Phenotypic and genetic antimicrobial resistance of the intestinal microbiota isolated from two alpacas (Vicugna pacos) post mortem

The alpaca (Vicugna pacos) is a domesticated species of camelid native to the Andean region. In Peru, alpacas possess important cultural value due to their historical significance and generate substantial economic benefits for communities involved in alpaca breeding by providing high-value meat with low cholesterol content, as well as skin and hypoallergenic fibre. In Poland, these animals are companion animals in petting zoos, or are used for therapeutic purposes and for wool production.

It is evident that gut-associated microbes have a significant impact on the nutritional status and health of their hosts, particularly where these hosts consume diets high in cellulose, as is the case with alpacas; unfortunately, little is known of their intestinal microbial population. There is one publication in which the effect of diet was investigated on the microbiota present in the forestomach compartment and the small and the large intestine of alpacas (5). Being frequent companion animals, alpacas are often in close contact with humans, and there is a need to enhance our understanding of the bacteria that constitute their gut microbiota and the potential drug resistance of the bacteria therein. The trend towards increasing drug resistance noted among microorganisms such as methicillin-resistant Staphylococcus aureus (MRSA) and totally drug-resistant and multidrug resistant Mycobacterium tuberculosis strains is an alarming one that demands urgent attention. In recent times, there have been concerted efforts to minimise the use of antibiotics in veterinary medicine, particularly with regard to farm animals, with a view to curbing the emergence of drug-resistant variants of pathogenic microorganisms. Drug resistance in environmental pathogens is a threat, it is essential to monitor continuously in application of the One Health principle, which is also the rationale for investigating innovative treatment options (17, 24, 28, 31, 32).

Bacteria have evolved various mechanisms for acquiring antimicrobial resistance (AMR), including the exchange and implementation of antimicrobial resistance genes. These resistance genes are frequently found on plasmids or transposons, which can be transferred between cells via conjugation, transformation or transduction. Bacterial communities (i.e. microbiota) rapidly acquire antibiotic resistance through such gene transfer, and this resistance spreads within and between different bacterial species inhabiting the same environment, such as the intestines (3, 20). One factor believed to have a significant impact on the transmission of antibiotic-resistant strains is the acquisition of antibiotic resistance by components of the microbiota of wild animals. It is conceivable that wild animals may act as carriers for these antibiotic-resistant strains, thereby contributing to the dissemination of resistance genes in the environment. From the soil and water environmental compartments, resistant strains may be transferred to other ecosystem members through contact.

The potential for the transmission of antibiotic-resistant microbes between humans and companion or wild animals is therefore a matter of concern. There is a need for greater monitoring of drug resistance in both pathogenic bacteria and in the natural microbiota and the environment, as this is a crucial step in identifying potential threats to both human and animal health (2, 18). Fortunately, recent years have seen a marked increase in our understanding of the role played by the bacterial microbiota in many animal species and in our awareness of microbiome elements’ potential to be shared among animals, humans and the environment. While alpacas are considered to be relatively disease resistant, they are believed to be at risk of parasitic infestations and diarrhoea (2).

Amikacin, ampicillin, ceftiofur, amoxicillin, amoxicillin and clavulanic acid, florfenicol, enrofloxacin, marbofloxacin, gentamicin, tetracycline, tobramycin and trimethoprim/sulfamethoxazole are used in alpaca treatment (15, 16). These drugs increasingly often fail against phenotypically and genotypically resistant bacteria, and it is strategically important to learn if strains of such bacteria are prevalent in particular species of farmed animals. Given that in Poland, the past two decades have demonstrated a growing trend in the breeding of newly introduced animal species including alpacas, which were officially recognised as farm animals in 2021, this study was planned to determine the phenotypic and genotypic drug resistance of bacteria isolated post mortem from the intestines of two alpacas with diarrhoea.

All bacteria were identified and characterised as described in a previous publication by the authors (23). Most of the bacteria isolated were Gram-positive, including dominant species such as Bacillus spp., Enterococcus spp. and Staphylococcus spp. Other Gram-positive bacteria identified included Cor. diphtheriae and Cor. stationis. Micrococcus luteus was also isolated, being variously indicated as Gram-positive or Gram-variable. Various Gram-negative bacteria were also identified, including Ent. cloacae, Ent. hormaechei, Ent. ludwigii and Ent. gergoviae; Citrobacter spp.; E. coli; and Stenotrophomonas maltophilia, Ser. liquefaciens, Ser. odorifera and Ser. marcescens. No resistance against IMI or COL was detected when all isolates of Citrobacter and Enterobacter (confirmed to be so by MALDI-TOF MS and biochemical tests) were subjected to microdilution MIC testing using the MICRONAUT plates as described above.

All C. freundii were resistant to AMP, AMO, AMC, first-generation cephalosporins, tetracyclines, FLO and SXT. Citrobacter freundii isolated from alpaca 1 was resistant to fluoroquinolones (ENR and NOR), while those isolated from the faeces of alpaca 2 were only resistant to NOR. All tested strains were resistant to second-generation fluoroquinolones (ENR and NOR), but not to the third-generation MAR. All Enterobacter strains were resistant to AMP and first-generation cephalosporins, seven Enterobacter strains were additionally resistant to AMO, three were to AMC and four were resistant to SXT. Escherichia coli was resistant to AMP, AMO and CF (Table 1). Judged against the TECOFF, E. coli showed resistance to STR (MIC > 16) (Supplementary Tables S1, S2 and S3). No IMI resistance was detected in any isolated species. No extended-spectrum β-lactamases (ESβLs) were detected by the screening in AST-GN96.

The TetA gene, encoding the tetracycline efflux pump, was found in C. freundii, Ent. cloacae and Ser. liquefaciens; all species exhibited phenotypic resistance to tetracyclines. In addition, Ser. marcescens was found to harbour tetB, encoding a tetracycline efflux protein which confers resistance to TET and DOX. Both the sul1 and sul2 genes, associated with sulphonamide resistance, were found in Ent. cloacae isolated from the colon swab of alpaca 1. In addition, sul1 was detected in all C. freundii, in Ent. cloacae isolated from the colon of alpaca 1 and the faeces of alpaca 2, and in SXT-resistant Ent. hormaechei. Interestingly, no tet or sul genes were detected in C. braakii, despite it demonstrating phenotypic resistance to tetracyclines and SXT. The aadA gene, encoding resistance to STR, was detected in sSTR-susceptible C. freundii and STR-resistant E. coli, together with the STR resistance genes strA and strB. All Enterococcus species were resistant to PG, BEN and ERY. Entc. gallinarum, Entc. faecium and Entc. hirae were resistant to aminoglycosides (GEN, NEO and STR), but Entc. casseliflavus was not. Enterococcus casseliflavus, Entc. gallinarum and Entc. hirae were resistant to ERY, but Entc. faecium was not. Four strains were TET resistant, and Entc. hirae and Entc. casseliflavus isolated from the colon and rectum of alpaca 2 were resistant to FLO. The analysis based on TECOFFs indicted that Entc. faecium showed resistance to GEN (MIC > 32 μg/mL), NEO (MIC > 64 μg/mL) and STR (MIC > 128 μg/mL). Enterococcus hirae showed resistance to GEN (MIC > 32 μg/mL), NEO (MIC > 128 μg/mL) and STR (MIC > 128 μg/mL). No resistance to vancomycin or teicoplanin was detected in any Enterococcus strains.

Four Staphylococcus species were resistant to AMO. Staphylococcus pseudintermedius and S. sciuri were resistant to PG and BEN, but S. aureus was not. Two S. aureus strains were resistant to ENR, and S. pseudintermedius was resistant to ERY. None of the Staphylococcus strains demonstrated methicillin resistance, as indicated by cefoxitin disc diffusion or AST-GP79 cards. Micrococcus luteus was resistant to PG, BEN, ERY and CLI. When cross-referenced with the TECOFF, S. aureus demonstrated resistance to NEO (MIC > 1 μg/mL) and STR (MIC > 16 μg/mL) and S. pseudintermedius to TYL (MIC >2 μg/mL). Micrococcus luteus was resistant to PG, BEN, ERY and CLI according to the CLSI M45 method standard (8) (Table 3).

All Bacillus species were resistant to PG and SXT. Thirteen of them were resistant to ERY, three were resistant to tetracyclines (DOX and OXY) and one (B. flexus) to GEN (Table 4, Supplemenatary Tables S4 and S5).

The antimicrobial resistance profiles of the remaining isolates were also analysed. The anatomical source of resistant strains and the corresponding profiles are presented in Table 5.

Both Cor. diphtheriae and Cor. stationis were resistant to penicillin, tetracyclines (DOX and OXY), ERY and SXT. Leuconostoc citreum was resistant only to PG and Stenotrophomonas maltophilia to SXT. (Supplementary Tables S6, S7, S8, S9, S10, S11, and S12).

Few works have examined the microorganisms in the microbiota of New World camelids (the guanaco, vicuña, llama and alpaca) (25). As these animals may have the potential to transmit zoonotic infections, it is crucial that veterinarians possess a comprehensive understanding of the bacterial infections suffered by alpacas, the species with which humans interact most. This need is even more pressing as alpaca breeding has recently gained popularity in regions where they are not indigenous, including Poland. Close interaction may also result in increased transmission of bacteria between humans and alpacas. Even if non-pathogenic bacteria are transferred between humans and animals, each new colonisation has the potential to modify the composition of the gastrointestinal microbiome, which may impair the ability of the host to resist colonisation by exogenous bacteria. If pathogenic and antibiotic-resistant strains are transferred, each cross-infection is a part of the global spread of AMR, which has been recognised as a matter of significant concern and a motive for continuous epidemiological surveillance in both human and non-human hosts.

The present study examined the intestinal bacterial composition of alpacas to evaluate the antibiotic-resistant profiles at both the genotypic and phenotypic level. Our findings confirmed the presence of antibiotic-resistant bacteria in animals that had not previously been exposed to antibiotics. Genes for AMR were identified in C. braakii, C. freundii, Ent. cloacae, Ent. hormaechei and E. coli. The identified bacteria also demonstrated varied resistance profiles, Citrobacter, Serratia and Enterococcus spp. exhibiting the highest level of resistance as determined by CLSI and EUCAST breakpoints.

It is possible that Enterobacterales may demonstrate resistance to first-line antibiotics such as ENR, GEN, OXY, penicillin, or SXT, because these antibiotics have been commonly used for many years by veterinarians in treating infections with bacteria in this taxonomic order in cattle, chickens and pigs. Enrofloxacin is used in the treatment of a wide range of farm and exotic animals because of its safety and efficacy against a variety of pathogens; however, our findings indicate that three C. freundii and two Ent. cloacae isolates were resistant to this antibiotic. All Enterobacter species are intrinsically resistant to AMP, AMO and first-generation cephalosporins owing to their production of constitutive AmpC, a chromosomal group 1 β-lactamase.

Our findings also indicate that all the Citrobacter isolates were resistant to FLO; however, while four of the five isolates carried the floR gene, it was not present in the C. freundii isolated from the faeces of alpaca 2. The same was noted for the Serratia strains. The bacteria positive for the floR gene had much higher MICs for FLO than the floR-negative strains (33). Escherichia coli isolated from the colon (1) was resistant to AMP, AMO, CF and STR. The aadA and strA/strB genes encode resistance to STR, and were detected in STR-resistant E. coli. No ESβL-producing strains were found in this study, unlike in the study of Cardenas-Arias et al. (4), which reported the presence of ESβL-producing E. coli ST10 in llamas.

Enterococcus hirae was the most frequently-detected species, followed by Entc. casseliflavus, Entc. gallinarum and Entc. faecium. This is consistent with data obtained for other camelids (14). It is well known that Enterococcus spp. exhibit decreased susceptibility to penicillin and AMP, as well as to semi-synthetic penicillins, because they express low-affinity penicillin-binding proteins. Our findings also identified resistance to aminoglycosides (GEN, NEO and STR), ERY and FLO; a similar profile of dissemination of antibiotic resistance was previously noted for Enterococcus spp. isolated from camelids (14). In contrast, Bacillus and Staphylococcus spp. exhibited the highest susceptibility to all tested antibiotics.

The findings of the present study on Staphylococcus are consistent with those reported in previous research on camelids (21). Nonetheless, several significant differences were noted, particularly in relation to the occurrence of drug resistance. Staphylococci are regarded as opportunistic pathogens, manifesting as colonisers and demonstrating the capacity to induce a broad spectrum of severe infections. Staphylococcus sciuri is often detected in animals, humans and the environment, where it is regarded as non-pathogenic; it has also been identified in diseased animals (21, 26). In our study, staphylococci showed resistance to AMO, PG, BEN, ENR and ERY. The TECOFFs imply that S. aureus resists NEO and STR, and that S. pseudintermedius resists TYL. Methicillin-resistant S. aureus has been identified as one of the most significant multidrug-resistant pathogens globally. The prevalence of MRSA among New World camelids is low but nevertheless alarming (26). In the current study, the tested Staphylococcus strains did not demonstrate methicillin resistance in cefoxitin disc diffusion or screening in AST-GP79 cards. However, previous research has identified β-lactam resistance in llamas and alpacas (27). Staphylococcus sciuri isolated from alpacas was found to display multiple resistance, with the bacteria being non-susceptible to at least one antimicrobial agent from three or more antibiotic classes (21).

Bacillus strains isolated from alpacas were susceptible to most tested antibiotics, but all Bacillus species were resistant to PG and SXT. Thirteen of the seventeen Bacillus isolates were resistant to ERY, and three to tetracyclines. Bacillus flexus was the only species resistant to GEN, which is consistent with the findings of another study (12).

Moraxella osloensis isolated from the small intestine of alpaca 2 showed resistance to PG and ERY. The prevalence of macrolide-resistant Moraxella has given rise to concerns regarding therapeutic interventions in cases of infection with species in this genus, particularly in children suffering from pneumonia caused by M. catarrhalis. Macrolide-sensitive Moraxella cells rapidly accumulate large amounts of macrolide antibiotics, whereas no such accumulation was observed in macrolide-resistant cells with smooth-type or rough chemotype c lipopolysaccharide (30).

Stenotrophomonas maltophilia has been identified as an opportunistic pathogen of significant concern to susceptible humans. It is known to play an important role as a nosocomial pathogen associated with infections, especially in immunocompromised or debilitated patients, and demonstrates remarkable intrinsic AMR and acquired resistance to multiple antimicrobial agents. In the present study, the Stenotrophomonas maltophilia isolated from the colon of alpaca 1 was found to be resistant to tetracyclines and SXT. However, the lack of reproducible AST, lack of clinical breakpoints and an absence of clinical outcome data rule out regarding these results as conclusive.

The Micrococcus luteus and Leuconostoc citreum isolated from the alpaca gut microbiota were different from those adapted to humans, and they remained free of antibiotic resistance mechanisms. Micrococcus luteus was resistant to PG, BEN and ERY, and one isolate was to CLI, while Leuconostoc citreum was only resistant to PG. Interestingly, while Corynebacterium spp. were resistant to penicillin, the tetracyclines, ERY and SXT in the present study, no such resistance was observed for Cor. pseudotuberculosis isolated from alpacas afflicted with lymphadenitis or subcutaneous abscesses by Anderson et al. (1). All strains were susceptible to tetracyclines, ERY and SXT.

A few studies have described the microbiota of alpacas and related camelids, including those of the eye (13, 29), ear (19), and skin (6). However, only a small number of studies have focused on the gut microbiota. Research into the microorganisms present in exotic animals such as alpacas and the antibacterial resistance profiles of those microorganisms is of critical importance, particularly in view of the dissemination of these bacteria both within the environment and between humans and other animal species.

Our findings indicate that post mortem sampled bacterial strains constituting the intestinal microbiota of alpacas had different antibiotic resistance profiles at the genotypic and phenotypic levels. Many of the bacteria isolated from the intestinal tracts of the alpacas demonstrated high antibiotic susceptibility, which is most likely due to low direct exposure to antibiotics. This absence of resistance could suggest that the capacity for transmission of antimicrobial resistance from alpacas to humans is limited. However, while most bacteria isolated from alpacas were different from those adapted to humans, our results indicated that potentially pathogenic isolates were also present in the alpaca intestine. Therefore, to confirm the risk of zoonoses and the potential transmission of AMR genes, it is recommended that monitoring should be extended over a longer period. As a response to the increase in the popularity of alpacas for recreation in recent years, more research on New World camelids should be carried out, especially when considering their increasing proximity to domestic and domiciled animals, as well as humans.