Phage cocktail to control the exponential growth of normal flora in processed sputum specimens grown overnight in liquid medium for rapid TB diagnosis

The mechanical pressure exerted during centrifugation and the chemical pressure experienced when sputum specimens are processed, leave the tubercle bacilli in the sputum unsuitable for rapid detection especially in phage based assays. Thus, growing Mycobacterium tuberculosis in broth, at least overnight, is mandatory for allowing the tubercle bacilli to recoup. During this time the surviving colonizing flora grow faster and overgrow tubercle bacilli interfering with TB diagnosis. In the present study normal flora surviving the action of 4% NaOH was isolated and characterized. Phages capable of killing 14 different species representing this normal flora were isolated from soil and sewage samples and characterized. A novel and bio-friendly approach to treat sputum samples with a cocktail of three phages capable of killing most of the 14 representative organisms and not infecting mycobacteria is explored to control the overgrowth of colonizing bacteria in broth culture. While 26 of the 100 sputum samples processed by modified Petroff's procedure showed growth of colonizing flora on blood agar, all of them when grown in broth overnight showed mixed, confluent growth. The addition of phagebiotics controlled them all, showing a significant reduction in colony forming units but resulting in few discrete colonies in 54 samples. Isolation of phages capable of controlling these surviving organisms and including them in the phagebiotics mixture should lead to the control of colonizing bacteria effectively.     

Comparison of Methods for Isolation of Anaerobic Bacteria from Clinical Specimens

Five different anaerobic culture methods and several different media were compared for their ability to recover anaerobes from clinical specimens. Specimens were obtained from patients with documented infections, avoiding contamination with normal flora, and immediately placed in an anaerobic transporter. Each specimen was cultured by all methods and on all the various media. The comparative data indicate that anaerobic jars (GasPak and evacuation-replacement types) are just as effective in the recovery of clinically significant anaerobes as the more complex roll-tube and chamber methods employing prereduced media. Liquid media were disappointing as a "back-up" system but chopped-meat glucose was superior to two thioglycolate formulations. Growth of all anaerobes was poorer on selective media, but these media were very helpful in the workup of specimens containing mixed growth of anaerobic and facultative organisms. A variety of different anaerobes was isolated, but no very fastidious or extremely oxygen-sensitive organisms were recovered. This suggests that such organisms may not play a significant role in causing clinical infections.     

Growth of Bacteria on Meat at Room Temperatures

The development of spoilage flora and the growth of individual psychrotrophs and pathogens on meat held at 20 or 30°C were studied. Under aerobic conditions psychrotrophic pseudomonads accounted for 60% of the spoilage flora at 20°C, but <20% at 30°C where they were displaced by species of Acinetobacter and Enterobacteriaceae which included both mesophilic and psychrotrophic strains. Mesophiles dominated the anaerobic spoilage flora at 30°C when clostridia were the major species, but at 20° the flora contained mesophiles and psychrotrophs in similar proportions and was dominated by Enterobacteriaceae. These results were largely predictable from the growth rate data for individual organisms.
Interactions between species occurred more frequently at 20°C than at 30°C. When pathogenic species were grown at 20 or 30°Cin competition with equal numbers of psychrotrophic spoilage organisms, no interactions were observed. When pathogens were grown in competition with high numbers of psychrotrophs, only Lactobacillus growing anaerobically inhibited Salmonella typhimurium and Escherichia coli , but other pathogens were inhibited to varying degrees depending on the competing species and the incubation conditions. In general, the degree of inhibition was greater at 20 than at 30°C and facultative organisms were more susceptible under anaerobic than aerobic conditions. It appears that the cumulative stresses of low pH, suboptimal temperatures and competition with large numbers of saprophytic organisms can inhibit many of the pathogens likely to be present on meat. The organisms least affected by the conditions on meat surfaces, Salmonella and Esch. coli , are likely to be the main hazards on meat of normal pH held at room temperatures.     

Occurrence of Mycobacterium Other than Mycobacterium tuberculosis in the Oral Cavity and in Sputum

Mycobacteria were cultured from 9% of 424 paired mouthwash-induced sputum specimens. The majority of the organisms were not Mycobacterium tuberculosis. Sputum cultures contributed 59% of these isolations. M. intracellulare was the species most frequently isolated. The non-M. tuberculosis mycobacteria may constitute part of the oral flora of the general population and are not more prevalent in hospitalized patients. Approximately one-third more isolations were made from the patients furnishing two or three pairs of specimens as compared to those patients providing one pair of specimens each. From the paired specimens of 4 of 113 patients, two species of Mycobacterium were isolated. M. intracellulare was isolated from two of three samples of tap water and from the fingers of 1 of 52 patients.     

The role of anaerobic bacteria in sinusitis

The normal oropharyngeal flora contained aerobic and anaerobic bacteria that can cause respiratory infections including sinusitis. Some of these bacteria can interfere with the growth of potential pathogens and may play a role in preventing infections. Anaerobic bacteria emerge as pathogens as the infection becomes chronic. This may be the result of the selective pressure of antimicrobial agents that enable resistant anaerobic organisms to survive, and from the development over time of conditions appropriate for anaerobic growth, which include the reduction in oxygen tension and an increase in acidity within the sinus cavity. Anaerobes were isolated in acute maxillary sinusitis of odontogenic origin and in over half of the patients with chronic sinusitis whenever proper techniques for their cultivation were employed. These organisms were also recovered in acute sinusitis that was associated with dental infections. The predominant isolates were pigmented Prevotella and Porphyromonas, Fusobacterium and Peptostreptococcus spp.     

慢性湿疹患者皮损、鼻粘膜需氧微生物群的定位、定量研究

本文首次应用定位、定量方法对23例小腿部慢性湿疹患者皮损、邻近皮肤及鼻孔的菌群,从微生态学的角度进行探讨,并将结果同24例正常人小腿皮肤及鼻孔的菌群做了比较。其结果表明:金黄色葡萄球菌为皮损及邻近皮肤的主要菌种,并且其分离率及密度有按正常皮肤、邻近皮肤、皮损依次增高现象;类白喉杆菌的数量在皮损部位明显低于正常皮肤与邻近皮肤;表皮葡萄球菌分离率皮损及邻近皮肤均明显降低。本组结果同正常对照组相比其皮肤微生物群菌谱虽无不同,但其数量的变异是显著的,这对探讨湿疹的病因及用生态制剂等调整皮肤菌群使其恢复微生态平衡达到防治目的有所裨益。     

Leptotrichia species in human infections

Leptotrichia species typically colonize the oral cavity and genitourinary tract. These anaerobic bacteria belong to the normal flora of humans and are seldom found in clinically significant specimens. However, on rare occasions, Leptotrichia has been isolated from blood cultures of patients with lesions in the oral mucosa, in particular from patients with neutropenia. These organisms should be considered potential pathogens in neutropenic patients, especially when breaks in the mucosal barriers are present through which they frequently spread to the bloodstream. Leptotrichia has also been recovered from immunocompetent persons, e.g. patients with endocarditis. Although their role in infections remains elusive and not much is known, they have been suggested as emerging pathogens. The present review deals with taxonomy, diagnosis, clinical importance, pathogenesis, host defence, infection control, and spectrum of Leptotrichia infections, and ends with a few typical case reports. Currently, six species have been validly published, but a number of yet uncultivable species exist. Molecular methods recovering uncultivable species should be used to get a real idea of their role as pathogens.     

Isolation and characterization of a spiral bacterium from the crypts of rodent gastrointestinal tracts

Spiral-shaped bacteria with a distinctive morphology were isolated from the intestinal mucosa of rats and mice on a campylobacter selective medium using microaerophilic incubation. These bacteria have been shown by other authors to be present in the intestinal tracts of several animal species but have not been cultured previously. The results of electron microscopic examinations and biochemical testing have shown that these organisms do not correspond to any known genus. Colonization experiments with pure cultures in gnotobiotic rodents have shown these bacteria to be mucosa associated, with a particular affinity for intestinal crypts. The pattern of colonization of the intestinal crypts in gnotobiotes known to be free of other mucosa-associated organisms differed from the colonization occurring in conventional animals that possess a normal mucosa-associated flora.     

Isolation and characterization of a spiral bacterium from the crypts of rodent gastrointestinal tracts.

Spiral-shaped bacteria with a distinctive morphology were isolated from the intestinal mucosa of rats and mice on a campylobacter selective medium using microaerophilic incubation. These bacteria have been shown by other authors to be present in the intestinal tracts of several animal species but have not been cultured previously. The results of electron microscopic examinations and biochemical testing have shown that these organisms do not correspond to any known genus. Colonization experiments with pure cultures in gnotobiotic rodents have shown these bacteria to be mucosa associated, with a particular affinity for intestinal crypts. The pattern of colonization of the intestinal crypts in gnotobiotes known to be free of other mucosa-associated organisms differed from the colonization occurring in conventional animals that possess a normal mucosa-associated flora.     

老年住院患者口咽部念珠菌定植状况及菌种分布调查

目的调查老年住院患者口咽部念珠菌定植状况及菌种分布特点,为有效预防念珠菌感染提供参考。方法对解放军第44医院收治的894例住院患者口咽部念珠菌定植状态进行调查分析。结果894例住院患者口咽部标本共培养出念珠菌121株,念珠菌定植率为13．5％,老年患者的定植率高于非老年患者。念珠菌定植以白色念珠菌所占比例最大,占74．5％。年龄≥80岁、肺部基础疾病的存在及使用抗菌药物患者口咽部念珠菌定植率高于普通患者,两者比较差异有统计学意义（P〈0．05）。结论老年患者口咽部念珠菌定植率较高,菌种分布以白色念珠菌最为常见。念珠菌定植与患者年龄、肺部基础疾病的存在及抗菌药物使用情况密切相关。     

Observations on the Microbiology of Cooked Chicken Carcasses

S ummary . The residual microbial flora and the flora developing during storage at 1–3° and at 16°, of chicken carcasses cooked in a circulating moist air oven operated at 85°, have been studied. All parts of the carcasses reached and maintained 85° for at least 50 min, and the residual flora consisted largely of spore forming bacteria. The predominant residual species were Bacillus subtilis and Clostridium bifermentans. Non-sporing bacteria were not detected after cooking nor after storage at 1–3° for up to 7 days. Storage at 16° for 3 days markedly increased the number of non-sporing organisms although off-odours typical of spoilage were not apparent until at least 10 days. Staphylococcus aureus and Salmonella spp. were not detected after cooking and storage and Cl. welchii was rarely isolated. It is concluded that poultry cooked by this method present a minimal risk of food-borne infection or intoxication by these organisms if contamination after cooking is avoided, the carcasses are cooled rapidly to c , 3° and stored at this temperature or frozen.     

Antibiotic decontamination of the dog and its consequences.

An isolation-decontamination regimen was developed which effectively reduced the numbers of resident flora of the dog. Bacterial counts in four dogs before treatment were 3.8 X 10(9) per gram of feces; no organisms were detectable in these same dogs after treatment, however, the intestinal flora had returned to slightly above normal levels 1 week after treatment. Decontamination was accomplished in a laminar air flow system designed to minimize the area that had to be under controlled conditions. By determining the antibiotic sensitivities of 67 isolated organisms representing eight species or groups of bacteria recovered from the four dogs, a standardized antibiotic regimen was developed consisting of bacitracin and neomycin administered as a dry powder in the food. The decontamination treatment apparently did not affect host metabolism because no alterations in serum levels of urea nitrogen, glucose, phosphate, total protein, chloride, sodium, potassium, serum glutamic oxalacetic transaminase, or serum glutamic pyruvic transaminase were found in the antibiotic-treated dogs. The decontamination process did, however, reduce normal granulopoietic stimulation.     

Oropharyngeal Cultures of Patients in Protected Environment Units: Evaluation of Semiquantitative Technique During Antibiotic Prophylaxis

A semiquantitative culture technique was used to monitor the microbial flora of the oropharynx of 30 patients receiving antibiotic prophylaxis in protected environment units. After institution of antibiotic prophylaxis, the median concentration of organisms in the oropharynx fell by 2 logs but gradually increased by 1 log and then remained stable. Neisseria spp., Micrococcus sp., and Streptococci were generally eradicated by the antibiotics but were replaced by Lactobacilli and yeast. Four of nine enteric organisms persisted despite in vitro sensitivity to the antibiotic regimens. Yeast were cultured from the initial specimens of only 17% of the 30 patients, but they were cultured subsequently from specimens of 80% of the 20 patients who remained in protected environment units for at least 8 weeks.     

Steroid desmolase synthesis by Eubacterium desmolans and Clostridium cadavaris.

The synthesis of a steroid desmolase was demonstrated in two obligate anaerobes: a new bacterial species, Eubacterium desmolans, isolated from cat fecal flora, and Clostridium cadavaris, recovered from sewage of New York City. The enzyme cleaves the C-17-C-20 bond of corticoids possessing hydroxyl functions at C-17 and C-21. The conversion is quantitative, provided the substrate concentration is less than 100 micrograms/ml and the organisms are in the log phase. The velocity of transformation parallels the bacterial growth curve and in the log phase is higher for E. desmolans than for C. cadavaris. In addition, both organisms synthesize a 20 beta-hydroxysteroid dehydrogenase.     

Steroid desmolase synthesis by Eubacterium desmolans and Clostridium cadavaris

The synthesis of a steroid desmolase was demonstrated in two obligate anaerobes: a new bacterial species, Eubacterium desmolans, isolated from cat fecal flora, and Clostridium cadavaris, recovered from sewage of New York City. The enzyme cleaves the C-17-C-20 bond of corticoids possessing hydroxyl functions at C-17 and C-21. The conversion is quantitative, provided the substrate concentration is less than 100 micrograms/ml and the organisms are in the log phase. The velocity of transformation parallels the bacterial growth curve and in the log phase is higher for E. desmolans than for C. cadavaris. In addition, both organisms synthesize a 20 beta-hydroxysteroid dehydrogenase.     

R Factor Transmission In Vivo

Experimental infections were induced in weanling pigs orally both with nalidixic acid (NA)-sensitive and -resistant strains of Salmonella choleraesuis var. kunzendorf, designated RC221 and RC221NA, respectively. Prior to the time of infection, cultures of normal bacterial flora were isolated from swine fecal matter and screened for the presence of R factors. A majority of these bacterial isolates harbored transferable resistances. Both strains RC221 and RC221NA have been shown to be competent recipients in vitro of the R factors present in the normal intestinal flora. The property of NA resistance greatly facilitated recovery of the infecting organism. After infection, salmonellae from liver, lung, spleen, lymph node, intestine, and feces were screened for the presence of R factors. Transfer of drug resistance in vivo was a rare occurrence; however, if infected specimens, particularly intestinal, were incubated in nutrient broth prior to plating, R factor transfer occurred, presumably in the test tube. Changes in recipient cultures were frequently observed after introduction of R factors from organisms of pig origin into the S. choleraesuis var. kunzendorf test organisms. Alterations include changes in typing reaction, granular growth in broth, differences in colony form, and reduction of virulence.     

Antibiotics and the Oral Streptococci of Man

The effects of 3 antibiotics, phenoxymethylpenicillin, cephalexin and clindamycin on the normal oral streptococcal flora in the region of the gingival crevice were investigated because these organisms are able to cause subacute bacterial endocarditis. Secretion of these antibiotics into the oral cavity was also examined. Penicillin and clindamycin exerted marked effects on the normal oral streptococci, whereas cephalexin did not cause any obvious change in the total flora. Following penicillin therapy, streptococci resistant to 1.5 μg/ml penicillin were observed and these organisms could be detected at least 8 weeks after the last dose of the antibiotic. They probably arose by selection from the mixed flora. Following cephalexin therapy, a much lower proportion of streptococci resistant to 15 μg/ml was found. The proportion of resistant strains fluctuated appreciably, however, probably due to their transient nature. Streptococci resistant to 1 μg/ml clindamycin were not observed in 10 out of 11 treated subjects.
Penicillin and clindamycin could be detected in the pooled saliva and gingival fluid after administering single doses of 500 mg and 300 mg, respectively. The peak levels were obtained between half and 1 h. The concentration of penicillin dropped rapidly within 3 h but clindamycin could be detected at significant levels for at least 6 h. No cephalexin could be detected in the pooled saliva or gingival fluid after a 500 mg dose. The implications of these findings in the prevention of subacute bacterial endocarditis are discussed.     

Normal vaginal aerobic and anaerobic bacterial flora of the rhesus macaque (Macaca mulatta).

The most common bacterial species isolated from the vaginas of 37 healthy rhesus macaques were Streptococcus viridans, coagulase negative Staphylococcus, Mobiluncus curtisii ss. curtisii, Corynebacterium renale-like organisms, Peptostreptococcus anaerobius, Gardnerella-like organisms, and other Corynebacterium species. The vaginal flora of the rhesus macaque differs from that previously reported for five other primate species. A two-year retrospective review of clinical cases of vaginitis and metritis found Escherichia coli and coagulase positive Staphylococci to be the most common pathogens isolated.     

Diagnostic value of Gram stain for assessment of vaginal smears during pregnancy

The main aim of this study was to compare the diagnostic value of Giemsa stained method with Gram stained method for the evaluation of vaginal smears among pregnant women. A study population comprised 111 pregnant between 6 and 30 weeks of gestation. The vaginal smears from every subject was diagnosed according to Giemsa and Gram stained method and micro-organisms were isolated by culture. In 29.3% cases diagnosed as normal flora (2a) on the basis of Giemsa method bacterial vaginosis was detected in Gram stains according to Spiegel's criteria and pathological microflora in concentration > or = 10(5) CFU/ml was cultured among 75.9% of them. Among 31.7% women who had grade 3a (abnormal) in Giemsa stains method normal flora was diagnosed on the basis on Gram's method and from 17.1% pregnant women from this group we did not isolated any pathogens. For evaluation of vaginal smears during pregnancy the Giemsa method should be replaced by Gram stained method.     

Rifampin-Blood-Agar as a Selective Medium for the Isolation of Certain Anaerobic Bacteria

A selective medium which allows detection of relatively small numbers of Fusobacterium varium in fecal specimens is described. Blood-agar containing 50 mug of rifampin per ml inhibits the growth of many species of Bacteriodes and of F. fusi-forme/nucleatum but allows good growth of F. varium and most strains of F. mortiferum. Quantitative cultures of 11 fecal specimens were done on rifampin and other selective and nonselective media. F. varium was recovered in counts of 10(6) and 10(7) per gram from two specimens on rifampin only. A third specimen yielded 10(10)F. varium on several media, including rifampin. Some Eubacterium and Clostridium species also grew on rifampin, and these ordinarily were distinguished from the Fusobacterium by colony morphology. This medium is of value in fecal flora studies and should be useful with other kinds of specimens where mixtures of organisms are common.