Improving the success of culturing Helicobacter pylori from gastric biopsies

Factors influencing the successful isolation of Helicobacter pylori from human gastric biopsies were studied. Within 24 h, each of the gastric biopsies was inoculated onto chocolate blood agar media and incubated for up to 2 weeks. Among 63 (70%) culture positive cases in 90 patients, 58 (64%) cases were culture positive for both specimens, while five (6%) cases were culture positive in only one biopsy. Of the 63 positive cultures, 51 H. pylori strains (81%) grew on both media with and without antibiotics. Eight strains (13%) grew only on medium without antibiotics, while four isolates (6%) were obtained only from medium with antibiotics. These results support the previous histological observation of patchy colonization of H. pylori in the stomach. The success rate for culture of H. pylori from gastric biopsies increased when two biopsies were taken and inoculated on chocolate blood agar media with and without antibiotics.     

Helicobacter canis bacteraemia in a 7-month-old child

On the basis of biochemical, phenotypic and 16S rRNA analyses, Helicobacter canis was isolated and identified from an otherwise healthy 7-month-old girl with intermittent fever. Blood cultures signalled bacterial growth after 5 days that was characterized as small gram-negative spiral rods. Subculturing on Colombia plates with 5% sheep blood, chocolate agar and brucella agar, aerobically and anaerobically as well as in a microaerophilic atmosphere, showed scanty growth after an additional 4 days. Secondarily seeded with fluid from the original bottle, the paediatric blood bottles repeatedly signalled growth after one night's incubation, whereas the conventially treated bottles did not support growth after 7 days' incubation. From the secondary seeded paediatric bottles a pure culture was isolated on chocolate agar plates, and identified as H. canis. This case indicates that blood culture systems should be compared and improved for their capacity to detect Helicobacter and related pathogenic bacteria species. Further studies are also needed to determine the importance of H. canis as a primary pathogen, and the role of cats in the possible zoonotic spread of H. canis to humans.     

Ways of improving the effectiveness of bacteriological diagnosis and treatment of nonspecific lung diseases

Isolation, identification and drug sensitivity assay of microorganisms from pathological materials of 177 patients with nonspecific diseases of the lungs, mainly pneumonia, were performed on blood and selective "chocolate" agars by using Baktofok-MK, a new dry nutrient basis developed by the authors. Blood and "chocolate" agars based on the Hottinger's hydrolysate were used as the control media. It was shown that with the quantitative procedure for inoculating the pathological material, the experimental media based on Baktofok-MK were much more sensitive to growth properties that the control media. That made it possible to detect larger numbers of etiologically important microbial species on the blood agar and to isolate clinical strains of Hemophilus spp. from a larger number of specimens on the "chocolate" agar.     

Transfer ofHaemophilus equigenitalis Taylor et al. 1978 to the genusTaylorella gen. nov. asTaylorella equigenitalis comb. nov.

The taxonomic position ofHaemophilus equigenitalis Taylor et al. 1978, the causative agent of contagious equine metritis, was studied in comparison with phenotypically similar organisms such asMoraxella, Legionella, and others.Haemophilus equigenitalis is a microaerophilic, Gram-negative, nonmotile, short rod; the mean base composition of deoxyribonucleic acid of this organism is 36.5±0.5 mol% G+C. It shows best growth on chocolate agar, but very poor or no growth on plain nutrient agar and blood agar, although it requires no X- or V-factors for the growth. It is positive in catalase, oxidase, phosphatase, and phosphoamidase tests, but very unreactive in other biochemical tests for routine use. It produces no acid from any carbohydrates nor glycosidase. Arylamidase activities of the organism to -naphtylamide derivatives of various amino acids, and di- and tripeptides were also compared with those of other taxa. The group of this organism was different from other known taxa in the numerical analysis of its phenotypic characteristics, DNA base composition, and DNA-DNA hybridization. These data indicate thatH. equigenitalis does not belong in the genusHaemophilus nor other known genera, but rather in a new genus. Therefore, we propose thatHaemophilus equigenitalis be reclassified in a new genusTaylorella asTaylorella equigenitalis.     

Comparison of Macroscopic Examination, Routine Gram Stains, and Routine Subcultures in the Initial Detection of Positive Blood Cultures

Blood was cultured in two vaccum bottles containing Columbia broth with sodium polyanethol sulfonate and CO₂. Filtered air was admitted to one bottle, and the bottles were incubated at 35 C until growth was detected or for a maximum of 7 days. Bottles were examined daily for macroscopic growth. Gram stains were made routinely on the 1st, 4th, and 7th days, and samples were routinely subcultured to sheep blood agar (incubated in GasPak jar) and chocolate agar (incubated in CO₂) on the 1st and 4th days of incubation. Of 1,127 positive blood cultures, 65% were first detected by macroscopic examination, 23% were first detected by Gram stain, and 12% were first detected only by subculture.     

Evaluation of negative results of BacT/Alert 3D automated blood culture system

Although automated continuous-monitoring blood culture systems are both rapid and sensitive, false-positive and false-negative results still occur. The objective of this study, then, was to evaluate negative results occurring with BacT/Alert 3D blood culture systems. A total of 1032 samples were cultured with the BacT/Alert 3D automated blood culture system, using both aerobic (FA) and anaerobic (FN) [corrected] media, and 128 of these samples yielded positive results. A total of 904 negative blood samples were then subcultured in 5% sheep blood agar, eosin methylene blue, chocolate agar, and sabouraud-dextrose agar. Organisms growing on these subcultures were subsequently identified using both Vitek32 (bioMerieux, Durham, NC) and conventional methods. Twenty four (2.6%) of the 904 subcultures grew on the subculture media. The majority (83.3%) of these were determined to be gram-positive microorganisms. Fourteen (58.3%) were coagulase-negative staphylococci, two (8.3%) were Bacillus spp., one (4.2%) was Staphylococcus aureus, and one (4.2%) was identified as Enterococcus faecium. Streptococcus pneumoniae and Neisseria spp. were isolated together in two (8.3%) vials. Gram-negative microorganisms comprised 12.5% of the subcultures, of which two (8.3%) were found to be Pseudomonas aeruginosa, and one (4.2%) was Pseudomonas fluorescens. The other isolate (4.2%) was identified as Candida albicans. We conclude that the subculture of negative results is valuable in the BacT/Alert 3D system, especially in situations in which only one set of blood cultures is taken.     

The fatty acid composition of Haemophilus and Pasteurella multocida cells as a phylogenetic marker

When grown on meat-peptone agar with heated blood, different Haemophilus species (H. influenzae, H. parahaemolyticus, H. parasuis, H. pleuropneumoniae), including different H. influenzae serovars (a, b, c, d, e, f), and Pasteurella multocida have identical fatty acid composition, characterized by the prevalence of fatty acids with 16 carbon atoms, constituting about 70% and more of the total number of fatty acids, and a low level of fatty acids with 18 carbon atoms. P. multocida strains cultivated on meat-peptone agar with unheated blood have a greatly increased content of fatty acids with 18 carbon atoms, while the content of fatty acids with 16 carbon atoms is much lower. The identity of fatty acid composition under similar cultivation conditions, together with their similarity in other phenetic signs, is indicative of close phylogenic relationship between bacteria belonging to the genus Haemophilus and P. multocida.     

Chrysosporium species, potential spoilage organisms of chocolate

Standard methods of analysing foods for the presence of moulds are inadequate for thedetection of genera such as Chrysosporium which do not grow at the high wateractivities of most mycological media. The use of malt, yeast, 50% glucose agar (MY50G) insealed containers as an enrichment medium allowed time for germination and growth ofheat-stressed spores. Three Chrysosporium spp., C. xerophilum Pitt, C. inops (Carmichael) and C. farinicola (Burnside) Skou, were isolated fromcommercial chocolate bars with a water activity (a _w ) of approximately0·28. Chrysosporium inops was isolated from commercial milk crumb and anew Chrysosporium sp. was isolated from Ghanaian cocoa beans. In chocolates madeby coating MY50G agar (a_w = 0·89) with chocolate (a_w = 0·27) containing C. inops arthroconidia, two types of deterioration were seenafter storage. The first was fat bloom due to recrystallization of the cocoa butter on the outer andinner chocolate surface. The second was growth of C. inops which occurred on theinside chocolate surface adjacent to the MY50G agar filling and on the outside surface afterholding at 92% equilibrium relative humidity (erh) for 12 d. There was some evidence that C. inops could grow on the outside of chocolates held at 5·7% erh after 4months' storage at 25 °C. The appearance of the white fungal growth was not unlikefat bloom to the naked eye but was clearly different with the electron microscope.     

Morphology of the type strain of Bacillus anthracis EY 3169T=ATCC 14578T grown either aerobically or anaerobically on agar plates--observation by light and laser microscopes

Growth characteristics including cell-arrangement of the type strain of Bacillus anthracis EY 3169T=ATCC 14578T grown on agar plates in level 3 laboratory were observed by both light and laser microscopes. Small daughter colonies appeared on parent colonies grown on 5% sheep blood or chocolate agar plates after 12 days incubation at room temperature. Daughter colonies, stained by Wirtz-Conklin method, were composed with vegetative cells and spores. Growth of daughter colonies might be supported by the debris of cells in the parent colony. Colonies grown under anaerobic conditions were flat with smooth edges, and the cells neither formed chains of any length, nor produced any spores after 25 days incubation at room temperature. It was thought that spores of B. anthracis were produced at the terminal stage of individual cell life instead of under unfavorable conditions for the organism. Air is needed for spore formation and cell-chain formation. More nutrients, probably amino acids, are needed for anaerobic growth rather than aerobic.     

Broth versus solid agar culture of swab samples of cadaveric allograft musculoskeletal tissue

As part of the donor assessment protocol, bioburden assessment must be performed on allograft musculoskeletal tissue samples collected at the time of tissue retrieval. Swab samples of musculoskeletal tissue allografts from cadaveric donors are received at the microbiology department of the South Eastern Area Laboratory Services (Australia) to determine the presence of bacteria and fungi. This study will review the isolation rate of organisms from solid agar and broth culture of swab samples of cadaveric allograft musculoskeletal tissue over a 6-year period, 2006–2011. Swabs were inoculated onto horse blood agar (anaerobic, 35 °C) and chocolate agar (CO₂, 35 °C) and then placed into a cooked meat broth (aerobic, 35 °C). A total of 1,912 swabs from 389 donors were received during the study period. 557 (29.1 %) swabs were culture positive with the isolation of 713 organisms, 249 (34.9 %) from solid agar culture and an additional 464 (65.1 %) from broth culture only. This study has shown that the broth culture of cadaveric allograft musculoskeletal swab samples recovered a greater amount of organisms than solid agar culture. Isolates such as Clostridium species and Staphylococcus aureus would not have been isolated from solid agar culture alone. Broth culture is an essential part of the bioburden assessment protocol of swab samples of cadaveric allograft musculoskeletal tissue in this laboratory.     

Evaluation of differential media for the identification of Corynebacterium genitalium and Corynebacterium pseudogenitalium (group JK corynebacteria)

Tween purple agar containing 1% fructose (TFP agar) differentiated Corynebacterium genitalium from C. pseudogenitalium, which respectively formed colorless and yellow colonies after 72 h incubation at 37 degrees C aerobically or in 5-10% CO2 in air. Thus TFP agar is a differential medium. Corynebacteria-like colonies grown on nonspecific urethritis (NSU) chocolate agar from urogenital material were identified as C. genitalium, C. pseudogenitalium, or commensals when subcultured on TPF agar. TFP agar was unsuitable for their primary isolation since the commensals turned the medium yellow with 24 h incubation. Gentamicin cannot be employed as a selective agent in medium for the isolation of these corynebacteria. TFP agar containing 10 micrograms/mL entamicin inhibited most strains of C. pseudogenitalium and C. genitalium isolated from urogenital infections. It did not inhibit isolates of these corynebacteria from cancer patients or suppress the normal bacterial flora of the urogenital tract. Evidence that gentamicin-resistant strains are characteristic of nosocomial infections is presented.     

A new MSPQC system for rapid detection of pathogens in clinical samples

A new multi-channel series piezoelectric quartz crystal (MSPQC) system for detection of pathogens in clinical sample was proposed. Some factors, which affect the detection of pathogens by using MSPQC, were all investigated. A total of 650 clinical samples were detected by MSPQC and compared with licensed BACTEC 9120 system (Becton Dickinson Diagnostic Instrument Systems, Sparks, MD, USA) simultaneously in the Third Xiangya Hospital of Central South University, China. When the incubation period was 5 days, two systems had similar detected results: the MSPQC system detected 123 growth of 650 (18.92%) bottles while the BACTEC 9120 detected 125 growth of 650 (19.23%) bottles. The MSPQC had 2 false-positive signals and 2 false-negative signals. However, BACTEC 9120 had 3 false-positive signals and 0 false-negative signals. Further identifications of bacteria were run by VITEK-2 (bioMérieux China Ltd.), 5% sheep blood trypticase soy agar (SBA) and chocolate agar (CA). Comparing with BACTEC 9120, MSPQC system possesses following advantages: shorter average detection time, less blood volume needed, less false-positive results and low cost. It can also provide information in real time. So MSPQC has a wonderful perspective in clinical application.     

Species-identification and antimicrobial susceptibility tests by the fully automated RAISUS using an early-harvested cell suspension]

We evaluated the usefulness of an early-harvested bacterial cell suspension to the fully automated RAISUS (Nissui Pharmaceuticals Co., Ltd., Tokyo) to provide the results of species-identification and antimicrobial susceptibility testings within a day after overnight-incubation of the primary cultures. A single, well-separated colony appeared on the primary culture plate was transferred onto a blood agar or chocolate agar plates, then incubated for 3 to 6 hours. The cell suspension to the RAISUS was properly prepared to the McFarland 0.5 turbidity from the early-harvested bacterial cells. When the five ATCC reference strains, consisting of Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Streptococcus pneumoniae ATCC 49619, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853, were repeatedly tested for the species-identification, all the identification results were acceptable. Antimicrobial susceptibility tests were evaluated with the above five strains and Haemophilus influenzae ATCC 49247. The results obtained indicated that the most susceptibility test results were comparable to those MICs obtained by the standard test procedure, but some strains, in particular, H. influenzae and P. aeruginosa gave significantly discrepant MICs for certain antimicrobial agents. The significant discrepancy in MIC determinations regarded the difference of viable cell concentrations in the cell suspension prepared respectively. Through the analysis of laboratory workflow, it became to apparent that 18S to 20S of the tests were completed by 5:00 p.m., and it required to wait until 3:00 a.m. to complete 90S of the tests. With these results, the early-harvested bacterial cell suspension is applicable to species-identification by RAISUS, but it is necessary to adjust viable cell concentrations to antimicrobial susceptibility test. Also, it is urgent to reconstitute a daily workflow to improve the rapidity of RAISUS test function.     

Serological relationship of some V-factor dependent Pasteurellaceae (Haemophilus sp.) from guineapigs and rabbits

Culture of guinea pig and rabbit respiratory tracts for bacteria using X- (haemin) and V- (NAD) factor in agar media detected infection by V-factor dependent Pasteurellaceae (Haemophilus sp.) in three colonies of guinea pigs and a group of rabbits. The 12 Haemophilus strains comprised three API NH codes classed as Haemophilus parainfluenzae and two codes classed as Haemophilus aphrophilus/paraphrophilus. Six cell wall lipid profiles were detected, but these were not related to API NH codes. Both bacteriological properties were used to select strains for serological studies but any relationship between bacteriological and serological properties of the Haemophilus strains was not evident. Varying serological relationships occurred between the newly isolated Haemophilus strains, [Pasteurella] pneumotropica NCTC 8284 and Haemophilus strains previously isolated from rats.     

Selective Culture Medium to Survey the Incidence of Haemophilus Species

A culture medium for the selective isolation of Haemophilus species is described. Bacitracin and nutritional supplements were incorporated in a rich basal agar medium to which rabbit blood was added to distinguish hemolytic species. Colony counts of seven typed strains of H. influenzae on this medium were within practical limits of counts on other media tested for clinical use. The bacitracin medium was as reliable as hemoglobin-agar for detecting H. influenzae and more sensitive for detecting other Haemophilus species in a clinical survey with the advantage of selectivity.     

Culture and morphological characteristics of Haemophilus influenzae and pneumococcus in bronchial infection]

In the cultures obtained by inoculating sputum samples faken from patients with bronchial infection into solid agar medium prepared on Hottinger's hydrolysate with fresh rabbit blood added Haemophilus influenzae produced colonies varying in their from (dome-shaped, conical, trapeziform), as well as in the morphology of the organisms. Pneumococci produced mainly flat colonies surrounded by the zone of alpha hemolysis. Along-side with isolated H. influenzae and pneumococcal colonies, symbiotic colonies could be observed. In these colonies pneumococci were diffused among H. influenzae.     

Rapid serotyping of campylobacters based on heat-stable antigens, using a combined passive haemagglutination/co-agglutination technique

Tryptic soy broth/agar, brain heart infusion agar and Columbia broth/agar, all widely used in the bacteriological laboratory, were radiation-sterilized at a dose of 10–15 kGy. The media were tested for the growth of 12 different micro-organisms, including fastidious pathogens such as Streptococcus pyogenes, Strep, pneumonias, Haemophilus influenzas and Neisseria meningitidis. Solid and fluid media supplemented with catalase before irradiation performed well in comparison with heat-sterilized media.     

Chocolate: (un)healthy source of polyphenols?

There is recent epidemiological evidence that chocolate consumption may improve vascular health. Furthermore, several small-scale human intervention studies indicate that habitual chocolate intake enhances the production of vasodilative nitric oxide and may lower blood pressure. It is hypothesized that potential beneficial effects of chocolate on vascular health are at least partly mediated by cocoa polyphenols including procyanidins. Based on cell culture studies, molecular targets of chocolate polyphenols are endothelial nitric oxide synthetase as well as arginase. However, human bioavailability studies suggest that the plasma concentrations of cocoa polyphenols are manifold lower than those concentrations used in cultured cells in vitro. The experimental evidence for beneficial vascular effects of chocolate in human interventions studies is yet not fully convincing. Some human intervention studies on chocolate and its polyphenols lack a stringent study design. They are sometimes underpowered and not always placebo controlled. Dietary chocolate intake in many of these human studies was up to 100 g per day. Since chocolate is a rich source of sugar and saturated fat, it is questionable whether chocolate could be recommended as part of a nutrition strategy to promote vascular health.     

利用鸡胚浸出液制作细菌培养基的研究

利用鸡胚浸出液制作的肉汤和琼脂平板培养基均能适合副鸡嗜血杆菌、大肠杆菌和沙门氏菌的生长,用平板计数法、麦氏比浊法和离心称重法分别对副鸡嗜血杆菌、大肠杆菌和沙门氏菌的培养产物进行含菌量、菌泥湿重测定,结果表明,鸡胚浸出济培养基培养的沙门氏菌产量是普通培养基培养的沙门氏菌产量的20倍以上,培养的副鸡嗜血杆菌产量是普通培养的3倍以上,而鸡胚浸出液培养基与普通培养基对大肠杆菌的培养数量则无明显差异。