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Interleukin-10 (IL-10) and IL-12 are two cytokines secreted by monocytes/macrophages in response to bacterial products which have largely opposite effects on the immune system. IL-12 activates cytotoxicity and gamma interferon (IFN-gamma) secretion by T cells and NK cells, whereas IL-10 inhibits these functions. In the present study, the capacities of gram-positive and gram-negative bacteria to induce IL-10 and IL-12 were compared. Monocytes from blood donors were stimulated with UV-killed bacteria from each of seven gram-positive and seven gram-negative bacterial species representing both aerobic and anaerobic commensals and pathogens. Gram-positive bacteria induced much more IL-12 than did gram-negative bacteria (median, 3,500 versus 120 pg/ml at an optimal dose of 25 bacteria/cell; P < 0.001), whereas gram-negative bacteria preferentially stimulated secretion of IL-10 (650 versus 200 pg/ml; P < 0.001). Gram-positive species also induced stronger major histocompatibility complex class II-restricted IFN-gamma production in unfractionated blood mononuclear cells than did gram-negative species (12,000 versus 3,600 pg/ml; P < 0.001). The poor IL-12-inducing capacity of gram-negative bacteria was not remediated by addition of blocking anti-IL-10 antibodies to the cultures. No isolated bacterial component could be identified that mimicked the potent induction of IL-12 by whole gram-positive bacteria, whereas purified LPS induced IL-10. The results suggest that gram-positive bacteria induce a cytokine pattern that promotes Th1 effector functions.
Human blood leukocytes and platelets and mouse peritoneal macrophages emit very rapid and very intense Luminol-dependent chemiluminescence (CL) signals when treated with streptococci, staphylococci, or with zymosan, which have been preopsonized with arginine-rich histone, dextran sulfate or polyanetholesulfonate (liquoid). Liquoid alone at 10-30 micrograms/2 X 10(5) leukocytes also triggers intense CL responses in the absence of a carrier. Strong CL can also be triggered, and at the same levels, when the various polyelectrolytes are simply mixed with the bacteria or zymosan and added to the leukocyte suspensions. The CL responses induced by the polyelectrolyte-bacteria complexes greatly exceed those triggered in leukocytes by antibody-complement-coated particles. Liquoid also shows a unique property of markedly augmenting CL signals which have already been induced by other ligand-coated bacteria or zymosan particles. Streptococci and staphylococci were found to be much superior to zymosan, Gram-positive bacilli, or E. coli as carriers for the various polyelectrolytes in the CL reaction. Neither protamine sulfate, lysozyme, myeloperoxidase, crystalline ribonuclease (all cationic in nature), chondroitin sulfate, heparin, nor alginate sulfate acted as ligands for triggering CL, when used to opsonize bacteria or zymosan. The induction of CL in blood leukocytes by the various ligand-coated bacteria is markedly inhibited by azide, KCN catalase, aminotriazole, and EDTA, agents known to inhibit the production of oxygen radicals following stimulation of leukocytes by opsonized bacteria. Two children diagnosed for chronic granulomatous diseases (CGD) of childhood and an apparently healthy sister of one of the male patients completely failed to respond with CL either to the polyelectrolyte-bacteria complexes, liquoid or antibody-coated bacteria and zymosan. It is proposed that liquoid be employed for the rapid screening of defects in certain oxygen-dependent metabolic processes in both PMNs and macrophages. It is also suggested that polyelectrolytes like the ones described in this study may markedly enhance the bactericidal properties of leukocytes and macrophages towards both extracellular and intracellular microorganisms and may perhaps also augment the tumoricidal effects of activated macrophages.
The relationship of mixotrophic and autotrophic Thiothrix species to morphologically similar chemoorganotrophic bacteria (e.g. Leucothrix species, Eikelboom type 021N bacteria) has been a matter of debate for some years. These bacteria have alternatively been grouped together on the basis of shared morphological features or separated on the basis of their nutrition. Many of these bacteria are difficult to maintain in axenic culture and, until recently, few isolates were available to allow comprehensive phenotypic and genotypic characterization. Several isolates of Thiothrix spp. and Eikelboom type 021N strains were characterized by comparative 16S rRNA sequence analysis. This revealed that the Thiothrix spp. and Eikelboom type 021N isolates formed a monophyletic group. Furthermore, isolates of Eikelboom type 021N bacteria isolated independently from different continents were phylogenetically closely related. The 16S rRNA sequence-based phylogeny was congruent with the morphological similarities between Thiothrix and Eikelboom type 021N. However, one isolate examined in this study (Ben47) shared many morphological features with the Thiothrix spp. and Eikelboom type 021N isolates, but was not closely related to them phylogenetically. Consequently, morphology alone cannot be used to assign bacteria to the Thiothrix/type 021N group. Comparative 16S rRNA sequence analysis supports monophyly of the Thiothrix/type 021N group, and phenotypic differences between the Thiothrix spp. and Eikelboom type 021N bacteria are currently poorly defined. For example, both groups include heterotrophic organisms that deposit intracellular elemental sulfur. It is therefore proposed that the Eikelboom type 021N bacteria should be accommodated within the genus Thiothrix as a new species, Thiothrix eikelboomii sp. nov., and three further new Thiothrix species are described: Thiothrix unzii sp. nov., Thiothrix fructosivorans sp. nov. and Thiothrix defluvii sp. nov.
Tendency of isolated bacteria from infections in general surgery and their antimicrobial susceptibilities during the period from April 2000 to March 2001 were investigated in a multicenter study in Japan, and the following results were obtained. The number of cases investigated as objectives was 234 for one year. A total of 388 strains (136 strains from primary infections and 252 strains from postoperative infections) were isolated from 165 cases (70.5% of total cases). In primary infections, anaerobic Gram-positive bacteria were predominant, while from postoperative infections, aerobic Gram-positive bacteria were predominant. Among aerobic Gram-positive bacteria, the isolation rate of Enterococcus faecalis was the highest, followed by that of Staphylococcus aureus from postoperative infections. Among anaerobic Gram-positive bacteria, the isolation rate of Peptostreptococcus spp. was the highest from both types of infections. Among aerobic Gram-negative bacteria, Escherichia coli was the most predominantly isolated from primary infections, followed by Klebsiella pneumoniae and Pseudomonas aeruginosa in this order, and from postoperative infections, P. aeruginosa was the most predominantly isolated, followed by Enterobacter spp. and Klebsiella spp. Among anaerobic Gram-negative bacteria, the isolation rate of Bacteroides fragilis group was the highest from both types of infections. There was no vancomycin-resistant S. aureus nor Enterococcus spp. Among anaerobic bacteria, there were many resistant strains against penicillins and cephems with MICs higher than 100 micrograms/ml, and the same trend was observed among other Bacteroides spp. and Prevotella spp.
Isolated bacteria from infections in general surgery during the period from July 1994 to June 1995 were investigated by a multicenter study in Japan, and the following results were obtained. One hundred and fifty-three strains were isolated from primary infections, and 143 strains were isolated from postoperative infections. From primary infections, both anaerobic Gram-positive and-negative bacteria were predominant, and from postoperative infections, aerobic Gram-positive bacteria were predominant. Among aerobic Gram-positive bacteria, the isolation rate of Enterococcus faecalis was highest, followed by that of Staphylococcus aureus from both types of infections. Among anaerobic Gram-positive bacteria, the isolation rate of Streptococcus intermedius was highest from primary infections, but from postoperative infections anaerobic Gram-positive bacteria was uncommon. Among aerobic Gram-negative bacteria, Escherichia coli was most predominantly isolated from primary infections, followed by Klebsiella pneumoniae and Pseudomonas aeruginosa in this order. From postoperative infections, P. aeruginosa was most predominantly isolated, followed by Serratia marcescens and E. coli. Among anaerobic Gram-negative bacteria, the isolation rate of Bacteroides fragilis group was the highest from both types of infections. We have noticed that resistant strains against imipenem and ofloxacin were increasing among P. aeruginosa and resistant strains against cefazolin were increasing among E. coli. MICs of cefazolin against four out of 30 strains of E. coli were higher than 100 micrograms/ml, and MICs of imipenem was higher than 50 micrograms/ml against 5 out of 22 strains of P. aeruginosa.
The annual multicenter studies on isolated bacteria from infections in general surgery and their antimicrobial susceptibility have been conducted in 19 facilities in Japan since July 1982. This paper describes the results obtained during the period from April 1997 to March 1998. The number of cases investigated as objectives was 215 for one year. A total of 420 strains (170 strains from primary infections and 250 strains from postoperative infections) were isolated from 174 cases (80.9% of total cases). In primary infections, the isolation rate of anaerobic bacteria was higher than in postoperative infections, while in postoperative infections, those of aerobic Gram-positive bacteria and Pseudomonas aeruginosa were higher than in primary infections. Among aerobic Gram-positive bacteria, the isolation rate of Enterococcus faecalis was the highest, followed by Staphylococcus aureus, which was frequently isolated from postoperative infections. Among anaerobic Gram-positive bacteria, Peptostreptococcus spp. and Streptococcus spp. were commonly isolated from both types of infections. Among aerobic Gram-negative bacteria, Escherichia coli was most predominantly isolated from primary infections, followed by P. aeruginosa, Klebsiella pneumoniae in this order, and from postoperative infections, P. aeruginosa was most predominantly isolated, followed by E. coli and K. pneumoniae. Among anaerobic Gram-negative bacteria, Bacteroides fragilis group was the majority of isolates from both types of infections. We found neither vancomycin nor arbekacin resistant strains of S. aureus, and found no vancomycin resistant strains of Enterococcus spp. The susceptibility of P. aeruginosa against carbapenems did not decline in the year 1997, while resistance of B. fragilis group against cephems advanced increasingly.
Isolated bacteria from infections in general surgery during the period from July 1994 to June 1995 were investigated in a multicenter study in Japan, and the following results were obtained. One hundred and sixty-four strains were isolated from primary infections, and 202 strains were isolated from postoperative infections. From primary infections, anaerobic Gram-positive bacteria were predominant, while from post operative infections, aerobic Gram-positive bacteria were predominant. Among aerobic Gram-positive bacteria, the isolation rate of Enterococcus faecalis was the highest, followed by that of Staphylococcus aureus from postoperative infections. Among anaerobic Gram-positive bacteria, the isolation rate of Peptostreptococcus spp. was the highest from both types of infections. Among anaerobic Gram-negative, Escherichia coli was the most predominantly isolated from primary infections, followed by Klebsiella pneumoniae and Pseudomonas aeruginosa in this order, and from postoperative infections, P. aeruginosa was the most predominantly isolated, followed by Enterobacter spp. and Klebsiella spp. Among anaerobic Gram-negative bacteria, the isolation rate of Bacteroides fragilis group was the highest from both types of infections. We noticed that MICs of cefazolin against three out of 23 strains of E. coli were higher than 100 micrograms/ml. Among anaerobic bacteria, there were many resistant strains against penicillins and cephems with MICs higher than 100 micrograms/ml, and the same trend was observed among other Bacteroides spp. and Prevotella spp.
Sample preparation for enumerating attached bacteria in turbid seawater by epifluorescence microscopy was improved by treating samples with a surfactant (Tween 80) followed by sonication. With optimal treatment with Tween 80 (final concentration, 10 ppm [10 mug/ml]) and sonication, as many as 10 times more attached bacteria were enumerated from turbid seawater relative to the number enumerated from an untreated control. Dispersion of bacteria by sonication alone resulted in the enumeration of only 42 to 72% of the attached bacteria. By this technique, fluctuations in the number of attached and free-living bacteria were determined in water from Aransas Pass, Tex., where surface sediments are resuspended on a regular basis by tidal currents. The abundance of attached bacteria increased in proportion to the seawater turbidity that resulted from sediment resuspension. The variation in abundance of free-living bacteria was not directly related to seawater turbidity. However, the magnitude of fluctuation in the abundance of free-living bacteria was related to the extent of turbidity variation during diurnal tides.
The minimum inhibitory concentrations of antibiotics for bacterial pathogens are derived from broth suspensions (broth dilution) and from nutrient surfaces (agar dilution). These concentrations may not apply when bacteria are on a nonnutrient surface such as in a foreign body infection. We compared bacteria (Staphylococcus epidermidis and Escherichia coli) broth suspension MBCs with MBCs of the same bacteria when on a nonnutrient surface in broth the growing and nongrowing phases. Bacteria growing on cotton surfaces were much less susceptible to antibiotic killing than when freely suspended in the liquid nutrient. These results alone, independent of host factors, would explain the failure of antibiotics to eradicate infections involving bacteria on foreign body surfaces. The resistance of bacteria to antibiotic killing is not caused by a lack of antibiotic penetration to the site of the bacteria, but by an altered state of the bacteria when they are associated with a surface.
The annual multicenter studies on isolated bacteria from infections in general surgery and their antimicrobial susceptibility have been conducted in 20 facilities in Japan since July 1982. This paper describes the results obtained during period from July 1996 to June 1997. The number of cases investigated as objectives was 217 for one year. A total of 406 strains were isolated from 177 cases (81.6% of total cases). From primary infections 162 strains were isolated, and from postoperative infections 244 strains were isolated, respectively. From primary infections, anaerobic bacteria were predominant, while from postoperative infections, aerobic Gram-positive bacteria were predominant. Among aerobic Gram-positive bacteria, the isolation rate of Enterococcus spp. was the highest. In postoperative infections, the majority of them were Enterococcus faecalis, while in primary infections, many of them were Enterococcus avium. The isolation rate of Staphylococcus spp., especially from postoperative infections, followed that of Enterococcus spp. Among anaerobic Gram-positive bacteria, Peptostreptococcus spp. and Streptococcus spp. were commonly isolated from both types of infections. Among aerobic Gram-negative bacteria, Escherichia coli was the most predominantly isolated from primary infections, followed by Klebsiella pneumoniae and Pseudomonas aeruginosa in this order, and from postoperative infections, P. aeruginosa was the most predominantly isolated, followed by E. coli and Enterobacter cloacae. Among anaerobic Gram-negative bacteria, Bacteroides fragilis group was the majority of isolates from both types of infections. The isolation rate of aerobic Gram-negative bacillus has decreased with time, while those of anaerobes like B. fragilis group and of aerobic Gram-positive bacteria have gradually increased in both types of infections. We found vancomycin-resistant strains of neither Staphylacoccus aureus nor Enterococcus spp.; however, the MIC of arbekacin for one of strains of S. aureus was 100 micrograms/ml. Both the MIC90's of meropenem and imipenem/cilastatin against P. aeruginosa isolated in this term were 25 micrograms/ml, which were higher than those against the strains isolated in the previous years. Compared with the isolated strains in the year 1995, progress of resistance against carbapenem antibiotics was confirmed.
One hundred sixty-one strains of adherent bacteria were isolated under anaerobic conditions from four sites on the rumen epithelial surface of sheep fed hay or a hay-grain ration. Before isolation of bacteria, rumen tissue was washed six times in an anaerobic dilution solution, and viable bacteria suspended in the washings were counted. Calculation indicated that unattached bacteria would have been removed from the tissue by this procedure, but a slow and progressive release of attached bacteria also occurred. Nevertheless, a wide range of characteristic morphological types remained associated with the epithelium as demonstrated by scanning electron microscopy. Most of these types were represented among the isolates. Characterization and presumptive identification of the isolates showed that 95.0% belonged to previously described genera of functionally significant rumen bacteria, including Butyrivibrio sp. (31.1%), Bacteroides sp. (22.4%), Selenomonas ruminantium (9.9%), Succinivibrio dextrinosolvens (8.7%), Streptococcus bovis (8.1%), Propionibacterium sp. (4.3%), Treponema sp. (3.1%), and Eubacterium sp., Lachnospira multiparus, and Ruminococcus flavefaciens (2.5% each). Eight isolates (5.0%) were not identified. L. multiparus was recovered only from hay-fed animals; all other genera were obtained from animals fed either ration. All S. bovis strains and two strains each of Bacteroides sp. and Butyrivibrio sp. were aerotolerant; all other strains were strictly anaerobic. Bacteria representing the gram-positive, facultatively anaerobic flora associated with rumen wall tissue (R. J. Wallace, K.-J. Cheng, D. Dinsdale, and E. R. Ørskov, Nature (London) 279:424-426, 1979) were therefore not recovered by the techniques used; instead a different fraction of the adherent population was isolated. The term "epimural" is proposed to describe the flora associated with the rumen epithelium.
Several common strains of bacteria have been studied to determine their influence on human and rabbit platelets in vitro. Bacteria at a nominal ratio of 1:1 were added to platelets in their native plasma or to platelets in a balanced salt solution. The platelet-bacterial interaction was examined by nephelometry and by sequential light microscopy of fixed specimens taken at key points in the reaction. The results support the following conclusions: (1) Bacteria that were potent stimuli of the platelet aggregation reaction were incorporated in large numbers into the forming platelet aggregates. (2) Bacteria sequestered within platelet aggregates remained in direct contact with the aggregated platelet mass for up to 1 hour in vitro and during this time showed no morphologic changes by light microscopy. (3) Plateletbacterial interaction produced clumping of the bacteria but no demonstrable killing of the microorganisms. (4) Collagen-platelet interaction resulted in release of platelet products that were capable of clumping bacteria but were not lethal to them. (5) Rabbit and human platelets showed no species differences in plateletbacterial interaction. (6) Washed platelets interacted with bacteria without added serum factors but the addition of serum accelerated the aggregation reaction.
Fourier transform-infrared (FT-IR) spectroscopy has been used to rapidly and nondestructively analyze bacteria, bacteria-polymer mixtures, digester samples and microbial biofilms. Diffuse reflectance FT-IR (DRIFT) analysis of freeze-dried, powdered samples offered a means of obtaining structural information. The bacteria examined were divided into two groups. The first group was characterized by a dominant amide I band and the second group of organisms displayed an additional strong carbonyl stretch at approximately 1740 cm-1. The differences illustrated by the subtraction spectra obtained for microbes of the two groups suggest that FT-IR spectroscopy can be utilized to recognize differences in microbial community structure. Calculation of specific band ratios has enabled the composition of bacteria and extracellular or intracellular storage product polymer mixtures to be determined for bacteria-gum arabic (amide I/carbohydrate C-O approximately 1150 cm-1) and bacteria-poly-beta-hydroxybutyrate (amide I/carbonyl approximately 1740 cm-1). The key band ratios correlate with the compositions of the material and provide useful information for the application of FT-IR spectroscopy to environmental biofilm samples and for distinguishing bacteria grown under differing nutrient conditions. DRIFT spectra have been obtained for biofilms produced by Vibrio natriegens on stainless steel disks. Between 48 and 144 h, an increase in bands at approximately 1440 and 1090 cm-1 was seen in FT-IR spectra of the V. natriegens biofilm. DRIFT spectra of mixed culture effluents of anaerobic digesters show differences induced by shifts in input feedstocks. The use of flow-through attenuated total reflectance has permitted in situ real-time changes in biofilm formation to be monitored and provides a powerful tool for understanding the interactions within adherent microbial consortia.
A novel method for the identification of bacteria in environmental samples by DNA hybridization is presented. It is based on the fact that, even within a genus, the genomes of different bacteria may have little overall sequence homology. This allows the use of the labeled genomic DNA of a given bacterium (referred to as a "standard") to probe for its presence and that of bacteria with highly homologous genomes in total DNA obtained from an environmental sample. Alternatively, total DNA extracted from the sample can be labeled and used to probe filters on which denatured chromosomal DNA from relevant bacterial standards has been spotted. The latter technique is referred to as reverse sample genome probing, since it is the reverse of the usual practice of deriving probes from reference bacteria for analyzing a DNA sample. Reverse sample genome probing allows identification of bacteria in a sample in a single step once a master filter with suitable standards has been developed. Application of reverse sample genome probing to the identification of sulfate-reducing bacteria in 31 samples obtained primarily from oil fields in the province of Alberta has indicated that there are at least 20 genotypically different sulfate-reducing bacteria in these samples.
For more than 100 years, investigators have tried to identify the bacteria responsible for dental caries formation and to determine whether their role is one of specificity. Frequent association of Lactobacillus acidophilus and Streptococcus mutans with caries activity gave credence to their being specific cariogens. However, dental caries occurrence in their absence, and the presence of other bacteria able to produce substantial amounts of acid from fermentable carbohydrate, provided arguments for non-specificity. In the 1940s, Stephan found that the mixed bacteria in dental plaque produced a rapid drop in pH following a sugar rinse and a slow pH return toward baseline. This response became a cornerstone of plaque and mixed-bacterial involvement in dental caries causation when Stephan showed that the pH decrease was inversely and clearly related to caries activity. Detailed examination of the pH (acid-base) metabolisms of oral pure cultures, dental plaque, and salivary sediment identified the main bacteria and metabolic processes responsible for the pH metabolism of dental plaque. It was discovered that this metabolism in different individuals, in plaque in different dentition locations within individuals, and in individuals of different levels of caries activity could be described in terms of a relatively small number of acid-base metabolic processes. This led to an overall bacterial metabolic vector concept for dental plaque, and helped unravel the bacterial involvement in the degradation of the carbohydrate and nitrogenous substrates that produce the acids and alkali that affect the pH and favor and inhibit dental caries production, respectively. A central role of oral arginolytic and non-arginolytic acidogens in the production of the Stephan pH curve was discovered. The non-arginolytics could produce only the pH fall part of this curve, whereas the arginolytics could produce both the fall and the rise. The net result of the latter was a less acidic Stephan pH curve. Both kinds of bacteria are numerous in dental plaque. By varying their ratios, we were easily able to produce Stephan pH curves indicative of different levels of caries activity. This and substantial related metabolic and microbial data indicated that it is the proportions and numbers of acid-base-producing bacteria that are at the core of dental caries activity. The elimination of S. mutans, as with a vaccine, was considered to have little chance of success in preventing dental caries in humans, since, in most cases, this would simply make more room for one or more of the many acidogens remaining. An understanding of mixed-bacterial metabolism, knowledge of how to manipulate and work with mixed bacteria, and the use of a bacterial metabolic vector approach as described in this article have led to (1) a more ecological focus for dealing with dental caries, and (2) new means of developing and evaluating anti-caries agents directed toward microbial mixtures that counter excess acid accumulation and tooth demineralization.