Validity of luminometry and bacteriological tests for diagnosing intramammary infection at dry-off in dairy cows.

The objective of this cross-sectional study was to estimate the validity of laboratory culture, Petrifilm and Tri-Plate on-farm culture systems, as well as luminometry to correctly identify IMI at dry-off in dairy cows, considering all tests to be imperfect. From September 2020 until December 2021, we collected composite milk samples from cows before dry-off and divided them into 4 aliquots for luminometry, Petrifilm (aerobic count), Tri-Plate, and laboratory culture tests. We assessed multiple thresholds of relative light units (RLU) for luminometry, and we used thresholds of ≥100 cfu/mL for the laboratory culture, ≥50 cfu/mL for Petrifilm, and ≥1 cfu for Tri-Plate tests. We fitted Bayesian latent class analysis models to estimate the sensitivity (Se) and specificity (Sp) for each test to identify IMI, with 95% credibility interval (BCI). Using different prevalence measures (0.30, 0.50, and 0.70), we calculated the predictive values (PV) and misclassification cost terms (MCT) at different false negative-to-false-positive ratios (FN:FP). A total of 333 cows were enrolled in the study from one commercial Holstein herd. The validity of the luminometry was poor for all thresholds, with an Se of 0.51 (95% BCI = 0.43-0.59) and Sp of 0.38 (95% BCI = 0.26-0.50) when using a threshold of ≥150 RLU. The laboratory culture had an Se of 0.93 (95% BCI = 0.85-0.98) and Sp of 0.69 (95% BCI = 0.49-0.89); the Petrifilm had an Se of 0.91 (95% BCI = 0.80-0.98) and Sp of 0.71 (95% BCI = 0.51-0.90); and the Tri-Plate had an Se of 0.65 (95% BCI = 0.53-0.82) and Sp of 0.85 (95% BCI = 0.66-0.97). Bacteriological tests had good PV, with comparable positive PV for all 3 tests, but lower negative PV for the Tri-Plate compared with the laboratory culture and the Petrifilm. For a prevalence of IMI of 0.30, all 3 tests had similar MCT, but for prevalence of 0.50 and 0.70, the Tri-Plate had higher MCT in scenarios where leaving a cow with IMI untreated is considered to have greater detrimental effects than treating a healthy cow (i.e., FN:FP of 3:1). Our results showed that the bacteriological tests have adequate validity to diagnose IMI at dry-off, but luminometry does not. We concluded that although luminometry is not useful to identify IMI at dry-off, the Petrifilm and Tri-Plate tests performed similarly to laboratory culture, depending on the prevalence and importance of the FP and FN results.

Comparison of PetrifilmTM AC and pour plate techniques used for the heterotrophic aerobic bacterial count in water.

Heterotrophic bacteria are commonly found in water samples. While these Heterotrophic Bacterial/Plate Counts (HPC) do not necessarily indicate a health hazard, high counts provide a good indication of the efficiency of water disinfection and integrity of distribution systems. The aim of this study was to compare the PetrifimTM AC method to the pour plate technique for the testing of HPC in water samples. Artificially contaminated (192 samples) and natural water samples (25) were processed using two methods. Both methods accurately detected high, medium and low counts of HPC, producing average Z scores between -2 and +2. Paired-wise student t-test and correlation coefficient showed nonsignificant differences between the results of two methods. Acceptable repeatability and reproducibility was obtained using both the methods. Uncertainty of measurement for PetrifilmTM AC and pour plate method was found to be 2.9% and 5.4%, respectively. PetrifilmTM AC proved to be robust at 33°C and 37°C. In conclusion, PetrifimTM AC, which is easy to process, read, and less time consuming, proved to be comparable to the conventional pour plate method in establishing HPC in water. In addition, PetrifimTM AC requires less space for the processing and incubation, generate small volume of waste for disposal, and requires no equipment, except for the incubator.

Microbial Burden Estimation of Food Items, Built Environments, and the International Space Station Using Film Media.

The use of film media involves considerably less preparation, waste, and incubator space than conventional agar-media-based assays and has proven in past studies to provide counts of cultivable microbes similar to those of traditional agar media. Film media also have the advantage of allowing sample volumes similar to those used in pour plates and, therefore, are well-suited for cultivable microbial counts in extremely low-biomass environments such as clean rooms or space habitats, particularly where the subsequent isolation of colonies is necessary. As the preparation of film media plates relies on water cohesion/adhesion rather than manual spreading, they may have future applications in low- or microgravity settings. In this study, cultivable microbial count performance was compared between agar media and film media in three kinds of samples: food items, surfaces in built environments on Earth (homes), and on the environmental surfaces of the International Space Station (ISS). Easy Plates (Kikkoman Corporation) and Petrifilm (3M) were compared with traditional agar plating for food and home surfaces, while only Easy Plates were compared with agar for ISS samples. For both food items and built environments on Earth, both types of film media performed comparably to agar media for bacterial counts, with R2 values of 0.94-0.96. Fungal counts for built-environment samples had a lower correlation between film and agar counts, with R2 values of 0.72-0.73. Samples from the ISS, which ranged from below detection to 103 CFU per 100 cm2, had R2 values of 0.80 for bacterial counts and 0.73 for fungal counts, partially due to multiple samples recording below the detection limit for agar or too numerous to count, and the growth of fungal species on R2A medium. The species compositions of isolates picked from agar vs. film media plates were similar; however, further phylogenetic analysis is needed to confirm the differential microbial diversity composition. Overall, film media such as Easy Plates and Petrifilm are viable alternatives to agar plates for low-biomass built environments as well as for food samples, and the two brands tested in this study performed equally well.

Seasonality in faecal contamination of drinking water sources in the Jirapa and Kassena-Nankana Municipalities of Ghana.

This paper examines seasonal variations in faecal contamination of drinking water sources in the Jirapa and Kassena-Nankana Municipalities of Ghana. Data collection involved a survey of 568 households, testing of faecal coliform concentrations in drinking water source samples (141 in the rainy season, 128 in the dry season), in-depth interviews with key water stakeholders, and field observation to identify sources of faecal contamination. From the water quality testing, faecal coliforms were detected in all source types, including 'treated' pipe-borne water. Contamination was significantly higher in the rainy season than in the dry season (P < 0.05) with 51.8% of water samples in the rainy season and 27.3% in the dry season failing to meet the World Health Organisation and Ghana Standard Authority guideline on faecal coliform concentrations in drinking water sources. The proportion of population at risk of faecal contamination in the rainy season was 41.5% compared to 33.1% in the dry season. We argue that in Ghana and Sub-Saharan Africa at large, water surveillance agencies risk underestimating population exposed to faecal contamination through drinking water sources if monitoring is only done in the dry season. To avoid this, we recommend seasonal monitoring of faecal concentration in drinking water sources. However, in periods of limited resources, monitoring is most appropriate in the rainy season when the risk of contamination is high.

Rapid estimation of Salmonella enterica contamination level in ground beef - Application of the time-to-positivity method using a combination of molecular detection and direct plating.

Little progress has been made in decreasing the incidence rate of salmonellosis in the US over the past decade. Mitigating the contribution of contaminated raw meat to the salmonellosis incidence rate requires rapid methods for quantifying Salmonella, so that highly contaminated products can be removed before entering the food chain. Here we evaluated the use of Time-to-Positivity (TTP) as a rapid, semi-quantitative approach for estimating Salmonella contamination levels in ground beef. Growth rates of 14 Salmonella strains (inoculated at log 1 to -2 CFU/g) were characterized in lean ground beef mTSB enrichments and time-to-detection was determined using culture and molecular detection methods. Enrichments were sampled at five timepoints and results were used to construct a prediction model of estimated contamination level by TTP (superscript indicates time in hours) defined as TTP4: ≥5 CFU/g; TTP6: ≤5, ≥1 CFU/g; TTP8: ≤1, ≥0.01 CFU/g; with samples negative at 8 h estimated ≤0.01 CFU/g. Model performance measures showed high sensitivity (100%) and specificity (83% and 93% for two detection methods) for samples with a TTP4, with false negative rates of 0%.

Short communication: Diagnostic accuracy of the Petrifilm culture system for identifying colostrum with excessive bacterial contamination in Quebec dairy herds.

The objective of this study was to validate the diagnostic accuracy of the Petrifilm culture system (3M, St. Paul, MN) for identifying colostrum with excessive bacterial contamination. An observational cross-sectional study was conducted between October 2015 and February 2016. Two colostrum aliquots were collected during the first meal of 332 calves (33 commercial Holstein dairy farms) in Quebec, Canada. One aliquot per calf was used to quantify the total bacteria count and the total coliform count using standard bacteriological laboratory testing (reference test). These results were dichotomized to identify colostrum with excessive bacterial contamination [aerobic count plate (AC) >100,000 cfu/mL; coliform count plate (CC) >10,000 cfu/mL]. The Petrifilm system was used to quantify both aerobic and coliform contamination of the other colostrum aliquot from each calf. As such, AC and CC were used according to the manufacturer's recommendations. The area under the curve of the receiver operating characteristic curve of AC and CC compared with the laboratory were 0.83, and 0.95, respectively. Using the optimal threshold of >24,000 cfu/mL for AC results, the Petrifilm system had a sensitivity (Se) of 69%, specificity (Sp) of 86%, and a kappa value of 0.54. Using the optimal threshold of >4,000 cfu/mL for CC results, the Petrifilm system had a Se of 93%, Sp of 90%, and kappa value of 0.64. Overall, these results suggest that the Petrifilm system is an appropriate alternative for identifying colostrum with excessive bacterial contamination.

Evaluation of 3M™ loop-mediated isothermal amplification-based kit and 3M™ ready-to-use plating system for detection of Listeria in naturally contaminated leafy vegetables, chicken, and their related processing environments.

The effectiveness of two different rapid methods involving the 3M™ molecular detection assay Listeria and the 3M™ Petrifilm environmental Listeria Plate were evaluated for the rapid detection of Listeria from naturally contaminated vegetables and chicken-processing environments against the standard culture-based method. A total of 178 samples were examined for the presence of Listeria. A total of 47/178 (26.4%) by standard ISO culture-based method (EN ISO 11290-1), 42/178 (23.6%) by 3M™ MDA Listeria and 40/178 (22.5%) by 3M™ Petrifilm EL Plate showed positive results, respectively. The accuracy, sensitivity, specificity, positive predictive value, and negative predictive value for 3M™ MDA Listeria and 3M™ Petrifilm EL Plate were 97.2, 89.4, 99.3, 97.7, 96.4% and 96.1, 85.1, 100.0, 100.0, 94.9%, respectively. Based on the Cohen's Kappa value, there was a complete and robust concordance between 3M™ MDA Listeria (0.911) and 3M™ Petrifilm EL Plates (0.894) as compared to the standard culture-based method.

3M Petrifilm Lactic Acid Bacteria Count Plate Is a Reliable Tool for Enumerating Lactic Acid Bacteria in Bacon.

ABSTRACT: This study aimed to evaluate the behavior of Petrifilm Lactic Acid Bacteria Count Plates (PLAB) as an alternative methodology to enumerate lactic acid bacteria (LAB) in bacon. Bacon samples (n = 40) were obtained from retail sale, 10-fold diluted with buffered peptone water (BPW, 0.2% [w/v]) and Letheen broth, and subjected to LAB enumeration according to four protocols: (i) de Man Rogosa Sharpe (MRS) agar, pH 5.7, 30°C; (ii) MRS, pH 5.7, 30°C, anaerobiosis; (iii) all-purpose Tween agar (APT), 25°C; and (iv) PLAB, 30°C. Colonies were enumerated at 24, 48, and 72 h, and the results expressed as log CFU per gram for comparison by analysis of variance and regression (P < 0.05). Furthermore, colonies were randomly selected and characterized as LAB (Gram staining and catalase). Mean LAB counts from MRS and PLAB did not present significant differences independently of incubation time or diluent (P > 0.05), whereas counts in APT with BPW after 24 h were significantly lower (P < 0.05). PLAB counts with BPW (24, 48, and 72 h) presented significant correlation with MRS (r ranging from 0.87 to 0.89; in anaerobiosis, r ranging from 0.94 to 0.95) and APT (r ranging from 0.84 to 0.86). With Letheen broth, PLAB (24, 48, and 72 h) presented significant correlation with MRS (r ranging from 0.92 to 0.94; in anaerobiosis, r ranging from 0.93 to 0.96) and APT (r ranging from 0.77 to 0.79). In total, 1,032 colonies (97%) from 1,063 colonies were characterized as LAB. Thus, PLAB can be considered as an alternative tool for enumerating LAB in bacon, with reliable results even after 24 h of incubation.

Evaluation of Petrifilm Lactic Acid Bacteria Plates for Counting Lactic Acid Bacteria in Food.

Although lactic acid bacteria (LAB) are used widely as starter cultures in the production of fermented foods, they are also responsible for food decay and deterioration. The undesirable growth of LAB in food causes spoilage, discoloration, and slime formation. Because of these adverse effects, food companies test for the presence of LAB in production areas and processed foods and consistently monitor the behavior of these bacteria. The 3M Petrifilm LAB Count Plates have recently been launched as a time-saving and simple-to-use plate designed for detecting and quantifying LAB. This study compares the abilities of Petrifilm LAB Count Plates and the de Man Rogosa Sharpe (MRS) agar medium to determine the LAB count in a variety of foods and swab samples collected from a food production area. Bacterial strains isolated from Petrifilm LAB Count Plates were identified by 16S rDNA sequence analysis to confirm the specificity of these plates for LAB. The results showed no significant difference in bacterial counts measured by using Petrifilm LAB Count Plates and MRS medium. Furthermore, all colonies growing on Petrifilm LAB Count Plates were confirmed to be LAB, while yeast colonies also formed in MRS medium. Petrifilm LAB Count Plates eliminated the plate preparation and plate inoculation steps, and the cultures could be started as soon as a diluted food sample was available. Food companies are required to establish quality controls and perform tests to check the quality of food products; the use of Petrifilm LAB Count Plates can simplify this testing process for food companies.

Early-lactation extended pirlimycin therapy against naturally acquired Staphylococcus aureus intramammary infections in heifers: A randomized controlled trial.

The primary objective of the current study was to evaluate cure rate following an early-lactation extended intramammary pirlimycin treatment on heifers naturally infected by Staphylococcus aureus. The secondary objective was to assess Petrifilm Staph Express (3M Microbiology, St. Paul, MN) count plate characteristics when used in a protocol for early-lactation detection of infected quarters in heifers. Milk samples were collected from heifers (n = 946) in the first few days following calving (mean = 5 d). Heifers with laboratory-confirmed S. aureus intramammary infection (n = 72) were randomly allocated into 2 groups. The treatment group (n = 54 quarters from 38 heifers) received an intramammary infusion of 50 mg of pirlimycin once per day for 8 consecutive days in infected quarters. The control group (n = 44 quarters from 34 heifers) did not receive any treatment. Treatment success was defined as having negative culture results for S. aureus in all 3 post-treatment quarter milk samples collected on d 17, 24, and 31 post-treatment. Treatment group mammary quarters showed a statistically significant higher cure rate (64.8%) compared with the control group (34.1%). A total of 38% of quarters identified as S. aureus-positive using the Petrifilm Staph Express count plate were in fact identified as non-aureus staphylococci on routine laboratory-based bacteriological culture. The current study demonstrates that a higher cure rate for S. aureus IMI can be achieved in dairy heifers if an extended treatment protocol is put in place soon after calving. Use of Petrifilm Staph Express count plate for identification of S. aureus-infected heifers could lead to unnecessary treatments because of false-positive results.

Impact of Mannanase-Producing Bacillus spp. on the Accuracy of the 3M Petrifilm Aerobic Count Method.

Consistent deviations of the 3M Petrifilm aerobic counts (AC) from the standard pour plate aerobic plate count (APC) were observed with dehydrated onion and garlic products. A large study was designed to determine the relationship of these two methods and the root cause for the deviations. A total of 3,800 dehydrated onion and garlic samples were analyzed by both the Petrifilm AC and the standard pour plate APC method. Large spreader-like liquefied areas were observed on numerous Petrifilm plates. These liquefied areas made enumeration inaccurate. "Liquefier" microorganisms from Petrifilm plates were isolated and identified to species level by 16S rRNA and gyrB gene sequencing. Enzyme diffusion assay was performed to determine potential enzymatic degradation of guar gum, the gelling agent used in Petrifilm plates. The results indicated that the correlation between Petrifilm AC and standard APC is relatively low. Paired t test results suggested that the Petrifilm AC method produced significantly different results compared with standard APC. The discrepancies were attributable at least partly to a liquefier organism that hydrolyzed guar gum, leading to liquefaction. Liquefaction of Petrifilm plates seems to have two effects on accuracy: (i) liquefied areas may allow motile organisms to move and multiply in the liquefied area during the incubation period, yielding more than one colony from one cell and, as a result, leading to overestimation of the microbial load and (ii) the blurred areas obscure other colonies, leading to potential underestimation. The liquefier organism was identified as Bacillus amyloliquefaciens , a potent mannanase producer and heat-resistant spore former. Enzyme diffusion assay confirmed that mannanase contained in the cell-free supernatant of B. amyloliquefaciens can hydrolyze the 1,4-ß-mannopyranosyl bond, the backbone of guar gum. This is the first report of the role of B. amyloliquefaciens in the liquefaction of Petrifilm plates and its negative impact on accuracy.

Enumeration of Escherichia coli in swab samples from pre- and post-chilled pork and lamb carcasses using 3M™ Petrifilm™ Select E. coli and Simplate® Coliforms/E. coli.

The aim of the study was to compare two analytical methods; 3M Petrifilm™ Select E. coli and SimPlate® Coliforms &E. coli, for detection and enumeration of E. coli using swab samples from naturally contaminated pork and lamb carcasses that were collected before and after chilling. Blast chilling was used for pork carcasses. Swab samples (n=180) were collected from 60 warm and 60 chilled pork carcasses, and 30 warm and 30 chilled lamb carcasses, and analysed in parallel. The concordance correlation coefficient between Petrifilm and SimPlate was 0.89 for pork and 0.81 for lamb carcasses. However, the correlation was higher for warm carcasses (0.90) than chilled carcasses (0.72). For chilled lamb carcasses, the correlation was only 0.50, and SimPlate gave slightly higher results than Petrifilm (P=0.09). Slower chilling gave slightly lesser agreement between methods than for blast chilling, however, both Petrifilm and SimPlate methodologies are suitable and recommended for use in small laboratories in abattoirs.

Application of a bacteriological on-farm test to reduce antimicrobial usage in dairy cows with purulent vaginal discharge.

The objective of this study was to assess the effect of a selective antibiotic treatment strategy based on a quick bacteriological on-farm test (Petrifilm, 3M Corp., St. Paul, MN) compared with the conventional antibiotic treatment of all cows having clinical endometritis (CE) defined by the presence of purulent vaginal discharge on both clinical cure rate and reproductive performance. The study was simultaneously conducted with dairy cows reared under a highly supplemented rotational grazing system in Argentina and in a freestall system in Slovakia. Cows having an abnormal vaginal discharge (VD, indicative of clinical endometritis) on 21 to 35 d in milk (DIM) were randomly allocated to 1 of 2 study groups: selective treatment (ST) or conventional treatment (CT). All cows in the CT group (n = 174) received a single intrauterine administration of 500 mg of cephapirin. In the ST group (n = 178), treatment decision was made according to the results of the bacteriological on-farm test. For this test, we collected intrauterine samples with the cytobrush technique and stroke the brushes onto 2 different Petrifilm plates, one for aerobic count and another for Enterobacteriaceae count, incubated the plates, and counted the number of colonies after 24 h. Positive cows (≥5 colonies in one or both plates) received a single intrauterine treatment with 500 mg of cephapirin, whereas negative cows (<5 colonies) remained untreated. Clinical cure rate was assessed by direct vaginal inspection at 14 d after treatment (VD-0). The odds for conception at first artificial insemination, artificial insemination by 80 DIM, pregnancy by 100 DIM, and for nonpregnancy by 200 DIM were estimated with mixed logistic regression models. The hazard of conception was also assessed with proportional hazard regression model. The selective antibiotic treatment strategy based on the outcome of Petrifilm test reduced the number of required treatments (57%) and maintained similar efficacy in terms of clinical cure and reproductive performance as the conventional antibiotic treatment of all endometritic cows.

Metagenomic analysis of medicinal Cannabis samples; pathogenic bacteria, toxigenic fungi, and beneficial microbes grow in culture-based yeast and mold tests.

Background: The presence of bacteria and fungi in medicinal or recreational Cannabis poses a potential threat to consumers if those microbes include pathogenic or toxigenic species. This study evaluated two widely used culture-based platforms for total yeast and mold (TYM) testing marketed by 3M Corporation and Biomérieux, in comparison with a quantitative PCR (qPCR) approach marketed by Medicinal Genomics Corporation. Methods: A set of 15 medicinal Cannabis samples were analyzed using 3M and Biomérieux culture-based platforms and by qPCR to quantify microbial DNA. All samples were then subjected to next-generation sequencing and metagenomics analysis to enumerate the bacteria and fungi present before and after growth on culture-based media. Results: Several pathogenic or toxigenic bacterial and fungal species were identified in proportions of >5% of classified reads on the samples, including Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Ralstonia pickettii, Salmonella enterica, Stenotrophomonas maltophilia, Aspergillus ostianus, Aspergillus sydowii, Penicillium citrinum and Penicillium steckii. Samples subjected to culture showed substantial shifts in the number and diversity of species present, including the failure of Aspergillus species to grow well on either platform. Substantial growth of Clostridium botulinum and other bacteria were frequently observed on one or both of the culture-based TYM platforms. The presence of plant growth promoting (beneficial) fungal species further influenced the differential growth of species in the microbiome of each sample. Conclusions: These findings have important implications for the Cannabis and food safety testing industries.

Adequacy of Petrifilm™ Aerobic Count plates supplemented with de Man, Rogosa & Sharpe broth and chlorophenol red for enumeration of lactic acid bacteria in salami.

The present study aimed to assess the performance of alternative protocols to enumerate lactic acid bacteria (LAB) in salami. Fourteen cultures and two mixed starter cultures were plated using six protocols: 1) Petrifilm™ Aerobic Count (AC) with MRS broth and chlorophenol red (CR), incubated under aerobiosis or 2) under anaerobiosis, 3) MRS agar with CR, 4) MRS agar with bromocresol purple, 5) MRS agar at pH5.7, and 6) All Purpose Tween agar. Samples of salami were obtained and the LAB microbiota was enumerated by plating according protocols 1, 2, 3 and 5. Regression analysis showed a significant correlation between the tested protocols, based on culture counts (p<0.05). Similar results were observed for salami, and no significant differences of mean LAB counts between selected protocols (ANOVA, p>0.05). Colonies were confirmed as LAB, indicating proper selectivity of the protocols. The results showed the adequacy of Petrifilm™ AC supplemented with CR for the enumeration of LAB in salami.

Evaluation of selective dry cow treatment following on-farm culture: Milk yield and somatic cell count in the subsequent lactation.

Compared with blanket dry cow therapy (DCT), the selective antimicrobial treatment of cows based upon on-farm culture results has the potential to reduce the amount of antimicrobials used in dairy production. The objective of the current study was to determine the effect of a Petrifilm (3M Canada, London, Ontario) on-farm culture-based selective DCT program on milk yield and somatic cell count (SCC) in the following lactation. A total of 729 low-SCC (<200,000 cells/mL) cows from 16 commercial dairy herds with a low bulk tank SCC (<250,000 cells/mL) were randomly assigned to receive either blanket DCT or Petrifilm-based selective DCT. Cows belonging to the blanket DCT group were infused with a commercial DCT product and an internal teat sealant (ITS) at drying off. Using composite milk samples collected on the day before drying off, cows in the selective DCT group were treated at drying off based on the results obtained by the Petrifilm on-farm culture system with DCT and ITS (Petrifilm culture positive) or ITS alone (Petrifilm culture negative). Milk test-day records for the following lactation were obtained from Dairy Herd Improvement for all cows enrolled in the trial. Repeated measures linear mixed models were used to assess the effect of study group (blanket or selective DCT) on test-day milk production and natural logarithm of SCC over the first 180 d of the subsequent lactation. According to the final multivariable models, when low-SCC cows were selectively treated with DCT at drying off based on results obtained using the Petrifilm on-farm culture system, no effect on milk production (least squares means for blanket DCT = 39.3 kg vs. selective DCT = 39.0 kg) or natural logarithm of SCC (least squares means for blanket DCT = 3.95 vs. selective DCT = 3.97) was observed in the subsequent lactation when compared with cows receiving blanket DCT. The results of this study indicate that selective DCT based on results obtained by the Petrifilm on-farm culture system enabled a reduction in the use of DCT without negatively affecting milk production and milk quality.

Avaliação do sistema Petrifilm™ HS na contagem de coliformes em leite pasteurizado / Evaluation of the Petrifilm™ HS system for counting coliforms in pasteurized milk

Os métodos microbiológicos alternativos apresentam vantagens sobre os ensaios convencionais, no entanto é preciso confirmar sua eficácia. O presente trabalho avaliou o sistema Petrifilm™ HS com o sistema Petrifilm™ EC, em comparação com a metodologia convencional na contagem de coliformes a 35 ºC em leite pasteurizado. Altas correlações foram encontradas entre as metodologias utilizadas para efetuar a contagem de coliformes a 35 ºC. O sistema Petrifilm™ HS para contagem de coliformes a 35 ºCem leite pasteurizado mostrou resultados satisfatórios...

Development of an alternative culture medium for the selective enumeration of Lactobacillus casei in fermented milk.

Monitoring the populations of probiotic strains of the species Lactobacillus casei in food is required by food industries in order to assure that a minimum concentration of these organisms will be ingested by consumers. In this context, Petrifilm™ AC plates can be used along with selective culture media to allow the enumeration of specific groups of lactic acid bacteria. The present study aimed to assess chemical substances as selective agents for Lb. casei in order to propose a selective culture medium to be used with Petrifilm™ AC plates as an alternative protocol for the enumeration of probiotic strains of this species in fermented milk. Twenty-six probiotic and starter cultures (including six strains of Lb. casei) were plated on de Man Rogosa and Sharpe (MRS) agar with distinct concentrations of nalidixic acid, bile, lithium chloride, metronidazole, sodium propionate, and vancomycin. Vancomycin at 10 mg/L demonstrated selective activity for Lb. casei. In addition, 2,3,5-triphenyltetrazolium chlorine was identified as a compound that did not inhibit Lb. casei, and Petrifilm™ AC plates used with MRS and vancomycin at 10 mg/L (MRS-V) demonstrated more colonies of this organism when incubated under anaerobic conditions than aerobic conditions. Acidophilus milk and yoghurt were prepared, added to Lb. casei strains, and stored at 4 °C. Lb. casei populations were monitored using MRS-V and MRTLV by conventional plating and associated with Petrifilm™ AC plates. All correlation indices between counts obtained by conventional plating and Petrifilm™ AC were significant (p < 0.05), but the best performance was observed for growth on MRS-V. The obtained data indicate the efficiency of using MRS-V associated with Petrifilm™ AC plates for the enumeration of Lb. casei strains in fermented milk. However, the selective potential of this culture medium must be evaluated considering the specific strains of Lb. casei and the starter cultures inoculated in the fermented milk that requires monitoring.

Development of a selective culture medium for bifidobacteria, Raffinose-Propionate Lithium Mupirocin (RP-MUP) and assessment of its usage with Petrifilm™ Aerobic Count plates.

This study aimed to develop a selective culture media to enumerate bifidobacteria in fermented milk and to assess this medium when used with Petrifilm™ AC plates. For this purpose, Bifidobacterium spp., Lactobacillus spp. and Streptococcus thermophilus strains were tested to verify their fermentation patterns for different carbohydrates. All bifidobacteria strains were able to use raffinose. Based on these characteristic, a selective culture medium was proposed (Raffinose-Propionate Lithium Mupirocin, RP-MUP), used with Petrifilm™ AC plates, and was used to enumerate bifidobacteria in fermented milk. RP-MUP performance was assessed by comparing the results with this medium to reference protocols and culture media for bifidobacteria enumeration. RP-MUP, whether used or not with Petrifilm™ AC, presented similar performance to TOS-MUP (ISO 29981), with no significant differences between the mean bifidobacteria counts (p < 0.05) and with high correlation indices (r = 0.99, p < 0.05). As an advantage, reliable results were obtained after just 48 h of incubation when RP-MUP was used with Petrifilm™ AC, instead of the 72 h described in the ISO 29981 protocol.

A field study evaluation of Petrifilm™ plates as a 24-h rapid diagnostic test for clinical mastitis on a dairy farm.

Clinical mastitis is one of the most common and expensive diseases of dairy cattle. To make an informed treatment decision, it is important to know the causative pathogen. However, no detection of bacterial growth can be made in approximately 30% of all clinical cases of mastitis. Before selecting the treatment regimen, it is important to know whether the mastitis-causing pathogen (MCP) is Gram-positive or Gram-negative. The aim of this field study was to investigate whether using two 3M Petrifilm™ products on-farm (which conveys a higher degree of sample freshness but also bears a higher risk for contamination than working in a lab) as 24-h rapid diagnostic of clinical mastitis achieved results that were comparable to the conventional microbiological diagnostic method. AerobicCount (AC)-Petrifilm™ and ColiformCount (CC)-Petrifilm™ were used to identify the total bacterial counts and Gram-negative bacteria in samples from clinical mastitis cases, respectively. Missing growth on both plates was classified as no bacterial detection. Growth only on the AC-Petrifilm™ was assessed as Gram-positive, and growth on both Petrifilm™ plates was assessed as Gram-negative bacterial growth. Additionally, milk samples were analysed by conventional microbiological diagnostic method on aesculin blood agar as a reference method. Overall, 616 samples from clinical mastitis cases were analysed. Using the reference method, Gram-positive and Gram-negative bacteria, mixed bacterial growth, contaminated samples and yeast were determined in 32.6%, 20.0%, 2.5%, 14.1% and 1.1% of the samples, respectively. In 29.7% of the samples, microbiological growth could not be identified. Using the Petrifilm™ concept, bacterial growth was detected in 59% of the culture-negative samples. The sensitivity of the Petrifilm™ for Gram-positive and Gram-negative MCP was 85.2% and 89.9%, respectively. The specificity was 75.4% for Gram-positive and 88.4% for Gram-negative MCP. For the culture-negative samples, sensitivity was 41.0% and specificity was 91.0%. The results indicate that the Petrifilm™ concept is suitable for therapeutic decision-making at the farm level or in veterinary practice. As this concept does not allow any statement about the genus or species of microorganisms, relevant MCP should be assessed periodically at the herd level with conventional microbiological diagnostics.