Comparison of CRISPR typing and conventional molecular methods for distinguishing Laribacter hongkongensis isolates from fish, frogs and humans.

High-resolution and efficient typing for Laribacter hongkongensis (L. hongkongensis) is essential for epidemiological investigation of such emerging foodborne pathogens. Clustered regularly interspaced short palindromic repeats (CRISPR) typing is an innovative molecular method that shows great promise for L. hongkongensis typing. Here, we explored the CRISPR typing method by combining CRISPR1 and CRISPR2 loci to characterize a collection of 109 L. hongkongensis isolates from humans and animals and compared it to current molecular methods such as pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The results showed that all three methods have high discriminatory power (diversity index was 0.9902 for PFGE, 0.9663 for CRISPR and 0.9562 for MLST); strong congruence was observed between them (Rand index was 0.969 between CRISPR and PFGE, 0.953 between CRISPR and MLST, 0.958 between PFGE and MLST). CRISPR typing could well distinguish the isolates in the same STs or PFGE profiles, and the genetic information contained by the CRISPR array is useful for deep phylogenetic typing. We demonstrate that rapid CRISPR typing is a practical genetic fingerprinting tool with high resolution, comparable ease of use and lower cost, ability to track the source of various groups of L. hongkongensis strains and indication of genetic characteristics.

Whole genome-based antimicrobial resistance, virulence, and phylogenetic characteristics of Trueperella pyogenes clinical isolates from humans and animals.

Trueperella pyogenes is an opportunistic zoonotic bacterial pathogen, whose antimicrobial resistance, virulence, and genetic relatedness between strains from animals and humans are barely studied. These characteristics were therefore analyzed for clinical T. pyogenes strains from 31 animals of 11 different species and 8 humans determining their complete circular genome sequence and antimicrobial susceptibility. The MICs of 19 antimicrobials including 3 antiseptics correlated to the resistance genes identified in silico within the genomes revealing a predominance of resistance to streptomycin (aadA9), sulfamethoxazole (sul1), and tetracycline (tet(33), tet(W/N/W)) among strains from humans and cattle. Additional resistance genes (erm(X), erm(56), cmx, drfA1, aadA1, aph(3'')-Ib (strA), aph(6)-Id (strB), aac(3)-IVa, aph(4)-Ia) were found only sporadically. The resistance genes were localized on genetic elements integrated into the chromosome. A cgMLST-based phylogenetic analysis revealed two major clusters each containing genetically diverse strains. The human strains showed the closest relatedness to strains from cattle. Virulence genes coding for fimbriae (fimA, fimC), neuroamidase (nanP, nanH), pyolysin (plo), and collagen binding protein (cbpA) were identified in strains from different hosts, but no correlation was observed between virulence factors and strain origin. The existence of resistance genes typically found in Gram-negative bacteria within the Gram-positive T. pyogenes indicates a wider capacity to adapt to antimicrobial selective pressure. Moreover, the presence of similar antimicrobial resistance profiles found in cattle and human strains as well as their closest relatedness suggests common zoonotic features and cattle as the potential source for human infections.

Antimicrobial efficiency of bromhexine hydrochloride against endometritis-causing Escherichia coli and Trueperella pyogenes in bovines.

In this study, the main agents associated with endometritis in cows in the state of Santa Catarina, Brazil, were identified and the resistance profile and virulence mechanisms of the bacterial isolates were evaluated. Isolates of Escherichia coli and Trueperella pyogenes were tested for their biofilm forming ability and the antimicrobial action of bromhexine hydrochloride in combination with other antimicrobials. A total of 37 uterine lavage samples were collected from cows with endometritis. Of the 55 bacteria isolated, 25.4% were identified as T. pyogenes and 16.3% as E. coli. The bacterial isolates showed greater resistance to sulfamethoxazole + trimethoprim (58.2%) and tetracycline (56.3%). Among the species, E. coli showed the highest resistance rates, with 100% of isolates showing resistance to amoxicillin, streptomycin, and gentamicin. The results of the minimum inhibitory concentration for the T. pyogenes isolates showed that 91.6% of the isolates were resistant to enrofloxacin and tetracycline, and 75% were resistant to ceftiofur and sulfamethoxazole + trimethoprim. All E. coli and T. pyogenes isolates showed biofilm forming ability. The plo, fimA, and nanH genes were identified in 100% of T. pyogenes isolates. In parallel, 100% of E. coli isolates had the fimH gene, and 11.1% had the csgD gene. Bromhexine hydrochloride showed antimicrobial activity against 100% of E. coli isolates and 66.6% of T. pyogenes isolates. Furthermore, when associated with antimicrobials, bromhexine hydrochloride has a synergistic and additive effect, proving to be an option in the treatment of endometritis in cows and an alternative for reducing the use of antimicrobials.

Multiphasic investigations imply transfer of orange-/red-pigmented strains of the bean pathogen Curtobacterium flaccumfaciens pv. flaccumfaciens to a new species as C. aurantiacum sp. nov., elevation of the poinsettia pathogen C. flaccumfaciens pv. poinsettiae to the species level as C. Poinsettiae sp. nov., and synonymy of C. albidum with C. citreum.

Curtobacterium flaccumfaciens (Microbacteriaceae), a plant-pathogenic coryneform species includes five pathovars with valid names and a number of proposed - but unvalidated - new members. In this study, phenotypic features and DNA similarity indexes were investigated among all C. flaccumfaciens members. Results showed that the C. flaccumfaciens pv. poinsettiae strains causing bacterial canker of Euphorbia pulcherrima in the USA as well as the orange-/red-pigmented strains of C. flaccumfaciens pv. flaccumfaciens pathogenic on dry beans in Iran are too distinct from each other and from the type strain of the species to be considered members of C. flaccumfaciens. Hence, the latter two groups were elevated at the species level as C. poinsettiae sp. nov. (ATCC 9682T = CFBP 2403T = ICMP 2566T = LMG 3715T = NCPPB 854T as type strain), and C. aurantiacum sp. nov. (50RT = CFBP 8819T = ICMP 22071T as type strain). Within the emended species C. flaccumfaciens comb. nov., yellow-pigmented strains causing bacterial wilt of dry beans and those causing bacterial canker of Euphorbia pulcherrima in Europe were retained as C. flaccumfaciens pv. flaccumfaciens and C. flaccumfaciens pv. poinsettiae, respectively; while taxonomic position of the sugar beet pathogen C. flaccumfaciens pv. beticola ATCC BAA144PT was confirmed. The newly described onion pathogen C. allii was also reclassified as C. flaccumfaciens pv. allii with the pathotype strain LMG 32517PT. Furthermore, C. flaccumfaciens pv. basellae causing bacterial leaf spot of malabar spinach (Basella rubra) was transferred to C. citreum pv. basellae with ATCC BAA143PT as pathotype.

Nanchangia anserum gen. nov., sp. nov., isolated from feces of greater white-fronted geese (Anser albifrons).

Two rod-shaped and Gram-stain-positive bacteria (strains C64T and C62) were isolated in 2020 from faeces of greater white-fronted geese (Anser albifrons) from Poyang Lake, PR China. Their optimal growth conditions were at 37 °C, pH 7.0 and with 0.5 % (w/v) NaCl. The two isolates showed a highest 16S rRNA gene sequence similarity to Bowdeniella nasicola DSM 19116T (92.1 %). Phylogenetic/phylogenomic analyses indicated that strains C64T and C62 clustered independently in the vicinity of the genera Varibaculum, Winkia and Mobiluncus within the family Actinomycetaceae, but could not be classified clearly as members of any of these known genera. The average amino acid identity values between our isolates and available genomes of members of the family Actinomycetaceae were around the genus threshold value (45-65 %). The major cellular fatty acids of the strains were C18 : 1ω9c and C16 : 0. The predominant polar lipids were phosphatidylinositol, phosphatidylglycerol, phosphatidylcholine, diacylglycerol, triacylglycerol and cardiolipin. The amino acid composition of peptidoglycan contained alanine, glutamic acid and glycine. The major respiratory menaquinones were MK-8(H4) and MK-9(H4). The whole cell sugars included galactose, arabinose and glucose. On the basis of the results of the 16S rRNA gene sequences comparison, whole-genome phylogenomic analysis, phenotypic and chemotaxonomic characteristics, we propose that strains C64T and C62 represent a novel species belonging to a novel genus within the family Actinomycetaceae, for which the name Nanchangia anserum gen. nov., sp. nov. is proposed. The type strain is Nanchangia anserum C64T (=CGMCC 1.18410T=GDMCC 1.1969T=KCTC 49511T=KACC 22143T).

Trueperella pecoris sp. nov. isolated from bovine and porcine specimens.

A novel Gram-stain-positive bacterium was isolated from a purulent bovine milk sample, the bovine placenta from an abortion, the udder secretion of a heifer and the lung of a pig that had succumbed from suppurative bronchopneumonia in Switzerland from 2015 to 2019. The strains grew best under aerobic conditions with 5 % CO2 and colonies were non-haemolytic and greyish-white. They were non-motile and negative for catalase and oxidase. The genomes of the four strains 19M2397T, 15A0121, 15IMD0307 and 19OD0592 were obtained by sequencing. The results of phylogenetic analyses based on the 16S rRNA gene grouped them within the genus Trueperella in the family Arcanobacteriaceae. The genomes had DNA G+C contents of 61.2-62.2 mol% and showed digital DNA-DNA hybridization (dDDH) values of 21.4-22.8 % and average nucleotide identity (ANI) values of approximately 77 % to their closest relatives Trueperella pyogenes and Trueperella bernardiae. With respect to the presence in different livestock species we propose the name Trueperella pecoris sp. nov. The type strain is 19M2397T (=CCOS 1952T=DSM 111392T), isolated from the udder secretion of a heifer diagnosed with summer mastitis in 2019.

Adapted Protocol for Saccharibacteria Cocultivation: Two New Members Join the Club of Candidate Phyla Radiation.

The growing application of metagenomics to different ecological and microbiome niches in recent years has enhanced our knowledge of global microbial biodiversity. Among these abundant and widespread microbes, the candidate phyla radiation (CPR) group has been recognized as representing a large proportion of the microbial kingdom (>26%). CPR are characterized by their obligate symbiotic or exoparasitic activity with other microbial hosts, mainly bacteria. Currently, isolating CPR is still considered challenging for microbiologists. The idea of this study was to develop an adapted protocol for the coculture of CPR with a suitable bacterial host. Based on various sputum samples, we tried to enrich CPR (Saccharibacteria members) and to cocultivate them with pure hosts (Schaalia odontolytica). This protocol was monitored by TaqMan real-time quantitative PCR (qPCR) using a system specific for Saccharibacteria designed in this study, as well as by electron microscopy and sequencing. We succeeded in coculturing and sequencing the complete genomes of two new Saccharibacteria species, "Candidatus Minimicrobia naudis" and "Candidatus Minimicrobia vallesae." In addition, we noticed a decrease in the CT values of Saccharibacteria and a significant multiplication through their physical association with Schaalia odontolytica strains in the enriched medium that we developed. This work may help bridge gaps in the genomic database by providing new CPR members, and in the future, their currently unknown characteristics may be revealed. IMPORTANCE In this study, the first TaqMan real-time quantitative PCR (qPCR) system, targeting Saccharibacteria phylum, has been developed. This technique can specifically quantify Saccharibacteria members in any sample of interest in order to investigate their prevalence. In addition, another easy, specific, and sensitive protocol has been developed to maintain the viability of Saccharibacteria cells in an enriched medium with their bacterial host. The use of this protocol facilitates subsequent studies of the phenotypic characteristics of CPR and their physical interactions with bacterial species, as well as the sequencing of new genomes to improve the current database.

Flaviflexus ciconiae sp. nov., isolated from the faeces of the oriental stork, Ciconia boyciana.

A novel Gram-stain-positive, non-motile, non-spore-forming, coccobacillus-shaped, strictly aerobic bacterium, designated strain H23T48T, was isolated from the faecal sample of an oriental stork collected from the Seoul Grand Park Zoo in Seoul, Republic of Korea. Optimal growth of strain H23T48T was observed at 30-37 °C, pH 8 and with 3 % (w/v) NaCl. 16S rRNA gene sequence-based phylogenetic analysis revealed that strain H23T48T was closely related to the genus Flaviflexus, with 97.0 and 96.7 % sequence similarities to Flaviflexus salsibiostraticola EBR4-1-2T and Flaviflexus huanghaiensis H5T, respectively. Strain H23T48T possessed MK-9(H4) as the major menaquinone and C16 : 0 (42.4 %), C18 : 1 ω9c (31.3 %) and C14 : 0 (17.7 %) as the major cellular fatty acids. The polar lipids included phosphatidylglycerol, two unidentified lipids, six unidentified phospholipids and two unidentified glycophospholipids. The amino acid composition of the cell-wall peptidoglycan was l-alanine, l-lysine, d-glutamic acid, l-aspartic acid and glycine. The genomic G+C content of strain H23T48T is 59.5 mol% and the average nucleotide identity value between H23T48T and F. salsibiostraticola KCT C33148T (=EBR4-1-2T) is 75.5 %. Based on the obtained data, strain H23T48T represents a novel species of the genus Flaviflexus, for which the name Flaviflexus ciconiae sp. nov. is proposed. The type strain is H23T48T (=KCTC 49253T=JCM 33282T).

Studies on Trueperella pyogenes isolated from an okapi (Okapia johnstoni) and a royal python (Python regius).

BACKGROUND: The present study was designed to characterize phenotypically and genotypically two Trueperella pyogenes strains isolated from an okapi (Okapia johnstoni) and a royal python (Python regius). CASE PRESENTATION: The species identity could be confirmed by phenotypic properties, by MALDI-TOF MS analysis and by detection of T. pyogenes chaperonin-encoding gene cpn60 with a previously developed loop-mediated isothermal amplification (LAMP) assay. Furthermore, sequencing of the 16S ribosomal RNA (rRNA) gene, the 16S-23S rDNA intergenic spacer region (ISR), the target genes rpoB encoding the ß-subunit of bacterial RNA polymerase, tuf encoding elongation factor tu and plo encoding the putative virulence factor pyolysin allowed the identification of both T. pyogenes isolates at species level. CONCLUSIONS: Both strains could be clearly identified as T. pyogenes. The T. pyogenes strain isolated in high number from the vaginal discharge of an okapi seems to be of importance for the infectious process; the T. pyogenes strain from the royal python could be isolated from an apparently non-infectious process. However, both strains represent the first isolation of T. pyogenes from these animal species.

Antimicrobial susceptibility of Trueperella pyogenes isolated from food-producing ruminants.

A total of 96 Trueperella pyogenes isolates, an opportunistic pathogen of food-producing ruminants, obtained from cattle (n = 34), sheep (n = 35) and goats (n = 27), and identified by Real Time PCR (qPCR), were analysed to determine the susceptibility to 12 antimicrobials commonly used in livestock, using a broth microdilution. The Minimal Inhibitory Concentration (MIC) distribution was unimodal for half of the antimicrobials tested with the exception of apramycin, gentamicin, streptomycin, oxytetracycline, tylosin, and erythromycin all of which showed bimodal MIC distributions. Low MIC90 values for penicillin, amoxicillin, ceftiofur, enrofloxacin, and gentamicin (<1 µg/ml) were obtained, suggesting that these antimicrobials would be the most effective first line empiric treatment for T. pyogenes infections in livestock. Furthermore, according to the specific T. pyogenes breakpoints for penicillin, sulfamethoxazole/trimethoprim and erythromycin, 93.7 % of isolates were susceptible to penicillin and 77.2 % to erythromycin, whereas 92.7 % were non-susceptible to sulfamethoxazole/trimethoprim. Significant differences were observed in the MIC distribution of almost all antimicrobials, except enrofloxacin, tylosin and erythromycin against cattle, sheep or goat isolates, although all antimicrobials showed similar MIC90 values, except apramycin and oxytetracycline that showed higher values when tested against cattle isolates. These data provide interesting information on the antimicrobials of choice for the treatment of infections caused by T. pyogenes in ruminants.

Development of a novel Trueperella pyogenes-specific PCR assay.

Trueperella pyogenes is an opportunistic pathogen that causes a wide variety of purulent infections. We recently isolated a T. pyogenes strain unable to be identified by the previously reported T. pyogenes pyolysin gene (plo)-specific PCR from the lung of a sheep with astasia. Sequence comparison of plo among representative strains revealed several nucleotide substitutions in the primer-annealing regions. As such substitutions were considered to be a reason for the low PCR specificity, we designed novel primers in conserved regions of plo. Under optimized conditions, the novel primers precisely identified all T. pyogenes strains tested, and no products were generated from any other bacterial strains, suggesting the usefulness of the novel PCR assay for the diagnosis of T. pyogenes infections.

Epidemiological analysis of Trueperella abortisuis isolated from cases of pig abortion of a single farm.

The present study was designed to characterize six Trueperella (T.) abortisuis strains, cultured over a period of 5 months from fetus and abortion material of six pigs of a single farm in Mecklenburg-West Pomerania federal state, Germany. It was of interest to investigate the epidemiological relationships of the six strains among each other and whether a single bacterial clone was responsible for the abortion situation of the single farm. All six strains were identified phenotypically, by MALDI-TOF MS analysis and by phylogenetic analysis based on 16S rRNA gene and gap (encoding the glyceraldehyde 3-phosphate dehydrogenase) and tuf (encoding elongation factor tu) gene sequencing. Further genotypic comparison was performed using different genomic DNA fingerprint methods including BOX-PCR, (GTG)5-PCR, and three RAPD-PCRs. The sequence analysis of the genes gap and tuf and the genomic DNA fingerprinting results revealed, as noval findings, that the six T. abortisuis strains cultured from a single farm represent six different bacterial clones showing a genetic variability of this bacterial species in the pig population. All six T. abortisuis strains were isolated in mixed culture with several other bacterial species. However, the T. abortisuis strain, generally found in high numbers, seemed to be responsible for the abortion situation in the farm.

Fudania jinshanensis gen. nov., sp. nov., isolated from faeces of the Tibetan antelope (Pantholops hodgsonii) in China.

Two hitherto unknown bacteria (strains 313T and 352) were recovered from the faeces of Tibetan antelopes on the Tibet-Qinghai Plateau, PR China. Cells were rod-shaped and Gram-stain-positive. The optimal growth conditions were at 37 °C and pH 7. The isolates were closely related to Actinotignum sanguinis (92.6 % 16S rRNA gene sequence similarity), Arcanobacterium haemolyticum (92.5 %), Actinotignum schaalii (92.4 %), Actinobaculum massiliense (92.2 %) and Flaviflexus huanghaiensis (91.6 %). Phylogenetic analyses showed that strains 313T and 352 clustered independently in the vicinity of the genera Actinotignum, Actinobaculum and Flaviflexus, but could not be classified clearly as a member of any of these genera. Phylogenomic analysis also indicated that strains 313T and 352 formed an independent branch in the family Actinomycetaceae. The major cellular fatty acids of the strains were C16 : 0 and C18 : 1ω9c. The polar lipids comprised diphosphatidylglycerol, phosphatidylinositol mannoside, phosphatidylglycerol, phosphatidylinositol and five unidentified components. The peptidoglycan contained lysine, alanine and glutamic acid. The respiratory quinone was absent. The whole-cell sugars included glucose and rhamnose. The DNA G+C content of strain 313T was 60.6 mol%. Based on the low 16S rRNA gene sequence similarities, its taxonomic position in the phylogenetic and phylogenomic trees and its unique lipid pattern, we propose that strains 313T and 352 represent members of a novel species in a new genus, for which the name Fudania jinshanensis gen. nov., sp. nov. is proposed. The type strain is 313T (=CGMCC 4.7453T=DSM 106216T).

Pathogenicity and Virulence of Trueperella pyogenes: A Review.

Bacteria from the species Trueperella pyogenes are a part of the biota of skin and mucous membranes of the upper respiratory, gastrointestinal, or urogenital tracts of animals, but also, opportunistic pathogens. T. pyogenes causes a variety of purulent infections, such as metritis, mastitis, pneumonia, and abscesses, which, in livestock breeding, generate significant economic losses. Although this species has been known for a long time, many questions concerning the mechanisms of infection pathogenesis, as well as reservoirs and routes of transmission of bacteria, remain poorly understood. Pyolysin is a major known virulence factor of T. pyogenes that belongs to the family of cholesterol-dependent cytolysins. Its cytolytic activity is associated with transmembrane pore formation. Other putative virulence factors, including neuraminidases, extracellular matrix-binding proteins, fimbriae, and biofilm formation ability, contribute to the adhesion and colonization of the host tissues. However, data about the pathogen-host interactions that may be involved in the development of T. pyogenes infection are still limited. The aim of this review is to present the current knowledge about the pathogenic potential and virulence of T. pyogenes.

Phenotypic, molecular and genomic characterization of Actinobaculum suis isolated from swine in Brazil.

Urinary tract infections (UTI) are considered one of the most important diseases of sows due to its close relationship with reproductive problems such as reduced litter size, increase in the rate of return to estrous, vulvar discharge, abortion, mastitis and anestrus. Actinobaculum suis is one of the main agents involved in porcine urinary tract infection and is responsible for the most severe and fatal cases in sows. In the present report, 23 A. suis strains isolated from a sow and boars in Brazil were identified by PCR and further characterized by broth microdilution, molecular typing by pulsed-field gel electrophoresis (PFGE), single-enzyme amplified fragment length polymorphism (SE-AFLP), and whole-genome sequencing. All strains were sensitive to ceftiofur, linezolid, nitrofurantoin, quinupristin-dalfopristin and vancomycin. Ciprofloxacin, daptomycin, lincomycin, erythromycin and tylosin resistance was observed in 100% of tested strains. Tetracycline and tigecycline also presented high resistance rates (87% and 30.4%, respectively). PFGE with eight different restriction enzymes and three programs did not enable strain characterization; however, all strains were typed by SE-AFLP that clustered strains according to their origin, thus proving an effective tool for A. suis genotyping. Whole-genome sequencing and comparative analysis enabled species differentiation from closely related genus. This is the first report of genomic characterization of A. suis.

The first reported case of pelvic inflammatory disease caused by Actinobaculum massiliense.

We report the first case of pelvic inflammatory disease (PID) caused by Actinobaculum massiliense. A 53-year-old woman attended the emergency department with symptoms compatible with a PID episode, finally resolved by intramuscular antibiotic treatment. Actinobaculum sp. was isolated by culture, and A. massiliense was confirmed by matrix assisted laser desorption time-of-flight mass spectrometry and 16S rRNA gene sequencing. Only a few cases of A. massiliense infections have been reported, and the pathogenesis of infections by these bacteria is poorly understood. The introduction of new diagnostic methods into hospital routines will improve the detection of new and little-studied pathogens.

Genomic characterisation, detection of genes encoding virulence factors and evaluation of antibiotic resistance of Trueperella pyogenes isolated from cattle with clinical metritis.

Trueperella pyogenes is one of the most important microorganisms causing metritis in post-partum cattle. Co-infection with other bacterial species such as Escherichia coli or Fusobacterium necrofurom increases the severity of the disease and the persistence of bacteria in utero. The aim of this study was to investigate the frequency of T. pyogenes strains, and their virulence and antimicrobial resistant profiles in metritis cases. The study was carried out on 200 samples obtained from metritis discharges of postpartum cattle on 18 farms around Tehran, Iran. Sixty-five T. pyogenes isolates (32.5%) were identified, of which 16 isolates were detected as pure cultures and the other 49 isolates from cultures most commonly mixed with E. coli or F. necrofurom. In terms of diversity in biochemical characteristic of T. pyogenes strains, 8 different biotypes were identified among the isolates. Single or multi antimicrobial resistance was observed in 48 isolates (73.9%), which was mostly against trimethoprim sulfamethoxazole, azithromycin, erythromycin and streptomycin. The tetracycline resistance gene tetW and macrolide resistance genes ermB and ermX were detected in 30, 18 and 25 isolates, respectively. In the screening of genes encoding virulence factors, fimA and plo genes were identified in all tested isolates. Genes encoding nanP, nanH, fimC, fimG, fimE and cbpA were detected in 50, 54, 45, 40, 50 and 37 of isolates, respectively. Thirteen different genotypes were observed in these T. pyogenes isolates. A significant association between clonal types and virulence factor genes, biochemical profile, CAMP test result, severity of the disease and sampling time was detected.

Significant increase in cultivation of Gardnerella vaginalis, Alloscardovia omnicolens, Actinotignum schaalii, and Actinomyces spp. in urine samples with total laboratory automation.

While total laboratory automation (TLA) is well established in laboratory medicine, only a few microbiological laboratories are using TLA systems. Especially in terms of speed and accuracy, working with TLA is expected to be superior to conventional microbiology. We compared in total 35,564 microbiological urine cultures with and without incubation and processing with BD Kiestra TLA for a 6-month period each retrospectively. Sixteen thousand three hundred thirty-eight urine samples were analyzed in the pre-TLA period and 19,226 with TLA. Sixty-two percent (n = 10,101/16338) of the cultures processed without TLA and 68% (n = 13,102/19226) of the cultures processed with TLA showed growth. There were significantly more samples with two or more species per sample and with low numbers of colony forming units (CFU) after incubation with TLA. Regarding the type of bacteria, there were comparable amounts of Enterobacteriaceae in the samples, slightly less non-fermenting Gram-negative bacteria, but significantly more Gram-positive cocci, and Gram-positive rods. Especially Alloscardivia omnicolens, Gardnerella vaginalis, Actinomyces spp., and Actinotignum schaalii were significantly more abundant in the samples incubated and processed with TLA. The time to report was significantly lower in the TLA processed samples by 1.5 h. We provide the first report in Europe of a large number of urine samples processed with TLA. TLA showed enhanced growth of non-classical and rarely cultured bacteria from urine samples. Our findings suggest that previously underestimated bacteria may be relevant pathogens for urinary tract infections. Further studies are needed to confirm our findings.

Cultivation of Peptidiphaga gingivicola from subgingival plaque: The first representative of a novel genus of Actinomycetaceae.

A novel bacterium was isolated from the subgingival plaque of a patient with periodontal disease. Bacterial strain BA112T is a facultative Gram-positive coccus. It metabolizes alanine, arginine, glycine, histidine, leucine, proline, serine and tyrosine, but does not appear to use carbohydrates. Urease, esculin, indole, catalase and nitrate reduction tests were all negative. Major cellular fatty acids were C18:0 , C12:0 , C16:0 , C18:1 w9c and C20:0 . The genome was sequenced and is 2.4 Mbp in length and has 64% GC content. Based on phylogenetics of the 16S rRNA sequence and concatenated alignments of 37 conserved proteins, BA112T belongs to the family Actinomycetaceae but is located on a branch of the tree without currently named members. Based on our phenotypic and phylogenetic studies, we propose that BA112T is the first known representative of a new genus, for which the name Peptidiphaga gingivicola gen. nov., sp. nov. is proposed. The type strain is BA112T .

Trueperella pyogenes isolated from a brain abscess of an adult roebuck (Capreolus capreolus).

The present study was designed to characterize phenotypically and genotypically a Trueperella pyogenes strain isolated from a brain abscess of an adult roebuck (Capreolus capreolus). The species identity could be confirmed by phenotypical investigations, by MALDI-TOF MS analysis, and by sequencing the 16S ribosomal RNA (rRNA) gene, the 16S-23S rRNA intergenic spacer region (ISR); by sequencing the target genes rpoB, gap, and tuf; and by detection of T. pyogenes chaperonin-encoding gene cpn60 with a previously developed loop-mediated isothermal amplification (LAMP) assay. The T. pyogenes strain could additionally be characterized by PCR-mediated amplification of several known and putative virulence factor-encoding genes which revealed the presence of the genes plo encoding pyolysin and nanH and nanP encoding neuraminidases; the genes fimA, fimC, and fimE encoding the fimbrial subunits FimA, FimC, and FimE; and the gene cbpA encoding collagen-binding protein CbpA. The present data give a detailed characterization of a T. pyogenes strain isolated from a brain abscess of a roebuck. However, the route of infection of the roebuck remains unclear.