Streptococcus agalactiae in elephants – A comparative study with isolates from human and zoo animal and livestock origin

Veterinary Microbiology 204 (2017) 141–150

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Streptococcus agalactiae in elephants – A comparative study with isolates from human and zoo animal and livestock origin

MARK

⁎

Tobias Eisenberga,f, , Jörg Raub, Uta Westerhüsc, Tobias Knauf-Witzensd, Ahmad Fawzya,e,f, Karen Schleza, Michael Zschöcka, Ellen Prenger-Berninghofff, Carsten Heydelf, Reinhard Stingb, Stefanie P. Glaeserg, Dipen Pulamig, Mark van der Lindenh, Christa Ewersf a

Hessian State Laboratory, Schubertstr. 60, 35392 Giessen, Germany Chemical and Veterinary Investigation Office Stuttgart, Schaflandstraße 3/2, 70736 Fellbach, Germany c Opel-Zoo, Königsteiner Straße 35, 61476 Kronberg, Germany d Wilhelma – Zoological and Botanical Gardens, Wilhelma 13, 70376 Stuttgart, Germany e Cairo University, Faculty of Veterinary Medicine, Department of Medicine and Infectious Diseases, Giza Square 12211, Egypt f Institute of Hygiene and Infectious Diseases of Animals, Justus-Liebig-University Giessen, Frankfurter Str. 85-87, 35392 Giessen, Germany g Institute of Applied Microbiology, Justus-Liebig-University Giessen, Heinrich-Buff-Ring 26, 35392 Giessen, Germany h National Reference Laboratory on Streptococcal Diseases, Abteilung Medizinische Mikrobiologie, Universitätsklinikum RWTH Aachen, Pauwelsstraße 30, 52074 Aachen, Germany b

A R T I C L E I N F O

A B S T R A C T

Keywords: Streptococcus agalactiae Elephant Loxodonta africana Elephas maximus FT-IR MALDI-TOF MS RAPD PFGE Capsule typing Persistent infection

Streptococcus (S.) agalactiae represents a significant pathogen for humans and animals. However, there are only a few elderly reports on S. agalactiae infections in wild and zoo elephants even though this pathogen has been isolated comparatively frequently in these endangered animal species. Consequently, between 2004 and 2015, we collected S. agalactiae isolates from African and Asian elephants (n = 23) living in four different zoos in Germany. These isolates were characterised and compared with isolates from other animal species (n = 20 isolates) and humans (n = 3). We found that the isolates from elephants can be readily identified by classical biochemistry and MALDI-TOF mass spectrometry. Further characterisations for epidemiological issues were achieved using Fourier transform-infrared spectroscopy, capsule typing and molecular fingerprinting (PFGE, RAPD PCR). We could demonstrate that our elephant isolate collection contained at least six different lineages that were representative for their source of origin. Despite generally broad antimicrobial susceptibility of S. agalactiae, many showed tetracycline resistance in vitro. S. agalactiae plays an important role in bacterial infections not only in cattle and humans, but also in elephants. Comparative studies were able to differentiate S. agalactiae isolates from elephants into different infectious clusters based on their epidemiological background.

1. Background Streptococcus (S.) agalactiae (group B Streptococcus, GBS) often asymptomatically colonise the human female genital tract (Da Cunha et al., 2014). Sequelae of human S. agalactiae colonisation include urinary-tract infection, diabetic foot infection, osteomyelitis and neonatal sepsis and meningitis, but streptococcal toxic shock syndrome,

necrotizing fasciitis, disseminated intravascular coagulopathy and renal impairment have also been reported (Sendi et al., 2008; Imöhl et al., 2011). Besides human infections, S. agalactiae is one of the most important bovine mastitis pathogens worldwide with significant economic impact for the dairy industry (Manning et al., 2010). The etiological agent of ‘Agalactia catarrhalis contagiosa’ is directly transmitted between cows during milking and its contagious role is also

Abbreviations: AVID, Arbeitskreis veterinärmedizinische Infektionsdiagnostik; ATCC, American Type Culture Collection; FT-IR, Fourier-transform infrared-spectroscopy; GBS, group B streptococci; GP, Gram-positive; MALDI-TOF MS, matrix-assisted laser desorption/ionization-time of flight mass spectrometry; MIC, minimum inhibitory concentration; MLST, multilocus sequence typing; PFGE, pulsed-field gel electrophoresis; RAPD, random amplified polymorphic DNA; S., Streptococcus ⁎ Corresponding author at: Hessisches Landeslabor, Abteilung Veterinärmedizin, Schubertstr. 60/Haus 13, 35392 Giessen, Germany. E-mail addresses: [email protected] (T. Eisenberg), [email protected] (J. Rau), [email protected] (U. Westerhüs), [email protected] (T. Knauf-Witzens), [email protected] (A. Fawzy), [email protected] (K. Schlez), [email protected] (M. Zschöck), ellen.prenger-berninghoff@vetmed.uni-giessen.de (E. Prenger-Berninghoff), [email protected] (C. Heydel), [email protected] (R. Sting), [email protected] (S.P. Glaeser), [email protected] (D. Pulami), [email protected] (M. van der Linden), [email protected] (C. Ewers). http://dx.doi.org/10.1016/j.vetmic.2017.04.018 Received 17 November 2016; Received in revised form 20 April 2017; Accepted 21 April 2017 0378-1135/ © 2017 Elsevier B.V. All rights reserved.

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and one post mortem examination (case no. 16) from elephants from four different zoos (A–D) in Germany between 2004 and 2015 as well as from various other isolations from zoo animals (A, E, F), livestock and humans (case no. 26–48; Supplementary Table S1).

reported for small ruminants and camelids (Tibary et al., 2006; Guerreiro et al., 2013) usually leading to outbreaks caused by one single infectious lineage within a herd (Brglez et al., 1979). Apart from udder infections S. agalactiae is only rarely reported as a veterinary pathogen in aquaculture as well as in wild fish (Bowater et al., 2012). Relatively few infections from wildlife implicating S. agalactiae have been reported. These include a fatal case with a phenotypically deviant S. agalactiae together with Lactococcus garvieae in a wild bottlenose dolphin (Evans et al., 2006) as well as in a dolphin from a zoo (Zappulli et al., 2005). Streptococcus agalactiae could also be isolated from a case series in African elephants [Loxodonta africana; (Keet et al., 1997)] as well as in a case from one female Asian elephant (Elephas maximus) with chronic endometritis and pododermatitis from a German zoo (Aupperle et al., 2008). Our laboratories have occasionally isolated S. agalactiae from African and Asian elephants kept in different zoos with pododermatitis as well as from other sites in recent years. The aim of this study was to further characterise respective isolates of S. agalactiae phenotypically and with molecular methods. Therefore, isolates from elephants were compared with isolates from humans, livestock as well as from wild and exotic animal species.

2.4. Phenotypic characterization Phenotypic characterization was performed by standard microbiological procedures: Haemolytic properties of the bacteria were examined on blood agar containing 5% sheep blood and microscopic examinations of fixed smears were performed using Gram staining. Bacterial colonies were tested for catalase activity with 3% H2O2 on microscopic slides. Since S. agalactiae displays a regular CAMP phenomenon (Hensler et al., 2008) when tested with an orthogonally growing Staphylococcus aureus (ATCC 25923, American Type Culture Collection; Manassas, VA, USA) this test was routinely carried out. Presumptive identification of streptococci based on the aforementioned criteria as well as on confirmation with Lancefield group-antigen specific Streptococcus antisera (Phadebact, MKL Diagnostics AB, Sollentuna, Sweden) was confirmed by standardized test systems, i.e. API Strep and Vitek2-compact, the latter with GP card system for Grampositive bacteria (all bioMérieux, Nürtingen, Germany). All tests were performed according to the manufacturer’s instructions.

2. Methods 2.1. Case descriptions

2.4.1. Identification by MALDI-TOF MS Bacterial isolates were selected from the culture plates and then transferred to steel-targets according to manufacturer’s instructions (BrukerBiotyper, BrukerDaltonics, Bremen, Germany). Isolates were prepared using the direct smear method and analysed by MALDI-TOF MS using Biotyper Version V3.3.1.0. The database used (DB 5989, BrukerDaltonics) comprised spectra from 294 Streptococcus reference spectra from 64 species including nine from different S. agalactiae strains.

Zoo A Zoo A houses female Asian elephants in an 830 m2 outdoor enclosure with sand and concrete flooring and a 96 m2 indoor enclosure with a Stallit® floor (Stallit, Gaishorn, Austria). Both areas include pools. The feed consists of hay, straw, branches, fruits and vegetables. The hoofs are cleaned daily and controlled and trimmed on a regular basis. 2.2. Auto-vaccination against S. agalactiae

2.4.2. Antimicrobial susceptibility testing All isolates were tested for antibiotic minimal inhibitory concentrations (MIC) using the broth microdilution method as recommended by the Clinical and Laboratory Standards Institute (CLSI). The microtiter plates (Sensititre NLMMCS10, TREK Diagnostic Systems Ltd., East Grinstead, UK) contained [with test ranges given in brackets (in mg/ mL)] amoxicillin (0.015–16), cefotaxime (0.015–16), chloramphenicol (2–16), clindamycin (0.12–256), erythromycin (0.12–256), levofloxacin (0.25–8), moxifloxacin (0.25–4), penicillin G (0.015–16) and tetracycline (0.12–256) with cation adjusted Mueller-Hinton broth (Oxoid, Wesel, Germany) and 5% lysed horse blood. The final inoculum was 5 × 105 CFU/ml. Incubation was at 37 °C for 24 h in ambient air. Streptococcus pneumoniae ATCC 49619 was used as a control strain. In default of appropriate validated clinical breakpoints for most animal species including elephants and for a better comparison with former studies the current CLSI criteria for clinical breakpoints for humans were applied [M100-S25; ‘non-meningitis’ interpretations].

To verify the possibility of S. agalactiae eradication in the two chronically infected female Asian elephants in zoo A, an autologous vaccine was produced. Briefly, isolate 151004802 that had been isolated from a necrotic ear wound margin from elephant “Pama” was grown in standard bouillon (VWR, Darmstadt, Germany) and harvested after 24 h incubation at 37 °C. For a local immunisation, a heat-killed formulation of S. agalactiae was administered by 5 mL spray inhalation into the trunk (density 5.7 × 107 cfu/mL; given once daily for ten days). A second route of application included parenteral administration by increasing doses of an inactivated S. agalactiae suspension mixed with phenol-sodium chloride solution (given subcutaneously every three to four days [0.5–2.5 mL] ten times). Harmlessness and success of these vaccinations were monitored by clinical examinations and repeated sampling of infectious sites known to harbour S. agalactiae (tip of the trunk nasal cavity, mouth, vulva, right and left elbow, toe of front foot, erosive skin lesion on left cheek) in at first weekly, later monthly intervals. Zoo B A group of three adult females and one sub-adult male African elephants was housed in an outdoor area of 6450 m2 with a 260 m2 water basin and an indoor compartment of 880 m2 in zoo B. Like in zoo A, animal husbandry fulfilled ethical standard guidelines according to the code of ethics and animal welfare of the world association of zoos and aquariums (WAZA; http://www.waza.org/ files/webcontent/1.public_site/5.conservation/code_of_ethics_and_ animal_welfare/Code%20of%20Ethics_EN.pdf).

2.4.3. Fourier transform-infrared spectroscopy (FT-IR) All S. agalactiae isolates were cultivated independently in 5–7 replicates at 37 °C for 24 h on sheep blood agar plates (Oxoid). Harvesting of cells, preparation of bacteria films on zinc selenide plates, drying and handling were performed as described previously (Eisenberg et al., 2015). The dried bacteria films were used directly for examination by FT-IR spectroscopy. Infrared spectra were recorded for each sample in a transmission mode from 500 to 4000 cm−1 with an FT-IR spectrometer (Tensor27 with HTS-XT-module, BrukerOptics, Ettlingen, Germany). Acquisition and first analysis of data were carried out using OPUS Software (vers. 4.2, BrukerOptics). Infrared spectra of isolates were compared by cluster analysis (cf. (Eisenberg et al., 2015)). Therefore, the second derivation of the vector normalized spectra in the wave number range of 500–1800 cm−1 and 2800–3000 cm−1 were

2.3. Isolation of bacteria Isolates of S. agalactiae were obtained during routine bacteriological investigations following skin swabbings (case no. 1–13, 17–25; Table 1) 142

151001913/2

151002450/1

151002450/2

151003337/1

151003337/2

18

19

20

21

P2528/11

11

17

10-7-D-02041

10

141014875

P6343/08

09

16

P5841/08

08

141011411/2 (no isolate)

P3602/08

07

15

P2843/08

06

141011411/1 (no isolate)

CVUAS 3181

05

14

CVUAS 1374

04

141000096/2

CVUAS 1342

03

13

CVUAS 5410

02

P6096/11

CVUAS 338

01

12

Isolate ID

Case no.

143

2015

2015

2015

2015

2015

2014

2014

2014

2014

2011

2011

2010

2008

2008

2008

2008

2008

2006

2006

2006

2004

Year of isolation

African elephant “Tamo” African elephant “Aruba” African elephant “Aruba“ African elephant “Wankie“ African elephant “Zimba“ African elephant “Tamo“ African elephant “Zimba“ African elephant “Tamo“ African elephant

Asian elephant “Ilona“

Asian elephant “Zella“ Asian elephant “Vilja” Asian elephant “Molly“ Asian elephant “Vilja” Asian elephant “Vilja“ Asian elephant “Shanti” Asian elephant – ”Shanti“ Asian elephant “Rani” Asian elephant “Shanti” African elephant “Aruba“ Asian elephant “Judy“

Animal species

F

F

F

F

M

F

B

B

B

B

B

B

B

M

M

M

B

B

F

C

F

F

B

D

F

F

F

C

C

C

F

F

A C

F

F

A A

F

F

Sex

A

A

Zoo

nail matrix

hollow foot wall, ventral fistula opening on coronary band

multiple abscesses

multiple abscesses

chronically inflamed temporal gland

skin abscess

abscess ear margin

abscess ear margin

abscess pus from multiple skin abscesses, especially on the rear discharge from temporal gland

(right and left ear ulcers, nasal cavity, spleen, urinary bladder), heart discharge from temporal gland

nail matrix

hollow foot wall, dorsal fistula opening on coronary band

multifocal chronic ulcerative dermatitis (ears), pronounces hyperkeratosis with rhagades on sole horn, slight spleen hyperplasia, catarrhal cystitis chronically inflamed temporal gland

abscess pus

+++

+++

(continued on next page)

St. xylosus +++, Bacillus sp. +, Pantoea agglomerans +

St. carnosus +

St. sciuri ++, C. parapsilosis +++, B. cereus +

St. aureus +++

+++

+

B. cereus +, C. albicans +, CNS +

various concomitant microbiota

+ − +++

+++

A. calcoaceticus ++, R. ornithinolytica ++, Citr. gillenii ++, mould fungi ++

E. coli ++, A. calcoaceticus ++, Proteus sp. +

St. aureus +++, Erwinia sp. +++, K. pneumoniae ++, Pseudomonas sp. +++, C. tropicalis +++, Alcaligenes sp. + +, Se. marcescens +++, E. coli (+), Corynebacterium sp. ++ E. coli ++, Acinetobacter sp. ++, St. warneri+, St. succinus + +, Pantoea sp. ++, F. necrophorum +++

E. coli, coliform bacteria, St. epidermidis, Micrococcus sp., Se. liquefaciens, Acinetobacter sp., Pseudomonas sp., Flavobacteria

Pseudomonas sp. +++, γ-haemolytic streptococci ++, Proteus sp. ++, coliform bacteria ++ Proteus sp. +++, St. aureus +++, Pseudomonas sp. +++, γhaemolytic streptococci +++ coliform bacteria ++, Acinetobacter sp. (+), γ-haemolytic streptococci +, St. epidermidis + Morganella morganii +++, E. coli +++, K. oxytoca +++, Alcaligenes faecalis + Poteus sp. ++, E. coli ++, CNS ++

–

K. pneumoniae +++

K. pneumoniae +++, E. coli +++

K. pneumoniae +++

–

Concomitant bacterial species*

++

+++

+

+++

+++

+++

fistula on nail matrix D2 at left front leg (trachea, spinal cord, uterus, urine, vagina, heart), lung skin

++

++

+++

++

+

++

+++

+

++++

S. agalactiae quantity+

foot

abscess

foot

nail matrix

nail matrix

nail matrix

nail matrix

nail matrix

nail matrix

Tissue with positive proof (isolate not stored)

abscess underneath right eye

erosive skin lesions

chronic infection of the urogenital tract, myocarditis

abscess

abscess

abscess foot

abscess foot

swab abscess nail matrix

abscess nail matrix

abscess nail matrix

abscess nail matrix

abscess nail matrix

Clinical presentation/ gross pathology

Table 1 Origin of Streptococcus agalactiae field isolates from elephants investigated in this study as well as clinical and gross pathology results from respective cases (M: male, F: female; –: none).

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2015 151004802 25

2015 151003441/3 24

2015 151003441/2 23

+: 1–10 colony forming units (cfu), ++: 11–50 cfu, +++: 51–200 cfu, ++++: > 200 cfu, * A.: Acinetobacter, B.: Bacillus, C.: Candida, Citr.: Citrobacter, CNS: Coagulase-negative staphylococci, E.: Escherichia, F.: Fusobacterium, R.: Raoultella, K.: Klebsiella, P.: Pseudomonas, Se.: Serratia, S.: Streptococcus, St.: Staphylococcus, Sten: Stenotrophomonas.

St. aureus +, Sten. maltophilia ++, P. aeruginosa ++, Acinetobacter sp. ++ +++ A

F

necrotic ear wound

necrotic ear wound margin

Kocuria kristinae + +++ M B

multiple abscesses

ulcer right ear

Kocuria kristinae + ++ M B

multiple abscesses

abscess right ear

Kocuria kristinae ++, Lactococcus raffinolactis + + multiple abscesses right ear M 22

151003441/1

2015

“Tamo“ African elephant “Tamo“ African elephant “Tamo“ African elephant “Tamo“ Asian elephant “Pama“

B

multiple abscesses

S. agalactiae quantity+ Isolate ID Case no.

Table 1 (continued)

Year of isolation

Animal species

Zoo

Sex

Clinical presentation/ gross pathology

Tissue with positive proof (isolate not stored)

Concomitant bacterial species*

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used for calculation with Ward’s algorithm (OPUS 4.2). The dendrogram obtained depicts the arrangement of isolates in groups according to their spectral differences (Fig. 2). 2.5. Molecular characterisation 2.5.1. PCR analysis of the capsule type A multiplex PCR for the determination of capsule types (serotypes) Ia, Ib and II–VIII was carried out as described by Poyart et al. (2007). The dltS gene encoding a histidine kinase that is specific to GBS was used as an internal marker during the same PCR procedure. 2.5.2. Genomic fingerprinting analysis 2.5.2.1. Randomly amplified polymorphic DNA (RAPD) PCRs. Comparative genomic fingerprint analysis of the isolates was performed with three different RAPD PCRs. These were performed with primers A (5′-CTGGCGGCTTG-3′) according to Glaeser et al. (2013), and OPB 17 (5′-AGGGAACGAG-3′), and OPB 18 (5′-CCACAGCAGT-3′) according to Martinez et al. (2000). The PCR master mix (15 μL) contained 1× Dream Taq Buffer (ThermoFisher Scientific, Dreieich, Germany), 1.0 μM primer, 200 μM dNTPs each, 3 μL 5× Q solution (Qiagen, Hilden, Germany), 0.8 U Dream Taq Polymerase (ThermoFisher) and 3 μL template DNA. The PCR conditions were as following: 1× (95 °C, 3 min), 45× (95 °C, 15 s; 34 °C, 1 min; 72 °C, 2 min), 1× (72 °C, 10 min). DNA fragments were separated in a 1.5% (w/v) agarose gel (90 V for 1.5 h), stained with ethidium bromide and documented with a gel documentation system (BioDoc-It, UVP, UK). Gels were processed with Photoshop and analysed in GelCompar II (Applied Maths). The fingerprint patterns were compared by clusteranalysis using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) and the curved based Pearson correlation as similarity coefficient. Analysis was performed using an optimization value and position tolerance of 5%. Each fingerprint type was first clustered individually. Thereafter a consensus matrix was calculated (including all three fingerprint types with equal contribution) and used for UPGMA based clustering. 2.5.3. Pulsed-field gel electrophoresis (PFGE) PFGE was performed using a modified protocol from Oliveira et al. (2005). Isolates were grown overnight (37 °C, 10% CO2) in BHI medium. Cells from 10 mL culture were harvested by centrifugation, washed with 5 mL and subsequently resuspended in 2 mL of ice cold TE buffer (10 mM Tris, 1 mM EDTA). 250 μL of the suspension were mixed with 375 μL of 1.2% Gold Agarose (Biozym, Germany) and pipetted into 100 μL plugs. These were incubated in 300 μL lysis buffer (1 M NaCl, 100 mM EDTA, 6 mM Tris, 0.5% Brij, 0.2% deoxycholate, 0.5% N-lauroyl sarcosine [pH 7.6]) with 30 μL lysozyme (10 mg/mL) first without (overnight, 37 °C) and then with 6 U proteinase K (Sigma; overnight, 56 °C). After washing 2-times with 1 mL TE buffer, 2-times with 300 μL TE buffer supplemented with 3 μL PMSF (100 mM in isopropanol) and again 2-times with 1 mL TE buffer, half agarose blocks were transferred into 200 μL Tango buffer and digested with 10 U SmaI restriction enzyme (both Thermo Scientific, USA) for 4 h at 30 °C. DNA was then separated by PFGE (angle 120; voltage 6 V/cm; pulsed-field times 1–13 s for 20 h; ramping, linear) in 1.2% agarose gels using a CHEF Mapper (Bio-Rad, Germany). PFGE patterns were analysed using the BioNumerics software (version 6.6, Applied Maths BVBA, Belgium). 3. Results 3.1. Case history and clinical examination Zoo A S. agalactiae has regularly been isolated in all four animals (Rietschel pers. communication), predominantly from the feet, especially from the nail matrix. Other regularly observed sites found to 144

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Fig. 2. Cluster analysis of respective spectra obtained by Fourier-transform infrared-spectroscopy (FT-IR) using OPUS Software (vers. 4.2, BrukerOptics). In each case three infraredspectra of Streptococcus agalactiae isolates from elephants and other species were used for calculation with Ward’s algorithm. The dendrogram obtained depicts the arrangement of isolates in groups according to their spectral differences.

well as treatment with local irrigations (e.g. Rivanol® [Dermapharm, Germany], Lotagen® [Essex, Germany], Betaisodona® [Albrecht, Germany], Novaderma® [WDT, Germany]). In cases of systemic infections antibiotics were administered intramuscularly (penicillin, streptomycin, cefquinome, ceftiofur, enrofloxacin). Even though aggressive treatment was performed in “Molly”, she had to be euthanised due to osteomyelitis in the digits of her front feet in 2011. Apart from other bacterial species other α- and γ-haemolytic streptococci could be isolated from her feet and inner organs. In all other animals the lesions

contain S. agalactiae were abscess contents, fistulas, skin lesions and vaginal discharge. In 2015 two females remained in the holding of which “Zella” suffered from an abscess on her right elbow. Apart from concomitant bacterial species, S. agalactiae has been repeatedly isolated. “Pama” also suffered from an ear thrombophlebitis in the same year and a part of her ear became necrotic, from which again S. agalactiae was isolated. Treatments included trimming of the nail and removal of necrotic tissues, washing with soaped water and disinfection with Chlorhexidin 0.05% or Lavasept® (Braun, Melsungen, Germany) as 145

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(Fig. 1C) and “Tamo’s” skin abscesses deteriorated involving further ulcers in the right ear’s surrounding. In all these lesions S. agalactiae could be isolated (Table 1). After sampling, the skin ulcers and the temporal gland were successfully treated with disinfectants and antimicrobials as mentioned above. Histories of the cases in zoos C and D are not available. 3.3. Bacterial isolates and biochemical identification

healed after varying treatment times, but despite treatments S. agalactiae could still be isolated from these chronic infectious sites.

In total 25 isolates of S. agalactiae could be cultivated from swab and organ samples originating from African and Asian elephants in four different zoos (Table 1), of which 23 had been stored and were further characterised in this study. Eleven isolates were originally cultured from African elephants (zoo B), six, five and one isolates came from Asian elephants from zoo A, zoo C and zoo D, respectively. For reasons of comparison, isolates from pigs, cows and humans (each n = 3), mice (n = 2) and one isolate each (n = 1) from a gorilla, chimpanzee, goat, capybara, dog, guinea pig, beaver, springhaas, jumping shrew, slender loris, coendou and capercaillie cock from zoos A, E, and F were also included (Supplementary Table S1). From the elephants S. agalactiae was mostly isolated in moderate to high numbers from swabs, which were taken from infected fistulae as well as from purulent discharge from the temporal glands. Normally, a rich mixture of aerobic, microaerophilic and anaerobic concomitant microbiota was also present (Table 1). Attempts to isolate S. agalactiae also from adjacent rhinoceroses in zoo A (n = 2) or from elephant faeces in zoos A and B (n = 4) were unsuccessful. Streptococcus agalactiae grew after 24 h on Columbia blood agar as whitish colonies, measuring approximately 1–2 mm in diameter. No growth was observed on Gassner agar. After further incubation for additional 24 h the regular, slightly moist colonies reached a size of 5 mm in diameter, surrounded by a narrow zone of β-haemolysis. Gram staining revealed regular gram-positive cocci. All isolates displayed the regular CAMP phenomenon with Staphylococcus aureus, thus indicating presence of the pore-forming cytotoxin (Hensler et al., 2008). All presumptively identified streptococci agglutinated only with group Bspecific Streptococcus antiserum. Despite some minor differences in the API 20 Strep profiles as well as Vitek2 GP biotype codes all but one GBS isolate examined were confirmed as S. agalactiae with excellent results. Tendentiously, identification results of the API 20 Strep suggested an even higher reliability than Vitek2 GP biotyping (Supplementary Table S2).

3.2. Auto-vaccination against S. agalactiae

3.4. Identification by MALDI-TOF MS

During the vaccination period the general health conditions in both animals never deteriorated. Following the higher concentrated equipotent doses, injection sites sometimes resulted in mild swellings and pain occasionally leading to reduced head movements for food collection on the same day. Swab samples taken in order to evaluate the shedding of S. agalactiae revealed only a slight decrease towards the end of the vaccination period as obtained by semi-quantitative evaluation of bacterial growth, but never a complete de-colonisation. Contrarily, respective sites were again heavily colonised with this microorganism several weeks later. Zoo B In 2010, female African elephant “Aruba” developed foot lesions on the left front leg consistent with pododermatitis (Table 1, Fig. 1A), which were successfully treated by regular warm ointment baths and pedicure. High amounts of S. agalactiae could be isolated. “Wankie” died four years later after a period of arthroses in several joints. During necropsy, a septicaemic cause of infection following acute arthritic episodes was diagnosed and S. agalactiae was isolated as predominant microorganism from nasal cavity, spleen, urinary bladder and heart. In 2014, “Tamo” developed skin abscesses (Fig. 1B). In 2015, a suppurative infection of the right temporal gland was noted in “Zimba”

All isolates of the present study could successfully be identified using MALDI-TOF MS and Bruker’s MALDI-Biotyper database in direct transfer protocol. Most identifications yielded score levels above 2.3 and all were classified in category A indicating high correctness of the species level and species consistency according to a scoring scheme provided by the manufacturer. In other cases, those values could be obtained after using an acetonitrile-formic acid extraction protocol provided by the manufacturer (data not shown). Furthermore, identification of the concomitant bacterial microbiota cultured from the elephants’ samples was also carried out by MALDI-TOF MS or – before its implementation – by commercially available biochemical assays (API systems, VITEK2-compact, both bioMérieux, Nürtingen, Germany; Table 1, Supplementary Table S1).

Fig. 1. Different clinical presentations of Streptococcus agalactiae infection in elephants: Pododermatitis on the medial digitus (D2) of the right forelimb in the African elephant “Aruba” (A); suppurative dermatitis and ulcers on the right ear margin in the African elephant “Tamo” (B) and purulent discharge secreted from the right temporal gland in the African elephant “Zimba” (C).

3.5. Antimicrobial susceptibility testing All S. agalactiae isolates were in vitro susceptible (in brackets minimum inhibitory concentrations [MIC] in μg/mL) to amoxicillin (≤0.12), cefotaxime (≤0.12), colistin (≤4), levofloxacin (≤1) and penicillin G (≤0.06). Most but not all isolates were susceptible to clindamycin, erythromycin and moxifloxacin, whereas most isolates 146

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isolates originating from zoo A. This is principally in accordance with results obtained by FT-IR and RAPD PCRs, where isolate CVUAS 338 is clearly separated from other zoo A isolates. Proximity to zoo C isolates – like in the RAPD PCRs – could not be observed.

showed resistance to tetracycline (Supplementary Table S2). 3.6. PCR analysis of the capsule type In contrast to all other isolates included in this study the majority of S. agalactiae derived from African and Asian elephants were nontypable with the method applied. This means that most S. agalactiae isolates from elephants from four zoos were indistinguishable based on capsule typing alone. Solely isolate CVUAS 338 from zoo A revealed capsule type Ia and four from the five isolates from Asian elephants from zoo C were determined as capsule type III. These latter capsule types were also merely found in livestock animals and humans. Notably, capsule type Ia occurred in a capibara and a coendou, both living in zoo A. All other zoo animal species were found to be infected with other capsule types.

4. Discussion Although only rarely reported in literature, pododermatitis is a common problem in zoo and circus elephants with as many as 50% of the captive population being affected, especially among Asian elephants (Fölsch, 1968; Fowler, 1993; Mikota et al., 1994; Keet et al., 1997; Lewis et al., 2010). The aetiology of pododermatitis in wild elephants was suspected to result from foot trauma by certain plant splinters following excessive drought and simultaneous overcrowding and contamination of soil near artificial water reservoirs (Keet et al., 1997). In this former study, a group of 13 wild male African elephants suffered from laminitis, sometimes involving all feet. A malodorous necropurulent exudate mixed with soil and environmental debris was regularly noticed covering raw, yellowish-red, pus-encrusted surfaces (up to 20 × 24 cm) that bled readily when lightly scarified (Keet et al., 1997). Locomotion of these animals was significantly reduced, sometimes ending up in extreme debilitation, septic arthritis and generally poor prognosis. In four out of 13 cases bacteriology was carried out and solely S. agalactiae was isolated from all four foot lesions and also from two axillary lymph nodes with varying amounts and composition of concomitant bacterial microbiota. On the other hand, Aupperle et al. (2008) found S. agalactiae in high (pododermatitis) and low (lymph nodes, kidneys, omentum) numbers in a female Asian elephant with endometritis accompanied by high growth rates of S. equi zooepidemicus and E. coli. Pathological findings included pododermatitis, leiomyoma and multiple fat tissue necrosis and were regarded as secondary issues beside severe chronic purulent endometritis. The microbiota of foot infections in elephants is not very well studied. Commonly, a high number of environmental contaminant bacteria can be isolated from foot lesions and members of Escherichia, Proteus, Pseudomonas (Fowler, 1993), Enterobacter, Klebsiella, Staphylococcus, Streptococcus (Mikota et al., 1994), Corynebacterium, Bacillus, Aeromonas and Mannheimia (Keet et al., 1997) have been encountered so far. Due to location and microclimate of feet ulcers anaerobic bacteria including Bacteroides fragilis, Eubacterium lentum, Clostridium tetani and Peptostreptococcus melanogenicus (Fowler, 1993; Keet et al., 1997) are also involved and thereby strikingly resemble etiopathological organisms of dermatitis digitalis in cattle and sheep (Sullivan et al., 2015). Interestingly, while regularly isolated as a mastitis pathogen in cattle, S. agalactiae has so far never been characterised as a contributor to dermatitis digitalis in cattle. On the other hand, spirochaetes have been found to contribute significantly to this disease in cattle, sheep and elk (Sullivan et al., 2015), but have so far never been reported from respective lesions in elephants. The most striking parallel can be seen between the involvement of S. agalactiae in diabetic feet in humans and similar lesions in elephants, which fuels the discussion concerning a common pathogenesis. Although continuous assessment of blood values, water uptake and urinalysis is uncommon in most ‘hands-off’ maintenance diabetes mellitus could be excluded for elephants from zoo A and generally seems to be a rare disease in elephants (Van der Kolk et al., 2011). Nevertheless, the authors mention also an involvement of a severe necrotic laminitis in their case; unfortunately, no bacteriological results are reported. Apart from foot lesions we have also isolated S. agalactiae for the first time from discharge of temporal glands, skin abscesses and ulcers as well as from inner organs in a fatal case. Attempts to isolate this organism also from adjacent rhinoceroses in zoo A or from elephant faeces in zoo B were unsuccessful. Detection of S. agalactiae in association with similar lesions in African and Asian elephants in at least four different zoos (without contact) and reports of two more cases in literature (Keet et al., 1997; Aupperle et al., 2008) suggest a special tropism of this pathogen for elephants or an increased susceptibility of

3.7. Cluster analysis of isolates created by infrared spectra using FT-IR The comparison of the infrared-spectra of the 23 isolates from elephants with a collection of field isolates originating from other species showed a separation into two main branches with ten separate clades in total. With few exceptions, there was a high correlation of these results with those obtained by capsule typing. One of the main branches was represented by all non-typable isolates derived from elephants. Only the isolate from the capercaillie cock clustered separate, but nearer to this group, although its capsule type III was clearly deviant. Moreover, non-typable isolates could be correctly assigned to the respective elephant host species by FT-IR analysis (Fig. 2). Isolates P6096/11 and P2528/11 from Asian elephants from zoos C and D, respectively, clustered within the same group like other isolates from Asian elephants from zoo A. However, the position of further typable elephant isolates was also merely influenced by their capsule types than by zoo origins or host species. This was also true for isolates from other host species from the same zoo. 3.8. Genomic fingerprinting analysis 3.8.1. RAPD PCRs All but one isolate (11-7-D-02410) could successfully be differentiated based on RAPD primers OPB17, OPB18 and A (Fig. 3). Interestingly, there was quite a good separation of elephant isolates from different zoos into different RAPD clusters. Most elephant isolates from zoos A, B, C each clustered together and separate from other groups. The first exceptions was isolate CVUAS 338 (represented by capsule type Ia) from an Asian elephant in zoo A that clustered among other zoo C isolates (all represented by capsule type III). The second outlier was again isolate P6096/11 (non-typable) from zoo C that clustered – with some distance – next to most zoo A isolates. Finally, isolate 10-7-D02041 (obtained 2010) lay apart from all other zoo B isolates (isolated 2014/2015). The majority of non-typable strains could also be differentiated with respect to zoo A and zoo B, but the single isolate from zoo D (P2528/11) was – in contrast to FT-IR results – found among other zoo B isolates. 3.9. Pulsed-field gel electrophoresis (PFGE) PFGE results resemble those of FT-IR and RAPD PCRs (Fig. 4). Clustering on a basis of 15% similarity grouped most elephant isolates depending on the zoo they originated from. An exception was P6096/ 11, that clustered nearer with zoo B isolates than with other zoo C isolates. The outlying position of P6096/11 in the context of zoo C is not surprising since it is the only “nt” isolate from this zoo that was also found to be unique by FT-IR and RAPD typing. The host of P6096/11 first moved to zoo C in 2009, one year after the other samples from there were taken, which in this case might explain the differences. Isolate CVUAS 338 (isolated in 2004) clusters only weakly with later 147

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Fig. 3. Cluster analysis based on genomic fingerprint pattern generated with three RAPD-PCRs (primers OPB17, OPB18 and A) of Streptococcus agalactiae isolates from elephants and other species. Fingerprint DNA pattern were separated on 1.5% (v/w) agarose gels and stained with ethidium bromide. Cluster analysis was performed with the UPGMA clustering method in GelCompar II using a consensus matrix based on the similarity matrices (all in equal contribution) which were calculated for the individual fingerprint types with the Pearson correlation similarity coefficient.

1261/52 and/76 from cattle. On the other hand, one has to take into account that all typing methods, RAPD-PCR, PFGE, and FT-IR, are based on different premises: The former two genotyping methods rely on isolated fragments or whole DNA, whereas the phenotypical FT-IR founds on biochemical cell components including storage vesicles, that do not necessarily share similarities with capsule and genotypes. Furthermore, these methods are strongly influenced by the selectivity applied. Although RAPD typing has previously been shown to have a similar high discriminatory index (> 0.95) as PFGE in GBS (Duarte et al., 2004), both methods will not produce identical results though. In humans, most S. agalactiae infections in neonates are caused by capsular types Ia, Ib, or III (Morozumi et al., 2015). In contrast to elephants and other animal species, non-typable isolates are uncommon in humans (Rosini et al., 2015). Based on the mentioned differences there is presently no clue that S. agalactiae isolates from elephants represent specific zoonotic pathogens. In Germany, a retrospective study revealed concordance of bovine with human sequence types worldwide as obtained by multilocus sequence typing (MLST) (Schlez et al., 2015). On the basis of identical or at least very similar capsule or sequence types further studies have supported the assumption of zoonotic transmission, especially between cattle and humans (Manning et al., 2010). Even though zoonotic human infections could not satisfactorily be proven, yet, bovine mastitis can be induced by human strains of S. agalactiae (Van den Heever and Giesecke, 1980). This would principally also include the possibility of transmission of S. agalactiae from humans, e.g. zookeepers, to elephants (anthropozoonosis).

these animals. Presently, due to occupational safety reasons the question remains to be answered whether the organism is asymptomatically colonising mucous membranes in elephants or whether it is introduced into the herd and then elephants themselves favour the spread this organism to other sites or mates by e.g. grooming or manipulating painful foot lesions with their trunks. Repeated treatment schemes and preliminary results from autologous vaccination trials indicate that colonisation of elephants with GBS and shedding cannot as easily be prevented as in cattle. Our molecular data analysis supports significant genotypic differences with respect to the zoo origin. Although the vast majority of elephant isolates was indistinguishable with respect to their capsule types, inside each zoo, isolates displayed considerable homogeneity as obtained by PFGE and RAPD analysis. However, a deviant type that was each represented by a single isolate only was also detected in zoo A (CVUAS 338) and zoo C (P6096/11), both of which were also characterised by different capsule types. This points towards one dominant infectious cluster of highly related isolates inside each cohort. The deviant distribution was also confirmed phenotypically by FT-IR spectroscopy, which indeed reflects a higher number of biomolecules than only surface proteins. However, S. agalactiae isolates from other animal species within the same zoo did not show any similarities with elephant isolates. However, the three presented typing methods also generated incongruent and – at first appearance – doubtful results and remarkable differences. Examples for an outlying position in one of the three methods were isolates 10-7-D-02041, P2825/11 and also 08-7-Mi148

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Fig. 4. CHEF-PFGE-electropherograms (angle 120; voltage 6 V/cm; pulsed-field times 1–13 s for 20 h; ramping, linear) of SmaI-restricted genomic Streptococcus agalactiae DNA from elephant and other species isolates. Cluster analysis of Dice similarity indices (UPGMA) was exerted to generate a dendrogram depicting the relationships among PFGE profiles using the BioNumerics 6.6 software (optimisation 1.5%, position tolerance 1.5%).

6. List of authors’ contributions

Interestingly, despite susceptibility to a wide spectrum of antimicrobials there is evidence for an increase of resistant isolates in humans (Shabayek and Abdalla, 2014; Teatero et al., 2015) and animals (Fischer et al., 2013; Pinto et al., 2014; Aisyhah et al., 2015) worldwide. To date resistance to clindamycin, tetracycline, and erythromycin, often in combination with a constitutive macrolide-lincosamidestreptogramin B resistance, is most commonly noted (Shabayek and Abdalla, 2014; Wang et al., 2015). Selective pressure of tetracyclines might have favoured a complete replacement of diversity of GBS by dissemination of few clones, which have acquired integrative and conjugative elements conferring tetracycline resistance (Da Cunha et al., 2014). The recently detected emergence of lineages of S. agalactiae that are resistant to quinolones (Piccinelli et al., 2015) and vancomycin (Srinivasan et al., 2014) is alarming. Presently, the majority of elephants’ isolates show antibiotic susceptibility offering good therapeutic options.

TE conceived the study. TE, AF, KS, MZ, JR, CH, MvdL and CE carried out diagnostics and experiments. TE, KS, RS and EPB isolated strains and gathered data from all zoos. UW and TKW were in charge of animal care, sample acquisition and therapy. SPG and DP performed GelCompar analyses. TE wrote the manuscript and all the authors read and approved the final manuscript. 7. Conflict of interest None. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Acknowledgements The authors like to thank Anna Mohr, Katharina Engel, Wolfgang Schmidt, Sabine Wolf, Barbara Depner, Anna Katharina Schmid, and Jana Kistenmacher for excellent technical assistance and Barbara Gamb for the unsurpassed literature service. The Hessian State Laboratory is supported by the Hessian Ministry for the Environment, Climate Change, Agriculture and Consumer Protection.

5. Conclusion S. agalactiae plays an important role in bacterial infections not only in cattle and humans, but also in elephants. Comparative studies based on differentiation of S. agalactiae isolates into different clusters with respect to elephant species and/or epidemiological background (zoo origin) as obtained by FT-IR spectroscopy, capsule typing, RAPD-PCR and PFGE are helpful for a better understanding of epidemiological links. Future studies should include further S. agalactiae isolates from wildlife in phylogenetic typing and in virulence profiling for better understanding pathogenic properties and possible zoonotic impact of this pathogen.

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