Identification of bacteria from clinical samples using Bartonella alpha-Proteobacteria growth medium

In an effort to overcome historical problems associated with the isolation of Bartonella species from animal and human blood samples, our laboratory developed a novel, chemically modified, insect-based, liquid culture medium (Bartonella alpha-Proteobacteria growth medium, BAPGM). In this study, we describe the isolation of non-Bartonella bacteria from aseptically obtained human blood and tissue samples that were inoculated into BAPGM pre-enrichment culture medium, and were obtained during attempts to define each individuals Bartonella infection status. After incubation for at least 7 days in liquid BAPGM, pre-enriched inoculums were sub-cultured onto a BAPGM/blood agar plate. Bacterial DNA was extracted from pooled plated colonies and amplified using conventional PCR targeting the 16S rRNA gene. Subsequently, amplicons were cloned, sequenced and compared to GenBank database sequences using the BLAST program. Regardless of the patient's Bartonella status, seventeen samples generated only one 16S rDNA sequence, representing the following genera: Arthrobacter, Bacillus, Bartonella, Dermabacter, Methylobacterium, Propionibacterium, Pseudomonas, Staphylococcus and bacteria listed as "non-cultured" in the GenBank database. Alkalibacterium, Arthrobacter, Erwinia, Kineococcus, Methylobacterium, Propionibacterium, Sphingomonas, and Staphylococcus were isolated from nine Bartonella-infected individuals. Co-isolation of Acinetobacter, Sphingomonas, Staphylococcus spp. and bacteria listed as "non-cultured" in the GenBank database was achieved for four samples in which Bartonella spp. were not detected. Despite the phylogenetic limitations of using partial 16S rRNA gene sequencing for species and strain identification, the investigational methodology described in this study may provide a complementary approach for the isolation and identification of bacteria from patient samples.     

巴尔通体液体培养条件简化及生长曲线观察

【目的】应用一种昆虫细胞培养基作为基础成分培养巴尔通体(Bartonella species),建立一种操作方便、高效稳定的巴尔通体液体培养方法。【方法】昆虫细胞培养基中添加10%胎牛血清,以此为基础培养液分别添加蔗糖和谷氨酰胺,比较这两种成分对汉赛巴尔通体(B.henselae)和五日热巴尔通体(B.quintana)生长的影响并观察其他10种巴尔通体在简化后的培养液中的生长特性。【结果】添加蔗糖和谷氨酰胺不会明显促进巴尔通体的生长,10种巴尔通体在简化后的培养液中均生长良好。不同种巴尔通体生长曲线不同,汉赛巴尔通体和五日热巴尔通体的世代时间分别为5.2 h和4.3 h,生长速度快于固体培养。【结论】以昆虫细胞培养基作为基础成分的培养液适于巴尔通体液体培养,特别是对一些更难培养的巴尔通体提供了一种较好的培养方法。     

Isolation of Salmonellae from Foods Samples: V. Determination of the Method of Choice for Enumeration of Salmonella

Comparison of three methods by which salmonellae may be isolated and enumerated from dried albumen, direct inoculation of enrichment media, centrifugation of samples, and pre-enrichment in noninhibitory media, reveals pre-enrichment to be the method of choice.

The superiority of pre-enrichment manifests itself in replicate aliquots of the same sample by producing a statistically significant increase in numbers of isolations of salmonellae and in empirical use with various albumen samples by consistently higher values of most probable numbers (MPN).

The primary factor involved in this superiority appears to be the greater ability of small numbers of salmonellae to initiate growth in the nonselective mannitol purple sugar broth than in the inhibitory enrichment media.

The method of analysis recommended entails inoculation of mannitol broth pre-enrichment medium, transfer of 24-hr culture aliquots to tetrathionate broth, and streaking on brilliant green agar for isolation of salmonellae.

     

人体肠道细菌寡营养培养组条件的优化研究

【目的】探讨寡营养对人体肠道细菌培养组的条件。【方法】通过稀释富集培养基、固体平板和增菌肉汤培养基成分获得寡营养培养基。对健康人粪便样本分别用原液(0)、5、10、20、30和40倍稀释的富集培养基(添加羊血和瘤胃液的血培养瓶)连续增菌,在不同时间点(第0、3、6、9、15、27、30天)吸取增菌液,用YCFA (yeast casitone fatty acid)固体培养平板分离菌落;用YCFA增菌肉汤增菌后再次挑取单菌落,利用基质辅助激光解吸/电离飞行时间(matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,MALDI-TOF)质谱和16S rRNA基因测序鉴定菌株。通过比较上述6种寡营养条件分离肠道菌群的效果,选取富集培养基原液、稀释10倍和30倍这3 种条件下分离效果较好的富集条件,与同样稀释倍数条件的固体平板和增菌肉汤分别组合成9种培养基条件,进一步优化肠道菌群的培养组条件。【结果】在6种寡营养富集培养基中,未稀释(原液)、10 倍和30倍稀释的富集培养基分离细菌的种类比其他...     

Performance of Selective and Differential Media in the Primary Isolation of Yeasts from Different Biological Samples

In view of the increase in yeast infections, especially polymicrobial ones, differential culture media have acquired increasing importance. The present study evaluated the Sabouraud chloramphenicol, Biggy agar, Pagano Levin agar and CHROMagar Candida media in terms of isolation, number of yeast colony forming units per plate, and inhibition of bacteria and filamentous fungi. To this end, we used 223 biological samples, including feces, and oral, vaginal and anal mucosae from 86 patients presenting or not symptoms of fungal infections. The four media did not differ significantly in terms of detection of yeast-positive cultures. The number of colony forming units per plate ranged from zero to 2.380, with a predominance of counts of 1 to 9 colonies per plate. No significant differences were observed among the four culture media in terms of number of colonies counted, for each kind of biological material. Fifteen species belonging to the genera Candida, Saccharomyces, Cryptococcus, Trichosporon and Rhodotorula were isolated, with C. albicans being the predominant species, followed by C. parapsilosis and R. rubra. CHROMagar Candida and Biggy agar were complementary in the isolation of the different species and favored a greater recovery of polymicrobial cultures. Pagano Levin agar isolated the smallest variety of species. Sabouraud chloramphenicol agar was the least effective in terms of bacterial inhibition and favored a greater development of filamentous fungi. The results suggest that more than one culture medium should be used for an adequate primary isolation.     

Bartonella spp. Bacteremia in Blood Donors from Campinas,Brazil

Bartonella species are blood-borne, re-emerging organisms, capable of causing prolonged infection with diverse disease manifestations, from asymptomatic bacteremia to chronic debilitating disease and death. This pathogen can survive for over a month in stored blood. However, its prevalence among blood donors is unknown, and screening of blood supplies for this pathogen is not routinely performed. We investigated Bartonella spp. prevalence in 500 blood donors from Campinas, Brazil, based on a cross-sectional design. Blood samples were inoculated into an enrichment liquid growth medium and sub-inoculated onto blood agar. Liquid culture samples and Gram-negative isolates were tested using a genus specific ITS PCR with amplicons sequenced for species identification. Bartonella henselae and Bartonella quintana antibodies were assayed by indirect immunofluorescence. B. henselae was isolated from six donors (1.2%). Sixteen donors (3.2%) were Bartonella-PCR positive after culture in liquid or on solid media, with 15 donors infected with B. henselae and one donor infected with Bartonella clarridgeiae. Antibodies against B. henselae or B. quintana were found in 16% and 32% of 500 blood donors, respectively. Serology was not associated with infection, with only three of 16 Bartonella-infected subjects seropositive for B. henselae or B. quintana. Bartonella DNA was present in the bloodstream of approximately one out of 30 donors from a major blood bank in South America. Negative serology does not rule out Bartonella spp. infection in healthy subjects. Using a combination of liquid and solid cultures, PCR, and DNA sequencing, this study documents for the first time that Bartonella spp. bacteremia occurs in asymptomatic blood donors. Our findings support further evaluation of Bartonella spp. transmission which can occur through blood transfusions.     

Establishment and follow-up of bifidobacterial species in the gut of healthy bottle-fed infants of 1–4 months age

Twenty-one healthy bottle-fed infants were screened monthly (1-4 months) for bifidobacteria in their stools. Bifidobacteria were detected by culture and isolates specified by PCR. Alternatively, direct PCR in undiluted fecal suspensions was carried out for detection of bifidobacteria under the cultural detection level. All infants harbored cultivable bifidobacteria throughout the study period. Beerens medium was shown to permit a better recovery of bifidobacteria than MRS and horse blood Columbia agar. Direct PCR detection proved valuable in detecting species for which no cultural isolate could be recovered since the species were under the cultural detection level. B. bifidum, B. longum-infantis and B. breve were confirmed as dominant and stable species in infant stools while B. adolescentis and B. catenulatum group exhibited unstable colonization profiles. A trend towards B. breve decrease began at month 3 while carriage of the B. catenulatum group and B. adolescentis was rising. This observation warrants further analysis to assess a possible switch occurring at month 3 in bottle-fed infants, between so-called infant and adult bifidobacterial species.     

Bartonella species in bat flies (Diptera: Nycteribiidae) from western Africa

Bat flies are obligate ectoparasites of bats and it has been hypothesized that they may be involved in the transmission of Bartonella species between bats. A survey was conducted to identify whether Cyclopodia greefi greefi (Diptera: Nycteribiidae) collected from Ghana and 2 islands in the Gulf of Guinea harbour Bartonella. In total, 137 adult flies removed from Eidolon helvum, the straw-coloured fruit bat, were screened for the presence of Bartonella by culture and PCR analysis. Bartonella DNA was detected in 91 (66·4%) of the specimens examined and 1 strain of a Bartonella sp., initially identified in E. helvum blood from Kenya, was obtained from a bat fly collected in Ghana. This is the first study, to our knowledge, to report the identification and isolation of Bartonella in bat flies from western Africa.     

A polymerase chain reaction for the detection of Brucella canis in semen of naturally infected dogs

The objective was to evaluate a PCR assay for the detection of Brucella canis in canine semen, comparing its performance with that of bacterial isolation, serological tests and PCR assay of blood. Fifty-two male dogs were examined clinically to detect reproductive abnormalities and their serum was tested by the rapid slide agglutination test, with and without 2-mercaptoethanol (2ME-RSAT and RSAT, respectively). In addition, microbiological culture and PCR assays were performed on blood and semen samples. The findings of the semen PCR were compared (Kappa coefficient and McNemar test) to those of blood PCR, culture of blood and semen, RSAT, and 2ME-RSAT. Nucleic acid extracts from semen collected from dogs not infected with B. canis were spiked with decreasing amounts of B. canis RM6/66 DNA and the resulting samples subjected to PCR. In addition, semen samples of non-infected dogs were spiked with decreasing amounts of B. canis CFU and the resulting suspensions were used for DNA extraction and amplification. Of the 52 dogs that were examined, the following tests were positive: RSAT, 16 (30.7%); 2ME-RSAT, 5 (9.6%); blood culture, 14 (26.9%); semen culture, 11 (21.1%); blood PCR, 18 (34.6%); semen PCR, 18 (34.6%). The PCR assay detected as few as 3.8 fg of B. canis DNA experimentally diluted in 444.9 ng of canine DNA (extracted from semen samples of non-infected dogs). In addition, the PCR assay amplified B. canis genetic sequences from semen samples containing as little as 1.0 x 10(0) cfu/mL. We concluded that PCR assay of semen was a good candidate as a confirmatory test for the diagnosis of brucellosis in dogs; its diagnostic performance was similar to blood culture or blood PCR. Furthermore, the PCR assay of semen was more sensitive than the 2ME-RSAT or semen culture. Examination of semen by PCR should be included for diagnosis of brucellosis prior to natural mating or AI; in that regard, some dogs that were negative on serological and microbiological examinations as well as blood PCR were positive on PCR of semen.     

Detection of Legionella pneumophila in bioaerosols by polymerase chain reaction

Most studies focusing on detecting microorganisms in air by polymerase chain reaction (PCR) have used a liquid impinger to sample bioaerosols, mainly because a liquid sample is easy to be processed by PCR analysis. Nevertheless, the use of multiple-hole impactors for the analysis of bioaerosols by PCR has not been reported despite its great utility in culture analysis. In this study we have modified the impaction onto an agar surface sampling method to impaction onto a liquid medium using the MAS-100 air sampler (Merck) (single-stage multiple-hole impactor). To evaluate the recovery of airborne microorganisms of both sampling methods, a suspension containing Escherichia coli was artificially aerosolized and bioaerosols were collected onto Tergitol-7 agar and phosphate-buffered saline (PBS) with the MAS-100. A linear regression analysis of the results showed a strong positive correlation between both sampling methods (r = 0.99, slope 0.99, and y intercept 0.07). Afterwards, the method of impingement into a liquid medium was used to study airborne Legionella pneumophila by PCR. A total of 64 samples were taken at a wastewater treatment plant, a chemical plant, and an office building and analyzed by culture and PCR. Results showed that three samples were positive both by PCR and plate culture, and that nine samples negative by plate culture were positive by PCR, proving that L. pneumophila was present in bioaerosols from these three different environments. The results demonstrate the utility of this single-stage multiple-hole impactor for sampling bioaerosols, both by culture and by PCR.     

Detection of Salmonella enterica in Naturally Contaminated Liquid Eggs by Loop-Mediated Isothermal Amplification, and Characterization of Salmonella Isolates

Loop-mediated isothermal amplification (LAMP) assay was effective in detecting Salmonella enterica in naturally contaminated liquid egg samples. Salmonella was detected in 110 samples taken from four egg-breaking plants. The egg samples were pre-enriched in buffered peptone water (BPW) at 37°C for 20 h. The selective enrichment was done in Rappaport-Vassiliadis or tetrathionate broth and plated onto xylose lysine deoxycholate agar and brilliant green agar, modified. In addition, the PCR assay was used to detect Salmonella after pre-enrichment in BPW at 37°C for 20 h. The culture method and PCR assay were compared to the LAMP assay, which was also performed after pre-enrichment in BPW. PCR failed to detect Salmonella in 10% of 110 samples, whereas the culture method and LAMP assay successfully identified Salmonella in all samples. However, the LAMP assay was found to be much more rapid than the culture method and as sensitive in detecting Salmonella from liquid eggs. In all of the egg-breaking plants studied, Salmonella was isolated on most tested days. The positive samples showed that more than 75% of the Salmonella strains had identical genetic patterns when analyzed by pulsed-field gel electrophoresis. This suggests that the same Salmonella strains having survived long periods of time in the plants were contaminating the production line. The LAMP assay is rapid, specific, and sensitive for Salmonella detection in liquid eggs and is able to monitor Salmonella contamination in egg-handling plants more reliably.     

Application of PCR for detection and identification of mycoplasma contamination in virus stocks

Summary A nested polymerase chain reaction (PCR) was used to detect and identify mycoplasma contaminants in viral stocks. The results of the PCR assay proved to be a sensitive and accurate indicator of the true status of the stock tested. Those samples positive by agar culture or Hoechst stain were also positive by PCR. Those samples that were inconclusive by Hoechst stain (10.05%) could be clearly determined to be mycoplasma positive or negative by PCR. The PCR assay also detected those fastidious species of mycoplasma that gave false negative results by the direct culture method. In many respects the PCR-based mycoplasma detection method described is superior to the agar culture and Hoechst staining detection methods. In this study, the PCR assay detected substantially more mycoplasma-positive viral stocks than did the agar culture assay. Due to its speed, sensitivity, and reliability, the PCR assay is of particular value in monitoring the process of removing mycoplasma from contaminated stocks. Furthermore, the PCR amplification products can be analyzed by restriction analysis to rapidly identify the species of the mycoplasma contaminating the stock tested.     

Development of real-time PCR methods for the rapid detection of low concentrations of Gluconobacter and Gluconacetobacter species in an electrolyte replacement drink

AIMS: To develop a rapid real-time polymerase chain reaction (PCR) method to detect Gluconobacter and Gluconacetobacter species in electrolyte replacement drinks. METHODS AND RESULTS: Samples of electrolyte replacement drinks were artificially contaminated with Gluconobacter species and then filtered to collect cells. DNA was extracted from the filters and analysed by real-time PCR on the ABI Prism 7000 system, using commercial detection kits for lactic and acetic acid bacteria. In addition, specific primers and Taqman probe were designed and used for the detection of seven Gluconobacter and Gluconacetobacter species. All the assays tested demonstrated a linear range of quantification over four orders of magnitude, suggesting detection levels down to 1 CFU ml(-1) in the original drink. CONCLUSIONS: A real-time PCR method was developed to detect low concentrations of Gluconobacter and Gluconacetobacter sp. in an electrolyte replacement drink. SIGNIFICANCE AND IMPACT OF THE STUDY: Real-time PCR methods allow a rapid, high throughput and automated procedure for the detection of food spoilage organisms. The real-time PCR assay described is as sensitive as the conventional method that involves pre-enrichment, enumeration on a selective agar (typically malt extract agar) and identification with a differential medium (typically Wallerstein nutrient agar). The real-time PCR assay also provides a more rapid rate of detection, with results in less than 24 h following enrichment for Gluconobacter and Gluconacetobacter species.     

Evaluation of a rapid and inexpensive liquid culture system for the detection of Mycobacterium avium subsp. paratuberculosis in bovine faeces

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of paratuberculosis, a chronic granulomatous enteric disease of ruminants. MAP detection by faecal culture provides the definitive diagnosis of the infection. Automated liquid systems for MAP culture are more sensitive and rapid than culture on solid media, but they are expensive and require specialised equipment. In this study, a non-automated culture method using a modified Middlebrook 7H9 liquid medium (7H9+) was compared with Herrold's solid medium (HEYM) and direct real-time PCR on dairy cattle faeces. MAP growth in 7H9+ was monitored weekly by real-time PCR until the 12th week post-inoculation. The analytical sensitivity of the three methods was evaluated using faecal samples from a healthy cow spiked with ten-fold dilutions of MAP organisms (10⁴-10^− 1) and naturally MAP-infected faeces serially diluted 1 to 10 in negative faecal samples. The limits of detection of the solid culture and direct real-time PCR were 10² and 10³ MAP/g, respectively. In comparison, the 7H9+ culture revealed as few as 1 MAP/g. A marked reduction in time to detection of the pathogen, compared with HEYM culture, was obtained. In addition, the three methods were applied to environmental faecal samples collected from a high- and a low-prevalence herd. The culture in 7H9+ showed to be the most sensitive test in the low-prevalence herd and provided faster results than HEYM. In the high-prevalence herd the three methods showed the same sensitivity and the real-time PCR had the shortest turnaround time. In conclusion, the use of 7H9+ for MAP-detection from cattle faeces maximizes diagnostic sensitivity and reduces turnaround time and, therefore, could replace culture in solid medium. Hence, we propose a two-step protocol for the assessment of MAP faecal excretion based on: 1) direct real-time PCR on all samples; and 2) inoculation of negative samples into 7H9+ and analysis after 3 and, if necessary, 6 weeks by real-time PCR.     

Selective media for Pasteurella pneumotropica

Bacteria from the subcutaneous abscesses which appeared in a laboratory colony of DS mouse since October of 1977 were identified as Pasteurella pneumotropica by various biological examinations. The abscess formation was limited to multiparous female mice over 100 day-age, but virgin females were free from the disease. The MIC of various antibacterial substances showed that potassium tellurite, kanamycin and bacitracin were effective to isolate the organism selectively from various infection sites harboring many other species of bacteria. A novel NKBT medium was prepared by adding these antibacterial substances to the heart infusion agar (HIA) supplemented by 10% Fildes digested blood. A fluid culture medium, TGN broth was prepared for multiplication of the organism by adding 10% Fildes digested blood and potassium tellurite to GN broth. To isolate the organism from the pharyngo-larynx a direct application of mucus wiped off the infection site onto the culture medium was sufficient, but pre-multiplication in the TGN broth was required for isolation of the organism from gut contents before inoculation onto the NKBT medium. The pre-cultivation in the TGN broth vastly improved the recovery of the organism especially from feces. Thereby we could easily detect the latent infection of this bacterium without sacrificing animals.     

Evaluation of different DNA extraction procedures for the detection of Salmonella from chicken products by polymerase chain reaction

Polymerase chain reaction (PCR) was used after a short pre-enrichment culture to detect Salmonella subspecies in chicken fillets. A direct PCR assay performed with chicken meat inoculated with Salmonella Typhimurium produced no PCR products. Six different DNA extraction protocols were tested to recover efficiently Salmonella DNA after a short incubation period. Three of them gave results that were reliable, rapid and sensitive. Successful protocols used Proteinase K and/or a centrifugation step to concentrate the samples. For reliable detection of Salmonella subspecies, a few thousand bacterial cells per ml must be present. To obtain this number of bacterial cells with an inoculation of about one cell in 25 g of ionized food products, it was necessary to incubate samples for at least 10 h before PCR. A larger inoculum of approximately 10 cells in 25 g of ionized food products, required 8 h in culture broth to give positive results by PCR-based assay.     

A comparison of isolation procedures for salmonellas from polluted water using two forms of Rappaport's medium

F ricker , C.R. 1984. A comparison of isolation procedures for salmonellas from polluted water using two forms of Rappaport's medium. Journal of Applied Bacteriology 56 , 305–309.
The efficiency of Rappaport's broth (RB10) and Rappaport's broth containing novobiocin (NRB10) were compared for the isolation of salmonellas from polluted water, both as direct enrichment media and after pre-enrichment in buffered peptone water. Ninety samples were examined and 41 were found to contain salmonellas by at least one of the procedures used. Direct inoculation of the sample into RB10 resulted in the recovery of salmonellas from only 29.3% of the samples found to be positive. The use of NRB10 as a direct enrichment medium increased the percentage recovery to 78.0% of the total positive samples. Pre-enrichment in buffered peptone water allowed the recovery of salmonellas from a total of 41 samples whereas direct enrichment recovered them from only 32. No significant difference was demonstrated in the efficiencies of RB10 and NRB10 in recovering salmonellas after pre-enrichment in buffered peptone water. Three selective agars were used; no significant difference in their ability to recover salmonellae was demonstrated.     

Improved Rapid Plate Method for the Isolation of Bacteriophages from Lysogenic Bacteria

A modification of a recently reported rapid plate method for the isolation of bacteriophages from lysogenic bacteria is described. The velveteen replica plate technique was used for inoculation of mitomycin C-induced colonies onto agar plates, and tetrazolium chloride was used to enhance detection of phage activity on replicated indicator plates.     

Choice of nutrient media for the laboratory diagnosis of Streptococcus group B

The comparative study of a large assortment of liquid and solid culture media used for the cultivation of streptococci in laboratory practice in the USSR and abroad was carried out with the aim of selecting the optimal media for the laboratory diagnosis of group B. streptococci. Liquid media were tested with the use of 7 streptococcal reference strains, and some of these media, found to yield the best results, were selected for tests on clinical material. The use of liquid accumulation media was shown to permit the isolation of group B. streptococcal strains which could not be detected by the direct inoculation of clinical material into dishes with blood agar. The character of hemolysis induced by group B. streptococci in solid media with 5% of blood added was found to depend on the composition of the medium and the conditions of cultivation.     

A comparison of isolation procedures for salmonellas from polluted water using two forms of Rappaport's medium

The efficiency of Rappaport's broth ( RB10 ) and Rappaport's broth containing novobiocin ( NRB10 ) were compared for the isolation of salmonellas from polluted water, both as direct enrichment media and after pre-enrichment in buffered peptone water. Ninety samples were examined and 41 were found to contain salmonellas by at least one of the procedures used. Direct inoculation of the sample into RB10 resulted in the recovery of salmonellas from only 29.3% of the samples found to be positive. The use of NRB10 as a direct enrichment medium increased the percentage recovery to 78.0% of the total positive samples. Pre-enrichment in buffered peptone water allowed the recovery of salmonellas from a total of 41 samples whereas direct enrichment recovered them from only 32. No significant difference was demonstrated in the efficiencies of RB10 and NRB10 in recovering salmonellas after pre-enrichment in buffered peptone water. Three selective agars were used: no significant difference in their ability to recover salmonellae was demonstrated.