The Influence of Specific Bacteria and a Filterable Agent on the Growth of Gnotobiotic Chicks

Growth depression was induced in chicks and a collection of aerobic and anaerobic bacteria was isolated from the crop and caecum. The collection of bacteria was tested in gnotobiotic chickens for its ability to depress growth with and without a bacteria-free faecal filtrate. None of the anaerobic bacteria depressed growth. Aerobic bacteria always depressed growth but only in the case of Streptococcus faecium was this statistically significant. The faecal filtrate also depressed growth. A statistically significant effect was obtained when faecal filtrate was combined with any of the following treatments: (a) the total collection of bacteria, (b) the aerobic group, (c) the anaerobic group, (d) Strep. faecalis var. liquefaciens , (e) Strep, faecium , (f) the unclassified streptococci, and (g) a group containing the lactobacilli and coliform organisms. The largest growth depression was obtained in chickens dosed with Strep, faecium and faecal filtrate.     

Composition of the enterococcal and streptococcal intestinal flora of poultry

Identification to species level showed that Enterococcus faecalis and Ent. faecium largely dominated the enterococcal and streptococcal gut flora of 1-d-old chicks. Enterococcus faecalis was rare in 3- to 5-week-old broilers. Two species, Ent. faecium and Streptococcus alactolyticus, were isolated from nearly all broilers examined. Enterococcus hirae and Ent. durans were found in the small intestines of this category of poultry. In layers and parent stock of over 12 weeks of age, Ent. cecorum dominated with Strep. alactolyticus ranking next. Other species were isolated irregularly. Enterococcus avium and Ent. gallinarum, originally described from chickens, were rarely found. These species did not appear to belong to the normal intestinal flora of poultry.     

The survival of streptococci and enterococci in animal feed and on straw with particular reference to Streptococcus uberis

The survival of Streptococcus uberis, Strep. bovis, Enterococcus faecalis spp. faecalis and Ent. faecium in animal feed and on wheat straw was studied for one month. Streptococcus bovis survived the best in feed and Strep. uberis the worst while on straw Ent. faecium was the most resilient species and there was little to choose between the other three. The recovery of all species, excepting Strep. bovis , on selective and non-selective agar was comparable. The growth of Strep. bovis , on the other hand, was inhibited on both Edward's medium and inulin agar.     

The survival of streptococci and enterococci in animal feed and on straw with particular reference to Streptococcus uberis

The survival of Streptococcus uberis, Strep. bovis, Enterococcus faecalis spp. faecalis and Ent. faecium in animal feed and on wheat straw was studied for one month. Streptococcus bovis survived the best in feed and Strep. uberis the worst while on straw Ent. faecium was the most resilient species and there was little to choose between the other three. The recovery of all species, excepting Strep. bovis, on selective and non-selective agar was comparable. The growth of Strep. bovis, on the other hand, was inhibited on both Edward's medium and inulin agar.     

Composition of the enterococcal and streptococcal intestinal flora of poultry

Identification to species level showed that Enterococcus faecalis and Ent. faecium largely dominated the enterococcal and streptococcal gut flora of 1-d-old chicks. Enterococcus faecalis was rare in 3- to 5-week-old broilers. Two species, Ent. faecium and Streptococcus alactolyticus , were isolated from nearly all broilers examined. Enterococcus hirae and Ent. durans were found in the small intestines of this category of poultry.
In layers and parent stock of over 12 weeks of age, Ent. cecorum dominated with Strep. alactolyticus ranking next. Other species were isolated irregularly. Enterococcus avium and Ent. gallinarum , originally described from chickens, were rarely found. These species did not appear to belong to the normal intestinal flora of poultry.     

Heat injury and recovery of Streptococcus faecium associated with the souring of chub-packed luncheon meat

The presence of NaCl in the heating medium provided some protection from lethal heat damage for cells of a Streptococcus faecium strain isolated from luncheon meat whereas the presence of NaNO2 either alone or in addition to NaCl, had no significant effect on cell survival. Subsequent recovery and growth of heat-damaged cells was retarded by the presence of NaCl. When NaNO2 was present in addition to NaCl the inhibitory effect of the latter was reduced. These principal components of the luncheon-meat-cure are apparently opposed in their activities on post-heating recovery and growth of Strep. faecium. Product stability, i.e. duration of the lag before growth occurs, is directly related to the severity of the heat treatment and to the concentration of NaCl in the product. Therefore the resistance of pasteurized chub-packed luncheon meat to streptococcal spoilage during storage at temperatures conducive to microbial growth results from a prolonged heat-induced salt-maintained pre-growth adjustment phase rather than to any inherent inhibitory property of the luncheon meat to the growth of non-heat-damaged Strep. faecium cells.     

Heat injury and recovery of Streptococcus faecium associated with the souring of chub-packed luncheon meat

The presence of NaCl in the heating medium provided some protection from lethal heat damage for cells of a Streptococcus faecium strain isolated from luncheon meat whereas the presence of NaNO₂ either alone or in addition to NaCl, had no significant effect on cell survival. Subsequent recovery and growth of heat-damaged cells was retarded by the presence of NaCl. When NaNO₂ was present in addition to NaCl the inhibitory effect of the latter was reduced. These principal components of the luncheon-meat-cure are apparently opposed in their activities on post-heating recovery and growth of Strep. faecium . Product stability, i.e. duration of the lag before growth occurs, is directly related to the severity of the heat treatment and to the concentration of NaCl in the product. Therefore the resistance of pasteurized chub-packed luncheon meat to streptococcal spoilage during storage at temperatures conducive to microbial growth results from a prolonged heat-induced salt-maintained pre-growth adjustment phase rather than to any inherent inhibitory property of the luncheon meat to the growth of non-heat-damaged Strep. faecium cells.     

Identification and composition of the streptococcal and enterococcal flora of tonsils, intestines and faeces of pigs

Streptococcus suis was the most frequent Streptococcus spp. in pig tonsils, followed by the beta-haemolytic porcine 'equisimilis'ecovar of Strep. dysgalactiae. The intestinal streptococcal flora was composed of Strep. bovis, Strep. hyointestinalis and Strep. suis. Many of these intestinal Strep. suis belonged to a beta-glucuronidase-negative biotype which is infrequent in lesions. Nearly half of the strains presumptively identified as Strep. alactolyticus produced acid from lactose. This species was not found in tonsils and intestines but was about equally prevalent as Strep. hyointestinalis in pig faeces and rectal swabs. Other streptococci were rare in this material.
Enterococci were much less frequently identified than streptococci in tonsils and faeces. In intestinal samples Enterococcus faecalis, Ent. faecium, Ent. hirae and Ent. cecorum were most frequently found. In faeces Ent. faecium was the most prevalent enterococcus.
The characteristics of the less well known species Strep. alactolyticus and Strep. hyointestinalis are described in detail, and guidelines for their differentiation from Strep. bovis and Strep. suis given.     

Ecology of Streptococcus faecium bacteriophage in chicken gut.

The interaction in the chick gut between Streptococcus faecium and its phage was examined. In conventional chicks, large numbers of S. faecium and phage were found in the cecum and smaller numbers were found in the anterior gut. In gnotobiotic chicks associated with S. faecium SY1 and its phage, there was no marked effect on bacterial numbers, but resistance to the phage rapidly developed. Depression of chick growth caused by S. faecium strain SY1 was partially reversed by its phage.     

Distribution and Properties of Serological Types of Streptococcus faecium, Streptococcus durans and Related Strains

S ummary . The distribution of 19 serological types of Streptococcus faecium and related organisms has been studied, using 367 strains isolated mainly from faecal samples. Several types occurred in man as well as in pigs, sheep, cattle or chickens.
Strains of the same serological type showed a diversity of fermentation reactions, so that organisms which could be identified as Streptococcus durans shared a common type antigen with Strep. faecium strains. The evidence given here supports the proposal that Strep. durans should in future be considered as a variant of Strep. faecium .
It has also been shown that, in common with those of Streptococcus faecalis , the type antigens of the Strep. faecium types are cell wall components.     

Effects of dietary chlortetracycline on the antimicrobial resistance of broiler faecal Streptococcaceae

A breeder flock and a control group of progeny birds were fed antimicrobial-free rations; a second group of progeny received rations supplemented with 50 g chlortetracycline (Ctc)/ton. Effects of dietary Ctc on the distribution of species and biotypes of faecal Gram-positive cocci and their relative resistance to 12 antimicrobial agents were studied. Antimicrobial resistance (AMR) pattern diversity and modal AMR patterns were determined for bacterial species common to all three groups. Numerical taxonomic analysis placed 1321 (97%) of 1360 isolates into eight species or biotypes. The largest cluster (n = 659, 48%) was a biotype of Streptococcus faecalis. Three clusters were biotypes of Streptococcus faecium and contained 580 isolates (42%). The isolates were susceptible to ampicillin and almost uniformly resistant to methicillin, neomycin, streptomycin, sulfadiazine and tetracycline. There were 54 and 47 different AMR patterns, including 0 to 11 and 1 to 11 resistance determinants, in isolates from control and Ctc-fed birds, respectively. Modal AMR patterns for Strep. faecalis and one biotype of Strep. faecium were very similar for all three groups of birds. However, modal patterns in a second biotype of Strep. faecium varied considerably for all three groups. Interpretation of AMR pattern diversities were equivocal among biotypes from both progeny groups. The variable distribution of isolates, proportions of resistant strains, modal patterns and diversity indices among the progeny were probably due to their exposure to different environmental sources of bacteria.     

Effects of dietary chlortetracycline on the antimicrobial resistance of broiler faecal Streptococcaceae

A breeder flock and a control group of progeny birds were fed antimicrobial-free rations; a second group of progeny received rations supplemented with 50 g chlortetracycline (Ctc)/ton. Effects of dietary Ctc on the distribution of species and biotypes of faecal Gram-positive cocci and their relative resistance to 12 antimicrobial agents were studied. Antimicrobial resistance (AMR) pattern diversity and modal AMR patterns were determined for bacterial species common to all three groups. Numerical taxonomic analysis placed 1321 (97%) of 1360 isolates into eight species or biotypes. The largest cluster ( n = 659, 48%) was a biotype of Streptococcus faecalis. Three clusters were biotypes of Streptococcus faecium and contained 580 isolates (42%). The isolates were susceptible to ampicillin and almost uniformly resistant to methicillin, neomycin, streptomycin, sulfadiazine and tetracycline. There were 54 and 47 different AMR patterns, including 0 to 11 and 1 to 11 resistance determinants, in isolates from control and Ctc-fed birds, respectively. Modal AMR patterns for Strep. faecalis and one biotype of Strep. faecium were very similar for all three groups of birds. However, modal patterns in a second biotype of Strep. faecium varied considerably for all three groups. Interpretation of AMR pattern diversities were equivocal among biotypes from both progeny groups. The variable distribution of isolates, proportions of resistant strains, modal patterns and diversity indices among the progeny were probably due to their exposure to different environmental sources of bacteria.     

THE DISTRIBUTION OF GROUP D STREPTOCOCCI IN CATTLE AND SHEEP

Numbers and types of Lancefield group D streptococci have been determined in samples from the colons of 17 cattle and 9 sheep. Mean total streptococcal counts of 8 × 10⁴/g in cattle and 2 × 10⁶/g in sheep were obtained. Streptococcus bovis was found in every sample and was the predominant species in 15 of the cattle and 6 of the sheep. Other group D streptococci ( Strep. faecalis, Strep. faecium and Strep. durans ) were rare in cattle, but in sheep they formed a significant proportion of the population. Of 60 Strep. faecium, Strep. durans and related strains, 51 fermented raffinose. Many of the strains of Strep. faecium were also atypical in that they fermented sorbitol and appreciably reduced tetrazolium in broth at pH 6.0.
Strep. bovis remained the predominant streptococcus in faeces samples from 4 dairy cows when they were tested again after an interval of 17 and 18 months.     

Influence of avilamycin administration and its subsequent withdrawal on emergence and disappearance of antimicrobial resistance in enterococci in the intestine of broiler chickens

AIMS: To investigate the influence of avilamycin (AVM) administration and its subsequent withdrawal on the emergence and disappearance of AVM-resistant enterococci in the intestine of broiler chickens. METHODS AND RESULTS: Five chicks each of C, L and H groups were given the basal diet, the basal diet supplemented with 5 g AVM/ton and the basal diet supplemented with 50 g AVM/ton, respectively. The AVM-resistant Enterococcus faecalis population did not emerge during 30 days of the AVM administration period, whereas the AVM-resistant Enterococcus faecium with a minimum inhibitory concentration of >512 microg ml(-1) in the faeces of chicks of the L and H groups emerged on 3 and 1 days after the AVM administration, respectively. Thereafter, the AVM-resistant Ent. faecium population density in both L and H groups maintained high levels during the AVM administration period. Twenty days after the AVM withdrawal, the AVM-resistant Ent. faecium population disappeared from the intestines of both four of five chicks of L group and three of five chicks of H group. The AVM-resistant Ent. faecium population density in one chick from each of the groups, L and H, did not change before and after the AVM removal. CONCLUSIONS: The AVM-resistant Ent. faecium emerged during the AVM administration, and disappeared from the intestine of most chicks after the AVM withdrawal. However, the AVM-resistant Ent. faecium persisted in some chicks 20 days after AVM withdrawal. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggest that introducing an AVM withdrawal period could minimize the risk of AVM-resistant Ent. faecium becoming carcass contaminants, and that prudent antibiotic use alone is not sufficient to stem emergence of the AVM-resistant Ent. faecium.     

The Role of Clostridium perfringens in the Growth Response of Chicks to Dietary Penicillin

S ummary . The previous observation that Clostridium perfringens is responsible for penicillin-relieved growth depression in chicks has been reassessed. The administration of Cl. perfringens to germfree chicks, to birds harbouring a non-growth depressing gut flora and to birds reared in an environment free from Cl. perfringens was without effect on growth rate. No correlation was observed in conventional chicks between the incidence of growth depression and the presence of Cl. perfringens in the gut of the chicks one day after food was first given.     

Effects of dietary chlortetracycline on the antimicrobial resistance of porcine faecal streptococcaceae

Breeding pigs and one-half of their progeny were fed antimicrobial-free rations; the other half of the progeny received rations supplemented with 100 g of chlortetracycline (Ctc)/ton. Effects of dietary Ctc with respect to the distribution of species and biotypes of faecal Gram-positive cocci and their relative resistance to 12 antimicrobial agents were studied. Diversity of antimicrobial resistance (AMR) patterns and modal AMR patterns were determined for bacterial species common to all three groups. Numerical taxonomic analysis placed 1140 of 1150 isolates (99%) into 10 groups. Three of these were biotypes of Streptococcus faecium and contained the largest number of isolates ( n = 934, 81%). Streptococcus faecalis, Strep. morbillorum, Pediococcus halophilus and Gemella haemolysans also were isolated. Generally, the proportion of tetracycline-resistant strains for a species or biotype was greater from pigs fed Ctc, although differences were not significant ( P > 0.05). There was a significant difference ( P > 0.05) among all the groups for the percentage of penicillin-resistant strains in a biotype of Strep. faecium. Overall, 57 and 43 different AMR patterns, including 2 to 11 and 1 to 11 resistance determinants, were demonstrated in isolates from control pigs and pigs fed Ctc, respectively. Modal AMR patterns in species and biotypes were the same from both progeny groups, except for Strep. faecium. AMR pattern diversity was decreased for strains from pigs fed Ctc. Similar proportions of resistant strains from each group of progeny pigs were accompanied by decreased AMR pattern diversity in strains from pigs fed Ctc. These results indicated a change in distribution of AMR phenotypical patterns, rather than a change in overall frequency of individual resistant phenotypes.     

Effects of dietary chlortetracycline on the antimicrobial resistance of porcine faecal streptococcaceae

Breeding pigs and one-half of their progeny were fed antimicrobial-free rations; the other half of the progeny received rations supplemented with 100 g of chlortetracycline (Ctc)/ton. Effects of dietary Ctc with respect to the distribution of species and biotypes of faecal Gram-positive cocci and their relative resistance to 12 antimicrobial agents were studied. Diversity of antimicrobial resistance (AMR) patterns and modal AMR patterns were determined for bacterial species common to all three groups. Numerical taxonomic analysis placed 1140 of 1150 isolates (99%) into 10 groups. Three of these were biotypes of Streptococcus faecium and contained the largest number of isolates (n = 934, 81%). Streptococcus faecalis, Strep. morbillorum, Pediococcus halophilus and Gemella haemolysans also were isolated. Generally, the proportion of tetracycline-resistant strains for a species or biotype was greater from pigs fed Ctc, although differences were not significant (P greater than 0.05). There was a significant difference (P less than 0.05) among all the groups for the percentage of penicillin-resistant strains in a biotype of Strep. faecium. Overall, 57 and 43 different AMR patterns, including 2 to 11 and 1 to 11 resistance determinants, were demonstrated in isolates from control pigs and pigs fed Ctc, respectively. Modal AMR patterns in species and biotypes were the same from both progeny groups, except for Strep. faecium. AMR pattern diversity was decreased for strains from pigs fed Ctc. Similar proportions of resistant strains from each group of progeny pigs were accompanied by decreased AMR pattern diversity in strains from pigs fed Ctc. These results indicated a change in distribution of AMR phenotypical patterns, rather than a change in overall frequency of individual resistant phenotypes.     

The distribution of homologous enterococcal plasmid DNA sequences in human faecal isolates.

Hybridization was used to investigate the distribution of enterococcal plasmid sequences among 306 strains of Enterococcus and Streptococcus spp. isolated from faeces of humans of various ages. As DNA probes for the survey three plasmids, whose DNAs did not hybridize each other and designated as pMS13, pTW34 and pHK30, were selected from plasmids borne in Ent. faecalis. pTW34 DNA hybridized only with DNAs from enterococci, with high frequency in Ent. faecalis and low frequency in Ent. faecium. pMS13 DNA hybridized with DNAs of all Enterococcus spp. tested and with Strep. bovis, Strep. equinus and Strep. salivarius. Eighty-five percent of Ent. faecium isolates had sequences homologous to pMS13 but in the other species the values were less than 60%. Some enterococci had DNAs which hybridized with the pHK30 probe. The different distribution of the three DNA sequences indicates the possibility that plasmid DNAs encode advantageous phenotypes for the colonization of bacteria in the lumen of the bowel.     

The distribution of homologous enterococcal plasmid DNA sequences in human faecal isolates

T. WATANABE, H. KUMATA, M. SASAMOTO AND M. SHIMIZU-KADOTA. 1992. Hybridization was used to investigate the distribution of enterococcal plasmid sequences among 306 strains of Enterococcus and Streptococcus spp. isolated from faeces of humans of various ages. As DNA probes for the survey three plasmids, whose DNAs did not hybridize each other and designated as pMS13, pTW34 and pHK30, were selected from plasmids borne in Ent. faecalis. pTW34 DNA hybridized only with DNAs from enterococci, with high frequency in Ent. faecalis and low frequency in Ent. faecium. pMS13 DNA hybridized with DNAs of all Enterococcus spp. tested and with Strep. bovis, Strep. equinus and Strep. salivarius. Eighty-five percent of Ent. faecium isolates had sequences homologous to pMS13 but in the other species the values were less than 60%. Some enterococci had DNAs which hybridized with the pHK30 probe. The different distribution of the three DNA sequences indicates the possibility that plasmid DNAs encode advantageous phenotypes for the colonization of bacteria in the lumen of the bowel.     

A Selective Medium for the Enumeration of Streptococcus bovis by Membrane Filtration

A new selective medium (membrane-bovis agar) for the detection and enumeration of Streptococcus bovis is described. It has been successfully used to quantify this organism in polluted waters, sewage and faeces of humans and farm animals. This medium is based on the ability of Strep. bovis to utilize ammonium sulphate as its sole source of nitrogen. Streptococcus faecalis, Strep. faecium, Strep. equinus, Strep. salivarius. Strep. mitis and other bacteria commonly found in water, sewage and faeces are completely inhibited.
Streptococcus bovis appear to be the predominant faecal streptococci in the faeces of farm animals and absent in the faeces of humans. A total of 541 characteristic colonies (on m-BA), isolated from various sources were identified to species level. Over 97% proved to be Strep. bovis. Therefore, routine confirmatory tests on colonies growing on this medium would appear to be unnecessary.