JCM Accepted Manuscript Posted Online 7 January 2015 J. Clin. Microbiol. doi:10.1128/JCM.03049-14 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1
Clinical and molecular epidemiology of Staphylococcus argenteus infections in Thailand
3
Janjira Thaipadungpanita#, Premjit Amornchaia, Emma K Nickersonb, Gumphol Wongsuvana,
4
Vanaporn Wuthiekanuna, Direk Limmathurotsakula,c, Sharon J Peacocka,b,d,e
5 6
Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol
8
Cambridge, United Kingdomb; Department of Tropical Medicine and Hygiene, Faculty of
10
and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailandd;
2
7
University, Bangkok, Thailanda; Cambridge University Hospitals NHS Foundation Trust,
9
Tropical Medicine, Mahidol University, Bangkok, Thailandc; Department of Microbiology
11
Department of Medicine, University of Cambridge, Addenbrooke’s Hospital, Cambridge,
12
United Kingdome
13
14
Running Head: S. argenteus infections in Thailand
15
16
#Address correspondence to Janjira Thaipadungpanit, [email protected]
17 18
Word count: abstract 50, text 1392, Table 1 and Figure 1, References 16, Supplementary
19
tables 2, Supplementary figures 3
1
20
Abstract
21
Molecular typing of 246 Staphylococcus aureus isolates from unselected patients in Thailand
22
showed that 10 (4.1%) were actually S. argenteus. Contrary to the suggestion that this is less
23
virulent compared with S. aureus, we demonstrated comparable morbidity, death and rates of
24
healthcare-associated infection in patients infected with the two species.
25 26
Keywords: Staphylococcus, argenteus, aureus, Thailand
2
27 28
Text The population genetic structure of Staphylococcus aureus has been extensively
29
described using multilocus sequence typing (MLST) (1). The original primers failed to
30
amplify all 7 MLST loci for a small proportion of presumptive S. aureus isolates, resulting in
31
a delayed description of an early branching lineage that was initially referred to as clonal
32
complex (CC) 75. Sequence type (ST) 1223 was subsequently described, which belongs to
33
the same lineage but does not cluster within CC75 (2,3). This lineage is genetically divergent
34
compared with S. aureus, with an average nucleotide divergence compared with S. aureus of
35
9.6% over all seven MLST loci (2). The new species name Staphylococcus argenteus was
36
proposed for CC75 and related STs (4), followed by formal taxonomic classification as S.
37
argenteus sp. nov. (5).
38
Using a single-nucleotide polymorphism (SNP) genotyping method based on MLST,
39
staphylococci belonging to CC75 were found to account for 71% of community-associated
40
methicillin-resistant S. aureus (MRSA) associated with skin and soft tissue infections in
41
indigenous communities in the Northern Territory of Australia (6). Further isolation of CC75
42
from these communities (3) was followed by reports of its isolation in Cambodia (2),
43
Indonesia (2), Fiji (7), Trinidad and Tobago (8), and from members of an isolated village in
44
the Amazonian forest in French Guinea (9). Information contained in the MLST database
45
suggests that S. argenteus is also present in Europe and Africa. Overall, this indicates
46
widespread dissemination of S. argenteus, and a report of its isolation from the faeces of the
47
Straw-Coloured Fruit Bat in Nigeria (10) indicates that this may be carried by other animal
48
species.
49
S. argenteus has not been reported from Thailand, but based on isolation elsewhere in
50
southeast Asia we presumed that it was present. This is made more plausible by the fact that
51
S. argenteus cannot be distinguished from S. aureus using routine diagnostic microbiology 3
52
identification (2,6,7,9). We investigated its presence in Thailand through a re-evaluation of S.
53
aureus isolated during a prospective study of 270 consecutive patients with invasive infection
54
presenting to Sappasithiprasong Hospital, Northeast Thailand between November 2006 and
55
November 2007 (11). All isolates were originally identified based on Gram stain and the
56
coagulase test, further evaluated for methicillin susceptibility and the presence of pvl and
57
mecA, and stored at -80°C (11).
58
Of the 270 isolates originally stored, 246 (91.1%) were available for genotyping using a
59
combination of PCR and multilocus sequence typing (MLST), of which 37 were MRSA. As
60
ST239 is the predominant MRSA lineage in Thailand (12), MRSA isolates were initially
61
screened using an ST239-specific PCR assay (12). All non-ST239 isolates were then typed
62
using MLST as described (1), except for minor modifications in primer design to improve
63
PCR amplification (Supplementary Table 1). Allele and ST assignment was performed using
64
the MLST database (http://saureus.mlst.net). S. argenteus isolates in the study collection were
65
identified based on phylogenetic clustering. To enrich the population for relevant genotypes,
66
38 STs were downloaded from the MLST database (http://www.mlst.net) as a reference
67
collection. These were selected following an initial analysis in which phylogenetic trees were
68
drawn for each S. aureus MLST locus using all of the data held in the MLST database (not
69
shown). This demonstrated that several arcC alleles (36, 151, 207 and 271) and pta alleles
70
(39, 107, 145, 175, 198, 256, 268 and 287) clustered in a single branch containing known S.
71
argenteus isolates and were not shared with S. aureus (Supplementary Figures 1 and 2). The
72
remaining five alleles were shared between both species. Based on this, we selected all STs
73
that contained these particular arcC and pta alleles. The 38 STs were visualised using
74
eBURST using a cut-off of 4 or more shared loci, which demonstrated that 33STs belonged
75
to four CCs: CC75 (n=4 STs), CC2198 (n=8 STs), CC2483 (n=10 STs) and CC1594 (n=11
76
STs). The remaining 5 STs were either singleton STs (ST2225, ST2351, and ST2470) or 4
77
paired (ST2596 and ST2793). A maximum likelihood tree was re-constructed from
78
concatenated sequences of 7 MLST loci using PhyML version 3.0.1 (13). The CLC Main
79
Workbench version 7.0 was used to edit and display the tree (Qiagen, USA).
80
A total of 40 STs were identified for the 246 Thai isolates. The predominant genotype
81
in the collection was ST121 (95/246, 38.6%); the frequency of STs for the collection is
82
summarised in Supplementary Figure 3. A maximum likelihood phylogenetic tree was
83
reconstructed based on concatenated sequences of 7 MLST loci for these STs, together with
84
the 38 reference S. argenteus STs from the MLST website (Figure 1). The phylogenetic tree
85
divided the Thai collection into two major branches containing 36 STs that were S. aureus,
86
and 4 STs that clustered with the S. argenteus reference sequences. Three groups were
87
observed for the entire S. argenteus data, one of which contained all four STs (representing
88
ten isolates) from the Thai collection. Three of these four STs have been identified previously
89
(ST1223 reported from Cambodia, Australia and French Guiana; ST2250 from the UK; and
90
ST2198 from Australia and Germany) (2,3,4,9). The fourth ST (ST2854) is novel. Based on
91
this phylogenetic analysis, we concluded that 10/246 isolates (4.1%) from an unselected,
92
consecutive collection of S. aureus from Thailand should be re-categorised as S. argenteus.
93
Our findings confirm that this species is endemic in Thailand, and provides additional
94
evidence to suggest that S. argenteus may be more widely distributed across southeast Asia.
95
S. argenteus isolates identified in Australia have been methicillin-resistant and
96
methicillin-susceptible, although methicillin-resistant isolates predominate and are
97
community-associated in this setting. Elsewhere, S. argenteus isolates are methicillin-
98
susceptible, but appear to be universally pvl-negative (6,7,8,9,14). All ten Thai S. argenteus
99
isolates were methicillin-susceptible, mecA negative and pvl negative. The frequency of
100
methicillin-resistance and pvl-positivity for the entire Thai collection is summarised in
101
Supplementary Figure 3. 5
102
S. argenteus isolates characteristically lack staphyloxanthin, a carotenoid pigment
103
responsible for the characteristic golden colony colour (4). All ten Thai S. argenteus grew on
104
blood agar as white colonies, which is consistent with this. Staphyloxanthin confers
105
resistance against oxidative stress and neutrophil killing (15), leading to the hypothesis that S.
106
argenteus may be less virulent (4). This is supported by the findings of a study showing that
107
an S. argenteus isolate (ST1850) was more susceptible to oxidative stress and neutrophil
108
killing in vitro, and was associated with reduced virulence in murine sepsis and skin infection
109
models compared with S. aureus (16). Transformation of this isolate with a plasmid carrying
110
the gene encoding staphyloxanthin led to increased resistance to oxidative stress in vitro, but
111
no change in resistance to neutrophil killing or in vivo virulence. A study that defined the
112
frequency of S. argenteus in three different clinical collections in northern Australia reported
113
that these were predominantly associated with skin and soft tissue infections, but rarely with
114
bacteraemia (1% (3/220) of methicillin-susceptible S. aureus (MSSA) bacteraemias, and 0%
115
(0/47) of MRSA bacteraemias) (16).
116
We tested the hypothesis that S. argenteus isolates were not associated with severe
117
disease in our Thai cohort. Clinical details and outcome from S. argenteus infection are
118
shown in Table 1. Most patients (8/10, 80%) had community-acquired infection but two were
119
healthcare-associated, indicating that S. argenteus may cause nosocomial infection. We noted
120
that S. argenteus was strongly associated with skin and soft tissue infection or superficial
121
abscesses (8/10 cases), which is consistent with previous reports. However, three patients
122
with S. argenteus infection had bacteraemia and two patients had bone and joint infection,
123
indicating an ability to cause invasive disease (Supplementary Table 2). Two patients
124
infected with S. argenteus died, one as a direct result of the infection. In general, disease
125
characteristics and outcome were not different between patients infected with S. argenteus
126
and MSSA. However, the median age of patients infected with S. argenteus was higher than 6
127
that of patients infected with MSSA (56.5 vs. 38, p=0.04), and patients infected with S.
128
argenteus were more likely to have diabetes and renal disease than patients infected with
129
MSSA (Table 1).
130
A limitation of this work is that the study cohort only included patients with S. aureus
131
isolated from a normally sterile site. This is likely to lead to under-representation of minor
132
skin and soft tissue infection, which could result in an underestimate of the true prevalence of
133
S. argenteus. Our data demonstrate, however, that S. argenteus may be associated with
134
serious morbidity, death, and nosocomial infection, particularly in patients with underlying
135
diseases such as diabetes mellitus and renal diseases.
136 137
Acknowledgements
138
This work was supported by the Wellcome Trust (089275/Z/09/Z). We thank the medical and
139
nursing staff at Sappasithiprasong Hospital and Mahidol-Oxford Tropical Medicine Research
140
Unit for their support.
141 142
References
143
1. Enright MC, Day NP, Davies CE, Peacock SJ, Spratt BG. 2000. Multilocus
144
sequence typing for characterization of methicillin-resistant and methicillin-
145
susceptible clones of Staphylococcus aureus. J Clin Microbiol 38:1008-1015.
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2. Ruimy R, Armand-Lefevre L, Barbier F, Ruppe E, Cocojaru R, Mesli Y, Maiga
147
A, Benkalfat M, Benchouk S, Hassaine H, Dufourcq JB, Nareth C, Sarthou JL,
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Andremont A, Feil EJ. 2009. Comparisons between geographically diverse samples
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of carried Staphylococcus aureus. J Bacteriol 191:5577-5583.
7
150
3. Ng JW, Holt DC, Lilliebridge RA, Stephens AJ, Huygens F, Tong SY, Currie BJ,
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Giffard PM. 2009. Phylogenetically distinct Staphylococcus aureus lineage prevalent
152
among indigenous communities in northern Australia. J Clin Microbiol 47:2295-2300.
153
4. Holt DC, Holden MT, Tong SY, Castillo-Ramirez S, Clarke L, Quail MA, Currie
154
BJ, Parkhill J, Bentley SD, Feil EJ, Giffard PM. 2011. A very early-branching
155
Staphylococcus aureus lineage lacking the carotenoid pigment staphyloxanthin.
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Genome Biol Evol 3: 881-895.
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5. Tong SY, Schaumburg F, Ellington MJ, Corander J, Pichon B, Leendertz F,
158
Bentley SD, Parkhill J, Holt DC, Peters G, Giffard PM. Novel staphylococcal
159
species that form part of a Staphylococcus aureus related complex: The non-
160
pigmented S. argenteus sp. nov. and the non-human primate associated S. schweitzeri
161
sp. nov. Int J Syst Evol Microbiol, in press (ijs.0.062752-0).
162
6. McDonald M, Dougall A, Holt D, Huygens F, Oppedisano F, Giffard PM,
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Inman-Bamber J, Stephens AJ, Towers R, Carapetis JR, Currie BJ. 2006. Use of
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a single-nucleotide polymorphism genotyping system to demonstrate the unique
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epidemiology of methicillin-resistant Staphylococcus aureus in remote aboriginal
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communities. J Clin Microbiol 44:3720-3727.
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7. Jenney A, Holt D, Ritika R, Southwell P, Pravin S, Buadromo E, Carapetis J,
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Tong S, Steer A. 2014. The clinical and molecular epidemiology of Staphylococcus
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aureus infections in Fiji. BMC Infect Dis 14:160.
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8. Monecke S, Stieber B, Roberts R, Akpaka PE, Slickers P, Ehricht R. 2014.
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Population structure of Staphylococcus aureus from Trinidad & Tobago. PLoS One
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9:e89120.
173 174
9. Ruimy R, Angebault C, Djossou F, Dupont C, Epelboin L, Jarraud S, Lefevre LA, Bes M, Lixandru BE, Bertine M, El Miniai A, Renard M, Bettinger RM,
8
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Lescat M, Clermont O, Peroz G, Lina G, Tavakol M, Vandenesch F, van Belkum
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A, Rousset F, Andremont A. 2010. Are host genetics the predominant determinant
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of persistent nasal Staphylococcus aureus carriage in humans? J Infect Dis 202:924-
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934.
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10. Akobi B, Aboderin O, Sasaki T, Shittu A. 2012. Characterization of Staphylococcus
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aureus isolates from faecal samples of the straw-coloured fruit bat (Eidolon helvum)
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in Obafemi Awolowo University (OAU), Nigeria. BMC Microbiol 12:279.
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11. Nickerson EK, Wuthiekanun V, Wongsuvan G, Limmathurosakul D, Srisamang
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P, Mahavanakul W, Thaipadungpanit J, Shah KR, Arayawichanont A,
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Amornchai P, Thanwisai A, Day NP, Peacock SJ. 2009. Factors predicting and
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reducing mortality in patients with invasive Staphylococcus aureus disease in a
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developing country. PLoS One 4:e6512.
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12. Feil EJ, Nickerson EK, Chantratita N, Wuthiekanun V, Srisomang P, Cousins R,
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Pan W, Zhang G, Xu B, Day NP, Peacock SJ. 2008. Rapid detection of the
189
pandemic methicillin-resistant Staphylococcus aureus clone ST 239, a dominant
190
strain in asian hospitals. J Clin Microbiol 46:1520-1522.
191 192 193
13. Guindon S, Gascuel O. 2003. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52:696-704. 14. Tong SY, Lilliebridge RA, Bishop EJ, Cheng AC, Holt DC, McDonald MI,
194
Giffard PM, Currie BJ, Boutlis CS. 2010. Clinical correlates of Panton-Valentine
195
leukocidin (PVL), PVL isoforms, and clonal complex in the Staphylococcus aureus
196
population of northern Australia. J Infect Dis 202:760-769.
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15. Liu GY, Essex A, Buchanan JT, Datta V, Hoffman HM, Bastian JF, Fierer J,
198
Nizet V. 2005. Staphylococcus aureus golden pigment impairs neutrophil killing and
199
promotes virulence through its antioxidant activity. J Exp Med 202:209-215.
9
200
16. Tong SY, Sharma-Kuinkel BK, Thaden JT, Whitney AR, Yang SJ, Mishra NN,
201
Rude T, Lilliebridge RA, Selim MA, Ahn SH, Holt DC, Giffard PM, Bayer AS,
202
Deleo FR, Fowler VG, Jr. 2013. Virulence of endemic nonpigmented northern
203
Australian Staphylococcus aureus clone (clonal complex 75, S. argenteus) is not
204
augmented by staphyloxanthin. J Infect Dis 208:520-527.
10
205
Table 1. Clinical manifestations and outcome of patients infected with methicillin-
206
susceptible S. aureus (MSSA) and S. argenteus Number of cases Age (years) median (interquartile range, range) Sex (male) Blood culture positive isolates Place of acquisition Community-acquired 2 Healthcare-associated 3 Underlying medical conditions 4 Cardiac disease Diabetes mellitus 5 Renal disease 6 Immunosuppression 7 Lung disease Pattern of disease No identified site, bacteraemia only 1 identified site >1 identified site
S. argenteus 10 56.5 (41-66, 11-73) 7 (70%) 3 (30%)
172 (86%) 27 (14%) 65 (33%) 12 (6%) 29 (15%) 15 (8%) 25 (13%) 9 (5%)
8 (80%) 2 (20%) 7 (70%) 1 (10%) 6 (60%) 6 (60%) 2 (20%) 1 (10%)
17 (9%) 155 (78%) 27 (13%)
2 (20%) 6 (60%) 2 (20%)
0.19
81 (41%) 45 (23%) 21 (11%) 24 (12%) 13 (7%) 14 (7%) 6 (3%) 7 (4%)
4 (40%) 0 4 (40%) 2 (20%) 0 0 0 0
0.15
150 (75%) 11 (6%) 26 (13%) 12 (6%)
7 (70%) 1 (10%) 1 (10%) 1 (10%)
0.58
Identifiable extra-vascular sites of infection Superficial abscesses Deep abscesses Other skin and soft tissue infections Bone and joint infections Prosthetic material infections Respiratory infection Endocarditis Other infections Outcome Cured Treatment failure Death due to S. aureus Death from other causes
207 208
1
209
2
210
3
211
notes.
212
4
213
disease, or arrhythmias including heart block requiring pacemaker.
214
5
215
mellitus, systematic lupus erythematous, multiple myeloma, glomerulonephritis or an unknown aetiology.
216
6
217
prednisolone more than 30 mg/day for more than 1 week.
218
7
219
obstructive pulmonary disease or asthma.
p values
1
MSSA 199 38 (14-59, 1-84) 118 (59%) 56 (28%)
0.04 0.74 >0.99 0.63 0.03 0.48 0.002
1
Clinical and molecular epidemiology of Staphylococcus argenteus infections in Thailand
3
Janjira Thaipadungpanita#, Premjit Amornchaia, Emma K Nickersonb, Gumphol Wongsuvana,
4
Vanaporn Wuthiekanuna, Direk Limmathurotsakula,c, Sharon J Peacocka,b,d,e
5 6
Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol
8
Cambridge, United Kingdomb; Department of Tropical Medicine and Hygiene, Faculty of
10
and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailandd;
2
7
University, Bangkok, Thailanda; Cambridge University Hospitals NHS Foundation Trust,
9
Tropical Medicine, Mahidol University, Bangkok, Thailandc; Department of Microbiology
11
Department of Medicine, University of Cambridge, Addenbrooke’s Hospital, Cambridge,
12
United Kingdome
13
14
Running Head: S. argenteus infections in Thailand
15
16
#Address correspondence to Janjira Thaipadungpanit, [email protected]
17 18
Word count: abstract 50, text 1392, Table 1 and Figure 1, References 16, Supplementary
19
tables 2, Supplementary figures 3
1
20
Abstract
21
Molecular typing of 246 Staphylococcus aureus isolates from unselected patients in Thailand
22
showed that 10 (4.1%) were actually S. argenteus. Contrary to the suggestion that this is less
23
virulent compared with S. aureus, we demonstrated comparable morbidity, death and rates of
24
healthcare-associated infection in patients infected with the two species.
25 26
Keywords: Staphylococcus, argenteus, aureus, Thailand
2
27 28
Text The population genetic structure of Staphylococcus aureus has been extensively
29
described using multilocus sequence typing (MLST) (1). The original primers failed to
30
amplify all 7 MLST loci for a small proportion of presumptive S. aureus isolates, resulting in
31
a delayed description of an early branching lineage that was initially referred to as clonal
32
complex (CC) 75. Sequence type (ST) 1223 was subsequently described, which belongs to
33
the same lineage but does not cluster within CC75 (2,3). This lineage is genetically divergent
34
compared with S. aureus, with an average nucleotide divergence compared with S. aureus of
35
9.6% over all seven MLST loci (2). The new species name Staphylococcus argenteus was
36
proposed for CC75 and related STs (4), followed by formal taxonomic classification as S.
37
argenteus sp. nov. (5).
38
Using a single-nucleotide polymorphism (SNP) genotyping method based on MLST,
39
staphylococci belonging to CC75 were found to account for 71% of community-associated
40
methicillin-resistant S. aureus (MRSA) associated with skin and soft tissue infections in
41
indigenous communities in the Northern Territory of Australia (6). Further isolation of CC75
42
from these communities (3) was followed by reports of its isolation in Cambodia (2),
43
Indonesia (2), Fiji (7), Trinidad and Tobago (8), and from members of an isolated village in
44
the Amazonian forest in French Guinea (9). Information contained in the MLST database
45
suggests that S. argenteus is also present in Europe and Africa. Overall, this indicates
46
widespread dissemination of S. argenteus, and a report of its isolation from the faeces of the
47
Straw-Coloured Fruit Bat in Nigeria (10) indicates that this may be carried by other animal
48
species.
49
S. argenteus has not been reported from Thailand, but based on isolation elsewhere in
50
southeast Asia we presumed that it was present. This is made more plausible by the fact that
51
S. argenteus cannot be distinguished from S. aureus using routine diagnostic microbiology 3
52
identification (2,6,7,9). We investigated its presence in Thailand through a re-evaluation of S.
53
aureus isolated during a prospective study of 270 consecutive patients with invasive infection
54
presenting to Sappasithiprasong Hospital, Northeast Thailand between November 2006 and
55
November 2007 (11). All isolates were originally identified based on Gram stain and the
56
coagulase test, further evaluated for methicillin susceptibility and the presence of pvl and
57
mecA, and stored at -80°C (11).
58
Of the 270 isolates originally stored, 246 (91.1%) were available for genotyping using a
59
combination of PCR and multilocus sequence typing (MLST), of which 37 were MRSA. As
60
ST239 is the predominant MRSA lineage in Thailand (12), MRSA isolates were initially
61
screened using an ST239-specific PCR assay (12). All non-ST239 isolates were then typed
62
using MLST as described (1), except for minor modifications in primer design to improve
63
PCR amplification (Supplementary Table 1). Allele and ST assignment was performed using
64
the MLST database (http://saureus.mlst.net). S. argenteus isolates in the study collection were
65
identified based on phylogenetic clustering. To enrich the population for relevant genotypes,
66
38 STs were downloaded from the MLST database (http://www.mlst.net) as a reference
67
collection. These were selected following an initial analysis in which phylogenetic trees were
68
drawn for each S. aureus MLST locus using all of the data held in the MLST database (not
69
shown). This demonstrated that several arcC alleles (36, 151, 207 and 271) and pta alleles
70
(39, 107, 145, 175, 198, 256, 268 and 287) clustered in a single branch containing known S.
71
argenteus isolates and were not shared with S. aureus (Supplementary Figures 1 and 2). The
72
remaining five alleles were shared between both species. Based on this, we selected all STs
73
that contained these particular arcC and pta alleles. The 38 STs were visualised using
74
eBURST using a cut-off of 4 or more shared loci, which demonstrated that 33STs belonged
75
to four CCs: CC75 (n=4 STs), CC2198 (n=8 STs), CC2483 (n=10 STs) and CC1594 (n=11
76
STs). The remaining 5 STs were either singleton STs (ST2225, ST2351, and ST2470) or 4
77
paired (ST2596 and ST2793). A maximum likelihood tree was re-constructed from
78
concatenated sequences of 7 MLST loci using PhyML version 3.0.1 (13). The CLC Main
79
Workbench version 7.0 was used to edit and display the tree (Qiagen, USA).
80
A total of 40 STs were identified for the 246 Thai isolates. The predominant genotype
81
in the collection was ST121 (95/246, 38.6%); the frequency of STs for the collection is
82
summarised in Supplementary Figure 3. A maximum likelihood phylogenetic tree was
83
reconstructed based on concatenated sequences of 7 MLST loci for these STs, together with
84
the 38 reference S. argenteus STs from the MLST website (Figure 1). The phylogenetic tree
85
divided the Thai collection into two major branches containing 36 STs that were S. aureus,
86
and 4 STs that clustered with the S. argenteus reference sequences. Three groups were
87
observed for the entire S. argenteus data, one of which contained all four STs (representing
88
ten isolates) from the Thai collection. Three of these four STs have been identified previously
89
(ST1223 reported from Cambodia, Australia and French Guiana; ST2250 from the UK; and
90
ST2198 from Australia and Germany) (2,3,4,9). The fourth ST (ST2854) is novel. Based on
91
this phylogenetic analysis, we concluded that 10/246 isolates (4.1%) from an unselected,
92
consecutive collection of S. aureus from Thailand should be re-categorised as S. argenteus.
93
Our findings confirm that this species is endemic in Thailand, and provides additional
94
evidence to suggest that S. argenteus may be more widely distributed across southeast Asia.
95
S. argenteus isolates identified in Australia have been methicillin-resistant and
96
methicillin-susceptible, although methicillin-resistant isolates predominate and are
97
community-associated in this setting. Elsewhere, S. argenteus isolates are methicillin-
98
susceptible, but appear to be universally pvl-negative (6,7,8,9,14). All ten Thai S. argenteus
99
isolates were methicillin-susceptible, mecA negative and pvl negative. The frequency of
100
methicillin-resistance and pvl-positivity for the entire Thai collection is summarised in
101
Supplementary Figure 3. 5
102
S. argenteus isolates characteristically lack staphyloxanthin, a carotenoid pigment
103
responsible for the characteristic golden colony colour (4). All ten Thai S. argenteus grew on
104
blood agar as white colonies, which is consistent with this. Staphyloxanthin confers
105
resistance against oxidative stress and neutrophil killing (15), leading to the hypothesis that S.
106
argenteus may be less virulent (4). This is supported by the findings of a study showing that
107
an S. argenteus isolate (ST1850) was more susceptible to oxidative stress and neutrophil
108
killing in vitro, and was associated with reduced virulence in murine sepsis and skin infection
109
models compared with S. aureus (16). Transformation of this isolate with a plasmid carrying
110
the gene encoding staphyloxanthin led to increased resistance to oxidative stress in vitro, but
111
no change in resistance to neutrophil killing or in vivo virulence. A study that defined the
112
frequency of S. argenteus in three different clinical collections in northern Australia reported
113
that these were predominantly associated with skin and soft tissue infections, but rarely with
114
bacteraemia (1% (3/220) of methicillin-susceptible S. aureus (MSSA) bacteraemias, and 0%
115
(0/47) of MRSA bacteraemias) (16).
116
We tested the hypothesis that S. argenteus isolates were not associated with severe
117
disease in our Thai cohort. Clinical details and outcome from S. argenteus infection are
118
shown in Table 1. Most patients (8/10, 80%) had community-acquired infection but two were
119
healthcare-associated, indicating that S. argenteus may cause nosocomial infection. We noted
120
that S. argenteus was strongly associated with skin and soft tissue infection or superficial
121
abscesses (8/10 cases), which is consistent with previous reports. However, three patients
122
with S. argenteus infection had bacteraemia and two patients had bone and joint infection,
123
indicating an ability to cause invasive disease (Supplementary Table 2). Two patients
124
infected with S. argenteus died, one as a direct result of the infection. In general, disease
125
characteristics and outcome were not different between patients infected with S. argenteus
126
and MSSA. However, the median age of patients infected with S. argenteus was higher than 6
127
that of patients infected with MSSA (56.5 vs. 38, p=0.04), and patients infected with S.
128
argenteus were more likely to have diabetes and renal disease than patients infected with
129
MSSA (Table 1).
130
A limitation of this work is that the study cohort only included patients with S. aureus
131
isolated from a normally sterile site. This is likely to lead to under-representation of minor
132
skin and soft tissue infection, which could result in an underestimate of the true prevalence of
133
S. argenteus. Our data demonstrate, however, that S. argenteus may be associated with
134
serious morbidity, death, and nosocomial infection, particularly in patients with underlying
135
diseases such as diabetes mellitus and renal diseases.
136 137
Acknowledgements
138
This work was supported by the Wellcome Trust (089275/Z/09/Z). We thank the medical and
139
nursing staff at Sappasithiprasong Hospital and Mahidol-Oxford Tropical Medicine Research
140
Unit for their support.
141 142
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Table 1. Clinical manifestations and outcome of patients infected with methicillin-
206
susceptible S. aureus (MSSA) and S. argenteus Number of cases Age (years) median (interquartile range, range) Sex (male) Blood culture positive isolates Place of acquisition Community-acquired 2 Healthcare-associated 3 Underlying medical conditions 4 Cardiac disease Diabetes mellitus 5 Renal disease 6 Immunosuppression 7 Lung disease Pattern of disease No identified site, bacteraemia only 1 identified site >1 identified site
S. argenteus 10 56.5 (41-66, 11-73) 7 (70%) 3 (30%)
172 (86%) 27 (14%) 65 (33%) 12 (6%) 29 (15%) 15 (8%) 25 (13%) 9 (5%)
8 (80%) 2 (20%) 7 (70%) 1 (10%) 6 (60%) 6 (60%) 2 (20%) 1 (10%)
17 (9%) 155 (78%) 27 (13%)
2 (20%) 6 (60%) 2 (20%)
0.19
81 (41%) 45 (23%) 21 (11%) 24 (12%) 13 (7%) 14 (7%) 6 (3%) 7 (4%)
4 (40%) 0 4 (40%) 2 (20%) 0 0 0 0
0.15
150 (75%) 11 (6%) 26 (13%) 12 (6%)
7 (70%) 1 (10%) 1 (10%) 1 (10%)
0.58
Identifiable extra-vascular sites of infection Superficial abscesses Deep abscesses Other skin and soft tissue infections Bone and joint infections Prosthetic material infections Respiratory infection Endocarditis Other infections Outcome Cured Treatment failure Death due to S. aureus Death from other causes
207 208
1
209
2
210
3
211
notes.
212
4
213
disease, or arrhythmias including heart block requiring pacemaker.
214
5
215
mellitus, systematic lupus erythematous, multiple myeloma, glomerulonephritis or an unknown aetiology.
216
6
217
prednisolone more than 30 mg/day for more than 1 week.
218
7
219
obstructive pulmonary disease or asthma.
p values
1
MSSA 199 38 (14-59, 1-84) 118 (59%) 56 (28%)
0.04 0.74 >0.99 0.63 0.03 0.48 0.002
