Detection of Antibody-Coated Bacteria in Expectorated Sputum for Diagnosis of Lower Respiratory Infections

We evaluated antibody-coated bacteria (ACB) in expectorated sputum to discriminate contaminating or colonizing organisms from true pathogens. We examined 60 expectorated sputum samples from 51 patients with lower respiratory infections (chronic obstructive pulmonary disease 25, pneumonia 20, purulent tracheobronchitis 6). All samples were examined with quantitative culture and immunofluorescent demonstration of ACB. From the results of quantitative culture, we divided specimens into pathogen-isolated and pathogen-free samples. Among pathogen-isolated samples, in which we isolated accepted pathogenic organisms at ≥ 10⁷ colony-forming units per ml, 16 of 23 samples were ACB-positive (69.5%). In contrast, among pathogen-free samples, in which we isolated accepted pathogens at < 10⁷ colony forming units per ml or only upper respiratory flora, only 3 of 37 samples were ACB-positive (8.1%). The ACB-positive rate was significantly higher in pathogen-isolated than in pathogen-free samples (P < 0.001). Consequently, detecting ACB in expectorated sputum shows good potential as another criterion for distinguishing contaminating or colonizing organisms from true pathogens.     

Survival of tubercle bacilli in heat-fixed and stained sputum smears

We used a slide culture technique to detect tubercle bacilli surviving in sputum smears (n=46) after conventional heat fixation and Ziehl-Neelsen staining. In all heat-fixed sputum smears, tubercle bacilli survived after time 0 (n=22), 24 h (n=7), 48 h (n=7), 72 h (n=4), and seven days (n=6). None of the stained sputum smears showed growth on slide cultures. Viable tubercle bacilli remaining in heat-fixed sputum smears for at least seven days may present an infection risk to laboratory staff. Thus, sputum smears should be stained immediately by the Ziehl-Neelsen method or stored in a safe container to avoid transmission of tuberculosis.     

Pulmonary Tuberculosis Apparently Caused by the Avian Tubercle Bacillus

Avian tubercle bacilli were repeatedly isolated from the sputum of a 65-year-old man who had cavitary disease in the upper lobe of the right lung. The clinical picture resembled that of pulmonary tuberculosis caused by the human type of tubercle bacilli, but the response to antituberculosis chemotherapy was unsatisfactory and the patient''s sputum remained positive. The bacilli were markedly pathogenic to chickens and rabbits but failed to produce progressive disease in guinea pigs. This is in accord with the properties of tubercle bacilli of the avian type.     

Automated identification of tubercle bacilli in sputum. A preliminary investigation

OBJECTIVE: To use an automated method to detect tubercle bacilli in sputum specimens. STUDY DESIGN: Using fluorescence microscopy, tubercle bacilli were identified on auramine-stained sputum specimens. Images were then captured with a digital camera and enhanced through imaging processing techniques. The bacilli were recognized using neural network classifiers. RESULTS: This preliminary investigation demonstrated a sensitivity of 94.1% for the identification of individual bacilli. As there are usually fairly numerous tubercle bacilli in the sputum of patients with active pulmonary tuberculosis, the overall diagnostic accuracy of sputum smear-positive patients can be expected to be very high. CONCLUSION: Potential benefits of automated screening for TB bacilli are: rapid, accurate, inexpensive diagnosis; the ability to screen larger numbers of people; increased resources to monitor patients; and reduction in health risks to staff.     

Rapid serodiagnosis of human mycobacteriosis by ELISA using cord factor (trehalose-6,6'-dimycolate) purified from Mycobacterium tuberculosis as antigen

IgG antibodies against purified cord factor (trehalose-6,6'-dimycolate, TDM) in sera of 99 patients infected with mycobacteria (42 patients with tuberculosis excreting tubercle bacilli in the sputum, 11 patients with non-tuberculous mycobacteriosis excreting acid-fast bacilli in the sputum, and 46 patients without bacilli in the sputum but diagnosed as having pulmonary tuberculosis by chest X-ray films and physical examination), five patients with lung cancer, and 100 healthy controls which included subjects positive and negative for the tuberculin test were tested by the ELISA with TDM purified from Mycobacterium tuberculosis H37Rv as the antigen. Of the 99 cases of mycobacteriosis, 83 patients (83.8%) had positive results (48 samples from 53 patients, or 90.5%, with bacilli in the sputum, and 35 samples from 46 patients (76%) with tuberculosis diagnosed clinically). The sera of the five patients with lung cancer and the 100 controls all gave negative results. Thus, the sensitivity and specificity were 83.8% and 100%, respectively. ELISA with TDM as the antigen is simple, reproducible, and useful for the rapid serodiagnosis of general mycobacterial infections including tuberculosis, because it does not involve the cultivation of bacteria.     

Detection of small numbers of Campylobacter jejuni and Campylobacter coli cells in environmental water, sewage, and food samples by a seminested PCR assay

A rapid and sensitive assay was developed for detection of small numbers of Campylobacter jejuni and Campylobacter coli cells in environmental water, sewage, and food samples. Water and sewage samples were filtered, and the filters were enriched overnight in a nonselective medium. The enrichment cultures were prepared for PCR by a rapid and simple procedure consisting of centrifugation, proteinase K treatment, and boiling. A seminested PCR based on specific amplification of the intergenic sequence between the two Campylobacter flagellin genes, flaA and flaB, was performed, and the PCR products were visualized by agarose gel electrophoresis. The assay allowed us to detect 3 to 15 CFU of C. jejuni per 100 ml in water samples containing a background flora consisting of up to 8, 700 heterotrophic organisms per ml and 10,000 CFU of coliform bacteria per 100 ml. Dilution of the enriched cultures 1:10 with sterile broth prior to the PCR was sometimes necessary to obtain positive results. The assay was also conducted with food samples analyzed with or without overnight enrichment. As few as 相似文献   

老年慢性支气管炎急性加重期105例痰需氧菌培养及药敏结果分析

目的分析慢性支气管炎急性加重期病原菌的类型、分布以及药物敏感性试验的情况。方法收集2004年1月至2007年12月我所住院的慢性支气管炎急性加重期患者共送检168例次痰标本进行培养和药物敏感试验。结果从痰液中共检出156株需氧菌,其中革兰阴性菌93株（59．62％）,革兰阳性菌41株（26．28％）,真菌22株（14．1％）;在革兰阴性菌中,以克雷伯菌属为主,占总分离率的21．15％（33／156）,其次是铜绿假单胞菌,分离出24株（15．38％）;革兰阳性菌则以金黄色葡萄球菌及肺炎链球菌居多。感染以革兰阴性杆菌为主,对11种临床常用的抗生素敏感率较高的是亚胺培南,其次是氨基糖苷类。结论本试验可提高对慢性支气管炎的临床诊断,根据其病原学特点合理使用抗菌药物。     

The antibacterial effect of river water onShigella Shigae in connection with the presence of corresponding antagonists and bacteriophages

Summary and conclusions It is emphasized that in the Tjiliwung river water no antagonists against the occurring typhoid bacteria are to be detected. Bacterial antagonists against dysentery bacteria can, however, easily be isolated, whereas the latter could not be isolated from the water by means of S.S. medium orLeifson plates. This elicited the question whether antibiotic substances in nature might cause a detrimental effect onShigella shigae.From 2054 colonies of water bacteria on broth agar 294 strains with an antagonistic effect on shigae bacteria were cultivated with methods based on the agar streak, the perforated Petri dish and that ofKelner. Among 3419 other colonies isolated from the same water samples no antagonists against typhoid bacteria could be detected. The method ofKelner proved 14% of the water bacteria growing on broth agar to be antagonistic against shigae bacteria. Some of these antagonists lost their activity soon after subinoculation on broth agar.In 14 out of 16 samples of Tjiliwung water a filtrable thermolabile agent could be detected, detrimental toSh. shigae when inoculated fairly profusely and in the presence of organic material (extract broth 1 : 10). The filtrated agent reduced the survival period ofSh. shigae to a maximum of 1/360 of the test period and inhibited the growth of these bacteria up to a dilution of 1 : 256. Heating at 100°C. destroyed the agent. In all cases this thermolabile anti-bacterial factor was associated with the presence of shigae phage and did not occur in two samples lacking the latter. The agent could not be separated from the phage particles by means of phage-tight glacial acid collodion filters through which human hemoglobine passes easily. The anti-shigae factor acted as a bacteriophage, not as an antibiotic. Propagation of the phage in three successive cultures could be demonstrated.No anti-typhoid agent was found in the filtrates lacking typhoid phages. It is concluded that the unheated river water filtrates owe their anti-shigae action mainly to the presence of corresponding phages. It is possible that in nature other factors may change the influence of the shigae-phage. Anti-bacterial substances produced by the numerous antagonists present are of no importance for the reduction in number of shigae bacteria in the river water.     

178例小儿肺炎分离菌分布和耐药性分析

目的 了解小儿肺炎病原菌谱及耐药性。方法 对2003～2004年178例小儿肺炎的痰标本进行细菌学培养,测定药物敏感性。结果 分离菌中,以革兰阴性菌居首位．占58．51％。肺炎克雷伯菌和大肠埃希菌为主琴芦种,对亚胺培南、美罗培南的耐药性最低;其次为革兰阳性菌,占40．43％,以金黄色葡萄球菌为主,耐甲氧西林的葡萄球菌为57．1％,对万古霉素、替考拉宁最敏感。结论 小儿肺炎病原菌以革兰阴性菌为主,并且出现多重耐药。临床上应避免盲目经验性用药,减少或减缓细菌耐药的发生。     

Obtaining hybridomas producing monoclonal antibodies with different specificities against Mycobacterium tuberculosis of the human and bovine types

A procedure for isolation of hybridomes producing monoclonal antibodies (McAB) to tubercle bacilli is described. Specificity of the McABs was studied with the solid phase radioimmune and immunoenzyme tests. Supernatant of tubercle bacilli destroyed with ultrasound was used as antigens. The McABs did not practically react with antigens of the tubercle bacilli atypical forms. Five ascitic monoclonal hybridomes were isolated. Four of them produced antibodies with selective specificity to antigens of bovine tubercle bacilli (M. bovis-8 and BCG) and one produced antibodies to antigens of human tubercle bacilli (H37Rv).     

Fate of Bacteria in Chicken Meat During Freeze-Dehydration, Rehydration, and Storage

Total plate counts were determined on boneless cooked, cubed chicken meat obtained from a commercial processor. Survival of the natural flora was determined after the meat was freeze-dehydrated and rehydrated at room temperature for 30 min and 50, 85, and 100 C for 10 min. Total counts of bacteria in the rehydrated samples were determined during storage of the meat at 4, 22, and 37 C until spoilage odor was detectable. Meat samples were inoculated with Staphylococcus aureus, then dried, rehydrated, and stored at the same temperatures. Numbers of surviving organisms in the inoculated samples were determined with use of both selective and nonselective media. Representative genera surviving the various rehydration treatments were determined. Approximately 32% of the bacteria in the meat survived during dehydration and rehydration at room temperature. Many numbers and types of vegetative bacteria also survived rehydration at 50 C. When meat was rehydrated at 85 or 100 C, the initial count was less than one per gram. The only organisms isolated from samples rehydrated at 85 or 100 C were of the genus Bacillus. S. aureus in inoculated samples survived dehydration and rehydration at 60 C. Storage of all rehydrated samples at 4 C gave a good shelf life (18 or more days). The study indicates that freeze-dehydrated meat should be produced with adequate microbiological control and that such meat should be rehydrated in very hot water.     

Detection of Small Numbers of Campylobacter jejuni and Campylobacter coli Cells in Environmental Water, Sewage, and Food Samples by a Seminested PCR Assay

A rapid and sensitive assay was developed for detection of small numbers of Campylobacter jejuni and Campylobacter coli cells in environmental water, sewage, and food samples. Water and sewage samples were filtered, and the filters were enriched overnight in a nonselective medium. The enrichment cultures were prepared for PCR by a rapid and simple procedure consisting of centrifugation, proteinase K treatment, and boiling. A seminested PCR based on specific amplification of the intergenic sequence between the two Campylobacter flagellin genes, flaA and flaB, was performed, and the PCR products were visualized by agarose gel electrophoresis. The assay allowed us to detect 3 to 15 CFU of C. jejuni per 100 ml in water samples containing a background flora consisting of up to 8,700 heterotrophic organisms per ml and 10,000 CFU of coliform bacteria per 100 ml. Dilution of the enriched cultures 1:10 with sterile broth prior to the PCR was sometimes necessary to obtain positive results. The assay was also conducted with food samples analyzed with or without overnight enrichment. As few as ≤3 CFU per g of food could be detected with samples subjected to overnight enrichment, while variable results were obtained for samples analyzed without prior enrichment. This rapid and sensitive nested PCR assay provides a useful tool for specific detection of C. jejuni or C. coli in drinking water, as well as environmental water, sewage, and food samples containing high levels of background organisms.     

Comparison of classical isolation protocols with a 24 h screen to detect viable salmonellas in faeces

A 24 h screen which detects three viable salmonella cells per g of faeces was compared with classical isolation procedures for their ability to identify salmonella-positive samples from a pig rearing unit. The screen involved an overnight enrichment in Muller-Kauffmann tetrathionate (MK) broth, subculture for 4 h in M broth containing 10 μg ml⁻¹ novobiocin, followed by detection of the presence of salmonellas by BacTrace and Salmonella-tek ELISAs. The classical protocols were: (1) an overnight and 48 h incubation in MK or selenite cysteine broth; or (2) overnight incubation in buffered peptone water and 24 h subculture in Rappaport-Vassiliadis broth (BPW-RV). Salmonellas were isolated from the broth cultures on xylose lysine deoxycholate and brilliant green agars. Thirty four of 100 samples were positive for salmonellas but no single isolation protocol identified all of them. The best of the classical isolation protocols, 48 h incubation in MK broth, identified 27 (79%) of the 34 positive samples whilst the screen identified 26 (76%) of the 34 positive samples. False-positive results were obtained from all isolation protocols except BPW-RV.     

768例肺结核患者下呼吸道感染致病菌耐药性探讨

目的探讨肺结核患者合并下呼吸道感染的致病菌及其耐药性。方法对768例确诊的肺结核患者进行痰和／或支气管肺泡灌洗液进行细菌培养。结果革兰阴性菌596株,占58．1％,对哌拉西林／舒巴坦及头孢哌N／舒巴坦耐药率在5．7％-14．3％。革兰阳性菌201株,占19．6％,对万古霉素耐药率为0。真菌228株,占22．2％,对伊曲康唑耐药,耐药率为10．6％。结论肺结核患者下呼吸道感染,以革兰阴性菌为主,真菌比例呈上升趋势。     

Cyclic AMP signalling in mycobacteria: redirecting the conversation with a common currency

cAMP is an ancient second messenger, and is used by many organisms to regulate a wide range of cellular functions. Mycobacterium tuberculosis complex bacteria are exceptional in that they have genes for at least 15 biochemically distinct adenylyl cyclases, the enzymes that generate cAMP. cAMP-associated gene regulation within tubercle bacilli is required for their virulence, and secretion of cAMP produced by M. tuberculosis bacteria into host macrophages disrupts the host's immune response to infection. In this review, we discuss recent advances in our understanding of the means by which cAMP levels are controlled within mycobacteria, the importance of cAMP to M. tuberculosis during host infection, and the role of cAMP in mycobacterial gene regulation. Understanding the myriad aspects of cAMP signalling in tubercle bacilli will establish new paradigms for cAMP signalling, and may contribute to new approaches for prevention and/or treatment of tuberculosis disease.     

Large intestine bacterial flora of nonhibernating and hibernating leopard frogs (Rana pipiens).

The bacteria in the large intestines of 10 northern leopard frogs (Rana pipiens) were enumerated and partially characterized. Four nonhibernating frogs were collected in the summer, four hibernating frogs were collected in the winter, and two frogs just emerged from hibernation were collected in the spring. All frogs had about 10(10) bacteria per g (wet weight) of intestinal contents and about 10(9) bacteria per g (wet weight) of mucosal scraping, although the counts from the winter frogs were slightly less than those from the other two groups of frogs. Another group of 14 summer frogs, after treatment to induce hibernation, showed a drop in bacterial counts accompanied by a change in the composition of the flora. In most frogs, Bacteroides was the dominant organism. Other bacteria repeatedly isolated at high dilutions were strict anaerobes, including butyrigenic and acetogenic helically coiled bacteria; fusobacteria; and acetogenic, small, gram-positive bacilli. These data indicate that the intestinal flora of frogs is similar to that of mammals and birds and that this flora can be maintained at temperatures close to freezing.     

Aerobic Oral Bacteria in Healthy Captive Snakes

Cotton swab samples were taken from the ventral surface of the mouth and from the proximal esophagus from 23 captive nonpoisonous snakes. The samples were cultured and investigated for aerobic bacteria. Both the mouth and the esophagus) samples of 6 snakes were negative. When the bacterial isolates of the mouth and the esophagus of the whole snake population were compared, it was found that the flora isolated from both locations were similar. However, when the samples of individual snakes were compared it was found that the same isolates were seldom found in both the mouth and the esophagus. The most common bacteria found were Pseudomonas sp., Alcaligenes-like organisms, Gram-positive rods and Gram-positive cocci belonging to the family Micrococcaceae. Important pathogens were seldom isolated. Salmonella virchow could be found from 2 snakes. The presence of bacteriologically negative samples, great variations in the composition of the flora between individual snakes, and the occurrence of typical environmental bacteria in the oral cavity all suggest that snakes lack a specific autochtonous flora: and the bacteria isolated from the oral cavity may be occasional environmental bacteria. The source of pathogens may be the environment, too.     

Spoilage association of chicken leg muscle.

The ability of pure cultures of bacteria isolated from spoiling chicken leg muscle to produce strong off-odors was tested by using sterile leg muscle sections. Changes in the flora during storage and the incidence and identity of organisms capable of producing strong off-odors were noted.     

Paraformaldehyde-Resistant Starch-Fermenting Bacteria in “Starch-Base” Drilling Mud

Starch-fermenting bacteria were found in each of 12 samples of nonfermenting starch-base drilling mud examined. Of the 12 samples, 3 contained very active starch-fermenting gram-positive spore-bearing bacilli closely resembling Bacillus subtilis. Similar active starch-fermenting bacteria were found in fermenting starch-base drilling mud and in corn starch and slough water used to prepare such mud. The active starch-fermenting microorganisms completely hydrolyzed 1% (w/v) corn starch within 24 hr at 37.5 C. The active starch-fermenting bacteria isolated from fermenting drilling mud were capable of surviving 12 hr of continuous exposure to 0.1% (w/w) paraformaldehyde or 1 hr of continuous exposure to 0.5% (w/w) paraformaldehyde, with no diminution in starch-fermenting ability. The same organisms fermented starch after 3 hr of continuous exposure to 0.5% (w/w) paraformaldehyde, but not after 4 hr of exposure. The phenomenon of rapid disappearance of paraformaldehyde from fermenting drilling mud was observed in the laboratory using a modified sodium sulfite test. Paraformaldehyde, initially present in a concentration of 0.192 lb per barrel of mud, completely disappeared in 9 hr at 22 to 23 C. A significant decrease in paraformaldehyde concentration was detected 0.5 hr after preparation of the mud. It is suggested that the presence of relatively high concentrations of ammonia and chloride in the mud may facilitate the disappearance of paraformaldehyde. The failure of 0.1% (w/w) paraformaldehyde to inhibit the strong starch-fermenting microorganisms isolated from fermenting drilling mud, and the rapid disappearance of paraformaldehyde from the mud, explains the fermentation of starch which occurred in this mud, despite the addition of paraformaldehyde.