Influence of oral doxycycline therapy on the diversity and antibiotic susceptibility of human intestinal bifidobacterial population

Aim:  To evaluate the influence of doxycycline therapy on the composition and antibiotic susceptibility of intestinal bifidobacteria.
Methods and Results:  Faecal samples were collected from nine subjects receiving doxycycline therapy and ten control subjects, and analysed for bifidobacteria by culturing and PCR–DGGE (denaturing gradient gel electrophoresis). A marked decrease in the diversity (average number of amplicons detected by PCR–DGGE 0·8 in the antibiotic vs 4·3 in the control group) of Bifidobacterium populations was observed during doxycycline therapy. The proportion of a tetracycline-resistant bifidobacterial population was higher in the antibiotic group than in the control group (83% vs 26%). Based on the tet gene PCR, resistance could be associated with the presence of tet (W). In two subjects, strains representing highly similar genetic fingerprints but different tetracycline susceptibilities were detected. A mutation causing lack of functionality in the tet (W) was observed in one of the susceptible strains.
Conclusions:  Doxycycline therapy had a drastic effect on the diversity and tetracycline susceptibility of intestinal Bifidobacterium populations.
Significance and Impact of the Study:  The use of broad-spectrum antibiotics increased the pool of tetracycline-resistant commensal bacteria in the intestine. The detection of resistance genes alone is not sufficient for the evaluation of bacterial antibiotic resistance.     

Antibiotic resistance of bacteria in raw and biologically treated sewage and in groundwater below leaking sewers

More than 750 isolates of faecal coliforms (>200 strains), enterococci (>200 strains) and pseudomonads (>340 strains) from three wastewater treatment plants (WTPs) and from four groundwater wells in the vicinity of leaking sewers were tested for resistance against 14 antibiotics. Most, or at least some, strains of the three bacterial groups, isolated from raw or treated sewage of the three WTPs, were resistant against penicillin G, ampicillin, vancomycin, erythromycin, triple sulfa and trimethoprim/sulfamethoxazole (SXT). Only a few strains of pseudomonads or faecal coliforms were resistant against some of the other tested antibiotics. The antibiotic resistances of pseudomonads, faecal coliforms and enterococci from groundwater varied to a higher extent. In contrast to the faecal coliforms and enterococci, most pseudomonads from all groundwater samples, including those from non-polluted groundwater, were additionally resistant against chloramphenicol and SXT. Pseudomonads from sewage and groundwater had more multiple antibiotic resistances than the faecal coliforms or the enterococci, and many pseudomonads from groundwater were resistant to more antibiotics than those from sewage. The pseudomonads from non-polluted groundwater were the most resistant isolates of all. The few surviving faecal coliforms in groundwater seemed to gain multiple antibiotic resistances, whereas the enterococci lost antibiotic resistances. Pseudomonads, and presumably, other autochthonous soil or groundwater bacteria, such as antibiotic-producing Actinomyces sp., seem to contribute significantly to the gene pool for acquisition of resistances against antibiotics in these environments.     

Effect of UV light disinfection on antibiotic-resistant coliforms in wastewater effluents.

Total coliforms and total coliforms resistant to streptomycin, tetracycline, or chloramphenicol were isolated from filtered activated sludge effluents before and after UV light irradiation. Although the UV irradiation effectively disinfected the wastewater effluent, the percentage of the total surviving coliform population resistant to tetracycline or chloramphenicol was significantly higher than the percentage of the total coliform population resistant to those antibiotics before UV irradiation. This finding was attributed to the mechanism of R-factor-mediated resistance to tetracycline. No significant difference was noted for the percentage of the surviving total coliform population resistant to streptomycin before or after UV irradiation. Multiple drug resistance patterns of 300 total coliform isolates revealed that 82% were resistant to two or more antibiotics. Furthermore, 46% of these isolates were capable of transferring antibiotic resistance to a sensitive strain of Escherichia coli.     

The isolation of tetracycline-resistant strains of strictly anaerobic bacteria from the rumen

Tetracycline resistant (Tc^R) strains of three of the major species of strictly anaerobic rumen bacteria Megasphaera elsdenii, Selenomonas ruminantium and Butyrivibrio fibrisolvens , were recovered with an isolation medium containing 20 μg/ml tetracycline. Only two of 14 strains of these species from other sources, isolated without antibiotic selection, showed tetracycline resistance. Evidence was found for the presence of plasmids in two tetracycline-resistant strains of M. elsdenii , and in some strains of S. ruminantium.     

Effect of antibiotics on the human intestinal flora in mice

Antibiotics used during selective decontamination were studied for their effect on the human intestinal flora in mice. Polymyxin B and neomycin were found to eliminate Escherichia coli from the gastrointestinal tract but did not alter total numbers of obligate anaerobes. Neomycin induced an increase of the percentage of gram-negative obligate anaerobes. Cephradine did not affect the numbers of obligate and facultative anaerobes but increased the percentage of gram-negative obligately anaerobic rods in the flora. The selective effect of polymyxin B and neomycin on the flora is accounted for by a relative insusceptibility of the anaerobic flora as compared with E. coli. Low concentrations of polymyxin B and neomycin were detected in caecal supernatants. This was found to be due to strong binding of both antibiotics to the solid fraction of intestinal contents.     

The aerobic and anaerobic microbiology of pustular acne lesions

Specimens from 32 pustular acne lesions that were inoculated on media supportive for the growth of aerobic and anaerobic bacteria showed bacterial growth. Only aerobic or facultative bacteria were recovered in 15 (47%) specimens, only anaerobic bacteria in 11 (34%) specimens, and mixed aerobic and anaerobic bacteria in 6 (18%) specimens. A total of 57 isolates, 31 anaerobes (1.0 per specimen) and 26 aerobes (0.8 per specimen) were recovered. The predominant isolates were Staphylococcus sp. (19 isolates), Peptostreptococcus sp. (15), and Propionibacterium sp. (10). Twelve (37.5%) of the comedones yielded only one organism. This retrospective study highlighted the polymicrobial nature of over two-thirds of culture positive pustular acne lesions and suggests the potential for pathogenic role of aerobic and anaerobic organisms other than P. acnes and Staphylococcus sp. in acne vulgaris.     

High prevalence and resistance rates to antibiotics in anaerobic bacteria in specimens from patients with chronic balanitis

Aim of the study was to assess both prevalence and antibiotic resistance in anaerobic bacteria from glans penis skin of 70 adults. Strain susceptibility was determined by breakpoint susceptibility test or E test. In 9 asymptomatic, 48 untreated and 13 treated symptomatic patients, anaerobes were found in 22.2%, 70.8% and 53.3%, respectively. Gram-positive strains (GPAs) were 2.2-fold more common than Gram-negative ones. Prevalent Gram-negative (GNAs) and GPAs were Prevotella spp. and anaerobic cocci, respectively. Clostridium difficile strain was found in an untreated patient. In GNAs, resistance rates to amoxicillin, metronidazole, clindamycin, tetracycline, levofloxacin, and amoxicillin/clavulanate were 42.1, 0, 52.6, 53.3, 86.7 and 5.2%, respectively. In GPAs, the resistance rates to metronidazole, clindamycin, tetracycline, levofloxacin and amoxicillin/clavulanate were 18.2, 34.1, 52.6, 36.8 and 0%, respectively. In conclusion, anaerobes were 1.6-fold more frequent in untreated symptomatic patients compared with other patients, suggesting their participation in development of chronic balanitis. GPAs were more common than GNAs. The resistance rates to amoxicillin, clindamycin, tetracycline, and levofloxacin were high. Most active agents were metronidazole and amoxicillin/clavulanate. Resistance in anaerobes varies according to sites of specimens and years of study.     

Ionophore resistance of ruminal bacteria and its potential impact on human health

In recent years, there has been a debate concerning the causes of antibiotic resistance and the steps that should be taken. Beef cattle in feedlots are routinely fed a class of antibiotics known as ionophores, and these compounds increase feed efficiency by as much as 10%. Some groups have argued that ionophore resistance poses the same public health threat as conventional antibiotics, but humans are not given ionophores to combat bacterial infection. Many ruminal bacteria are ionophore-resistant, but until recently the mechanism of this resistance was not well defined. Ionophores are highly lipophilic polyethers that accumulate in cell membranes and catalyze rapid ion movement. When sensitive bacteria counteract futile ion flux with membrane ATPases and transporters, they are eventually de-energized. Aerobic bacteria and mammalian enzymes can degrade ionophores, but these pathways are oxygen-dependent and not functional in anaerobic environments like the rumen or lower GI tract. Gram-positive ruminal bacteria are in many cases more sensitive to ionophores than Gram-negative species, but this model of resistance is not always clear-cut. Some Gram-negative ruminal bacteria are initially ionophore-sensitive, and even Gram-positive bacteria can adapt. Ionophore resistance appears to be mediated by extracellular polysaccharides (glycocalyx) that exclude ionophores from the cell membrane. Because cattle not receiving ionophores have large populations of resistant bacteria, it appears that this trait is due to a physiological selection rather than a mutation per se. Genes responsible for ionophore resistance in ruminal bacteria have not been identified, but there is little evidence that ionophore resistance can be spread from one bacterium to another. Given these observations, use of ionophores in animal feed is not likely to have a significant impact on the transfer of antibiotic resistance from animals to man.     

Participation of gram-negative anaerobes in infections in hospitalized patients

The aim of the study was to assess the prevalence and antibiotic susceptibility profiles of strictly anaerobic Gram-negative bacteria isolated from clinical samples taken from hospitalized patients from 01.01.2007 to 31.12.2008. The specimens were cultured using media, incubated at 37 degrees C under anaerobic conditions. Biochemical identification and antibiotic susceptibility were done in an automated system ATB Expression (bioMerieux S.A, France). For selected strains of Bacteroides sp. sensitivity was determined using E-test (AB BIODISK, Sweden). Overall 1274 strains of obligate anaerobes were isolated. Gram-negative bacteria were cultured in number of 333 strains. Most frequently isolated was Bacteroides sp. (46,9%) and Prevotella sp. (29,7%). Isolated bacteria are still susceptible to imipenem (100%), metronidazole (100%) and beta-lactam antibiotics with beta-lactamase inhibitors: amoxicillin/clavulanate (97,8%) piperacillin/tazobactam (99,1%), ticarcillin/clavulanate (99,1%).     

Effect of age and housing location on antibiotic resistance of fecal coliforms from pigs in a non-antibiotic-exposed herd

The relationship of age and housing location to single antibiotic resistance, multiple antibiotic resistance, and resistance patterns of fecal coliforms obtained during a 20-month period from pigs in a herd that was not exposed to antibiotics for 126 months was determined. Bacteria resistant to single and multiple antibiotics were isolated more frequently (P less than 0.01) from pigs under 7 months of age. A greater proportion of isolates from pigs over 6 months of age was sensitive to the 13 antimicrobial agents tested (P less than 0.01), while a smaller proportion showed resistance to single (P less than 0.05) and multiple (P less than 0.01) antibiotics. More than 80% of the resistant isolates were resistant to tetracycline, streptomycin, or sulfisoxazole. Resistance was greater (P less than 0.01) for pigs in the finishing unit than for those on pasture. Resistance to ampicillin, carbenicillin, and tetracycline was greater (P less than 0.05) for pigs in the finishing unit than for those in the farrowing house. More isolates from pigs on pasture were sensitive to all antimicrobial agents tested (P less than 0.01). A greater proportion of isolates from pigs in the finishing unit showed resistance to a single antibiotic (P less than 0.01). The data from this study suggest that exposure to antibiotics is not the only factor that influences the prevalence of bacteria that are resistant to single and multiple antibiotics in the feces of domestic animals and that considerable research is needed to define the factors influencing antibiotic resistance in fecal bacteria.     

Effect of age and housing location on antibiotic resistance of fecal coliforms from pigs in a non-antibiotic-exposed herd.

The relationship of age and housing location to single antibiotic resistance, multiple antibiotic resistance, and resistance patterns of fecal coliforms obtained during a 20-month period from pigs in a herd that was not exposed to antibiotics for 126 months was determined. Bacteria resistant to single and multiple antibiotics were isolated more frequently (P less than 0.01) from pigs under 7 months of age. A greater proportion of isolates from pigs over 6 months of age was sensitive to the 13 antimicrobial agents tested (P less than 0.01), while a smaller proportion showed resistance to single (P less than 0.05) and multiple (P less than 0.01) antibiotics. More than 80% of the resistant isolates were resistant to tetracycline, streptomycin, or sulfisoxazole. Resistance was greater (P less than 0.01) for pigs in the finishing unit than for those on pasture. Resistance to ampicillin, carbenicillin, and tetracycline was greater (P less than 0.05) for pigs in the finishing unit than for those in the farrowing house. More isolates from pigs on pasture were sensitive to all antimicrobial agents tested (P less than 0.01). A greater proportion of isolates from pigs in the finishing unit showed resistance to a single antibiotic (P less than 0.01). The data from this study suggest that exposure to antibiotics is not the only factor that influences the prevalence of bacteria that are resistant to single and multiple antibiotics in the feces of domestic animals and that considerable research is needed to define the factors influencing antibiotic resistance in fecal bacteria.     

The effect of different oral antibiotics on the gastrointestinal microflora of a wild rodent (Aethomys namaquensis)

Gut sterilization via the oral administration of antibiotics facilitates physiological studies of the nutritionally important relationship between intestinal microflora and the host. However, the composition of gut flora is extremely variable, and as a result, the efficacy of antibiotics in achieving gut sterilization varies considerably between species. We tested the effectiveness of three antibiotic cocktails in sterilizing the gut of a rodent pollinator, the Namaqua rock mouse (Aethomys namaquensis). The cocktails were (1) streptomycin sulfate and bacitracin (previously used with domestic mice and rats), (2) chloramphenicol and bacitracin (based on antibiotic screening tests performed on faecal flora) and (3) Baytril 10% oral solution (a veterinary antimicrobial agent containing enrofloxacin). We tested for antibiotic inactivation by determining bacterial viability through fluorescence staining of faecal samples. We also tested techniques to maintain sterility during antibiotic treatment without the benefit of a laminar flow cabinet. Antibiotics were administered orally in food and water consumed ad libitum over 4 consecutive days. Antibiotic effectiveness was assessed by culturing anaerobic bacteria from faecal samples collected before and after each antibiotic treatment. Treatment with Baytril 10% oral solution eliminated or significantly reduced faecal flora, whereas other antibiotics did not. This study clearly demonstrates the importance of testing the effectiveness of antibiotics before their use in studies that involve antibiotic-treated subjects, particularly if these are species previously untested.     

感染根管的厌氧菌培养结果分析

从64只感染根管中的58只根管分离到144株无芽胞厌氧菌,其中类杆菌54株,厌氧性链球菌23株,韦荣氏球菌17株,真杆菌11株,梭杆菌10株,放线菌8株,双岐杆菌2株,消化链球菌和消化球菌19株。40只根管为厌氧菌和兼性厌氧菌或需氧菌混合感染,18只根管和6只根管分别为单独厌氧菌和兼性厌氧菌感染。33只根尖周炎根管分别采集牙髓和根尖渗出物样本进行培养,实验结果表明牙髓样本中革兰氏阳性厌氧杆菌检出率较高,根尖渗出物中以产黑素类杆菌属的细菌检出率较高。根尖周炎和牙槽脓肿患者的感染根管中产黑素类杆菌属的细菌检出率明显高于蜂窝组织炎患者。     

The intestinal microflora during the first weeks of life

Bacterial colonization of the neonatal gastrointestinal tract begins during birth when the neonate comes into contact with the maternal cervical and vaginal flora. In infants delivered by Caesarean section, bacteria colonizing the neonate gastrointestinal tract are provided by the environment. The first bacteria encountered in the majority of healthy infants, are facultative anaerobes, which remain predominant during the first 2 weeks of life. Among them, Staphylococcus, Enterobacteriaceae and Streptococcus were the genera most commonly isolated from the newborn faecal flora at birth. Facultative anaerobic bacteria are followed closely by Bifidobacterium sp. Clostridium perfringens is present within 2 days with an increase incidence in newborns delivered by a Caesarean section. Clostridium perfringens seems to be the precursor for installation of other anaerobic putrefactive bacteria, as Bacteroides and other Clostridium sp. The impact of Caesarean section and the period and quality of hospitalization are mainly implicated in changes of the normal newborn flora. Feeding seems to modulate the colonization pattern. In humans, breast milk plays a role in passive immunization of the neonatal intestine, and contains factors that promote the growth of Bifidobacterium bifidum in the intestinal flora. Formula feeding seems to promote implantation and persistence of Clostridium perfringens, and clearly enhances intestinal colonization of C. difficile in newborns.     

Characteristics of bacteria isolated by the anaerobic roll-tube method from cheeses and ground beef.

In this study the methods of Hungate were used to quantitate the anaerobic bacteria present in commercially available ground beef, cheddar cheese, and German hand cheese. Of 235 anaerobic roll-tube isolates from ground beef and German hand cheese, all were facultative anaerobes. Of 213 anaerobic roll-tube isolates from cheddar cheese, 91% were facultative anaerobes and 9% were obligate anaerobes. Using results of biochemical tests, 14 or the 17 obligately anaerobic isolates from cheddar cheese were Propionibacterium acnes, two were strains of Propionibacterium that could not be speciated, and one was tentatively identified as a strain of Streptococcus evolutus. Obligate anaerobes were estimated to be present in the cheddar cheese at a level of about 10(6)/g. The possible significance of these levels of P. acnes in nonsterile foods is discussed.     

Phenotypic diversity and antibiotic resistance in soil bacterial communities

The community structure in two different agricultural soils has been investigated. Phenotypic diversity was assessed by applying BIOLOG-profiles on a total of 208 bacterial isolates. Diversity indices were calculated from cluster analysis of the BIOLOG data. The bacterial isolates were also evaluated for resistance towards six different antibiotics, mercury resistance and the presence of plasmids. The presence of tetracycline-resistant determinants class A to E among Gram-negative bacteria was analysed with DNA probes. The distribution of tetracycline resistance markers among colonies growing on non-selective and tetracycline-selective plates were compared. The phenotypic approach demonstrated some difference in the diversity within the two soils. The frequency of antibiotic resistance isolates was high in both soils, whereas the frequency of mercury resistance differed significantly. We found no correlation between plasmid profiles and antibiotic resistance patterns. We found all the tetracycline resistance determinants except class B, indicating that the diversity of the tetracycline resistance determinants was complex in populations of resident soil bacteria under no apparent selective pressure for the genes in question.     

The effects of the novel bifidogenic trisaccharide,neokestose, on the human colonic microbiota

The potential prebiotic effect of the fructo-trisaccharide, neokestose, on intestinal bacteria was investigated. Bifidobacterium sp. utilized neokestose to a greater extend and produced more biomass from neokestose than facultative anaerobes under anaerobic conditions in batch culture. Lactobacillus salivarius utilized glucose but negligible amounts of neokestose. L. salivarius and the facultative anaerobes produced significantly more biomass from glucose than from neokestose, whereas the biomass yields obtained with bifidobacteria on neokestose and glucose, respectively, were not significantly different. Static batch cultures inoculated with faeces supported the prebiotic effect of neokestose, which had been observed in the pure culture investigations. Bifidobacteria and lactobacilli were increased while potentially detrimental coliforms, clostridia and bacteroides, decreased after 24 h fermentation with neokestose. In addition, this effect was more pronounced with neokestose than with a commercial prebiotic fructo-oligosaccharide. It was concluded that neokestose has potential as a novel bifidogenic substance and that it might have advantages over the commercially available sources currently used.     

Antibiotic resistant bacteria in Windermere and two remote upland tarns in the English Lake District

The incidence of antibiotic resistance was determined in over 2000 bacteria which were divided into the following groups: faecal streptococci, coliforms (excluding Escherichia coli), E. coli, Pseudomonas spp. and aquatic bacteria (i.e. bacteria predominant in the lake water which were excluded from the previous four categories). The isolates were obtained from the water of Windermere (English Lake District) and from a sewage effluent which entered the lake. With the exception of the faecal streptococci, the incidence of antibiotic resistance was higher in the bacteria isolated from the lake water than in those from the effluent, and ranked according to groups Pseudomonas spp. greater than E. coli greater than aquatic bacteria greater than coliforms greater than faecal streptococci. The highest incidence of multiple resistance was found among the pseudomonads. When corrected for the relative size of each population the pool of antibiotic resistance in the aquatic bacteria was by far the largest. The incidence of antibiotic resistance in aquatic bacteria isolated from Windermere was, however, lower than in those isolated from two remote upland tarns. This finding may have been due to differences in the species composition of the three sites except that the same results were obtained when only fluorescent pseudomonads were tested. The upland tarns were not totally isolated from man and other animals but did not receive any sewage or other effluents and therefore the results were surprising. Possible explanations include a lack of susceptibility in aquatic bacteria and increased resistance associated with growth in nutrient poor environments.     

Mixed continuous culture experiments with an antibiotic-producing streptomycete andEscherichia coli

Four strains ofStreptomyces aureofaciens capable of producing different amounts of tetracycline have been grown in continuous culture with either of two strains ofEscherichia coli orBacillus pumillus. Each of the bacteria had faster specific growth rates than any of the streptomycetes.E. coli NTCT 5993 had a higher affinity for sucrose than didS. aureofaciens SR 11. In mixed culture experiments, the bacteria displaced the streptomycete under the following conditions: (a) when the streptomycete produced no tetracycline; (b) in tetracycline-producing cultures after a fraction of the bacteria had died—the bacterial population that subsequently developed was resistant to the drug; and (c) when using tetracycline-resistant bacteria. Under nutrient conditions leading to high antibiotic levels, it was possible to kill all the bacteria, and the streptomycete survived after a transient fall in mycelium and tetracycline levels. No stable mixed population was ever seen. Once tetracycline-resistant bacteria had displaced the streptomycete, and tetracycline concentrations had fallen, a sensitive bacterial population reappeared. Competition between sensitive and resistantE. coli NCTC 5993 in the chemostat confirmed the selective advantage of the sensitive strain. Results were discussed in terms of the role of antibiotics in nature.