Enterobacteriaceae, coliforms, faecal coliforms and salmonellas in raw ewes'milk

Thirty-nine samples of freshly drawn ewes' milk collected at 13 farms, and 120 samples of raw ewes' milk collected on arrival at a dairy were examined. Farm samples had geometric mean counts of 4.4 × 10² Enterobacteriaceae/ml, 3.9 × 10² coliforms/ml and 2.0 × 10² faecal coliforms/ml, whereas the respective mean counts were 6.2 × 10³/ml, 5.4 × 10³/ml and 1.3 × 10³/ml for dairy samples. Salmonellas were not detected by enrichment procedures in any of the 159 samples examined. Escherichia coli (47.5% strains), Enterobacter cloacae (17.7%), Ent. agglomerans (11.3%), Hafnia alvei (6.5%) and Klebsiella oxytoca (6.0%) were the predominant species in 434 Enterobacteriaceae strains isolated from farm samples. Levels and species of Enterobacteriaceae found in the present work in raw ewes' milk imply a considerable risk of early blowing in cheese-making from unpasteurized milk.     

Incidence of Staphylococcus aureus, coliforms and antibiotic-resistant strains of Escherichia coli in rural water supplies in Port Harcourt

The bacteriological quality of some rural water supplies in Port Harcourt was monitored over a 3 month period. The supplies were unsatisfactory as judged by standard plate counts (10(3)/ml) and the presence of presumptive and faecal coliforms and Staphylococcus aureus. The recovery of potentially pathogenic bacteria (e.g. Pseudomonas aeruginosa) further substantiated the existence of health hazards. The most frequently isolated coliforms were Escherichia coli, Enterobacter aerogenes and Klebsiella pneumoniae. Coliform contamination was greater in well water than in river or stream water samples. An antibiotic sensitivity test revealed that 17.5-27.2% of E. coli strains were resistant to three or more antibiotics. Escherichia coli isolated from well water samples exhibited the greatest degree of multiple resistance. Some strains were resistant to all the six antibiotics tested. The danger of an epidemic of waterborne diseases in the communities as a result of drinking water from these non-potable sources is noted.     

Incidence of pathogenic bacteria in raw milk in Ireland.

Raw milk from 70 farms was sampled over 13 months for salmonellas, listerias, Escherichia coli, Staphylococcus aureus and mastitic streptococci; total bacterial counts (TBC), coliforms and somatic cells were also counted. TBC < or = 30,000/ml were obtained in 63% of samples. High count milks were found mainly during the winter months: 13% of samples had > 10(4) mastitis pathogens/ml of milk. The mean somatic cell count varied from 4.0 x 10(5) to 8.0 x 10(5)/ml throughout the year with highest counts during the late lactation period. Coliforms were present in all samples, but 65-71% of samples had < 100 coliforms/ml. Up to 60% of supplies had < or = 10 E. coli/ml. One of the 589 samples tested (0.1%) was positive for salmonellas. Yersinia enterocolitica and Y. enterocolitica-like organisms were isolated from 39% of samples with up to 68% of samples positive at some sampling periods. A total of 222 strains of yersinias were isolated; Y. enterocolitica (59%) was the most common strain followed by Y. fredriksenii (35%), Y. kristensenii (1.0%), Y. intermedia (4.5%) and Y. aldovae (0.5%). Listerias were isolated from 8.3% of samples tested; 4.9% were Listeria monocytogenes and 3.4% were L. innocua. There was a significant rise in the isolation rate between December and April from a base line of 0-5% during the spring and summer to 35-37% during the winter months while the cows were indoors. Of 66 silage samples tested from the farms involved in the survey 9% of samples were positive for listerias; 3% of these were L. monocytogenes and 6% were L. innocua.(ABSTRACT TRUNCATED AT 250 WORDS)     

Injured coliforms in drinking water

Coliforms were enumerated by using m-Endo agar LES and m-T7 agar in 102 routine samples of drinking water from three New England community water systems to investigate the occurrence and significance of injured coliforms. Samples included water collected immediately after conventional treatment, during the backwash cycle, at various points in the distribution system, and 1 week after the break and subsequent repair of a distribution main. Injured coliforms in these samples averaged greater than 95%. m-T7 agar yielded 8- to 38-fold more coliforms than did m-Endo agar LES. The geometric mean of coliforms recovered by m-Endo agar LES was less than 1 confirmed coliform per 100 ml, although m-T7 agar yielded 5.7 to 67.5 confirmed coliforms per 100 ml. In addition, the majority of these samples giving positive results on m-T7 agar produced no detectable counts on m-Endo agar LES. These findings indicated that coliforms were injured and largely undetected by use of accepted analytical media in the systems examined.     

Injured coliforms in drinking water.

Coliforms were enumerated by using m-Endo agar LES and m-T7 agar in 102 routine samples of drinking water from three New England community water systems to investigate the occurrence and significance of injured coliforms. Samples included water collected immediately after conventional treatment, during the backwash cycle, at various points in the distribution system, and 1 week after the break and subsequent repair of a distribution main. Injured coliforms in these samples averaged greater than 95%. m-T7 agar yielded 8- to 38-fold more coliforms than did m-Endo agar LES. The geometric mean of coliforms recovered by m-Endo agar LES was less than 1 confirmed coliform per 100 ml, although m-T7 agar yielded 5.7 to 67.5 confirmed coliforms per 100 ml. In addition, the majority of these samples giving positive results on m-T7 agar produced no detectable counts on m-Endo agar LES. These findings indicated that coliforms were injured and largely undetected by use of accepted analytical media in the systems examined.     

Microbiological characteristics of anevato: a traditional greek cheese

Nine batches of Anevato, raw goat milk cheese, were examined throughout a 60 day storage time at three different periods within the lactation season of the goat. High mean log counts per gram of cheese for aerobic bacteria (7·92–9·56), lactic acid bacteria (7·78–9·32), Gram-negative organisms 5·64–9·67), psychrotrophs (7·90–11·79) and proteolytic bacteria (7·57–9·36) were found. Enterobacteriaceae, coliforms and yeasts were considerably lower. Enterobacteriaceae and coliforms in the curd of cheese made in May were lower by approximately 3·0 log₁₀ cfu g⁻¹ than counts in curd made in January, and were lower by about 2·5 log₁₀ cfu g⁻¹ than those in cheese made in March. This coincided with lower pH and higher counts of lactic acid bacteria in cheese made in March and May. Yeast populations were affected by the season and were higher in May than March and/or January. Lactococci dominated in the cheese until 15 days, but lactobacilli became predominant after 30 days. Lactococcus lactis was the most abundant species of lactic acid bacteria found in Anevato cheese. Results suggest the need for improving milk quality and/or using heat-treated milk to produce Anevato cheese; the use of L. lactis as a starter would possibly eliminate or suppress the growth of undesirable organisms.     

Incidence of Staphylococcus aureus, coliforms and antibiotic-resistant strains of Escherichia coli in rural water supplies in Port Harcourt

The bacteriological quality of some rural water supplies in Port Harcourt was monitored over a 3 month period. The supplies were unsatisfactory as judged by standard plate counts (10³/ml) and the presence of presumptive and faecal coliforms and Staphylococcus aureus. The recovery of potentially pathogenic bacteria (e.g. Pseudomonas aeruginosa ) further substantiated the existence of health hazards. The most frequently isolated coliforms were Escherichia coli, Enterobacter aerogenes and Klebsiella pneumoniae. Coliform contamination was greater in well water than in river or stream water samples. An antibiotic sensitivity test revealed that 17.5–27.2% of E. coli strains were resistant to three or more antibiotics. Escherichia coli isolated from well water samples exhibited the greatest degree of multiple resistance. Some strains were resistant to all the six antibiotics tested. The danger of an epidemic of waterborne diseases in the communities as a result of drinking water from these non-potable sources is noted.     

Psychrotrophic bacterial flora of raw ewes' milk, with particular reference to gram negative rods

The microbial flora of 141 samples of raw ewes' milk was determined, before and after storage for 72 h at 4 degrees and 7 degrees C. Penicillin-resistant bacteria represented ca 61% of 1760 psychrotrophic isolates from refrigerated milk samples. Pseudomonas fluorescens and Pseudomonas fluorescent group-related strains predominated (ca 86%) in the Gram negative psychrotrophic microflora. Leuconostoc dextranicum was the most frequent Gram positive psychrotrophic species isolated.     

Incidence of pathogenic bacteria in raw milk in Ireland

Raw milk from 70 farms was sampled over 13 months for salmonellas, listerias, Escherichia coli, Staphylococcus aureus and mastitic streptococci; total bacterial counts (TBC), coliforms and somatic cells were also counted. TBC ≤30000/ml were obtained in 63% of samples. High count milks were found mainly during the winter months: 13% of samples had > 10⁴ mastitis pathogens/ml of milk. The mean somatic cell count varied from 4.0 × 10⁵ to 8.0 × 10⁵/ml throughout the year with highest counts during the late lactation period. Coliforms were present in all samples, but 65–71% of samples had < 100 coliforms/ml. Up to 60% of supplies had ≤10 E. coli /ml. One of the 589 samples tested (0.1%) was positive for salmonellas. Yersinia enterocolitica and Y. enterocolitica -like organisms were isolated from 39% of samples with up to 68% of samples positive at some sampling periods. A total of 222 strains of yersinias were isolated; Y. enterocolitica (59%) was the most common strain followed by Y. fredriksenii (35%), Y. kristensenii (1.0%), Y. intermedia (4.5%) and Y. aldovae (0.5%). Listerias were isolated from 8.3% of samples tested; 4.9% were Listeria monocytogenes and 3.4% were L. innocua. There was a significant rise in the isolation rate between December and April from a base line of 0–5% during the spring and summer to 35–37% during the winter months while the cows were indoors. Of 66 silage samples tested from the farms involved in the survey 9% of samples were positive for listerias; 3% of these were L. monocytogenes and 6% were L. innocua. Only half of the farms feeding contaminated silage produced milk containing listerias.     

Contamination of milk by enterococci and coliforms from bovine faeces

AIM: To determine the contribution of enterococci and coliforms from bovine faeces and teats to contamination of raw milk. Methods: Putative enterococci (n = 301) and coliforms (n = 365) were isolated from bovine faeces (n = 20), cows' teats (n = 20), the raw milk (n = 1) and the milking environment (n = 4) on one farm. The clonal relationships of each bacterial group were investigated using Pulsed-Field Gel Electrophoresis of genomic macrorestriction fragments. Representatives of the different clusters of enterococci were identified by molecular techniques including rep-PCR, SDS protein profiling, Fluorescent Amplified Fragment Length Polymorphism (FAFLP), phenylalanyl-tRNA synthase (pheS) sequence analysis and/or 16S rDNA gene sequencing. Coliforms were identified by API 20E strips. RESULTS: The majority of the bovine faecal enterococcal isolates were identified as a potential new species of Aerococcus (100 isolates); E. faecium (28 isolates), and Aerococcus viridans (28 isolates) were also found. All coliform isolates from the bovine faeces were identified as Escherichia coli. The coliforms present in the milk were Hafnia alvei, Serratia liquefaciens, Yersinia enterocolitica and Enterobacter amnigenus. No E. coli, Enterococcus or Aerococcus from the bovine faeces were found in the milk. A single clone of H. alvei was found in the water, the milking equipment and the milk, suggesting that the water was the source of the organism in the milk. No vancomycin-resistant aerococci or enterococci were found while most of the isolates tested showed the presence of at least one virulence gene. The milk-sock retained strains that adhered to particulate faecal material. Coliforms were present at approx. 2 orders of magnitude greater than enterococci in the bovine faeces. CONCLUSIONS: The results imply that bovine faeces are not an important source of contamination of raw milk with enterococci or coliforms. SIGNIFICANCE AND IMPACT OF THE STUDY: The results confirm those of two previous studies (Gelsomino et al. 2001, Int J Food Microbiol71, 177-188 and Kagkli et al. 2007, Int J Food Microbiol114, 243-251) on two other farms. The three studies show that contamination of milk by enterococci, lactobacilli and coliforms of bovine faecal origin is extremely low. The results also suggest that where raw milk is implicated in food infection, other factors in addition to faecal contamination must be involved.     

Evaluation of four membrane filter media in anaerobic-MF recovery of faecal coliforms from freshwater in Nigeria.

MacConkey (MC), membrane lauryl sulphate (MIS), membrane faecal coliform amended with rosolic acid (MFC + R) and without the acid (MFC - R) were evaluated in the anaerobic membrane filtration (anaerobic-MF) recovery of faecal coliform populations (FCs), genera and faecal coliform positive (FC-positive) strains isolated from various sources of freshwater, i.e., rivers, rural wells, unchlorinated distributive supplies and hand pumps. Mean counts (x 10(2)/100 ml) of presumptive (typical) FCs varied from 13.69 (MC) to 40.81 (MLS) in river samples, and from 2.0 (MC) to 4.19 (MFC + R) in wells. The proportion of FC-positive, typical FCs ranged from 48.66 (MIS) to 66-67% (MC) in rivers, and from 50 (MC) to 90.22% (MFC + R) in wells. More than 30% of the typical FCs from all sources on each medium was FC-negative. These usually formed small (ca 1.0 mm diam.) colonies on the test agar, and were prevalent in wells. Typical FCs and FC-positive strains were not recovered from piped supplies and hand pumps. In spite of anaerobic incubation, non-faecal coliforms (NFCs) were often higher than the FCs; the FC:NFC ratios for rivers ranged from 1.65 (MC) to 7.65 (MLS) and (MFC + R) but were < 1.0 for wells on each medium. Escherichia coli, Klebsiella and Enterobacter species were recovered on all media: approximately 35-64% of the strains confirmed as FCs were E. coli, 20-42% were Kl. pneumoniae. The FC counts on the media were variable, but the overall performance in recovering 'true' FCs was similar; < 70% of strains per medium were FC-positive. None could count E. coli exclusively.     

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.     

Characterization of lactic acid bacteria isolated from the one humped camel milk produced in Morocco

One hundred and twenty (120) strains of lactic acid bacteria (LAB) were enumerated and isolated from raw dromedary milk in Morocco using various cultured media. Strains isolated were characterized by phenotypic, physiological and biochemical properties. Results showed that high counts of LAB were found. Presumptive lactobacilli counts ranged from 2.5x10(2) to 6x10(7)cfu/ml, presumptive lactococci levels varied from 5x10(2) to 6x10(7)cfu/ml, presumptive streptococci counts varied from 4.2x10(2) to 8x10(7)cfu/ml, presumptive leuconostoc levels ranged from 5.4x10(2) to 5.4x10(7)cfu/ml. Results showed also that Lactobacillus and Lactococcus were the predominant genera with 37.5% and 25.8%, respectively. The dominated species found were Lactococcus lactis subsp. lactis (17.5%), Lactobacillus helveticus (10%), Streptococcus salivarius subsp. thermophilus (9.20%), Lactobacillus casei subsp. casei (5.80%) and Lactobacillus plantarum (5%). This is the first report on the characterization of LAB strains isolated from the one humped camel milk produced in Morocco.     

Staphylococcus aureus, thermostable nuclease and staphylococcal enterotoxins in raw ewes' milk Manchego cheese

Growth and survival of two enterotoxigenic strains of Staphylococcus aureus were studied during manufacture and ripening of eight batches of raw ewes' milk Manchego cheese. Only 2-3 generations of Staph. aureus occurred in the vat and during pressing. The death rate of Staph. aureus (mean decrease in log cfu/g/week of ripening) from day 1 to day 60 was 0.421 in cheese made with 1% Streptococcus lactis starter and 0.404 in cheese made without starter. Thermostable nuclease was produced in the vat by growing Staph. aureus cells; it was inactivated by rennet during the first 24 h and synthesized again by surviving cells of Staph. aureus from day 1 to day 60. Staphylococcal enterotoxins A, B, C and D were not detected in any batches of cheese, even though Staph. aureus counts exceeded 10(7) cfu/g.     

Staphylococcus aureus, thermostable nuclease and staphylococcal enterotoxins in raw ewes' milk Manchego cheese

Growth and survival of two enterotoxigenic strains of Staphylococcus aureus were studied during manufacture and ripening of eight batches of raw ewes' milk Manchego cheese. Only 2–3 generations of Staph. aureus occurred in the vat and during pressing. The death rate of Staph. aureus (mean decrease in log cfu/g/week of ripening) from day 1 to day 60 was 0.421 in cheese made with 1% Streptococcus lactis starter and 0.404 in cheese made without starter. Thermostable nuclease was produced in the vat by growing Staph. aureus cells; it was inactivated by rennet during the first 24 h and synthesized again by surviving cells of Staph. aureus from day 1 to day 60. Staphylococcal enterotoxins A, B, C and D were not detected in any batches of cheese, even though Staph. aureus counts exceeded 10⁷ cfu/g.     

Hydrolysis of caseins and formation of hydrophilic and hydrophobic peptides by wild Lactococcus lactis strains isolated from raw ewes'' milk cheese

AIMS: To investigate the hydrolysis of alphaS1-, alphaS0-, betaB-, betaA1- and betaA2-caseins by 32 wild lactococci of different randomly amplified polymorphic DNA (RAPD) patterns, isolated from raw ewes' milk cheese, and the production of hydrophilic and hydrophobic peptides from whole casein by those strains. METHODS AND RESULTS: Most strains hydrolysed all caseins, and degraded beta-caseins to a larger extent than alphaS-caseins, when the proteolytic activity of whole cells was determined by capillary electrophoresis. Higher levels of hydrophilic than of hydrophobic peptides were produced from whole casein by all strains, according to reverse-phase high performance liquid chromatography analyses. CONCLUSIONS: Cell envelope proteinases of most lactococci isolated from raw ewes' milk cheese were CEPII, CEPII/III or CEPIII (classification of Exterkate et al. 1993). A negative correlation was found between degraded alphaS- and beta-caseins and a highly positive correlation between hydrophilic and hydrophobic peptides. SIGNIFICANCE AND IMPACT OF THE STUDY: Fast acid-producing lactococci from raw ewes' milk cheese have considerable and diverse caseinolytic activities. Their peptide production patterns do not reveal serious risks of bitter-flavour defect in cheeses if used as components of dairy starters.     

Rapid, single-step most-probable-number method for enumerating fecal coliforms in effluents from sewage treatment plants.

A single-step most-probable-number method for enumerating fecal coliforms in sewage treatment plant effluents is described. The method requires the use of only one lactose-based medium and a single incubation temperature of 44.5 degrees C, and it can be completed in 18 h or less, as compared with up to 72 h for the standard most-probable-number method. The appearance of growth is the sole criterion used for designating positives, which can be determined either by increases in the electrical impedance ratio of inoculated medium, as compared to an uninoculated control using a Bactometer model 32, or by visual examination of inoculated medium for turbidity. In trials with 53 samples of unchlorinated sewage treatment plant effluent, fecal coliform counts by the single-step most-probable-number method, throughout a range of less than 10 to almost 10(7) fecal coliforms per 100 ml of effluent, were in excellent agreement with counts abtained by the standard most-probable-number procedure. Similar agreement was obtained in comparative trials with 31 chlorinated effluent samples from two sewage treatment plants. Overall, 87% of 452 positives were confirmed as containing fecal coliforms. The applicability of the single-step most-probable-number method to automated sewage treatment plant operations is discussed.     

Rapid, single-step most-probable-number method for enumerating fecal coliforms in effluents from sewage treatment plants.

A single-step most-probable-number method for enumerating fecal coliforms in sewage treatment plant effluents is described. The method requires the use of only one lactose-based medium and a single incubation temperature of 44.5 degrees C, and it can be completed in 18 h or less, as compared with up to 72 h for the standard most-probable-number method. The appearance of growth is the sole criterion used for designating positives, which can be determined either by increases in the electrical impedance ratio of inoculated medium, as compared to an uninoculated control using a Bactometer model 32, or by visual examination of inoculated medium for turbidity. In trials with 53 samples of unchlorinated sewage treatment plant effluent, fecal coliform counts by the single-step most-probable-number method, throughout a range of less than 10 to almost 10(7) fecal coliforms per 100 ml of effluent, were in excellent agreement with counts abtained by the standard most-probable-number procedure. Similar agreement was obtained in comparative trials with 31 chlorinated effluent samples from two sewage treatment plants. Overall, 87% of 452 positives were confirmed as containing fecal coliforms. The applicability of the single-step most-probable-number method to automated sewage treatment plant operations is discussed.     

Effect of seawater storage on coliforms, faecal coliforms and Escherichia coli.

The effect of refrigeration of seawater samples for 24 h prior to assaying coliforms, faecal coliforms and Escherichia coli was assessed. When the initial coliform counts were low, the amount of bacteria in refrigerated samples decreased. When the number of initial total coliforms was high, there was an increase following cold storage. E. coli counts also decreased. Faecal coliforms, when in the initial 200-500 count range, showed a decrease during cold storage but with good correlation (r = 0.9). In all other groups, no correlation between counts of before versus after storage was found. Assessment of seawater pollution following 24 h cold storage should not be made.     

Evaluation of four membrane filter media in anaerobic-MF recovery of faecal coliforms from freshwater in Nigeria

M.T. OGAN. 1992. MacConkey (MC), membrane lauryl sulphate (MIS), membrane faecal coliform amended with rosolic acid (MFC + R) and without the acid (MFC — R) were evaluated in the anaerobic membrane filtration (anaerobic-MF) recovery of faecal coliform populations (FCs), genera and faecal coliform positive (FC-positive) strains isolated from various sources of freshwater, i.e. rivers, rural wells, unchlorinated distributive supplies and hand pumps. Mean counts (x 10²/100 ml) of presumptive (typical) FCs varied from 13.69 (MC) to 40.81 (MLS) in river samples, and from 2.0 (MC) to 4.19 (MFC + R) in wells. The proportion of FC-positive, typical FCs ranged from 48.66 (MIS) to 66.67% (MC) in rivers, and from 50 (MC) to 90.22% (MFC + R) in wells. More than 30% of the typical FCs from all sources on each medium was FC-negative. These usually formed small ( ca 1.0 mm diam.) colonies on the test agar, and were prevalent in wells. Typical FCs and FC-positive strains were not recovered from piped supplies and hand pumps. In spite of anaerobic incubation, non-faecal coliforms (NFCs) were often higher than the FCs; the FC: NFC ratios for rivers ranged from 1.65 (MC) to 7.65 (MLS) and (MFC + R) but were < 1.0 for wells on each medium. Escherichia coli, Klebsiella and Enterobacter species were recovered on all media: approximately 35–64% of the strains confirmed as FCs were E. coli, 20–42% were Kl. pneumoniae. The FC counts on the media were variable, but the overall performance in recovering 'true' FCs was similar; < 70% of strains per medium were FC-positive. None could count E. coli exclusively.