Skip to main content
PMC home page
PLOS ONE logoLink to PLOS ONE
. 2009 Dec 14;4(12):e8286. doi: 10.1371/journal.pone.0008286

Molecular Characterization of Pneumococcal Isolates from Pets and Laboratory Animals

Mark van der Linden 1,*, Adnan Al-Lahham 2, Werner Nicklas 3, Ralf René Reinert 1,¤
Editor: Stefan Bereswill4
PMCID: PMC2788425  PMID: 20011527

Abstract

Background

Between 1986 and 2008 Streptococcus pneumoniae was isolated from 41 pets/zoo animals (guinea pigs (n = 17), cats (n = 12), horses (n = 4), dogs (n = 3), dolphins (n = 2), rat (n = 2), gorilla (n = 1)) treated in medical veterinary laboratories and zoos, and 44 laboratory animals (mastomys (multimammate mice; n = 32), mice (n = 6), rats (n = 4), guinea pigs (n = 2)) during routine health monitoring in an animal facility. S. pneumoniae was isolated from nose, lung and respiratory tract, eye, ear and other sites.

Methodology/Principal Findings

Carriage of the same isolate of S. pneumoniae over a period of up to 22 weeks was shown for four mastomys. Forty-one animals showed disease symptoms. Pneumococcal isolates were characterized by optochin sensitivity, bile solubility, DNA hybridization, pneumolysin PCR, serotyping and multilocus sequence typing. Eighteen of the 32 mastomys isolates (56%) were optochin resistant, all other isolates were optochin susceptible. All mastomys isolates were serotype 14, all guinea pig isolates serotype 19F, all horse isolates serotype 3. Rats had serotypes 14 or 19A, mice 33A or 33F. Dolphins had serotype 23F, the gorilla serotype 14. Cats and dogs had many different serotypes. Four isolates were resistant to macrolides, three isolates also to clindamycin and tetracyclin. Mastomys isolates were sequence type (ST) 15 (serotype 14), an ST/serotype combination commonly found in human isolates. Cats, dogs, pet rats, gorilla and dolphins showed various human ST/serotype combinations. Lab rats and lab mice showed single locus variants (SLV) of human STs, in human ST/serotype combinations. All guinea pig isolates showed the same completely new combination of known alleles. The horse isolates showed an unknown allele combination and three new alleles.

Conclusions/Significance

The isolates found in mastomys, mice, rats, cats, dogs, gorilla and dolphins are most likely identical to human pneumococcal isolates. Isolates from guinea pigs and horses appear to be specialized clones for these animals. Our data redraw attention to the fact that pneumococci are not strictly human pathogens. Pet animals that live in close contact to humans, especially children, can be infected by human isolates and also carriage of even resistant isolates is a realistic possibility.

Introduction

Streptococcus pneumoniae is the major causative pathogen of community-acquired respiratory tract infections (RTIs) in humans, including community-acquired pneumonia, acute otitis media and acute maxillary sinusitis. It is also the cause of severe invasive infections like meningitis and bacteremia in both children and adults. Most humans, especially young children (0–5 years of age), are likely to be colonized at least once during their lifetime with pneumococci that are spread by droplet infection [1]. Pneumococcal-related disease is usually preceded by colonization of the nasopharynx. Therefore, pneumococcal carriage is believed to play an important role in horizontal spread of S. pneumoniae in the community.

In general, S. pneumoniae is considered to be a human pathogen only, despite there being established mouse and rat models for various S. pneumoniae-caused diseases [2]. For example, BALB/c mice are used as models for pneumococcal pneumonia and meningitis [3], and rats for pneumococcal otitis media [4]. Transmission of other streptococcal species from animals to humans is well-documented, in particular for S. equi subsp. zooepidemicus, S. canis, S. suis, S. porcinus and S. phocae [5], [6], [7], [8], [9]. Pneumococci are naturally competent organisms and can easily exchange DNA in their natural habitat, the human mouth and throat. This habitat is populated by various streptococcal species, which form a ‘gene pool’ out of which the pneumococci can recruit their resistance genes. Gene transfer and mosaic genes have been intensively reported for S. pneumoniae, S. oralis, S. sanguis and S. mitis [10].

Multilocus sequence typing (MLST) produces unambiguous molecular typing data [11], [12]. The MLST technique is highly portable as any laboratory can compare the sequences of the seven loci in their isolates with those in a central database (www.mlst.net) and thereby obtain the allelic profile of each isolate. MLST results in data giving insight into the clonal relatedness of bacterial isolates.

In the present study, we characterized a large collection of S. pneumoniae from pets and laboratory animals including mastomys, guinea pigs, cats, mice, rats, horses, dogs, dolphins and a gorilla. Our findings show that S. pneumoniae is capable of colonizing and infecting animal hosts and raises the question whether these animals, which as pets are often in close contact to children, serve as an extra-human reservoir for S. pneumoniae.

Results

During routine checks of animals in the animal facilities of the German Cancer Research Center (DKFZ) in Heidelberg, Germany, α-hemolysing streptococci were isolated from multimammate mice (Mastomys coucha, n = 32), rats (Rattus norwegicus F344, n = 4), mice (Mus musculus C57BL/6, n = 6) and guinea pigs (Cavia porcellus, n = 2). Isolates from 41 pets comprising guinea pigs (n = 17), cats (Felis domesticus, n = 12), dogs (Canis lupus familiaris, n = 3), horses (Equus caballus, n = 4) and rats (Rattus rattus, n = 2) were received from a veterinary medical laboratory (Vet Med Labor GmbH) in Ludwigsburg, Germany and the Department of Infectious Diseases and Immunology (VMDC) of the Faculty of Veterinary Medicine of the University of Utrecht, the Netherlands. Two isolates from dolphins (Tursiops truncatus) were obtained from a zoo in Nuremberg, Germany and one isolate from a gorilla (Gorilla gorilla) was from a zoo in Kerkrade, the Netherlands.

Two mastomys suffered from conjuntivitis. Two guinea pigs (laboratory animals) were C4-immune deficient, and one of them suffered from peritonitis and sepsis and died. All (n = 17) pet guinea pigs were obviously ill: RTI (n = 10), otitis media (n = 1), abcesses (n = 2), undefined illness (n = 4). Both pet rats were ill (one with RTI, one with unknown disease). Seven pet cats and three pet dogs suffered from RTI. The five other pet cats suffered from otitis media, otitis externa, conjunctivitis, polyarthritis and meningitis respectively. The latter animal died. Three horses had RTI, for one there were no detailed diagnostic data available. The two dolphins were zoo animals that died without showing clear symptoms of disease, but autopsy showed pneumococci in lung biopsies. The gorilla suffered from acute infection with inflammated tonsilli with purulent excretion.

Mastomys isolates were obtained from swabs taken from nose (n = 14), eye (n = 10), nose and eye (n = 2), lung (n = 1) and trachea (n = 1). Carriage isolates were obtained from eye swabs. Guinea pig isolates were from swabs from nose (n = 10), ear (n = 2) and wound, abcess, lung, trachea, abdomen, bulla tympanica (one each). For one animal the origin of the material could not be traced anymore. Isolates from laboratory mice an rats were all from nose swabs. For the two pet rats isolates wereobtained from lung and wound swabs (one animal each). For the cats islates were obtained from swabs from nose (n = 5), thorax (n = 2), and ear, wound, throat, synovia (one animal each). For the cat that suffered from meningitis and died the isolate was obtained from a brain biopsy. The three dog isolates were obtained from nose swabs. Horse isolates were obtained from nose swabs (n = 3) and BAL (n = 1). The two isolates from dolphins were obtained from lung-tissue biopsies. The gorilla isolate was obtained from purulent secretion material ( Table 1 ).

Table 1. Characteristics of 85 Streptococcus pneumoniae isolates from pets and laboratory animals.

Isolate number Animal Year of isolation country origin Diagnosis Site of isolation Opto Test Serotype MLST
24073 mastomys 1 2003 Germany DKFZ no clinical symptoms nose R 14 15
24074 mastomys 2 2003 Germany DKFZ no clinical symptoms nose S 14 15
24080 mastomys 3 2003 Germany DKFZ no clinical symptoms nose S 14 15
22877 mastomys 4 2003 Germany DKFZ no clinical symptoms nose R 14 15
22878 mastomys 5 2003 Germany DKFZ no clinical symptoms nose R 14 15
22879 mastomys 6 2003 Germany DKFZ no clinical symptoms trachea R 14 15
22875 mastomys 7 2004 Germany DKFZ no clinical symptoms nose R 14 15
22876 mastomys 8 2004 Germany DKFZ no clinical symptoms nose R 14 15
22881 mastomys 9 2004 Germany DKFZ no clinical symptoms nose R 14 15
22882 mastomys 10 2004 Germany DKFZ no clinical symptoms lung S 14 15
22883 mastomys 11 2004 Germany DKFZ no clinical symptoms nose R 14 15
22884 mastomys 12 2004 Germany DKFZ no clinical symptoms nose R 14 15
27110 mastomys 13 2005 Germany DKFZ no clinical symptoms nose S 14 15
22955 mastomys 14 2005 Germany DKFZ no clinical symptoms nose R 14 15
22956 mastomys 15 2005 Germany DKFZ no clinical symptoms nose R 14 15
26267 mastomys 16 2005 Germany DKFZ no clinical symptoms eye R 14 15
27072 mastomys 17 2006 Germany DKFZ no clinical symptoms nose/eye S 14 15
27673 mastomys 18 2006 Germany DKFZ no clinical symptoms eye R 14 15
27857 mastomys 19 2006 Germany DKFZ no clinical symptoms eye R 14 15
28293 mastomys 20 2006 Germany DKFZ no clinical symptoms nose/eye R 14 15
28417 mastomys 21 2006 Germany DKFZ no clinical symptoms eye R 14 15
28418 mastomys 22 2006 Germany DKFZ no clinical symptoms eye S 14 15
28419 mastomys 23 2006 Germany DKFZ no clinical symptoms eye R 14 15
28420 mastomys 24 2006 Germany DKFZ no clinical symptoms eye S 14 15
28421 mastomys 25 2006 Germany DKFZ no clinical symptoms eye S 14 15
28422 mastomys 26 2006 Germany DKFZ no clinical symptoms eye S 14 15
29791 mastomys 27 2006 Germany DKFZ conjunctivitis eye S 14 15
33432 mastomys 28 2008 Germany DKFZ conjunctivitis nose R 14 15
27703 mouse (C57BL/6) 1 2006 Germany DKFZ no clinical symptoms nose S 33A SLV100
28448 mouse (C57BL/6) 2 2006 Germany DKFZ no clinical symptoms nose S 33A SLV100
33645 mouse (C57BL/6) 3 2008 Germany DKFZ no clinical symptoms nose S 33F SLV100
32276 mouse (C57BL/6) 4 2007 Germany DKFZ no clinical symptoms nose S 33A SLV100
32969 mouse (C57BL/6) 5 2007 Germany DKFZ no clinical symptoms nose S 33A SLV100
32970 mouse (C57BL/6) 6 2007 Germany DKFZ no clinical symptoms nose S 33A SLV100
24072 rat (F344) 1 2003 Germany DKFZ no clinical symptoms nose S 14 SLV 124
24075 rat (F344) 2 2003 Germany DKFZ no clinical symptoms nose S 14 SLV 124
24077 rat (F344) 3 2003 Germany DKFZ no clinical symptoms nose S 14 SLV 124
24079 rat (F344) 4 2003 Germany DKFZ no clinical symptoms nose S 14 SLV 124
32965 rat 5 2007 Austria VML respiratory problems, sudden death lung S 19A 3546
34828 rat 6 2008 Monaco VML no data wound S 19A SLV 3546
24070 guinea pig 1 1986 Germany DKFZ peritonitis, sepsis lung S 19F new
24071 guinea pig 2 1986 Germany DKFZ peritonitis abdomen S 19F new
28614 guinea pig 3 2006 The Netherlands VML severe respiratory problems nose S 19F new
28976 guinea pig 4 2006 Germany VML head wound wound S 19F new
31598 guinea pig 5 2007 Germany VML umbillical abcess, jaw abcess abcess S 19F new
32066 guinea pig 6 2007 Germany VML respiratory problems, clotted nose nose S 19F new
32449 guinea pig 7 2007 Germany VML purulent efflux nose, eyes nose S 19F new
32668 guinea pig 8 2007 Germany VML coughing, nasal discharge, swollen lymphnodes nose S 19F new
32669 guinea pig 9 2007 Germany VML coughing, nasal discharge nose S 19F new
33098 guinea pig 10 2007 France VML tired, tilted head ear S 19F new
33099 guinea pig 11 2007 Germany VML otitis media ear S 19F new
34603 guinea pig 12 2008 The Netherlands VML nasal discharge, tiredness nose S 19F new
34719 guinea pig 13 2008 Germany VML sneezing, nasal discharge nose S 19F new
34728 guinea pig 14 2008 Germany VML Abortion of offspring, dead Trachea, uterus, liver S 19F new
35456 guinea pig 15 2008 Peru VML no data nose S 19F new
34950 guinea pig 16 2008 Germany VML recidiving rhinitis nose S 19F new
35100 guinea pig 17 2008 Germany VML coughing, purulent nasal discharge nose S 19F new
35489 guinea pig 18 2006 The Netherlands VMDC no data no data S 19F new
35494 guinea pig 19 2007 The Netherlands VMDC respiratory problems bulla tympanica S 19F new
28615 cat 1 2006 The Netherlands VML severe respiratory problems nose S 3 180
31057 cat 2 2007 Germany VML otitis media wound S 19F 177
32317 cat 3 2007 Germany VML conjunctivitis, strong sniffing throat S 6A 473
32667 cat 4 2007 Germany VML rhinitis, conjunctivitis nose S 19F 1815
32827 cat 5 2007 Germany VML dyspnoea, lungoedema, thorax puncture S 22F 819
33211 cat 6 2008 Germany VML purulent otitis externa ear S 14 9
34829 cat 7 2008 Deutschland VML nasal efflux nose S 31 1994
35135 cat 8 2008 The Netherlands VML sneezing, nasal discharge nose S 19A 3017
35488 cat 9 2003 The Netherlands VMDC purulent meningitis, died brain S 23F 311
35490 cat 10 2003 The Netherlands VMDC chronic rhinitis nose S 6B 176
35492 cat 11 1997 The Netherlands VMDC polyarthritis synovia S 22A 3705
35493 cat 12 2004 The Netherlands VMDC liquothorax, respiratory problems thorax fluid S 19F 177
32966 dog 1 2007 Netherlands VML coughing, sneezing, nasal discharge nose S 14 124
33437 dog 2 2008 Germany VML chronic recidivating rhinitis nose S 3 180
33770 dog 3 2008 Germany VML sinusitis nose S 10A SLV1551
29800 horse 1 2006 Germany VML sinusitis nose S 3 new
32450 horse 2 (pony) 2007 Germany VML coughing, nasal discharge nose S 3 new
34368 horse 3 2008 Germany VML no data nose, tracheallavage S 3 new
34604 horse 4 (racing horse) 2008 Germany VML coughing, nasal discharge, swollen lymphnodes BAL S 3 new
24186 dolphin 1 1986 Germany ZN died withour disease symptoms biopsy lung S 23F 440
24187 dolphin 2 1986 Germany ZN died withour disease symptoms biopsy lung S 23F 440
35491 ape (gorilla) 2008 The Netherlands ZK acute infection, inflammated tonsilli with pus excretion pus S 14 124
Open in a new tab
a

DKFZ: German Cancer Research Center (DKFZ), Heidelberg, Germany; VML: Vet Med Labor GmbH, Ludwigsburg, Germany, VMDC: Department of Infectious Diseases and Immunology (VMDC) of the Faculty of Veterinary Medicine of the University of Utrecht, the Netherlands, ZN: Zoo Nuremberg, Germany, ZK: Zoo Kerkrade, The Netherlands.

Further characterization of the streptococcal isolates at the German National Reference Center for Streptococci confirmed identification of all isolates as Streptococcus pneumoniae. All isolates were bile soluble, showed hybridization with a species-specific gene probe and had positive pneumolysin PCRs. Eighteen isolates, all from mastomys, were optochin-resistant.

All mastomys isolates (n = 32) were serotype 14. Guinea pig isolates were 19F, mice isolates were 33A or 33F, laboratory rat isolates were14, pet rat isolates were 19A, horse isolates were 3, dolphin isolates were 23F and the gorilla isolate was 14. Cats and dogs had a large variety of different serotypes (3 (n = 2), 6A, 6B, 10A, 14 (n = 2), 19A, 19F (n = 3), 22A, 22F, 23F, 31) ( Table 1 ).

To exclude a possible transfer of S. pneumoniae from the animal facility staff members to the laboratory animals, throat swabs of animal facility staff members of the German Cancer Research Center (DKFZ) were taken. None of the seven staff members were found to carry pneumococci. All obtained isolates belonged to normal throat flora (Streptococcus oralis, Streptococcus mitis, Streptococcus parasanguis and Gemella morbillorum). No indications for the presence of pathogenic species were found.

To determine whether mastomys could carry pneumococcal isolates asymptomatically, four animals were followed over periods of several weeks. S. pneumoniae could be isolated from the same obviously healthy animal over periods of 6, 7, 17 and 22 weeks respectively ( Table 2 ). All isolates were serotype 14.

Table 2. Carriage of S. pneumoniae by mastomys.

Isolate number Animal 1st sample (week of life) last sample (week of life) Carriage length (weeks) Year of isolation country origin Diagnosis Site of isolation Opto Test Serotype MLST
31578 mastomys #18 52 74 22 2007 Germany DKFZ no clinical symptoms eye S 14 n.d.
32161 mastomys #29 66 73 7 2007 Germany DKFZ no clinical symptoms eye S 14 n.d.
31467 mastomys #33 55 72 17 2007 Germany DKFZ no clinical symptoms eye S 14 n.d.
31580 mastomys #168 45 51 6 2007 Germany DKFZ no clinical symptoms eye S 14 n.d.
Open in a new tab
a

DKFZ: German Cancer Research Center (DKFZ), Heidelberg, Germany; n.d.: not done.

MIC analysis of the isolates showed that four isolates were resistant to clarithromycin, three of them were also resistant to clindamycin and tetracyclin. The isolates were from two cats and two pet rats. The three multiresistant isolates showed an iMLSB phenotype and an ermB genotype, the single resistant isolate had the M-phenotype, and mef(A) subclass mef(A) ( Table 3 ). All other isolates were susceptible for all tested substances (penicillin G, amoxicillin, cefotaxime, cefpodoxime, cefuroxime, clarithromycin, clindamycin, tetracycline, levofloxacin, trimethoprim-sulfamethoxazole and vancomycin).

Table 3. Macrolide resistant isolates found in pet cats and rats.

Animal Diagnosis MLST Serotype Macrolide resistance genotype Macrolide resistance phenotype CLA (μg/ml) CLI (μg/ml) TET (μg/ml) PEN (μg/ml) CEF (μg/ml)
cat 4 rhinitis, conjunctivitis 1815 19F ermB iMLSB 16 16 16 0,015 0,015
cat 6 otitis externa eitrig 9 14 mefA M 8 0,12 0,25 0,015 0,015
rat 5 Breathing problems, sudden death 3546 19A ermB iMLSB 16 8 16 0,015 0,015
rat 6 no data SLV 3546 19A ermB iMLSB 16 16 16 0,06 0,015
Open in a new tab

CLA: clarithromycin, CLI: clindamycin, TET: tetracyclin, PEN: penicillin, CEF: cefotaxime.

MLST analysis revealed that all mastomys isolates were sequence type (ST)15. All 19 guinea pigs had STs with the same new combination of existing alleles, which has not been found previously, nor have single or double locus variants ( Table 4 ). Mice isolates showed two new sequence types, both single locus variants (SLV) of ST100, one harbouring a variant spi allele, with 99% identity to allele 9, and the other a variant recP allele (99% identity to allele 12). The four laboratory rats had the same SLV of ST124, showing a variant gki allele (99% identity to allele 1). The cats and dogs showed a large variety of STs (9, 124, 176, 177 (n = 2), 180 (n = 2), 311, 473, 819, 1815, 1994, 3017, 3705 and an SLV of 1551). Three horses had the same new ST, with new alleles for spi (99% identity to allele 10), xpt (99% identity to allele 1) and ddl (99% identity to allele 6). The fourth horse (a pony horse) had the same ST, except for the spi allele which was an exact match with allele 10. Both dolphin isolates had ST 440, the gorilla isolate ST 124 ( Table 4 ).

Table 4. MLSTs of 81 Streptococcus pneumoniae isolates from pets and laboratory animals in Germany.

Animal number of animals Year Serotype MLST aroE gdh gki recP spi xpt ddl
mastomys 28 2003–2006 14 15 1 5 4 5 5 3 8
guinea pig 19 1986/2006/2007/2008 19F new 2 5 4 5 27 20 5
mouse (C57BL/6) 5 2006 33A/33F new (SLV 100) 5 12 29 12 9 (99%) 39 18
mouse (C57BL/6) 1 2006 33A new (SLV 100) 5 12 29 12 (99%) 9 39 18
rat (F344) 4 2003 14 new (SLV 124) 7 5 1 (99%) 8 14 11 14
rat (pet) 1 2007 19A 3546 1 5 41 5 10 28 8
rat (pet) 1 2008 19A new (SLV 3546) 1 5 41 5 10 99% 28 8
cat 1 2008 14 9 1 5 4 5 5 1 8
cat 1 2003 6B 176 7 13 8 6 10 6 14
cat 2 2004/2007 19F 177 7 14 4 12 1 1 14
cat 1 2006 3 180 7 15 2 10 6 1 22
cat 1 2003 23F 311 1 8 9 1 6 4 6
cat 1 2007 6A 473 7 25 4 4 15 20 28
cat 1 2007 22F 819 1 1 4 1 18 58 18
cat 1 2007 19F 1815 1 5 4 12 5 3 159
cat 1 2008 31 1994 1 2 29 1 111 14 18
cat 1 2008 19A 3017 8 11 14 1 17 230 14
cat 1 1997 22A 3705 2 194 53 18 10 1 1
dog 1 2007 14 124 7 5 1 8 14 11 14
dog 1 2008 3 180 7 15 2 10 6 1 22
dog 1 2008 10A SLV 1551 5 7 4 6 10 1 6
horse 3 2006 3 new 10 9 4 12 10 (99%) 10 (99%) 6 (99%)
horse (pony) 1 2007 3 new 10 9 4 12 10 10 (99%) 6 (99%)
dolphin 2 1986 23F 440 7 5 1 1 13 31 14
gorilla 1 2008 14 124 7 5 1 8 14 11 14
Open in a new tab

ST 15/serotype 14, ST 100/33F, ST 124/14, ST 3546/19A and ST 440/23F found in mastomys, mice, rats, dog, gorilla and dolphins respectively are common sequence type/serotype combinations found in human pneumococcal isolates (www.mlst.net). This is also the case for the combinations ST 9/14, ST 176/6B, ST 177/19F, ST 180/3, ST 311/23F, ST 473/6A, ST 819/22F, ST 1551/10A, ST 1815/19F, ST 1994/31, and ST 3017/19A found in cats and dogs. The combination ST 3705/22A found in one cat has so far never been encountered in humans.

Discussion

It is generally accepted that Streptococcus pneumoniae is strictly a human pathogen despite there being animal models (mouse, rat) available to study pneumococcal infections. However, on the other hand, recent literature describing naturally occurring carriage of or infections by pneumococci in animal hosts is scarce [13], [14]. A report from 1988 describes the isolation of S. pneumoniae serotype 3 from a racehorse [15]. Other pneumococcal isolates from racehorses also appear to have serotype 3 [16], [17]. In a recent report from Chi et al. S. pneumoniae isolated from Chimpansees are described [13]. Notably, anecdotal reports of S. pneumoniae isolated from animals can be found as early as in the 1940s ( Table 5 ).

Table 5. Anecdotal reports of Streptococcus pneumoniae isolated from different mammalian species.

Animals Number of infected animals Outbreak Serotypes Laboratory animals Year Source
guinea pigs 39 yes 19 yes 1945 Homburger et al. [33]
rats 71 yes 2 yes 1950 Mirick et al. [18]
rats 254 yes 8 yes 1965 Ford [19]
rats 156 no 2, 3, 19 yes 1969 Weisbroth and Freimer [20]
rats 32 no 3 yes 1971 Mitruka [21]
mice, rats 9,10 yes 35 yes 1988 Fallon et al. [34]
horses 10 no 3 no 1988 Huber and Willoughby [15]
horses 11 no 3 no 1999 Whatmore et al. [16]
cat 1 no n.d. no 2006 Zhang et al. [14]
Open in a new tab

n.d: no data.

Outbreaks of infections with S. pneumoniae of serotypes 2, 3, 8 and 19 in laboratory rats in the US have been described in several publications [18], [19], [20], [21]. Furthermore, veterinary textbooks describe pneumococcal infections to be not only present in rodents (rats, mice, guinea pigs) but also in larger domestic animals (calves, horses) [22], [23].

Since all pneumococcal isolates obtained from animals in this study were bile soluble, and showed hybridization with a species specific DNA probe, they were unambiguously identified as S. pneumoniae. Moreover, the fact that serotyping and MLS typing gave positive results confirms that the isolates are pneumococci. However, 18 out of 32 mastomys isolates were optochin-resistant. Since pneumococcal isolates are almost always optochin susceptible and optochin resistance is usually a trait of other viridance group streptococci, this could be one of the reasons why pneumococci are being overlooked and therefore not reported in animal isolates, thereby leading to underrepresentation of information on the extent of pneumococci colonization in the literature. Finally, it is noteworthy that optochin-resistant pneumococci are being increasingly reported from human sources [24].

The most obvious cause of infection for the laboratory animals would be the animal facility staff. For the mastomys colony in the German Cancer Research Center this could be verified. None of the staff carried pneumococci at the moment of sampling, making it less likely that animals had been infected recently. However, it cannot be excluded that the colony got infected at an earlier point in time since carriage in humans is transient. For the other laboratory animals, which came from large commercial facilities, a possible infection by facility staff members could not be tested.

Most ST/serotype combinations identified in the isolates in this study are commonly found among human pneumococcal isolates (www.mlst.net). The combination ST 15/serotype 14, found in 28 mastomys is commonly found among human isolates in association with meningitis and bacteremia and has been reported from the UK, the Netherlands, Portugal, Germany, Italy, Brazil and Hong Kong (www.mlst.net).

The combination ST 100/33F is commonly encountered among human isolates and has been reported from Spain, Germany, Poland and USA. The combination with serotype 33A has not been reported so far (www.mlst.net). This could indicate that a serotype switch from 33F to 33A has occurred in mice, as well as a mutation in the spi or the recP allele.

The combination ST 124/serotype 14 found in the gorilla, a dog and, as an SLV, in the laboratory rats has been reported from Germany, Sweden, UK, Denmark, Finland, Norway, The Netherlands, Australia, Canada, and Poland and is associated with meningitis, bacteremia, pneumonia and otitis media in human isolates, as well as with carriage (www.mlst.net).

The combination ST 3546/19A found in two pet rats has been reported only once, from Norway, and was isolated from blood (www.mlst.net).

The combination serotype ST 440/23F (UK, Poland, Czech Republic, Switzerland) found in the two dolphins is also commonly encountered among human isolates where it is associated with meningitis and bacteremia as well as being found in carriage (www.mlst.net).

The combination ST 180/serotype 3 found in a cat and in a dog isolate has been reported from several countries (UK, Spain, Poland, Portugal, Italy, Taiwan, Canada, Denmark, Netherlands) and is associated with invasive disease (meningitis, bacteremia) but also with community acquired pneumonia, otitis media and carriage (www.mlst.net). The combinations ST 9/14, ST 176/6B, ST 177/19F, ST 311/23F, ST 473/6A, ST 819/22F, ST 1551/10A, and ST 1994/31 found in cats and dogs are all well known human ST/serotype combinations associated with both invasive disease and carriage (www.mlst.net). Two isolates, ST 1815/19F and ST 3017/19A, both found in cats have been reported only once to the MLST database (www.mlst.net). ST 1815/19F was from a case of bacteremia from the Czech Republic. ST 3017/19A was isolated from blood from a patient from the Netherlands, which is also the country of origin of the cat. The combination ST 3705/22A has not been reported to the MLST database. The only isolate with ST 3705 is a carriage isolate from the Netherlands with serogroup 19. Again this isolate is from the same country as the cat in this study.

eBURST analyses revealed that STs 15, 124, 177, 180, 473 and 1994 are all predicted founders of clonal complexes (CC). ST 124 is the predicted founder of a group of 78 STs and is one of the original penicillin-resistant clones described in South Africa [25]. CC124 also comprises ST 440. ST9 and ST1815 belong to CC15. STs 176, 311, 819, 1551, and 3705 are members of different clonal complexes. STs 3546 and 3017 are singletons, i.e. they are not related to any other STs in the database. Taken together, our data show that the pneumococcal isolates from mastomys, mice, rats, cats, dogs, dolphins and gorilla analysed in this study are highly similar, if not identical, to human isolates related to invasive pneumococcal disease and carriage.

Serotype 19F, found in all four guinea pig isolates, is a common serotype found in human isolates (www.mlst.net), where it is associated with invasive pneumococcal disease as well as carriage. It occurs in combination with large numbers of different STs. However, the STs of all 19 guinea pig isolates consist of a new allele combination never encountered in human isolates. From these findings it may be interpreted that the guinea pigs did not pick up the pneumococcal infections from humans. Moreover, the fact that the same new allele combination has been found in isolates from guinea pigs from different locations (pets, laboratory animals), different countries (France, Germany, Peru, the Netherlands) and widely separated in time (1986, 2006) seems to indicate that this clone is typical for guinea pigs and that these animals are a reservoir for S. pneumoniae of this specific sequence type. As apparent from our diagnostic data, this clone is clearly pathogenic for guinea pigs.

The four horse isolates described here have serotype 3 and a new ST, with three or two new alleles. Whatmore et al. report on a collection of horse pneumococcal isolates of serotype 3 and conclude on the basis of RFLP analyses that these isolates are closely related to, but distinguishable from human pneumococcal isolates [16]. Showing a new sequence type, our data confirm that the horse isolates form a population different from human isolates. Pneumococci found in guinea pigs and horses seem to belong to separate host specific populations. The isolates found in mastomys, mice, rats, cats, dogs, dolphins and gorilla are most likely identical to human pneumococcal isolates.

Four animals, two cats and two rats, had macrolide resistant isolates. One cat isolate showed moderate resistance to clarithromycin and carried the mefA subclass mef(A) gene. The isolate had ST15 combined with serotype 14, which is the most common macrolide resistant clone (PMEN England14-9) in Germany [26], [27]. The other isolates all contained the ermB resistance marker, which results in higher MICs for clarithromycin and resistance to clindamycin. These isolates were also resistant to tetracyclin. The ST/serotype combinations were 1815/19F and (SLV)3546/19A. As discussed above these are rather rare combinations. However, serotypes 19A and 19F are strongly associated with multidrug resistance.

Our findings are unequivocal proof that pneumococci are capable of causing severe disease in a number of different animal species. One of the pet guinea pigs was a member of a larger colony in which the animals suffered from respiratory problems and of which several animals died. Further, the cats, dogs and horses also suffered from severe respiratory problems, and the two dolphins were likely to have died from pneumococcal infections.

On the other hand, laboratory animals (mastomys, mice and rats) from which pneumococci were isolated showed no disease symptoms. For two animals from the mastomys colony that suffered from conjunctivitis, another potential causative agent for the clinical signs (Pasteurella pneumotropica biotype Heyl) was found in large numbers in addition to S. pneumoniae. This finding indicates that the rodents may carry S. pneumoniae in their normal flora without suffering from pneumococcal disease, much like in the case of humans. Indeed, for four individual mastomys, we were able to isolate the same isolate over a period of up to 22 weeks, showing that the animals are carriers.

This is the first time that a larger collection of S. pneumoniae isolated from a range of animals has been characterized on a molecular basis using MLST, enabling a solid comparison to human isolates present in the MLST database. The results show that on one hand animals can be infected with human pneumococcal clones. On the other hand animals like horses and guinea pigs seem to be affected by their ‘own’ specific pneumococcal clones. The fact that pets like cats, dogs, guinea pigs, rats and mice are possible carriers of pneumococcal strains, and in many cases of the same ST/serotype combinations as human isolates, is worrisome. Even more alarming is the fact that antibiotic resistant strains, on one occasion even the most common macrolide resistant clone in Germany (England14-9), could be isolated from pets. Our data imply that there is an infection route for pneumococci from humans to pet animals that live in close contact to humans, especially children. By residing in animals, pneumococci would not only enlarge their gene-pool with other streptococcal species found more commonly in animals, but would also be able to avoid the negative selective pressure of vaccination in children. The question remains if pneumococci residing in animals can re-infect humans. On basis of the present data no conclusion on this can be drawn and further research is needed.

Materials and Methods

Study material

The German National Reference Center for Streptococci (GNRCS) received a total of 85 bacterial isolates from animals in Austria, France, Germany, Monaco, The Netherlands and Peru in 1986 (n = 4) and during 2003–2008 (n = 81). Fourty-four isolates were from the animal facility of the German Cancer Research Center (DKFZ) in Heidelberg, Germany, 32 isolates were from pets treated at a commercial veterinary medical laboratory (Vet Med Labor GmbH) in Ludwigsburg, Germany, 6 isolates were obtained from the Department of Infectious Diseases and Immunology (VMDC) of the Faculty of Veterinary Medicine of the University of Utrecht, the Netherlands, two isolates were from a zoo in Nuremberg and one from a zoo in Kerkrade, the Netherlands. All samples were sent to the GNRCS as isolated bacteria, no primary material was received.

Testing of animal facility staff

Throat swaps were obtained in May 2006 from seven members of the animal facility staff of the German Cancer Research Center and tested for bacterial content using routine microbiological methods. Throat swabs were taken by the institute medical officer of the DKFZ, after verbal consent of each of the animal facility staff members. Isolated bacteria were analysed both at the DKFZ as well as at the GNRCS.

Characterization of isolates

Streptococcus pneumoniae isolates were characterized by optochin susceptibility and bile solubility testing. Optochin susceptibility testing was carried out in a 5% CO2 atmosphere on sheep blood agar [28]. Bile solubility testing was performed by preparing a bacterial suspension in 1 mL 0.85% NaCl (McFarland standard 1.0) adding four drops of 10% sodium deoxycholate. Complete lysis after incubation for 2 h at 35°C was taken as a positive result. A commercially available DNA hybridization test (AccuProbe® Streptococcus pneumoniae identification test; bioMérieux, Germany) was used according to the manufacturer's instructions.

Serotyping

Pneumococcal strains were serotyped by the Neufeld Quellung reaction using type and factor sera provided by the Statens Serum Institut, Copenhagen, Denmark [29].

Susceptibility testing

Minimal inhibitory concentration (MIC) testing was performed using the broth microdilution method as recommended by the Clinical Laboratory Standards Institute [30]. Microtiter plates containing penicillin G, amoxicillin, cefotaxime, cefpodoxime, cefuroxime, clarithromycin, clindamycin, tetracycline, levofloxacin, trimethoprim-sulfamethoxazole and vancomycin with cation-adjusted Mueller-Hinton broth (Oxoid, Wesel, Germany) plus 5% lysed horse blood (Oxoid) were used. The final inocculum was 5×105 CFU/ml. MICs were determined following incubation for 24 h at 35°C in ambient air. S. pneumoniae ATCC 49619 was used as a control strain. Current Clinical Laboratory Standards Institute interpretive criteria were used to define antimicrobial resistance [30].

DNA extraction

Isolates were inoculated from agar plates (5% sheep blood) into a sterile culture tube containing 10 mL Todd-Hewitt broth (Oxoid Limited Basingstoke, Hampshire, UK) and incubated over night at 37°C. After centrifugation, the chromosomal DNA was isolated using a DNA extraction kit (Qiagen, Hilden, Germany).

Pneumolysin PCR

Analysis of the pneumolysin gene (ply) was performed using real-time PCR on a Light Cycler (Roche Diagnostics GmbH, Penzberg, Germany) according to the LightCycler Operator's Manual Version 3.5. PCR was carried out using the Light Cycler Faststart DNA Master SYBR Green I – kit according to the manufacturer's instructions. The reaction mixture of 20 µl contained 2 µl SYBR Green I, 2 µl MgCl2, 12 µl H20, 1.5 µl of each primer and 200 ng of DNA. The oligonucleotide primers pair was ply fwd 894–915 5′ TGC AGA GCG TCC TTT GGT CTA T 3′ and ply rev 974–950 5′ CTC TTA CTC GTG GTT TCC AAC TTG A 3′ [31]. PCR cycling comprised initial denaturation for 10 min at 95°C and 35 amplification cycles for 0 s at 95°C, 2 s at 62°C and 4 s at 72°C with temperature transition rates of 20°C/s. This was followed by a melting programme of 30 s at 95°C, 30 s at 67°C and 0 s at 95°C at rates of 20, 20, and 0.1°C/s, respectively, and cooling at 40°C for at least 30 s.

Multilocus sequence typing

Multilocus sequence typing (MLST) of 81 isolates was carried out as described previously. Briefly, internal fragments of the aroE, gdh, gki, recP, spi, xpt and ddl genes were amplified by PCR from chromosomal DNA with the primer pairs described by Enright and Spratt [12]. The alleles at each of the seven loci provide the allelic profile of each isolate and also define their sequence type (ST). Allelic profiles are shown as the alleles at each of the seven loci, in the order aroE, gdh, gki, recP, spi, xpt and ddl.

MLST analysis

STs were compared to the pneumococcal MLST database on www.mlst.net. The STs were analysed using the program eBURST. This program is able to predict and display the relationships between closely-related isolates of a bacterial species or population. eBURST, unlike cluster diagrams, trees or dendrograms, uses a simple but appropriate model of bacterial evolution in which an ancestral (or founding) genotype increases in frequency in the population, and while doing so, begins to diversify to produce a cluster of closely-related genotypes that are all descended from the founding genotype. This cluster of related genotypes is referred to as a ‘clonal complex’ (eburst.mlst.net) [32].

Acknowledgments

The authors thank Sandra Barbosa, Ingrid Breuer, Meryem Karanfil, Natalia Levina and Nelli Neuberger for excellent technical assistance. We are grateful to Peter Kopp from Vet Med Labor GmbH, Ludwigsburg and Engeline van Duijkeren from Department of Infectious Diseases and Immunology (VMDC) of the Faculty of Veterinary Medicine of the University of Utrecht, the Netherlands for providing pet isolates. We thank Gerhard Haase for critically reading the manuscript.

Footnotes

Competing Interests: Author ML is a member of advisory boards of Wyeth and GSK. Author RRR has been the head of the German National Reference Center for Streptococci (GNRCS) until 2007, at which time he accepted a position in Wyeth. The study was then continued by ML, who is the current head of the GNRCS. This work has not been funded by Wyeth. The funding was from the yearly budget of the national reference center, obtained from RKI and the German ministry of health, both public funders of the national reference center. AA and WN have no competing interests.

Funding: The work was funded, in part, by grant 1369–235 from the Robert Koch Institute and the German Ministry of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

  • 1.Bogaert D, De Groot R, Hermans PW. Streptococcus pneumoniae colonisation: the key to pneumococcal disease. Lancet Infect Dis. 2004;4:144–154. doi: 10.1016/S1473-3099(04)00938-7. [DOI] [PubMed] [Google Scholar]
  • 2.Chiavolini D, Pozzi G, Ricci S. Animal models of Streptococcus pneumoniae disease. Clin Microbiol Rev. 2008;21:666–685. doi: 10.1128/CMR.00012-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Gerber J, Raivich G, Wellmer A, Noeske C, Kunst T, et al. A mouse model of Streptococcus pneumoniae meningitis mimicking several features of human disease. Acta Neuropathol (Berl) 2001;101:499–508. doi: 10.1007/s004010000326. [DOI] [PubMed] [Google Scholar]
  • 4.van der Ven LT, van den Dobbelsteen GP, Nagarajah B, van Dijken H, Dortant PM, et al. A new rat model of otitis media caused by Streptococcus pneumoniae: conditions and application in immunization protocols. Infect Immun. 1999;67:6098–6103. doi: 10.1128/iai.67.11.6098-6103.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Michaud S DR, Higgins R. Streptococcus suis meningitis: first case reported in Québec. Can J Infect Dis. 1996;7:329–331. doi: 10.1155/1996/354693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Whatmore AM, Engler KH, Gudmundsdottir G, Efstratiou A. Identification of isolates of Streptococcus canis infecting humans. J Clin Microbiol. 2001;39:4196–4199. doi: 10.1128/JCM.39.1.4196-4199.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Kuusi M, Lahti E, Virolainen A, Hatakka M, Vuento R, et al. An outbreak of Streptococcus equi subspecies zooepidemicus associated with consumption of fresh goat cheese. BMC Infect Dis. 2006;6:36. doi: 10.1186/1471-2334-6-36. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Duarte RS, Barros RR, Facklam RR, Teixeira LM. Phenotypic and genotypic characteristics of Streptococcus porcinus isolated from human sources. J Clin Microbiol. 2005;43:4592–4601. doi: 10.1128/JCM.43.9.4592-4601.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Higgins R. Bacteria and fungi of marine mammals: a review. Can Vet J. 2000;41:105–116. [PMC free article] [PubMed] [Google Scholar]
  • 10.Hakenbeck R, Balmelle N, Weber B, Gardes C, Keck W, et al. Mosaic Genes and Mosaic Chromosomes: Intra- and Interspecies Genomic Variation of Streptococcus pneumoniae. Infect Immun. 2001;69:2477–2486. doi: 10.1128/IAI.69.4.2477-2486.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Maiden MC, Bygraves JA, Feil E, Morelli G, Russell JE, et al. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc Natl Acad Sci U S A. 1998;95:3140–3145. doi: 10.1073/pnas.95.6.3140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Enright MC, Spratt BG. A multilocus sequence typing scheme for Streptococcus pneumoniae identification of clones associated with serious invasive disease. Microbiology. 1998;144:3049–3060. doi: 10.1099/00221287-144-11-3049. [DOI] [PubMed] [Google Scholar]
  • 13.Chi F, Leider M, Leendertz F, Bergmann C, Boesch C, et al. New Streptococcus pneumoniae clones in deceased wild chimpanzees. J Bacteriol. 2007;189:6085–6088. doi: 10.1128/JB.00468-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Zhang S, Wilson F, Pace L. Streptococcus pneumoniae-associated cellulitis in a two-month-old Domestic Shorthair kitten. J Vet Diagn Invest. 2006;18:221–224. doi: 10.1177/104063870601800217. [DOI] [PubMed] [Google Scholar]
  • 15.Huber L, Willoughby R, Lynch J. Ontario. Streptococcus pneumoniae Type 3 in an Ontario racehorse. Can Vet J. 1988;29:665–666. [PMC free article] [PubMed] [Google Scholar]
  • 16.Whatmore AM, King SJ, Doherty NC, Sturgeon D, Chanter N, et al. Molecular characterization of equine isolates of Streptococcus pneumoniae: natural disruption of genes encoding the virulence factors pneumolysin and autolysin. Infect Immun. 1999;67:2776–2782. doi: 10.1128/iai.67.6.2776-2782.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Chanter N. Streptococcus pneumoniae and equine disease. Equine Vet J. 1994;26:5–6. doi: 10.1111/j.2042-3306.1994.tb04319.x. [DOI] [PubMed] [Google Scholar]
  • 18.Mirick GS, Richter CP, Schaub IG, Franklin R, Mac CR, et al. An epizootic due to pneumococcus type II in laboratory rats. Am J Hyg. 1950;52:48–53. doi: 10.1093/oxfordjournals.aje.a119408. [DOI] [PubMed] [Google Scholar]
  • 19.Ford TM. An outbreak of pneumonia in laboratory rats associated with diplococcus pneumoniae type 8. Lab Anim Care. 1965;15:448–451. [Google Scholar]
  • 20.Weisbroth SH, Freimer EH. Laboratory rats from commercial breeders as carriers of pathogenic pneumococci. Lab Anim Care. 1969;19:473–478. [PubMed] [Google Scholar]
  • 21.Mitruka BM. Biochemical aspects of Diplococcus pneumoniae infections in laboratory rats. Yale J Biol Med. 1971;44:253–264. [PMC free article] [PubMed] [Google Scholar]
  • 22.Baker DG. Natural Pathogens of Laboratory Animals: Their Effects on Research. 2003. Washington: American Society for Microbiology. [DOI] [PMC free article] [PubMed]
  • 23.Amtsberg G, Blobel H, Boehm KH, Bollwahn W, Brueckler J, et al. Stuttgart: Gustav Fischer Verlag; 1980. Handbuch der bakteriellen Infektionen bei Tieren Band II; Blobel H, Schliesser T, editors. [Google Scholar]
  • 24.Aguiar SI, Frias MJ, Santos L, Melo-Cristino J, Ramirez M. Emergence of optochin resistance among Streptococcus pneumoniae in Portugal. Microb Drug Resist. 2006;12:239–245. doi: 10.1089/mdr.2006.12.239. [DOI] [PubMed] [Google Scholar]
  • 25.Reinert RR, Jacobs MR, Appelbaum PC, Bajaksouzian S, Cordeiro S, et al. Relationship between the original multiply resistant South African isolates of Streptococcus pneumoniae from 1977 to 1978 and contemporary international resistant clones. J Clin Microbiol. 2005;43:6035–6041. doi: 10.1128/JCM.43.12.6035-6041.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Reinert RR, Franken C, van der Linden M, Lutticken R, Cil M, et al. Molecular characterisation of macrolide resistance mechanisms of Streptococcus pneumoniae and Streptococcus pyogenes isolated in Germany, 2002-2003. Int J Antimicrob Ag. 2004;24:43–47. doi: 10.1016/j.ijantimicag.2004.02.020. [DOI] [PubMed] [Google Scholar]
  • 27.van der Linden M, Al-Lahham A, Haupts S, Reinert RR. Clonal spread of mef-positive macrolide-resistant Streptococcus pneumoniae isolates causing invasive disease in adults in Germany. Antimicrob Agents Chemother. 2007;51:1830–1834. doi: 10.1128/AAC.01453-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Arbique JC, Poyart C, Trieu-Cuot P, Quesne G, Carvalho Mda G, et al. Accuracy of phenotypic and genotypic testing for identification of Streptococcus pneumoniae and description of Streptococcus pseudopneumoniae sp. nov. J Clin Microbiol. 2004;42:4686–4696. doi: 10.1128/JCM.42.10.4686-4696.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Neufeld F. Über die Agglutination der Pneumokokken und über die Theorie der Agglutination. Zeitschrift für Hygiene und Infektionskrankheiten. 1902;34:54–72. [Google Scholar]
  • 30.Clinical Laboratory Standards Institute. 2006. Performance standards for antimicrobial susceptibility testing; sixteenth informational supplement; Institute CLS, editor. Wayne, PA.
  • 31.Corless CE, Guiver M, Borrow R, Edwards-Jones V, Fox AJ, et al. Simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in suspected cases of meningitis and septicemia using real-time PCR. J Clin Microbiol. 2001;39:1553–1558. doi: 10.1128/JCM.39.4.1553-1558.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Feil EJ, Li BC, Aanensen DM, Hanage WP, Spratt BG. eBURST: inferring patterns of evolutionary descent among clusters of related bacterial genotypes from multilocus sequence typing data. J Bacteriol. 2004;186:1518–1530. doi: 10.1128/JB.186.5.1518-1530.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Homburger F, Wilcox C, Barnes MW, Finland M. An epizootic of pneumococcus type 19 infections in guinea pigs. Science. 1945;102:449–450. doi: 10.1126/science.102.2653.449. [DOI] [PubMed] [Google Scholar]
  • 34.Fallon MT, Reinhard MK, Gray BM, Davis TW, Lindsey JR. Inapparent Streptococcus pneumoniae type 35 infections in commercial rats and mice. Lab Anim Sci. 1988;38:129–132. [PubMed] [Google Scholar]

Articles from PLoS ONE are provided here courtesy of PLOS

RESOURCES