Abstract

1. Introduction

Decades after the emergence of Methicillin resistant Staphylococcus aureus (MRSA) in humans, first in health care centers and then also in the community, livestock associated MRSA (LA-MRSA) were discovered. Marked geographical differences were found in the prevalence of LA-MRSA, with the European strains differing significantly from Asian and North American strains [1]. The body of evidence that MRSA has also emerged in wildlife is ever increasing. A recent review showed the importance of the wildlife reservoir of MRSA and potential sentinels for antimicrobial resistance and the Clonal Complexes (CCs) 398 and 130 were identified as the most prevalent with an over-representation of the mecC gene [2]. However, it should be pointed out that there are few studies available, and those may represent a bias towards Europe as most studies were performed in this region.

The small Indian mongoose was first introduced into the Caribbean in the 1800s to control rats and snakes on sugar cane plantations and are regarded as an invasive species. While the rats could not be controlled, the snakes disappeared from the island and now the mongooses endanger mainly birds nesting on the ground.

St Kitts and Nevis are two small Caribbean islands with approximately 57,000 inhabitants. We recently observed a high prevalence of MRSA US300 clone amongst human clinical infections in St. Kitts [3]. Since the island is also home to a large monkey population, we recently applied a One Health approach to investigate staphylococcal diversity in vervet monkeys where we detected only methicillin resistant coagulase negative strains [4]. In our efforts to determine the role of invasive animals in antimicrobial resistance, we investigated the microbiomes of the small Indian mongoose on the island of St Kitts [5]. Mongooses have the typical microbiota of a carnivorous feeding pattern with a dominant abundance of Firmicutes (54.96%), followed by Proteobacteria (13.98%) and Fusobacteria (12.39%), and a relatively minor proportion of Actinobacteria (10.4%) and Bacteroidetes (6.40%). Some differences were observed based on the area where the animals were captured and between male and female animals [5]. While S. aureus is not a major component of the microbiome [5], we decided to further characterize the S. aureus isolates as there is a major problem with MRSA infections in humans on the island [3]. This One Health approach will allow us to determine potential reservoirs and determine the spread of strains in wildlife. Out of samples from the former study [5], we isolated 5 S. aureus and analyzed them by whole genome sequencing. Here we report the presence of a novel sequence type (ST) of methicillin susceptible S. aureus (MSSA) and MRSA ST5 in the intestines of mongooses in St Kitts.

2. Results

Out of 81 samples, five S. aureus isolates were obtained from five animals. Table 1 shows the details of the isolates. Two isolates were identified as MRSA and three as MSSA. Of the three MSSA, two belonged to a new sequence type, ST7434, as a single locus variant within the clonal complex 30, with spa type t019, while the third MSSA isolate was a ST5 t002. The two MRSA isolates, originating from two different animals caught in the same region (Paradise Heights, peri-urban area), were both ST5 spa type t002. The difference between both MRSA ST5 isolates was a single SNP. The two MSSA ST7434 isolates originated from different animals and different neighborhoods, but were very similar, showing only four SNPs difference. The difference between the MSSA ST5 and MRSA ST5 was substantial with about 524 SNPs difference. The ST5 MRSA were compared to two MRSA ST5, one spa type t002 and one t2235 isolated from human infections in St. Kitts [3]. The human strains differed from each other by 71 SNPs, and differed 530 SNPs from the closest mongoose strain, which was the MSSA ST5 (Figure 1).

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Figure 1

SNP tree of the 2 human MRSA and 3 mongoose S. aureus ST5 strains.

The MSSA ST7434 had two exoenzymes, three genes of the gamma-hemolysin, six enterotoxins and the toxic shock syndrome toxin-1. Both the MSSA and MRSA ST5 strains, had substantially more virulence genes including host immune evasion genes and leucocidins. The virulence profile between the MRSA and MSSA ST5 strains were, however, not identical (Table 1).

In the MSSA strains, only the blaZ gene, encoding resistance against penicillin was detected and was associated with Tn552. The blaZ gene in strain M30-PB was present on a plasmid with rep20, together with the Superantigen enterotoxin SEA. BLAST search demonstrated that this is a common plasmid in staphylococci. In the MRSA strains; two blaZ genes were present, one in the SCCmec element and one on another location in the chromosome. The MRSA strains all carried the SCCmec type Iva (2B) and harbored resistance genes against macrolide antibiotics and aminoglycosides alongside the mecA gene as well as other plasmids (Table 1).

3. Discussion

This is the first description of S. aureus and MRSA isolates obtained from the small Indian mongoose (Urva auropunctata). Previously, MRSA has been isolated from captive Cynenicillateillata and Helogale parvulaor or the yellow and dwarf mongoose, respectively, both belonging to the same Herpestidae family. Those strains belong to the classical LA-MRSA CC398 and mecC carrying ST130 [6]. The captured mongooses were mainly from residential areas where they frequently roam among human garbage. In this study, the strains were isolated from fecal samples which is the most common staphylococcal carriage site. It remains to be determined what the prevalence in the nose or on the skin is. A larger study, including samples from different body sites in mongooses should be carried out to determine the actual prevalence and thus also the potential burden for human health on the island. Nevertheless, gastrointestinal carriage of MRSA in humans has been shown to be epidemiologically and clinically important [7,8]. The role of MRSA in the intestines of mongooses may have its importance in the general epidemiology and spread of MRSA at the human-wildlife interface.

MSSA were included in this study as we performed a non-selective isolation aim to investigate the diversity of S. aureus in the samples. Apart from a single MSSA ST5 we found two MSSA strains with a ST, ST7434, belonging to CC30, which is commonly found in humans but also in several animal species including marmots, camels, pigs, white stork and Portuguese buzzard [9]. It remains unclear whether ST7434 is specifically associated with mongooses as two very similar strains from different neighborhoods were isolated.

MRSA ST5 has previously been reported in animals [1], though not very often and is mainly found in swine in the US [10]. It is one of the most frequently detected MRSA STs in humans globally, especially in Asia, and has been mainly associated with SCCmecII [11,12]. Our strains, however, possessed the SCCmec type IVa (2B), which is the same cassette found in the predominant ST8 as well as the ST5 strains from St Kitts [3]. The strains were clonally highly related, but were isolated from different animals, but from the same geographical region, suggesting that this strain is spreading amongst mongooses or that the strain was picked up from a common source.

Since the mongoose MSSA strain, and not the mongoose MRSA strain, was the closest relative to the human MRSA ST5 strains, there is little indication for a direct transmission of MRSA between mongooses and humans. Nevertheless, host jumps of MSSA ST5 from humans to poultry has been described [13], though there is no information on potential host jumps from poultry to mongooses. The presence of MRSA in the poultry of St. Kitts is unknown and the current collection of ST5 strains from mongooses is also too limited to demonstrate potential relations with ST5 strains from other origins. We postulate however that the ST5 strains were originally from mongooses and acquired the methicillin resistance from potential human origin.

Of major relevance is the presence of the susceptible ST5 t002 strain. Since the strain differs substantially from the mongoose ST5 MRSA strains in SNPs, resistance genes, virulence genes, and plasmids, this leads to the assumption that the SCCmec element might have been acquired locally from human MRSA strains of St. Kitts. Further investigations isolating MSSA and MRSA from mongooses or other sources may bring clarity.

4. Materials and Methods

5. Conclusions

MRSA ST5 t002 SCCmec IVa (2B) was isolated from wild invasive mongooses on a small Caribbean island. This reflects the broad host range of S. aureus and the potential reservoir of these clones that have been known to cause infections in humans. At the same time a new type of MSSA, ST7434, was present in mongooses, a single locus variant of CC30, commonly found in humans. Further studies are necessary to demonstrate the One Health aspect of MRSA in wildlife.

Acknowledgments

Funding Statement

This study was financed by an intramural grant of RUSVM (PD-2018) and the MALDI-TOF MS was financed by the Research Foundation Flanders (FWO-Vlaanderen) as a Hercules project (G0H2516N, AUGE/15/05).

Author Contributions

Conceptualization, P.B., A.H. and A.A.M.J.B.; methodology, P.B., A.H. and A.A.M.J.B.; validation, P.B., A.H., A.M., F.B. and A.A.M.J.B.; formal analysis, P.B. and A.H.; investigation, A.H.; resources, P.B.; data curation, P.B.; writing—original draft preparation, A.H.; writing—review and editing, P.B., A.H., A.M., F.B. and A.A.M.J.B.; supervision, P.B.; project administration, P.B.; funding acquisition, P.B. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

This study was approved by the Ross University Institutional Animal Care and Use Committee under the IACUC Number 17.04.13.

Data Availability Statement

Sequences are submitted to NCBI as PRJNA777776 with SRR16820787 to SRR16820793.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

References