Rapid bacterial identification and antimicrobial susceptibility testing assay in positive blood cultures / 预防医学

Objective@#To establish a rapid bacterial identification and antimicrobial susceptibility testing assay in positive blood cultures, so as to provide insights into timely diagnosis and treatment of bloodstream infections.@*Methods@#A total of 1 154 blood culture samples were collected from inpatients in Zhejiang Hospital from February to May, 2022. The bacterial isolates were enriched and purified using improved separation gel method, and bacterial identification and antimicrobial susceptibility tests were performed using VITEK2 mass spectrometry system and VITEK2 Compact automated microbiology system. The accuracy of the new assay for bacterial identification and antimicrobial susceptibility tests was evaluated with the conventional VITEK 2 compact system as the standard. @*Results@#Of 1 154 blood culture specimens, the conventional VITEK 2 compact system detected 174 positives and 980 negatives. The new assay and the conventional VITEK 2 compact system identified consistent bacterial isolates in 165 out of 174 positive blood culture samples, and the accuracy of bacterial identification was 94.83% for the new assay, with a 99.21% accuracy for identifying Gram-negative bacteria and 82.22% for Gram-positive bacteria. Antimicrobial susceptibility tests were performed in 158 bacterial isolates, and the new assay presented a 90.17% accuracy, with a 90.27% accuracy for Gram-negative bacteria and 89.74% for Gram-positive bacteria. The conventional VITEK 2 compact system required 30 hours and longer to complete bacterial identification and antimicrobial susceptibility tests, and the new assay required 9 to 18 hours.@*Conclusions@#The new rapid bacterial identification and antimicrobial susceptibility testing assay shortens the time of bacterial culture, achieves rapid bacterial identification and antimicrobial susceptibility testing in blood culture specimens and has a high accuracy that meets clinical needs, which facilitates rapid diagnosis and treatment of bloodstream infections.

Primer probes for targeted identification and detection of Bifidobacterium animalis and their application / 中国药科大学学报

@#In this study, in order to overcome the shortcomings of the current methods used to identify Bifidobacterium animalis, such as long time, complicated operation and low adaptability of experimental environment, specific primer probes were designed based on ERIC-PCR technology to identify and detect B.animalis.Based on the genomic DNA of B.animalis HP-B1124, the ERIC-PCR reaction conditions of B.animalis HP-B1124 were optimized, and the ERIC-PCR fragments were obtained one by one and sequenced.Two pairs of specific primer probes were designed.The accuracy, specificity, limitation and universality of the two pairs of primer probes were evaluated, and the two pairs of specific primer probes were used for testing the products containing B.animalis in the commercially published formula.The two pairs of specific primer probes designed in this study could be used for identified strains of B.animalis more simply, quickly and targeted.This method has optimized the current relatively traditional methods of pure culture and plate counting identification of B.animalis, and has solved the high requirements of SNP genotyping technology and real-time fluorescence quantitative PCR method for experimental equipment and reagents in the identification of B.animalis to a certain extent.It has the characteristics of low cost, high specificity and earn a broad market development prospect.

Identificación de especies de micobacterias mediante espectrometría de masas (MALDI-TOF) / Identification of mycobacteria species through mass spectrometry (MALDI-TOF)

Resumen Introducción: Las enfermedades producidas por micobacterias son de gran importancia clínica y epidemiológica presentando el complejo Mycobacterium tuberculosis (MTBc) una morbi-mortalidad mayor que la producida por micobacterias no tuberculosas (MNTB). La identificación tradicional está basada en sus características fenotípicas mediante procesos laboriosos e incapaces en algunos casos de distinguir entre especies. Actualmente, la mayoría de las técnicas utilizadas se basan en métodos moleculares que tienen alta veracidad, pero son complejas y de alto costo. La espectrometría de masas con desorción/ionización láser asistida por una matriz asociada a tiempo de vuelo (MALDI-TOF MS) se basa en la comparación del espectro proteico producido con respecto al de una base de datos de referencia. Objetivo: Evaluar el rendimiento de MALDI-TOF MS en la identificación de micobacterias comparado con métodos moleculares: Material y Métodos: Se analizaron 28 aislados de nueve especies distintas mediante MALDI-TOF MS. Resultados: Se identificó correctamente 78,5% de las aislados (22/28), concordante en 100% (9/9) de MNTB de crecimiento rápido, 60% (9/15) en las MNTB de crecimiento lento y 100% (4/4) de MTBc. Todas las especies no identificadas (6/6) pertenecen al complejo M. avium/intracellulare. Conclusión: MALDI-TOD MS es una metodología rápida, fácil y de bajo costo, con adecuada veracidad respecto a los métodos moleculares.

Abstract Background: Mycobacterial diseases are very important both clinically and epidemiologically. Mycobacterium tuberculosis complex (MTBc) infections confer higher morbidity and mortality rate than non-tuberculous mycobacteria (NTM) infections. Traditional species identification techniques are based on phenotypic characteristics which take a long time by laborious processes and in occasions are no conclusive. Currently, most used techniques are based on molecular methods, which are accurate but are expensive and complex. Matrix Assisted Laser Desorption/Ionization Time-of-Flight mass spectrometry (MALDI-TOF MS) is a simple, cheap and fast identification method based on comparing protein spectra with a reference database. Aim: To assess the performance of MALDI-TOF MS in the identification of MTBc and NTM, compared with molecular methods. Methods: For that purpose, 28 isolates of 9 different species were analyzed through MALDI-TOF MS. Results: 78.5% (22/28) of isolates were correctly identified, 100% (9/9) of rapidly growers NTM, 60% (9/15) of slow growing NTM and 100% (4/4) of MTBc. Every unidentified isolate (6/6) corresponded to M. avium/intracellulare complex. Conclusion: MALDI-TOF MS is fast, simple and cheaper than molecular methods and also has adequate accuracy.

Bacterial identification, somatic cell count, antimicrobial profile and toxigenic Staphylococcus strains search from mastitic cow milk samples on small farms properties / Identificação bacteriana, contagem de células somáticas, perfil antimicrobiano e pesquisa de linhagens toxigênicas de Staphylococcus em amostras de leite bovino de pequenas propriedades rurais

Bovine mastitis has a negative impact on milk production and can pose risks to public health. The present study aimed to evaluate the quality of bovine milk from small farms in the Botucatu/SP region. Somatic cell counts (SCC), identification of pathogens involved in mastitis, and sensitivity antimicrobial profile of staphylococci isolated were performed. The presence of enterotoxin encoding genes in isolates of staphylococci obtained from milk was investigated. Milk samples from individual mammary quarters of cows were submitted to the California mastitis test (CMT) and SCC. Of the 239 dairy cows from 21 dairy herds evaluated (mean = 11.4 animals/property), two cows (0.8%) presented clinical mastitis and 86 (35.9%) subclinical mastitis. Bacterial culture was performed in 177 quarter milk samples. Staphylococci were identified in 55 (31.1%), corynebacteria in 45 (25.4%), streptococci in 25 (14.1%) and coliforms in four (2.3%) milk samples. Average SCC from culture-positive samples was 1598x103 cells/mL, in case of staphylococci was 1362x103 cells/ml, streptococci was 2857x103 cells/mL, corynebacteria was 976x103 cells/mL and in the cases of coliforms 1161x103 cells/mL were obtained. Staphylococci showed a high sensitivity (>95%) to cephalothin, cotrimoxazole, enrofloxacin, and gentamicin, with a 41.2% resistance to penicillin and 11.8% to oxacillin. Both coagulase positive (CPS) and negative staphylococci (CNS) carried genes encoding enterotoxins in 21.6% of the first group and 41.9% in the second. The sea gene was the most detected 45.8% (n=24) between them, followed by seb with 29.2% and sec with 25.0%. The sed gene was not identified. We highlight the potential risk to public health in the possibility of strains of Staphylococcus spp. enterotoxin-producing genes that can cause staphylococcal food poisoning.(AU)

A mastite bovina impacta negativamente a produção leiteira e pode acarretar riscos à saúde pública. O presente estudo teve como objetivo a avaliação da qualidade do leite bovino proveniente de pequenas propriedades na região de Botucatu/SP. Foi realizada a contagem de células somáticas (CCS), identificação dos patógenos envolvidos nas mastites, e realizado o perfil de sensibilidade aos antimicrobianos dos estafilococos isolados. Pesquisou-se a presença de genes codificadores de enterotoxinas em isolados de estafilococos obtidos a partir do leite mastítico. Amostras de leite de quartos mamários individuais de vacas foram submetidas ao "California mastitis test" (CMT) e à CCS. Das 239 vacas em lactação provenientes de 21 rebanhos leiteiros avaliados (média = 11,4 animais/propriedade), dois (0,8%) animais apresentaram mastite clínica e, 86 (35,9%) mastite subclínica. 177 amostras de leite foram cultivadas em ágar sangue bovino 5% e ágar MacConckey e obteve-se 55 (31,1%) Staphylococcus spp., 25 (14,1%) Streptococcus spp., 45 (25,4%) Corynebacterium spp. e quatro (2,3%) coliformes. A média da CCS das amostras procedentes de todos os quartos mamários infectados avaliados foi de 1598x103 células/mL, enquanto que nos casos que foram isolados Staphylococcus spp. foi de 1362x103 células/mL, Streptococcus spp. 2857x103 células/mL, Corynebacterium spp. de 976x103 células/mL e nos casos de coliformes 1161x103 células/mL. Os estafilococos revelaram grande sensibilidade (>95%) à cefalotina, cotrimoxazol, enrofloxacina e gentamicina, com resistência de 41,2% à penicilina e 11,8% à oxacilina. Tanto estafilococos coagulase positivos (ECP) como negativos (ECN) revelaram genes codificadores de enterotoxinas em 21,6% do primeiro grupo e 41,9% no segundo. O gene sea foi o mais detectado 45,8% (n=24), seguido pelo seb com 29,2% e sec com 25,0%. O gene codificador da sed não foi identificado. Frente aos resultados, destaca-se o risco potencial à saúde pública pela possibilidade de veiculação de linhagens de Staphylococcus spp. carreadores de genes produtores de enterotoxinas, podendo ocasionar toxi-infecções alimentares.(AU)

Análise bacteriológica e físico-química de leite cru resfriado, proveniente de posto de refrigeração, sob inspeção federal, em Rio Bonito, RJ

Com o objetivo de avaliar a qualidade microbiológica e fisico-química do leite cru de propriedades rurais do município de Rio Bonito-RJ e arredores, foram analisadas 20 amostras de leite provenientes de Posto de Refrigeração, subsidiado à Indústria Nestlé, coletadas de latões e de tanque de refrigeração. Realizaram-se análises microbiológicas como Contagem Total de Bactérias Heterotróficas Aeróbias Mesófilas (B.H.A.M), Contagem total de Bactérias Heterotróficas Aeróbias Psicotrófilas (B.H.A.P) seguindo as análises fisico-químicas, tais como: temperatura, acidez titulável, prova alizarol, lactofermentação, prova da redução e contagem de células somáticas. Para as variáveis da prova do alizarol houve reprovação em 20% das amostras, já para acidez 95% estavam dentro dos padrões normais. A temperatura das amostras, no ato da coleta, apresentou grandes variações em decorrência da distância e transporte inadequados. No teste da redutase apenas 15% foram consideradas boas ou ótimas e na lactofermentação 100% das amostras formaram algum tipo de coágulo. Em relação à contagem de bactérias, as B.H.A.M foram encontradas fora dos padrões em 65% das amostras e nas B.H.A.P em 80% dos resultados encontrados. Quanto à contagem de células somáticas apenas uma amostra apresentou-se fora do padrão. Conclui-se que a qualidade do leite pode ser melhorada por meio de assistência técnica e instrucional aos produtores, considerando os aspectos da legislação vigente associados à higienização adequada dos latões e utensílios de ordenha. O leite deve ser resfriado na fazenda e transportado sem delongas até a cooperativa.

With the objective of evaluating the microbiology and physicochemical quality of raw milk from rural properties in the city of Rio Bonito-RJ and surrounding areas, were analyzed 20 samples of raw milk from Refrigeration Station, subsidized by Nestlé, collected from bulk milk collectors and cooling tank. Microbiological analyzes were performed as Total Counting of Mesophilic Aerobic Heterotrophic Bacteria (MA.H.B), Total Counting of Heterotrophic Aerobic Psychophotrophic Bacteria (H.A.P.B) and physicochemical analyzes: temperature, titratable acidity, alizarol test, lactofermentation, reduction test and cell count somatic cells. For the variables of the alizarol test there was reprobation in 20% of the samples, already for acidity 95% were within normal standards. The temperature of the samples, at the time of collection, presented great variations due to inadequate distance and transport. In the reductase test only 15% were considered good or optimal and in the lactofermentation 100% of the samples formed some type of clot. Regarding bacterial counts, M.A.H.B. were found out of standards in 65% of samples and in H.A.P.B in 80% of the results found. As for somatic cell count, only one sample was out of standard. It is concluded that the quality of milk can be improved by providing technical and instructional assistance to producers and by appropriate hygiene of brass and milking utensils. The milk should be cooled on the farm and transported without delay to the Milk Collection Centers.

Análise bacteriológica e físico-química de leite cru resfriado, proveniente de posto de refrigeração, sob inspeção federal, em Rio Bonito, RJ

Com o objetivo de avaliar a qualidade microbiológica e físico-química do leite cru de propriedades rurais do município de Rio Bonito-RJ e arredores, foram analisadas 20 amostras de leite provenientes de Posto de Refrigeração, subsidiado à Indústria Nestlé, coletadas de latões e de tanque de refrigeração. Realizaram-se análises microbiológicas como Contagem Total de Bactérias Heterotróficas Aeróbias Mesófilas (B.H.A.M), Contagem total de Bactérias Heterotróficas Aeróbias Psicotrófilas (B.H.A.P) seguindo as análises físico-químicas, tais como: temperatura, acidez titulável, prova alizarol, lactofermentação, prova da redução e contagem de células somáticas. Para as variáveis da prova do alizarol houve reprovação em 20% das amostras, já para acidez 95% estavam dentro dos padrões normais. A temperatura das amostras, no ato da coleta, apresentou grandes variações em decorrência da distância e transporte inadequados. No teste da redutase apenas 15% foram consideradas boas ou ótimas e na lactofermentação 100% das amostras formaram algum tipo de coágulo. Em relação à contagem de bactérias, as B.H.A.M foram encontradas fora dos padrões em 65% das amostras e nas B.H.A.P em 80% dos resultados encontrados. Quanto à contagem de células somáticas apenas uma amostra apresentou-se fora do padrão. Conclui-se que a qualidade do leite pode ser melhorada por meio de assistência técnica e instrucional aos produtores, considerando os aspectos da legislação vigente associados à higienização adequada dos latões e utensílios de ordenha. O leite deve ser resfriado na fazenda e transportado sem delongas até a cooperativa.

With the objective of evaluating the microbiology and physicochemical quality of raw milk from rural properties in the city of Rio Bonito-RJ and surrounding areas, were analyzed 20 samples of raw milk from Refrigeration Station, subsidized by Nestlé, collected from bulk milk collectors and cooling tank. Microbiological analyzes were performed as Total Counting of Mesophilic Aerobic Heterotrophic Bacteria (M.A.H.B), Total Counting of Heterotrophic Aerobic Psychophotrophic Bacteria (H.A.P.B) and physicochemical analyzes: temperature, titratable acidity, alizarol test, lactofermentation, reduction test and cell count somatic cells. For the variables of the alizarol test there was reprobation in 20% of the samples, already for acidity 95% were within normal standards. The temperature of the samples, at the time of collection, presented great variations due to inadequate distance and transport. In the reductase test only 15% were considered good or optimal and in the lactofermentation 100% of the samples formed some type of clot. Regarding bacterial counts, M.A.H.B were found out of standards in 65% of samples and in H.A.P.B in 80% of the results found. As for somatic cell count, only one sample was out of standard. It is concluded that the quality of milk can be improved by providing technical and instructional assistance to producers and by appropriate hygiene of brass and milking utensils. The milk should be cooled on the farm and transported without delay to the Milk Collection Centers.

Direct identification from positive blood broth culture by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS)

ABSTRACT Bloodstream infections (BSIs) are among the most concerning bacterial infections. They are one of the leading causes of morbidity and mortality, and occur in 30-70% of critical care patients. The prompt identification of the causative microorganism can help choosing the appropriate antimicrobial therapy that will lead to better clinical outcomes. Blood culture is one of the most relevant tests for microbiological diagnosis of bacterial infections. The introduction of the MALDI-TOF microbiological diagnosis significantly decreased the time of identifying microorganisms. However, it depends on the growth on solid culture medium. In this study, 538 bottles of positive blood cultures were evaluated to test the accuracy of an in house modified protocol. The study sample consisted of 198 Gram-negative and 350 Gram-positive bacteria. In all, 460 (83.94%) species were identified based on the direct plate findings. The protocol allowed the identification of 185/198 (93.43%) of the Gram-negative bacteria, including aerobes, anaerobes, and non-fermenters, and 275/350 (78.85%) of the Gram-positive bacteria. The proposed method has the potential to provide accurate results in comparison to the traditional method with the potential to reduce the turnaround time for the results and optimize antimicrobial therapy in BSI.

Advances of molecular biology techniques in rapid bacterial identification / 国际儿科学杂志

Sepsis is one of the most important causes of death in critically ill children.The leading cause of sepsis is bacterial infection.The early diagnosis of bacterial infection allows initiation of appropriate antimicrobial therapy that strongly correlates with positive outcomes.At present,the methods of detection and identification of bacteria in our country are still at the level of blood culture and biochemistry,and they have the weakness of time-consuming process and lower positive rate.Therefore,it is very important to establish a rapid and sensitive diagnostic method for early diagnosis of bacterial infection.In recent years,with the rapid development of molecular detection technology,some new molecular biological technologies can not only improve the detection accuracy and sensitivity,but also reduce the detection hours and expand the detection pathogen spectrum.Nowadays,they have been applied to bacteria classification and identification.There are two kinds of molecular biological technology that are applied at present.One is based on nucleic acid detective technology,such as nucleic acid amplification,DNA sequencing,gene chips,etc.The other is based on proteome technologies,such as biological mass spectrometry.In this paper,the application of recent molecular biological techniques in the rapid detection of bacteria is reviewed.

Identification of a newly reported Francisella species by average nucleotide identity based on high-throughput whole genome sequencing technology / 临床检验杂志

Objectives To identify the Francisella strain isolated from blood of a patient with drowning-associated pneumonia.Methods The whole genome of the strain,designated Wenzhou1,was sequenced using the high throughput sequencing technology by 2000/miSeq system of Illumina platform,and the obtained genome draft was assembled by MicrobeTrakr Plus software.The phylogenetic neighbors of Wenzhou1 were obtained by NCBI BLAST analysis from GenBank database for the gene sequences of 16S rRNA,malate dehydrogenase(mdh),DNA-directed RNA polymerase subunit beta (rpoB) and succinate dehydrogenase subunit alpha (sdhA).The average nucleotide identity(ANI) between Wenzhou1 and its phylogenetic neighbors was analyzed by the software OrthoANI using NCBI BLAST search under the Java Runtime Environment Version 8.Results The genome size of Wenzhou1 was 1.96 × 106 bp,containing 74 contigs.The genomic G + C mol％ of Wenzhou1 was 32.1％,which was similar to the other species of genus Francisella and Allofranicella.Based on the analysis of NCBI BLAST of GenBank for the similarities of 16S rRNA gene,mdh gene,rpoB gene and sdbA gene sequences,Wenzhou1 was most closely related to F.hispaniensis FSC454 and Francisella cf.novicida 3523.The ANI of Wenzhou1 was 97.8％ to F.hispaniensis FSC454,97.5％ to 97.6％ to Francisella cf.novicida 3523,but only 91.3％ to 91.5％ to the four subspecies of F.tularensis.Conclusion ANI analysis based on whole genome sequence should be an accurate,effective method for bacterial identification.Wenzhou1 could be identified as F.hispaniensis by ANI with high-throughput whole genome sequencing technology.

Surveillance of antibiotic resistance in the clinical isolates collected from Shanghai Xinhua Hospital Chongming Branch during 2015 / 中国感染与化疗杂志

Objective To analyze the antimicrobial resistance profile of clinical isolates in Shanghai Xinhua Hospital Chongming Branch affiliated to Shanghai Jiaotong University School of Medicine , a member of China Antimicrobial Resistance Surveillance System, during 2015, for the purpose to facilitate rational antimicrobial therapy. Methods Strain identification?and?susceptibility?testing?were?carried?out?for?the?clinical?isolates?using?MicroScan?WalkAway?96?Automated?Systems and Kirby-Bauer method. Results In 2015, a total of 1815 isolates were collected, including gram-negative bacteria (73.2 %) and gram-positive bacteria (26.8 %). The top three frequently isolated species were Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. ESBL-producing strains were found in 36.3 % of the Escherichia coli isolates, 12.6 % of the Klebsiella (K. pneumoniae and K. oxytoca) isolates, and 28.0 % of the Proteus mirabilis isolates. The prevalence of carbapenem-resistant strains was 0.69 % in Enterobacteriaceae isolates. The prevalence of methicillin-resistant strain was 29.1 % in S. aureus, and 61.4 % in coagulase-negative Staphylococcus isolates. No more than 15 % of the Enterobacteriaceae isolates and no more than 20 % of the P. aeruginosa and Acinetobacter isolates were resistant to carbapenems. No vancomycin-or linezolid-resistant strains were found in Enterococcus or Staphylococcus. Conclusions Antibiotic-resistant clinical isolates are a serious threat for clinical antimicrobial treatment. We should pay more attention to such urgent situation and rational use of antibiotics.

Comparative analysis of anti-Helicobacter pylori activities of FEMY-R7 composed of Laminaria japonica and Oenothera biennis extracts in mice and humans / 한국실험동물학회지

Helicobacter pylori-eliminating effects of FEMY-R7, composed of Laminaria japonica and Oenothera biennis extracts, were investigated in mice and humans. Male C57BL/6 mice were infected with the bacteria by intragastric inoculation (1x10(9) CFU/mouse) 3 times at 2-day intervals, and simultaneously, orally treated twice a day with total 20, 64 or 200 mg/kg/day FEMY-R7 for 2 weeks. In Campylobcter-like organism (CLO)-detection tests on gastric mucosa and feces, FEMY-R7 reduced the urease-positive reactivity in a dose-dependent manner; i.e., the positivity ratios were decreased to 70, 20, and 10% for gastric mocosa and to 80, 50, and 20% for feces. In a clinical sudy, human subjects, confirmed to be infected with Helicobacter pylori, were orally administered twice a day with capsules containing total 100, 320 or 1,000 mg/man/day FEMY-R7 (matching doses for 20, 64 or 200 mg/kg/day, respectively, in mice from a body surface area-based dose translation) for 8 weeks. FEMY-R7 decreased the positivity ratios in feces to 70, 40, and 30%, respectively. In bacterial culture, H. pylori was identified from the CLO-positive stools of mice and humans. The bacterial identification ratios exhibited a good correlation between the matching doses in mice and humans. It is suggested that FEMY-R7 could be a promising functional food without tolerance as an adjunct to reduce the dosage of antibiotics for the treatment of recurrent H. pylori infection.

Use of MALDI-TOF MS technique for rapid identification of bacteria from positive blood cultures.

We evaluated the feasibility of same‑day routine aerobic bacterial identification using the following procedures: Picking colonies from 4 and 6 h incubated subculture from positive blood culture bottle and analyzing them by matrix‑assisted laser desorption/ionisation time‑of‑flight mass spectrometry (MALDI‑TOF MS). The matched identification rate of this procedure at the species level was 80.6% (141/175) for the 4‑h cultures compared with overnight cultures and 90.9% (159/175) for the 6‑h cultures. Thus, our technique provides an easy and rapid method for identification of aerobic bacteria in routine clinical microbiology laboratories.

Identifying 1 strain of conditional pathogenic Ochrobactrum by 16S rRNA gene sequencing / 国际检验医学杂志

Objective To explore a rapid bacterial identifying method based on the 16S rRNA gene sequence analysis technology to provide the scientific basis for the diagnosis and treatment of unknown pathogenic bacteria.Methods The pure colonies were iso-lated and cultured directly from a clinical patient′s sputum sample.The colony as a template for PCR amplification with universal primers to amplify 16S rRNA gene fragments of unknown bacteria.The product of PCR was sequenced directly,then the sequence result was compared by using the BLAST of NCBI and the pathogen was identified based on the sequence homology.Results 1 strain of unknown pathogen was identified as ochrobactrum by this test and confirmed by ABI bacterial rapid identification sys-tem.Conclusion This study simplifies the isolation and identification procedures of unknown pathogen from the clinical samples and establishes a simple method for the rapid identification of pathogens by using 16S rRNA gene amplification.

Recent advances on chlamydial taxonomy and identification / 中国人兽共患病学报

ABSTRACT:Chlamydiae are obligate intracellular bacteria with a unique biphasic developmental cycle ,and have close rela‐tionships with human .Emerging Chlamydia species such as Chlamydia ibidis ,Chlamydia avium and Chlamydia gallinacea changed the chlamydial taxonomy .The biggest change is the abandon of genus Chlamydophilaand its previous six species were recombined into genus Chlamydia ,which in together with the emerging species ,expands genus Chlamydia to 12 species .This paper briefly reviewed the latest taxonomy of Chlamydiae and their identification methods ,which include the classical biological characteristics and physico‐chemical properties ,modern molecular genetics ,and the newly developed whole genome analysis . Presently ,molecular genetics methods ,including sequence analysis of 16S rRNA ,ompA and other Chlamydia‐specific genes , are commonly used for chlamydial identification .

Development of rapid phenotypic system for the identification of Gram-negative oxidase-positive bacilli in resource-limited settings.

Rapid and accurate identification of bacterial pathogens is a fundamental goal of clinical microbiology. The diagnosis and surveillance of diseases is dependent, to a great extent, on laboratory services, which cannot function without effective reliable reagents and diagnostics. Despite the advancement in microbiology diagnosis globally, resourcelimited countries still struggle to provide an acceptable diagnosis quality which helps in clinical disease management and improve their mortality and morbidity data. During this study an indigenous product, Quick Test Strip (QTS) NE, was developed for the rapid identification of biochemically slower group of Gram-negative oxidase-positive bacilli that covers 19 different bacterial genera. Some of the members belonging to these groups are well-established human pathogens, e.g. various species of Vibrio, Pseudomonas, Burkholderia, Aeromonas, Achromobacter and Stenotrophomonas. This study also evaluates the performance of QTS-NE by comparing with genotypic characterization methods. A total of 232 clinical and reference bacterial isolates were tested by three different methods. QTSNE provides 100% concordant results with other rapid identification and molecular characterization methods and confirms the potential to be used in clinical diagnosis.

Evaluation on the capability of CHROMagar orientation medium combined with simple biochemical tests for identificaction of common oxidase-negtive gram-negative bacilli / 中华微生物学和免疫学杂志

Objective To evaluate the practicability of CHROMagar orientation medium combined with simple biochemical tests for identification of common oxidase-negtive gram-negative bacilli.Methods The CHROMagar orientation medium was used together with biochemical tests including indole test , ornithine decarboxylase test and lysine decarboxylase test for identification of common oxidase -negtive gram-negative bacilli.The sensitivity, specificity, likelihood ratio, Youden index and Kappa value of the diagnostic assays were evaluated .McNemar test was performed to evaluate facticity, accuracy and cost of the method in com-parison with the Vitek-2 system as reference method .Results The identification of oxidase-negtive gram-negative bacilli from 318 bacterial strains showed that the sensitivities and specificities of CHROMagar orien-tation mediumm in combination with simple biochemical tests to Serratia marcescens, Stenotrophomonas mal-tophilia and Acinetobacter baumannii reached 100%, and for Escherichia coli, Enterobacter aerogenes and Klebsiella pneumoiae were above 90%.The specificities for identification of Enterobacter cloacae, Klebsiella oxytoca, Citrobacter freundii and Proteus mirabilis were all above 90%, but the sensitivities were around 75%-90%.Kappa values of the assays were above 0.85, howerer, which was only 0.5947 for Citrobacter freundii.McNemar test showed that all P values were above 0.05, and cost of the assays was reduced by 90%.Conclusion CHROMagar orientation medium in combination with simple biochemical tests is a cost-effective assay for identification of common oxidase-negtive gram-negative bacilli .

Evaluation of the capability of three phenotypic tests used alone or in combination for identification of Staphylococcus aureus / 中华微生物学和免疫学杂志

Objective To evaluate the capability of three tests used alone or in combination for identification of Staphylococcus aureus.Methods Identification of Staphylococcus aureus by the detection of spa gene with PCR and the Vitek-2 system were selected as the reference methods.Comparison of three phenotypic tests including DNase,mannitol fermentation and tube coagulase test was carried out to analyze the sensitivity,specificity,positive/negative predictive value,positive/negative likelihood ratio and Youden index.The consistency,cost and related indexes of the assays were analyzed between the combined phenotypic tests and the reference methods.Results In the present study,324 isolates of Staphylococci,including 293 Staphylococcus aureus and 31 non-Staphylococcus aureus,were collected.Single biochemical test could not identify Staphylococcus aureus efficiently.Comparison between the reference methods and the combined three biochemical tests by Kappa statistic analysis indicated that an overall Kappa value was 0.9441,and the algorithm of combined test was less costly.The sensitivity and specificity of this algorithm were 100％ and 90.3％,respectively.Conclusion The cost-effective algorithm of combined DNase,mannitol fermentation and tube coagulase test could efficiently distinguish Staphylococcus aureus from other Staphylococcus species.

Identificación bacteriana basada en el espectro de masas de proteínas: Una nueva mirada a la microbiología del siglo XXI / Bacterial identification based on protein mass spectrometry: A new insight at the microbiology of the 21st century

Bacterial identification is important for the proper treatment of infected patients hospitalized with serious infections especially in critical care units. Identification by conventional methods used in microbiology laboratories takes at least 16 hours since a culture is positive. The introduction of mass spectrometry, specifically MALDI-TOF MS (matrix-assisted laser desorption/ ionization time-of-flight mass spectrometer) in the microbiology laboratory could mean a radical change in the identification accuracy, turn around time (6 minutes per bacteria) and cost (about 5 times cheaper than conventional identification). Since its introduction in clinical microbiology laboratories in 2008, many reports about its usefulness in identifying microorganisms from colonies, as well as directly from positive blood cultures and urine samples have been published. This review describes MALDI-TOF MS methodology, its identification performance for bacteria (aerobic and anaerobic), mycobacterium and yeasts, its future applications in microbiology and its main disadvantages.

La identificación bacteriana es muy importante en el manejo adecuado de los pacientes infectados, especialmente aquellos con infecciones graves hospitalizados en unidades de pacientes críticos. La identificación por los métodos convencionales utilizados en los laboratorios de microbiología clínica demora al menos 16 horas desde que un cultivo es positivo. La introducción de la espectrometría de masas, específicamente del espectrómetro de masas por tiempo de migración (tiempo de vuelo) con desorción/ionización laser asistida por una matriz (MALDI-TOF MS, por su sigla en inglés matrix-assisted laser desorption/ionization time-of-flight mass spectrometer), en el laboratorio de microbiología podría significar un cambio radical en la precisión de la identificación, el tiempo de detección (6 minutos por bacterias) y el costo (aproximadamente 5 veces más económico que la identificación convencional). Desde su introducción en los laboratorios de microbiología clínica en el año 2008, se han escrito numerosas publicaciones sobre su utilidad en la identificación de microorganismos desde colonias, así como directamente desde hemocultivos positivos y de muestras de orina. Esta revisión describe la metodología de MALDI-TOF MS, su rendimiento en la identificación de bacterias aerobias, anaerobias, micobacterias y levaduras, sus futuras aplicaciones en microbiología y sus principales desventajas.

Bacterial identification in the diagnostic laboratory: How much is enough.

The major impetus for bacterial identification came after the advent of solid culture media. Morphological appearance of bacterial colonies was often sufficient for their identification in the laboratory. Even in modern times, preliminary identification of most cultivable bacteria is based on such morphological characters. Advances have been made media for the presumptive identifi cation of common organisms encountered in clinical samples. Phenotypic characterisation of bacteria with, physiological tests with a battery of biochemical tests differentiate related bacterial genera as well as confirm their identity. . Each laboratory can select its own method(s) of identification, provided they are based on scientific / epidemiological evidence; clinical laboratory and standards institute (CLSI) is a widely accepted organization and laboratories in many parts of the world follow its recommendations for bacterial identification. Some of the latest advances in identification include Matrix Assisted Laser Desorption Ionization - Time of Flight Mass Spectroscopy (MALDI-TOF) is a state of art facility used for fast and reliable species-specific identification of bacteria including Mycobacteria and fungi including yeasts. However the single most important factor that decides the method of bacterial identification in any laboratory is the cost involved. In the final analysis, selection of tests for bacterial identification should be based on their standardization with proper scientific basis. Considering the cost and lack of easy availability of commercial kits, we have put forward a simplified and rapid method of identification for most commonly encountered bacterial pathogens causing human infection in India

Isolation, characterization and identification of bacteria from coolant oils.

Coolants are hydrocarbons, used to lubricate parts of machines for smooth performance. While in use, a coolant quickly gets contaminated with foreign materials, making it less effective and unpleasant odors are developed due to microbial action. Hence coolants need to be replaced frequently. The expense of disposing used coolants and replacing it with fresh coolants adds significantly to the manufacturing cost. The present study is focused on isolation, identification and characterization of coolant oil contaminating bacteria as an initial step to solve these contamination problems. Used and unused samples of coolant were collected from oil stations, auto mechanic workshops and steel industry for the isolation of the contaminants. Ten dominant bacterial isolates of the genus Pseudomonas, Staphylococcus, Micrococcus, Salmonella, Cellobiococcus and Pneumonia were identified by morphology, biochemical tests and PIB tool. Isolates were subjected to four different media, various pH and temperatures for characterization of optimal conditions of growth. Pseudomonas pseudomallei, Micrococcus luteus 3, Micrococcus varians and Salmonella ferlac were observed in mineral, synthetic and aerobic media, Staphylococcus hyicus, Cellobiococcus species and Staphylococcus intermedius in synthetic and aerobic fermentation media and Pseudomonas cepacia, Pseudomonas piketti in mineral salt and aerobic fermentation media. The ten isolates showed optimal growth at different temperatures between 20°C and 90°C and different pH, ranging from acidic to alkaline. In conclusion, the used coolants harbor hazardous pathogens such as Pseudomonas species which multiply rapidly and survive high temperatures. These isolates could be targeted for further studies on development of antidotes as a solution to the coolant contamination problems.