The storage and transportation of raw milk at low temperatures promote the growth of psychrotrophic bacteria and the production of thermo-stable enzymes, which pose great threats to the quality and shelf-life of dairy products. Though many studies have been carried out on the spoilage potential of psychrotrophic bacteria and the thermo-stabilities of the enzymes they produce, further detailed studies are needed to devise an effective strategy to avoid dairy spoilage. The purpose of this study was to explore the spoilage potential of psychrotrophic bacteria from Chinese raw milk samples at both room temperature (28 °C) and refrigerated temperature (7 °C). Species of Yersinia, Pseudomonas, Serratia, and Chryseobacterium showed high proteolytic activity. The highest proteolytic activity was shown by Yersinia intermedia followed by Pseudomonas fluorescens (d). Lipolytic activity was high in isolates of Acinetobacter, and the highest in Acinetobacter guillouiae. Certain isolates showed positive β-galactosidase and phospholipase activity. Strains belonging to the same species sometimes showed markedly different phenotypic characteristics. Proteases and lipases produced by psychrotrophic bacteria retained activity after heat treatment at 70, 80, or 90 °C, and proteases appeared to be more heat-stable than lipases. For these reasons, thermo-stable spoilage enzymes produced by a high number of psychrotrophic bacterial isolates from raw milk are of major concern to the dairy industry. The results of this study provide valuable data about the spoilage potential of bacterial strains in raw milk and the thermal resistance of the enzymes they produce.
Animals ; Bacteria/genetics* ; Bacterial Proteins/chemistry* ; Biofilms ; Cold Temperature ; Dairy Products ; Endopeptidases/chemistry* ; Enzyme Stability ; Food Microbiology ; Hot Temperature ; Lipase/chemistry* ; Milk/microbiology* ; Peptide Hydrolases/chemistry* ; Phospholipases/chemistry* ; RNA, Ribosomal, 16S/genetics* ; Raw Foods/microbiology* ; beta-Galactosidase/chemistry*

Objective: To understand the etiological characteristics and drug susceptibility of Mycobacterium thermoresistibile and Mycobacterium elephantis isolated from a cow with mastitis and provide evidence for the prevention and control of infectious mastitis in cows. Methods: The milk sample was collected from a cow with mastitis, which was pretreated with 4% NaOH and inoculated with L-J medium for Mycobacterium isolation. The positive cultures were initially identified by acid-fast staining and multi-loci PCR, then Mycobacterium species was identified by the multiple loci sequence analysis (MLSA) with 16S rRNA, hsp65, ITS and SodA genes. The drug sensitivity of the isolates to 27 antibiotics was tested by alamar blue assay. Results: Two anti-acid stain positive strains were isolated from the milk of a cow with mastitis, which were identified as non-tuberculosis mycobacterium by multi-loci PCR, and multi-loci nucleic acid sequence analysis indicated that one strain was Mycobacterium thermoresistibile and another one was Mycobacterium elephantis. The results of the drug susceptibility test showed that the two strains were resistant to most antibiotics, including rifampicin and isoniazid, but they were sensitive to amikacin, moxifloxacin, levofloxacin, ethambutol, streptomycin, tobramycin, ciprofloxacin and linezolid. Conclusions:Mycobacterium thermoresistibile and Mycobacterium elephantis were isolated in a cow with mastitis and the drug susceptibility spectrum of the pathogens were unique. The results of the study can be used as reference for the prevention and control the infection in cows.
Animals ; Anti-Bacterial Agents/pharmacology* ; Antitubercular Agents/pharmacology* ; Cattle ; Drug Resistance, Bacterial ; Female ; Humans ; Mastitis, Bovine/microbiology* ; Microbial Sensitivity Tests ; Milk/microbiology* ; Mycobacterium/isolation & purification* ; Mycobacterium Infections/veterinary* ; Mycobacterium tuberculosis/drug effects* ; Nontuberculous Mycobacteria/isolation & purification* ; Polymerase Chain Reaction ; RNA, Ribosomal, 16S/genetics*

OBJECTIVEIn this study, milk from a cow with mastitis was analyzed to determine the presence of mycobacterial infection. Milk quality and security problems pertaining to the safe consumption of dairy products were also discussed in this study.

METHODSMilk was preprocessed with 4% NaOH. Then, mycobacteria were isolated from the milk sample on L-J medium. The isolate was identified using multiple loci Polymerase Chain Reaction (PCR) and multi-locus sequence analysis with 16S rRNA, sodA, hsp65, and ITS genes. The drug sensitivity of the isolate to 27 antibiotics was tested through alamar blue assay.

RESULTSSmooth, moist, pale yellow colonies appeared on the L-J medium within a week after inoculation. Based on the results of multiple loci PCR analysis, the isolate was preliminarily identified as non-tuberculous mycobacteria. The 16S rRNA, SodA, hsp65, and ITS gene sequences of the isolate exhibited 99%, 99%, 99%, and 100% similarities, respectively, with those of the published reference strains of Mycobacterium elephantis (M. elephantis). The drug sensitivity results showed that the strain is resistant to isoniazid, p-aminosalicylic acid, and trimesulf but is sensitive to ofloxacin, rifampicin, amikacin, capreomycin, moxifloxacin, kanamycin, levofloxacin, cycloserine, ethambutol, streptomycin, tobramycin, rifabutin, ciprofloxacin, linezolid, cefoxitin, clarithromycin, and minocycline.

CONCLUSIONTo the best of our knowledge, this study is initially to report the isolation of M. elephantis from the milk of a cow with mastitis in China.

Animals ; Anti-Bacterial Agents ; pharmacology ; Cattle ; China ; Drug Resistance, Bacterial ; Female ; Mastitis, Bovine ; epidemiology ; microbiology ; Milk ; microbiology ; Mycobacterium ; drug effects ; genetics ; isolation & purification ; Mycobacterium Infections ; epidemiology ; microbiology ; veterinary ; Phylogeny ; Polymerase Chain Reaction

OBJECTIVETo study the bacterial culture results of expressed breast milk.

METHODSA total of 1178 expressed breast milk samples were collected for bacterial culture. The breast milk sampled from the mothers of preterm neonates (n=615) and term neonates (n=563) who were hospitalized between May 2014 and April 2015.

RESULTSThere was no significant difference in bacterial counts between the preterm and term sample groups (P>0.05). Potential intestinal pathogens were found in 55 samples (4.63%) of the 1178 samples, with no significant difference between the preterm and term sample groups (P>0.05). The second expressed milk samples from 33 mothers were cultured. Only 10 samples (30%) were found to have the same bacteria as the first time. The detection rate of bacterial load of ≥ 10⁵ CFU/mL was higher in those samples with potential intestinal pathogens, as compared with those samples without potential intestinal pathogens (43.64% vs 14.87%; P<0.05). There was no correlation between the incidence of neonatal infections and potential intestinal pathogens in breast milk.

CONCLUSIONSBreast milk is not sterile. Bacterial loads and phylotypes are variable. Random breast milk cultures can neither describe bacterial colonies in breast milk, nor be a predictor of neonatal infection.

Bacterial Load ; Female ; Humans ; Male ; Milk, Human ; microbiology ; Pilot Projects

OBJECTIVEThis study is aimed to develop a two-tube melting curve-based multiplex real time PCR assay (MCMRT-PCR) for the simultaneous detection of six common foodborne pathogenic bacteria (diarrhoeagenic Escherichia coli, Salmonella, and Shigella in tube 1, Staphylococcus aureus, Vibrio parahaemolyticus, and Listeria monocytogenes in tube 2).

METHODSA two-tube MCMRT-PCR assay was performed on 7900HT Fast Real-Time PCR System (Applied Biosystems, USA). Amplification by PCR was optimized to obtain high efficiency. The sensitivity and specificity of assays were investigated.

RESULTSThe detection limit of optimized MCMRT-PCR assay was 3.9×102 CFU/mL for S. aureus, 4.4×102 CFU/mL for L. monocytogenes, 3.0×102 CFU/mL for Salmonella, 2.5×102 CFU/mL for Shigella, 2.1×102 CFU/mL for V. parahaemolyticus, and 1.2×102 CFU/mL for E. coli. The feasibility of MCMRT-PCR was further evaluated using artificially contaminated milk, the sensitivity was at the level of 105 CFU/mL.

CONCLUSIONA two-tube MCMRT-PCR assay using six primer sets was developed for detection of multiple pathogens. Our findings demonstrates that the proposed two-tube assay is reliable, useful and rapid for simultaneous detection of six foodborne pathogenic bacteria with an intended application in provincial Centers for Diseases Control and Prevention (CDC).

Animals ; Bacteria ; genetics ; isolation & purification ; Food Microbiology ; methods ; Milk ; microbiology ; Multiplex Polymerase Chain Reaction ; Real-Time Polymerase Chain Reaction ; Sensitivity and Specificity

This research uses six Agrobacterium rhizogenes R1601, R15384, R1000, A4, R1025 and R1 to infect silymarin explants to induce hairy roots and silibin. All of the six A. rhizogenes can induce Silybum marianum to generate hairy roots and the A. rhizogene A4 shows comparatively high infection on the plant. This research determines the condition to induce silymarin hairy roots by the factors of infection time, pre-culturing, co-culturing and pH value. The fact that MS liquid medium fits the proliferation of silymarin hairy roots is determined. Through PCR molecular identification, it can be seen that the DNA plasmids in the A. rhizogenes are successfully integrated into the genome of transformed roots. Using liquid chromatography, it is determined that the silibin content in silymarin hairy roots is 2.5 times that in the plant In this research, the silymarin hairy roots culturing system is established, which lays a foundation for the study of culturing silymarin hairy roots and producing silibin.
Agrobacterium ; genetics ; physiology ; Cell Culture Techniques ; methods ; Milk Thistle ; chemistry ; genetics ; growth & development ; microbiology ; Plant Roots ; chemistry ; genetics ; growth & development ; microbiology ; Silymarin ; analysis ; Transformation, Genetic

The sequences of the ccrAB genes from bovine-, canine- and chicken-originating methicillin-resistant Staphylococcus (S.) epidermidis (MRSE) and bovine methicillin-resistant Staphylococcus (S.) aureus (MRSA) were compared to investigate the frequency of intra-species horizontal transfer of the staphylococcal cassette chromosome mec (SCCmec) complex. Nineteen MRSE strains were isolated from bovine milk, chickens, and dogs, and their genetic characteristics were investigated by multilocus sequence typing and SCCmec typing. Among the animal MRSE strains, the most frequent SCCmec type was type IV, which consisted of the type B mec complex and ccrAB type 2. The ccrA2 and ccrB2 genes were sequenced from the bovine, chicken and canine MRSE strains and compared with those of the bovine MRSA strains. The sequences generally clustered as MRSA and MRSE groups, regardless of the animal source. Additionally, no bovine MRSE sequence was associated with the bovine MRSA groups. Although most of the bovine MRSE and MRSA isolates possessed SCCmec type IV sequences, our results suggest that the intra-species gene transfer of the SCCmec complex between bovine S. aureus and bovine S. epidermidis strains is not a frequent event.
Animals ; Anti-Bacterial Agents/*pharmacology ; Bacterial Proteins/*genetics/metabolism ; Bacterial Typing Techniques/veterinary ; Cattle ; Cattle Diseases/epidemiology/metabolism ; Chickens ; Dog Diseases/epidemiology/metabolism ; Dogs ; *Drug Resistance, Bacterial ; *Gene Transfer, Horizontal ; Methicillin/*pharmacology ; Methicillin-Resistant Staphylococcus aureus/genetics/isolation & purification ; Milk/microbiology ; Multilocus Sequence Typing/veterinary ; Poultry Diseases/epidemiology/metabolism ; Prevalence ; Republic of Korea/epidemiology ; Staphylococcal Infections/epidemiology/microbiology/*veterinary ; Staphylococcus epidermidis/genetics/isolation & purification

OBJECTIVETo determine the presence and levels of microbes in unexpired pasteurized milk from randomly selected supermarkets in Kingston, Jamaica.

METHODSThe quantitative study used a stratified random sampling technique in the selection of the 20 representative milk samples from six (6) supermarkets. Microbiological tests such as methylene blue reduction, standard plate count (SPC), coliform plate count (CPC), purity plate culture, gram staining and biochemical tests were performed to examine the microbes in purchased unexpired pasteurized milk.

RESULTSOne sample (BCr016) had a pH of 4.0, a rancid odour and curdled appearance. It decolourized within one hour during the methylene blue reduction test and was classified as class 4 milk. Seven of the samples were sterile with no microbe growth on the plate count agar and violet red bile salt agar (VRBA). The milk samples that appeared to be safe for consumption were all 10, 11, 12 and 13 days before expiration. The VRBA sample BCr016, had a colony count of 13 400 CFU/ mL. There was the presence of Escherichia coli in sample LCr021 which had a standard plate count of 1 580 SPC/mL and a coliform count of 500 CFU/mL. Enterobacter sp. was present in colonies from BCr016 and all the other milk samples.

CONCLUSIONSUnacceptable levels of Enterobacter spp. and Escherichia coli were found in most of the samples. Effective measures to ensure safe milk for human consumption such as the phosphatase test and methylene blue reduction test should be routinely performed on each batch of milk processed by dairy plants.

Animals ; Colony Count, Microbial ; Developing Countries ; Food Microbiology ; Humans ; Jamaica ; Milk ; microbiology

A simplified polymerase chain reaction (PCR) assay was developed for fast and easy screening of mycoplasma mastitis in dairy cattle. Species of major mycoplasma strains [Mycoplasma (M.) bovis, M. arginini, M. bovigenitalium, M. californicum, M. bovirhinis, M. alkalescens and M. canadense] in cultured milk samples were detected by this simplified PCR-based method as well as a standard PCR technique. The minimum concentration limit for detecting mycoplasma by the simplified PCR was estimated to be about 2.5 x 10(3) cfu/mL and was similar to that of the standard PCR. We compared the specificity and sensitivity of the simplified PCR to those of a culture method. Out of 1,685 milk samples cultured in mycoplasma broth, the simplified PCR detected Mycoplasma DNA in 152 that were also positive according to the culture assay. The sensitivity and specificity of the simplified PCR were 98.7% and 99.7%, respectively, for detecting mycoplasma in those cultures. The results obtained by the simplified PCR were consistent with ones from standard PCR. This newly developed simplified PCR, which does not require DNA purification, can analyze about 300 cultured samples within 3 h. The results from our study suggest that the simplified PCR can be used for mycoplasma mastitis screening in large-scale dairy farms.
Animals ; Cattle ; Colony Count, Microbial/veterinary ; DNA, Bacterial/chemistry/genetics ; Disease Outbreaks/prevention & control/veterinary ; Female ; Mastitis, Bovine/diagnosis/*microbiology ; Milk/cytology/*microbiology ; Mycoplasma/genetics/*isolation & purification ; Mycoplasma Infections/diagnosis/microbiology/*veterinary ; Polymerase Chain Reaction/veterinary

Staphylococcus aureus is a major etiological pathogen of bovine mastitis, which triggers significant economic losses in dairy herds worldwide. In this study, S. aureus strains isolated from the milk of cows suffering from mastitis in Korea were investigated by spa typing and staphylococcal enterotoxin (SE) gene profiling. Forty-four S. aureus strains were isolated from 26 farms in five provinces. All isolates grouped into five clusters and two singletons based on 14 spa types. Cluster 1 and 2 isolates comprised 38.6% and 36.4% of total isolates, respectively, which were distributed in more than four provinces. SE and SE-like toxin genes were detected in 34 (77.3%) isolates and the most frequently detected SE gene profile was seg, sei, selm, seln, and selo genes (16 isolates, 36.3%), which was comparable to one of the genomic islands, Type I nuSabeta. This is a first report of spa types and the prevalence of the recently described SE and SE-like toxin genes among S. aureus isolates from bovine raw milk in Korea. Two predominant spa groups were distributed widely and recently described SE and SE-like toxin genes were detected frequently.
Animals ; Cattle ; Cluster Analysis ; DNA, Bacterial/chemistry/genetics ; Enterotoxins/chemistry/*genetics ; Female ; Genotype ; Mastitis, Bovine/epidemiology/genetics/*microbiology ; Microsatellite Repeats ; Milk/*microbiology ; Polymerase Chain Reaction/veterinary ; Prevalence ; Republic of Korea/epidemiology ; Staphylococcal Infections/epidemiology/microbiology/*veterinary ; Staphylococcus aureus/classification/genetics/*isolation & purification