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Keywords:

  • Chimpanzee;
  • colonization;
  • furunculosis;
  • gorilla;
  • great apes;
  • interspecies transmission;
  • sepsis;
  • species barrier;
  • Staphylococcus aureus

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References

The risk of serious infections caused by Staphylococcus aureus is well-known. However, most studies regarding the distribution of (clinically relevant) S. aureus among humans and animals took place in the western hemisphere and only limited data are available from (Central) Africa. In this context, recent studies focused on S. aureus strains in humans and primates, but the question of whether humans and monkeys share related S. aureus strains or may interchange strains remained largely unsolved. In this study we aimed to evaluate the distribution and spread of human-like S. aureus strains among great apes living in captivity. Therefore, a primate facility at the International Centre for Medical Research of Franceville (Gabon) was screened. We detected among the primates a common human S. aureus strain, belonging to the spa-type t148. It was isolated from three different individuals of the western lowland gorilla (Gorilla gorilla gorilla), of which one individual showed a large necrotizing wound. This animal died, most probably of a staphylococcal sepsis. Additionally, we discovered the t148 type among chimpanzees (Pan troglodytes) that were settled in the immediate neighbourhood of the infected gorillas. A detailed analysis by pulsed field gel electrophoresis showed that the gorilla and chimpanzee isolates represented two closely related strains. To our knowledge, this is the first report of a human-associated S. aureus strain causing disease in great apes. The simultaneous detection in gorillas and chimpanzees indicated an interspecies transmission of this S. aureus strain. Our results recommend that protection of wild animals must not only be based on habitat conservation, but also on the assessment of the risk of contact with human pathogens.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References

Staphylococcus aureus is a leading human and veterinary pathogen. Its pathogenicity is based on a variety of virulence factors such as toxins and tissue-destroying enzymes [1]. Staphylococcus aureus causes a number of diseases ranging from local to systemic infections, which often lead to sepsis. In the last three decades, evolution of resistance, e.g. to methicillin, has become an enormous problem for treatment of S. aureus infections, turning this bacterium into one of the most prominent pathogens in hospital-associated infections [2].

In recent years, several studies have focused on animal colonization/infection by S. aureus, especially in pets [3, 4] and farm animals [5, 6]. In this context, a high prevalence of methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) strains in livestock species, e.g. cattle and pigs, became prominent [7]. These studies clearly demonstrated the occurrence of interspecies transmission, which in turn highlights the possibility of zoonotic infections of humans by livestock-associated S. aureus. On the other hand, this also opens the possibility of epidemics in animal populations caused by human-associated strains [8].

Recently, Schaumburg et al. [9] demonstrated that human-associated S. aureus strains are widely distributed on wild great apes. In contrast, in the same study, monkeys were mainly colonized by strains belonging to new multilocus sequence-types or spa-types, which had only rarely been isolated from humans [9]. However, our knowledge about interspecies transmission of pathogens from humans to primates is scarce.

In this study, the transmission of a widely distributed human-associated S. aureus strain (spa-type t148) and its species barrier transgression are described. This strain was able to colonize three individuals of a gorilla population; one of these gorillas died of an infection that was most likely caused by this strain, starting with a furuncle and ending in sepsis. A closely related variant of the same spa-type was also isolated from chimpanzees living in the direct neighbourhood of the infected gorilla, so that an interspecies transmission of the strain between the two great apes populations seems to be likely.

To our knowledge, this is the first report clearly demonstrating a direct correlation between a lethal infection of a great ape caused by a well-known and widely distributed human S. aureus strain and interspecies transmission of the causative bacteria.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References

Sampling

All individuals at the Centre of Primatology were routinely checked by a general medical examination, including biological and clinical diagnostics in November and December 2011. Primate samples were obtained non-invasively during routine health surveys, as nasal, oral, vaginal and rectal swabs; this procedure is not considered to be an animal experiment. The samples were immediately frozen at −80°C and afterwards delivered for bacteriological analysis. All samples were collected in accordance with international guidelines applied at the International Centre for Medical Research of Franceville Centre of Primatology.

Microbiological analyses

Isolated bacteria were cultivated on Columbia agar plates (Becton and Dickinson, Heidelberg, Germany), Mueller Hinton II (Difco, Detroit, MI, USA) or in tryptic soy broth (Oxoid, Wesel, Germany) at 37°C. Gram-positive and catalase-positive cocci were identified as S. aureus using BD Aureus select agar (Becton and Dickinson). Bacterial species identification was confirmed by standard slide tests for the clumping factor and the 4- to 24-h tube test for free coagulase in rabbit–citrate–plasma (Becton and Dickinson). Staphylococcus aureus ATCC 33592 (MRSA; American Type Culture Collection, Wesel, Germany) and Staphylococcus epidermidis DSM 20044 (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) served as positive and negative controls.

Molecular typing

Staphylococcus aureus isolates were discriminated by multilocus sequence-typing (MLST) [10], pulsed field gel electrophoresis (PFGE) [11] and spa-typing [12].

For spa-typing, five colonies were resuspended in 100 μL sterile water (Ampuwa, Fresenius, Bad Homburg, Germany). The cell suspension was heated for 10 min at 95°C. Cell debris was pelleted by centrifugation (5 min, 20 000 g, at room temperature). An appropriate volume of the resulting supernatant served as template for PCR. The repeat region of the spa gene was amplified by PCR [13]. Resulting PCR products were sequenced (Seqlab, Göttingen, Germany) and spa-types were determined using the StaphType software and the Ridom SpaServer (www.spaserver.ridom.de).

The MLST was performed as described previously [10]. Chromosomal DNA was purified using the PrestoSpinD BUG kit (Molzym, Bremen, Germany) according to the supplier's instructions. Cell lysis was achieved by supplementation of 10 μL lysostaphin (5 mg/mL; Genmedics, Reutlingen, Germany). Sequencing was performed by Seqlab (Göttingen, Germany). Analyses were performed employing the S. aureus MLST site (http://saureus.mlst.net/).

Pulsed-field gel electrophoresis was performed as previously described [11]. Briefly, chromosomal DNA was purified and digested using the restriction enzyme SmaI (Roche, Mannheim, Germany) and analysed on the DRIII contour-clamped homogeneous electric field system (Bio-Rad, Munich, Germany) by using pulsed field gel electrophoresis (1%; Bio-Rad), 6 V/cm, a field angle of 120°, a switch time of 5–15 s for 7 h, and a switch time of 15–60 s for a further 19 h. Staphylococcus aureus NCTC 8325 served as standard.

All strains were analysed for antimicrobial susceptibility by the agar disk-diffusion method including cefoxitin, oxacillin, ampicillin, ampicillin + sulbactam, cefazolin and penicillin (Oxoid). The latex-agglutination-based test systems (Oxoid) were used to test for production of the staphylococcal enterotoxins A, B, C and D and the toxic shock syndrome toxin (TSS). Expression of the exfoliative toxins A and B was detected by specific polyclonal antibodies in Ouchterlony immunodiffusion tests. The strains were tested for the presence of the mecA and the Panton–Valentine leukocidin encoding genes (lukSF) employing the Genotype Staphylococcus test kit (Hain Lifescience, Nehren, Germany).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References

Bacterial strains were isolated from three captive western lowland gorillas (Gorilla gorilla gorilla) and six chimpanzees (Pan troglodytes) in the Centre of Primatology at the International Centre for Medical Research of Franceville, Gabon. A total of 13 S. aureus strains, belonging to three spa-types (t148, t56 and t5017), were identified. All strains were confirmed as MSSA, but were resistant against penicillin and ampicillin (Table 1). A prevalence of one specific spa-type (t148) was obvious. Initially, S. aureus strain t148 was isolated from a wound on the back of an infected gorilla. This gorilla, which had reached an advanced age (38 years) even for captive gorillas [13], had been in good health at previous medical checks. During an ensuing health check, the veterinarians confirmed a weight loss of 12 kg from its initial weight of 80 kg. A large lesion on the back of the gorilla with an approximate size of 4 × 6 cm and about 1-cm in depth (Fig. 1) was also observed. Swabs were taken from this lesion for microbial identification and the t148 spa-type was isolated. After the sudden death of this gorilla, different tissue samples were taken from heart and lung during autopsy. The spa-typing of the isolated S. aureus strains revealed that these isolates also belonged to the t148 spa-type. In a PFGE analysis, the strains showed an identical restriction pattern (Fig. 2, lanes 10–13) that was indistinguishable from the strain that had previously been isolated from the infected wound of the same individual (Fig. 2, lane 5). These findings indicate that the individual most probably died of a staphylococcal sepsis, caused by the t148 strain. Yet, S. aureus could not be isolated from the nose and oral cavity, indicating that the great ape was not colonized by this strain previous to the fatal infection.

Table 1. Staphylococcus aureus isolates identified from gorillas and chimpanzees. Thirteen S. aureus strains of three spa-types (t148, t52 and t5017) were isolated from three gorillas and six chimpanzees
Species, nameSample locationS. aureus presentspa-typeST-typeaAntibiotic resistanceToxinEnclosurePFGE lane
  1. PFGE, pulsed field gel electrophoresis; ST, sequence type.

  2. a

    n.d., not determined.

Gorilla TyphenLesionYest148ST72Ampicillin and penicillinEnterotoxin CB5
HeartYesn.d.n.d.10
HeartYesn.d.n.d.11
LungYesn.d.n.d.12
LungYesn.d.n.d.13
Gorilla TaniVaginalYesn.d.n.d.A6
VaginalYesn.d.n.d.7
Gorilla CarolineOralYesn.d.n.d.2
Gorilla Cola

Rectal

nasal

oral

No S. aureus isolatedB 
Gorilla Djoutou

Rectal

nasal

oral

No S. aureus isolatedA 
Chimpanzee NzelaLesionYest148ST72Ampicillin and penicillinEnterotoxin CC + D3
Chimpanzee PatOralYesn.d.n.d.C4
Chimpanzee JeffNasalYesn.d.n.d.D9
Chimpanzee Nuria

Nasal

oral

Yest56n.d.Sensitive to ampicillin and penicillinn.d.C 
Chimpanzee CharlesOralYesn.d.n.d.
Chimpanzee MebaleNasalYest5017n.d.n.d.
image

Figure 1. The lesion on the dead gorilla. The lesion infected by the t148 Staphylococcus aureus strain was found on the back of the gorilla ‘Typhen’ (size of 4 × 6 × 1 cm).

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image

Figure 2. Pulsed field gel electrophoresis (PFGE) analysis of the t148 strains isolated from gorillas and chimpanzees. All t148 strains isolated from great apes were analysed by PFGE. An identical restriction pattern was observed for strains that had been isolated from different body sites of the deceased gorilla ‘Typhen’ (lane 5 = wound isolate, lane 10 + 11 heart isolates, lane 12 + 13 lung isolates) and of the colonized gorillas ‘Caroline’ (lane 2) and ;Tani' (lanes 6 + 7). A nearly identical PFGE pattern was observed for all analysed chimpanzee isolates (lanes 3, 4, 9). The methicillin-sensitive Staphylococcus aureus strain NCTC 8325 served as standard (lanes 1 + 8).

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The t148 spa-type was identified in different body samples taken from two other gorillas and three chimpanzees (Table 1), including one isolate that was swabbed from a lesion of a healthy chimpanzee. All veterinarians, monkey carers and scientists who were involved in this project, were also swabbed. However, neither this spa-type nor the other spa-types identified on great apes in this study could be isolated from these humans. For the two gorillas ‘Cola’ and ‘Djoutou’ that were housed together with the other gorillas, colonization with S. aureus could not be detected (Table 1).

The isolated strains of spa-type t148 were analysed by PFGE analysis (Fig. 2). Thereby, an identical PFGE pattern was observed for all gorilla isolates (lanes 2, 5–7). Also, strains that were isolated from chimpanzees shared a similar PFGE pattern (lanes 3, 4 and 9). Interestingly, the PFGE restriction patterns showed a slight deviation that appeared as a shift of one band.

Two of the strains with the almost similar PFGE pattern were isolated from wound lesions of a gorilla and a chimpanzee, respectively, and additionally analysed for their ability to produce staphylococcal toxins. For both strains enterotoxin C production was observed, but tests for the enterotoxins A, B and D, the TSS toxin and for the genes (lukSF) encoding for the Panton–Valentine leukocidin yielded negative results. For a closer analysis, both strains were subsequently included in an MLST analysis. Here, a common allelic profile (1-4-1-8-4-4-3) was determined, identifying both strains as the sequence type 72 that belongs to the clonal complex 8 [14], which is widespread in humans.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References

In this study we aimed to evaluate the distribution and spread of human-like S. aureus strains among great apes living in captivity. Previously, two studies reported infections of two great apes, an orang-utan [15] and a chimpanzee [16], with S. aureus causing oral ulcers and septicaemia or staphylococcal meningitis, respectively.

We identified common human S. aureus strains among captive gorilla and chimpanzee populations at the Centre of Primatology of the Centre of Medical Research in Franceville (Gabon). A prevalence of one specific S. aureus type became obvious here. By spa- and MLST-typing the dominant strain was assigned to the spa-type t148 and the sequence type ST72, respectively, and is hence characterized as a widely distributed and human-associated strain.

This particular strain was initially isolated from the inflamed wound of an individual gorilla, which later died, most likely from a staphylococcal septicaemia caused by this specific strain. In agreement with our findings, the recent literature demonstrates that strains of the type t148 are able to cause bloodstream infections as well as to colonize healthy human beings [17, 18].

Strains of the t148 spa-type are documented as human isolates on the Ridom spa-server homepage and were detected in 207 cases worldwide. More than 50% (107 of 207) of the isolates are MRSA. Human t148 spa-type MSSA have previously been isolated in nine cases in Gabon. In line with this, S. aureus of the ST72 type (CC8) appears to be among the predominant strains found in the nasal flora of the Gabonese Babongo pygmies [19]. Colonization of wild non-human primates by human-related S. aureus strains of the MLST types ST1 (CC1), ST9 (CC9) and ST601 was recently reported for great apes and for an unknown MLST type isolated from monkeys [9]. Also, colonization of captive macaques by human-associated staphylococcal strains belonging to new ST- and spa-types was described [20].

Beside the deceased gorilla ‘Typhen’, in this study, S. aureus strains of the t148 spa- and ST72-types were isolated from two other gorillas and three chimpanzees. For two of the chimpanzees, the strains were isolated from noses, indicating colonization. One chimpanzee isolate was obtained from an infected wound, whereas no S. aureus could be isolated from the mouth and nose of this individual.

In a PFGE analysis the chimpanzee-associated and gorilla-associated t148 strains were shown to represent two closely related variants of the same PFGE type with a shift of one band in their PFGE patterns. These differences are minor (i.e. single genetic events) and the isolates are considered, by definition, to be closely related and belong probably to the same strain [21]. In fact, it has been previously shown that S. aureus can show certain variability in the genomic DNA fragment pattern during the course of an epidemic [22]. Based on these results, we assume an interspecies transmission of the t148 strain between chimpanzees and gorillas, which resulted in the establishment of two different variants of the same MSSA strain.

For three more chimpanzees that were living in the neighbouring enclosure, cross-colonization with S. aureus was also determined. However, these isolates belonged to two other human-related spa-types, t56 and t5017. These findings are in line with the observations published by van den Berg et al. [20] for the colonization of macaques. The authors described a frequent transmission of S. aureus between individuals that live in the same animal room, resulting in colonization of the individual with up to two epidemic strains in each enclosure.

In conclusion, although the origin of the t148 strain could not be determined in this study, the spread of the strain can be hypothesized as follows: the dead gorilla ‘Typhen’ was settled in an enclosure (B in Fig. 3) next to two gorillas ‘Caroline’ and ‘Tani’ that lived in a neighbouring enclosure (A in Fig. 3). Although both outside lawn areas are separated by a 4-m high concrete wall, the inside sleeping areas (A to C in Fig. 3) are connected and the gorillas can come into contact with each other through the separating fences; in this way bacterial transmission can occur. In the right wing of the building (C in Fig. 3) the two chimpanzees, ‘Pat’ and ‘Nzela’ were housed and also colonized by the t148 strain. The barrier situation within this enclosure is the same as described above. Although no direct contact is possible, bacterial transmission may readily occur by different means (e.g. by spitting) or by chewing fruits and throwing the fruit skins, contaminated with bacteria from the oral cavity [9]. The colonized chimpanzee ‘Nzela’ (initially within section C) had been settled into the quarantine station (D) where the apes have close contact with each other and where another chimpanzee, ‘Jeff’, was already housed. In the end, the t148 strain was isolated from both chimpanzees during the analysis.

image

Figure 3. The Centre of Primatology facilities at the International Centre for Medical Research of Franceville, Gabon. Gorillas were located in section A: ‘Caroline’, ‘Tani’ and ‘Djoutou’; in section B: ‘Typhen’ and ‘Cola’. Chimpanzees ‘Pat’, ‘Nuria’, ‘Charles’, ‘Mebale’ and initially ‘Nzela’ were housed in section C. Finally, ‘Nzela’ was located with ‘Jeff’ in the quarantine section D.

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The primate species that were examined within this study, the western lowland gorilla and the chimpanzees, are two threatened species of the Central African region, categorized as critically endangered (CR) and endangered (EN) species [23], respectively. A zoonotic risk resulting from human to wild animal transmission and vice versa has been largely documented [24]. In this context, it has been shown that (i) human retroviral infections, e.g. human immunodeficiency virus and human T-lymphotropic virus, are rooted in simian zoonotic transmissions through handling and butchering primates or by close contact with them [25]; and that (ii) gorilla and chimpanzee populations have suffered from several often devastating infections, e.g. Ebola virus [26] or anthrax [27]. A transmission of (antibiotic-resistant) S. aureus of human origin might represent a potential threat to populations of great apes, especially as it has been demonstrated that transmissions of MRSA and community-acquired MRSA between humans and animals are possible [28]. Also the transmission of community-acquired MRSA could increase as a result of it pathogenicity [29], even though this remains a hypothesis [9, 19].

The impact of infectious diseases on the health and ecology of wild species needs challenging multidisciplinary and long-term surveys, in particular in the equatorial African rain forest with its exceptional biodiversity. Taken together, the potential of interspecies transmission has been underestimated so far and needs to be studied extensively on both animal and human grounds of conservation medicine, risk assessment of emerging zoonotic diseases and health ecology including environmental factors [30].

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References

Funding was provided by the French Embassy through the French Ministry of Foreign and European Affairs, Libreville, Gabon; Global Virus Forecasting Initiative, San Francisco, CA, USA. This publication made use of the spa-typing website http://www.spaserver.ridom.de/) that is developed by Ridom GmbH and curated by (http://www.SeqNet.org/).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References
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