A global perspective on the use, sales, exposure pathways, occurrence, fate and effects of veterinary antibiotics (VAs) in the environment

Almost immediately after a human being is born, so too is a new microbial ecosystem, one that resides in that person's gastrointestinal tract. Although it is a universal and integral part of human biology, the temporal progression of this process, the sources of the microbes that make up the ecosystem, how and why it varies from one infant to another, and how the composition of this ecosystem influences human physiology, development, and disease are still poorly understood. As a step toward systematically investigating these questions, we designed a microarray to detect and quantitate the small subunit ribosomal RNA (SSU rRNA) gene sequences of most currently recognized species and taxonomic groups of bacteria. We used this microarray, along with sequencing of cloned libraries of PCR-amplified SSU rDNA, to profile the microbial communities in an average of 26 stool samples each from 14 healthy, full-term human infants, including a pair of dizygotic twins, beginning with the first stool after birth and continuing at defined intervals throughout the first year of life. To investigate possible origins of the infant microbiota, we also profiled vaginal and milk samples from most of the mothers, and stool samples from all of the mothers, most of the fathers, and two siblings. The composition and temporal patterns of the microbial communities varied widely from baby to baby. Despite considerable temporal variation, the distinct features of each baby's microbial community were recognizable for intervals of weeks to months. The strikingly parallel temporal patterns of the twins suggested that incidental environmental exposures play a major role in determining the distinctive characteristics of the microbial community in each baby. By the end of the first year of life, the idiosyncratic microbial ecosystems in each baby, although still distinct, had converged toward a profile characteristic of the adult gastrointestinal tract.

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Development of the human infant intestinal microbiota.

Background: Recent evidence suggests that there is a link between metabolic diseases and bacterial populations in the gut The aim of this study was to assess the differences between the composition of the intestinal microbiota in humans with type 2 diabetes and non-diabetic persons as control Methods and Findings: The study included 36 male adults with a broad range of age and body-mass indices (BMIs), among which 18 subjects were diagnosed with diabetes type 2 The fecal bacterial composition was investigated by real-time quantitative PCR (qPCR) and in a subgroup of subjects (N=20) by tag-encoded amplicon pyrosequencing of the V4 region of the 16S rRNA gene The proportions of phylum Firmicutes and class Clostridia were significantly reduced in the diabetic group compared to the control group (P=003) Furthermore, the ratios of Bacteroidetes to Firmicutes as well as the ratios of Bacteroides-Prevotella group to C coccoides-E rectale group correlated positively and significantly with plasma glucose concentration (P=004) but not with BMIs Similarly, class Betaproteobacteria was highly enriched in diabetic compared to non-diabetic persons (P=002) and positively correlated with plasma glucose (P=004) Conclusions: The results of this study indicate that type 2 diabetes in humans is associated with compositional changes in intestinal microbiota The level of glucose tolerance should be considered when linking microbiota with metabolic diseases such as obesity and developing strategies to control metabolic diseases by modifying the gut microbiota

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Gut Microbiota in Human Adults with Type 2 Diabetes Differs from Non-Diabetic Adults

Dynamics and Stabilization of the Human Gut Microbiome during the First Year of Life.

OBJECTIVE.The aim of this study was to examine the contribution of a broad range of external influences to the gut microbiotic composition in early infancy. METHODS.Fecal samples from 1032 infants at 1 month of age, who were recruited from the KOALA Birth Cohort Study in the Netherlands, were subjected to quantitative real-time polymerase chain reaction assays for the enumeration of bifidobacteria, Escherichia coli, Clostridium difficile, Bacteroides fragilis group, lactobacilli, and total bacterial counts. Information on potential determinants of the gut microbiotic composition was collected with repeated questionnaires. The associations between these factors and the selected gut bacteria were analyzed with univariate and multivariate analyses. RESULTS.Infants born through cesarean section had lower numbers of bifidobacteria and Bacteroides, whereas they were more often colonized with C difficile, compared with vaginally born infants. Exclusively formula-fed infants were more often colonized with E coli, C difficile, Bacteroides, and lactobacilli, compared with breastfed infants. Hospitalization and prematurity were associated with higher prevalence and counts of C difficile. Antibiotic use by the infant was associated with decreased numbers of bifidobacteria and Bacteroides. Infants with older siblings had slightly higher numbers of bifidobacteria, compared with infants without siblings. CONCLUSIONS.The most important determinants of the gut microbiotic composition in infants were the mode of delivery, type of infant feeding, gestational age, infant hospitalization, and antibiotic use by the infant. Term infants who were born vaginally at home and were breastfed exclusively seemed to have the most “beneficial” gut microbiota (highest numbers of bifidobacteria and lowest numbers of C difficile and E coli).

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Factors influencing the composition of the intestinal microbiota in early infancy

The prevalence of resistance in enterococci to antibiotics, commonly used for therapy in poultry or as antimicrobial growth promoters (AMGPs), was determined in faecal samples of two chicken populations: broilers in which antibiotic and AMGP use is common and laying-hens with a low antibiotic usage. In addition faecal samples were examined from three human populations: broiler farmers, laying-hen farmers and poultry slaughterers. MICs of an extended panel of antibiotics for a randomly chosen gentamicin- or vancomycin-resistant enterococcal isolate from each faecal specimen were also determined. The prevalence of resistance for all antibiotics tested was higher in broilers than in laying-hens. Resistance in faecal enterococci of broiler farmers was for nearly all antibiotics higher than those observed in laying-hen farmers and poultry slaughterers. The overall resistance in broilers was correlated with the resistance in broiler farmers and in poultry slaughterers. No correlation between the results obtained in the laying-hens with any of the other populations was found. The 27 gentamicin-resistant isolates all showed high-level resistance to gentamicin and two of these isolates, both Enterococcus faecium, were resistant to all antibiotics tested, except vancomycin. The 73 vancomycin-resistant enterococci (VRE) isolated from the five populations belonged to four different species and in all isolates the vanA gene cluster was detected by blot hybridization. The pulsed-field gel electrophoresis (PFGE) patterns of these vancomycin-resistant enterococci were quite heterogeneous, but Enterococcus hirae isolates with the same or a closely related PFGE pattern were isolated at two farms from the broiler farmer and from broilers. Molecular characterization of vanA-containing transposons of these isolates showed that similar transposon types, predominantly found in poultry, were present. Moreover, similar vanA elements were not only found in isolates with the same PFGE pattern but also in other VRE isolated from both humans and chickens. The results of this study suggest transmission of resistance in enterococci from animals to man. For VRE this might be clonal transmission of animal strains, but transposon transfer seems to occur more commonly.

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Antibiotic resistance of faecal Escherichia coli in poultry, poultry farmers and poultry slaughterers

To determine the influence of either exclusive breast-feeding or formula feeding on both composition and quantity of the gut microbiota in infants, we have developed real-time, quantitative PCR assays for the detection of Bifidobacterium spp. and Clostridium difficile. Furthermore, we have monitored the prevalence and counts of Escherichia coli by applying a previously described real-time PCR assay. We found all 100 infants tested to be colonized by Bifidobacterium spp. The bifidobacterial counts were comparable between the 50 breast-fed and 50 formula-fed infants with median values of 10.56 log10 and 10.24 log10 CFU g(-1) wet weight faeces, respectively. C. difficile was detected in 14% of the breast-fed and 30% of the formula-fed infants. In addition, the C. difficile counts were significantly lower in breast-fed infants than in the formula-fed group (median values of 3.28 log10 and 7.43 log10 CFU g(-1), respectively; p=0.03). The prevalence of E. coli in the breast-fed and formula-fed group was 80% and 94%, respectively. Also, the E. coli counts in colonized infants was significantly lower in the breast-fed infants than in the formula-fed group (median values of 9.11 log10 and 9.57 log10 CFU g(-1), respectively; p=0.004). We conclude that the prevalence and counts of C. difficile as well as E. coli are significantly lower in the gut microbiota of breast-fed infants than in that of formula-fed infants, whereas the prevalence and counts of Bifidobacterium spp. is similar among both groups.

Quantification of Bifidobacterium spp., Escherichia coli and Clostridium difficile in faecal samples of breast-fed and formula-fed infants by real-time PCR.

Vancomycin-resistant enterococci (VRE) were isolated for the first time in 1986 in Europe and in 1987 in the United States. Since then VRE infections have been reported from all over the world. In 1993 enterococci accounted for 12% and VRE accounted for 1% of all hospital infections in the United States (10). In Europe the rate of fecal carriage of VRE in the community is much higher (e.g., 2 to 28%) than that in the United States, where VRE seem to be more or less absent outside hospitals (10, 17). In Europe a diversity of VRE types has also been isolated from sewage, animal waste, meat and meat products, and feces of healthy persons, suggesting a heterogeneous pool of VRE outside hospitals (7, 8, 25, 26). The observation that in the United States VRE have not been isolated from animal sources (6, 10) might be due to the fact that in the United States glycopeptides were never approved for use in animal feeds as antimicrobial performance enhancers (APEs) for growth promotion (28). Until recently, avoparcin, a glycopeptide that shows complete cross-resistance with vancomycin, was commonly used in The Netherlands and in most other countries of the European Union for this purpose (23). The use of avoparcin as an APE has been suggested as a risk factor for carriage of VRE in food animals, as the usage of any antibiotic produces a selective pressure in favor of resistance (5, 12, 26, 27, 29, 34, 39). Therefore, its use as an APE was suspended in the European Union in 1996.

Four main types of resistance to glycopeptides have been described: VanA, VanB, VanC, and VanD (4, 18, 30, 31). The vanA gene cluster is the one most commonly found and encodes inducible high-level resistance to both vancomycin and teicoplanin. Dissemination of vancomycin resistance can occur through both clonal expansion of resistant enterococci and horizontal transmission of resistance genes (21, 22). Horizontal transfer of vancomycin resistance is explained by the fact that the genetic determinant for the VanA type of vancomycin resistance typically resides on a mobile DNA element such as Tn1546 (3), which can be transferred to enterococci by plasmids. Recently, in several studies, the heterogeneity of the VanA resistance determinant was described. In those studies, indistinguishable Tn1546-like elements could be found in VRE isolated from farm animals and humans, suggesting transfer of vancomycin resistance elements from farm animals and humans (31, 38, 40).

In a previously published report of a study in which the prevalence of VRE in turkeys and farmers was assessed, we found that VRE could be detected in 50% of the fecal samples from turkeys (n = 47) and in 39% of the fecal samples from turkey farmers (n = 47) (33). Furthermore, VRE were isolated from 20% of the fecal samples from turkey slaughterers (n = 48) and 14% of the fecal samples from (sub)urban residents from the same area (n = 117). In addition, an indistinguishable VRE and a vanA-containing element were found in one sample from a turkey and one from a turkey farmer, suggesting animal-to-human transmission of vancomycin resistance as a likely route (33).

In the present study we analyzed in more detail the epidemiology of VRE in the context of the influence of avoparcin use in turkey feed. The relative amount of VRE in fecal samples from turkeys and three groups of healthy volunteers with different risks of exposure to fecal bacteria from turkeys, i.e., turkey farmers, turkey slaughterers, and a control group of (sub)urban residents, was determined by calculating the number of VRE relative to the total number of enterococci. The VRE that were isolated were identified and classified with respect to the type of vancomycin resistance. In addition, the antibiotic susceptibilities to several antimicrobial agents of enterococci isolated from vancomycin-free and vancomycin-containing selective agar plates were determined. Possible sharing of VRE between turkeys and humans was assessed by genotyping by pulsed-field gel electrophoresis (PFGE). In addition, the similarity of vancomycin resistance elements found in animal and human isolates was assessed by comparing Tn1546 derivatives of isolates of VRE from turkeys and the three groups of healthy human volunteers.

(Parts of this study were presented at the 37th Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Ontario, Canada, 28 September to 1 October 1997.)

Enterococci with Glycopeptide Resistance in Turkeys, Turkey Farmers, Turkey Slaughterers, and (Sub)Urban Residents in the South of The Netherlands: Evidence for Transmission of Vancomycin Resistance from Animals to Humans?

The prevalence and degree of antibiotic resistance of faecal indicator bacteria, Escherichia coli and enteroccoci, were determined in 1321 faecal samples collected from pigs at five abattoirs in The Netherlands and in 100 samples from Swedish pigs. In the Dutch samples a high prevalence of resistance was observed in E. coli for three commonly used antibiotics in pig medicine, amoxycillin (70-94%), oxytetracycline (78-98%) and trimethoprim (62-96%). Also, the prevalence for chloramphenicol (55-67%) and neomycin (38-67%) was relatively high. For the other compounds tested the prevalence was less than 10%. The percentage of samples with a high degree of resistant E. coli showed the same tendency in all Dutch abattoirs although significant differences between the abattoirs were observed. The percentage of Swedish samples with a high degree of resistant E. coli was significantly lower for all antibiotics except nitrofurantoin, gentamicin, flumequin and ciprofloxacin. All enterococci were susceptible to amoxycillin and high-level resistance to gentamicin was observed in 4-6% of the Dutch samples. A high prevalence of resistance and a high degree of resistance was found for erythromycin and oxytetracycline. The prevalence of resistance to dalfopristin-quinupristin ranged from 6 to 8% and for vancomycin from 24 to 46%. Significant differences between the abattoirs were found for all compounds tested except amoxycillin. In the Swedish population both the prevalence and degree of resistance in enterococci were significantly lower except for amoxycillin and gentamicin. This point prevalence study showed that the prevalence and degree of antibiotic resistance in indicator bacteria, E. coli and enterococci, in faecal samples from pigs differed between two countries and reflected differences in antibiotic usage in pigs. To analyse the differences observed between the slaughterhouses, additional information about the farms of origin and antibiotic consumption is necessary.

Antimicrobial resistance in pig faecal samples from The Netherlands (five abattoirs) and Sweden

High prevalence of colonization with vancomycin- and pristinamycin-resistant enterococci in healthy humans and pigs in The Netherlands: is the addition of antibiotics to animal feeds to blame?

N. London

Papers

Antibiotic resistance of faecal Escherichia coli in poultry, poultry farmers and poultry slaughterers

Quantification of Bifidobacterium spp., Escherichia coli and Clostridium difficile in faecal samples of breast-fed and formula-fed infants by real-time PCR.

Enterococci with Glycopeptide Resistance in Turkeys, Turkey Farmers, Turkey Slaughterers, and (Sub)Urban Residents in the South of The Netherlands: Evidence for Transmission of Vancomycin Resistance from Animals to Humans?

Antimicrobial resistance in pig faecal samples from The Netherlands (five abattoirs) and Sweden

High prevalence of colonization with vancomycin- and pristinamycin-resistant enterococci in healthy humans and pigs in The Netherlands: is the addition of antibiotics to animal feeds to blame?