Coliforms and enterococci isolated from the intestinal tract of conventional mice

Coliforms and enterococci were isolated from the intestinal tract of infant (12-day-old) and adult (6-to 8-week-old) conventional mice. Eighty coliform isolates and eighty enterococcal strains were grouped according to their ability to ferment or hydrolyze various substrates. Sixty-one of the coliform isolates were identified asEscherichia coli. The remaining 19 strains were similar toE. coli, but did not produce-galactosidase. The enterococci belonged to two species:Streptococcus faecium andS. faecalis. Four biotypes ofS. faecium and two biotypes ofS. faecalis were detected. Xylosefermenting enterococci were isolated with a higher frequency from infant mice than from adults.     

Nucleic acid hybridization of group N and group D streptococci

Streptococci of serological groups N and D were found to belong to three different 23S ribosomal RNA (rRNA) homology clusters which are not closely related to each other. One cluster includes the group N streptococci:Streptococcus lactis, S. cremoris, S. diacetylactis, and, in addition, two lactobacilli, Lactobacillus hordniae andL. xylosus. The second one is composed ofStreptococcus faecium, S. durans, andS. faecalis with its subspecies. All are members of serological group D. The third group includes the group D streptococciStreptococcus bovis andS. equinus together withS. thermophilus andS. salivarius. DNA-DNA hybridization studies confirm the close genetic relationship within each of the rRNA homology clusters.     

Studies on Streptococci: I. Distribution of Fecal Streptococci in Man

To understand the significance to the host of streptococci as part of the intestinal microflora, we first tried to investigate the distribution of human fecal streptococci on the species level. Of the selective media compared, KMN agar was more effective than the other media for the isolation of streptococci from human feces. We made an effort to improve streptococcal classification. Especially we used utilization of 1% pyruvate, 1% arginine, and 1% citrate for differentiation between Streptococcus faecalis and S. faecium. In a tellurite tolerance test, S. faecalis was distinguished more clearly from S. faecium in the medium containing 0.16 or 0.32% tellurite. We devised methods of presumptive identification of fecal streptococci from the results of the characteristics of 1,442 isolates. These methods enabled us to identify many strains rapidly. Different results in the distribution of species of streptococci between children and adults were observed. S. faecalis and S. faecium were isolated constantly from both groups. S. bovis and S. avium were isolated frequently from the feces of children. On the other hand, “viridans” streptococci, e.g. S. salivarius, S. mitis and S. MG-intermedius were present at a high frequency in, and no S. avium could be isolated from, the feces of adults.     

Recognition of Group D Streptococcal Species of Human Origin by Biochemical and Physiological Tests

The speciation of 262 strains of group D streptococci isolated from human sources is described. One hundred forty-two isolates from blood cultures were included; 96 of these were submitted as isolates from clinical cases of subacute bacterial endocarditis. The results show that 98 Streptococcus faecalis, 29 S. faecalis var. zymogenes, 44 S. faecalis var. liquefaciens, 27 S. faecium, 13 S. durans, 44 S. bovis, and 7 unspeciated S. bovis-like group D isolates were identified. No S. faecium var. casseliflavus, S. equinus, or S. avium (group Q streptococci) were identified among the human isolates. The speciation procedures and techniques are detailed. The procedures and limitations of the tests used are discussed. Ninety-eight percent of the 262 strains were speciated by a spectrum of tests that allowed us to recognize atypical as well as typical strains within species.     

Enterococci in Insects

Enterococci were obtained from 213 of 403 insects cultured during a 14-month period, in numbers from 10³ to 3 × 10⁷/g of insect. Insects were taken only from nonurban, wild, and cultivated fields and woods. In species of insects carrying them, enterococci were not always present in every individual cultured, and often more than one species of enterococcus occurred within a species. Enterococci were obtained from certain insects taken in the field during the dormant season, suggesting their role as overwintering agents. They were generally present in species feeding on nectar, succulent plant parts, and on and ir forest litter, but not from insects feeding on less succulent leaves and stems. Streptococcus faecalis was recovered from 32%, Streptococcus faecium from 22.4%, and Streptococcus faecium var. casseliflavus from 43.5% of members of the 37 taxa of insects. S. faecalis and S. faecium var. casseliflavus exhibit a high percent of conformity to the properties published for them. The heterogeneity in properties of S. faecium is similar to that found for the species taken from plants. Many fail to grow in broth at 45 C or in broth containing 6.5% NaCl; 50% of the cultures ferment both melezitose and melibiose, and a few ferment neither sugar. The remainder ferment melibiose only. Failure to reduce methylene blue in milk by S. faecalis and S. faecium is correlated with the inability to ferment lactose. More than 93% of the cultures of S. faecalis digest casein in milk from the top downward, following the production of a soft, flowing curd. Because this property is not characteristic of S. faecalis taken from humans, the reaction in litmus milk is suggested as a means of differentiation between cultures of remote and innocent origin in nature and recent, human pollution.     

Influence of β-lactam antibiotics in vitro on mixed cultures ofEscherichia coli andStreptococcus faecalis

The effect of -lactams in vitro on mixed cultures ofEscherichia coli andStreptococcus faecalis was studied by monitoring the time-dependent course of antibacterial action of ampicillin, mezlocillin, piperacillin, and cefotaxime. These tests revealed that ampicillin and especially mezlocillin acted bactericidally againstE. coli andS. faecalis; piperacillin and cefotaxime selectedS. faecalis from mixed cultures. This behavior suggests interference with the natural balance between the two species.     

Fish skin bacteria: Colonial and cellular hydrophobicity

Dental plaque is a complex community of bacteria coexisting in an environment frequently limited by carbon and energy sources. UnlikeStreptococcus mutans, other oral streptococci such asS. milleri andS. sanguis have an absolute requirement for and actually consume all available arginine when grown glucose limited in a chemically defined medium. The conditions, particularly in terms of arginine concentration, under which the dental plaque bacteriaS. mutans andS. milleri would coexist under glucose-limiting conditions were investigated. The minimum level of arginine supporting optimal growth ofS. milleri was found to be ca. 50M, and above this level these strains outcompetedS. mutans. However, coexistence withS. mutans could be achieved at arginine levels of 14–40M, depending upon theS. milleri andS. mutans strains used. Under such dual limitation,S. milleri was unable to respond to glucose pulses but did respond to pulses of arginine and arginine plus glucose. One of the twoS. milleri strains did not tolerate low pH. In contrast,S. mutans did not tolerate high pH whereasS. milleri was unaffected. This is relevant to dental plaque where arginine catabolism produces a pH rise. Additionally, arginine is an important nutrient since it can be used as an energy source by some oral streptococci.     

Lysozymelike lytic enzyme of Streptococcus faecalis and its role in the larval development of wax moth, Galleria mellonella

The predominant type of bacteria present in the gut of larval Galleria mellonella were streptococci group D identified as Streptococcus faecalis which showed bacteriolytic activity. Young larvae usually contained mixed populations with a marked dominance of fecal streptococci while normally developed mature larvae most frequently contained large uniform populations of S. faecalis. Pupal stages were found to contain the highest percentage of individuals with pure cultures of fecal streptococci.The author suggests a hypothesis that, owing to its bacteriolytic properties, S. faecalis can be considered as a component of the natural, nonspecific defense mechanism of G. mellonella against bacterial infections. The lytic enzyme released in the exponential growth phase of S. faecalis participates in the selection process stabilizing the microbial flora of wax moth larvae; it limits the population of other forms of bacteria. Larval resistance to bacterial infections to a large extent depends on the magnitude of the populations and thus on S. faecalis muramidase concentration. Bacterial lysozyme inhibited the growth of the ingested organisms and in consequence it prevented the proliferation of undesired bacteria in the digestive tract of Galleria larvae.The lytic enzyme proved to be identical with autolysin, a β-N-acetylmuramide glycanhydrolase (EC 3.2.1.17) which has been isolated from trypsin-speeded wall autolysates of S. faecalis by Shockman and Cheney (1969).     

Species Differentiation of Group D Streptococci

Three hundred and fourteen strains of group D streptococci were studied by means of a number of tests. The majority of the strains were identified as Streptococcus faecalis (83 strains), Streptococcus faecium (131 strains), or Streptococcus bovis (32 strains). Several strains (47 or nearly 15%) either shared characteristics of two species or were completely atypical. S. faecalis and S. bovis were more easily identified than S. faecium, which is not sharply defined from the other species and could be subdivided into several fermentative types on the basis of fermentation of arabinose, mannitol, sorbitol, glycerol, and sucrose. The value of some characteristics in species identification is discussed. Growth in the presence of potassium tellurite 1:2,500 and in the presence of 6.5% NaCl and fermentation of arabinose, glycerol, and raffinose are very important tests for the identification of the three species. The reduction of tetrazolium salts, the reduction of litmus milk, and the fermentation of sorbitol may serve as complementary tests for the same purpose. For the differentiation of these three species the “pattern of reactions” is more important than single tests.     

Pulsed-Field Gel Electrophoresis Is More Discriminating Than Multilocus Enzyme Electrophoresis and Random Amplified Polymorphic DNA Analysis for Typing Pyogenic Streptococci

The SmaI restriction endonuclease digestion patterns of chromosomal DNAs from 99 pyogenic streptococci belonging to Lancefield group A (41 Streptococcus pyogenes), group C (seven S. dysgalactiae, 11 \QS. equisimilis\W, three S. equi, eight S. zooepidemicus) and group G (25 human group G Streptococcus, four S. canis) were analyzed by pulsed-field gel electrophoresis (PFGE), and the results were compared with those previously obtained by multilocus enzyme electrophoresis (MLEE) and random amplified polymorphic DNA analysis (RAPD). PFGE revealed 93 distinct types among the 99 strains, and no patterns were common to strains of different species. The discriminatory power of PFGE was greater than that of MLEE and RAPD for groups A and G streptococci. The polymorphism among group C streptococci was similar with the three techniques. PFGE is, therefore, the most efficacious method for epidemiological typing of pyogenic streptococci. Received: 31 July 1996 / Accepted: 17 August 1996     

Fatty acid composition of membranes of L-forms ofStreptococcus faecalis andProteus mirabilis cultured at different osmolalities

The fatty acid composition of membranes of L-forms ofStreptococcus faecalis andProteus mirabilis cultured at different osmolalities and in different osmotic stabilizers was examined.S. faecalis L-forms cultured with sucrose in the medium showed a decrease in the unsaturated fatty acid C₁₈₁ and an increase in the C₁₈ fatty acid and C₁₉ cyclopropane fatty acid. Fatty acid composition ofS. faecalis L-forms cultured in medium containing 1.8% NaCl was similar to the fatty acid composition of L-forms cultured in brain-heart infusion broth (BHI) without osmotic stabilizer and was between the composition of fatty acids of L-forms in BHI with sucrose and that in BHI without 0.5 M sucrose. InProteus mirabilis L-forms, there were differences between L-forms cultured with and without sucrose, but these differences were not comparable to the changes observed inS. faecalis L-forms.P. mirabilis L-forms cultured with and without NaCl in the medium had similar fatty acid compositions.     

Spherical Lactic Acid-producing Bacteria of Southern-grown Raw and Processed Vegetables

The frequency and levels of population of the spherical lactic acid-producing bacteria were determined on raw and processed yellow summer and zucchini squash, a variety of greens, green beans, okra, southern peas, and butter and lima beans, and on fresh cucumbers and corn flowers. Six taxa occurred consistently: Leuconostoc mesenteroides, yellow-pigmented streptococci, Streptococcus faecium, Aerococcus viridans, and S. faecalis and S. faecalis var. liquefaciens. The same taxa occurred with the same order of frequency on processed, frozen vegetables, but with a marked decrease in the occurrence of S. faecalis var. liquefaciens. S. lactis, S. cremoris, S. equinus, S. bovis, and pediococci were isolated infrequently. No other member of the viridans group of the streptococci and no member of the pyogenic group was isolated. Approximately 88% of the cultures were identified. Total counts of the lactic-acid-producing bacteria rarely exceeded 10⁵ per gram of sample, and there was a reduction by 90% during the second year of study, probably because of drought. Only one bacterial species was found on 40% of the raw and 34% of the processed vegetable samples. Two or more species or taxa were present on the remainder of 153 raw and 56 processed vegetable samples. A. viridans was present on squash, greens, okra, and southern peas, and its frequency of occurrence on vegetables suggests that plants are its natural habitat.     

Comparison of conventional and miniaturized biochemical techniques for identification of animal streptococcal isolates

Human clinical streptococcal isolates can be identified rapidly by means of commercially available miniaturized biochemical systems, in contrast to animal and environmental isolates which may require extensive characterization using conventional methods. Streptococcal isolates (n=548) from fresh animal feces of cattle, swine, and broiler chickens were tested by means of conventional biochemical and physiological techniques, and also with a miniaturized technique in which conventional formulations were dispensed in 0.1 ml volume into microtiter plates. Agreement of the positive feature frequencies of the two methods were compared. Results from the tolerance tests in the two methods were generally in good agreement. However, the miniaturized method tended to give false negative results in some carbohydrate fermentation tests. Agreement between the 2 methods ranged from 100% for bile esculin tests to 71% for raffinose fermentation. Cluster analysis of the conventional method data indicated that there were 11 biochemically related groups of isolates, 2 of which were identified asStreptococcus faecalis, andS. morbillorum. Half of the isolates biochemically resembledS. faecium. Errors of miniaturized tests occurred mainly in certain tests and in certain biochemically related clusters of isolates. The data indicate that further investigation of experimental conditions such as medium formulation and inoculum size could lead to a successful miniaturized technique for testing animal streptococcal isolates.     

Drug susceptibility testing of clinical isolates of streptococci and enterococci by the Phoenix automated microbiology system

Background  

Drug resistance is an emerging problem among streptococcal and enterococcal species. Automated diagnostic systems for species identification and antimicrobial susceptibility testing (AST) have become recently available. We evaluated drug susceptibility of clinical isolates of streptococci and enterococci using the recent Phoenix system (BD, Sparks, MD). Diagnostic tools included the new SMIC/ID-2 panel for streptococci, and the PMIC/ID-14 for enterococci. Two-hundred and fifty isolates have been investigated: β-hemolytic streptococci (n = 65), Streptococcus pneumoniae (n = 50), viridans group streptococci (n = 32), Enterococcus faecium (n = 40), Enterococcus faecalis (n = 43), other catalase-negative cocci (n = 20). When needed, species ID was determined using molecular methods. Test bacterial strains were chosen among those carrying clinically-relevant resistance determinants (penicillin, macrolides, fluoroquinolones, glycopeptides). AST results of the Phoenix system were compared to minimal inhibitory concentration (MIC) values measured by the Etest method (AB Biodisk, Solna, Sweden).     

Histological and microbiological aspects of the vagina in captive howler monkeys (Alouatta caraya)

The histology and the glycogen content of the vaginal epithelium was studied in 15 adultAlouatta caraya monkeys. The histological sections revealed a thin epithelium without cornification. The application of the PAS-amylase method showed absence of glycogen. The microbial flora of the vagina was examined in eight adultAlouatta caraya. The vaginal smears showed a mixed flora with a predominance of Gram+cocci. Haemolytic streptococci were found in 100% of the tested monkeys. Other cocci, such asStaphylococcus albus, Streptococcus faecalis andMicrococcus sp. were less frequent. Of the fecal organisms,Proteus sp. was the most frequently isolated. Neither ureaplasms nor any species ofLactobacillus could be isolated. Positive cultures for nonpathogenic yeasts was a common feature. The relation between the absence of glycogen in the vaginal epithelium and the impossibility to isolate lactobacilli is discussed.     

Development of lactic acid bacteria during early stages of fermentation in fish silage

Summary The development of various lactic acid bacteria during the early stages of fermentation (1–6 days after ensiling) in fish silage was studied. The first type of organisms that grew fastest was the oval cocci (most of them resembledLeuconostoc mesenteroides andStreptococcus lactis) followed by round cocci (mostlyS. faecalis). The number of oval cocci increased rapidly one day after ensiling and then decreased after 2–3 days. The round cocci increased first after 2–3 days and then decreased slowly after 4–5 days. Lactobacilli began to increase in number (more than 10¹⁰ per g silage) first after 6 days. Thus the pH in the silage was mainly lowered by the action of streptococci. Also in MRS medium the pH was more rapidly lowered byS. faecalis than byLactobacillus plantarum and other rods.     

Fine structure and fatty acid composition of a motile streptococcus

The fine structure and fatty acid composition of a motile yellow pigmented streptococcus, isolated from gypsy moth (Porthetria dispar) larvae, was compared to that ofStreptococcus faecalis. The yellow streptococcus differed fromS.faecalis in the following manner: (1) absence of19:0 cyclic acid in its fatty acid complement, (2) the presence of a carotenoid, and (3) flagellation. Additionally its fine structure exhibited cores (microbial organelles) and mesosomes similar to those previously observed in other members of group D.This work was supported in part by U.S. Department of Agriculture, Forest Service Grant No. 4000-01.     

Survival and activity ofStreptococcus faecalis andEscherichia coli in tropical freshwater

The survival ofStreptococcus faecalis andEscherichia coli was studied in situ in a tropical rain forest watershed using membrane diffusion chambers. Densities were determined by acridine orange direct count and Coulter Counter. Population activity was determined by microautoradiography, cell respiration, and by nucleic acid composition. Densities ofS. faecalis andE. coli decreased less than 1 log unit after 105 hours as measured by direct count methods. Activity as measured by respiration, acridine orange activity, and microautoradiography indicated that both bacteria remained moderately active during the entire study. After 12 hours,E. coli was more active thanS. faecalis as measured by nucleic acid composition. In this tropical rain forest watershed,E. coli andS. faecalis survived and remained active for more than 5 days; consequently, both would seem to be unsuitable as indicators of recent fecal contamination in tropical waters.     

New perspectives on bacterial ferredoxin evolution

Summary Recent evidence indicates that a gene transposition event occurred during the evolution of the bacterial ferredoxins subsequent to the ancestral intrasequence gene duplication. In light of this new information, the relationships among the bacterial ferredoxins were reexamined and an evolutionary tree consistent with this new understanding was derived. The bacterial ferredoxins can be divided into several groups based on their sequence properties; these include the clostridial-type ferredoxins, theAzotobacter-type ferredoxins, and a group containing the ferredoxins from the anaerobic, green, and purple sulfur bacteria. Based on sequence comparison, it was concluded that the amino-terminal domain of theAzotobacter-type ferredoxins, which contains the novel 3Fe3S cluster binding site, is homologous with the carboxyl-terminal domain of the ferredoxins from the anaerobic photosynthetic bacteria.A number of ferredoxin sequences do not fit into any of the groups described above. Based on sequence properties, these sequences can be separated into three groups: a group containingMethanosarcina barkeri ferredoxin andDesulfovibrio desulfuricans ferredoxin II, a group containingDesulfovibrio gigas ferredoxin andClostridium thermoaceticum ferredoxin, and a group containingDesulfovibrio africanus ferredoxin I andBacillus stearothermophilus ferredoxin. The last two groups differ from all of the other bacterial ferredoxins in that they bind only one FeS cluster per polypeptide, whereas the others bind two. Sequence examination indicates that the second binding site has been either partially or completely lost from these ferredoxins.Methanosarcina barkeri ferredoxin andDesulfovibrio desulfuricans ferredoxin II are of interest because, of all the ferredoxins whose sequences are presently known, they show the strongest evidence of internal gene duplication. However, the derived evolutionary tree indicates that they diverged from theAzotobacter-type ferredoxins well after the ancestral internal gene duplication. This apparent discrepancy is explained by postulating a duplication of one halfchain sequence and a deletion of the other halfchain. TheClostridium thermoaceticum andBacillus stearothermophilus groups diverged from this line and subsequently lost one of the FeS binding sites.It has recently become apparent that gene duplication is ubiquitous among the ferredoxins. Several organisms are now known to have a variety of ferredoxins with widely divergent properties. Unfortunately, in only one case are the sequences of more than one ferredoxin from the same organism known. Thus, although the major features of the bacterial ferredoxin tree are now understood, a complete bacterial phylogeny cannot be inferred until more sequence information is available.     

Narrow host range of some streptococcal R plasmids

Nine R plasmids originally harbored by Streptococcus faecalis (pIP614, pIP655, pIP685, pIP686, pIP 1075, pIP1017),S. faecium (pIP716, pIP991), and group B Streptococcus (pMV120) wild-type hosts were transferred by conjugation into various recipients in order to study the extent of their intraspecies, interspecies, and intergeneric host range. Recipients were streptococci of groups A, B, C, D (S. faecalis, S. faecium, S. durans, S. bovis), and G, S. sanguis, two S. pneumoniae strains (encapsulated and nonencapsulated), and two strains of different genera, Staphylococcus aureus and Listeria inocua. The plasmids carried different antibiotic resistance markers: tetracycline, high levels of gentamicin and kanamycin or of streptomycin and kanamycin, and chloramphenicol. These R plasmids displayed narrow host ranges. They transferred into S. faecalis recipients and plasmid DNA could be detected in these transconjugants. Occasionally, the R plasmids also transferred into one or more other recipients, but no detectable plasmid DNA could be demonstrated in the new hosts.