinker surface		S	R	R	R	R	R	vanC-1
25	E. avium	Balanced feed		S	R	R	R	R	S	vanC-2/C-3
26	E. avium	Barn bed		S	S	R	R	I	S	vanC-1

R, resistant; I, intermediate; S, susceptible by the disk diffusion test: AMN (ampicillin), PEN (penicillin), VAN (vancomycin), ERY (erythromycin), CHL (chloramphenicol), LVX (levofloxacin).

Table 2.

Antibiotic susceptibility percentages of vancomycin-resistant Enterococcus (VRE) isolates obtained from a poultry farm in Argentina.

Antibiotic	Susceptible	Intermediate	Resistant
	n (%)
Ampicillin	24 (92.3)	–	2 (7.7)
Penicillin	20 (76.9)	–	6 (23.1)
Vancomycin	–	1 (3.8)	25 (96.2)
Erythromycin	4 (15.4)	1 (3.8)	21 (80.8)
Chloramphenicol	17 (65.4)	2 (7.7)	7 (26.9)
Levofloxacin	6 (23.1)	5 (19.2)	15 (57.7)

Table 3.

Distribution of van genotypes in vancomycin-resistant Enterococcus (VRE) isolates obtained from a poultry farm in Argentina according to the production phase.

	n (%)
Henhouse 1 isolates
vanC-1	3 (11.5)
vanC-2/C-3	1 (3.8)
vanC-1, vanC-2/C-3	1 (3.8)
Without detected van genes	3 (11.5)
Henhouse 2 isolates
vanC-1	2 (7.7)
vanC-2/C-3	1 (3.8)
Without detected van genes	3 (11.5)
Slaughter room isolates
vanC-1	9 (34.6)
Without detected van genes	3 (11.5)

In Argentina, Delpech et al.³ isolated E. faecalis and E. faecium with high levels of AMR to glycopeptides from chicken feces. In the present study, most of the isolates showed phenotypic total resistance to vancomycin and were MDR–ERV. Moreover, it was determined that of the isolates analyzed, 57.7% carried the gene vanC-1 and 11.5%, vanC-2/C-3. These genes, unlike vanA and vanB, would be associated with low levels of resistance to vancomycin⁶. In China, Yu et al.¹⁵ found E. faecalis in poultry production, but did not detect vanA or vanB genes. In contrast, Osman et al.¹² found vancomycin resistance genes (vanA, vanB, vanC) in Enterococcus obtained from chickens in Egypt. Regarding local epidemiology, the vanA gene was detected in E. faecium strains previously obtained from Argentinian chickens³ and, vancomycin-resistant E. faecium strains harboring either vanA or vanB were recovered from patients with invasive infections (2010–2014) at the Public Hospital of Tandil¹³.

Although conclusions should be interpreted with caution due to the limited number of samples studied, our results suggest that healthy chickens from the studied region may serve as a reservoir of MDR–VRE and this information is a call for further surveillance in the poultry sector. MDR Enterococcus represents a therapeutic threat to the community, and controlling its spread from poultry to humans is crucial¹⁰. The presence of a van gene pool in chickens, as observed in this study, and the colonization of humans by glycopeptide-resistant strains highlight the need to control this reservoir so that these glycopeptide-resistant strains that colonize the chicken intestine are not selected and spread through the poultry production chain into the community. The emergence of resistance is a global concern for human and animal health. For this reason, coordinated action between both sectors is essential to preserve the therapeutic efficacy of treatments used for human infections in the future.

CRediT authorship contribution statement

Juliana González: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing – original draft, Writing – review & editing, Project administration, Funding acquisition. Constanza Pifano García: Validation, Investigation, Formal analysis. Juliana Cantón: Investigation, Writing – review & editing. María José Izaguirre: Investigation, Writing – review & editing. María Soledad Ríos: Investigation, Writing – review & editing. Andrea Mariel Sanso: Conceptualization, Methodology, Writing – original draft, Writing – review & editing, Supervision.

Funding

This work was supported by Fondo para la Investigación Científica y Tecnológica (FONCYT, PICT 2020 serie A No. 02851).

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

We thank M. Rosa Ortiz and Victoria Ballesteros for technical assistance.