Inhibition of ruminal cellulose fermentation by extracts of the perennial legume cicer milkvetch (Astragalus cicer)

Cicer milkvetch (Astragalus cicer L.) is a perennial legume used as a pasture or rangeland plant for ruminants. A study was undertaken to determine whether reported variations in its ruminal digestibility may be related to the presence of an antinutritive material. In vitro fermentation of neutral detergent fiber (NDF) of cicer milkvetch by mixed rumen microflora was poorer than was the fermentation of NDF in alfalfa (Medicago sativa L.). Fermentation of cicer milkvetch NDF was improved by preextraction of the ground herbage with water for 3 h at 39 degrees C. Such water extracts selectively inhibited in vitro fermentation of pure cellulose by mixed ruminal microflora and by pure cultures of the ruminal bacteria Ruminococcus flavefaciens FD-1 and Fibrobacter succinogenes S85. Inhibition of the cellulose fermentation by mixed ruminal microflora was dependent upon the concentration of cicer milkvetch extract and was overcome upon prolonged incubation. Pure cultures exposed to the extract did not recover from inhibition, even after long incubation times, unless the inhibitory agent was removed (viz., by dilution of inhibited cultures into fresh medium). The extract did not affect the fermentation of cellobiose by R. flavefaciens but did cause some inhibition of cellobiose fermentation by F. succinogenes. Moreover, the extracts did not inhibit hydrolysis of crystalline cellulose, carboxymethyl cellulose, or p-nitrophenylcellobioside by supernatants of these pure cultures of cellulolytic bacteria or by a commercial cellulase preparation from the fungus Trichoderma reesei. The agent caused cellulose-adherent cells to detach from cellulose fibers, suggesting that the agent may act, at least in part, by disrupting the glycocalyx necessary for adherence to, and rapid digestion of, cellulose.     

Strategies that ruminal bacteria use to handle excess carbohydrate

When ruminal bacteria have insufficient nitrogen and other nutrients, excess carbohydrate can be toxic. Pure cultures that are nitrogen-limited can convert only some of the excess carbohydrate to intracellular polysaccharide, but this pool can be quickly saturated. Fibrobacter succinogenes cultures that have excess cellobiose secrete glucose and cellotriose into the culture medium, and Prevotella ruminicola produces methylglyoxal, a highly toxic substance that causes a dramatic decrease in viability. Some ruminal bacteria (e.g., Streptococcus bovis and Selenomonas ruminantium) have mechanisms to decrease ATP production or spill the ATP that has already been produced. These mechanisms of decreasing intracellular ATP seem to protect the cell. Most ruminal bacteria can use ammonia as a nitrogen source, but amino nitrogen increases the growth efficiency of mixed ruminal bacteria. Amino nitrogen-dependent improvements in growth efficiency can be explained by an increase in growth rate and a decrease in energy spilling. Amino nitrogen is only beneficial if the rate of carbohydrate fermentation is rapid and carbohydrate is in excess.     

Microbiology of liver and spleen abscesses

To study the aerobic and anaerobic microbiology of liver and spleen abscesses and correlate the results with predisposing factors, potential causes and routes of infection, clinical and laboratory data of 48 patients with liver abscesses and 29 with spleen abscesses treated between 1970 and 1990 were reviewed retrospectively. In liver abscesses, a total of 116 isolates (2.4 isolates/specimen) was obtained; 43 were aerobic and facultative species (0.9 isolates/specimen) and 73 were anaerobic species or microaerophilic streptococci (1.5 isolates/specimen). Aerobic bacteria only were isolated from 12 (25%) abscesses, anaerobic bacteria only from eight (17%), and mixed aerobic and anaerobic bacteria from 28 (58%); polymicrobial infection was present in 38 (79%). The predominant aerobic and facultative isolates were Escherichia coli (11 isolates), Streptococcus group D (8), Klebsiella pneumoniae (5) and Staphylococcus aureus (4). The predominant anaerobes were Peptostreptococcus spp. (18 isolates), Bacteroides spp. (13), Fusobacterium spp. (10), Clostridium spp. (10) and Prevotella spp. (4). There were 12 isolates of micro-aerophilic streptococci. S. aureus and beta-haemolytic streptococci were associated with trauma; Streptococcus group D, K. pneumoniae and Clostridium spp. with biliary disease; and Bacteroides spp. and Clostridium spp. with colonic disease. In splenic abscesses, a total of 56 isolates (1.9 isolates/specimen) was obtained; 23 were aerobic and facultative species (0.8 isolates/specimen), 31 were anaerobic species or micro-aerophilic streptococci (1.1 isolates/specimen) and two were Candida albicans. Aerobic bacteria only were isolated from nine (31%) abscesses, anaerobic bacteria from eight (28%), mixed aerobic and anaerobic bacteria from 10 (34%) and C. albicans in two (7%); polymicrobial infection was present in 16 (55%). The predominant aerobic and facultative isolates were E. coli (5 isolates), Proteus mirabilis (3), Streptococcus group D (3), K. pneumoniae (3) and S. aureus (4). The predominant anaerobes were Peptostreptococcus spp. (11 isolates), Bacteroides spp. (5), Fusobacterium spp. (3) and Clostridium spp. (3). S. aureus, K. pneumoniae and Streptococcus group D were associated with endocarditis, E. coli with urinary tract and abdominal infection, Bacteroides spp. and Clostridium spp. with abdominal infection and Fusobacterium spp. with respiratory infection.     

Bacteriological study of oral open abscesses

Although studies of bacteriology of closed oral abscesses have been extensively done, there are few studies on microorganisms involving open oral abscesses. We examined bacteriologically three open abscesses with precaution against bacterial contamination with oral normal flora and saliva, when sampling. The specimens were subjected to aerobic and anaerobic cultures within 2 hours after sampling. All three cases were infected with 5 to 14 species of aerobic and anaerobic bacteria; Streptococcus spp., Prevotella intermedia and other Prevotella spp. were predominant in all three cases. All six Prevotella spp. isolated were beta-lactamase producers, being resistant to beta-lactam antibiotics. These results emphasize the importance of prompt anaerobic culture for the bacteriological study of open oral abscess and the significance of nitrocefin test to detect beta-lactamase produced by oral isolates, especially Prevotella spp.     

Aerobic and anaerobic microbiology of axillary hidradenitis suppurativa

A retrospective review of the microbiological and clinical data of 17 specimens obtained from axillary hidradenitis suppurativa (HS) over a period of 6 years was undertaken to study the aerobic and anaerobic microbiology of this condition. A total of 42 bacterial isolates (2.5 per specimen) were obtained, 12 aerobic or facultative (0.7 per specimen) and 30 anaerobic or micro-aerophilic (1.8 per specimen). Aerobic and facultative bacteria only were isolated in six (35%) cases, anaerobic bacteria only in seven (41%) and mixed aerobic and anaerobic bacteria in four (24%). The predominant aerobic bacteria were Staphylococcus aureus (six isolates), Streptococcus pyogenes (three) and Pseudomonas aeruginosa (two). The most frequently isolated anaerobes were Peptostreptococcus spp. (10), Prevotella spp. (seven), micro-aerophilic streptococci (four), Fusobacterium spp. (three) and Bacteroides spp. sensu stricto (three). This study highlights the polymicrobial nature and predominance of anaerobic bacteria in axillary HS and the need for antimicrobial thereby to reflect this.     

The effect of dietary fatty acids on lactic acid bacteria associated with the epithelial mucosa and from faecalia of Arctic charr, Salvelinus alpinus (L.)

Arctic charr, Salvelinus alpinus (L.), held in fresh water, were fed four experimental diets containing different polyunsaturated fatty acids (PUFA). In addition, one group fed a diet containing only coconut oil as sole lipid source served as control. The population of aerobic heterotrophic bacteria associated with the epithelial mucosa and the faecalia was estimated using the dilution plate technique. Generally, the population level of adherent bacteria increased along the digestive tract (stomach, small intestine and large intestine). Adherent Gram-negative and Gram-positive bacteria seemed to be present at equal levels in all parts of the alimentary tract. Lactic acid bacteria dominated among the Gram-positive bacteria, and they were detected in all regions of fish fed the PUFA supplemented diets. The frequency of lactic acid bacteria was highest in the digestive tract of fish fed diets with added 7.0% linolenic acid (18:3 n-3) or 4% of a PUFA mix. A lower frequency of lactic acid bacteria was found in fish fed dietary linoleic acid (18:2 n-6), and they were absent or present in low numbers in fish fed the coconut oil diet. It is suggested that dietary fatty acids affect the attachment sites for the gastrointestinal microbiota, possibly by modifying the fatty acid composition of the intestine wall. Numerical taxonomy procedures showed that the lactic acid bacteria Carnobacterium spp. and a Carnobacterium piscicola-like strain were predominant, with smaller numbers of Lactobacillus plantarum, Streptococcus spp. and Leuconostoc mesenteroides present. Seven strains of Carnobacterium spp. were further identified on the basis of 16S rDNA sequence analysis, and all these strains were identified as Carnobacterium piscicola.     

Production of amino acids by yogurt bacteria

The dynamics of free amino acid production by the selected strains Streptococcus thermophilus 13a and Lactobacillus bulgaricus 2-11 were studied in pure and mixed cultivations during yogurt starter culture manufacture. L. bulgaricus 2-11 showed the highest activity for producing free amino acids with high individual concentrations over the first hour of growth (50% of the total amount). By the end of milk's full coagulation (4.5 h), 70% of the total amount of amino acids was released. S. thermophilus 13a showed poor proteolytic properties and consumed up to 70% of the free amino acids produced by L. bulgaricus 2-11 in the process of coagulation of milk with the mixed culture.     

Detection and quantification of poultry probiotic bacteria in mixed culture using monoclonal antibodies in an enzyme-linked immunosorbent assay

Murine monoclonal antibodies were used in an enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of selected probiotic bacteria present in a continuous-flow competitive exclusion culture known to be effective at reducing chicken cecal and crop colonization by Salmonella typhimurium. Veillonella, Enterococcus avium and S. typhimurium were grown anaerobically in batch culture of Viande Levure broth in pure culture and mixed culture. The mixed cultures produced significantly more acetate and propionate than any of the pure cultures with acetate and propionate being the predominant volatile fatty acids. The association in mixed culture resulted in a significant increase in cell numbers compared to the respective pure cultures. The ELISA was capable of detecting 10(4) cells per ml of the bacteria. The plots of cell numbers determined by the ELISA versus direct plating increased in accordance with increases in cell numbers with r2 values of 0.950, 0.922 and 0.940 for the pure culture incubations and 0.901, 0.924 and 0.905 in the mixed culture incubation for E. avium, S. typhimurium and Veillonella, respectively. The results indicate that the monoclonal antibodies can be used to quantitatively assay individual probiotic bacterial species grown in a mixed culture incubation.     

An rRNA approach for assessing the role of obligate amino acid-fermenting bacteria in ruminal amino acid deamination

Ruminal amino acid degradation is a nutritionally wasteful process that produces excess ruminal ammonia. Monensin inhibited the growth of monensin-sensitive, obligate amino acid-fermenting bacteria and decreased the ruminal ammonia concentrations of cattle. 16S rRNA probes indicated that monensin inhibited the growth of Peptostreptococcus anaerobius and Clostridium sticklandii in the rumen. Clostridium aminophilum was monensin sensitive in vitro, but C. aminophilum persisted in the rumen after monensin was added to the diet. An in vitro culture system was developed to assess the competition of C. aminophilum, P. anaerobius, and C. sticklandii with predominant ruminal bacteria (PRB). PRB were isolated from a 10(8) dilution of ruminal fluid and maintained as a mixed population with a mixture of carbohydrates. PRB did not hybridize with the probes to C. aminophilum, P. anaerobius, or C. sticklandii. PRB deaminated Trypticase in continuous culture, but the addition of C. aminophilum, P. anaerobius, and C. sticklandii caused a more-than-twofold increase in the steady-state concentration of ammonia. C. aminophilum, P. anaerobius, and C. sticklandii accounted for less than 5% of the total 16S rRNA and microbial protein. Monensin eliminated P. anaerobius and C. sticklandii from continuous cultures, but it could not inhibit C. aminophilum. The monensin resistance of C. aminophilum was a growth rate-dependent, inoculum size-independent phenomenon that could not be maintained in batch culture. On the basis of these results, we concluded that the feed additive monensin cannot entirely counteract the wasteful amino acid deamination of obligate amino acid-fermenting ruminal bacteria.     

Modeling of the competitive growth of Listeria monocytogenes and Lactococcus lactis in vegetable broth

Current mathematical models used by food microbiologists do not address the issue of competitive growth in mixed cultures of bacteria. We developed a mathematical model which consists of a system of nonlinear differential equations describing the growth of competing bacterial cell cultures. In this model, bacterial cell growth is limited by the accumulation of protonated lactic acid and decreasing pH. In our experimental system, pure and mixed cultures of Lactococcus lactis and Listeria monocytogenes were grown in a vegetable broth medium. Predictions of the model indicate that pH is the primary factor that limits the growth of L. monocytogenes in competition with a strain of L. lactis which does not produce the bacteriocin nisin. The model also predicts the values of parameters that affect the growth and death of the competing populations. Further development of this model will incorporate the effects of additional inhibitors, such as bacteriocins, and may aid in the selection of lactic acid bacterium cultures for use in competitive inhibition of pathogens in minimally processed foods.     

Aerobic and anaerobic microbiology of empyema. A retrospective review in two military hospitals

The microbiology and clinical features of empyema were studied retrospectively in 197 patients whose specimens yielded bacterial growth after inoculation for aerobic and anaerobic bacteria. Three hundred forty-three organisms (216 aerobic or facultative and 127 anaerobic organisms) were isolated. Aerobic bacteria were isolated in 127 (64 percent) patients, anaerobic bacteria in 25 (13 percent), and mixed aerobic and anaerobic bacteria in 45 (23 percent). The predominant aerobic or facultative organisms were Streptococcus pneumoniae (70 isolates), Staphylococcus aureus (58), Escherichia coli (17), Klebsiella pneumoniae (16), and Haemophilus influenzae (12). The predominant anaerobes were pigmented Prevotella and Porphyromonas species (24), Bacteroides fragilis group (22), anaerobic cocci (36), and Fusobacterium species (20). beta-Lactamase-producing organisms were recovered in 49 (38 percent) of 128 tested specimens. These included all 42 tested S aureus and 15 B fragilis group, 4 of 9 K pneumoniae, 3 of 9 H influenzae, 3 of 8 pigmented Prevotella and Porphyromonas species, and 2 of 6 E coli. Most patients from whom S pneumoniae and H influenzae were recovered had pneumonia, and most patients with S aureus had pneumonia, aspiration pneumonia, and lung abscesses. The recovery of anaerobic bacteria was mostly associated with the concomitant diagnosis of aspiration pneumonia, and lung, subdiaphragmatic, dental, and oropharyngeal abscesses. These data highlight the importance of anaerobic bacteria in selected cases of empyema.     

Use of exogenous ribonuclease for improvement of Lactobacillus biological properties

The influence of exogenous RNAse on the dynamics of the acid formation by the industrial strain 8R-A3 of Lactobacillus plantarum, its antibiotic sensitivity and antagonistic activity was studied. In the presence of the RNAase growth stimulating dose both a decrease of the culture lag-phase and a more intensive accumulation of lactic acid in the incubation medium resulting in an increase of the Lactobacillus antagonistic activity were observed. It was shown that RNAase increased the Lactobacillus stability to tetracycline and erythromycin by 32 to 57 per cent as compared to the control.     

A Darwinian theory for the origin of cellular differentiation

The use of starter cultures to control and run the fermentative process is a usual way of manufacturing sausages in meat industries. The first stage in the starter culture designing process is to characterize the lactic acid bacteria isolated from these meat products, in order to select the best strains. The strains used for this study were isolated from different dry fermented sausages, obtained during the manufacturing process. The main tests used to identify the isolated bacteria were: microscopic-morphologic characteristics, catalase activity, production of gas, growth at 8, 15 and 45 degrees C, fermentation of carbohydrates and production of lactic acid isomers. A total of 194 strains were identified. Lactobacillus sake and Lactobacillus plantarum were the most frequent species. Other microbiological tests were also performed, and three strains of Lactobacillus sake were found which did not produce dextran from sucrose.     

Microbiology of perianal cellulitis in children: comparison of skin swabs and needle aspiration

OBJECTIVE: To establish the aerobic and anaerobic microbiology of perianal cellulitis in children, comparing skin swab and needle aspirate methodology. METHOD: Swabs of involved skin and needle aspirates of cellulitis were studied for aerobic and anaerobic bacteria. RESULTS: Specimens obtained from 10 patients with perianal cellulitis showed bacterial growth. Polymicrobial aerobic-anaerobic flora was found in all skin surface cultures, where the predominate isolates were Peptostreptococcus spp., Escherichia coli, and alpha hemolytic streptococci. The number of isolates in needle aspirates varied between one and two. The predominant ones were E. coli (3), Peptostreptococcus spp. (3), Staphylococcus aureus (2), and Bacteroides fragilis group (2). Complete or partial concordance in microbiology between skin swabs and needle aspirates was present in six instances. In four instances, isolates recovered from needle aspirates were not isolated from the skin surface. CONCLUSIONS: This study demonstrates the diversity of aerobic and anaerobic organisms isolated from perianal cellulitis, and the superiority of needle aspirates in establishing the microbiology of the infection.     

Evaluation of universal preenrichment broth for the recovery of foodborne pathogens from milk and cheese

The use of universal preenrichment broth for the recovery of verotoxigenic Escherichia coli, Salmonella spp., and Listeria monocytogenes from milk and cheese was examined. Universal preenrichment broth supported the growth of low inoculum levels (10 cfu/ml) of these organisms in pure cultures and in mixed cultures containing higher levels of other pathogens or bacterial flora from raw milk. This medium also supported the recovery and growth of heat-injured Salmonella spp., L. monocytogenes, and verotoxigenic E. coli at inoculum levels of 10(2) cfu/ml to yield cell levels of 10(8) cfu/ml in pure cultures and at least 10(5) cfu/ml in the presence of high levels of known competitive pathogens or microflora of cheese samples after 24 h of incubation. Universal preenrichment broth performed better than Listeria enrichment broth in supporting the recovery and growth of heat-injured L. monocytogenes and equally as well as buffered peptone water or trypticase soy broth in supporting the growth of uninjured L. monocytogenes, Salmonella spp., and verotoxigenic E. coli. Coenrichment of these pathogens in universal preenrichment broth reduced the quantity of milk or cheese samples that were required for analysis and also reduced the cost and labor involved in preparing and processing separate preenrichment media.     

Augmentation of macrophage phagocytic activity by cell-free extracts of selected lactic acid-producing bacteria

Oral and intraperitoneal administration of lactic acid-producing bacteria can significantly augment the immune response in murine models; however, the immunopotentiating effects in these studies differ significantly. Murine macrophagelike cell line J774 was cultured in the presence of cell-free extracts of Lactobacillus acidophilus and Bifidobacterium longum, and the effect on macrophage function was evaluated by measurement of synthesis of selected enzymes and their ability to take up either acrylamide particles or live Salmonella typhimurium. Lysozyme activity of J774 cells was significantly decreased by cell-free extracts of B. longum, but not of L. acidophilus, whereas extracts of both strains induced morphological changes and significantly enhanced phagocytosis of inert particles or viable Salmonella. Whole cell extracts of lactic acid-producing bacteria are therefore capable of altering macrophage function in a strain-dependent manner.     

Effect of nutrient composition on the in vitro growth of urogenital lactobacilli and uropathogens

Previous clinical studies have shown that nutrients and probiotic agents can alter the composition of the vaginal flora. The present in vitro study has shown that uropathogens have a growth advantage over lactobacilli, but potentially there are natural substances that could be applied vaginally to stimulate lactobacilli growth to the detriment of the pathogens. When chemically defined medium representative of vaginal fluid at pH 5.5 was supplemented with skim milk, it acted as a better substrate for Lactobacillus rhamnosus GR-1 than for uropathogenic bacteria and Candida albicans. Lactobacillus MRS medium, even at pH 4.5, supports the growth of pathogens, but when supplemented with ascorbic acid or EDTA, Lactobacillus growth was significantly higher. When L. rhamnosus GR-1 was coincubated in a combined nutrient composition of vitamins and lactose, it survived better than Escherichia coli and Enterococcus faecalis. These in vitro results provide a basis for testing nutritional supplements to alter the urogenital flora in an attempt to enhance restoration and maintenance of a normal disease-free state.     

Organisms isolated from dogs and cats with anaerobic infections and susceptibility to selected antimicrobial agents

OBJECTIVE: To determine the prevalence of obligate anaerobic bacteria in bacterial infections in dogs and cats and susceptibility to selected antimicrobial agents. DESIGN: Case series. SAMPLE POPULATION: Specimens from 1,267 dogs and 243 cats. PROCEDURE: Standard anaerobic and aerobic bacterial culture methods were used. Anaerobic isolates were tested for susceptibility to selected antimicrobial agents. RESULTS: Obligate anaerobic bacteria were isolated from 199 (15.7%) and 69 (28.4%) specimens obtained from dogs and cats, respectively. More than half of the specimens that contained obligate anaerobic bacteria were from draining tracts (exclusively dogs), pleural fluid, abscesses, bones, the respiratory tract, or the abdominal cavity. The most commonly isolated obligate anaerobic bacteria (approx 70% of all isolates) were Bacteroides spp, Peptostreptococcus spp, Fusobacterium spp, and Porphyromonas spp. Eighty percent of the specimens that contained obligate anaerobic bacteria also contained facultative anaerobic or aerobic organisms. The organisms most commonly isolated in association with obligate anaerobic bacteria were members of the family Enterobacteriaceae (Escherichia coli was the most common), Pasteurella spp, and Staphylococcus intermedius. Ninety-seven obligate anaerobic isolates were tested for susceptibility to ampicillin, amoxicillin-clavulanic acid, chloramphenicol, clindamycin, and metronidazole. All were susceptible to amoxicillin-clavulanic acid and chloramphenicol, and most were susceptible to metronidazole. Only 71% of the Bacteroides isolates were susceptible to ampicillin, and only 83% were susceptible to clindamycin. Only 80% of the Clostridium isolates were susceptible to clindamycin, but all were susceptible to ampicillin. CLINICAL IMPLICATIONS: Data on sites and conditions from which anaerobic bacteria are commonly isolated, along with results of susceptibility testing, may be useful in designing antimicrobial treatment regimens.     

Effect of ruminal lactic acid-utilizing bacteria on adaptation of cattle to high-energy rations

Heifers, unadapted to a concentrate ration, were intraruminally inoculated (1 dose) with cultures of ruminal lactic acid-utilizing bacteria or with ruminal fluid from a steer adapted to a concentrate ration. Inoculation with cultures (1 L) of Selenomonas ruminantium or Megasphaera elsdenii did not produce better average daily weight gains or feed efficiency of heifers fed a high-energy ration for 21 days, if these values were compared with the performance of noninoculated heifers. Average daily weight gain and feed efficiency of heifers inoculated with 1 L of Peptococcus asaccharolyticus culture or with 1 L of adapted ruminal fluid and fed a high-energy ration for 21 days were better if these values were compared with the performance of noninoculated heifers.     

Aerobic and anaerobic bacteriology of cutaneous abscesses in children

Specimens from 209 cutaneous abscesses in children were cultured for aerobic and anaerobic microorganisms. Of these, nine (4%) were sterile and 51 (24%) yielded pure cultures that were predominantly Staphylococcus aureus. The rest of the abscesses yielded growth of two or more aerobic and/or anaerobic organisms. The data were organized according to these anatomic locations: head, neck, trunk, finger, nailbed, hand, leg, buttocks, perirectal, and vulvovaginal areas. Aerobic bacteria only were present in 92 specimens (46%), anaerobes only were isolated in 52 (26%), and mixed aerobic and anaerobic bacteria were present in 56 abscesses (28%). A total of 467 isolates (270 anaerobes and 197 aerobes) were recovered, accounting for 2.3 isolates per specimen (1.3 anaerobes and 1.0 aerobes). The presence of more than one anaerobe per abscess was obtained from the vulvo-vaginal, buttocks, perirectal, finger, nailbed, and head areas. Aerobes were more prevalent in the neck, hand, leg, and trunk areas. The predominant aerobes recovered were: S aureus (89 isolates), alpha- and nonhemolytic streptococci (29), group A beta-hemolytic streptococci (16), Enterobacter (10), and Escherichia coli (8). The predominant anaerobes recovered were anaerobic Gram-positive cocci (79 isolates), Bacteroides sp (116, including 31 B melaninogenicus group and 29 B fragilis group), and Fusobacterium sp (39). Our findings indicate the polymicrobial nature and predominance of anaerobes in cutaneous abscesses in children in perirectal, head, finger, and nailbed areas.