Aerococcus urinae infection of the pubic symphysis: A rare case report.

Aerococcus urinae is a Gram-positive, catalase-negative coccus, rarely responsible for urinary tract infections and seldom described for musculoskeletal infections like spondylodiscitis. An 86-year-old man presented to our hospital for groin pain without fever. Pelvic CT-guided biopsy revealed an A. urinae pubic symphysis osteomyelitis. He received a treatment by amoxicillin per os for six weeks, and did not need any surgery. An eight -month- follow-up showed a favorable evolution. Pubic symphysis infection can be induced by a wide variety of pathogens, and may have very different clinical presentations. Some authors recommend systematic surgery, but in case of susceptible pathogen associated with a low level of joint destruction, medical treatment alone should be sufficient to cure and make surgery unnecessary.

Clinical, Bacteriological, and Genetic Characterization of Bone and Joint Infections Involving Linezolid-Resistant Staphylococcus epidermidis: a Retrospective Multicenter Study in French Reference Centers.

The choice of the best probabilistic postoperative antibiotics in bone and joint infections (BJIs) is still challenging. Since the implementation of protocolized postoperative linezolid in six French referral centers, linezolid-resistant multidrug-resistant Staphylococcus epidermidis (LR-MDRSE) strains were isolated in patients with BJI. We aimed here to describe clinical, microbiological, and molecular patterns associated with these strains. All patients with at least one intraoperative specimen positive for LR-MDRSE between 2015 and 2020 were included in this retrospective multicenter study. Clinical presentation, management, and outcome were described. LR-MDRSE strains were investigated by MIC determination for linezolid and other anti-MRSA antibiotics, characterization of genetic determinants of resistance, and phylogenetic analysis. Forty-six patients (colonization n = 10, infection n = 36) were included in five centers, 45 had prior exposure to linezolid, 33 had foreign devices. Clinical success was achieved for 26/36 patients. Incidence of LR-MDRSE increased over the study period. One hundred percent of the strains were resistant to oxazolidinones, gentamicin, clindamycin, ofloxacin, rifampicin, ceftaroline, and ceftobiprole, and susceptible to cyclins, daptomycin, and dalbavancin. Susceptibility to delafloxacin was bimodal. Molecular analysis was performed for 44 strains, and the main mutation conferring linezolid resistance was the 23S rRNA G2576T mutation. All strains belonged to the sequence type ST2 or its clonal complex, and phylogenetic analysis showed emergence of five populations corresponding geographically to the centers. We showed the emergence of new clonal populations of highly linezolid-resistant S. epidermidis in BJIs. Identifying patients at risk for LR-MDRSE acquisition and proposing alternatives to systematic postoperative linezolid use are essential. IMPORTANCE The manuscript describes the emergence of clonal linezolid-resistant strains of Staphylococcus epidermidis (LR-MDRSE) isolated from patients presenting with bone and joint infections. Incidence of LR-MDRSE increased over the study period. All strains were highly resistant to oxazolidinones, gentamicin, clindamycin, ofloxacin, rifampicin, ceftaroline, and ceftobiprole, but were susceptible to cyclins, daptomycin, and dalbavancin. Susceptibility to delafloxacin was bimodal. The main mutation conferring linezolid resistance was the 23S rRNA G2576T mutation. All strains belonged to the sequence type ST2 or its clonal complex, and phylogenetic analysis showed emergence of five populations corresponding geographically to the centers. LR-MDRSE bone and joint infections seem to be accompanied by an overall poor prognosis related to comorbidities and therapeutic issues. Identifying patients at risk for LR-MDRSE acquisition and proposing alternatives to systematic postoperative linezolid use become essential, with a preference for parenteral drugs such as lipopeptids or lipoglycopeptids.

Biofilm Formation in Streptococcus agalactiae Is Inhibited by a Small Regulatory RNA Regulated by the Two-Component System CiaRH.

Regulatory small RNAs (sRNAs) are involved in the adaptation of bacteria to their environment. CiaR-dependent sRNAs (csRNAs) are controlled by the regulatory two-component system (TCS) CiaRH, which is widely conserved in streptococci. Except for Streptococcus pneumoniae and Streptococcus sanguinis, the targets of these csRNAs have not yet been investigated. Streptococcus agalactiae, the leading cause of neonatal infections, has four conserved csRNA genes, namely, srn015, srn024, srn070, and srn085. Here, we demonstrate the importance of the direct repeat TTTAAG-N5-TTTAAG in the regulation of these csRNAs by CiaRH. A 24-nucleotide Srn024-sap RNA base-pairing region is predicted in silico. The sap gene encodes a LPXTG-cell wall-anchored pullulanase. This protein cleaves α-glucan polysaccharides such as pullulan and glycogen present in the environment to release glucose and is involved in adhesion to human cervical epithelial cells. Inactivation of S. agalactiae pullulanase (SAP) leads to no bacterial growth in a medium with only pullulan as a carbon source and reduced biofilm formation, while deletion of ciaRH and srn024 genes significantly increases bacterial growth and biofilm formation. Using a new translational fusion vector, we demonstrated that Srn024 is involved in the posttranscriptional regulation of sap expression. Complementary base pair exchanges in S. agalactiae suggest that Srn024 interacts directly with sap mRNA and that disruption of this RNA pairing is sufficient to yield the biofilm phenotype of Srn024 deletion. These results suggest the involvement of Srn024 in the adaptation of S. agalactiae to environmental changes and biofilm formation, likely through the regulation of the sap gene. IMPORTANCE Although Streptococcus agalactiae is a commensal bacterium of the human digestive and genitourinary tracts, it is also an opportunistic pathogen for humans and other animals. As the main cause of neonatal infections, it is responsible for pneumonia, bacteremia, and meningitis. However, its adaptation to these different ecological niches is not fully understood. Bacterial regulatory networks are involved in this adaptation, and the regulatory TCSs (e.g., CiaRH), as well as the regulatory sRNAs, are part of it. This study is the first step to understand the role of csRNAs in the adaptation of S. agalactiae. This bacterium does not currently exhibit extensive antibiotic resistance. However, it is crucial to find alternatives before multidrug resistance emerges. Therefore, we propose that drugs targeting regulatory RNAs with Srn024-like activities would affect pathogens by reducing their abilities to form biofilm and to adapt to host niches.

Distribution of Achromobacter Species in 12 French Cystic Fibrosis Centers in 2020 by a Retrospective MALDI-TOF MS Spectrum Analysis.

Achromobacter spp. are nonfermenting Gram-negative bacilli mainly studied among cystic fibrosis (CF) patients. The identification of the 19 species within the genus is time-consuming (nrdA-sequencing), thus data concerning the distribution of the species are limited to specific studies. Recently, we built a database using MALDI-TOF mass spectrometry (MS) (Bruker) that allows rapid and accurate species identification and detection of the multiresistant epidemic clones: A. xylosoxidans ST137 spreading among CF patients in various French and Belgium centers, and A. ruhlandii DES in Denmark. Here, we first assessed whether species identification could be achieved with our database solely by analysis of MS spectra without availability of isolates. Then, we conducted a multicentric study describing the distribution of Achromobacter species and of the clone ST137 among French CF centers. We collected and analyzed with our local database the spectra of Achromobacter isolates from 193 patients (528 samples) from 12 centers during 2020. In total, our approach enabled to conclude for 502/528 samples (95.1%), corresponding to 181 patients. Eleven species were detected, only five being involved in chronic colonization, A. xylosoxidans (86.4%), A. insuavis (9.1%), A. mucicolens (2.3%), A. marplatensis (1.1%) and A. genogroup 3 (1.1%). This study confirmed the high prevalence of A. xylosoxidans in chronic colonizations and the circulation of the clone A. xylosoxidans ST137 in France: four patients in two centers. The present study is the first to report the distribution of Achromobacter species from CF patients samples using retrospective MALDI-TOF/MS data. This easy approach could enable future large-scale epidemiological studies.

Genotypic Characterization and Biofilm Production of Group B Streptococcus Strains Isolated from Bone and Joint Infections.

Bone and joint infections (BJI) represent the second cause of invasive Group B Streptococcus (GBS) infections. Biofilm formation plays a major role in BJI. This study's aim was to analyze the genetic features and biofilm production of GBS strains. In six French laboratories, 77 GBS strains isolated from BJI and 57 strains from vaginal human colonization (Hcol) were characterized and compared by Multi-Locus Sequence Typing (MLST). PCR was used to search for the adhesins (bsaB, lmb, scpB, fbsA, fbsB, hvgA, bibA, bca, srr-1, and srr-2) and Pilus Islands (PI) related genes (PI-1, PI-2a, PI-2b). Biofilm production was studied by crystal violet assay. Strains were categorized into three groups, based on Specific Biofilm Formation (SBF) values defined as: weak, moderate, or strong producers. Molecular study revealed three major clonal complexes (CC) in BJI strains: CC1 (42%), CC23 (22%) and CC10 (14%). Several associations between CC and adhesin/pili were identified: CC1 with srr2, PI-1 + 2a; CC10 with srr-1, bca, PI-1 + 2a; CC17 with fbsB, hvgA, srr-2, PI-1+PI-2b; CC19 with bibA, srr-1, PI-1 + 2a; CC23 with fbsB, bibA, srr-1, PI-2a. The biofilm production was significantly different according to CC, adhesins and pili gene detection. CC10, CC23 and strains harboring fbsB produce more biofilm than CC1, PI-1 + 2a (independently). Finally, SBF values were significantly stronger for Hcol strains rather than for BJI strains (76% versus 40%). This study revealed that Hcol strains appeared to produce stronger biofilm than BJI strains, though they belonged to similar CCs and had the same adhesin and pili content. IMPORTANCE Bone and joint infections (BJI) are pathologies that can be life-threatening and result in compromised functional prognosis for patients. Relapses are common and often related to biofilm formation. Group B streptococci (GBS) BJI increased since the last decade. However, few data are available on this subject in the literature. Our study aims to highlight genotype and biofilm production of GBS isolates from BJI. Seventy-seven GBS strains isolated from BJI and 57 from asymptomatic human vaginal colonization were characterized by multilocus sequence typing (MLST), adhesins content, nature of the pili and the ability to form biofilm. Our results revealed that vaginal human colonization strains produced stronger biofilm than BJI strains, despite belonging to the same phylogenetic lineage and having the same adhesin and pili content.

An Inventory of CiaR-Dependent Small Regulatory RNAs in Streptococci.

Bacteria adapt to the different environments encountered by rapid and tightly controlled regulations involving complex networks. A first line of control is transcriptional with regulators such as two-component systems (TCSs) that respond to physical and chemical perturbations. It is followed by posttranscriptional regulations in which small regulatory RNAs (sRNAs) may affect RNA translation. Streptococci are opportunistic pathogens for humans and farm animals. The TCS CiaRH is highly conserved among this genus and crucial in bacterial survival under stressful conditions. In several streptococcal species, some sRNAs belong to the CiaRH regulon and are called csRNAs for cia-dependent sRNAs. In this review, we start by focusing on the Streptococcus species harboring a CiaRH TCS. Then the role of CiaRH in streptococcal pathogenesis is discussed in the context of recent studies. Finally, we give an overview of csRNAs and their functions in Streptococci with a focus on their importance in bacterial adaptation and virulence.

Regulatory RNAs in the Less Studied Streptococcal Species: From Nomenclature to Identification.

Streptococcal species are Gram-positive bacteria involved in severe and invasive diseases in humans and animals. Although, this group includes different pathogenic species involved in life-threatening infections for humans, it also includes beneficial species, such as Streptococcus thermophilus, which is used in yogurt production. In bacteria virulence factors are controlled by various regulatory networks including regulatory RNAs. For clearness and to develop logical thinking, we start this review with a revision of regulatory RNAs nomenclature. Previous reviews are mostly dealing with Streptococcus pyogenes and Streptococcus pneumoniae regulatory RNAs. We especially focused our analysis on regulatory RNAs in Streptococcus agalactiae, Streptococcus mutans, Streptococcus thermophilus and other less studied Streptococcus species. Although, S. agalactiae RNome remains largely unknown, sRNAs (small RNAs) are supposed to mediate regulation during environmental adaptation and host infection. In the case of S. mutans, sRNAs are suggested to be involved in competence regulation, carbohydrate metabolism, and Toxin-Antitoxin systems. A new category of miRNA-size small RNAs (msRNAs) was also identified for the first time in this species. The analysis of S. thermophilus sRNome shows that many sRNAs are associated to the bacterial immune system known as CRISPR-Cas system. Only few of the other different Streptococcus species have been the subject of studies pointed toward the characterization of regulatory RNAs. Finally, understanding bacterial sRNome can constitute one step forward to the elaboration of new strategies in therapy such as substitution of antibiotics in the management of S. agalactiae neonatal infections, prevention of S. mutans dental caries or use of S. thermophilus CRISPR-Cas system in genome editing applications.

Single nucleotide resolution RNA-seq uncovers new regulatory mechanisms in the opportunistic pathogen Streptococcus agalactiae.

BACKGROUND: Streptococcus agalactiae, or Group B Streptococcus, is a leading cause of neonatal infections and an increasing cause of infections in adults with underlying diseases. In an effort to reconstruct the transcriptional networks involved in S. agalactiae physiology and pathogenesis, we performed an extensive and robust characterization of its transcriptome through a combination of differential RNA-sequencing in eight different growth conditions or genetic backgrounds and strand-specific RNA-sequencing. RESULTS: Our study identified 1,210 transcription start sites (TSSs) and 655 transcript ends as well as 39 riboswitches and cis-regulatory regions, 39 cis-antisense non-coding RNAs and 47 small RNAs potentially acting in trans. Among these putative regulatory RNAs, ten were differentially expressed in response to an acid stress and two riboswitches sensed directly or indirectly the pH modification. Strikingly, 15% of the TSSs identified were associated with the incorporation of pseudo-templated nucleotides, showing that reiterative transcription is a pervasive process in S. agalactiae. In particular, 40% of the TSSs upstream genes involved in nucleotide metabolism show reiterative transcription potentially regulating gene expression, as exemplified for pyrG and thyA encoding the CTP synthase and the thymidylate synthase respectively. CONCLUSIONS: This comprehensive map of the transcriptome at the single nucleotide resolution led to the discovery of new regulatory mechanisms in S. agalactiae. It also provides the basis for in depth analyses of transcriptional networks in S. agalactiae and of the regulatory role of reiterative transcription following variations of intra-cellular nucleotide pools.

A tetracycline-inducible expression vector for Streptococcus agalactiae allowing controllable gene expression.

Streptococcus agalactiae is the principal cause of neonatal infections; however, for its study, genetic tools are scarce. We have developed pG+off, an expression vector with a tetracycline inducible promoter that allows a tightly-controllable gene expression in S. agalactiae. Using a reporter gene, we have shown that pG+off is characterized by tight repression under non-induced condition.

The transcription of the neuD gene is stronger in serotype III group B streptococci strains isolated from cerebrospinal fluid than in strains isolated from vagina.

Group B streptococci (GBS) are a major cause of neonatal meningitis, and sialic acid is a determinant of the development of meningitis. The transcription level of the neuD gene, used as a marker of neu gene expression and capsular production, was significantly higher in serotype III GBS strains isolated from meningitis than from vaginal carriage. This was irrespective both of the phylogenetic position of strains and of the presence of a thymine at position 264 in the neuD gene. Differences in neuD gene transcription may explain in part why particular isolates among the GBS strains colonizing the vagina can cause meningitis.

Characterization of an extended-spectrum class A ß-lactamase from a novel enterobacterial species taxonomically related to Rahnella spp./Ewingella spp.

OBJECTIVES: To characterize the naturally occurring ß-lactamase gene identified from a clinical isolate belonging to a novel enterobacterial species that is closely related to Rahnella spp. and Ewingella spp. METHODS: Shotgun cloning and expression in Escherichia coli were performed in order to characterize this resistance determinant. Enzymatic activities were measured by UV spectrophotometry after an ion-exchange chromatography purification procedure. RESULTS: A chromosomal gene coding for the extended-spectrum ß-lactamase (ESBL) SMO-1 was identified from a novel enterobacterial species that is taxonomically related to Rahnella aquatilis and Ewingella americana. The ß-lactamase efficiently hydrolysed penicillins and cefotaxime, and shared 75% amino acid identity with the plasmid-mediated ß-lactamase SFO-1 from Serratia fonticola, 74% amino acid identity with the plasmid-mediated ESBL CTX-M-2 originating from Kluyvera spp. and 72% amino acid identity with the chromosomally encoded and intrinsic RAHN-1 from R. aquatilis. CONCLUSIONS: We have identified a novel enterobacterial species recovered from a clinical specimen, constituting another potential source of acquired ESBL. The ESBL shared significant similarities with the CTX-M-type enzymes.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry for bacterial strain characterization.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a new technique for rapid and effective bacterial species identification in clinical microbiology laboratories. Conventional bacterial identification is based essentially on biochemical tests and requires lengthy incubation procedures. By contrast, MALDI-TOF MS can identify bacterial species in a few minutes, directly from a freshly grown colony. Various approaches have been developed for bacterial identification and the efficacy of this method has been demonstrated in several studies. This technique should also be suitable for the classification of organisms to subspecies level and for the phenotypic detection of certain antibiotic resistance mechanisms, such as ß-lactamases production.

Variability of neuD transcription levels and capsular sialic acid expression among serotype III group B Streptococcus strains.

Serotype III group B Streptococcus (GBS) is the major cause of neonatal meningitis, but the risk of infection in the colonized neonates is variable. Capsular sialic acid (Sia), whose synthesis is encoded by neu genes, appears to be a major virulence factor in several bacterial species able to reach the cerebrospinal fluid. Therefore, variations of Sia expression related to the genetic diversity of strains may have an impact on the risk of meningitis in colonized neonates. We characterized by MLST the phylogenetic diversity of 64 serotype III GBS strains isolated from vaginal flora and randomly selected. These strains mostly belonged to three major sequence types (STs): ST1 (11%), ST17 (39%) and ST19 (31%). The genetic diversity of strains of these lineages, characterized by PFGE, allowed the selection of 17 representative strains, three ST1, six ST17 and eight ST19, with NEM316 as reference, in order to evaluate (i) by quantitative RT-PCR, the level of transcription of the neuD gene as a marker for the transcription of neu genes and (ii) by enzymological analysis, the expression of Sia. The mean transcription level of neuD was higher for ST17 strains than for ST1 and ST19 strains in the early, mid- and late exponential growth phases, and was maximum in the early exponential growth phase for ST17 strains and in the mid-exponential growth phase for ST1 and ST19 strains. Mean Sia concentration was higher for ST17 than for ST1 and ST9 strains in all three growth phases. For the total population, Sia concentration varied notably in the stationary phase, from 0.38 to 9.30 nmol per 10(8) viable bacteria, with a median value of 2.99 nmol per 10(8) bacteria. All ST17 strains, only one-third of the ST19 strains and none of the ST1 strains had Sia concentrations higher than the median Sia concentration. Therefore, differences in the level of expression of Sia by strains of the major serotype III GBS phylogenetic lineages might be one of the factors that explain the leading role of ST17 strains in neonatal meningitis.

A multi locus variable number of tandem repeat analysis (MLVA) scheme for Streptococcus agalactiae genotyping.

BACKGROUND: Multilocus sequence typing (MLST) is currently the reference method for genotyping Streptococcus agalactiae strains, the leading cause of infectious disease in newborns and a major cause of disease in immunocompromised children and adults. We describe here a genotyping method based on multiple locus variable number of tandem repeat (VNTR) analysis (MLVA) applied to a population of S. agalactiae strains of various origins characterized by MLST and serotyping. RESULTS: We studied a collection of 186 strains isolated from humans and cattle and three reference strains (A909, NEM316 and 2603 V/R). Among 34 VNTRs, 6 polymorphic VNTRs loci were selected for use in genotyping of the bacterial population. The MLVA profile consists of a series of allele numbers, corresponding to the number of repeats at each VNTR locus. 98 MLVA genotypes were obtained compared to 51 sequences types generated by MLST. The MLVA scheme generated clusters which corresponded well to the main clonal complexes obtained by MLST. However it provided a higher discriminatory power. The diversity index obtained with MLVA was 0.960 compared to 0.881 with MLST for this population of strains. CONCLUSIONS: The MLVA scheme proposed here is a rapid, cheap and easy genotyping method generating results suitable for exchange and comparison between different laboratories and for the epidemiologic surveillance of S. agalactiae and analyses of outbreaks.

Rapid detection of "highly virulent" Group B Streptococcus ST-17 and emerging ST-1 clones by MALDI-TOF mass spectrometry.

MALDI-TOF MS identified a 6250-Da protein specific to Sequence Type-1 (ST-1) strains and a 7625-Da protein specific to ST-17 strains when used for identification of Group B streptococci. The strains of these STs are major causes of meningitis and late-onset-disease in neonates. This rapid method of identification could thus be valuable in the evaluation of risk of neonatal diseases.

Two-component system RgfA/C activates the fbsB gene encoding major fibrinogen-binding protein in highly virulent CC17 clone group B Streptococcus.

Group B streptococcus (GBS) strains with the highest ability to bind to human fibrinogen belong to the highly invasive clonal complex (CC) 17. To investigate the fibrinogen-binding mechanisms of CC17 strains, we determined the prevalence of fibrinogen-binding genes (fbsA and fbsB), and fbs regulator genes (rogB encoding an fbsA activator, rovS encoding an fbsA repressor and rgf encoding a two-component system [TCS] whose role on fbs genes was not determined yet) in a collection of 134 strains representing the major CCs of the species. We showed that specific gene combinations were related to particular CCs; only CC17 strains contained the fbsA, fbsB, and rgf genes combination. Non polar rgfAC deletion mutants of three CC17 serotype III strains were constructed. They showed a 3.2- to 5.1-fold increase of fbsA transcripts, a 4.8- to 6.7-fold decrease of fbsB transcripts, and a 52% to 68% decreased fibrinogen-binding ability, demonstrating that the RgfA/RgfC TCS inhibits the fbsA gene and activates the fbsB gene. The relative contribution of the two fbs genes in fibrinogen-binding ability was determined by constructing isogenic fbsA, fbsB, deletion mutants of the three CC17 strains. The ability to bind to fibrinogen was reduced by 49% to 57% in ΔfbsA mutants, and by 78% to 80% in ΔfbsB mutants, suggesting that FbsB protein plays a greater role in the fibrinogen-binding ability of CC17 strains. Moreover, the relative transcription level of fbsB gene was 9.2- to 12.7-fold higher than that of fbsA gene for the three wild type strains. Fibrinogen-binding ability could be restored by plasmid-mediated expression of rgfAC, fbsA, and fbsB genes in the corresponding deletion mutants. Thus, our results demonstrate that a specific combination of fbs genes and fbs regulator genes account for the high fibrinogen-binding ability of CC17 strains that may participate to their enhanced invasiveness for neonates as compared to strains of other CCs.

Molecular characterization of temporally and geographically matched Streptococcus agalactiae strains isolated from food products and bloodstream infections.

In a defined geographic area, during a 3-month period, 914 food products were screened for Streptococcus agalactiae, and S. agalactiae strains isolated from bloodstream infections (BSI) in nonpregnant adults were collected. Eleven S. agalactiae strains were isolated from 1.2% of food products, with high rates in pastries (7.0%) and seafood products (11.8%). These findings indicate that S. agalactiae is a food product contaminant. Seven S. agalactiae BSI were observed in nonpregnant adults representing an incidence of 0.015/100 admissions. The distribution of strains in serotypes did not differ according to origin of the strains; food products and clinical strains were of serotypes Ia (22%), Ib (11%), II (5%), III (22%), IV (5%), and V (33%). The strains isolated from seafoods were of serotypes Ia and Ib. The distribution of strains in Sequence Types differed according to their origin; food strains were equally distributed between the major clonal complex (CC), CC1 (27%), CC9 (18%), CC17 (18%), and CC23 (27%), whereas a high proportion of BSI strains belonged to CC1 (57%). DNA macrorestriction using SmaI revealed diversity; nine different patterns were found for the 11 food strains and seven for the 7 BSI strains. One pattern was similar for two food strains and one BSI strain. On account of the molecular characteristics previously described for S. agalactiae strains of human carriage and fish and mice infections, the serotype characteristics of seafood strains suggest contamination by aquatic S. agalactiae, whereas the molecular characteristics of strains from pastries suggest human contamination, but may also originate from rodents. Indeed, serotype V CC1 strains, found in food and responsible for a high percentage of BSI in nonpregnant adults, belong to a known clone spreading worldwide, and have also been described in mice.

Identification of Streptococcus agalactiae isolates from various phylogenetic lineages by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Variations in proteins related to bacterial diversity may affect species identification performed using matrix-assisted laser desorption ionization (MALDI)-time of flight mass spectrometry. Using this method, we identified 110 Streptococcus agalactiae isolates characterized by serotyping and multilocus sequence typing. Serotype III and sequence type 23 strains expressed the widest variation in molecular weight of putative "species-identifying" biomarker ions. Recognition of the diversity of MALDI patterns observed in strains that represent all major intraspecies lineages assists in the constitution of an optimal reference database.