Upload
others
View
0
Download
0
Embed Size (px)
Citation preview
1
Re-Identification of Streptococcus bovis Isolates Causing Bacteraemia 1
According with the New Taxonomy Criteria: Still an Issue? 2
3
Beatriz Romero1,2
, María-Isabel Morosini1,2
, Elena Loza1,2
, 4
Mercedes Rodríguez-Baños1, Enrique Navas
3, Rafael Cantón
1,2 and Rosa del Campo
1,2* 5
6
7
1Servicio de Microbiología, Hospital Universitario Ramón y Cajal and IRYCIS; Unidad 8
de Resistencia a Antibióticos y Virulencia Bacteriana asociada al Consejo Superior de 9
Investigaciones Científicas (CSIC). 2CIBER en Epidemiología y Salud Pública 10
(CIBERESP). 3Servicio de Enfermedades Infecciosas, Hospital Universitario Ramón y 11
Cajal. Madrid, Spain 12
13
*Corresponding author: Mailing address: Servicio de Microbiología, Hospital 14
Universitario Ramón y Cajal. Ctra. Colmenar, Km 9,1. 28034 Madrid. Spain. 15
Phone: 34-91 3368542; Fax: 34-91 3368809; e-mail: [email protected] 16
17
Short title: S. bovis re-identification. 18
19
Key words: Streptococcus bovis, bacteraemia, 16S rDNA, sodA 20
Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.00524-11 JCM Accepts, published online ahead of print on 13 July 2011
on Septem
ber 27, 2020 by guesthttp://jcm
.asm.org/
Dow
nloaded from
2
ABSTRACT 21
All Streptococcus bovis blood culture isolates recovered from Jan-2003 to Jan-2010 22
(n=52) in our institution were re-identified using new taxonomic criteria based upon 23
their genetic traits. Initial identification was performed by the semiautomatic WIDER 24
System (Fco. Soria-Melguizo, Spain) and the API 20 Strep (BioMérieux, France). All 25
isolates were re-identified by PCR amplification and sequencing of both 16S rDNA and 26
sodA genes and by mass spectrometry using MALDI-TOF MS (Bruker, Germany). 27
Results of 16s rDNA/sodA gene sequencing were as follows: Streptococcus gallolyticus 28
subsp. gallolyticus 14/14, Streptococcus gallolyticus subsp. pasteurianus 24/24, 29
Streptococcus spp. 7/0, Streptococcus infantarius subsp. infantarius 0/2, Streptococcus 30
lutetiensis 0/5, Leuconostoc mesenteroides 4/0, and Lactococcus lactis 3/3. The 31
MALDI-TOF MS identified 27 S. gallolyticus without sub-speciation, 4 L. 32
mesenteroides, 3 L. lactis, 6 S. lutetiensis whereas 12 isolates rendered a “non reliable 33
identification” result. Pulsed-field gel electrophoresis grouped all S. gallolyticus subsp. 34
gallolyticus into 3 major clusters clearly different from those of the subsp. pasteurianus 35
which, in turn, exhibited no clonal relationship. Resistance percentages of tested 36
antimicrobials were: 38% erythromycin, 23% fosfomycin, 10% levofloxacin, 6% 37
tetracycline, and 4% cotrimoxazole. The most frequent underlying diseases were 38
hepatobiliar disorders (53%), endocarditis (17%) and malignancies (12%). We conclude 39
that sequencing of sodA gene was the most discriminatory method, and that S. 40
gallolyticus subsp. pasteurianus appear to have a higher genetic diversity than S. 41
gallolyticus subsp. gallolyticus. 42
on Septem
ber 27, 2020 by guesthttp://jcm
.asm.org/
Dow
nloaded from
3
INTRODUCTION 43
Streptoccocus bovis, a non-enterococcal group D Streptococcus, can be found as part of 44
human gastrointestinal microbiota in 5-16% of individuals (17). However, it causes 45
bacteraemia and endocarditis, particularly in men and in the elderly (7-8). S. bovis 46
bacteraemia and colon tumours association was established in the late 1970s (14). 47
Moreover, recent studies have described a frequent association between its isolation 48
with chronic liver and biliary tract disorders (10). 49
Streptococcal taxonomy has progressively changed according to the description of 50
new species originally grouped as S. bovis. During the late 1990s and at the beginning 51
of this decade, several authors re-named the S. bovis biotype I as Streptococcus 52
gallolyticus subsp. gallolyticus (21); the biotype II/1 as Streptococcus lutetiensis (18) 53
and finally the biotype II/2 as S. gallolyticus subsp. pasteurianus (20). 54
Clinicians still remain unfamiliar with the new taxonomy of S. bovis species, 55
mostly due to the complexity of current nomenclature and specific identification 56
requirements based on molecular microbiology not available in routine clinical 57
laboratories. Nevertheless, due to their specific disease association and their 58
microbiology features, a proper identification of the S. bovis isolates is needed. The aim 59
of this study was to review all S. bovis bacteraemic episodes documented along the last 60
seven years in our hospital, focusing on the new taxonomy and the probable association 61
of different subspecies and pathologies. 62
63
PATIENTS AND METHODS 64
Bacterial identification. All S. bovis isolates (n= 52, from 51 patients) causing 65
bacteraemia recovered from blood cultures between January 2003 and January 2010 66
were studied. Initially, S. bovis identification was routinely performed using the 67
on Septem
ber 27, 2020 by guesthttp://jcm
.asm.org/
Dow
nloaded from
4
commercial API 20 Strep gallery (bioMérieux, Marcy l´Etoile, France) and the 68
semiautomated WIDER system (Fco. Soria Melguizo, Madrid, Spain) (5). The ability to 69
grow on bile esculin agar (BD, New York, US) was determined after 24 h of incubation 70
at 37ºC. Isolates were tested for the presence of Lancefield streptococcal antigen D by 71
agglutination using the SLIDEX® Strepto Plus Kit (BioMérieux, Marcy l'Etoile, 72
Francia). 73
Subsequently, both 16S rDNA PCR with universal primers (16-F: 5’-AGGAT 74
TAGATACCCTGGTAGTCCA-3’; and 16-R: 5’-AGGCCCGGGAACGTATTCAC-3’) 75
and sodA PCR with degenerate primers (dl: 5´-CCITAYICITAYGAYG 76
CIYTIGARCC-3´; and d2: 5´-ARRTARTAIGCRTGYTCCCAIACRTC-3´) were 77
performed (18). Amplicons (500 bp for 16S rDNA and 609 bp for sodA, respectively) 78
were sequenced using an ABI prism 377 automated sequencer (PE, Norwalk, Conn, US) 79
after ExoSAP-IT (Amersham, Bucks, UK) purification. Sequences were aligned using 80
the ClustalW tool from the website www.ebi.ac.uk, and phylogenetic trees were 81
constructed with the TreeView software (Michael Eisen, Stanford University, 82
California, USA). 83
Mass spectrometry using the Bruker Biotyper MALDI78 TOF MS system (Bruker 84
Daltonics, Germany) was also performed as part of the bacterial re-identification 85
scheme (3). 86
Antimicrobial susceptibility testing. Susceptibility (microdilution method) was 87
performed using the WIDER panels for Gram-positive organisms, and interpreted 88
according to Clinical and Laboratory Standards Institute criteria (5-6).
89
Genetic diversity. Clonal relatedness was determined by pulsed-field gel 90
electrophoresis (PFGE) using a protocol initially described for Streptococcus suis 91
on Septem
ber 27, 2020 by guesthttp://jcm
.asm.org/
Dow
nloaded from
5
serotype 2 (15). A dendrogram was constructed using the Phoretix 5.0 software 92
(Nonlinear Dynamics Ltd., United Kingdom) based on the Dice’s coefficient. 93
Patients. Clinical charts of all patients were reviewed with the approval of the Ethic 94
Committee of our institution to assess both demographic and clinical data as well as 95
diagnostics and treatments. All patients in whom colonic pathologies were suspected 96
were submitted to colonoscopy examination. A possible biliary source of bacteraemia 97
was assigned if there was a clinical or surgical diagnosis of acute cholecystitis or 98
cholangitis, after the exclusion of other possible focus of infection. 99
100
RESULTS 101
Isolates previously identified as Streptococcus bovis were re-identified using the current 102
nomenclature. All 52 isolates, including the non-streptococcal ones, had a clear positive 103
reaction with latex agglutination test for group D Streptococcus. API 20 Strep initial 104
data were not available, and contemporary profiles corresponded to 29 S. bovis II/2 105
(which included 24 S. gallolyticus subsp. pasteurianus and 5 S. lutetiensis isolates, 106
according with the sodA identification), 14 S. bovis I, 2 S. bovis II/4, 3 L. lactis and 4 107
Leuconostoc spp. 108
Nucleotide sequences of 16S rDNA amplicons (500 bp) allowed the classification 109
of the isolates as Streptococcus gallolyticus subsp. gallolyticus (n=14); Streptococcus 110
gallolyticus subsp. pasteurianus (n=24); Streptococcus spp. (n=7); Lactococcus lactis 111
(n=3) and Leuconostoc mesenteroides (n=4) (Figure 1). 112
Isolates were also re-classified based on the nucleotide sequence of an internal 113
fragment of the sodA gene as: S. gallolyticus subsp. gallolyticus (n=14), S. gallolyticus 114
subsp. pasteurianus (n=24), Streptococcus infantarius subsp. infantarius (n=2); 115
Streptococcus lutetiensis (n=5), and L. lactis (n=3). The seven Streptococcus spp. 116
on Septem
ber 27, 2020 by guesthttp://jcm
.asm.org/
Dow
nloaded from
6
isolates, not identified at species level by the 16S rDNA sequence analysis, were 117
characterized as S. infantarius subsp. infantarius and S. lutetiensis using the sodA gene 118
amplification and sequencing. Consistently negative results for sodA amplification were 119
obtained in the 4 isolates previously identified as L. mesenteroides by 16s rDNA. The 120
sodA nucleotide phylogenetic analysis discriminated the S. gallolyticus subsp. 121
pasteurianus isolates in two clusters (Figure 2), although aminoacidic sequences 122
remained identical (Figure 3). However, several aminoacidic differences were detected 123
in the subsp. gallolyticus group. 124
Finally, the MALDI TOF MS identified 27 S. gallolyticus without no sub-125
speciation, 4 L. mesenteroides, 3 L. lactis, 6 S. lutetiensis whereas in 12 isolates a “non 126
reliable identification” result was obtained. 127
PFGE-SmaI analysis grouped all S. gallolyticus subsp. gallolyticus into 3 major 128
clusters clearly different from those of the subsp. pasteurianus which, in turn, exhibited 129
no PFGE related patterns among them (Figure 4). 130
Antimicrobial susceptibility results are shown in Table 1. All isolates remained 131
susceptible to penicillin, ampicillin, amoxicillin/clavulanate, oxacillin, 132
quinupristin/dalfopristin, linezolid, and rifampicin. Glycopeptides resistance, 133
vancomycin and teicoplanin, was only observed on L. mesenteroides isolates. As 134
expected, all streptococcal isolates (n=45) showed low level resistance to 135
aminoglycosides. Additionally, 16 isolates exhibited high level resistance to 136
streptomycin (>1,000 mg/l), whereas high level of resistance to gentamicin was not 137
observed. Resistance percentages for other antimicrobial tested varied according with 138
bacterial species (Table 1). 139
When considering clinical data from all 51 patients with documented S. bovis 140
bacteraemia, patient’s median age was 73.5±15 years (range from 27 to 98 years, mode 141
on Septem
ber 27, 2020 by guesthttp://jcm
.asm.org/
Dow
nloaded from
7
82 years). Gender distribution was 28 male (54.9%) and 23 female (45.1%). A mean of 142
6.5 episodes per year was documented, being the maximum (n=10 episodes) in 2005. 143
An accumulation of cases between April-June (n=21) were observed, being October-144
December the months with lower incidence (n=7). Blood cultures were obtained in 145
different wards, being the Emergency Unit the most frequent (51%) followed by 146
General Medicine (16%), ICU (10%), Gastroenterology (8%) and others (15%). The 147
most frequent symptoms referred at hospital admission were fever and abdominal pain. 148
Patient’s underlying diseases are showed in Table 2. The most common feature 149
detected was hepatobiliary disorder (53%), followed by endocarditis (17.6%), 150
cardiovascular disease (11.7%), diverticulitis and/or colon polyps (11.7%), digestive 151
tract carcinomas (8%), ferropenic anemia (6%), and other malignancies (4%). Only one 152
patient died during the bacteraemic episode. Interestingly, a female had two different 153
bacteraemic episodes separated by 3 years and caused by genetically different S. 154
gallolyticus subsp. pasteurianus isolates. 155
156
DISCUSSION 157
The former S. bovis group is, at present, divided in four major subspecies (12), being 158
two of them genetically close, S. gallolyticus subsp. gallolyticus, formerly S. bovis 159
biotype I, and S. gallolyticus subsp. pasteurianus, formerly S. bovis biotype II/2, and the 160
other two more distantly related, S. infantarius subsp. infantarius, and S. lutetiensis 161
(named as S. infantarius subsp coli by some authors) (2), formerly S. bovis biotype II/1. 162
Streptococcus gallolyticus subsp. macedonicus, is also member of the group but it is 163
generally consider nonpathogenic for humans. However, and mainly due to 164
discrepancies still remaining, a deeply genetically-based classification is awaited. 165
on Septem
ber 27, 2020 by guesthttp://jcm
.asm.org/
Dow
nloaded from
8
It has been reported a clear association between S. gallolyticus subsp. gallolyticus 166
causing bactearemia and/or endocarditis and presence of colon cancer (1,4,11). 167
Nevertheless, the basis of this association is still not completely understood. In this 168
study, half of the patients had hepatobiliary disorders, whereas endocarditis or 169
cardiovascular diseases were only observed in 17.6 and 11.7% of the patients, 170
respectively. Colonic polyps and carcinomas of gastrointestinal tract were diagnosed in 171
11.7 and 8% of the patients, respectively. When considering only those patients with S. 172
gallolyticus subsp. gallolyticus bacteraemia (n=14), malignances were previously 173
diagnosed in 7 of them (50%) although some of them non-related to the gastrointestinal 174
tract (lymphoma, leukemia or prostate carcinoma), even though colonoscopy 175
exploration was not performed in all patients. 176
Different authors suggest a positive association between S. gallolyticus subsp. 177
gallolyticus isolation with male and elderly patients and endocarditis (7,18). In our case, 178
gender distribution was balanced (55% of male individuals), age of presentation was 179
around 70 years old, and endocarditis was only detected in 17% of patients. Our results 180
are in concordance with those recently published by other Spanish group (9). 181
During the studied period, 76,089 blood samples from patients with suspected 182
bacteraemia were processed in our laboratory. From these, 6,935 (9.1%) were positive, 183
and 52 of them rendered of S. bovis identification (0.74%), although considering the 184
present data of our work, only 45 isolates corresponded in fact to this specie (0.64%). In 185
the same period, 270 infective endocarditis were diagnosed in our center, mostly due to 186
Staphylococcus aureus (30.7%), coagulase-negative staphylococci (17.4%), and 187
Enterococcus (13.3%). “S. bovis” was identified in 7 cases (6 S. gallolyticus sbsp. 188
pasteurianus, and 1 S. gallolyticus sbsp gallolyticus); representing the 2.6% of the total 189
endocarditis (Navas et al. unpublished results). 190
on Septem
ber 27, 2020 by guesthttp://jcm
.asm.org/
Dow
nloaded from
9
Surprisingly, 4 Leuconostoc mesenteroides and 3 Lactococcus lactis causing 191
bacteraemia were initially identified as S. bovis by the semi-automated system used in 192
our laboratory. When re-identification was obtained by 16S rDNA sequence, bacterial 193
identification were retested using the same system, observing that the misleading 194
identification was due to their slow biochemical reactivity; at 24 h of incubation the 195
identification was again S. bovis whereas at 48 h, the identification was correct. 196
Moreover, vancomycin resistance in Leuconostoc isolates was only observed at 48 197
hours. Another important factor to be considered is the positive agglutination of our 198
Leuconostoc and Lactococcus isolates for the Lancefield antigen D. We were unable to 199
find similar references, and this fact has been only demonstrated for Pediococcus spp. 200
isolates (16). 201
As it has been described by other authors (13,19), we detected differences for the 202
isolates identification between the 16S rDNA and the sodA gene sequences methods, 203
being the sodA gene partial sequence considered as the most accurate identification. 204
Recently it has been described high concordance between MALDI-TOF MS and sodA 205
nucleotide sequenciation (12), although in our experience, Bruker technology is unable 206
to identified Streptococcus isolates yet, especially S. gallolyticus subsp. pasteurianus. 207
Contemporary API 20 Strep results agree with the soda classifications, although 5 S. 208
lutetiensis showed the same profile that S. gallolyticus subsp. pasteurianus. 209
Nevertheless, API 20 Strep can be considered a useful method to discriminate the two 210
main S. gallolyticus subspecies, but from a clinical and epidemiological point of view it 211
could be also of interest to further discriminate S. infantarius subsp. infantarius, and S. 212
lutetiensis using the sodA analysis. 213
In spite of many efforts have been direct to improve the S. bovis group 214
identification, data about genetic studies remain unknown. MLST tool has not yet 215
on Septem
ber 27, 2020 by guesthttp://jcm
.asm.org/
Dow
nloaded from
10
developed, and only one reference of high genetic diversity among Italian S. bovis 216
isolates by PFGE (22). In our experience, also high genetic diversity was observed 217
among the 45 streptococcal isolates. 218
Our study demonstrated that in a routine clinical microbiology laboratory, 219
incorporation of a MALDI TOF MS system will partially solve identification problems 220
of S. bovis group. Nevertheless, this system will advise the presence of S. gallolitycus 221
and further molecular testing can be performed if needed. Data entries in the data base 222
for different S. gallolitycus subspecies should be a future goal of MALDITOF MS 223
system as performed with other species. 224
225
226
ACKNOWLEDGMENTS 227
RdC has a “Miguel Servet” contract (CB05/137) from the Instituto de Salud Carlos III-228
FIS. MR-B has a contract from the Instituto de Salud Carlos III-FIS, project 229
AI07/90034. This work was partially funded by the European Project TROCAR 230
(HEALTH-F3-2008-223031). 231
232
233
234
235
236
237
238
239
240
on Septem
ber 27, 2020 by guesthttp://jcm
.asm.org/
Dow
nloaded from
11
REFERENCES 241
1. Abdulamir, A. S., R. R. Hafidh, and F. A. Bakar. 2011. The association of 242
Streptococcus bovis/gallolyticus with colorectal tumors: The nature and the 243
underlying mechanisms of its etiological role. J. Exp. Clin. Cancer Res. 30:11. 244
2. Beck, M., R. Frodl, and G. Funke. 2008. Comprehensive study of strains 245
previously designated Streptococcus bovis consecutively isolated from human 246
blood cultures and emended description of Streptococcus gallolyticus and 247
Streptococcus infantarius subsp. coli. J. Clin. Microbiol. 46:2966-2972. 248
3. Bizzini, A., C. Durussel, J. Bille, G. Greub, and G. Prod’hom. 2010. 249
Performance of matrix-assisted laser desorption ionization-time of flight mass 250
spectrometry for identification of bacterial strains routinely isolated in a clinical 251
microbiology laboratory. J. Clin. Microbiol. 48:1549–1554. 252
4. Boleij, A., R. M. Schaeps, and H. Tjalsma. 2009. Association between 253
Streptococcus bovis and colon cancer. J. Clin. Microbiol. 47:516. 254
5. Cantón, R., et al. 2000. Evaluation of the Wider system, a new computer-assisted 255
image-processing device for bacterial identification and susceptibility testing. J. 256
Clin. Microbiol. 38:1339-1346. 257
6. Clinical and Laboratory Standards Institute. 2007. Performance standards for 258
antimicrobial susceptibility testing; 17th
informational supplement. Document 259
M100-S17, CLSI, Wayne, PA. 260
7. Corredoira, J., et al. 2008. Characteristics of Streptococcus bovis endocarditis 261
and its differences with Streptococcus viridans endocarditis. Eur. J. Clin. 262
Microbiol. Infect. Dis. 27:285-291. 263
on Septem
ber 27, 2020 by guesthttp://jcm
.asm.org/
Dow
nloaded from
12
8. Durante-Mangoni, E., et al. 2008. Current features of infective endocarditis in 264
elderly patients: results of the International Collaboration on Endocarditis 265
Prospective Cohort Study. Arch. Intern. Med. 168:2095–2103. 266
9. Fernández-Ruiz, M., et al. 2010. Streptococcus bovis bacteraemia revisited: 267
clinical and microbiological correlates in a contemporary series of 59 patients. J. 268
Infect. 61:307-313. 269
10. González-Quintela, A., C. Martínez-Rey, J. C. Castroagudin, M. C. Rajo-270
Iglesias, and M. J. Domínguez-Santalla. 2001. Prevalence of liver disease in 271
patients with Streptococcus bovis bacteraemia. J. Infect. 42:116–119. 272
11. Gupta, A., R. Madani, and H. Mukhtar. 2010. Streptococcus bovis 273
endocarditis, a silent sign for colonic tumour. Colorectal Dis. 12:164-171. 274
12. Hinse, D., et al. 2011. Differentiation of species of the Streptococcus 275
bovis/equinus-complex by MALDI-TOF Mass Spectrometry in comparison to 276
sodA sequence analyses. Syst. Appl. Microbiol. 34:52-57. 277
13. Hoshino, T., T. Fujiwara, and M. Kilian. 2005. Use of phylogenetic and 278
phenotypic analyses to identify nonhemolytic streptococci isolated from 279
bacteremic patients. J. Clin. Microbiol. 43:6073-6085. 280
14. Klein, R., et al. 1977. Association of Streptococcus bovis with carcinoma of the 281
colon. N. Engl. J. Med. 297:800-802. 282
15. Luey, C.K., et al. 2007. Rapid pulsed-field gel electrophoresis protocol for 283
subtyping of Streptococcus suis serotype 2. J. Microbiol. Methods. 68:648-650. 284
16. Murray, P. R., E. J. Baron, J. H. Jorgensen, M. A. Pfaller, and R. H. Yolken 285
(ed.). 2003. Manual of Clinical Microbiology. 8th
ed. American Society for 286
Microbiology, Washington, D.C. 287
on Septem
ber 27, 2020 by guesthttp://jcm
.asm.org/
Dow
nloaded from
13
17. Noble, C. J. 1978. Carriage of group D streptococci in the human bowel. J. Clin. 288
Pathol. 31:1182–1186. 289
18. Poyart, C., G. Quesne, and P. Trieu-Cuot. 2002. Taxonomic dissection of the 290
Streptococcus bovis group by analysis of manganese-dependent superoxide 291
dismutase gene (sodA) sequences: reclassification of 'Streptococcus infantarius 292
subsp. coli as Streptococcus lutetiensis sp. nov. and of Streptococcus bovis 293
biotype II.2 as Streptococcus pasteurianus sp. nov. Int. J. Syst. Evol. Microbiol. 294
52:1247-1255. 295
19. Sasaki, E., R. Osawa, Y. Nishitani, and R. A. Whiley. 2004. Development of a 296
diagnostic PCR assay targeting the Mn-dependent superoxide dismutase gene 297
(sodA) for identification of Streptococcus gallolyticus. J. Clin. Microbiol. 298
42:1360-1362. 299
20. Schlegel, L., et al. 2000. Streptococcus infantarius sp. nov., Streptococcus 300
infantarius subsp. infantarius subsp. nov. and Streptococcus infantarius subsp. 301
coli subsp. nov., isolated from humans and food. Int. J. Syst. Evol. Microbiol. 302
50:1425–1434. 303
21. Schlegel, L., F. Grimont, E. Ageron, P. A. D. Grimont, and A. Bouvet. 2003. 304
Reappraisal of the taxonomy of the Streptococcus bovis/Streptococcus equinus 305
complex and related species: description of Streptococcus gallolyticus subsp. 306
gallolyticus subsp. nov., S. gallolyticus subsp. macedonicus subsp. nov. and S. 307
gallolyticus subsp. pasteurianus subsp. nov. Int. J. Syst. Evol. Microbiol. 53:631–308
45. 309
22. Tripodi, M. F., R. Fortunato, R. Utili, M. Triassi, and R. Zarrilli. 2005. 310
Molecular epidemiology of Streptococcus bovis causing endocarditis and 311
bacteraemia in Italian patients. Clin. Microbiol. Infect. 11:814-819. 312
on Septem
ber 27, 2020 by guesthttp://jcm
.asm.org/
Dow
nloaded from
Table 1. Antibiotic susceptibility for the different species studied. 1
Antibiotic Resistance Percentage (MIC50)
Stra Lev Ery Cln Fos SxT
S. gallolyticus
subsp. gallolyticus n=14
42.8
(≤1000)
14.2
(2)
35.7
(≤0.5)
35.7
(≤0.5)
35.7
(≤32)
35.7
(≤1/19)
S. gallolyticus
subsp. pasteurianus n=24
20.8
(≤1000)
8.3
(≤1)
37.5
(≤0.5)
25.0
(≤0.5)
29.1
(≤32)
25.0
(≤1/19)
S. infantarius
subsp. infantarius n=2
0
(≤1000)
0
(≤1)
0
(≤0.5)
0
(≤0.5)
0
(≤32)
0
(≤1/19)
S. lutetiensis n=5
60.0
(>1000)
40.0
(2)
60.0
(>2)
60.0
(>2)
20.0
(≤32)
20.0
(≤1/19)
L. lactis n=3
66.6
(>1000)
0
(2)
33.3
(≤0.5)
33.3
(≤0.5)
66.6
(>64)
66.6
(>2/38)
L. mesenteroides n=4
0
(≤0.25)
0
(2)
50.0
(≤0.5)
25.0
(≤0.5)
25.0
(≤0.32)
25.0
(≤1/19)
a: High Level Resistance (>1000 mg/L). Str: streptomycin; Lev: levofloxacin; Ery: erythromycin; Cln: 2
clindamycin; Fos: fosfomycin; SxT: trimethropim sulfamethoxazole. 3
on Septem
ber 27, 2020 by guesthttp://jcm
.asm.org/
Dow
nloaded from
Table 2. Patient’s clinical features.
S. gallolyticus
subsp.
gallolyticus
S. gallolyticus subsp.
pasteurianus S. lutetiensis
S. infantarius
subsp.
infantarius
L. lactis L. mesenteroides
Cholecystitis / Cholangitis 3 5 1
Cirrhosis 2 10 1 1 1
HVC 5 1 1
Acute appendicitis 1
Colonic adenoma 1 4 1
Colonic polyps 2 7 1
Rectal carcinoma 1
Diabetes 2
Isquemic Cardiopathy 3 1 1 1
Endocarditis 1 6
Mitral stenosis 1
Renal Insufficiency 1
Urinary tract infection 1 1
Bladder cancer 1
Prostate cancer 1
Pulmonary cancer 1
MALT lymphoma 1
Leukemia 1
on Septem
ber 27, 2020 by guesthttp://jcm
.asm.org/
Dow
nloaded from
0.1
52
50
45
36
32
23
16
11
1
5
49
44
34
30
20
14
6
18
47
46
37
3
9
24
38
42
39
8
35
27
19
22
51
41
40
31
28
26
25
21
13
10
4
7
48
43
33
29
17
12
2
15
Group B. S. gallolyticus sbsp. gallolyticus/pasteurianus
Group C. S. gallolyticus sbsp. pasteurianus
Group D. S. lutetiensis/S. infantarium sbsp. infantarium
Group E. L. lactis
Group F. L. mesenteroides
Group A. S. gallolyticus sbsp. gallolyticus/pasteurianus
1
2
Figure 1. Clustering of 16S rDNA nucleotide sequences obtained fron all 52 isolates, 3
and constructed by the TreeView sowftare. 4
on Septem
ber 27, 2020 by guesthttp://jcm
.asm.org/
Dow
nloaded from
Group 1. S. gallolyticus sbsp. pasteurianus
Group 2. S. gallolyticus sbsp. pasteurianus
Group 3. S. gallolyticus sbsp. gallolyticus
Group 5. S. infantarium sbsp. infantarium
Group 4. S. lutetiensis
Group 6. L. lactis
0.1
41
51
40
31
28
25
21
10
7
4
52
30
20
15
1
12
14
36
6
11
29
50
23
43
37
47
46
3
38
9
24
19
22
27
18
34
32
17
5
16
2
49
48
45
44
33
13
26
5
6
Figure 2. Clustering of sodA nucleotide sequences obtained from non-Leuconostoc 7
isolates (n=48) and constructed by the TreeView sowftare.8
on Septem
ber 27, 2020 by guesthttp://jcm
.asm.org/
Dow
nloaded from
1 NVNAALEKHPEIGDDLEALLADVDSIPTDIRQAVINNGGGHLNHALFWELLSPEKQEPTTQVLAAIEEAFGSFDEFKAAFTQAATTRFGSGWAWLVVNENGKLEVLSTANQDTPISQGKAP
d E A A D
e F E A T A D
a E A A
c E A A
g E A A V
b EA N A A
f E A V
a A L VI L K A A S F ADV V K KDD I N E K
A L VI L K A A S F ADV V K KDD I N E K
4 A L VI EL K A A S AD ED T KD T T L E K
41
18
34
2
49
52
30
14
6
29
23
43
37
46
9
9
10
Figure 3. Aminoacidic sequences comparison for the SodA protein from the different sodA alleles detected. 11
12
on Septem
ber 27, 2020 by guesthttp://jcm
.asm.org/
Dow
nloaded from
Figure 4. Dendrograme obtained for the 24 S. gallolyticus subsp. pasteurianus and 14
S. gallolyticus subsp. gallolyticus isolates using the UPGMA method and the Phoretix
5.0 sowftare.
S. gallolyticus sbsp. pasteurianus
S. gallolyticus sbsp. gallolyticus
S. gallolyticus sbsp. pasteurianus
S. gallolyticus sbsp. gallolyticus
on Septem
ber 27, 2020 by guesthttp://jcm
.asm.org/
Dow
nloaded from