Staphylococcus hominis subspecies can be identified by SDS-PAGE or MALDI-TOF MS profiles.
ABSTRACT: Staphylococcus hominis is part of the normal human microbiome. Two subspecies, S. hominis hominis (Shh) and S. hominis novobiosepticus (Shn), have clinical significance. Forty-nine S. hominis isolates were analyzed by the MicroScan automated system, SDS-PAGE and MALDI-TOF methods, followed by partial sequencing of the 16S rDNA gene. The trehalose fermentation test, disk diffusion and broth microdilution tests were used to identify (novobiocin test) and access the susceptibility to oxacillin and vancomycin of isolates. The SCCmec elements and genomic diversity were evaluated by PCR and PFGE methods, respectively. Profiles of 28 (57%; 8 Shh and 20 Shn) isolates corroborated with the results found in all the applied methods of identification. The remaining 21 (43%) isolates were phenotypically identified as Shh by MicroScan; however, they were identified as Shn by SDS-PAGE and mass spectral, and confirmed by 16S rDNA sequencing. Among 41 isolates identified as Shn by the molecular and mass spectrometry methods, 19 (41%) were novobiocin-sensitive, and the trehalose test indicated 11 positive isolates, which are considered atypical phenotypic results for this subspecies. In addition, 92.7% of the isolates identified as Shn by these methods carried mecA gene, while only 12.5% of the Shh isolates were positive. Together, the results highlighted the SDS-PAGE and MALDI-TOF MS methods as promising tools for discriminating S. hominis subspecies.
Project description:From 2002 to 2003, 32 isolates of Staphylococcus hominis subsp. novobiosepticus (SHN) were recovered from 21 patients, 18 of whom were neonates, with 13 considered to have late-onset SHN sepsis. All isolates from neonates had an indistinguishable pulsed-field gel electrophoresis pattern. Our data support SHN as an important nosocomial pathogen in neonates.
Project description:Staphylococcus hominis is a commensal resident of human skin and an opportunistic pathogen. The species is subdivided into two subspecies, S. hominis subsp. hominis and S. hominis subsp. novobiosepticus, which are difficult to distinguish. To investigate the evolution and epidemiology of S. hominis, a total of 108 isolates collected from 10 countries over 40 years were characterized by classical phenotypic methods and genetic methods. One nonsynonymous mutation in gyrB, scored with a novel SNP typing assay, had a perfect association with the novobiocin-resistant phenotype. A multilocus sequence typing (MLST) scheme was developed from six housekeeping gene fragments, and revealed relatively high levels of genetic diversity and a significant impact of recombination on S. hominis population structure. Among the 40 sequence types (STs) identified by MLST, three STs (ST2, ST16 and ST23) were S. hominis subsp. novobiosepticus, and they distinguished between isolates from different outbreaks, whereas 37 other STs were S. hominis subsp. hominis, one of which was widely disseminated (ST1). A modified PCR assay was developed to detect the presence of ccrAB4 from the SCCmec genetic element. S. hominis subsp. novobiosepticus isolates were oxacillin-resistant and carriers of specific components of SCCmec (mecA class A, ccrAB3, ccrAB4, ccrC), whereas S. hominis subsp. hominis included both oxacillin-sensitive and -resistant isolates and a more diverse array of SCCmec components. Surprisingly, phylogenetic analyses indicated that S. hominis subsp. novobiosepticus may be a polyphyletic and, hence, artificial taxon. In summary, these results revealed the genetic diversity of S. hominis, the identities of outbreak-causing clones, and the evolutionary relationships between subspecies and clones. The pathogenic lifestyle attributed to S. hominis subsp. novobiosepticus may have originated on more than one occasion.
Project description:Ninety-two strains of lactic acid bacteria (LAB) were isolated from a Malaysian food ingredient, chili bo, stored for up to 25 days at 28 degreesC with no benzoic acid (product A) or with 7,000 mg of benzoic acid kg-1 (product B). The strains were divided into eight groups by traditional phenotypic tests. A total of 43 strains were selected for comparison of their sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) whole-cell protein patterns with a SDS-PAGE database of LAB. Isolates from product A were identified as Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus farciminis, Pediococcus acidilactici, Enterococcus faecalis, and Weissella confusa. Five strains belonging to clusters which could not be allocated to existing species by SDS-PAGE were further identified by 16S rRNA sequence comparison. One strain was distantly related to the Lactobacillus casei/Pediococcus group. Two strains were related to Weissella at the genus or species level. Two other strains did not belong to any previously described 16S rRNA group of LAB and occupied an intermediate position between the L. casei/Pediococcus group and the Weissella group and species of Carnobacterium. The latter two strains belong to the cluster of LAB that predominated in product B. The incidence of new species and subspecies of LAB in chili bo indicate the high probability of isolation of new LAB from certain Southeast Asian foods. None of the isolates exhibited bacteriocin activity against L. plantarum ATCC 14917 and LMG 17682.
Project description:Synthetic nitrite is considered an undesirable preservative for meat products; thus, controlling synthetic nitrite concentrations is important from the standpoint of food safety. We investigated 1,000 species of microorganisms from various kimchi preparations for their potential use as a starter culture for the production of nitrites. We used 16S rRNA gene sequence analysis to select a starter culture with excellent nitrite and nitric oxide productivity, which we subsequently identified as Staphylococcus hominis subspecies hominis WiKim0113. That starter culture was grown in NaCl (up to 9%; w/v) at 10°C-40°C; its optimum growth was observed at 30°C at pH 4.0-10.0. It exhibited nonproteolytic activity and antibacterial activity against Clostridium perfringens, a bacterium that causes food poisoning symptoms. Analysis of Staphylococcus hominis subspecies hominis WiKim0113 with an API ZYM system did not reveal the presence of ?-glucuronidase, and tests of the starter culture on 5% (v/v) sheep blood agar showed no hemolytic activity. Our results demonstrated the remarkable stability of coagulase-negative Staphylococcus hominis subspecies hominis WiKim0113, especially in strain negative for staphylococcal enterotoxins and sensitive to clinically relevant antibiotics. Moreover, Staphylococcus hominis subspecies hominis WiKim0113 exhibited a 45.5% conversion rate of nitrate to nitrite, with nitrate levels reduced to 25% after 36 h of culturing in the minimal medium supplemented with nitrate (200 ppm). The results clearly demonstrated the safety and utility of Staphylococcus hominis subspecies hominis WiKim0113, and therefore its suitability as a starter culture.
Project description:To detect the outer membrane protein (OMP), which plays a key role in carbapenem resistance, whole-genome and transcriptome analysis of the clinical carbapenem-resistant Klebsiella pneumoniae was carried out. The index strain lacked both OmpK35 and OmpK36, whereas the other strains lacked only OmpK35. After SDS-PAGE, the putative OMP bands were excised and identified as OmpA and OmpK36. MALDI-TOF MS showed peaks at ?36 and ?38 kDa that corresponded to OmpA and OmpK36, respectively. In all the strains except YMC2014/03/P345, the ?38 kDa peaks were present. The K. pneumoniae ATCC 13883 isolate showed three bands on SDS-PAGE and three corresponding peaks on MALDI-TOF MS. The additional third peak at ?37 kDa corresponding to OmpK35 was observed. To verify OmpK35 peak detection in other K. pneumoniae isolates by MALDI-TOF MS, we analyzed six strains from our laboratory's strain bank. Whole genome sequence indicated that only two isolates had intact OmpK35. Both MALDI-TOF MS and SDS-PAGE did not show a ?37 kDa peak or an OmpK35 band as observed in the K. pneumoniae ATCC 13883 isolate. Separation using SDS-PAGE showed a single peak representing OmpA. Therefore, both SDS-PAGE and MALDI-TOF MS were not completely reliable for OMP detection because they fail to detect OmpK35. To the best of our knowledge, this is the first report on the performance of SDS-PAGE and MALDI-TOF MS for the detection of OMP's using whole-genome and RNA sequencing analyses.
Project description:The genotyping of numerous isolates of Cryptosporidium parasites has led to the definition of new species and a better understanding of the epidemiology of cryptosporidiosis. A single-locus genotyping method based on the partial sequence of a polymorphic sporozoite surface glycoprotein gene (GP60) has been favored by many for surveying Cryptosporidium parvum and C. hominis populations. Since genetically distinct Cryptosporidium parasites recombine in nature, it is unclear whether single-locus classifications can adequately represent intraspecies diversity. To address this question, we investigated whether multilocus genotypes of C. parvum and C. hominis cluster according to the GP60 genotype. C. hominis multilocus genotypes did not segregate according to this marker, indicating that for this species the GP60 sequence is not a valid surrogate for multilocus typing methods. In contrast, in C. parvum the previously described "anthroponotic" genotype was confirmed as a genetically distinct subspecies cluster characterized by a diagnostic GP60 allele. However, as in C. hominis, several C. parvum GP60 alleles did not correlate with distinct subpopulations. Given the rarity of some C. parvum GP60 alleles in our sample, the existence of additional C. parvum subgroups with unique GP60 alleles cannot be ruled out. We conclude that with the exception of genotypically distinct C. parvum subgroups, multilocus genotyping methods are needed to characterize C. parvum and C. hominis populations. Unless parasite virulence is controlled at the GP60 locus, attempts to find associations within species or subspecies between GP60 and phenotype are unlikely to be successful.
Project description:Four colistin susceptibility testing methods were compared with the standard broth microdilution (BMD) in a collection of 75 colistin-susceptible and 75 <i>mcr</i>-positive <i>E. coli</i>, including ST131 isolates. Taking BMD as reference, all methods showed similar categorical agreement rates (CA) of circa 90%, and a low number of very major errors (VME) (0% for the MicroScan system and Etest<sup>®</sup>, 0.7% for UMIC<sup>®</sup>), except for the disc diffusion assay (breakpoint ? 11 mm), which yielded false-susceptible results for 8% of isolates. Of note is the number of <i>mcr</i>-positive isolates (17.3%) categorized as susceptible (?2 mg/L) by the BMD method, but as resistant by the MicroScan system. ST131 <i>mcr</i>-positive <i>E. coli</i> were identified as colistin-resistant by all MIC-based methods. Our results show that applying the current clinical cut-off (>2 mg/L), many <i>mcr</i>-positive <i>E. coli</i> remain undetected, while applying a threshold of >1 mg/L the sensitivity of detection increases significantly without loss of specificity. We propose two possible workflows, both starting with the MicroScan system, since it is automated and, importantly, it categorized all <i>mcr</i>-positive isolates as colistin-resistant. MicroScan should be followed by either BMD or MIC-based commercial methods for colistin resistance detection; or, alternatively, MicroScan, followed by PCR for the <i>mcr</i> screening.
Project description:The rapid and cost-efficient determination of carbapenem resistance is an important prerequisite for the choice of an adequate antibiotic therapy. A MALDI-TOF MS-based assay was set up to detect porins in the current study. A loss of the components of porin alone such as OmpK35/OmpK36 or together with the production of carbapenemases will augment the carbapenem resistance. Ten strains of Escherichia coli and eight strains of Klebsiella pneumoniae were conducted for both sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and MALDI-TOF MS analysis. MALDI-TOF/TOF MS analysis was then performed to verify the correspondence of proteins between SDS-PAGE and MALDI-TOF MS. The results indicated that the mass spectrum of ca. 35,000, 37,000, and 38,000-m/z peaks of E. coli ATCC 25922 corresponded to OmpA, OmpC, and OmpF with molecular weight of approximately ca. 38, 40, and 41 kDa in SDS-PAGE gel, respectively. The band of OmpC and OmpF porins were unable to be distinguished by SDS-PAGE, whereas it was easy to be differentiated by MALDI-TOF MS. As for K. pneumoniae isolates, the mass spectrum of ca. 36,000 and 38,600-m/z peaks was observed corresponding to OmpA and OmpK36 with molecular weight of approximately ca. 40 and 42 kDa in SDS-PAGE gel, respectively. Porin OmpK35 was not observed in the current SDS-PAGE, while a 37,000-m/z peak was found in K. pneumoniae ATCC 13883 and carbapenem-susceptible strains by MALDI-TOF MS which was presumed to be the characteristic peak of the OmpK35 porin. Compared with SDS-PAGE, MALDI-TOF MS is able to rapidly identify the porin-deficient strains within half an hour with better sensitivity, less cost, and is easier to operate and has less interference.
Project description:Endophytic bacteria play an important role in agriculture by improving plant performance and adaptation against biotic and abiotic stresses. In the present study molecular methods were used for identifying Bacillus endophytic bacteria isolated from Brazilian sweet corn. SDS-PAGE of whole-cell protein extract of forty-two isolates revealed a high number of scrutinable bands. Twenty-four isolates were identified in nine different groups of duplicated bacteria and eighteen were identified as unique. Some high-accumulated polipeptides with variable length were observed in almost isolates. Partial sequencing of 16S ribosomal gene revealed that all isolates are Bacillus sp. and among thirteen isolates with similar protein profiles, two were different strains. Among the forty-two isolates identified by rDNA sequencing, Bacillus subitilis and B. pumilus were the most frequenty species (15 and 12 isolates, respectively) followed by B. licheniformes (7 isolates), B. cereus (5 isolates) and B. amiloliquefascens (3 isolates). According to present results, SDS-PAGE technique could be used as a fast and cheap first tool for identifying inter-specific variation in maize endophytic bacterial collections while rDNA sequencing could be applied for analyzing intra-specific variation among isolates with similar protein profile as well as for taxonomic studies.
Project description:Bacterial identification is challenging in low-resource settings (LRS). We evaluated the MicroScan identification panels (Beckman Coulter, Brea, CA, USA) as part of Médecins Sans Frontières' Mini-lab Project. The MicroScan Dried Overnight Positive ID Type 3 (PID3) panels for Gram-positive organisms and Dried Overnight Negative ID Type 2 (NID2) panels for Gram-negative organisms were assessed with 367 clinical isolates from LRS. Robustness was studied by inoculating Gram-negative species on the Gram-positive panel and vice versa. The ease of use of the panels and readability of the instructions for use (IFU) were evaluated. Of species represented in the MicroScan database, 94.6% (185/195) of Gram-negative and 85.9% (110/128) of Gram-positive isolates were correctly identified up to species level. Of species not represented in the database (e.g., <i>Streptococcus suis</i> and <i>Bacillus</i> spp.), 53.1% out of 49 isolates were incorrectly identified as non-related bacterial species. Testing of Gram-positive isolates on Gram-negative panels and vice versa (<i>n</i> = 144) resulted in incorrect identifications for 38.2% of tested isolates. The readability level of the IFU was considered too high for LRS. Inoculation of the panels was favorably evaluated, whereas the visual reading of the panels was considered error-prone. In conclusion, the accuracy of the MicroScan identification panels was excellent for Gram-negative species and good for Gram-positive species. Improvements in stability, robustness, and ease of use have been identified to assure adaptation to LRS constraints.