Application of broad-spectrum PCR amplification and direct sequencing for identification of the infrequent bacterial cultures from clinical sources, targeting the bacterial 16S rRNA gene with universal primes

Objective To identify one runny mucoid-like Gram-negative bacteria with pink pigment isolated from clinical pus sample. Methods The pus sample was aseptically extracted from a deep lesions of one patient, then stored in Amies medium at room temperature for transportation. One sheep blood plate and one chocolate plate were used to detect the possible pathogens from the specimens. After inoculation, the plates were placed in a humidified incubator with 5% CO2 at 35 ℃. To identify the obtained isolates, we used the commercial Vitek2 and API systems, combining some traditional morphological examination and classical biochemical and physiological characteristics. For pure cultures, the cellular fatty acids were extracted, methylated, and determined by gas chromatography method. The 16S rRNA gene was amplified and sequenced by a commercial broad-spectrum PCR primers. The phylogenetic tree based on 16S rRNA gene was constructed by Mega 4.1 software using the neighbour-joining methods with 1 000 bootstrap replications. Results One runny mucoid-like Gram-negative bacterium, named K8756, was isolated both on sheep blood and chocolate plates after 72 h incubation. The API 20NE profile was 1245045 after a 3-day culture, which would be identified as Ochrobactrum anthropi with a good confidence of 98% probability. It was identified as Ralstonia pickettii and Bordetella bronchiseptica by VITEK 2 GN kits. However, further comparative 16S rRNA gene sequences showed that strain K8756 was closely related to the valid published Roseomonas mucosa MDA 5527 with 100% identity. Colonial morphologic features, phenotypic characteristics and major cellular fatty acid composition were also with high similarity to Roseomonas mucosa. Conclusions Strain K8756( = GIMCC 1.0030 ) is identified as Roseomonas mucosa by the polyphasic phenotypic and genotypic characteristics. The comparative analysis based on 16S rRNA gene sequences is a useful method for identifying the problematic and newly named bacteria. Key words: RNA,ribosomal,16S; Sequence analysis,RNA

Objective To analyze the molecular epidemiology and resistant mechanisms of macrolide-nonsusceptible Moraxella catarrhalis. Methods A total of 383 strains of Moraxella catarrhalis were collected from nasopharynx of children under 2 years old.The minimum inhibition concentration (MIC) values were determined by Etest method, and the production of β-lactamase was examined by using a nitrocefin-based test.The pulsed-field gel electrophoresis (PFGE) method was used to analyze the type of different isolates.Polymerase chain reaction (PCR) and sequencing were performed for the resistance mechanism of macrolide resistance in Moraxella catarrhalis.The non-susceptibility rates of six cities(Beijing, Shanghai, Jinan, Nanjing, Wuhan and Dongguan) were compared by χ2 test. Results According to Clinical and Laboratory Standards Institute(CLSI) breakpoints, the non-susceptibility rates for erythromycin and azithromycin in 383 strains of Moraxella catarrhalis were 40% and 23%, respectively.Whereas, the non-susceptibility rates were 59% and 60% based on pharmacokinetics/pharmacodynamics (PK/PD) breakpoints.Significant differences in non-susceptibility rates to macrolide were observed in different cities, and the higher non-susceptibility rates were determined in relatively northern cities, such as Beijing and Jinan.Among the 383 strains of Moraxella catarrhalis, 92%(353/383) of isolates produced β-lactamase.A total of 14 patterns of groups were generated by PFGE in 37 high-level macrolide resistant Moraxella catarrhalis, and 43%(16/37) of isolates could be considered to be related or indistinguishable to group A.In this study, the ermA, ermB, mefA, and mefE genes were not detected, while the A2982T, A2796T, A2983T mutations in 23S rRNA gene may be related to macrolide resistance in Moraxella catarrhalis.The A2982T and A2796T mutations conferred high-level macrolide resistance, while the A2983T mutation conferred low-level resistance. Conclusions A large number of macrolide-nonsusceptible Moraxella catarrhalis isolates are detected in the study.The macrolide resistance is probably related to the mutations in 23S rRNA gene, and the relationship between MIC values of macrolide and mutations has been determined.（Chin J Lab Med,2012,35:247-252） Key words: Macrolides; Moraxella(branhamella) catarrhalis; Drug resistance，bacterial; RNA，ribosomal，23S; Mutation

Distinctive Molecular typing of 16S rRNA of Bacillus species isolated from farm settlement.

Genomic characterisation of linezolid-resistant Enterococcus faecalis from Western Australia 2016–2021

Purpose: Wounds infection is frequent hospital acquired infections which is caused by a wide variety of microorganism. Inappropriate treatment could be more hazardous to suffering patients and could lead to death. This manuscript is aimed to identify the existence of Pseudomonas aeruginosa from the clinical samples and determine the molecular evolution and population structure of Pseudomonas species using bioinformatics tools Method: The specimens were swiped from the bandage of infected patients, who were admitted for the therapeutic intervention in the hospitals, localized in Delhi-NCR region. Primarly, specimens were cultured in cetrimide broth for specific growth of Pseudomonas sp. Single colony isolation on cetrimide agar plate and characterization of bacterial colony by producing fluorescence under UV light was done to confirm Pseudomonas aeruginosa (P. aeruginosa). Furthermore, phylogenetic analysis was performed on the basis of PCR and sequencing of 16s rRNA sequence of P. aeruginosa. Results: Assay based on bacterial culture, biochemical and 16S rRNA gene analysis methods confirm the presence of P. aeruginosa in collected wound specimen. Basic local alignment search tool analysis indicates that newly sequenced 16S rRNA gene sequence was shown 99% similarity to P. aeruginosa species, analyzed using NCBI-BLAST tool. The phylogenetic analysis and nucleotide base composition studies performed using 45 sequences of 16S rRNA gene from 18 different species of Pseudomonas, including P. aeruginosa. The phylogenetic analysis was performed using Maximum Likelihood method for evolutionary relationships. Conclusion: The biochemical and molecular characterization revealed that the clinical samples were infected with P. aeruginosa. Further, phylogenetic analysis of 16S RNA sequences revealed evolutionary conservation among different Pseudomonas species.

임상검체에서 분리되는 그람음성 막대균의 제 3세대 cephalosporin에 대한 내성율의 증가는 임상적으로 심각한 문제가 되고 있다. 3세대 cephalosporin 및 monobactam계 항균제에 대한 내성은 주로 Extended-Spectrum <TEX>${\beta}$</TEX>-Lactamase(ESBL)의 생성에 기인한다. 따라서 ESBL 유전자의 정확한 검출은 병원내의 감염경로 파악을 위한 감시 및 역학조사를 위해 필수적이다. 본 연구는 2012년 2월부터 8월까지 대전, 충남, 충북지역의 대학병원으로부터 ESBL 생성 Klebsiella penumoniae 46균주를 분리하여 Clinical and Laboratory Standards Institute (CLSI)에 따라 ceftazidime (CAZ)과 CAZ/clavulanate (CLA)를 이용한 combination disk test (CDT) 방법에 의해 표현형을 조사하고, 유전형 특이 프라이머를 이용한 multiplex PCR을 수행하여 유전형을 검출하였다. CDT 결과 42균주가 ESBL생성균주로 확인되었다. PCR 결과, 46균주 모두 TEM형이었으며, 37균주는 SHV형, 14균주는 CTX-M형으로 나타났으며 10균주가 TEM, SHV, CTX-M 유전자를 모두 가지고 있었다. Multiplex PCR에 의한 유전형 검출 방법은 임상에서 분리한 ESBL생성 K. penumoniae균주의 감별과 검출에 유용한 방법으로 사료된다. Among Gram-negative pathogens in Korea, the incidence of resistance to third generation cephalosporins is becoming an ever-increasing problem. The production of extended-spectrum <TEX>${\beta}$</TEX>-lactamase (ESBL) is the main mechanism of bacterial resistance to a third-generation cephalosporins and monobactams. Accurate identification of the ESBL genes are necessary for surveillance and epidemiological studies of the mode of transmission in the hospital. This study was conducted to detect the genes encoding ESBL of 46 K. penumoniae isolated from Daejeon, Chungnam and Chungbuk regional university hospitals from February to August in 2012. The phenotypes of the isolated specimens were examined according to the combination disc test (CDT) by the Clinical and Laboratory Standards Institute (CLSI). Forty two ESBL producing K. penumoniae isolates could be detected using ceftazidime (CAZ) discs with and without clavulanate (CLA). By CDT, 42 K. pneumoniae strains were confirmed to be ESBL strains. Genotyping was performed by multiplex PCR with type-specific primers. By PCR analysis, TEM gene in 46 strains, SHV gene in 37 strains and CTX-M genes in 14 strains were identified. Ten isolates did carry genes encoding ESBLs of all types TEM, SHV and CTX-M. The multiplex polymerase chain reaction (PCR) analysis was better to detect and differentiate ESBL producing K. penumoniae strains in clinical isolates.

ObjectiveThe purpose of this study was to study the intestinal microbial diversity of the caracal cats (Caracal caracal). MethodsFecal samples from 7 wild adult caracal cats (2 males and 5 females) were collected, 2 of them were raised in Ji'nan Wildlife Park and 5 of them were raised in Weihai Wildlife Park. High-throughput sequencing of the 16S rRNA gene V3-V4 region of caracal cats was carried out. ResultsA total of 1458741 valid sequences of the V3-V4 region of the 16S rRNA gene was obtained, with an average of 208392 valid sequences and an average sequence length of 433 bp. By classifying with 97% sequence similarity, an average of 233 operational taxonomic units (OTUs) were obtained. These OTUs were all classified into the bacterial domain, including 13 phyla, 26 classes, 43 orders, 75 families, and 119 genera. Among them, the most abundant bacteria are Firmicutes (accounting for 61.7% of all OTUs), Actinobacteria (12.42%), Bacteroidetes (7.79%), Fusobacteroidetes (7.79%) and Proteobacteria (7.53%). The most abundant families were Peptostreptococcaceae (accounting for about 16.15% of all OTUs), Clostridiaceae_I (14.78%), Lachnospiraceae (13.13%) and Coriobacteriaceae (12.31%), etc. The most abundant genera were Collinsella (11.44%), Peptoclostridium (10.91%), Clostridium sensu stricto 1 (10.3%), Bacteroides (7.41%) and Peptostreptococcus (5.21%), etc. An average of 15.35% of the OTUs in the gut microbiota of the 7 caracal cats was unclassified at the genus level. Cluster analysis showed that the samples from the same park were clustered into one branch. ConclusionCharacteristics of the intestinal microbiota of the caracal cats were described and the results would provide scientific information for the rescue feeding and digestive physiology research of the caracal cats.

BLAST (Basic Local Alignment Search Tool) designed by Altschul and coworkers has become one of the most, if not the most, popular bioinformatics tools in the world. As amount of molecular data grows exponentially, refinements in the BLAST programs as well as data filtration at pre- or post-database search have been implemented with the BLAST server at NCBI (National Center of Biotechnology Information, http://www.ncbi.nih.gov/). However, such refinements with the BLAST programs at NCBI still do not satisfy all users at specific circumstances. Here we designed BLASTextend, a web-based tool, to help users to further ”mine” the data in a lengthy BLAST outcome and furthermore, to be able to enhance BLAST searching efficiency with a custom database. This tool, powered by the BLAST programs downloaded from NCBI, consists of three major parts: (1) a database management system for users to upload or to delete a custom database for BLAST searching, (2) a BLAST result parsing tool to tabulate the resulted alignment information and (3) an integrated system to provide a single entry to run BLAST analysis, to manage custom database and to parse the BLAST results in one streamline. BLASTextend is made freely available to users at http://abrc.sinica.edu.tw:218/blast/.

Amplification and sequence analysis of the 16S rRNA gene can be applied to detect and identify bacteria in clinical samples. We examined 75 clinical samples (17 culture-positive, 58 culture-negative) prospectively by two different PCR protocols, amplifying either a single fragment (1343 bp) or two fragments (762/598 bp) of the 16S rRNA gene. The 1343 bp PCR and 762/598 bp PCRs detected and identified the bacterial 16S rRNA gene in 23 (31 %) and 38 (51 %) of the 75 samples, respectively. The 1343 bp PCR identified 19 of 23 (83 %) PCR-positive samples to species level while the 762/598 bp PCR identified 14 of 38 (37 %) bacterial 16S rRNA gene fragments to species level and 24 to the genus level only. Amplification of shorter fragments of the bacterial 16S rRNA gene (762 and 598 bp) resulted in a more sensitive assay; however, analysis of a large fragment (1343 bp) improved species discrimination. Although not statistically significant, the 762/598 bp PCR detected the bacterial 16S rRNA gene in more samples than the 1343 bp PCR, making it more likely to be a more suitable method for the primary detection of the bacterial 16S rRNA gene in the clinical setting. The 1343 bp PCR may be used in combination with the 762/598 bp PCR when identification of the bacterial rRNA gene to species level is required.

Structure and function of methanogenic microbial communities in soils from flooded rice and upland soybean fields from Sanjiang plain, NE China

Many weevils are known as notorious devastating agricultural pests and generally associated with symbiotic bacteria. Here, we investigated the following pest and non-pest weevils collected in Japan for their bacterial associates: the banana stem weevil Odoiporus longicollis (Olivier); the Asiatic palm weevil Rhabdoscelus lineaticollis (Heller); the red palm weevil Rhynchophorus ferrugineus (Olivier); the Japanese giant weevil Sipalinus gigas (Fabricius); the olive weevil Pimelocerusperforatus (Roelofs); the black hard weevil Pachyrhynchus infernalis Fairmaire; and the Yonaguni hard weevil Metapocyrtus yonagunianus Chujo. Bacterial 16S rRNA gene was amplified by a polymerase chain reaction (PCR) from all the weevils, and genotyping and sequencing of the PCR products revealed that Nardonella, an ancient weevil-associated endosymbiont lineage, is the dominant bacterial associate for them. Molecular phylogenetic analyses based on bacterial 16S rRNA and groEL gene sequences showed that the weevil endosymbionts are placed within the Nardonella clade in the γ-Proteobacteria. The phylogenetic relationship of the Nardonella endosymbionts was concordant with the systematics of the weevil hosts, favoring the hypothesis of weevil-Nardonella co-speciation over evolutionary time. In situ hybridization visualized localization of the Nardonella endosymbionts in the larval bacteriome at the foregut-midgut junction in R. ferrugineus and S. gigas, and in the ovarial tips of adult females in O. longicollis. Our results highlight the general relevance of the Nardonella endosymbionts to the biology, control and management of these and other pest weevils.

Objective To investigate the bacterial resistance in nationwide and understand the distribution of bacterial and resistance trend. Methods The 6507 clinical isolates were collected from 19 hospitals in 17 cities.The susceptibility tests were performed using agar dilution method recommended by Clinical and Laboratory Standards Institute （CLSI） in central laboratory.The values of MIC50，MIC90 and MICrange were calculated by SPSS 17.0 and the susceptibilities of isolates to antimicrobial agents were determined by using CLSI (2010) guideline.Of all 6507 isolates，4691 strains were collected from target wards and 1816 were isolated from others wards. Results Among 4691 strains, 1156 were Gram-positive (24.6%) and 3535 were Gram-negative (75.4%).Based on the minimum inhibitory concentration results, the prevalence of methicillin resistant Stapylococcus aureus and methicillin resistant Stapylococcus epidermidis are 51.6% (325/630) and 87.0% (228/262) respectively.Staphylococci showing intermediate or full resistance to vancomycin were not observed. Coagulase negative Staphylococci showed 2.5% (16/642) intermediate rate and 1.6% (10/642) full resistance rate to teicoplanin, and showed 0.5% (3/642) resistance rate to linezolid.Antibiotic resistance rate of Enterococcus faecalis to ampicillin was 17.1% (19/111), while the resistance rate of Enterococcus faecium to ampicillin reached up to 85.0% (164/193).Three Enterococcus faecium were resistant to glycopeptides.The prevalence of penicillin resistance Streptococcus pneumoniae and penicillin intermediate Streptococcus pneumoniae were 41.2% (145/352) and 37.2% (131/352) respectively based on oral penicillin criterion, while the prevalence were 0.0% (0/352) and 6.0% (21/352) based on vein to non-meningitis criterion.A vast majority of Enterobacteriaceae maintained high susceptibility to carbapenems, with resistance rate less than 2.0%. In addition, tigecycline, moxalactam, fosfomycin and amikacin displayed desirable antibacterial activity against Enterbacteriaceae, and resistance rates to these drugs were all less than 10.0%.For non-fermenting Gram-negative isolates, resistance rate of Pseudomonas aeruginosa and Acinetobacter baumannii to imipenem were 23.1% (139/601) and 53.5% (419/784) respectively.Resistance rate of Acinetobacter baumannii was much higher than that during the period 2007-2008.Colistin, tigecycline，minocycline and fosfomycin demonstrated good antibacterial activity against Acinetobacter baumanniiin vitro. Conclusions Compared with MOHNARIN 2007-2008 year surveillance results, significant increase in resistance rate of Acinetobacter baumannii was demonstrated.Resistant strains to linezolid and tigecycline were found.Bacterial resistance has been a widespread problem in our country, which requires much more attention.（Chin J Lab Med,2012,35:70-90） Key words: Drug resistance，bacterial; Microbial sensitivity tests; Enterobacteriaceae; Staphyloccocus aureus

Recent major changes and updates of Clinical and Laboratory Standards Institute(CLSI) document M100 for performance standards for antimicrobial susceptibility testing were introduced in the article, which include changes of interpretive criteria and comments, antimicrobial susceptibility testing of infrequently isolated or fastidious bacteria, changes of appendixes and others.Brief comments were made for these changes.（Chin J Lab Med,2012,35:673-675） Key words: Anti-bacterial agents; Microbial sensitivity tests; Reference standards

Objective To investigate the antibacterial resistance of Enterobacteriaceae and Haemophilus in nationwide's tertiary hospitals and understand the trend of antimicrobial resistance. Methods The clinical isolates were collected from 19 tertiary hospitals in 19 cities nationwide over the period from July 2013 to June 2014 according to the same protocol. About 190 strains were collected from each hospital.The minimal inhibitory concentrations (MICs) were tested using agar dilution method recommended by Clinical and Laboratory Standards Institute (CLSI) in the central laboratory (Institute of Clinical Pharmacoloty, Peking University First Hospital). The susceptibilities of isolates to antimicrobial agents were determined using CLSI or European Committee on Antimicrobial Susceptibility Testing (EUCAST) 2015 guideline. SPSS 17.0 was used to caculated MIC50, MIC90, MICrange, susceptible rate and resistance rate. Results A total of 2 762 pathogenic isolates were studied. Based on the MIC results, extended spectrum β-lactamase (ESBLs) phenotype rates of Escherichia coli and Klebsiella pneumoniae were 66.0% (532/806) and 38.7% (226/584), respectively. Carbapenems, tigecycline, moxalactam, β-lactam/β-lactamase inhibitor combinations, amikacin and fosfomycin displayed desirable antibacterial activity against Enterbacteriaceae, but a significant increase in carbapenems resistant K. pneumoniae was noted (from 3.5%-5.9% in last surveillance raise to 9.1%-12.5%). About 70% K. pneumoniae isolated from children (≤14 years) were resistant to penicillins and cephalosporin, which was more than those from old people (≥65 years, t=2.085, P=0.042). Haemohpilus influenzae isolated from children were more resistant to some β-lactam than those from old people (21.9%-43.8% vs 3.3%-19.7%, t=2.229, P=0.031), so bacterial resistance in children is an important problem in China. Conclusions The resistance rates for E. coli to many antimicrobial agents were higher in China than foreign countries, which was one of the main resistance problem in our country. Compared with past surveillance results, growth of carbapenem resistant K. pneumoniae should be payed more attention.Haemohpilus spp.resistance was more serious in children in China.(Chin J Lab Med, 2016, 39: 103-119) Key words: Drug Resistance, Microbial; Enterobacteriaceae; Haemophilus; Microbial Sensitivity Tests; Drug Monitoring

Objective To investigate the antimicrobial susceptibility of nonfermenting gram-negative bacteria in nationwide's tertiary hospitals and understand the trend of antimicrobial resistance. Methods The clinical isolates were collected from 19 tertiary hospitals in 19 cities nationwide over the period from July 2013 to June 2014 according to the same protocol. About 100 strains were collected from each hospital. The minimal inhibitory concentrations (MICs) were tested using agar dilution method recommended by Clinical and Laboratory Standards Institute (CLSI) in the central laboratory (Institute of Clinical Pharmacoloty, Peking University First Hospital). The susceptibilities of isolates to antimicrobial agents were determined using CLSI or European Committee on Antimicrobial Susceptibility Testing (EUCAST) 2015 guideline. SPSS 17.0 was used to caculated MIC50, MIC90, MICrange, susceptible rate and resistance rates. Results A total of 1 535 pathogenic isolates were studied. Based on the MIC results, resistance rate of Pseudomonas aeruginosa and Acinetobacter baumannii to imipenem were 28.0% (180/644) and 70.5% (456/647) . Multidrug resistance rate of P. aeruginosa and A. baumannii were 34.9% (225/644) and 79.6% (515/647), and extensively drug-resistance rate were 9.8% (63/644) and 72.2% (467/647), respectively. Resistance rate of strains isolated from intensive care unit (ICU) were higher than those from non-ICU, especially in A. baumannii (about 52.4%-90.9% vs 34.0%-73.3%, t=2.209, P=0.032). Relevance ratio and resistance rate of nonfermenting gram-negative bacteria in children (≤14 years) were significantly lower than those from adults and old people (≥65 years) (about 3.2%-50.0% vs 7.1%-81.3%, t=-2.800--5.671, P<0.01). Resistance rate of P. aeruginosa isolated from sputum were higher than those isolated from blood and drainage fluid (about 18.7%-74.2% vs 0.0%-75.4%, t=2.337-2.707, P<0.05), and resistance rate of A. baumannii isolated from drainage fluid were higher than urine (about 19.7%-88.2% vs 6.0%-66.0%, t=2.884, P=0.006). Conclusions Compared with past surveillance results, resistance rates of.P. aeruginosa, Stenotrophomonas maltophilia, and Burkholderia cepacia were steady. Sustained growth of resistance rate of A. baumannii was identified, especially to some antibiotics showing good in vitro activity former, such as minocyclin et al. Multidrug resistance nonfermenting gram-negative bacterial has been a worldwide challenge for healthcare management.(Chin J Lab Med, 2016, 39: 130-138) Key words: Drug Resistance, Microbial; Gram-Negative Bacteria; Microbial Sensitivity Tests; Drug Monitoring