First human case of severe septicaemia associated with Mycoplasma capricolum subsp. capricolum infection

The wall-less bacterium Mycoplasma capricolum subsp. capricolum is known as an agent of respiratory disease, mastitis and arthritis in small ruminants. It has also been associated with contagious agalactia of goats, a syndrome affecting mammary glands, joints and eyes, which occurs in small ruminant flocks of Mediterranean countries and many other regions (DaMassa et al., 1987, 1992; Bergonier et al., 1997; Gómez-Martı́n et al., 2013). As a species, M. capricolum subsp. capricolum is highly adapted to goats, even though there are also reports on findings in sheep (Al-Momani et al., 2006) and South American camelids (Pitcher & Nicholas, 2005). Here, to the best of our knowledge, we report the first case of a human infection with M. capricolum subsp. capricolum.


Introduction
The wall-less bacterium Mycoplasma capricolum subsp.capricolum is known as an agent of respiratory disease, mastitis and arthritis in small ruminants.It has also been associated with contagious agalactia of goats, a syndrome affecting mammary glands, joints and eyes, which occurs in small ruminant flocks of Mediterranean countries and many other regions (DaMassa et al., 1987(DaMassa et al., , 1992;;Bergonier et al., 1997;Go ´mez-Martı ´n et al., 2013).As a species, M. capricolum subsp.capricolum is highly adapted to goats, even though there are also reports on findings in sheep (Al-Momani et al., 2006) and South American camelids (Pitcher & Nicholas, 2005).Here, to the best of our knowledge, we report the first case of a human infection with M. capricolum subsp.capricolum.

Case report
A 62-year-old man presented symptoms of recurrent fever and severe limb pain, which was diagnosed as septicaemia and meningoencephalitis combined with bilateral asymptomatic pneumonia and pleural effusion.The symptoms had emerged at the end of a tourist visit to the Cape Verde Islands around New Year's Day 2014.After his return to Germany, the patient was hospitalized for 2 weeks.On day 6 after admission, sinusitis and blepharoconjunctivitis were additionally diagnosed; bacterial infection was identified (see below) and meningitis was suspected.Therefore, combined antibiotic treatment with ceftriaxon (for 8 days), roxythromycin and doxycyclin (each for 3 weeks) was started.At the time of discharge from hospital, 2 weeks after admission, the symptoms had disappeared.
Blood examination revealed thrombocytopenia and several elevated inflammation parameters, such as leukocytes, monocytes, neutrophils, erythrocyte sedimentation rate and C-reactive protein.The following viral infections could be excluded after nucleic acid testing and serology: influenza A and B, Dengue, Chikungunya and cytomegalovirus.Antibody ELISAs for Brucella and Coxiella proved negative.No malaria antigen was detected.Whilst bacterial culture from cerebrospinal fluid was negative, initial blood culture on Columbia Agar containing 7 % sheep blood (Oxoid) was weak, but could be improved on Mycoplasma/Ureaplasma Agar (Oxoid), as well as in liquid culture media (BD Bactec Plus Aerobic/Anaerobic; Becton Dickinson).Partial sequencing of the isolates from 10 experiments revealed identical results.Based on the 16S rRNA locus (GenBank accession number KP718739) and a partial sequence of the 23S rRNA (GenBank accession The M. capricolum subsp.capricolum strain isolated was further characterized using multilocus sequence typing (MLST), which is based on partial sequences of seven housekeeping genes (Fischer et al., 2012).The sequences were concatenated and aligned to reference sequences of members of the 'Mycoplasma mycoides cluster'.We reconstructed a maximum-likelihood phylogeny tree based on the 3816 nt sequence alignment using PhyML 3.0 (Guindon et al., 2010).To assess statistical support for the resulting phylogeny, we performed 1000 bootstrap replicates assuming a GTR+G+I model of nucleotide substitution.The tree was drawn using FigTree 1.4.2(http://tree.bio.ed.ac.uk/software/figtree/) and is shown in Fig. 1.The patient isolate 14DL0024 clearly clustered within the clade of M. capricolum subsp.capricolum and was separated from its closest relative M. capricolum subsp.capripneumoniae.
To find out whether specific antibodies had been generated, we examined a patient serum sample taken 3 weeks after the end of antibiotic treatment using Western blotting.A serum sample from an earlier time point was not available.Whole-cell antigens used included the patient strain 14DL0024, the type strains of closely related members of the 'Mycoplasma mycoides cluster', i.e.M. mycoides subsp.mycoides, M. mycoides subsp.capri and M. capricolum subsp.capripneumoniae, Mycoplasma agalactiae, as well as Mycoplasma pneumoniae.The results in Fig. 2 provide evidence of the patient serum containing antibodies reacting with a number of antigens of M. capricolum subsp.capricolum, but also with those of the related mycoplasmal agents.The serum of a randomly chosen healthy blood donor, which served as a control, failed to show any reactive bands (data not shown).

Discussion
The relatively low specific antibody level in the patient's serum could be due to immune suppression during sepsis (Monserrat et al., 2013) or a consequence of early antibiotic treatment.The fact that a custom-made ELISA failed to unambiguously reveal differences between serum antibody levels of the patient and control is probably also a consequence of the weak immune response and indicates insufficient sensitivity of the assay.
Although it has been impossible to identify the source of the infection, an association with the patient's 2-week stay on the Cape Verde Islands appears very likely, even more so as M. capricolum subsp.capricolum has not been encountered in his home country (Germany) to date.The patient had stayed in local tourist hotels and eaten in restaurants predominantly serving foreign tourists.Non-pasteurized milk products or raw meat were reportedly not served, but food products from goats figure prominently in the Cape Verdean diet.Moreover, the tourist group spent the major part of its time in the countryside.Even though the patient reported no direct contact with animals, it seems certain that the tourists passed by goat herds during their hiking tours.The patient's known heavy-smoker status may have contributed to the severity of the infection.It is also pertinent to note that the goat population on the Cape Verde Islands is large, and that both the number of goats and goat milk production have doubled in the last 10 years (http://faostat3.fao.org/home/E).Therefore, the probability of human exposure to goat-adapted bacteria, such as M. capricolum subsp.capricolum, should have increased as well.