A guide to human microbiome research: study design, sample collection, and bioinformatics analysis.
Qian Xu-Bo,Chen Tong,Xu Yi-Ping,Chen Lei,Sun Fu-Xiang,Lu Mei-Ping,Liu Yong-Xin
Chinese medical journal
The purpose of this review is to provide medical researchers, especially those without a bioinformatics background, with an easy-to-understand summary of the concepts and technologies used in microbiome research. First, we define primary concepts such as microbiota, microbiome, and metagenome. Then, we discuss study design schemes, the methods of sample size calculation, and the methods for improving the reliability of research. We emphasize the importance of negative and positive controls in this section. Next, we discuss statistical analysis methods used in microbiome research, focusing on problems with multiple comparisons and ways to compare β-diversity between groups. Finally, we provide step-by-step pipelines for bioinformatics analysis. In summary, the meticulous study design is a key step to obtaining meaningful results, and appropriate statistical methods are important for accurate interpretation of microbiome data. The step-by-step pipelines provide researchers with insights into newly developed bioinformatics analysis methods.
10.1097/CM9.0000000000000871
Prevalence and Characterization of Isolated From Pasteurized Milk in China.
Dai Jingsha,Wu Shi,Huang Jiahui,Wu Qingping,Zhang Feng,Zhang Jumei,Wang Juan,Ding Yu,Zhang Shuhong,Yang Xiaojuan,Lei Tao,Xue Liang,Wu Haoming
Frontiers in microbiology
is one of the most important food-borne pathogens globally. It produces various toxins and invasive enzymes and can be found in numerous food products. Milk is an important source of staphylococcal food poisoning. After pasteurization, this microorganism or its enterotoxins might still remain in pasteurized milk. Therefore, this study was to investigate the contamination of in 258 pasteurized milk from 39 cities of China. The prevalence and levels of in these samples as well as antibiotic susceptibility profiles, virulence genes, biofilm formation, and biofilm related genes, typing and MLST were used to determine the characterization among the isolates. It was found 3.9% of samples were detected in 8 of 39 cities in China. The contaminated level were not very excessive which showed the MPN values of the most positive samples (9/10) were less than 1 MPN/g. All pasteurized milk-related isolates have ability to produce biofilm and harbored , genes, other biofilm related genes were showed in 91.7% of isolates and gene were showed in 50%, except gene which were free in all isolates. The antibiotic susceptibility test showed that all isolates were resistant or intermediate-resistant to different concentrations of the antibiotics. Furthermore, 75.0% of the isolates were resistant to three or more antibiotic classes, which indicated multidrug resistance. The isolates had virulence potential, which showed 66.7% (8/12) of the isolates carried one or more virulence-associated genes. Molecular typing by MLST and typing enabled classification of these isolates into a total of 11 sequence types (STs) and types, which indicated high genetic diversity. Most of these types were related to various clinical infections. Thus, the findings of this study reflect the potential risk of infection in China. Our study also provides comprehensive analysis of the prevalence of in pasteurized milk and helps ensure more accurate treatment of human infection with effective antibiotics.
10.3389/fmicb.2019.00641
Metagenomic analysis reveals the influences of milk containing antibiotics on the rumen microbes of calves.
Li Wei,Han Yunsheng,Yuan Xue,Wang Guan,Wang Zhibo,Pan Qiqi,Gao Yan,Qu Yongli
Archives of microbiology
Milk containing antibiotics is used as cost-effective feed for calves, which may lead to antibiotic residues-associated food safety problems. This study aims to investigate the influence of antibiotics on rumen microbes. Through metagenomic sequencing, the rumen microbial communities of calves fed with pasteurized milk containing antibiotics (B1), milk containing antibiotics (B2) and fresh milk (B3) were explored. Each milk group included calves in 2 (T1), 3 (T2) and 6 (T3) months of age. Using FastQC software and SOAPdenovo 2, the filtered data, respectively, were performed with quality control and sequence splicing. Following KEGG annotation was conducted for the uploaded sequences using KAAS software. Using R software, both species abundance analysis and differential abundance analysis were performed. In the B1 samples, the species abundance of Bacteroidetes gradually decreased along with the extension of feeding time, while that of Fibrobacteres gradually increased. The species abundances of Proteobacteria (p value = 0.01) and Spirochaetes (p value = 0.03) had significant differences among T1, T2 and T3 samples. Meanwhile, only the species abundance of Spirochaetes (p value = 0.04) had significant difference among B1, B2 and B3 samples. Cell cycle involving GSK3β, CDK2 and CDK7 was significantly enriched for the differentially expressed genes in the T1 versus T2 and T1 versus T3 comparison groups. Milk containing antibiotics might have a great influence on these rumen microbes and lead to antibiotic residues-associated food safety problems. Furthermore, GSK3β, CDK2 and CDK7 in rumen bacteria might affect milk fat metabolism in early growth stages of calves.
10.1007/s00203-016-1311-8
Daily ingestion of tetracycline residue present in pasteurized milk: a public health problem.
de Albuquerque Fernandes Sergio Augusto,Magnavita Ana Prudencia Assis,Ferrao Sibelli Passini Barbosa,Gualberto Simone Andrade,Faleiro Amanda Santos,Figueiredo Abdias Jose,Matarazzo Soraia Vanessa
Environmental science and pollution research international
The objective of this study was to evaluate (qualitatively and quantitatively) the occurrence of antibiotic residue in pasteurized milk in Brazil. Pasteurized milk samples (n = 252) were collected monthly from Nov. 2010-Oct. 2011 from 21 commercial establishments (brands). A screening test (Delvotest® SP-NT) was applied to those samples. In positive (n = 19) and/or suspect samples (n = 24), we quantified oxytetracycline (OTC) and tetracycline (TC) by high-performance liquid chromatography with diode array detector (HPLC-DAD). OTCs were detected in all positive samples and TCs in six. In the 24suspected samples, OTCs were detected in 23 and TCs were not found in 8. Of the milk brands evaluated (n = 21), the presence of antibiotic residue was not detected in 4; in the other brands, both positive and suspect samples were verified. Results indicate the presence of antibiotic residue above legal limits. According to actual milk consumption in Brazil (441 mL/kg BW/day), in only 9 of the 17 brands of milk with antibiotic residue, the estimated daily intake was at or less than the maximum recommended by the European Union. The screening test used was effective to identify the presence of antibiotic residue (OTC and TC), confirmed by HPLC-DAD. The OTC is the predominant antimicrobial used by dairy farmers. Ingestion of contaminated milk by OTC and TC can increase the resistance of microorganisms to antibiotics.
10.1007/s11356-013-2286-5
Feeding untreated and pasteurized waste milk and bulk milk to calves: effects on calf performance, health status and antibiotic resistance of faecal bacteria.
Aust V,Knappstein K,Kunz H-J,Kaspar H,Wallmann J,Kaske M
Journal of animal physiology and animal nutrition
Non-saleable milk (waste milk, WM) is contaminated with an undefined spectrum of potentially harmful pathogens and antimicrobial residues. The objective of this study was to determine the impact of feeding bulk milk (BM) or WM - both pasteurized or not - on calf performance, health and the antibiotic resistance of specific faecal bacteria. A total of 114 calves from a large-scale dairy were housed outdoors in individual hutches and were randomly assigned to one of four feeding groups. The calves were fed either WM, pasteurized WM (pWM), BM or pasteurized BM (pBM) from day 3 to 56 of life. Milk samples taken from the pasteurizer and calves' nipple buckets were investigated at regular intervals for total plate count and counts of thermoduric bacteria, coliforms and mastitis pathogens. Faecal samples were taken on days 2, 14, 28 and 56 of life from randomly selected calves of the WM, pWM and BM groups (each N = 8-9) and processed to obtain from each sample preferably two isolates of Escherichia (E.) coli and Enterococcus spp. respectively. Isolates were tested for their antimicrobial susceptibility to 25 antimicrobial agents by broth microdilution. Daily weight gain, milk and calf starter intake and health parameters did not differ significantly between the calves of the four feeding groups. The proportion of resistant E. coli isolates was significantly higher in calves fed WM and in calves fed pWM (most pronounced for cephalosporins) than in calves receiving BM. No differences in resistance were found for Enterococus spp. Thus, the concerns for selecting resistant faecal bacteria by feeding WM seem to be justified. Nonetheless, pasteurized WM of cows not treated with antimicrobials represents an acceptable feed for young calves.
10.1111/jpn.12019
Occurrence and transformation of veterinary antibiotics and antibiotic resistance genes in dairy manure treated by advanced anaerobic digestion and conventional treatment methods.
Wallace Joshua S,Garner Emily,Pruden Amy,Aga Diana S
Environmental pollution (Barking, Essex : 1987)
Manure treatment technologies are rapidly developing to minimize eutrophication of surrounding environments and potentially decrease the introduction of antibiotics and antibiotic resistant genes (ARGs) into the environment. While laboratory and pilot-scale manure treatment systems boast promising results, antibiotic and ARG removals in full-scale systems receiving continuous manure input have not been evaluated. The effect of treatment on ARGs is similarly lacking. This study examines the occurrence and transformation of sulfonamides, tetracyclines, tetracycline degradation products, and related ARGs throughout a full-scale advanced anaerobic digester (AAD) receiving continuous manure and antibiotic input. Manure samples were collected throughout the AAD system to evaluate baseline antibiotic and ARG input (raw manure), the effect of hygenization (post-pasteurized manure) and anaerobic digestion (post-digestion manure) on antibiotic and ARG levels. Antibiotics were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the ARGs tet(O), tet(W), sul1 and sul2 were analyzed by quantitative polymerase chain reaction (Q-PCR). Significant reductions in the concentrations of chlortetracycline, oxytetracycline, tetracycline and their degradation products were observed in manure liquids following treatment (p < 0.001), concomitant to significant increases in manure solids (p < 0.001). These results suggest sorption is the major removal route for tetracyclines during AAD. Significant decreases in the epimer-to-total residue ratios for chlortetracycline and tetracycline in manure solids further indicate degradation is desorption-limited. Moreover, sul1 and sul2 copies decreased significantly (p < 0.001) following AAD in the absence of sulfonamide antibiotics, while tetracyclines-resistant genes remained unchanged. A cross-sectional study of dairy farms utilizing natural aeration and liquid-solid separation treatments was additionally performed to compare levels of antibiotics and ARGs found in AAD with the levels in common manure management systems. The concentration of antibiotics in raw manure varied greatly between farms while minimal differences in ARGs were observed. However, significant (p < 0.01) differences in the levels of antibiotics and ARGs (except tet(W)) were observed in the effluents from the three different manure management systems.
10.1016/j.envpol.2018.02.024
Effects of feeding pasteurized waste milk to dairy calves on phenotypes and genotypes of antimicrobial resistance in fecal Escherichia coli isolates before and after weaning.
Maynou G,Migura-Garcia L,Chester-Jones H,Ziegler D,Bach A,Terré M
Journal of dairy science
The aim of this study was to evaluate the effects of feeding pasteurized waste milk (pWM) to calves on antimicrobial resistance of fecal Escherichia coli at both phenotypic and genotypic levels. Fifty-two Holstein female calves (3 ± 1.3 d of age) were fed 1 of the 2 different types of milk: milk replacer (MR) without antimicrobials or pWM with β-lactam residues until weaning at 49 d of age. Fecal swabs of all calves were obtained on d 0, 35, and 56 of the study and 3 E. coli isolates per sample were studied. Phenotypic resistance was tested by the disk diffusion method against a panel of 12 antimicrobials. A total of 13 resistance genes consisting of β-lactam, sulfonamide, tetracycline, and aminoglycoside families were examined by PCR. Feeding pWM to calves increased the presence of phenotypic resistance to ampicillin, cephalotin, ceftiofur, and florfenicol in fecal E. coli compared with MR-fed calves. However, the presence of resistance to sulfonamides, tetracyclines, and aminoglycosides was common in dairy calves independent of their milk-feeding source, suggesting other factors apart from the feeding source are involved in the emergence of antimicrobial resistance.
10.3168/jds.2017-13040
Comparative metagenomics and characterization of antimicrobial resistance genes in pasteurized and homemade fermented Arabian laban.
Yasir Muhammad,Bibi Fehmida,Hashem Anwar M,Azhar Esam I
Food research international (Ottawa, Ont.)
The aim of this study was to investigate bacterial diversity and function in a fermented milk drink called laban, which is traditionally served in the Middle East, Africa, and Indian subcontinent. Pasteurized laban (LBP) and unpasteurized, homemade, raw laban (LBR) underwent 16S rRNA gene amplicon and shotgun sequencing to analyze their bacterial community, presence of antimicrobial resistance genes (ARGs), and metabolic pathways. This study highlighted relatively greater diversity in LBR bacterial populations compared to LBP, despite containing similar major taxa that consisted primarily of Firmicutes followed by Proteobacteria, Bacteroidetes, and Actinobacteria. The dominant species, Streptococcus thermophilus, was relatively more abundant in LBP (80.7%) compared to LBR (47.9%). LBR had increased diversity and higher relative abundance of several known probiotic bacteria, such as Streptococcus salivarius and Lactococcus lactis, whereas Lactobacillus acidophilus was detected at a higher abundance in LBP. Pathogens like Acinetobacter baumannii, Streptococcus pneumoniae, Streptococcus pyogenes, and Escherichia coli had lower abundance in LBP compared to LBR. Thirty-three ARGs were detected in LBR compared to nine in LBP and are responsible for resistance to 11 classes of antibiotics. A significant proportion of the metagenomes from both types of laban were assigned to housekeeping functions, such as amino acid metabolism, translation, membrane transport, and carbohydrate metabolism. LBR demonstrated increased diversity in probiotics and metabolic functions compared to LBP. However, the relatively high diversity of pathogenic and opportunistic bacteria and ARGs in LBR raises safety concerns and highlights the need for a more hygienic environment for the processing of homemade fermented dairy foods.
10.1016/j.foodres.2020.109639