Colonization of the botanical environment by Klebsiella isolates of pathogenic origin.

Growth, survival, and pathogenicity of Klebsiella growing in and on environmental foci were examined. Total coliforms present in raw wastes from pulp mills were in excess of 10(5)/ml, and 60 to 80% were Klebsiella. Fecal coliform counts ranged from 10(1) to 10(5)/ml. Klebsiella isolates from industrial effluents and a variety of human and bovine mastitis origins multiplied in pulp waste and commonly exceeded 10(6) cells per ml. Pathogenic isolates also multiplied in dilute aqueous extracts of sawdust to comparable levels. Klebsiella strains from vegetable surfaces and human infections grew rapidly on the surfaces of potatoes and lettuce and exceeded 10(3) organisms per g of surface peel and leaf after a 24h incubation at room temperature. After 7 weeks on potatoes stored at 5 degrees C, some 10 to 30% of the day 1 Klebsiella counts were recoverable. Three Klebsiella isolates of pathogenic origin were passed 45 times through sterile pulp effluent (270 generations), and mean lethal dose levels in mice were periodically monitored. In two instances, a significant decrease in virulence was noted after 15 to 26 passes (90 to 156 generations). The third culture, of bovine mastitis origin, retained its original mean lethal dose value. Botanical milieu provided suitable habitats for the multiplication and colonization of Klebsiella isolates of disease origins in the same manner as indigenous isolates. Aquatic environments polluted with botanical material served as potential reservoirs for perpetuating the growth and spread of opportunistic Klebsiella pathogens that may ultimately colonize animals, humans, and aquatic organisms.     

Antibiotic Resistance and Its Transfer Among Clinical and Nonclinical Klebsiella Strains in Botanical Environments

A total of 183 isolates of Klebsiella from drinking water, market vegetables, wood, sawdust, industrial effluents, and human and animal origin were examined for susceptibility to 10 antibacterial agents. Incidence of resistance to two or more antibiotics tested was: 65% of the human clinical isolates, 59% among bovine mastitis, and 24% among the nonclinical isolates. The five different multiple resistance patterns among nonclinically derived Klebsiella were also found among the human and bovine mastitis isolates. Statistical analyses revealed that patterns of resistance among Klebsiella isolates from drinking water, market vegetables, and industrial effluents were highly correlated with each other and with resistance patterns of human clinical isolates. Antibiotic resistance was transferred between Klebsiella growing in two habitat-simulated environments (growing radish plants and aqueous sawdust suspensions). Transconjugants were detected in 5 of 21 and 6 of 21 mating pairs, respectively. Average transconjugants/donor ranged from 10⁻³ to 10⁻⁶ in Penassay broth, from 10⁻⁶ to 10⁻⁷ on radish plants, and from 10⁻⁵ to 10⁻⁸ in sawdust suspensions. Although antibiotic resistance transfer under simulated environmental conditions can occur, regrowth of clinical strains is probably the major cause for the widespread occurrence of antibiotic-resistant Klebsiella in the nonclinical environment.     

Significance of Fecal Coliform-Positive Klebsiella

A total of 191 Klebsiella pneumoniae isolates of human clinical, bovine mastitis, and a wide variety of environmental sources were tested for fecal coliform (FC) response with the membrane filtration and most probable number techniques. Twenty-seven Escherichia coli cultures of human clinical and environmental origins were also tested. Eighty-five percent (49/58) of known pathogenic K. pneumoniae were FC positive, compared with 16% (19/120) of the environmental strains. E. coli results indicated 93% (13/14) of the clinical and 85% (11/13) of the environmental strains as FC positive. There was no significant difference in the incidence of FC-positive cultures between pathogenic Klebsiella and E. coli. pH measurements of K. pneumoniae and E. coli cultures growing in m-FC broth at 44.5°C revealed three distinct pH ranges correlating with colony morphology. β-Galactosidase assays of Klebsiella and E. coli cultures at 44.5°C indicated all were able to hydrolyze lactose, even if they were FC negative by the membrane filtration or most probable number techniques. The FC response pattern appears stable in K. pneumoniae. Three pathogenic cultures showed no change in FC responses after 270 generations of growth in sterile pulp mill effluent. Since K. pneumoniae is carried in the gastrointestinal tract of humans and animals and 85% of the tested pathogenic strains were FC positive, the isolation of FC-positive Klebsiella organisms from the environment would indicate their fecal or clinical origin or both. The added fact that K. pneumoniae is an opportunistic pathogen of increasing importance makes the occurrence of FC-positive environmental Klebsiella, particularly in large numbers, a potential human and animal health hazard.     

Klebsiella Biotypes Among Coliforms Isolated from Forest Environments and Farm Produce

Samples of water, soil, needles, and bark were collected from three different forest environments and from a pulp and paper mill. In addition, samples of fresh produce were obtained from a local supermarket. All samples were examined for total and fecal coliforms. The counts obtained from the forestrelated samples did not correlate with sample type or location. When 123 isolates were identified biochemically, 71% were Klebsiella, 19% Enterobacter, 8% Citrobacter, and 2% Escherichia. All the Citrobacter, 75% of the Enterobacter, and 65% of the Klebsiella were negative for growth in elevated coliform (EC) broth. All the Escherichia were EC positive. The counts obtained from the fresh produce were generally higher than the forest counts, but the distribution of biotypes was similar. Of the 146 isolates examined 64% were Klebsiella, 14% were Escherichia, 14% were Enterobacter, and 8% were Citrobacter. All the Enterobacter and Citrobacter were EC negative, whereas 25% of the Klebesiella and 80% of the Escherichia were EC positive.     

Impact of microbial tolerance in persistent secondary Klebsiella pneumoniae peritonitis

Purpose and methodsMicrobial tolerance represents a diminished pro-inflammatory response following repeated stimulation by a host of pathogen-associated molecular patterns (PAMPs) of varying origins. Toll-like receptors (TLRs) have been centrally implicated in the development of tolerance. The purpose of this study was to investigate the impact of tolerance in a previously described murine model.C57BL/6 mice were pretreated intraperitoneally with phosphate buffered saline (PBS), heat-killed Klebsiella 2 × 10⁸ CFU (hkKlebsiella), LPS 10 mg/kg (LPS 10), or BLP 10 mg/kg (BLP 10). Following pretreatment, peritonitis was induced 24 h later using 10³ intraperitoneal Klebsiella CFU. Peritoneal concentrations of TNF-α, IL-10 and nitric oxide (NO), as well as characteristic cell patterns, were determined. Long-term consequences of microbial tolerance were assessed by measuring survival and weight-loss.ResultsFollowing in vitro stimulation with Klebsiella 10⁵ and 10³ CFU, TNF-α and IL-10 secretion were diminished in macrophages harvested from mice pretreated with hkKlebsiella, LPS 10 and BLP 10. Pretreated animals had significantly lower bacterial counts. Conversely, local NO levels were elevated. Survival was not different between the groups.ConclusionPretreatment with TLR ligands induced microbial tolerance, with reduced peritoneal cytokine concentrations and enhanced early bacterial clearance. However, this did not translate into improved survival.     

Colonization of growing radish plants by clinical and nonclinical isolates ofKlebsiella inoculated onto seeds

Klebsiella was found to multiply and colonize growing radish root bulb surfaces following the inoculation of seeds with 10¹–10⁴ cells. All 29 cultures ofKlebsiella originally isolated from 5 different sources were capable of growth to 10⁶–10⁷ colony-forming units/g of root bulb within 1 week after seed germination. Linear regression analysis illustrated differences inKlebsiella survival rates over 4 weeks of radish plant development. Analysis of covariance showed the survival ability wasKlebsiella from vegetables>mastitis>human, water, and pulp mill isolates. It was also shown thatKlebsiella species 2 had a significantly higher survival rate than the otherKlebsiella species. This finding correlates well with the observation thatKlebsiella species 2 is the most commonKlebsiella species isolated from vegetables. Average densities for allKlebsiella groups at plant harvest (5 weeks) ranged from 10³–10⁵ colony-forming units/g of radish plant. The possible health significance of these densities ofKlebsiella on vegetables consumed raw by humans is discussed.     

Characterization of aerobic and facultative anaerobic bacteria from the liquid phase of an anaerobic fixed-bed digester treating a cheese whey substrate

Bacterial counts on the liquid phase of an anaerobic, fixed-bed digester, treating a deproteinated, prefermented cheese whey substrate, were conducted on two different media under aerobic and facultative conditions. Average counts of 16.6×10⁶ and 26.5×10⁶ ml^–1 were obtained on the two media, with the nutritionally poorer of the two media giving the highest average count. Seventy-five isolates from both media, incubated aerobically as well as in anaerobic jars, were obtained. These isolates as well as 13 reference strains were phenotypically characterized. The similarities between cultures were calculated using the similarity coefficient of Sokal and Michener [16]. The organisms were clustered using the unweighted pair group method, and the results presented as a simplified dendrogram. The isolates could be divided into 3 main groups: gram-negative fermentative rods, mainlyEnterobacter, Klebsiella, andCitrobacter, withKlebsiella as the predominant genus; gram-positive bacteria, mainly enterococci; and gram-negative nonfermentive rods of the generaPseudomonas, Alcaligenes, andAcinetobacter. All the enterobacteria and enterococci were able to produce acetic acid, an intermediate in methanogenesis.     

Link between genotype and antimicrobial resistance in bovine mastitis-related Staphylococcus aureus strains, determined by comparing Swiss and French isolates from the Rhône Valley

Staphylococcus aureus is a major bovine mastitis pathogen. Although the reported antimicrobial resistance was generally low, the emergence of new genetic clusters in bovine mastitis requires examination of the link between antimicrobial resistance and genotypes. Here, amplified fragment length polymorphism (AFLP) profiles and standard antimicrobial resistance profiles were determined in order to characterize a total of 343 S. aureus cow mastitis isolates from two geographically close regions of Switzerland and France. AFLP profiles revealed similar population compositions in the two regions, with 4 major clusters (C8, C20, C97, and C151), but the proportions of isolates in each cluster significantly diverged between the two countries (P = 9.2 × 10⁻⁹). Antimicrobial resistance was overall low (<5% resistance to all therapeutically relevant molecules), with the exception of penicillin resistance, which was detected in 26% of the isolates. Penicillin resistance proportions differed between clusters, with only 1 to 2% of resistance associated with C20 and C151 and up to 70% associated with bovine C97. The prevalence of C20 and C8 was unexpectedly high and requires further investigation into the mechanism of adaptation to the bovine host. The strong association of penicillin resistance with few clusters highlights the fact that the knowledge of local epidemiology is essential for rational choices of antimicrobial treatment in the absence of susceptibility testing. Taken together, these observations argue in favor of more routine scrutiny of antimicrobial resistance and antibiotic-resistant clones in cattle and the farm environment.     

Acetylene Reduction (Nitrogen Fixation) by Pulp and Paper Mill Effluents and by Klebsiella Isolated from Effluents and Environmental Situations

High rates of acetylene (C₂H₂) reduction (nitrogenase activity) were observed in woodroom effluent from a neutral sulfite semi-chemical mill under aerobic (up to 644 nmol of C₂H₄ produced per ml per h) and under anaerobic (up to 135 nmol of C₂H₄ produced per ml per h) conditions. Pasteurized effluent developed C₂H₂ reduction activity when incubated under anaerobic but not under aerobic conditions. Activities were increased by addition of 0.5 to 3.0% glucose or xylose. Enrichment and enumeration studies showed that N₂-fixing Azotobacter and Klebsiella were abundant, and N₂-fixing Bacillus was present. Of 129 isolates of Klebsiella from pulp mills, lakes, rivers, and drainage and sewage systems, 32% possessed nitrogen-fixing ability.     

A culture and biochemical assay system for Trypanosoma lewisi

Trypanosoma lewisi was cultivated as forms which appeared to be physiologically similar to those found in vivo. The medium consisted of 1.0 g peptone, 1.0 g glucose, 10 ml rat serum, 10,000 units penicillin G, 10,000 μg streptomycin and 90 ml Hank's Balanced Salt Solution. It was supplemented with 8.0 × 10⁸ rat erythrocytes per milliliter. In the complete medium trypanosomes multiplied for 48–72 hr. Cultured forms were lethal to newborn rats and infective to adults.Adsorbed early immune serum inhibited the growth of the trypanosomes in vitro and the percentage of reproductives declined from 66 to 45%. The cultured trypanosomes were also susceptible to both trypanocidal antibodies.     

Oral Administration of Lactobacillus Strains Isolated from Breast Milk as an Alternative for the Treatment of Infectious Mastitis during Lactation

In this study, 20 women with staphylococcal mastitis were randomly divided in two groups. Those in the probiotic group daily ingested 10 log₁₀ CFU of Lactobacillus salivarius CECT5713 and the same quantity of Lactobacillus gasseri CECT5714 for 4 weeks, while those in the control one only ingested the excipient. Both lactobacillus strains were originally isolated from breast milk. On day 0, the mean staphylococcal counts in the probiotic and control groups were similar (4.74 and 4.81 log₁₀ CFU/ml, respectively), but lactobacilli could not be detected. On day 30, the mean staphylococcal count in the probiotic group (2.96 log₁₀ CFU/ml) was lower than that of the control group (4.79 log₁₀ CFU/ml). L. salivarius CECT5713 and L. gasseri CECT5714 were isolated from the milk samples of 6 of the 10 women of the probiotic group. At day 14, no clinical signs of mastitis were observed in the women assigned to the probiotic group, but mastitis persisted throughout the study period in the control group women. In conclusion, L. salivarius CECT5713 and L. gasseri CECT5714 appear to be an efficient alternative for the treatment of lactational infectious mastitis during lactation.     

Promoting effects of organic medium supplements on the micropropagation of promising ornamental Daphne species (Thymelaeaceae)

Three Daphne species (Thymelaeaceae) were propagated in vitro using media enriched with natural ingredients including coconut water, pineapple pulp, arabinogalactan, chitosan, and conditioned medium containing exudates of the green alga Desmodesmus subspicatus. High vigor of proliferative shoots and enhanced rooting efficiency were obtained. The propagation rate for shoot cultures of Daphne caucasica and Daphne tangutica increased significantly when cultured in the presence of 10 ml?L^?1 coconut water or 10 ml?L^?1 pineapple pulp. Addition of 10 ml?L^?1 pineapple pulp, 10 ml?L^?1 coconut water, or 20% conditioned medium to the culture medium stimulated organogenesis in D. caucasica. The percentage of rooted shoots in this difficult-to-root species reached 80% in enriched medium. Daphne jasminea plants rooted efficiently on media without growth regulators but supplemented with 10 ml?L^?1 pineapple pulp or 10 ml?L^?1 coconut water. Plants of D. caucasica and D. jasminea were successfully acclimatized to greenhouse conditions. Biochemical evaluation of pineapple pulp using thin-layer chromatography revealed the absence of natural auxins. However, the low-molecular-weight fraction (<500 Da) obtained via dialysis significantly stimulated rhizogenesis in each species tested.     

Treatment with Gentamicin on a Murine Model of Protothecal Mastitis

The aim of this study was to establish a murine protothecal mastitis model and to evaluate the treatment efficiency of gentamicin. Challenge routes were determined with a pathogenic Prototheca zopfii genotype 2 (P. zopfii) strain. 25 BALB/c mice were inoculated in mammary glands with graded dosages (10³, 10⁴, 10⁵, 10⁶, 10⁷ CFU of P. zopfii) and killed on the 7th day. Another 25 animals were also killed at 1, 3, 5, 7, and 9 days after inoculation of 1 × 10⁶ CFU of P. zopfii, the milk somatic cell counts, pathological section of mammary glands, and P. zopfii burden were observed. The antimicrobial activity was tested using disc diffusion test and minimum inhibitory concentrations. Gentamicin was given intramuscularly to analyze the therapeutic effect. The results showed that the best infection route was intra-mammary gland, and the mastitis model was established with 1 × 10⁶ CFU of P. zopfii. After infection, the somatic cell counts increased significantly. The pathological reaction mainly consisted of infiltration of inflammatory cells, destruction of acini, accumulation of lymphocyte cells and the severity of the changes was dosage and time-dependent. The P. zopfii burden revealed that P. zopfii continuously replicated. In vitro susceptibility tests indicated that the Prototheca strains were antimicrobial susceptible to gentamicin at concentrations between 0.03 and 4 μg/ml. In vivo therapeutic assay demonstrated that high concentrations of gentamicin (≥20 mg/kg) could inhibit the growth of P. zopfii. We conclude that the murine model of protothecal mastitis was established successfully and gentamicin may be an effective choice for treatment of P. zopfii.     

Genomic Comparative Study of Bovine Mastitis Escherichia coli

Escherichia coli, one of the main causative agents of bovine mastitis, is responsible for significant losses on dairy farms. In order to better understand the pathogenicity of E. coli mastitis, an accurate characterization of E. coli strains isolated from mastitis cases is required. By using phylogenetic analyses and whole genome comparison of 5 currently available mastitis E. coli genome sequences, we searched for genotypic traits specific for mastitis isolates. Our data confirm that there is a bias in the distribution of mastitis isolates in the different phylogenetic groups of the E. coli species, with the majority of strains belonging to phylogenetic groups A and B1. An interesting feature is that clustering of strains based on their accessory genome is very similar to that obtained using the core genome. This finding illustrates the fact that phenotypic properties of strains from different phylogroups are likely to be different. As a consequence, it is possible that different strategies could be used by mastitis isolates of different phylogroups to trigger mastitis. Our results indicate that mastitis E. coli isolates analyzed in this study carry very few of the virulence genes described in other pathogenic E. coli strains. A more detailed analysis of the presence/absence of genes involved in LPS synthesis, iron acquisition and type 6 secretion systems did not uncover specific properties of mastitis isolates. Altogether, these results indicate that mastitis E. coli isolates are rather characterized by a lack of bona fide currently described virulence genes.     

Molecular and Phenotypic Characterization of Aerococcus viridans Associated with Subclinical Bovine Mastitis

Aerococcus viridans is a wide spread bacterium in the environment and clinically this organism is associated with different diseases in animals and humans. However, the geno- and phenotypic characterization of A. viridans associated with bovine mastitis has not yet been reported. The objectives of this study were to investigate the genetic and phenotypic diversity of A. viridans isolates using three different molecular methods including 16S rRNA gene sequencing, pulsed-field gel electrophoresis and random amplified polymorphic DNA (RAPD) along with biochemical tests, including antimicrobial susceptibility test. In total, 60 A. viridans strains were cultured from dairy herds presenting with subclinical mastitis. The results of biochemical tests revealed that most of the isolates (75.0%) were accurately identified by API Rapid 20 Strep system and the majority of A. viridans strains (96.7%) were found to be catalase negative, while two (3.3%) isolates were weakly positive. All isolates were resistant to trimethoprim-sulfamethoxazole, followed by streptomycin (96.7%), tetracycline (65.0%) and clindamycin (56.7%) by minimum inhibition concentration-determining broth microdilution technique. As compared to the sequence of 16S rRNA gene, both PFGE and RAPD showed their capacities to discriminate the intra-species diversity of A. viridans. Furthermore, most of the isolates obtained from the same herd or region belonged to the same major RAPD group, which indicated that RAPD is an appropriate assay for tracking the origins of isolates and epidemiological studies of A. viridans. This is a novel approach to use three molecular techniques and to compare their efficiency regarding the genetic diversity of A. viridans. The data suggest that A. viridans associated with subclinical mastitis has a considerable phenotypic and genotypic diversity.     

Microbial Diversity of Bovine Mastitic Milk as Described by Pyrosequencing of Metagenomic 16s rDNA

Dairy cow mastitis is an important disease in the dairy industry. Different microbial species have been identified as causative agents in mastitis, and are traditionally diagnosed by bacterial culture. The objective of this study was to use metagenomic pyrosequencing of bacterial 16S rRNA genes to investigate bacterial DNA diversity in milk samples of mastitic and healthy dairy cows and compare the results with those obtained by classical bacterial culture. One hundred and thirty-six milk samples were collected from cows showing signs of mastitis and used for microbiological culture. Additionally, 20 milk samples were collected from healthy quarters. Bacterial DNA was isolated from the same milk samples and the 16S rRNA genes were individually amplified and pyrosequenced. Discriminant analysis showed that the groups of samples that were most clearly different from the rest and thus easily discriminated were the normal milk samples from healthy cows and those characterised by culture as Trueperella pyogenes and Streptococcus spp. The mastitis pathogens identified by culture were generally among the most frequent organisms detected by pyrosequencing, and in some cases (Escherichia coli, Klebsiella spp. and Streptococcus uberis mastitis) the single most prevalent microorganism. Trueperella pyogenes sequences were the second most prevalent sequences in mastitis cases diagnosed as Trueperella pyogenes by culture, Streptococcus dysgalactiae sequences were the second most prevalent sequences in mastitis cases diagnosed as Streptococcus dysgalactiae by culture, and Staphyloccocus aureus sequences were the third most prevalent in mastitis cases diagnosed as Staphylococcus aureus by culture. In samples that were aerobic culture negative, pyrosequencing identified DNA of bacteria that are known to cause mastitis, DNA of bacteria that are known pathogens but have so far not been associated with mastitis, and DNA of bacteria that are currently not known to be pathogens. A possible role of anaerobic pathogens in bovine mastitis is also suggested.     

INSULIN PRODUCES A BIPHASIC RESPONSE INTETRAHYMENA THERMOPHILABY STIMULATING CELL SURVIVAL AND ACTIVATING PROLIFERATION IN TWO SEPARATE CONCENTRATION INTERVALS

Cells ofTetrahymenamay produce autocrine signal molecules with effects on survival and proliferation. Here we have tested the effects of human recombinant and bovine insulin, and the B22–B30 fragment of bovine insulin over a wide range of concentrations (10^?5–10^?18m ) on cell survival and proliferation in a synthetic nutrient medium. The cells were grown in conical flasks at low initial cell densities (40 and 400cells/ml). Insulin prevented rapid cell death and/or promoted cell proliferation over two separate concentration ranges: down to nanomolar levels and again in the low pico- and femtomolar range. At an initial population density of 400cells/ml the cells multiplied at both concentration intervals. At 40 or fewer organisms/ml the cells multiplied in the high concentration interval, whereas in the low interval they survived for about four times longer than those in the control cultures. B22–B30 added to cultures of 40 initial cells/ml produced a stimulation of cell survival in the low pico- and high femtomolar range. In the presence of hemin (50nm ) cells at 400 initial organisms/ml multiplied at insulin concentrations down to about 3nm and again from 300am to 10pm . In some cases, hemin plus insulin activated cell proliferation between the two concentration intervals as well. At 40cells/ml the cells not only survived but proliferated in the femtomolar range. Cells in cultures supplemented with both hemin and B22–B30 multiplied at the low concentration interval (from about 100fm to 10pm ).     

Characterization of Staphylococcus aureus isolates recovered from dairy sheep farms (agr group,adherence, slime,resistance to antibiotics)

Staphylococcus aureus is one of the major causes of dairy sheep mastitis. The S. aureus agr locus (accessory gene regulator) regulates the production of most staphylococcal exoproteins, including exoenzymes, toxins, surface proteins, and other virulence factors. S. aureus have four agr groups (alleles) determined by PCR. In this study, 46 S. aureus isolates, recovered in south-east of France, were also characterized by their properties of adherence to smooth surfaces, slime production and resistance to 10 antibiotics. For 46 S. aureus associated with dairy sheep mastitis (subclinical mastitis, clinical mastitis, environment of dairy sheep farm), 80% (37/46) belonged to agr group 3, 39% (18/46) were adherent (adherent, strongly adherent or with maximal adherence). For the same isolates, 26% (12/46) were slime producers (moderate or strong producers). All the 46 isolates were susceptible to oxacillin, except for two isolates including two sheep subclinical mastitis isolates. The dairy sheep subclinical mastitis isolates were for 79% (22/28), susceptible to nine other antibiotics tested.     

Genetic Variation among Staphylococcus aureus Strains from Norwegian Bulk Milk

Strains of Staphylococcus aureus obtained from bovine (n = 117) and caprine (n = 114) bulk milk were characterized and compared with S. aureus strains from raw-milk products (n = 27), bovine mastitis specimens (n = 9), and human blood cultures (n = 39). All isolates were typed by pulsed-field gel electrophoresis (PFGE). In addition, subsets of isolates were characterized using multilocus sequence typing (MLST), multiplex PCR (m-PCR) for genes encoding nine of the staphylococcal enterotoxins (SE), and the cloverleaf method for penicillin resistance. A variety of genotypes were observed, and greater genetic diversity was found among bovine than caprine bulk milk isolates. Certain genotypes, with a wide geographic distribution, were common to bovine and caprine bulk milk and may represent ruminant-specialized S. aureus. Isolates with genotypes indistinguishable from those of strains from ruminant mastitis were frequently found in bulk milk, and strains with genotypes indistinguishable from those from bulk milk were observed in raw-milk products. This indicates that S. aureus from infected udders may contaminate bulk milk and, subsequently, raw-milk products. Human blood culture isolates were diverse and differed from isolates from other sources. Genotyping by PFGE, MLST, and m-PCR for SE genes largely corresponded. In general, isolates with indistinguishable PFGE banding patterns had the same SE gene profile and isolates with identical SE gene profiles were placed together in PFGE clusters. Phylogenetic analyses agreed with the division of MLST sequence types into clonal complexes, and isolates within the same clonal complex had the same SE gene profile. Furthermore, isolates within PFGE clusters generally belonged to the same clonal complex.     

Microbiota of Cow’s Milk; Distinguishing Healthy,Sub-Clinically and Clinically Diseased Quarters

The objective of this study was to use pyrosequencing of the 16S rRNA genes to describe the microbial diversity of bovine milk samples derived from clinically unaffected quarters across a range of somatic cell counts (SCC) values or from clinical mastitis, culture negative quarters. The obtained microbiota profiles were used to distinguish healthy, subclinically and clinically affected quarters. Two dairy farms were used for the collection of milk samples. A total of 177 samples were used. Fifty samples derived from healthy, culture negative quarters with a SCC of less than 20,000 cells/ml (group 1); 34 samples derived from healthy, culture negative quarters, with a SCC ranging from 21,000 to 50,000 cells/ml (group 2); 26 samples derived from healthy, culture negative quarters with a SCC greater than 50,000 cells/ml (group 3); 34 samples derived from healthy, culture positive quarters, with a SCC greater than 400,000 (group 4, subclinical); and 33 samples derived from clinical mastitis, culture negative quarters (group 5, clinical). Bacterial DNA was isolated from these samples and the 16S rRNA genes were individually amplified and pyrosequenced. All samples analyzed revealed great microbial diversity. Four bacterial genera were present in every sample obtained from healthy quarters (Faecalibacterium spp., unclassified Lachnospiraceae, Propionibacterium spp. and Aeribacillus spp.). Discriminant analysis models showed that samples derived from healthy quarters were easily discriminated based on their microbiota profiles from samples derived from clinical mastitis, culture negative quarters; that was also the case for samples obtained from different farms. Staphylococcus spp. and Streptococcus spp. were among the most prevalent genera in all groups while a general multivariable linear model revealed that Sphingobacterium and Streptococcus prevalences were associated with increased 10 log SCC. Conversely, Nocardiodes and Paenibacillus were negatively correlated, and a higher percentage of the genera was associated with a lower 10 log SCC.