Milk Isolates Research Articles

Detection of presumptive Bacillus cereus in the Irish dairy farm environment

AbstractThe objective of the study was to isolate potentialBacillus cereussensu lato (B.cereus s.l.)from a range of farm environments. Samples of tap water, milking equipment rinse water, milk sediment filter, grass, soil and bulk tank milk were collected from 63 farms. In addition, milk liners were swabbed at the start and the end of milking, and swabs were taken from cows’ teats prior to milking. The samples were plated on mannitol egg yolk polymyxin agar (MYP) and presumptiveB. cereus s.l. colonies were isolated and stored in nutrient broth with 20% glycerol and frozen at -80 °C. These isolates were then plated on chromogenic medium (BACARA) and colonies identified as presumptiveB. cereus s.l. on this medium were subjected to 16S ribosomal RNA (rRNA) sequencing. Of the 507 isolates presumed to beB. cereus s.l. on the basis of growth on MYP, only 177 showed growth typical ofB. cereus s.l. on BACARA agar. The use of 16S rRNA sequencing to identify isolates that grew on BACARA confirmed that the majority of isolates belonged toB. cereus s.l. A total of 81 of the 98 isolates sequenced were tentatively identified as presumptiveB. cereus s.l. Pulsed-field gel electrophoresis was carried out on milk and soil isolates from seven farms that were identified as having presumptiveB. cereus s.l. No pulsotype was shared by isolates from soil and milk on the same farm. PresumptiveB. cereus s.l. was widely distributed within the dairy farm environment.

Occurrence of methicillin-resistant Staphylococcus aureus in dairy cattle herds, related swine farms, and humans in contact with herds

In this study we investigated the circulation of methicillin-resistant Staphylococcus aureus (MRSA) in 2 dairy cattle farms (farm A and B), previously identified as MRSA-positive in bulk tank milk samples, and epidemiologically related to swine farms. Collected specimens included quarter milk samples and nasal swabs from dairy cows, pig nasal swabs collected at both the farm and slaughterhouse level, environmental dust samples, and human nasal swabs from the farms’ owners and workers. The prevalence of MRSA was estimated at the herd level by testing quarter milk samples. The prevalence of MRSA was 4.8% (3/63; 95% confidence interval=0–10.2%) and 60% (33/55; 95% confidence interval=47.05–72.95) in farm A and B, respectively. In farm A, MRSA was also isolated from humans, pigs sampled at both farm and slaughterhouse level, and from environmental samples collected at the pig facilities. The dairy cattle facilities of farm A tested negative for MRSA. In farm B, MRSA was isolated from environmental dust samples in both the cattle and pig facilities, whereas nasal swabs collected from cows and from humans tested negative. Sixty-three selected MRSA isolates obtained from different sources in farm A and B were genetically characterized by multilocus sequence typing, spa-typing, ribosomal spacer-PCR, and also tested for the presence of specific virulence genes and for their phenotypical antimicrobial susceptibility by broth microdilution method. Different clonal complex (CC) and spa-types were identified, including CC398, CC97, and CC1, CC already reported in livestock animals in Italy. The MRSA isolates from quarter milk of farm A and B mostly belonged to CC97 and CC398, respectively. Both lineages were also identified in humans in farm A. The CC97 and CC398 quarter milk isolates were also identified as genotype GTBE and GTAF by ribosomal spacer-PCR respectively, belonging to distinct clusters with specific virulence and resistance patterns. The GTBE and GTAF clusters also included swine, environmental, and human isolates from both farms. A high heterogeneity in the genetic and phenotypic profiles was observed in environmental isolates, in particular from farm B. These results demonstrate the possibility of a dynamic sharing and exchange of MRSA lineages or genotypes between different species and farm compartments in mixed-species farms. The risk of transmission between swine and related dairy cattle herds should be considered. Our findings also confirm the zoonotic potential of livestock-associated MRSA and underline the importance of applying biosecurity measures and good hygiene practices to prevent MRSA spread at the farm level and throughout the food production chain.

Behavior of artificial listerial contamination in model Greek Graviera cheeses manufactured with the indigenous nisin A‐producing strain Lactococcus lactis subsp. cremoris M104 as costarter culture

AbstractGrowth of Listeria monocytogenes during processing of traditional Greek Graviera cheese is inhibited, but the pathogen may survive ripening. Therefore, this study used Lactococcus lactis subsp. cremoris M104, a nisin A‐producing (NisA+) raw milk isolate, to enhance inactivation of a nonpathogenic Listeria cocktail contaminated in model Graviera mini cheeses. Cheeses were manufactured from thermized milk with a commercial starter culture (CSC) or the CSC plus strain M104 (CSC + M104), ripened at 18 °C and 90% RH for 20 days and stored at 4 °C in vacuum for 60 days. Listeria populations declined 10‐fold in all fermenting cheeses but then survived with little death during ripening and storage. NisA+ M104 colonies were prevalent and the nisA gene was detected, but nisin activity was weak to undetectable, in all CSC + M104 cheeses. Thus, Graviera cheese processing should be optimized to ensure nisA gene expression by strain M104 at levels sufficient to increase inactivation of L. monocytogenes.Practical applicationsThis study provided important preliminary evidence that the in situ NisA+ antilisterial activity of L. lactis subsp. cremoris M104 during Graviera cheese fermentation increased by reducing the curd cooking temperature from 48 °C to 42 °C. This indicated the feasibility to increase nisin production and activity by suitably modifying selected technological factors during traditional cooked hard cheese processing. Apart from its bioprotective potential, this NisA+ L. lactis subsp. cremoris genotype (represented by the indigenous strains M104 and M78) possesses desirable sugar fermentation capabilities and enzymatic activities similar to those of the best performing industrial L. lactis or L. cremoris strains in commercial starters (Parapouli et al., ). Since 2015 this novel lactococcal genotype has been applied as hand‐made, costarter culture for the production of commercial Greek Graviera cheeses characterized by specific flavor characteristics and improved textural properties than counterpart cheeses produced with CSCs only (Pappas Bros. collaborating cheese plant, Epirus; private communication).

Molecular Characterization of Corynebacterium bovis causing Clinical Mastitis and Increasing Somatic-Cell Count

Bovine mastitis remains the most economically important disease affecting dairy cows. Bacteria of the genus Corynebacterium spp., especially Corynebacterium bovis ( C. bovis ), are frequently isolated from bovine mastitis although there are divergent reports about the role of this pathogen in bovine mastitis. In the present study we report the presence of new C. bovis strains from milk exhibiting increased SCC of 12 cows with subclinical and clinical mastitis. Irregular Gram-positive bacilli isolated in pure cultures underwent conventional biochemical tests which generated indefinite phenotypic profiles ( Corynebacterium mastitidis/C. bovis ). API-Coryne System 3.0 identified all the milk isolates as C. bovis (code number 4501014; 99.9% confidence level). Phylogenetic analysis based on 16S rRNA and rpo B sequences confirmed the identification of the strains as C. bovis .

Characterization of Bacillus cereus isolates from local dairy farms in China.

Bacillus cereus is an important opportunistic foodborne pathogen. In the present work, a total of 306 milk and environmental samples were collected from 10 local dairy farms in Beijing, China. Of the 92 B. cereus-like isolates, 88 and 4 belonged to B. cereus and B. thuringiensis, respectively. The prevalence of B. cereus isolates in bedding, feces, feed, liquid manure and raw milk was 93.3%, 78.9%, 41.2%, 100.0% and 9.8%, respectively. Three main toxin genes nhe, hbl and ces were detected with rates of 100.0%, 78.3% and 1.1%, but no strain harbored cytK1 The production of Nhe, Hbl and cereulide could be confirmed by specific monoclonal antibodies-based enzyme immunoassays in 94.6%, 70.7% and 1.1% of all isolates, respectively. Cytotoxicity tests were used to further corroborate the results of genetic and protein-based assays; 91.3% of the isolates showed cytotoxicity to Vero cells. All isolates were tested for antimicrobial resistance against 17 antibiotics. All isolates were resistant to lincomycin, retapamulin, tiamulin and valnemulin, while two strains were susceptible to ampicillin and ceftiofur. A total of 16 isolated strains were resistant to tetracycline. Since spores of B. cereus are not inactivated during manufacturing of most milk products, contamination of milk with B. cereus on the farm level may represent a potential hazard, particularly with respect to emetic toxin-producing strains.

In vitro selection, characterization and cytotoxic effect of bacteriocin of Lactobacillus sakei GM3 isolated from goat milk

Goat milk isolate Lactobacillus sakei GM3 exhibited acid and bile tolerance and showed antagonistic activity against food borne pathogens. Bacteriocin produced by L. sakei GM3 reached maximum production at stationary phase after 18 h. Bacteriocin was found to be retained active after treatment with different pH, temperature and organic solvents but showed sensitivity to some proteolytic enzymes. The molecular mass was found to be 4.8 kDa after subjecting to salting out, size exclusion chromatography, reversed-phase HPLC on C18 column and MALDI-TOF-MS system. The cytotoxicity on HT29 cell line by bacteriocin GM3 showed maximum survival inhibition (45.60 ± 0.5%) at 2240 AU/ml concentration. The results suggest that the L. sakei GM3 has the potential to be used in food industry as a biopreservative.

Identification and characterization of a heat-resistant protease from Serratia liquefaciens isolated from Brazilian cold raw milk

The cold storage of raw milk before heat treatment in dairy industry promotes the growth of psychrotrophic microorganisms, which are known for their ability to produce heat-resistant proteolytic enzymes. Although Pseudomonas is described as the main causative genus for high proteolytic spoilage potential in dairy products, Serratia liquefaciens secretes proteases and may be found in raw milk samples as well. However, at the present there is no information about the proteolytic spoilage potential of S. liquefaciens in milk after heat-treatment. The main aim of this research was to assess the proteolytic spoilage potential of S. liquefaciens isolated from Brazilian raw milk and to characterize the involved protease. S. liquefaciens was shown to secrete one heat-resistant spoilage metalloprotease of, approximately, 52kDa encoded by the ser2 gene. The heat-resistance of Ser2 was similar to the aprX encoded metalloprotease produced by Pseudomonas. Although the ser2 gene was detected in all S. liquefaciens isolates tested in this study, the proteolytic activity of the isolates in milk was highly heterogeneous. Since nucleotide and deduced amino acid sequences of ser2 of all tested isolates are identical, this heterogeneity may be attributed to differences in enzyme expression levels or post-translational modifications.

Occurrence and antibiotic resistance of enterotoxigenic Staphylococcus aureus in raw ovine and caprine milk in Greece

Ovine (n = 140) and caprine (n = 35) raw bulk tank milk samples from farms in central Greece were examined for the occurrence of enterotoxigenic Staphylococcus aureus. The S. aureus isolates were screened for staphylococcal enterotoxin (SE) production, the presence of enterotoxin genes, antibiotic resistance (AR), and methicillin resistance. S. aureus was isolated from 24.3% and 31.4% of ovine and caprine milk samples, respectively. Among the S. aureus isolates of ovine milk (n = 34) and caprine (n = 11) milk, the enterotoxigenic (SEA-SED) isolates were 21 (61.8%) and 7 (63.6%) for the ovine and caprine milk, respectively. Most toxigenic isolates harbored more than one toxin gene and a total of 11 distinct toxinotypes were detected. The most frequent toxin-gene combinations were “sec, tst” (8 isolates), “seb, seg, sei, tst” (4), “seb, seg, sei” (3), and “seb” (3). Six isolates displayed multiple AR towards up to five antimicrobials. Among ovine milk isolates, the highest resistance frequency was observed towards erythromycin (11.8% of the isolates) and tetracycline (8.8%). Among caprine milk isolates, the most frequent resistance was observed towards erythromycin (18.2%). One methicillin-resistant S. aureus (MRSA) isolate was detected in an ovine milk sample and belonged to spa type t4038. This spa type has been isolated for the first time in Greece and, to our knowledge, has not been previously reported among MRSA isolates from raw milk or dairy products worldwide.

Pseudomonas aeruginosa in Dairy Goats: Genotypic and Phenotypic Comparison of Intramammary and Environmental Isolates

Following the identification of a case of severe clinical mastitis in a Saanen dairy goat (goat A), an average of 26 lactating goats in the herd was monitored over a period of 11 months. Milk microbiological analysis revealed the presence of Pseudomonas aeruginosa in 7 of the goats. Among these 7 does, only goat A showed clinical signs of mastitis. The 7 P. aeruginosa isolates from the goat milk and 26 P. aeruginosa isolates from environmental samples were clustered by RAPD-PCR and PFGE analyses in 3 genotypes (G1, G2, G3) and 4 clusters (A, B, C, D), respectively. PFGE clusters A and B correlated with the G1 genotype and included the 7 milk isolates. Although it was not possible to identify the infection source, these results strongly suggest a spreading of the infection from goat A. Clusters C and D overlapped with genotypes G2 and G3, respectively, and included only environmental isolates. The outcome of the antimicrobial susceptibility test performed on the isolates revealed 2 main patterns of multiple resistance to beta-lactam antibiotics and macrolides. Virulence related phenotypes were analyzed, such as swarming and swimming motility, production of biofilm and production of secreted virulence factors. The isolates had distinct phenotypic profiles, corresponding to genotypes G1, G2 and G3. Overall, correlation analysis showed a strong correlation between sampling source, RAPD genotype, PFGE clusters, and phenotypic clusters. The comparison of the levels of virulence related phenotypes did not indicate a higher pathogenic potential in the milk isolates as compared to the environmental isolates.

Coagulase‐Negative Staphylococci Collected from Bovine Milk: Species and Antimicrobial Gene Diversity

AbstractThe aim of this study was to examine the genetically mediated antimicrobial resistance in 94 coagulase‐negative staphylococcal (CNS) milk isolates (buffalo, n = 88, and cow, n = 6), and to determine whether antimicrobial resistance profiles differed between bacterial species. Our analysis of 94 CNS isolates from milk confirmed the well‐established multiresistant character of staphylococci in the dairy setting. Resistance against oxacillin, ciprofloxacin and cefoxitin was most frequently observed. Eleven CNS species isolated from buffalo's and cow's milk samples were 100% sensitive to gentamicin, erythromycin, clindamycin and ciprofloxacin. Resistance to oxacillin was attributed to the mecA gene in 44.7% of the oxacillin‐resistant isolates. The mecA gene was detected in Staphylococcus intermedius, epidermidis, hominis, hyicus, caprae, sciuri, lugdunensis and xylosus while totally absent in chromogenes, simulans and lentus. Of the 11 CNS species, S. epidermidis, S. lugdunensis, S. hominis, S. xylosus and S. intermedius were the only species that exhibited multiple resistance.Practical ApplicationsStudying coagulase‐negative staphylococci (CNS) at the species level can provide valuable information about species‐specific differences that can be vital data to prevent the dissemination of antimicrobial‐resistant genes and resistant pathogens to the community. CNS have long been regarded as pathogenic, but their important role as colonizers of the bovine udder has been recognized and studied in recent years. CNS have become increasingly resistant to multiple antibiotics. In recent years, increasing numbers of reports have shown that the mecA gene is present in CNS strains, including hospital‐acquired infections, community‐associated methicillin‐resistant Staphylococcus aureus infections, animal epidermis, beaches and public transportation systems. Therefore, it is highly important to detect the mecA gene as a marker to investigate antibiotic resistance in milk that may represent a serious health and economic concern.

Quantification of the proteolytic and lipolytic activity of microorganisms isolated from raw milk

Heat-resistant enzymes of psychrotolerant microorganisms are associated with the spoilage of UHT-milk and milk products. In this study, we investigated the extracellular peptidase, esterase and lipase activity after submerged cultivation at 6 °C of 231 recently isolated microorganisms in milk medium. In contrast to the widely used agar diffusion tests for secretion of hydrolases, here a more realistic liquid screening approach was used. The advantages of the latter are the possibility of quantifying the enzyme activity in volumetric units and the opportunity to simulate the growth conditions of the microorganisms to the storage conditions of raw milk. The majority of enzymatically active isolates belonged to Gram-negative bacteria, especially the genus Pseudomonas. Surprisingly, among them, twelve novel Pseudomonas species were discovered. In this study, we demonstrated that numerous raw milk isolates, including bacteria and yeasts, produce extracellular enzymes that may cause spoilage problems for the dairy industry.

Optimization of Lactobacillus brevis NS01 Brevicin Production and Its Application in Apple Juice Biopreservation Using Food-Grade Clarifying Agent Silica as a Carrier

Bacteriocins are nowadays recognized as good biopreservatives that are able to contain microbial contaminant development in several food products. In this study, the goat milk isolate Lactobacillus brevis NS01 brevicin activity on common foodborne pathogenic organisms and its characterization, stability, and statistical response surface methodology (RSM) optimization of media components for enhanced production were determined. Its adsorption onto common stabilizing agent silica and applicability in apple juice preservation were also determined. The L. brevis NS01 produced 3.2 kDa common foodborne pathogen inhibitory brevicin having more polar (68.6 %) than non-polar (30.8 %) amino acids. Optimization of growth conditions by RSM demonstrated that glucose, sodium acetate, and triammonium sulfate concentration of 33.41, 8.36, and 2.22 g/L, respectively, increased its production. Brevicin activity was constant over a broad range of pH (3–8) and temperature (20–100 °C) and reached a maximum at pH 6 and 40 °C. Maximum adsorption of brevicin onto silica took place at pH 6–7, 40 °C for 240 min. The carrier silica exhibited significant Fourier transform infrared (FTIR) spectral changes upon brevicin adsorption and presented good biopreservative activity. This study demonstrates the potential large-scale use of brevicin adsorbed silica for shelf life increase and clarification at a same time.

SCREENING OF LACTIC ACID BACTERIA FROM SUDANESE FERMENTED FOODS FOR BACTERIOCIN PRODUCTION

Forty isolates of lactic acid bacteria (LAB) were isolated from different types of fermented foods consumed in Sudan. Phenotypic tests revealed that all isolates were homofermentative LAB. Twenty-four isolates produced inhibitory substances primarily active against Staphylococcus aureus ATCC 2818 and Escherichia coli ATCC 29522. The inhibitory activity of 88% of enterococci and 58% of lactobacilli was recorded from meat isolates, whereas all activity of pediococcal isolates came from fermented milk isolates. The cell-free cultures of 18 isolates exhibiting inhibitory activity was chosen for further investigation such as sensitivity to proteolytic enzyme (pepsin), effect of heat treatment (60°C for 60min, 100°C for 20min and 121°C for 15min) and effect of pH ( pH 2.0, pH 6.5, and pH 9.0). The inhibitory activity was eliminated upon treatment with pepsin. The bacteriocin-like substances lost their activity after heating at all temperatures used and at alkaline pH (9.0), whereas they were active at acidic pH (2.0). The antimicrobial activity of bacteriocin-like substances produced by the isolated LAB could prevent spoilage and/or pathogenic microorganism in Sudanese fermented food. Further study should be related with species identification of the producer strains and with the purification and characterization of these becteriocin-like substances in order to explore them in food industry.

Lactococcus hircilactis sp. nov. and Lactococcus laudensis sp. nov., isolated from milk.

Two strains of lactic acid bacteria, designated 117(T) and 4195(T), were isolated from goat milk in Valtellina, Italy and from cow milk in Valle Trompia, Italy, respectively, and characterized taxonomically by a polyphasic approach. The strains were Gram-stain-positive, coccoid, non-spore-forming and catalase-negative bacteria. Morphological, physiological and phylogenetic data indicated that these isolates belonged to the genus Lactococcus. Strain 117(T) was closely related to Lactococcus fujiensis, Lactococcus lactis subsp. lactis, L. lactis subsp. cremoris, L. lactis subsp. hordniae, L. lactis subsp. tructae and Lactococcus taiwanensis, showing 93-94% and 82-89% 16S rRNA and rpoB gene sequence similarities, respectively. Strain 4195(T) was closely related to Lactococcus chungangensis, Lactococcus raffinolactis, Lactococcus plantarum and Lactococcus piscium, showing 92-98% and 86-99% 16S rRNA and rpoB gene sequence similarities, respectively. Based on this evidence and the data obtained in the present study, the milk isolates represent two novel species of the genus Lactococcus, for which the names Lactococcushircilactis sp. nov., and Lactococcuslaudensis sp. nov. are proposed. The respective type strains are 117(T) ( = LMG 28352(T) = DSM 28960(T)) and 4195(T )( = LMG 28353(T) = DSM 28961(T)).

Isolation and molecular characterization of Brucella isolates in cattle milk in Uganda.

Brucellosis is endemic in livestock and humans in Uganda and its transmission involves a multitude of risk factors like consumption of milk from infected cattle. To shed new light on the epidemiology of brucellosis in Uganda the present study used phenotypic and molecular approaches to delineate the Brucella species, biovars, and genotypes shed in cattle milk. Brucella abortus without a biovar designation was isolated from eleven out of 207 milk samples from cattle in Uganda. These isolates had a genomic monomorphism at 16 variable number tandem repeat (VNTR) loci and showed in turn high levels of genetic variation when compared with other African strains or other B. abortus biovars from other parts of the world. This study further highlights the usefulness of MLVA as an epidemiological tool for investigation of Brucella infections.

Genetic relatedness and virulence factors of bovine Staphylococcus aureus isolated from teat skin and milk

The objective of this study was to assess the role of teat skin colonization in Staphylococcus aureus intramammary infections (IMI) by evaluating genetic relatedness of Staph. aureus isolates from milk and teat skin of dairy cows using pulsed-field gel electrophoresis and characterizing the isolates based on the carriage of virulence genes. Cows in 4 known Staph. aureus-positive herds were sampled and Staph. aureus was detected in 43 quarters of 20 cows, with 10 quarters positive in both milk and skin (20 isolates), 18 positive only in milk, and 15 only on teat skin. Quarters with teat skin colonized with Staph. aureus were 4.5 times more likely to be diagnosed with Staph. aureus IMI than quarters not colonized on teat skin. Three main clusters were identified by pulsed-field gel electrophoresis using a cutoff of 80% similarity. All 3 clusters included both milk and skin isolates. The majority of isolates (72%) belonged to one predominant cluster (B), with 60% of isolates in the cluster originating from milk and 40% from teat skin. Genotypic variability was observed within 10 pairs (formed by isolates originating from milk and teat skin of the same quarter), where isolates in 5 out of the 10 pairs belonged to the same cluster. Forty-two virulence factors were screened using PCR. Some virulence factors were carried more frequently by teat skin isolates than by milk isolates or isolates from quarters with high somatic cell counts. Isolates in the predominant cluster B carried virulence factors clfA and clfB significantly more often than isolates in the minor clusters, which may have assisted them in becoming predominant in the herds. The present findings suggest that teat skin colonization with Staph. aureus can be an important factor involved in Staph. aureus IMI.

Addition to Thermized Milk of Lactococcus lactis subsp. cremoris M104, a Wild, Novel Nisin A–Producing Strain, Replaces the Natural Antilisterial Activity of the Autochthonous Raw Milk Microbiota Reduced by Thermization

Recent research has shown that mild milk thermization treatments routinely used in traditional Greek cheese production are efficient to inactivate Listeria monocytogenes and other pathogenic or undesirable bacteria, but they also inactivate a great part of the autochthonous antagonistic microbiota of raw milk. Therefore, in this study, the antilisterial activity of raw or thermized (63°C, 30 s) milk in the presence or absence of Lactococcus lactis subsp. cremoris M104, a wild, novel, nisin A-producing (Nis-A+) raw milk isolate, was assessed. Bulk milk samples were taken from a local cheese plant before or after thermization and were inoculated with a five-strain cocktail of L. monocytogenes (approximately 4 log CFU/ml) or with the cocktail, as above, plus the Nis-A+ strain (approximately 6 log CFU/ml) as a bioprotective culture. Heat-sterilized (121°C, 5 min) raw milk inoculated with L. monocytogenes was used as a control treatment. All milk samples were incubated at 37°C for 6 h and then at 18°C for an additional 66 h. L. monocytogenes grew abundantly (>8 log CFU/ml) in heat-sterilized milk, whereas its growth was completely inhibited in all raw milk samples. Conversely, in thermized milk, L. monocytogenes increased by 2 log CFU/ml in the absence of strain M104, whereas its growth was completely inhibited in the presence of strain M104. Furthermore, nisin activity was detected only in milk samples inoculated with strain M104. Thus, postthermal supplementation of thermized bulk milk with bioprotective L. lactis subsp. cremoris cultures replaces the natural antilisterial activity of raw milk reduced by thermization.

Cyclic lipodepsipeptides produced by Pseudomonas spp. naturally present in raw milk induce inhibitory effects on microbiological inhibitor assays for antibiotic residue screening.

Two Pseudomonas strains, identified as closely related to Pseudomonas tolaasii, were isolated from milk of a farm with frequent false-positive Delvotest results for screening putative antibiotic residues in raw milk executed as part of the regulatory quality programme. Growth at 5 to 7°C of these isolates in milk resulted in high lipolysis and the production of bacterial inhibitors. The two main bacterial inhibitors have a molecular weight of 1168.7 and 1140.7 Da respectively, are heat-tolerant and inhibit Geobacillus stearothermophilus var. calidolactis, the test strain of most of the commercially available microbiological inhibitor tests for screening of antibiotic residues in milk. Furthermore, these bacterial inhibitors show antimicrobial activity against Staphylococcus aureus, Bacillus cereus and B. subtilis and also interfere negatively with yoghurt production. Following their isolation and purification with RP-HPLC, the inhibitors were identified by NMR analysis as cyclic lipodepsipeptides of the viscosin group. Our findings bring to light a new challenge for quality control in the dairy industry. By prolonging the refrigerated storage of raw milk, the keeping quality of milk is influenced by growth and metabolic activities of psychrotrophic bacteria such as pseudomonads. Besides an increased risk of possible spoilage of long shelf-life milk, the production at low temperature of natural bacterial inhibitors may also result in false-positive results for antibiotic residue screening tests based on microbial inhibitor assays thus leading to undue production loss.

Short communication: Comparison of virulence factors in Klebsiella pneumoniae strains associated with multiple or single cases of mastitis

Klebsiella pneumoniae mastitis in dairy cattle is generally due to an opportunistic infection from the environment, resulting in large heterogeneity among mastitis-causing strains within a herd. However, in mastitis outbreaks in 4 herds, several strains of K. pneumoniae were identified as the cause of infection in multiple cows, suggesting increased ability to either cause disease or evade host defenses. In this study, differences in capsule formation and immune evasion were compared in 5 pairs of K. pneumoniae strains, where one strain in each pair was associated with multiple cases of mastitis and the other with a single case of mastitis. Production of capsular polysaccharide, ability to evade killing by polymorphonuclear neutrophilic leukocytes (PMNL), and the relationship between the 2 were evaluated for each strain grown in broth or milk. Growth of isolates in skim milk increased capsule size and ability to evade killing by PMNL, depending on strain type. Specifically, strains associated with multiple cases of mastitis had increased capsule size in skim milk. Strains associated with single cases of mastitis were better able to evade killing by PMNL when grown in skim milk. Our results, although preliminary, suggest that the 2 groups of strains may constitute different subpopulations of K. pneumoniae. However, our findings do not indicate that capsule or evasions of killing by PMNL explain increased mastitis outbreaks with Klebsiella. Further work will explain the enhanced ability of some strains to cause mastitis in dairy cows.

Enumeration and Characterization of Aeromonas spp. Isolated from Milk and Some Dairy Products in Sharkia Governorate, Egypt

A survey was conducted to determine the prevalence of Aeromonas spp. in 75 random samples (25 each of raw cow\'s milk, local plain yoghurt and Domiati cheese) collected from different dairy shops, supermarket and street peddlers in Diarb Negm and Zagazig cities, Sharkia Governorate, Egypt. Investigations involved proteolytic and lipolytic activities of isolated Aeromonas spp. and the effect of heat- treatment, acidity, pH and sodium chloride concentration on prevalence of Aeromonas bacteria. Prevalence of Aeromonas spp. was proved in 32.0%, 44.0% and 20.0% of examined raw cow\'s milk, local plain yoghurt and Domiati cheese samples with a mean count of 9.8 x103, 1.4 x105 and 6.9 x103/ml, respectively. Classification of confirmed raw cow\'s milk isolates revealed that A. trota, A. hydrophila, A. janda and A. caviae were the predominant strains with percentages of 40.0%, 25.0%, 25.0% and 10.0%, respectively. While, local plain yoghurt isolates could be classified as A. caviae, A. sobria, A. hydrophila, A. trota and A. schubertii with percentages of 36.4%, 22.7%, 18.2%, 13.6% and 9.1%, respectively. Meanwhile, Classification of 10 confirmed Domiati cheese cultures revealed that the predominant strains were A. hydrophila, A. caviae and A. trota with percentages of 30%, 50% and 20%, respectively. All laboratory pasteurized milk samples revealed no count and there is marked decrease in the count of Aeromonas spp. as the acidity % of the examined raw cow’s milk samples increase, the pH value of the examined local plain yoghurt samples decrease and the NaCl % of the examined Domiati cheese samples increase. Characterization of isolated Aeromonas strains pointed that 50% of A. hydrophila, 60% of A. caviae, 40% of A. sobria, 53.8% of A. trota, 100% of A. janda and 50% of A. schubertii were psychrotrophic. A. hydrophila exhibited proteolytic and lipolytic activities at the percentage of 41.7% and 16.7%, respectively but in case of A. caviae strains the percentages were 46.7% and 20%, respectively and with A. trota were 30.8% and 15.4%, respectively. 60% of A. sobria and 100% of A. janda and A. schubertii strains showed proteolytic activity only. The public health importance and economic significance of existing microorganisms as well as the suggestive measures for improving the quality of raw milk and dairy products were discussed.