Paenalcaligenes faecalis sp. nov., a novel species of the family Alcaligenaceae isolated from chicken faeces.

A Gram-negative, motile, rod-shaped aerobic and alkalogenic bacterium, designated as strain YLCF04T, was isolated from chicken faeces. Its growth was optimal at 28 °C (range, 10-40 °C), pH 8 (range, pH 6-9) and in 1 % (w/v) NaCl (range, 0-10 %). It was classified to the genus Paenalcaligenes and was most closely related to Paenalcaligenes hominis CCUG 53761AT (97.5 % similarity) based on 16S rRNA gene sequence analysis. Average nucleotide identity and digital DNA-DNA hybridization values between YLCF04T and P. hominis CCUG 53761AT were 76.3 and 18.2 %, respectively. Strain YLCF04T has a genome size of 2.7 Mb with DNA G+C content of 46.3 mol%. Based on its phylogenetic, genomic, phenotypic and biochemical characteristics, strain YLCF04T represents a novel species of the genus Paenalcaligenes, for which the name Paenalcaligenes faecalis sp. nov. is proposed. The type strain is YLCF04T (=CCTCC AB 2022359T= KCTC 92789T).

Analysis of authorized coccidiostats in chicken feces and environmental water by ultra-performance liquid chromatography-tandem mass spectrometry based on dispersive solid-phase extraction and lyophilization.

A simple, sensitive, and efficient method based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for the determination of 8 coccidiostats in chicken feces and environmental water (including sewage, pond water, and lake water) surrounding the farm. Target analytes in chicken feces were extracted with 2% acetic acid in acetonitrile solution, followed by a dispersive solid-phase extraction (DSPE) cleanup step using the mixture of PSA and C18 adsorbents. Environmental water samples were pretreated using a lyophilization approach. Analysis was carried out on a UPLC-MS/MS with the combination of methanol and 0.1% formic acid aqueous solution as the mobile phase under multiple reaction monitoring in positive and negative ionization modes. Results showed that 8 coccidiostats were linear with correlation coefficients higher than 0.99. Method validation was performed using fortified samples, reaching satisfactory recoveries of 75.9%-97.8% in chicken feces and 71.9%-108.2% in environmental water. Limits of detection for 8 analytes in chicken feces and environmental water were 0.03∼2 µg/kg and 0.005∼1 µg/L, respectively. Matrix effects were calculated and strong signal suppression (>50%) for some coccidiostats was observed. The developed method was successfully applied to analyze coccidiostats in chicken feces and environmental water collected from local chicken farms.

Storage media and RNA extraction approaches substantially influence the recovery and integrity of livestock fecal microbial RNA.

Background: There is growing interest in understanding gut microbiome dynamics, to increase the sustainability of livestock production systems and to better understand the dynamics that regulate antibiotic resistance genes (i.e., the resistome). High-throughput sequencing of RNA transcripts (RNA-seq) from microbial communities (metatranscriptome) allows an unprecedented opportunity to analyze the functional and taxonomical dynamics of the expressed microbiome and emerges as a highly informative approach. However, the isolation and preservation of high-quality RNA from livestock fecal samples remains highly challenging. This study aimed to determine the impact of the various sample storage and RNA extraction strategies on the recovery and integrity of microbial RNA extracted from selected livestock (chicken and pig) fecal samples. Methods: Fecal samples from pigs and chicken were collected from conventional slaughterhouses. Two different storage buffers were used at two different storage temperatures. The extraction of total RNA was done using four different commercially available kits and RNA integrity/quality and concentration were measured using a Bioanalyzer 2100 system with RNA 6000 Nano kit (Agilent, Santa Clara, CA, USA). In addition, RT-qPCR was used to assess bacterial RNA quality and the level of host RNA contamination. Results: The quantity and quality of RNA differed by sample type (i.e., either pig or chicken) and most significantly by the extraction kit, with differences in the extraction method resulting in the least variability in pig feces samples and the most variability in chicken feces. Considering a tradeoff between the RNA yield and the RNA integrity and at the same time minimizing the amount of host RNA in the sample, a combination of storing the fecal samples in RNALater at either 4 °C (for 24 h) or -80 °C (up to 2 weeks) with extraction with PM kit (RNEasy Power Microbiome Kit) had the best performance for both chicken and pig samples. Conclusion: Our findings provided a further emphasis on using a consistent methodology for sample storage, duration as well as a compatible RNA extraction approach. This is crucial as the impact of these technical steps can be potentially large compared with the real biological variability to be explained in microbiome and resistome studies.

Active Yeast but Not Henhouse Environment Affects Dropping Moisture Levels in Egg-Laying Hens.

Dropping moisture (DM) refers to the water content in feces. High DM negatively affects poultry production, environment, production costs, and animal health. Heredity, nutrition, environment, and disease may affect DM level. DM has medium inheritability and is related to cage height in henhouses. We examined the relationship among DM level, production performance, and environmental factors at different locations at the same henhouse height and effects of three types of additives. We measured the correlation between environmental factors including temperature, humidity, CO2 concentration, absolute pressure, and DM levels and laying performance of 934 Rhode Island Red hens. DM level was not significantly associated with environmental factors or production performance. We divided 64 persistently high DM hens into control and treatment groups supplied with different additives (probiotics, anisodamine, and antibiotics). DM levels, laying performance, egg quality, and serum biochemical indices were determined. Compared with the control and antibiotics, probiotics significantly reduced DM levels and eggshell strength while improving yolk color but did not significantly affect production performance. The additives reduced the b value of eggshell color; compared with probiotics, anisodamine decreased serum globulin levels. Exogenous active yeast supplementation can significantly reduce DM levels.

Complete genome sequence of Streptococcus hyointestinalis B19, a strain producing bacteriocin, isolated from chicken feces.

Streptococcus hyointestinalis B19 was isolated from chicken feces collected from local farm in Anseong, Korea. S. hyointestinalis B19 was shown to produce bacteriocin-like compounds exhibiting inhibitory activities against several pathogens including strains of Clostridium perfringens and Listeria monocytogenes. The whole genome of S. hyointestinalis B19 strain was sequenced using PacBio RS II platform. The genome comprised four contigs with a size of 2,217,061 bp. The DNA G + C content was found to be 42.95 mol%. Annotation results revealed 2,266 coding sequences (CDSs), 18 rRNAs, and 61 tRNA genes. Based on genome analysis, we found that the strain B19 possessed various genes associated with bacteriocin synthesis, modification, and transport.

Exploring the profile of antimicrobial resistance genes harboring by bacteriophage in chicken feces.

Bacteriophage may play an important role in antimicrobial resistance genes (ARGs) transmission. However, the contribution of bacteriophage to the spread of ARGs in environment, especially in poultry farm environment, is rarely known. In this study, the prevalence of ARGs in bacteriophage DNA was investigated in chicken feces from 30 different poultry farms in China. Then the abundance of the aac(6')-Ib-cr, blaCTX-M, ermB, floR, mcr-1, sul1, tetM and intI1 genes was determined by qPCR in bacteriophage and compared with certain representative plasmid DNA samples. The results showed that 12 ARGs (aac(6')-Ib-cr, aph(3')-IIIa, blaCTX-M, ermB, ermF, floR, mcr-1, qnrS, sul1, sul2, vanA, tetM genes) and class 1 integron gene intI1 were detected in bacteriophage DNA fraction. The sul1, tetM and aac(6')-Ib-cr genes were most prevalent with high detection rates of 77%, 61% and 55%, respectively. To our best knowledge, this study firstly reported the presence of the mcr-1 gene in bacteriophage DNA derived from farms environments. We found that the gene copy (GC) numbers of the aac(6')-Ib-cr, ermB and sul1 genes were as high as 5.47, 5.22 and 5.54 log10 GC/g, respectively. Both the prevalence and abundance of ARGs in broiler fecal wastes were also generally higher than in laying hens. In addition, although the GC numbers of the aac(6')-Ib-cr, floR and tetM genes in plasmid DNA was higher than that in phage DNA fraction by 4.68, 3.59 and 3.9 orders of magnitude, respectively, the absolute abundances of the blaCTX-M and mcr-1 genes in phage DNA were close to or even higher than that in plasmid DNA at farm SIL2, SIL4 and SIB1. As potential vessels for ARGs, bacteriophage could not be ignored due to their unique extracellular persistence in environments. Overall, this is the first comprehensive survey about bacteriophage carried ARGs from farms in different regions in China.

Characterization of Resistance Patterns and Detection of Apramycin Resistance Genes in Escherichia coli Isolated from Chicken Feces and Houseflies after Apramycin Administration.

The aim of this study was to evaluate the influence of apramycin administration on the development of antibiotic resistance in Escherichia coli (E. coli) strains isolated from chicken feces and houseflies under field conditions. Chickens in the medicated group (n = 25,000) were given successive prophylactic doses (0.5 mg/l) of apramycin in their drinking water from Days 1 to 5, while no antibiotics were added to the un-medicated groups drinking water (n = 25,000). Over 40 days, a total of 1170 E. coli strains were isolated from fecal samples obtained from medicated and un-medicated chickens and houseflies from the same chicken farm. Apramycin MIC90 values for E. coli strains obtained from the medicated group increased 32-128 times from Days 2 to 6 (256-1024 µg/ml) when compared to those on Day 0 (8 µg/ml). Strains isolated from un-medicated chickens and houseflies had consistently low MIC90 values (8-16 µg/ml) during the first week, but showed a dramatic increase from Days 8 to 10 (128-1024 µg/ml). The apramycin resistance gene aac(3)-IV was detected in E. coli strains from medicated (n = 71), un-medicated (n = 32), and housefly groups (n = 42). All strains positive for aac(3)-IV were classified into 12 pulsed-field gel electrophoresis (PFGE) types. PFGE types A, E, and G were the predominant types in both the medicated and housefly groups, suggesting houseflies play an important role in spreading E. coli-resistant strains. Taken together, our study revealed that apramycin administration could facilitate the occurrence of apramycin-resistant E. coli and the apramycin resistance gene acc(3)-IV. In turn, these strains could be transmitted by houseflies, thus increasing the potential risk of spreading multi-drug-resistant E. coli to the public.

A purple non-sulfur bacterium producing polyhydroxybutyrate and the conserved region of pha synthase gene / Bactéria púrpura não sulfurosa produtora de polihidroxibutirato e a região conservada do gene pha sintase

This study aimed to screen purple non-sulfur bacteria capable of accumulating granules or polyhydroxybutyrate (PHB) inside the cells, identify the potent strain, assay the enzyme or PHA synthase, and compare the PHB synthase gene with that of related strains. A total of 58 strains of purple non-sulfur bacteria were isolated from 108 samples of chicken feces in the chicken-egg farm of the Department of Animal Science, Faculty of Natural Resources at Prince of Songkla University, Hat Yai, Thailand. After cultivating the bacteria in glutamate malate (GM) medium without added glutamic acid under light (3,000 Lux) at 35oC for 5 days, the intracellular biopolymer granules of the bacteria were observed by using a Confocal Laser Scanning Microscope (CLSM) with excitation and emission wavelength of 530 and 605 nm, respectively. Gas chromatography (GC) was carried out for quantitative analysis of PHB. There were five strains, CH12, CH52, CH72, CH90 and CH92, showed biopolymer granules under CLSM, and accumulated PHB 5, 1.7, 1.5, 1.4 and 1.8% (w w-1) of the cell dry weight (CDW), respectively. The 16S rDNA sequence analysis of CH12 strain showed a high homology of 100% correlation to that of Rhodopseudomonas palustris strain NCIB8288. Regarding the taxonomic characteristics and 16S rDNA sequence analysis, CH12 strain was identified as Rps. palustris NCIB8288. The PHA synthase activity of the crude extract from CH12 strain was 25 units/mL. The conserved regions could be aligned and selected among 5 strains of Rhodopseudomonas palustris (strains BisA53, TIE-1, CGA009, HaA2 and BisB18). The purified PCR product was obtained for further studies.

Este estudo teve como objetivo rastrear bactérias púrpuras não sulfurosas capazes de acumular grânulos ou polihidroxibutirato (PHB) dentro das células, identificar a estirpe potente, ensaiar a enzima ou PHA sintaxe, e comparar com o gene PHB sintase com aquele de estirpes relacionadas. Um total de 58 estirpes de bactérias púrpuras não sulfurosas foram isoladas a partir de 108 amostras de fezes de galinhas na granja produtora de ovos do Departamento de Ciência Animal, Faculdade de Recursos Naturais da Universidade Prince of Songkla, Hat Yai, Tailândia. Depois de cultivar as bactérias em um substrato de glutamato/malato (GM), sem ácido glutâmico adicionado, sob luz (3000 lux) a 35 ºC durante 5 dias, os grânulos de biopolímeros intracelulares das bactérias foram observados utilizando um microscópio confocal (do inglês Confocal Laser Scanning Microscope - CLSM) com comprimentos de onda de excitação e emissão de 530 e 605 nm, respectivamente. A cromatografia gasosa (do inglês Gas chromatography - GC) foi realizada para uma análise quantitativa de PHB. Havia 5 estirpes, CH12, CH52, CH72, CH90 e CH92, que mostraram grânulos biopoliméricos quando submetidos ao CLSM, e PHB-5 acumulado de 1.7, 1.5, 1.4 and 1.8% (w w-1) do peso celular seco (do inglês cell dry weight - CDW), respectivamente. A análise da sequência do rDNA 16S da estirpe CH12 demonstrou uma alta correlação de homologia de 100% para aquela da estirpe NCIB8288 da Rhodopseudomonas palustris. Em relação às características taxonômicas e da análise da sequência do rDNA 16S, a estirpe CH12 foi identificada como Rps. palustris NCIB8288. A atividade da PHA sintase do extrato bruto da estirpe CH12 foi de 25 unidades/mL. As regiões conservadas puderam ser alinhadas e selecionadas entre 5 estirpes de Rhodopseudomonas palustris (BisA53, TIE-1, CGA009, HaA2 e BisB18). O produto purificado da reação em cadeia da polimerase - PCR foi obtido para estudos futuros.

Fate of mesophilic aerobic bacteria and Salmonella enterica on the surface of eggs as affected by chicken feces, storage temperature, and relative humidity.

We compared the microbiological quality of chicken eggshells obtained from a traditional wholesale market and a modern supermarket. We also determined the survival and growth characteristics of naturally occurring mesophilic aerobic bacteria (MAB) and artificially inoculated Salmonella enterica on eggshells under various environmental conditions (presence of chicken feces, temperature [4, 12, or 25 °C], and relative humidity [RH; 43 or 85%]). The populations of MAB, coliforms, and molds and yeasts on eggshells purchased from a traditional wholesale market were significantly (P ≤ 0.05) higher than those from a modern supermarket. In the second study, when we stored uninoculated eggs under various storage conditions, the population of MAB on eggshells (4.7-4.9 log CFU/egg) remained constant for 21 days, regardless of storage conditions. However, when eggshells were inoculated with S. enterica and stored under the same conditions, populations of the pathogen decreased significantly (P ≤ 0.05) under all tested conditions. Survival of S. enterica increased significantly (P ≤ 0.05) in the presence of feces, at low temperatures, and at low RH. These observations will be of value when predicting the behavior of microorganisms on eggshells and selecting storage conditions that reduce the populations of S. enterica on eggshells during distribution.

Ocorrência de Campylobacter em carne de frango, fezes de frango e humanas e pesquisa dos genes cdt / Occurrence of Campylobacter in poultry, meat chicken and human feces, and cdt GENES research

Foram coletadas 100 amostras de conteúdo fecal de aves de corte, 100 de produtos de frango (coxa, sobrecoxa, asa, dorso, carne moída e fígado) e 100 de fezes de humanos, e analisadas para pesquisa de Campylobacter. Realizou-se a determinação da espécie e da presença dos genes cdt, responsáveis pela codificação da toxina citoletal distensiva (CDT), através da técnica da PCR. A bactéria foi isolada de 61% das amostras de fezes de frango, 20% de produtos de frango e 3% de fezes de humanos. A maioria dos isolados foi determinada como C. jejuni . Destes, 93,5% apresentaram os genes para a toxina CDT. Apesar de a ocorrência de Campylobacter em fezes de humanos ter sido baixa, a prevalência em frangos de corte e produtos de frango foi elevada, fato que, aliado à presença dos genes cdt na maioria dos isolados, representa risco potencial para os consumidores. Esses resultados são indicativos da necessidade de medidas preventivas no sistema de produção e de boas práticas de fabricação na indústria, de forma a minimizar a contaminação dos produtos e diminuir o risco para os consumidores.

A hundred chicken fecal samples, a hundred samples of retail poultry products and a hundred samples of human feces were collected and tested for the presence of Campylobacter. The species identification and the analysis for the presence of cdt genes, responsible for encoding the cytolethal distending toxin, were performed by PCR. Campylobacter was found in 61% of the chicken fecal samples, in 20% of the poultry products and in 3% of the human feces. Most isolates were identified as C. jejuni. In 93.5% of these isolates, the cdt genes have been detected. Despite the occurrence of Campylobacter in feces of humans has been low, the prevalence in broilers and poultry products was high, which, combined with the presence of cdt genes in most isolates, represents a potential risk to consumers. These results suggest there is a need for preventive measures in the production system and good manufacturing practices in the industry so as to minimize contamination of products and reduce the risk to consumers.