Isolates and typing. The isolates characterised as well as strain affiliations, geographic origins and clinical presentations are summarised in Table 1. Autopsy images showing typical aspects of putrid infections in some animals are shown in Fig. 1. The complete microarray hybridisation patterns are provided as Supplemental file 2 and some relevant features will be discussed in the descriptions of the respective strains. While all German isolates yielded hybridisation signals for lukF/S-PV, frequently only weak positive or ambiguous results for the lukS-PV probe were observed. This prompted further investigations, including the detection of PVL by lateral flow assay 21 (Table 1) and whole genome sequencing (see below).
Phenotypic and genotypic resistance properties of the S. aureus isolates. Antimicrobial susceptibility testing revealed that all beaver isolates from Germany were susceptible to all antimicrobial agents tested. The distribution of minimal inhibitory concentration (MIC) values and test ranges are displayed in Supplemental File 3a. The phenotypic data corresponded well with microarray data, since none of the corresponding resistance genes was identified.
The chromosomal variant of the metallothiol transferase gene fosB was present in all CC1956 isolates. Sequence analysis revealed a frame shift at position 108 creating a stop codon at positions (pos.) 146..148 compared to the reference sequence (N315, GenBank BA000018.3 [2,389,328..2,389,747]). This resulted in a truncated protein of 48 amino acids (aa) rather than 139 aa as for the original fosB gene product. The mutation was present in all available sequences (i.e., Oxford Nanopore and Illumina of WT19 as well as Illumina of WT63, WT64, WT66, WT67a, WT67b, WT68, WT69, WT70 and WT71). While fosB was originally implicated in fosfomycin resistance, it appears to be linked to certain CCs. Indeed, it was also present in the CC8 and CC12 beaver isolates (B2, B3, B4) as well as in the reference sequences of the respective CCs (Supplemental File 2). The fosB gene was absent from the CC49 isolate WT65 and from the CC49 reference sequence of Tager 104, GenBank CP012409.1, as well as from the CC398 isolate B1. Moreover, all sequenced isolates (from animals A to F) harboured a gene designated tet(38), encoding a major facilitator superfamily permease. While this gene was implicated in low-level tetracycline resistance when overexpressed 22, its mere presence certainly is not associated with tetracycline resistance as it can be found in virtually every S. aureus genome.
Biocide susceptibility testing of the CC49/1956 isolates revealed unimodal MIC distributions (Supplemental File 3b), with ranges encompassing not more than three to four dilution steps for each of the biocides (benzalkonium chloride, 0.00003-0.000125%; polyhexanide, 0.000125-0.0005%; chlorhexidine, 0.00006-0.00025% and octenidine, 0.00006-0.00025% with percentages given as mass per volume).
The chromosomal heavy metal resistance markers arsB/R and czrB were detected by hybridisation in all four CC1956 isolates tested as well as in the CC49 isolate. This was confirmed by sequencing. There was no evidence for plasmid- or SCC-borne heavy metal resistance markers.
The sequence of the phage-borne leukocidin genes in WT19 and WT65. As mentioned above, CC49/CC1956 beaver isolates yielded occasionally ambiguous hybridisation intensities for lukS-PV probes prompting further investigation assuming that the specifically designed oligonucleotides were not able to bind optimally at the target due to mismatches, i.e., allelic variants. Sequencing revealed the presence of distinct alleles of phage-borne leukocidin genes (Figs. 2a/b and 3a/b). The sequences from the two sequenced beaver isolates were identical to each other despite their origin from different prophages in different CCs. In general, the beaver alleles, hitherto referred to as “Beaver Leukocidin” or BVL, lukF/S-BV, appeared to be closer related to the PVL genes from human strains of S. aureus than to those from ruminants and horses. There was no evidence for recombination/chimerism in lukF-BV and lukS-BV as mismatches compared to other sequences were evenly distributed across the entire sequences. Sequences of lukF-BV and lukS-BV were also related but clearly distinct from core genomic lukF/S-int of S. intermedius/pseudintermedius.
Core genome and genomic islands of the CC1956 isolate WT19. As revealed by array experiments (Supplemental File 1) and confirmed by genome sequencing of WT19, CC1956 isolates presented with agr IV alleles and capsule type 5. They were positive for cna, but they lacked seh and egc enterotoxin genes, ORF CM14 as well as sasG. Leukocidin genes lukX/Y, lukD/E and lukF/S-hlg were present. This is also in accordance with previously sequenced BVL-negative CC1959 isolates (SAMEA3251370, SAMEA3251372, SAMEA3251377, SAMEA3251376, SAMEA3251380; Supplemental File 2)
The WT19 genome (Supplemental Files 4a and 4b) harboured two uncharacterised enterotoxin genes (pos. 1,940,148..1,940,900 and pos. 1,939,378..1,940,121). Both were also found in DAR4145 (CC772) where they also formed a genomic island at approximately the same position within the genome (GenBank CP010526.1: RU53_RS09775, pos. 1,968,336..1,969,061 and RU53_RS09780, pos. 1,969,088..1,969,840). One of these two genes (“seu2”= RU53_RS09780) was covered by the second array-based assay 23 and it was found in all four isolates tested with this array.
Mobile genetic elements in the CC1956 isolate WT19. The lukF/S-BV prophage was integrated into the lipase 2 gene (lip2, “geh”, “sal3”, “salip35”, CP000253.1 [314,326:316,398]), and spanned pos. 322,629 to 365,636. Besides leukocidin genes, it also included genes associated with the different modules of a typical Siphoviridae genome (lysogeny, DNA metabolism, packaging and capsid morphogenesis, tail morphogenesis, host cell lysis; 24,25; see Supplemental File 5/Fig. 4).
Furthermore, there was a small pathogenicity island at pos. 869,706 to 884,748 that included pif encoding a phage interference protein, a gene for a small terminase subunit, genes for “putative proteins” as well as a paralog of a complement inhibitor SCIN family protein (scn2) and a variant of the von Willebrand factor binding protein Vwb (vwb3). Thus, it is considered a staphylococcal pathogenicity island (SaPI) related to the one in S0385, AM990992.1.
Another prophage integrated between rpmF and isdB, pos. 1,107,447 to 1,146,132. A third prophage was located between a truncated nikB and Q5HG37, pos. 1,425,279 to 1,481,870. Finally, there was a forth prophage between Q5HDU4 and sarV (actually interrupting an MFS transporter between those genes), pos. 2,340,832 to 2,386,591. This prophage sequence corresponded to the phage that was detected by nanopore sequencing after induction by Mitomycin C (see below and Supplemental File 6).
Phage morphology and sequencing of phages from the CC1956 isolate WT19. In three separate preparations, large number of phages were observed that were well contrasted with uranyl acetate and with phosphotungstic acid. Phages had elongated capsids. The non-contractile, thin tails were straight or slightly curved and ended in a bulb-shaped base plate. Based on these characteristics, they were assigned to the order Caudovirales, family Siphoviridae.
Capsids were measured in 40 phages, tails in 34 and base plates in 33 phages. Based on these measurements, two distinct populations emerged (Fig. 5). In one (Fig. 5A), the prolate, distinctly pentagonal capsids averaged 39 ± 5 nm (range 32–46 nm) in diameter and 92 ± 8 nm (range 80–104 nm) in length. Tails were 276 ± 20 nm (range 243–310 nm) long, had a diameter of 11 ± 1 nm (range 10–12 nm) and had a stacked discs appearance. Their baseplates were 16 nm (range 16–31 nm) by 27 nm (range 19–33 nm). The other population (Fig. 5B) had elongated oval capsids with a maximal diameter of 55 ± 2 nm (range 51–60 nm) diameter and 93 ± 5nm (range 85–100 nm) length. Their tails measured 287 ± 12 nm (range 275–313 nm) in length and 9 ± 1 nm (8–10) in diameter and had a rail-road-track morphology. Dimensions of baseplates were 25 nm (range 21–30 nm) by 29 nm (range 23–39 nm).
Oxford Nanopore sequencing of one of these phage preparations (Supplemental File 6) yielded just one circular contig with a coverage of 724. Its sequence was identical to forth prophage, between Q5HDU4 and sarV, except for a loss of a single triplet out of a total length of 46,387 nt.
Core genome and genomic islands of the CC49 isolate WT65. The CC49 isolate carried agr group II alleles and capsule type 5. It was positive for sasG. It lacked seh and egc enterotoxin genes, ORF CM14 and the collagen adhesion gene cna. A truncated copy of the enterotoxin S gene (GenBank CP000046, pos. 2,203,972..2,204,196) was found as well as leukocidin genes lukG/H = lukX/Y, lukD/E and lukF/S-hlg. With regard to presence and alleles of chromosomal markers such as MSCRAMM or ssl genes, the genome of WT65 (Supplemental Files 7a and 7b) is closely related to the CC49 reference sequences such as Tager 104, GenBank CP012409.1 (Supplemental File 2).
Mobile genetic elements in the CC49 isolate WT65. One prophage was integrated into the lip2 gene spanning pos. 311,401 to 354,724. The prophage included the lukF/S-BV genes as well as genes associated with the different modules of a typical Siphoviridae genome (Supplemental File 5/Fig. 4). Sequences corresponding to the lysogeny and replication modules were clearly different compared to the lukF/S-BV-prophage in the CC1956 isolate WT19 while approximately the second half of the two respective prophage sequences (the lower part of the alignment in Fig. 4) were virtually identical in gene content, order and orientation.
Other mobile genetic elements (Supplemental File 7a/b) included a small pathogenicity island, pos. 402,133 to 416,237 (between rpsR encoding 30S ribosomal protein S18 and its terminator), that included hypothetical proteins, a gene of a terminase small subunit, vwb3, the scn2 gene (putative paralog of complement inhibitor) encoding a "van Willebrand factor" binding protein. Between genes ktrB and groL, pos. 2,029,208 to 2,042,866, another SaPI was identified that contained additional, slightly different copies of vwb3 and scn2 gene as well as terminase small subunit, integrase and excisionase (xis-AIO21657) genes.
Finally, five genes between pos.1,334,169 and 1,339,503 were annotated as phage capsid genes although no other phage-related genes were found in this region.
Phage morphology and sequencing of phages from the CC49 isolate WT65. Four separate phage preparations were examined. In one of them, few phage-like structures were detected. These findings could not be confirmed in the following preparations. Thus, they were interpreted as artefacts, also given that it was not possible to induce a sufficient amount of phages for Oxford Nanopore sequencing.
Table 1: Details of animals, isolates and strains.
Animal
|
Geographic origin
|
Isolate
|
Pathology
|
MLST
|
spa
|
Strain ID according to array
|
PVL by lateral flow
|
A
|
Berlin
|
WT19
|
Multifocal moderate to severe suppurative necrotising pneumonia and suppurative pyelonephritis. Found dead.
|
ST4614a
|
t3058
|
CC1956-MSSA (lukF-PV+/lukS-PV?)b
|
Positive
|
B
|
Berlin
|
WT63
|
Severe suppurative necrotising pneumonia and multiple small abscesses in spleen, kidney, caecum, mesenteric lymph nodes and intercostal muscles. Found dead.
|
ST4614
|
t3058
|
CC1956-MSSA (lukF-PV+/lukS-PV?)
|
Positive
|
WT64
|
ST4614
|
t3058
|
CC1956-MSSA (lukF-PV+/lukS-PV?)
|
Positive
|
C (“Tiffy”)
|
Bavaria
|
WT65
|
Severe abscessing mandibular lymphadenitis, severe suppurative cystitis. Died in a wildlife rehabilitation centre.
|
ST49
|
t208
|
CC49-MSSA (lukF-PV+/lukS-PV?)
|
Positive
|
D
|
Berlin
|
WT66
|
Severe fibrinous-purulent myocarditis, suppurative pyelonephritis (right kidney only) and prostatitis. Found dead.
|
ST4614
|
t3058
|
CC1956-MSSA (lukF-PV+/lukS-PV?)
|
Positive
|
WT67a
|
ST4614
|
t3058
|
CC1956-MSSA (lukF-PV+/lukS-PV?)
|
Positive
|
WT67b
|
ST4614
|
t3058
|
CC1956-MSSA (lukF-PV+/lukS-PV?)
|
Positive
|
WT68
|
ST4614
|
t3058
|
CC1956-MSSA (lukF-PV+/lukS-PV?)
|
Positive
|
WT69
|
ST4614
|
t3058
|
CC1956-MSSA (lukF-PV+/lukS-PV?)
|
Positive
|
E
|
Berlin
|
WT70
|
Suppurative necrotising pneumonia, suppurative pyelonephritis and lymphadenitis (popliteal lymph nodes, multiple abscesses in caecum wall. Found dead.
|
ST4614
|
t3058
|
CC1956-MSSA (lukF-PV+/lukS-PV?)
|
Positive
|
F
|
Berlin
|
WT71
|
Abscesses subcutaneous (chest wall/axilla) and in popliteal lymph nodes. Euthanised due to poor physical condition.
|
ST4614
|
t3058
|
CC1956-MSSA (lukF-PV+/lukS-PV?)
|
Positive
|
G
|
Berlin
|
WT110
|
Suppurative necrotising pneumonia, enlarged thyroid gland containing small yellowish abscesses, suppurative pyelonephritis, splenomegaly, multiple abscesses in caecum and colon walls as well as in skin. Cachexia. Found dead.
|
N/A
|
N/A
|
CC1956-MSSA (lukF-PV+/lukS-PV?) c
|
Positive
|
WT111
|
N/A
|
N/A
|
CC1956-MSSA (lukF-PV+/lukS-PV?) c
|
Negative
|
H
|
Marchfeld Channel, Austria
|
B1
|
Nasal Swab. Good physical condition except for gunshot traumata including parts of a bullet in the right hindleg with surrounding acute necrotizing inflammation. All other organs unremarkable.
|
N/A
|
t085
|
CC398-MSSA
|
N/A
|
I
|
Lower Austria
|
B2
|
Abscessing lymph node, knee. Shot dead.
|
N/A
|
t008
|
CC8-MSSA
|
N/A
|
J
|
Eisenstadt region, Austria
|
B3
|
Pneumonia (Streptococcus pyogenes, Morganella morganii). Splenomegaly. S. aureus originated from enrichment culture of lung tissue. Found dead.
|
N/A
|
t394
|
CC8-MSSA (sed/j/r+)
|
N/A
|
K
|
Lower Austria
|
B4
|
Nasal Swab. Healthy animal shot dead.
|
N/A
|
t156
|
CC12-MSSA (sep+)
|
N/A
|
a ST4614 is a single locus variant of ST1956; arc-6, aroE-291, glpF-6, gmk-2, pta-7, tpi-225, yqil-585
b Clearly positive for lukF-PV, weekly positive or ambiguous signal for lukS-PV
c WT110 and WT111 differed in haemolysis on Columbia blood agar and were thus handled separately although array analysis eventually revealed identical strain affiliations.