The taxonomic position of a group of seven closely related lactose-negative enterobacterial strains, which were isolated from fresh faecal samples of Marmota himalayana collected from the Qinghai-Tibetan plateau, China, was determined by using a polyphasic approach. Cells were Gram-reaction-negative, non-sporulating, non-motile, short rods (0.5–1 × 1–2.5 μm). By 16S rRNA gene sequences, the representative strain, HT073016^T, showed highest similarity values with Escherichia fergusonii ATCC 35469^T at 99.3 %, Escherichia coli ATCC 11775^T at 99.2 %, Escherichia albertii LMG 20976^T at 98.9 %, Escherichia hermannii CIP 103176^T at 98.4 %, and Escherichia vulneris ATCC 33821^T at 97.7 %. Phylogenetic analysis based on the 16S rRNA gene sequences showed that the seven strains formed a monophyletic group with five other species of the genus Escherichia. Digital DNA–DNA hybridization studies between strain HT073016^T and five other species of the genus Escherichia showed that it shared less than 70 % DNA–DNA relatedness with all known species of the genus Escherichia, supporting the novel species status of the strain. The DNA G+C content of strain HT073016^T was 53.8 mol%. On the basis of phenotypic and phylogenetic characteristics, strain HT073016^T and the six other HT073016^T-like strains were clearly distinct from the type strains of other recognized species of the genus Escherichia and represent a novel species of the genus Escherichia, for which the name Escherichia marmotae sp. nov. is proposed, with HT073016^T ( = CGMCC 1.12862^T = DSM 28771^T) as the type strain.

Klebsiella pneumoniae (K. pneumoniae) is a frequent pathogen causing nosocomial infections and outbreaks. We developed a multiple cross displacement amplification (MCDA) assay for the detection of K. pneumoniae, which can get the positive results within 40 minutes’ isothermal amplification. Gold-nanoparticle lateral flow biosensor (LFB) and colorimetric indicators were used for the rapid readouts of MCDA amplification. The detection limit of this assay was 100 fg per reaction at 65°C, which was confirmed to be the optimal amplification temperature according to the real time turbidimeters. For specificity, all of the 30 clinical-source K. pneumoniae strains were positive for the MCDA, and all of the non-K. pneumoniae strains belonging to 31 different species were negative for this MCDA assay. To evaluate the practical applicability of this method, we assessed its detection limit for K. pneumoniae strains in sputum samples (24 CFU per reaction), and DNA templates of 100 sputum samples further underwent the MCDA-LFB tests. All of the sputum samples being positive for K. pneumoniae (30/100) with the culture method were successfully identified with the MCDA assay, the detection power of which was higher than that of polymerase chain reaction (PCR) (25/100). Thus, the MCDA test for K. pneumoniae combined with the gold nanoparticle LFB as the results readout scheme, are simple, specific, and sensitive methods for the rapid diagnosis of K. pneumoniae in clinical samples.

Shigella and Salmonella are frequently isolated from various food samples and can cause human gastroenteritis. Here, a novel multiple endonuclease restriction real-time loop-mediated isothermal amplification technology (MERT-LAMP) were successfully established and validated for simultaneous detection of Shigella strains and Salmonella strains in only a single reaction. Two sets of MERT-LAMP primers for 2 kinds of pathogens were designed from ipaH gene of Shigella spp. and invA gene of Salmonella spp., respectively. Under the constant condition at 63˚C, the positive results were yielded in as short as 12 minutes with the genomic DNA extracted from the 19 Shigella strains and 14 Salmonella strains, and the target pathogens present in a sample could be simultaneously identified based on distinct fluorescence curves in real-time format. Accordingly, the multiplex detection assay significantly reduced effort, materials and reagents used, and amplification and differentiation were conducted at the same time, obviating the use of postdetection procedures. The analytical sensitivity of MERT-LAMP was found to be 62.5 fg and 125 fg DNA/reaction with genomic templates of Shigella strains and Salmonella strains, which was consist with normal LAMP assay, and at least 10- and 100-fold more sensitive than that of qPCR and conventional PCR approaches. The limit of detection of MERT-LAMP for Shigella strains and Salmonella strains detection in artificially contaminated milk samples was 5.8 CFU and 6.4 CFU per vessel. In conclusion, the MERT-LAMP methodology described here demonstrated a potential and valuable means for simultaneous screening of Shigella and Salmonella in a wide variety of samples.

Shigella flexneri is the major cause of shigellosis in developing countries. A new S. flexneri serotype, Xv, appeared in 2000 and replaced serotype 2a as the most prevalent serotype in China. Serotype Xv is a variant of serotype X, with phosphoethanolamine modification of its O antigen mediated by a plasmid that contained the opt gene. Serotype Xv isolates belong to sequence type 91 (ST91). In this study, whole-genome sequencing of 59 S. flexneri isolates of 14 serotypes (serotypes 1 to 4, Y, Yv, X, and Xv) indicated that ST91 arose around 1993 by acquiring multidrug resistance (MDR) and spread across China within a decade. A comparative analysis of the chromosome and opt -carrying plasmid pSFXv_2 revealed independent origins of 3 serotype Xv clusters in China, with different divergence times. Using 18 cluster-dividing single-nucleotide polymorphisms (SNPs), SNP typing divided 380 isolates from 3 provinces (Henan, Gansu, and Anhui) into 5 SNP genotypes (SGs). One SG predominated in each province, but substantial interregional spread of SGs was also evident. These findings suggest that MDR is the key selective pressure for the emergence of the S. flexneri epidemic clone and that Shigella epidemics in China were caused by a combination of local expansion and interregional spread of serotype Xv.

Two bacterial strains were isolated from faecal samples of the Tibetan antelopes. The isolates were Gram-positive, catalase-negative, coccus-shaped organisms that were tentatively identified as a new streptococcal species based on their morphological features, biochemical test results and phylogenomic findings. Comparative 16S rRNA gene sequencing studies confirmed that the organisms were members of the genus Streptococcus, but they did not correspond to any recognized species of the genus. The nearest phylogenetic relative of the unknown coccus was Streptococcus ursoris NUM 1615T (93.4% 16S rRNA gene sequence similarity). groEL and rpoB sequence analysis of the novel isolates showed interspecies divergence of 26.9% and 22.1%, respectively, from the type strain of its closest 16S rRNA gene phylogenetic relative, S. ursoris. The complete genome of the type strain TA 26T has been sequenced. Digital DNA-DNA hybridization studies between strain TA 26T and other species of the genus Streptococcus deposited in GenBank database showed less than 70 % DNA-DNA relatedness, supporting a novel species status of the strain. On the basis of their genotypic and phenotypic differences to other existing Streptococcus species, the two isolates represent a novel species of the genus Streptococcus, for which the name Streptococcus pantholopis sp. nov. (type strain TA 26T= CGMCC 1.15667T = DSM 102135T) is proposed.