Performance assessment of the Bruker Biotyper MALDI-TOF MS for the identification of difficult-to-identify viridans group streptococci.

Differentiating Streptococcus pneumoniae among nonpneumococcal viridans group streptococci (VGS) is challenging in conventional laboratories. Therefore, we aimed to evaluate the performance of the latest Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system in identifying VGS by comparing the results to those of the specific gene sequencing approach. Clinical isolates were initially identified using the BD Phoenix system to identify Streptococcus species. The optochin test was used to distinguish nonpneumococcal VGS from S. pneumoniae. The species of individual reference strains and clinical isolates were determined by comparing the sequences of the 16S rDNA, gyrB, sodA, groESL, or coaE genes with those in the GenBank sequence databases. We evaluated the performance of the Bruker Biotyper MALDI-TOF MS in VGS identification using two different machines with three databases. We collected a total of 103 nonpneumococcal VGS and 29 S. pneumoniae blood isolates at a medical center in northern Taiwan. Among these isolates, only seven could not be identified at the species level by the specific gene sequencing approach. We found that none of the nonpneumococcal VGS isolates were misidentified as pneumococci by the latest Biotyper system, and vice versa. However, certain strains, especially those in the mitis and bovis groups, could still not be correctly identified. The latest Bruker Biotyper 4.1 (DB_10833) showed significant improvement in identifying VGS strains. However, a specific gene sequencing test is still needed to precisely differentiate the species of strains in the mitis and bovis groups.

SERS-based rapid susceptibility testing of commonly administered antibiotics on clinically important bacteria species directly from blood culture of bacteremia patients.

Bloodstream infections are a growing public health concern due to emerging pathogens and increasing antimicrobial resistance. Rapid antibiotic susceptibility testing (AST) is urgently needed for timely and optimized choice of antibiotics, but current methods require days to obtain results. Here, we present a general AST protocol based on surface-enhanced Raman scattering (SERS-AST) for bacteremia caused by eight clinically relevant Gram-positive and Gram-negative pathogens treated with seven commonly administered antibiotics. Our results show that the SERS-AST protocol achieves a high level of agreement (96% for Gram-positive and 97% for Gram-negative bacteria) with the widely deployed VITEK 2 diagnostic system. The protocol requires only five hours to complete per blood-culture sample, making it a rapid and effective alternative to conventional methods. Our findings provide a solid foundation for the SERS-AST protocol as a promising approach to optimize the choice of antibiotics for specific bacteremia patients. This novel protocol has the potential to improve patient outcomes and reduce the spread of antibiotic resistance.

Staphylococcus taiwanensis sp. nov., isolated from human blood.

A novel coagulase-negative Staphylococcus strain (NTUH-S172T) was isolated from human blood culture in Taiwan with preliminary identification of Staphylococcus saprophyticus. 16S rRNA gene analysis and multilocus sequence analysis (MLSA) showed that NTUH-S172T was most closely related to Staphylococcus haemolyticus. The average nucleotide identity and digital DNA-DNA hybridization values with the whole genome sequence were <95 % and<70 % when compared to the related species. Strain NTUH-S172T could be distinguished from S. haemolyticus by urease production and from Staphylococcus borealis by nitrate reduction. In addition, the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) spectrum of NTHU-S172T was significantly different from that of S. haemolyticus, which could be used in clinical identification. In conclusion, it is proposed that this isolate represents a novel species, named Staphylococcus taiwanensis sp. nov., with type strain NTUH-S172T (=BCRC 81315T=JCM 34726T).

The phenomenon of staphylococcal interbacterial aggregate and net structures (SIAN) in community-associated methicillin-resistant Staphylococcus aureus CA-MRSA/J.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) usually causes skin and soft tissue infections (SSTIs), but occasionally causes invasive infections with a broad range of manifestations. Virulence factors and pathogenesis of CA-MRSA have been investigated in details with genotype ST8/SCCmecIVa (USA300), which first emerged in the United States. However, CA-MRSA evolves rapidly, with different clones dominating in different world regions; their pathogenesis remains unclear. CA-MRSA with genotype ST8/SCCmecIVl (CA-MRSA/J) emerged in 2003 in Japan, spreading widely with a fatal case. We have studied the genetic characteristics of CA-MRSA/J, and during the course of this study, we found that CA-MRSA/J has bacteriophage-like spikes with or without a hexagonal cap (spikes X and Xc). Here, we report that CA-MRSA/J strain NN55 has non-phage-like, one-µm-long/jerky spikes with or without a hexagonal cap (LSX/LSXc), and also that LSX/LSXc forms (staphylococcal) interbacterial aggregate/net structures (SIAN). Regarding the phenomenon of SIAN, NN55 first formed single short spike X, followed by multiple molecules of long and jerky LSX/LSXc, leading to the interbacterial construction of SIAN, in colonies with high cell densities. The LSX/LSXc and SIAN structures have not been reported in S. aureus. NN55 was invasive in a HEp-2 cell assay, exhibiting SIAN. The novel SIAN structures may be foci-skeletons or toxic aggregates in NN55's invasive infections. The phenomenon of SIAN suggests novel staphylococcal cell-surface dynamism, providing a new structure-and-function relationship model and advancing the understanding of CA-MRSA pathogenesis.

Structures of a highly variable cell-wall anchored protein-encoding the spj gene from ST8/SCCmecIVl community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA/J) isolated from 2003 onwards: An indicator of a strongly invasive pathotype.

The cell wall-anchored protein-encoding spj gene on staphylococcal cassette chromosome mec IVl (SCCmecIVl) was found to vary in size because of its 22- and 86-aa repeat domains. The 22-aa repeats are the more flexible of the two repeats, comprising three 11-aa units, and were classified into three groups with eleven types. The 11/22-aa repeats are longer in individuals with bullous impetigo, shorter in those with invasive disease and were absent in a fatal case, this last one having been rapidly diagnosed by PCR. IS431-flanking pUB110 (bleO, aadD) is present on SCCmecIVl at 90%. The bacterial surface has the spj product and a unique surface layer.

Molecular characterization of Streptococcus pneumoniae, particularly serotype19A/ST320, which emerged in Krasnoyarsk, Russia.

Streptococcus pneumoniae, a common human pathogen, colonizes the nasopharynx and causes diseases including acute otitis media (AOM). Herein, pneumococcal serotype distributions in children before and after PCV7 vaccination and in patients with pneumococcal disease in Siberian Russia (Krasnoyarsk) are reported. Analyses included antimicrobial susceptibility testing, sequence typing (ST), pulsed field gel electrophoresis, virulence-related surface protein gene (VSG) typing with novel primers and structural analysis by scanning electron microscopy. In healthy children (HC) prior to administration of PCV7, drug-susceptible serotype23F/ST1500 was a major pneumococcal genotype. In the PCV7 trial, multidrug-resistant serotype19A/ST320 emerged in vaccinees after PCV7, exhibiting a PCV7-induced serotype replacement. Multidrug-resistant serotype19A/ST320 was evident in patients with AOM. Community-acquired pneumonia (CAP) isolates showed genetic similarities to the AOM (ST320) genotype, constituting a common non-invasive AOM-CAP group. In contrast, meningitis isolates were more divergent. Overall, 25 ST types were identified; five (20%) of which were Krasnoyarsk-native. Regarding VSGs, PI-1 (rlrA/rrgB), PI-2 (pitA/B), psrP and cbpA were present at 54.3%, 38.6%, 48.6%, and 95.7%, respectively, with two major VSG content types, PI-1- /PI-2- /psrP+ /cbpA+ and PI-1+ /PI-2+ /psrP- /cbpA+ , being found for HC and non-invasive diseases, respectively. A major clone of serotype19A/ST320 (PI-1+ /PI-2+ ) produced the longest pneumococcal wire (pilus) structures in colonies. ST1016 (PI-1- /PI-2- ) in HC had HEp-2 cell-adherent pili. These results suggest that serotype19A/ST320 and related genotypes, with the VSG content type PI-1+ /PI-2+ /psrP- /cbpA+ , emerged in vaccinees after PCV7 in Siberia, accompanying diseases in non-vaccinated children, and that some genotypes (serotypes19A/ST320 and 18/ST1016) produced novel pneumococcal structures, predicting their roles in colony formation and adherence.

Distribution of Staphylococcal Cassette Chromosome (SCC) mec Element Types in Fusidic Acid-Resistant Staphylococcus epidermidis and Identification of a Novel SCC7684 Element.

We analyzed the staphylococcal cassette chromosome mec (SCCmec) types of 143 fusidic acid- and methicillin-resistant Staphylococcus epidermidis isolates. The most frequent SCCmec type was SCCmec III/SCCHg (53%), followed by SCCmec IV (29%). Clonal spreading of SCCmec III/SCCHg strains contributed to the increased prevalence of SCCmec III. A novel non-mec SCC structure, SCC7684, adjacent to SCCmec III, which carries a new ccrC allotype (ccrC3 allele 1) and contains heavy metal resistance genes, was identified in 14 isolates.

Novel Structure of Enterococcus faecium-Originated ermB-Positive Tn1546-Like Element in Staphylococcus aureus.

We determined the resistance determinants in 274 erythromycin-resistant methicillin-susceptible Staphylococcus aureus (MSSA) isolates during a 13-year period, 2000 to 2012. The resistance phenotypes, inducible macrolide-lincosamide-streptogramin (iMLS), constitutive MLS (cMLS), and macrolide-streptogramin (MS) resistance phenotypes, were examined by a double-disk diffusion D test. The ermB gene was more frequent (35%; 97/274) than ermC (27%; 75/274) or ermA (21%; 58/274). All 97 ermB-positive isolates harbored Tn551 and IS1216V The majority (89/97) of ermB-positive isolates displayed the cMLS phenotype and carried mobile element structure (MES)-like structures, which has been previously reported in sequence type 59 (ST59) methicillin-resistant S. aureus (MRSA). The remaining 8 ermB-carrying isolates, belonging to ST7 (n = 4), ST5 (n = 3), and ST59 (n = 1), were sasK intact and did not carry MES-like structures. Unlike a MES-like structure that was located on the chromosome, the ermB elements on sasK-intact isolates were located on plasmids by S1 nuclease pulsed-field gel electrophoresis (PFGE) analysis and conjugation tests. Sequence data for the ermB-containing region (14,566 bp) from ST59 NTUH_3874 revealed that the best match was a Tn1546-like element in plasmid pMCCL2 DNA (GenBank accession number AP009486) of Macrococcus caseolyticus Tn1546 is recognized as an enterococcal transposon and was known from the vancomycin resistance gene cluster in vancomycin-resistant Enterococcus (VRE). So far, acquisitions of Tn1546 in S. aureus have occurred in clonal complex 5 (CC5) MRSA, but not in MSSA. This is the first report that MSSA harbors an Enterococcus faecium-originated ermB-positive Tn1546-like element located on a plasmid.

Emergence of a small colony variant of vancomycin-intermediate Staphylococcus aureus in a patient with septic arthritis during long-term treatment with daptomycin.

OBJECTIVES: Small colony variants (SCVs) of Staphylococcus aureus are associated with persistent and drug-resistant infections. We demonstrated for the first time the emergence of SCVs in a patient with vancomycin-intermediate S. aureus (VISA) infection during long-term treatment with daptomycin. METHODS: A 73-year-old man with septic arthritis was infected with VISA. The patient was treated with daptomycin; however, the patient remained infected with VISA, with continuous isolation of VISA from his blood during long-term treatment. Five VISA isolates were characterized by: PFGE; genotyping including staphylococcal cassette chromosome mec (SCCmec), spa and MLST; antimicrobial susceptibility testing; and scanning and transmission electron microscopy. WGS and fatty acid analysis were also performed. RESULTS: The five VISA isolates were from a single clone of ST239/spa3(t037) and, of these, the first three were SCCmecIII positive and daptomycin susceptible, whereas the last two were SCCmecIII negative and daptomycin resistant and exhibited the characteristics of SCVs. The first and last isolates showed 13 remarkable genetic differences in SCCmec and the mprF, cls2, clpX and fabF genes. Of these, mutation of fabF (encoding the fatty acid synthase) seemed to be partially responsible for the slow growth and ultrastructural features, including an abnormal intercellular substance, and for the daptomycin resistance of SCVs. CONCLUSIONS: For the first time, we identified SCVs of VISA in a patient with septic arthritis during long-term treatment with daptomycin. Daptomycin-resistant SCVs of VISA were evolved in a stepwise manner and the mutation of fabF is likely responsible for the physical and ultrastructural characteristics and daptomycin resistance.

Evaluation of the Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry system for identification of blood isolates of Vibrio species.

Among 56 blood isolates of Vibrio species identified by sequencing analysis of 16S rRNA and rpoB genes, the Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system correctly identified all isolates of Vibrio vulnificus (n = 20), V. parahaemolyticus (n = 2), and V. fluvialis (n = 1) but none of the isolates of serogroup non-O1/O139 (non-serogroup O1, non-O139) V. cholerae (n = 33) to the species level. All of these serogroup non-O1/O139 V. cholerae isolates were correctly identified using the newly created MALDI-TOF MS database.

A novel fusidic acid resistance determinant, fusF, in Staphylococcus cohnii.

OBJECTIVES: To determine MICs of fusidic acid for and identify genetic determinants of resistance in Staphylococcus cohnii isolates. METHODS: Susceptibility to fusidic acid was determined by the standard agar dilution method in 24 S. cohnii subsp. urealyticus clinical isolates, 7 S. cohnii subsp. cohnii clinical isolates and 2 reference strains. Sequencing of a novel resistance determinant, fusF, and its flanking regions was performed by long and accurate PCR and inverse PCR. To evaluate the function of fusF, the MIC of fusidic acid was determined for recombinant Staphylococcus aureus carrying a plasmid expressing fusF. RESULTS: A total of 25 S. cohnii subsp. urealyticus (24 clinical isolates and 1 reference strain) and 2 S. cohnii subsp. cohnii displayed low-level resistance to fusidic acid (MICs 2-16 mg/L). Sequencing of a 4259 bp fragment from S. cohnii subsp. urealyticus ATCC 49330 revealed a novel resistance gene, designated fusF, which displayed 70.5% nucleotide and 67.3% amino acid identity to fusD. Expression of fusF in S. aureus confers resistance to fusidic acid. CONCLUSIONS: A novel FusB-family gene, fusF, was identified as a major resistance determinant in S. cohnii clinical isolates resistant to fusidic acid.

A novel staphylococcal cassette chromosomal element, SCCfusC, carrying fusC and speG in fusidic acid-resistant methicillin-resistant Staphylococcus aureus.

A high prevalence of fusC (16/46, 59%) was found in fusidic acid-resistant methicillin-resistant Staphylococcus aureus isolates collected from 2008 to 2010. Nucleotide sequencing of fusC and flanking regions revealed a novel staphylococcal cassette chromosome (SCC) structure, SCCfusC, which was integrated into rlmH and located upstream from SCCmec. The SCCfusC element contained speG, which may contribute to the polyamine resistance.

Evaluation of the Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry system for identification of blood isolates of Acinetobacter species.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (Bruker Biotyper) was able to accurately identify 98.6% (142/144) of Acinetobacter baumannii isolates, 72.4% (63/87) of A. nosocomialis isolates, and 97.6% (41/42) of A. pittii isolates. All Acinetobacter junii, A. ursingii, A. johnsonii, and A. radioresistens isolates (n = 28) could also be identified correctly by Bruker Biotyper.

Bruker biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry system for identification of Nocardia, Rhodococcus, Kocuria, Gordonia, Tsukamurella, and Listeria species.

We evaluated whether the Bruker Biotyper matrix-associated laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system provides accurate species-level identifications of 147 isolates of aerobically growing Gram-positive rods (GPRs). The bacterial isolates included Nocardia (n = 74), Listeria (n = 39), Kocuria (n = 15), Rhodococcus (n = 10), Gordonia (n = 7), and Tsukamurella (n = 2) species, which had all been identified by conventional methods, molecular methods, or both. In total, 89.7% of Listeria monocytogenes, 80% of Rhodococcus species, 26.7% of Kocuria species, and 14.9% of Nocardia species (n = 11, all N. nova and N. otitidiscaviarum) were correctly identified to the species level (score values, ≥ 2.0). A clustering analysis of spectra generated by the Bruker Biotyper identified six clusters of Nocardia species, i.e., cluster 1 (N. cyriacigeorgica), cluster 2 (N. brasiliensis), cluster 3 (N. farcinica), cluster 4 (N. puris), cluster 5 (N. asiatica), and cluster 6 (N. beijingensis), based on the six peaks generated by ClinProTools with the genetic algorithm, i.e., m/z 2,774.477 (cluster 1), m/z 5,389.792 (cluster 2), m/z 6,505.720 (cluster 3), m/z 5,428.795 (cluster 4), m/z 6,525.326 (cluster 5), and m/z 16,085.216 (cluster 6). Two clusters of L. monocytogenes spectra were also found according to the five peaks, i.e., m/z 5,594.85, m/z 6,184.39, and m/z 11,187.31, for cluster 1 (serotype 1/2a) and m/z 5,601.21 and m/z 11,199.33 for cluster 2 (serotypes 1/2b and 4b). The Bruker Biotyper system was unable to accurately identify Nocardia (except for N. nova and N. otitidiscaviarum), Tsukamurella, or Gordonia species. Continuous expansion of the MALDI-TOF MS databases to include more GPRs is necessary.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry can accurately differentiate Aeromonas dhakensis from A. hydrophila, A. caviae, and A. veronii.

Among 217 Aeromonas isolates identified by sequencing analysis of their rpoB genes, the accuracy rates of identification of A. dhakensis, A. hydrophila, A. veronii, and A. caviae were 96.7%, 90.0%, 96.7%, and 100.0%, respectively, by the cluster analysis of spectra generated by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Gemella parahaemolysans sp. nov. and Gemella taiwanensis sp. nov., isolated from human clinical specimens.

Four Gram-staining-positive, catalase-negative, coccoid isolates, designated NTUH_1465(T), NTUH_2196, NTUH_4957 and NTUH_5572(T), were isolated from human specimens. The four isolates displayed more than 99.6% 16S rRNA gene sequence similarity with Gemella haemolysans ATCC 10379(T), and 96.7 to 98.6% similarity with Gemella sanguinis ATCC 700632(T), Gemella morbillorum ATCC 27824(T) or Gemella cuniculi CCUG 42726(T). However, phylogenetic analysis of concatenated sequences of three housekeeping genes, groEL, rpoB and recA, suggested that the four isolates were distinct from G. haemolysans ATCC 10379(T) and other species. Isolates NTUH_2196, NTUH_4957 and NTUH_5572(T) clustered together and formed a stable monophyletic clade. DNA-DNA hybridization values among strains NTUH_1465(T) and NTUH_5572(T) and their phylogenetically related neighbours were all lower than 49%. The four isolates could be distinguished from G. haemolysans and other species by phenotypic characteristics. Based on the phylogenetic and phenotypic results, two novel species Gemella parahaemolysans sp. nov. (type strain NTUH_1465(T) = BCRC 80365(T) = JCM 18067(T)) and Gemella taiwanensis sp. nov. (type strain NTUH_5572(T) = BCRC 80366(T) = JCM 18066(T)) are proposed.

New structure of phage-related islands carrying fusB and a virulence gene in fusidic acid-resistant Staphylococcus epidermidis.

Nucleotide sequencing of the fusB-flanking regions in two fusidic acid-resistant Staphylococcus epidermidis isolates with the type IV aj1-leader peptide (LP)-fusB structure (lacking aj1) revealed that their fusB gene was located on novel phage-related islands inserted downstream of smpB and are here referred to as SeRIfusB-3692 and SePIfusB-857. The novel SePIfusB-857 structure was followed by SeCI857, forming a composite pathogenicity island which contained a putative virulence gene, vapE. The linkage of fusB and vapE may contribute to bacterial adaption.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry can accurately differentiate between Mycobacterium masilliense (M. abscessus subspecies bolletti) and M. abscessus (sensu stricto).

Among 36 Mycobacterium masilliense and 22 M. abscessus isolates identified by erm(41) PCR and sequencing analysis of rpoB and 23S rRNA genes, the rate of accurate differentiation between these two subspecies was 100% by cluster analysis of spectra generated by Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Comparison of the accuracy of matrix-assisted laser desorption ionization-time of flight mass spectrometry with that of other commercial identification systems for identifying Staphylococcus saprophyticus in urine.

Among 30 urinary isolates of Staphylococcus saprophyticus identified by sequencing methods, the rate of accurate identification was 100% for Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), 86.7% for the Phoenix PID and Vitek 2 GP systems, 93.3% for the MicroScan GP33 system, and 46.7% for the BBL CHROMagar Orientation system.

Applicability of an in-house extraction protocol in a Bruker Biotyper matrix-assisted laser desorption/ionization time-of-flight mass spectrometry system for the identification of Streptococcus agalactiae from broth-enriched vaginal/rectal swab specimens.

BACKGROUND AND PURPOSE: Early laboratory identification of group B Streptococcus (GBS, Streptococcus agalactiae) in the birth canal of pregnant women is critical for prompt administration of antimicrobial therapy and may further reduce the mortality rate due to GBS neonatal infection. METHODS: A total of 164 vaginal/rectal swab specimens collected from pregnant women at 35-37 weeks of gestation were screened for GBS vaginal colonization. The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS, Bruker Biotyper, Bruker Daltonik GmbH, Bremen, Germany) system was used to detect GBS from Carrot broth and LIM broth enrichment using an in-house extraction protocol. The results were compared to those by conventional broth-enriched culture/identification methods as the gold standard. BD MAX™ GBS assay (Becton Dickinson, Sparks, MD, USA) was also performed for Carrot broth-enriched specimen. Discordant results were investigated using the GeneXpert® GBS PCR assay (Cepheid Inc., Sunnyvale, CA, USA). RESULTS: Using the extraction protocol, 33 (20.1%) of the 164 specimens were positive in Carrot broth, and 19 (11.6%) were positive in LIM broth. Using the culture protocol, 38 (23.2%) samples in Carrot broth and 35 (21.3%) in LIM broth were positive. The sensitivity, specificity, and positive and negative predictive values using the extraction protocol in Carrot broth and LIM broth compared to the gold standard conventional culture/identification method were 86.8% and 50.0%, 100% and 100%, 100% and 100%, and 96.2% and 86.9%, respectively. CONCLUSIONS: The extraction protocol with MALDI-TOF MS from Carrot broth-enriched samples provides a more rapid turnaround time, lower cost, and acceptable sensitivity and specificity to correctly identify pathogens when compared to conventional culture/identification methods.