Antimicrobial Resistance and Extended-Spectrum Beta-Lactamase Production in Enterobacteria Isolated from Free-Living Primates

Abstract

The aim of this study was to evaluate the profile of antimicrobial resistance in members of the family Enterobacteriaceae and to detect strains of extended-spectrum beta-lactamases (ESBL) in free-living Sapajus nigritus. Oral and rectal swabs were collected from 15 primates to isolate and identify bacteria, conduct phenotypic tests to detect antimicrobial sensitivity and ESBL-producing strains, and calculate the multiple antimicrobial resistance index. The least effective antimicrobial was amoxicillin (72.72%) and ampicillin (57.57%), and the samples were considered as high risk for public health. No sample was positive for ESBL production. The results show that this study is relevant to One Health initiatives, considering the possibility of transmission of bacterial resistance and resistant genes originating from direct or indirect interaction of S. nigritus with parkgoers. However, these animals cannot be considered as the only reservoirs and transmitters of resistant bacteria as they can be contaminated by human-origin bacteria that can be extremely harmful and often prove lethal to them that play an important role in relationship to the ecology, spreading seeds and controlling insects.

Introduction

Cebus is one of the New World primates with a large geographical distribution, being second only to Alouatta, occurring from Central America to northern Argentina (Silva Junior 2001). In Brazilian Portuguese, this primate is popularly known as macaco prego (nail monkey) due to the conformation of the adult male reproductive organ that has the shape of a nail (Rocha 1992). Ecologically, these animals have a role in seed dispersal and in the control of potential agricultural and pests (Mikich et al. 2015).

Black capuchin monkeys are widely distributed and live in different types of environments that can be dry, such as the forests along the coast of Colombia and Venezuela; very humid, such as the Amazon and Atlantic forests; and Brazilian biomes, such as the Cerrado and Caatinga (Bicca-Marques et al. 2006).

Semideciduous forests, such as ecological parks, offer less pulp fruits than humid forests. Reduced food availability makes black capuchin monkeys seek new alternatives to supply their physiological and metabolic needs (Vieira and Oliveira 2014).

An important effect seen in habitat fragmentation is the presence of high primate density in small forest fragments (Jardim 2005). Owing to this situation, primate populations have been considered an important element in the transmission chain of emerging infectious diseases as reservoirs that allow the emergence of different zoonosis and the possibility of the presence and transmission of bacterial resistance making them important to the One Health (Garcia et al. 2005).

The most frequent bacteria found in nonhuman primates are enterobacteria of the family Enterobacteriaceae, such as Escherichia coli (Sobreira 2018, Zaniolo et al. 2018). Some microorganisms such as Salmonella spp. and the species Shigella or Yersinia pestis are always associated with human diseases, whereas, others such as E. coli, Klebsiella pneumoniae, and Proteus mirabilis are commensal members of the normal microbiota that can cause opportunistic infections (Pignatelli et al. 2014, Cruz et al. 2016).

Companion and wild animals can become ill when exposed to humans, but wild animals are more likely to become ill because they are less frequently exposed to human microorganisms.

Study by Zaniolo et al. (2018) researched enterobacteria samples in Sapajus nigritus from a municipal urban park located in a city in the northern region of Paraná state and identified only the bacterial species present in the samples, however, research related to antimicrobial resistance of bacteria in these same samples are essential, because of the lack of literature (possibly due to the difficulty of capturing this animal species) and the lack of data on the consequences of this interaction with humans, as well as the importance of free-living monkeys in urban parks as possible carriers of disease and bacterial resistance. Due to this situation, the aim of this study was to evaluate the antimicrobial resistance profile in members of the Enterobacteriaceae family and to detect extended-spectrum beta-lactamase (ESBL) producing strains in free-living S. nigritus.

Materials and Methods

The study was conducted on rectal and oral samples previously collected from primates identified as S. nigritus from a municipal urban park located in a city in the northern region of the state of Paraná from November 2014 to August 2015 (Zaniolo et al. 2018).

In brief, the primates were captured in Tomahawk traps, distributed in the park and checked every day. The usual elements of S. nigritus diet such as fruits and eggs were used as baits. After capture, the animals were physically contained with hand nets and anesthetized, then these animals were clinically evaluated and the rectal and oral samples were collected using sterile swabs (Zaniolo et al. 2018). The samples were cultured and isolated according to Zaniolo et al. (2018). Bacteria of the family Enterobacteriaceae were biochemically identified using an enterobacterial kit (NewProv^®, Paraná, Brazil) according to the manufacturer's recommendations.

The agar diffusion method was used to determine the bacterial resistance profile according to the Clinical and Laboratory Standards Institute (CLSI 2013, 2015) recommendations. The antimicrobials tested were gentamicin (10 μg), ciprofloxacin (5 μg), sulfatrim (25 μg), ceftazidime (30 μg), amikacin (30 μg), aztreonam (30 μg), chloramphenicol (30 μg), ampicillin (10 μg), tobramycin (10 μg), cefoxitin (30 μg), ceftriaxone (30 μg), cefotaxime (30 μg), tetracycline (30 μg), amoxicillin (10 μg), amoxicillin + clavulanate (30 μg), imipenem (10 μg), meropenem (10 μg), and norfloxacin (10 μg). As the samples were isolated from animals, the veterinary antimicrobial agents enrofloxacin (5 μg) and ceftiofur (30 μg) were also tested. After incubating the dish, the diameter of the inhibition halos around each antibiotic disk was measured to confirm sensitivity or resistance.

To determine resistance to multiple antibiotics resistance (MAR) index was used, defined as a/b, where a was the number of antimicrobials, to which the isolates were resistant, and b the number of antimicrobials, to which the isolates were exposed. Values above 0.200 suggest samples of high risk to public health (Krumperman 1983).

A double-disk synergy test was used to detect ESBL-producing strains, in which disks containing cefotaxime (30 μg), ceftazidime (30 μg), ceftriaxone (30 μg), and aztreonam (30 μg) were distributed at a distance of 20 mm from a disk containing amoxicillin + clavulanate (20/10 μg). Any increase or distortion in the inhibition halo of one of the antibiotics toward the amoxicillin + clavulanate disk was considered suggestive of ESBL production (Brun-Buisson et al. 1987).

Results

Samples were collected from the oral and rectal cavity of 15 young adult S. nigritus, 13 (86.66%) males, and 2 (13.33%) females, all weighing between 1.7 and 3.8 kg and clinically healthy, totaling 30 swabs.

Regarding bacterial resistance, Table 1 shows the resistance profile of all bacterial isolates of the family Enterobacteriaceae, showing that the least effective antibiotics were amoxicillin (72.72%), followed by ampicillin (57.57%), cefoxitin (45.45%), imipenem (42.42%), amoxicillin + clavulanate (23.33%), and aztreonam (23.33%). The other antibiotics presented values below 10% resistance.

Table 1.

Resistance Profile of Bacterial Isolates of the Family Enterobacteriaceae Collected from Oral and Rectal Swabs of Sapajus Nigritus Captured in an Urban Park of a City in the Northern Region of the State of Paraná, Brazil, 2014–2015

Antimicrobials	Rectal samples		Oral samples		Rectal + oral samples
Antimicrobials	R	%	R	%	Total	%
Norfloxacin	—	—	—	—	—	—
Enrofloxacin	—	—	—	—	—	—
Nalidixic acid	—	—	02	13.33	02	6.06
Ciprofloxacin	—	—	—	—	—	—
Tobramycin	01	5.55	—	—	—	—
Amikacin	—	—	—	—	—	—
Gentamicin	—	—	—	—	—	—
Meropenem	—	—	—	—	—	—
Imipenem	08	44.44	06	40.00	14	42.42
Ertapenem	—	—	—	—	—	—
Ceftazidime	—	—	01	6.66	01	3.03
Ceftriaxone	—	—	—	—	—	—
Cefotaxime	01	5.55	—	—	01	3.03
Ceftiofur	—	—	02	13.33	02	6.06
Cefoxitin	08	44.44	07	46.66	15	45.45
Cefepime	—	—	—	—	—	—
Ampicillin	08	44.44	11	73.33	19	57.57
Amoxicillin	11	61.11	13	86.66	24	72.72
Amoxicillin + clavulanate	04	22.22	03	20.00	07	21.21
Aztreonam	02	11.11	05	33.33	07	21.21
Sulfatrim	—	—	—	—	—	—
Tetracycline	03	16.66	03	20.00	06	18.18
Chloramphenicol		—	—	—	—	—

The antibiotic multiresistant index (MAR) showed that six (40.00%) samples were confirmed as high risk to public health, three (20.00%) oral cavity samples and three (20.00%) rectal.

Regarding ESBL production, no samples showed a distortion in the inhibition halo, which indicates no ESBL production.

Discussion

A previous study (Zaniolo et al. 2018) using the samples of the same origin reported that E. coli, a bacteria of great importance for human and animal health, was detected in 41.38% of the oral and rectal samples collected from 15 S. nigritus. These results suggested the importance of evaluating the presence or absence of antimicrobial resistance and also of detecting ESBL-producing strains due to the possibility of direct or indirect interaction between park visitors (garbage collectors, passersby, elderly people exercising, and elementary and middle school students participating in environmental classes among others) and monkeys.

In 2017, Medina et al. studied bacterial resistance in rectal swabs collected from nonhuman primate species living in the Primate Rescue and Rehabilitation Center of Peru and reported the prevalence of E. coli (42.5%), followed by bacteria of the genus Serratia (27.4%), with higher sensitivity to gentamicin (97.2%) and ceftriaxone (88.7%), similar to the present study. Even though the most resistant drugs were gentamicin and ceftriaxone, which was not corroborated by this study, it was probably because a different primate species was studied. However, Sobreira (2018) analyzed oral samples of free-living monkeys at an environmental park located in Anápolis (Goiás), corroborating the results of the present study, which showed that the most prevalent enterobacteria was E. coli and ampicillin and tetracycline were the antimicrobials to which resistance had developed the most.

Antimicrobial resistance refers to the ability of bacteria to multiply in the presence of antimicrobial agents at doses higher than those indicated for animal or human patients. It is a natural biological process resulting from the use of these drugs to treat infections (Wannmacher 2004) and accelerated by their irrational and indiscriminate use (Martins et al. 2015).

Regarding the multiple resistance index (MAR), the high risk to public health was found in six samples, indicating that these animals can become potential transmitters of resistance to humans and also to other species that inhabit the park.

The emergence and spread of ESBL-producing bacteria have been reported as an important public health problem especially related to pathogens associated with health care-related infections associated with human health care (Silva and Lincopan 2012). In addition, ESBL-producing strains are a therapeutic challenge due to their resistance to penicillin, cephalosporin, and monobactam antibiotics, reducing the number of therapeutic options (Silva and Lincopan 2012).

This study does not report the detection of ESBL-producing strains, which is an excellent finding, given the high interaction of these animals with the community. To our knowledge, there are no studies reporting the presence of ESBL-producing strains in nonhuman primates, but they have been detected in different animal species such as dogs, pigs, cattle, horses, and rodents among others (Carvalho et al. 2016, Fernandes et al. 2016, Arias et al. 2018).

This study shows the importance of conducting sanitary studies on this animal species, especially free-living animals in a municipal park in an urban area, where direct or indirect interaction with the community (public cleaners, hikers, elderly people exercising, and elementary and high school students to participate in environmental knowledge classes among others) is very intense. Due to this close interaction between primates and humans, molecular studies should be conducted for the detection and confirmation of possible resistance genes in these animals because of the possibility of this transmission among animals, parkgoers, and the environment itself.

Conclusion

The results show that this study is relevant to One Health initiatives, considering the possibility of bacterial resistance transmission and possible resistant genes originating from direct or indirect interaction of S. nigritus with parkgoers. However, we cannot consider only these animals as reservoirs and transmitters of resistant bacteria, because in addition to being important in the ecological maintenance of their habitat, they must also be considered as potential victims because they can be contaminated by bacteria of human origin that can be extremely harmful and often prove lethal to them.

Bacterial resistance can be better prevented by the integrated action between veterinary medicine and local health services where it is necessary to study the diseases in a broad way connecting the environment, human, and animal health and avoiding the importance of these results for the unique health. In dealing with free-living primates in an urban municipal park, new phenotypic and molecular studies should be conducted on this animal species and also on parkgoers to infer the potential genetic relationship between multiresistant primate bacteria and humans.

Footnotes

Acknowledgments

The authors are grateful to the owners for the constant collaboration, to UNIPAR for the financing awarded to this research, to CNPq, CAPES, and Araucária Foundation for the various study grants. We are also grateful to the pregnant women who participated for their constant collaboration.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

We are thankful to UNIPAR and the Araucária Foundation (CP 09/2016 Institutional Program for Basic and Applied Research – Protocol: 47661.491.48325.12122016 for funding this research, to CAPES for concession of the PROSUP school fee.

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