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Acinetobacter baumannii is a frequent cause of the nosocomial infections. Herein, a novel isothermal amplification technique, multiple cross displacement amplification (MCDA) is employed for detecting all A. baumannii strains and identifying the strains harboring blaOXA-23-like gene. The duplex MCDA assay, which targets the pgaD and blaOXA-23-like genes, could identify the A. baumannii isolates and differentiate these isolates harboring blaOXA-23-like gene. The disposable lateral flow biosensors (LFB) were used for analyzing the MCDA products. A total of sixty-eight isolates, include fifty-three A. baumannii strains and fifteen non-A. baumannii strains, were employed to optimize MCDA methods and determine the sensitivity, specificity and feasibility. The optimal reaction condition is found to be 63 °C within 1 h, with limit of detection at 100 fg templates per tube for pgaD and blaOXA-23-like genes in pure cultures. The specificity of this assay is 100%. Moreover, the practical application of the duplex MCDA-LFB assay was evaluated using clinical samples, and the results obtained from duplex MCDA-LFB method were consistent with conventional culture-based technique. In sum, the duplex MCDA-LFB assay appears to be a reliable, rapid and specific technique to detect all A. baumannii strains and identify these strains harboring blaOXA-23-like gene for appropriate antibiotic therapy.

Five strains of Gram-positive-staining, catalase-negative, coccus-shaped, chain-forming organisms isolated separately from the respiratory tracts of five Marmota himalayana animals in the Qinghai-Tibet Plateau of China were subjected to phenotypic and molecular taxonomic analyses. Comparative analysis of the 16S rRNA gene indicated that these singular organisms represent a new member of the genus Streptococcus , being phylogenetically closest to Streptococcus marmotae DSM 101995T (98.4 % similarity). The groEL, sodA and rpoB sequence analysis showed interspecies similarity values between HTS2T and Streptococcus . marmotae DSM 101995T, its closest phylogenetic relative based on 16S rRNA gene sequences, of 98.2, 78.8 and 93.7 %, respectively. A whole-genome phylogenetic tree built from 82 core genes of genomes from 16 species of the genus Streptococcus validated that HTS2T forms a distinct subline and exhibits specific phylogenetic affinity with S. marmotae . In silico DNA–DNA hybridization of HTS2T showed an estimated DNA reassociation value of 40.5 % with Streptococcus . marmotae DSM 101995T. On the basis of their phenotypic characteristics and phylogenetic findings, it is proposed that the five isolates be classified as representatives of a novel species of the genus Streptococcus , Streptococcus himalayensis sp. nov. The type strain is HTS2T (=DSM 101997T=CGMCC 1.15533T). The genome of Streptococcus himalayensis sp. nov. strain HTS2T contains 2195 genes with a size of 2 275 471 bp and a mean DNA G+C content of 41.3 mol%.

Five strains of a Gram-stain-positive, catalase-negative, α-haemolytic, coccus-shaped chain-forming organism were isolated separately from the lower respiratory tracts of five animals of Marmota himalayana in the endemic area of plague, the Qinghai-Tibet Plateau, China. Based on their morphological characteristics, biochemical features and molecular phylogenetic studies, the strains were placed as representing a new member of the genus Streptococcus . Comparative 16S rRNA gene sequence studies indicated that strain HTS5T shared 96.5, 96.2 and 96.0 % similarity with Streptococcus gallinaceus CCUG 42692T, Streptococcus parasanguinis ATCC 15912T and Streptococcus suis ATCC 43765T, respectively. Sequence analysis of its rpoB and sodA genes showed that strain HTS5T was most closely related to Streptococcus cuniculi CCUG 65085T with 9.2 and 10.9 % interspecies divergence, respectively. The whole genome phylogenetic tree based on 339 core genes of 65 Streptococcus genomes confirmed that HTS5T belongs to a distinct lineage that is well separated from recognized species of the genus Streptococcus . In silico DNA–DNA hybridization using 65 available genomes from GenBank showed that HTS5T displayed less than 70 % DNA–DNA relatedness with the other 65 species of the genus Streptococcus deposited in the GenBank database. The genome of strain HTS5T (2 322 791 bp) contained 2377 genes and had a G+C content of 41.6 mol%. Therefore, the five strains are considered to represent a novel species of the genus Streptococcus for which the name Streptococcus marmotae sp. nov. is proposed. The type strain is HTS5T (=DSM 101995T=CGMCC 1.15534T).

Two strains (NLN63T and NLN82) of Gram-stain-negative, oxidase- and catalase-positive, bacilli-shaped organisms were isolated from the faecal samples of two separate Rattus norvegicus in Baisha county of Hainan Province, Southern PR China. Phylogenetic analysis based on the near full-length 16S rRNA sequences revealed that strain NLN63T belongs to the genus Pelistega , having maximum similarity to Pelistega suis CCUG 64465T (97.1 %), Pelistega europaea CCUG 39967T (96.2 %) and Pelistega indica DSM 27484T (96.2 %), respectively. The phylogenomic tree built on 553 core genes from genomes of 20 species in the genus Pelistega and other adjacent genera further confirmed that strains NLN63T and NLN82 form a distinct subline and exhibit specific phylogenetic affinity with P. europaea CCUG 39967T. In digital DNA–DNA hybridization analyses, strain NLN63T showed low estimated DNA reassociation values (21.4–22.6 %) with the type strains of the species in the genus Pelistega . The DNA G+C contents of strains NLN63T and NLN82 were 37.3 and 37.1 mol%, respectively. Strain NLN63T had a unique MALDI-TOF MS profile, contained Q-8 as the major quinone and C16 : 0, summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c or both) and summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c or both) as the dominant fatty acids. Based upon these polyphasic characterization data obtained from the present study, a novel species of the genus Pelistega , Pelistega ratti sp. nov., is proposed with NLN63T (=GDMCC 1.1697T=JCM 33788T) as the type strain.

Staphylococcus aureus (S. aureus), including methicillin-resistant S. aureus (MRSA), is one of the most important human pathogens, which is responsible for bacteremia, soft-tissue infections, and food poisoning. Hence, multiple cross displacement amplification (MCDA) is employed to detect all S. aureus strains, and differentiates MRSA from methicillin-sensitive S. aureus. Multiplex MCDA (m-MCDA), which targets the nuc gene (S. aureus-specific gene) and mecA gene (encoding penicillin-binding protein-2′), could detect S. aureus strains and identify MRSA within 85 min. Detection of the m-MCDA products is achieved using disposable lateral flow biosensors. A total of 58 strains, including various species of Gram-positive and Gram-negative strains, are used for evaluating and optimizing m-MCDA assays. The optimal amplification condition is found to be 63°C for 40 min, with detection limits at 100 fg DNA/reaction for nuc and mecA genes in the pure cultures, and 10 CFU/tube for nuc and mecA genes in the blood samples. The analytical specificity of m-MCDA assay is of 100%, and no cross-reactions to non-S. aureus strains are produced according to the specificity testing. Particularly, two additional components, including AUDG enzyme and dUTP, are added into the m-MCDA amplification mixtures, which are used for eliminating the unwanted results arising from carryover contamination. Thus, the m-MCDA technique appears to be a simple, rapid, sensitive, and reliable assay to detect all S. aureus strains, and identify MRSA infection for appropriate antibiotic therapy.

Two bacterial strains were individually isolated from Marmota himalayana respiratory tracts; the animals were from the Tibet–Qinghai Plateau, PR China. The isolates were Gram-stain-positive, catalase-negative, coccus-shaped, chain-forming organisms. Analysis of 16S rRNA gene sequences indicated that the type strain HTS25T shared 98.0, 97.4, 97.2 and 97.1 % similarity with Streptococcus cuniculi , Streptococcus acidominimus , Streptococcus marmotae and Streptococcus himalayensis respectively. Sequence analysis of the sodA and rpoB genes indicated that HTS25T was closely related to S. marmotae (similarities of 94.7 and 91.4 % respectively). Analysis of groEL sequences showed interspecies similarity of 84.8 % between HTS25T and S. himalayensis . A whole-genome phylogenetic tree reconstructed from 81 core genes from the genomes of 17 members of the genus Streptococcus was used to validate that HTS25T forms a distinct subline from other recognized species of the genus Streptococcus . DNA–DNA hybridization of HTS25T showed a maximum estimated DNA reassociation value of 32.1 % to Streptococcus cuniculi CCUG 65085T. On the basis of the results of phenotypic and phylogenetic analyses, we propose that the two isolates be classified as representing a novel species of the genus Streptococcus , named Streptococcus respiraculi sp. nov. The type strain is HTS25T (=DSM 101998T=CGMCC 1.15531T). The genome of Streptococcus respiraculi sp. nov. strain HTS25T (2 067 971 bp) contains 2001 genes with an average DNA G+C content of 42.7 mol%.

Pseudomonas aeruginosa causes nosocomial infections of burn patients and other immunocompromised individuals, but the conventional diagnosis of P. aeruginosa infection depends on time-consuming culture-based methods. Hence, a simple, fast, sensitive technique for detection of P. aeruginosa using multiple cross displacement amplification (MCDA) and gold nanoparticle-based lateral flow biosensors (LFB) was developed. By using this technique, the reaction could be completed at an optimized constant temperature (67°C) within only 40 min. The reaction product could be detected visually using an LFB, eliminating the need for special equipment. The P. aeruginosa-MCDA-LFB method was highly specific, and accurately distinguished P. aeruginosa from other pathogens. Just 10 fg of genomic DNA template (from pure culture) could be detected. The assay could also detect P. aeruginosa in clinical sputum samples and showed the same sensitivity and specificity as the reference (culture-biochemical) method. In the future, this rapid, simple and accurate P. aeruginosa-MCDA-LFB technique might be applied in clinical practice.

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.

A Gram-stain-negative, microaerophilic strain, 80(YS1)T, with a spiral-shaped morphology and 1–2 sheathed flagella at each end of the cells was isolated from the gastric mucosa of Marmota himalayana, the animal reservoir of Yersinia pestis in China, on the Qinghai-Tibet Plateau. The strain grew at 30, 35 and 42 °C, but not at 25 °C. Growth was in the form of a thinly spreading film on brain heart infusion agar containing 8 % sheep blood under microaerobic conditions. The strain did not hydrolyse urea or hippurate, and did not grow on media containing 1 % glycine. It reduced nitrate to nitrite, and was catalase- and alkaline-phosphatase-positive, susceptible to nalidixic acid and resistant to cefalotin. It was positive for genus-specific PCR for the genus Helicobacter , but could not be classified to any recognized species according biochemical tests results. Therefore, a phylogenetic study based on 16S rRNA, 23S rRNA, 60 kDa heat-shock protein (hsp60) and gyrase subunit B (gyrB) genes was conducted. The 16S rRNA gene sequence (1468 bp) analysis showed that strain 80(YS1)T was most closely related to Helicobacter marmotae (96.7 % similarity). The 23S rRNA gene sequence (2879 bp) analysis showed that the strain was most closely related to Helicobacter canis (96 % similarity). The complete gyrB gene sequence (2325 bp) analysis showed that it was related phylogenetically to Helicobacter cinaedi (79.4 % similarity) and H. marmotae (79.1 % similarity). Analysis of the partial sequence of the hsp60 gene of strain 80(YS1)T showed closest similarity to the sequences of Helicobacter equorum (82 %) and H. cinaedi (81 %), respectively. However, there was no hsp60 sequence of H. marmotae available for analysis. The data of morphological, biochemical and phylogenetic characteristics all supported that this strain represents a novel species. The name Helicobacter himalayensis sp. nov. is proposed for this novel species with the type strain 80(YS1)T ( = CGMCC 1.12864T = DSM 28742T)

A survey was conducted to detect the distributing situation of Staphylococcus aureus in 6 kinds of food and the molecular biological characteristics of the isolates, so as to provide theoretical basis for our food risk monitoring. A total of 205 samples of 6 kinds of food were collected and examined for S. aureus. The GB 4789-2010 (food hygienic inspection method of China), mini VIDAS auto analysis, KB method and pulsed-field gel electrophoresis (PFGE) method were used to detect S. aureus, enterotoxin, drug sensitivity and homology analysis, respectively. The positive rates of S. aureus in the 205 samples were 15.6 % (32 samples), and the positive rates in raw milk were highest (30.0 %), with raw meat and aquatic products next and third (25.0and 12.0 %), cooked meat and milk products (10.0 % and 7.5 %, respectively). No S. aureus was detected in ice creams. With shock culturing in 37 °C for 24 h, 14 strains (43.7 %) produced enterotoxin in the 32 strains of S. aureus. In the drug sensitive tests, in the 32 isolates, 87.5 % were resistant to penicillin; 84.4 % were resistant to ampicillin; 26 (81.3 %) were resistant to tetracycline; 20 (62.5 %) showed resistance to erythromycin. All of the 32 strains were sensitive to trimethoprim sulfamethoxazole, oxacillin, cefoxitin, gentamicin, and vancomycin. No methicillin-resistant S. aureus was isolated from our food samples. PFGE typing showed that S. aureus genotypes profile had significant difference between the strains, indicating diversity contamination of foodborne S. aureus.

Two bacterial strains were isolated from faecal samples of Tibetan antelopes. The isolates were Gram-stain-positive, catalase-negative, coccus-shaped organisms that were tentatively identified as representing a novel 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). Analysis of groEL and rpoB gene sequences of the novel isolates showed interspecies divergence of 27.0 and 22.2 %, respectively, from the type strain of its closest 16S rRNA gene phylogenetic relative, S. ursoris. The complete genome of strain TA 26T has been sequenced. Digital DNA–DNA hybridization studies between strain TA 26T and other species of the genus Streptococcus deposited in the 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 from recognized Streptococcus species, the two isolates represent a novel species of the genus Streptococcus , for which the nameStreptococcus pantholopis sp. nov. (type strain TA 26T=CGMCC 1.15667T=DSM 102135T) is proposed.

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.

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, HT073016T, showed highest similarity values with Escherichia fergusonii ATCC 35469T at 99.3 %, Escherichia coli ATCC 11775T at 99.2 %, Escherichia albertii LMG 20976T at 98.9 %, Escherichia hermannii CIP 103176T at 98.4 %, and Escherichia vulneris ATCC 33821T 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 HT073016T 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 HT073016T was 53.8 mol%. On the basis of phenotypic and phylogenetic characteristics, strain HT073016T and the six other HT073016T-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 HT073016T ( = CGMCC 1.12862T = DSM 28771T) as the type strain.

To the Editor: Neisseria meningitidis colonizes the nasopharynx of humans and can cross the epithelial barrier of the nasopharynx, causing septicemia, meningitis, or both (1,2). In Anhui Province, China, there has been a previously high risk for epidemic cerebrospinal meningitis. Before 2012, all meningococcal diseases were caused by N. meningitidis serogroups A, B, and C, and the unique sequence type (ST) 4821 clone of serogroup C was first identified in this region during 2003–2004 (3). No widespread epidemics of cerebrospinal meningitis and no N. meningitidis–associated deaths have occurred in Anhui since bivalent meningococcal vaccines against serogroups A and C were first used in 2003 (4). During 2011–2013, however, 15 infections caused by N. meningitidis serogroup W135 ST11, which belongs to a hyperinvasive lineage (5), were reported in Hefei, Anhui Province. Two of the cases (1 each in 2012 and 2013) were fatal and occurred in patients who denied having recently traveled, which suggests that the clone may have spread in an endemic fashion. The 2 patients also had no history of vaccination with tetravalent polysaccharide vaccine (serogroups A/C/Y/W). The other 13 cases occurred in close contacts of the patients who died. The fatal cases of serogroup W135 infection were in 14- and 17-year-old boys. One of the boys had dropped out of school and worked in a hotel. He sought medical care for a headache with sudden onset, vomiting, and high fever (temperature 40°C). The other boy was a junior college student. At hospital admission, he had vomiting, diarrhea, and high fever (temperature 39°C). According to the Chinese surveillance system, meningococcal disease is reported by local hospitals to the local Center for Disease Control and Prevention and then to the provincial Center for Disease Control and Prevention, where specific measures are taken to control and prevent the disease. Serogroup W135 infection in the 2 boys in Hefei was identified and reported by different hospitals. Both boys reported that they had not traveled outside Hefei in the 2 months before illness onset or had any contact with persons with meningococcal disease. A total of 61 close contacts were identified for the boys. Despite treatment, the 2 boys died of disseminated intravascular coagulation and multiple organ failure. Cerebrospinal fluid and blood specimens were cultured on chocolate agar for 24 h; results showed bacterial growth consistent with the features of Neisseria spp. Four isolates were identified as serogroup W135 by using specific antiserum (Remel, Lenexa, KS, USA). Throat swab specimens were collected from the 61 close contacts of the 2 boys; all contacts were asymptomatic. Gram staining and biochemical tests confirmed the presence of N. meningitidis in 13 of the 61 samples, and slide agglutination, using specific antiserum, showed that all 13 isolates from contacts were serogroup W135. Pulsed-field gel electrophoresis (PFGE) (6), multilocus sequence typing, outer membrane protein gene (porA) variant region subtyping, and antimicrobial drug susceptibility tests were used to characterize the 17 isolates. PFGE patterns (pulse types 1 and 2) for the isolates were indistinguishable and shared >96% similarity with the dominant patterns, including patterns for isolates from Guangdong, Guangxi, and Jiangsu Provinces, China (Figure). All isolates had the same multilocus ST (ST11) and PorA subtype (P1.5,2), which belong to the multilocus ST11/electrophoretic type 37 complex (7). Results of Kirby-Bauer testing showed that all 17 isolates were resistant to sulfamethoxazole and sensitive to penicillin, ampicillin, ceftriaxone, cefotaxime, meropenem, minocycline, chloramphenicol succinate, and rifampin. Resistance to ciprofloxacin, which, to our knowledge, had not previously been reported for N. meningitidis from mainland China, was shown for 70.6% (12/17) of the isolates. Figure Pulsed-field gel electrophoresis (PFGE) pattern–based cluster analysis of 27 N. meningitidis serogroup W135 isolates from China: 17 isolates were collected from persons in Hefei City, Anhui Province, and 10 were collected from persons from other ... Since 2000, N. meningitidis W135 ST11 disease has become a serious problem worldwide (8,9). In mainland China, serogroup W135 ST11 cases have recently been reported in Guangdong, Guangxi, Zhejiang, Jiangsu, and Henan Provinces (10), but no deaths were reported until 2012, when the first of the 2 boys died in Anhui Province (Technical Appendix). According to the PFGE, multilocus sequence typing, and PorA typing results, the dominant clonal complex detected in Hefei matches the dominant type recently detected in other Chinese provinces. Pathogenicity characteristics of the isolates from Hefei and epidemiologic investigations indicate that the N. meningitidis W135 ST11 clone has emerged in Hefei, raising the possibility of its introduction into other regions. Meningococcal polysaccharide vaccines A and C are being used for routine vaccination in China. However, meningococcal diseases caused by N. meningitidis serogroups other than A and C, especially those belonging to hyperinvasive lineages, are an emerging problem that must be addressed. The 2 fatal meningitis cases in Hefei highlight the need for further epidemiologic surveillance to monitor the incidence of meningococcal disease caused by serogroup W135 and the need for better public health strategies to control the disease. Testing of the 17 isolates from Hefei for antimicrobial drug susceptibility indicated that sulfamethoxazole is not effective against serogroup W135 ST11 infection and that 70.6% of isolates were resistant to ciprofloxacin. Our findings indicate that changes in the molecular and epidemiologic characteristics of N. meningitidis in China should be monitored to enhance our ability to respond to emerging meningococcal disease. Technical Appendix: Cases of meningococcal disease caused by serogroup W135, China, January 2006–March 2013. Click here to view.(98K, pdf)