Histamine-forming Bacteria Research Articles

Inhibitory activity of natural antimicrobial compounds against histamine-forming bacterial isolates from cheese

This study was conducted to investigate the presence of histamine-forming bacteria in cheeses, and to assess the impact of naturally-occurring antimicrobials on isolates' viability and histamine-forming potential. Cheese isolates' ability to produce histamine was evaluated by incubation in the presence of the histamine precursor; qualitative detection with a pH indicator and a quantitative analysis by high-performance liquid chromatography. Eight of the 114 isolates were able to produce histamine, but only one was capable of generating histamine at high concentrations. Histamine-producing isolates were identified by matrix-assisted laser desorption ionisation-time of flight mass spectrometry. Isolated strain C_MRSV_25A_4 was the microorganism with the highest capacity to produce histamine, and was identified as Lacticaseibacillus rhamnosus. The antimicrobial potential of natural antimicrobials was tested against this isolate. The results showed that carvacrol, cinnamaldehyde, eugenol, thymol and nisin exhibited remarkable bactericidal activity. These compounds’ inhibitory potential was evaluated against the histamine-generating ability of strain C_MRSV_25A_4 at three concentrations of interest: minimum bactericidal concentration (MBC), MBC/2 and MBC/4. The results on histamine-forming capability at sub-bactericidal concentrations were promising. Given these results, we propose novel antimicrobial formulations based on natural antimicrobials as efficient strategies to reduce potential microbial contamination and histamine accumulation throughout cheese production.

Physicochemical and microbiological attributes of dried anchovies (Stolephorus commersonnii) in the formation of histamine along the supply chain.

Food safety along the supply chain must be safeguarded to protect the public's health from any foodborne disease. Despite the consumption of fish products, scombroid poisoning is not a wellknown occurrence. This study was formulated to evaluate at which stage of the supply chain the increase in the histamine content of dried anchovies (Stolephorus commersonnii) occurs. The study showed that histamine substances and Staphylococcus aureus were detected at all stages of the supply chain. It was also found that the concentration of histamine generally increased from the fisherman to the processors and retailers. Post-hoc analysis using the Fisher test showed no significant difference in the histamine content of dried anchovies from the processing centers (𝑥̅ =5 4.07 mg/kg) and retailers (𝑥̅ = 67.63 mg/kg), but it was significantly higher than in the raw anchovies (𝑥̅ = 8.29 mg/kg). S. aureus remains low at <10 colony-forming units (CFU/g). The aerobic plate count supports this conclusion with a 90.29% increase from the processing centers to the retailers. However, it is still significant as a source of histamine formation influenced by water activity and salt content. A correlation between salt content and water activity with histamine content was identified with a Pearson correlation coefficient of 0.8357. It is recommended to review the processing method to control the salt content and the handling and storage at the retailers, as they showed the highest potential for histamine-forming bacteria growth, leading to an increase in histamine levels in dried anchovies.

Prevalence, isolation and identification of histamine forming bacteria in selected stages of supply chain of Yellowfin tuna export industry in Sri Lanka

This study aimed to identify histamine-forming bacteria (HFB) and the sources of introduction of such bacteria to recommend control measures to mitigate histamine formation in yellowfin tuna (YFT). Field samples were collected from multi-day boats that landed at Dikkowita, Negombo, Trincomalee and Dondra fishery harbours. Ice from the fish holds (n=63) and chill transport vehicles (n=63), and swabs from the fish holds (n=63), the boat decks (n=63) and the skin of YFT (n=63) were collected. Fish loin samples (n=15), ice samples (n=36) and swabs from the skin of YFT (n=18), floor (n=18) and chill transport vehicles (n=18) were collected from fish processing plants. Presumptive HFB isolated from Nivens medium and Violet Red Bile Glucose (VRBG) agar were screened for histamine forming ability in Tripticase soy broth (TSB) supplemented with 1.0% L-histidine. HFB isolates were characterized by sequencing approximately 1400 bp of the 16S rDNA. Seven isolates that produced histamine in the range of 3000–4000 ppm in TSB isolated from ice samples, and a swab sample collected from the boat deck, were confirmed as Klebsiella aerogens (n=6) and Morganella morganii (n=1) respectively. Hafnia alvei (n=1), Serratia sp. (n=2), Citrobacter freundii (n=1), Rahnella sp. (n=1) and Aeromonas salmonicida (n=8) were also among the isolated histamine forming bacteria. Pseudomonas sp. (n=24) and Shewanella baltica (n=7), which are known as spoilage bacteria were also isolated and showed weak histamine formation. Hence, it is evident that histamineforming bacteria could be introduced into the fish from ice and contacting surfaces. This necessitates the practice of rigorous cleaning procedures and adaptation of proper postharvest handling procedures to minimize contamination of the fish.

HISTAMINE LEVEL AND HISTAMINE-PRODUCING BACTERIA ISOLATED FROM SALTED AND FREEZE SARDINE FISH (Sardina spp.)

Histamine is the biogenic amine responsible for scombroid poisoning in various types of fish and fish products. The study evaluated histamine content and bacteriological quality of salted and freeze sardine samples. All fish samples were cultured on Reinforced Closterdial medium; mannitol salt (MS) agar and violet red bile glucose (VRBG) agar, the positive colonies were sub-cultured on histamine-forming bacterium agar, followed by biochemical and PCR identification. Production of histamine in the fish muscles was measured by high performance liquid chromatography. The mean histamine values were about; 8.25 mg/100g. The mean of total aerobic counts was; 4×104 ± 2.1×104 while, the total anaerobic count; 5×104 ±1.2×103 Log CFU/g. Enterobacteriacea spp. about; 5×104 ±2.5×104 Log CFU/g while, the mean value of Staphylococcus spp. about; 3×104 ±3.7×104 Log CFU/g. The mean value of histamine-forming bacterial were; 3.3×106 Log CFU/g. Molecular Identification For Enterobacteriaceae were Klebsiella pneumonia, Staphylococcus xylosus, Escherichia coli and Enterobacter cloacae. We concluded that during storage, there was a positive relationship between the histamine levels and the histamine-forming bacteria (HFB) numbers, while, histamine and the bacteria forming histamine were recorded in all examined sardine samples. Key words: histamine; Escherichia coli; Klebsiella pneumonia; molecular identification; scombroid; Staphylococcus xylosus

Prevalence of Histamine-Forming Bacteria in Two Kinds of Salted Fish at Town Markets of Guangdong Province of South China

Histamine-forming bacteria (HFB) were isolated from the 70 salted fish samples bought from town markets of Guangdong Province of south China. In addition, the histamine-forming ability of HFB was analyzed. There were 31 strains of HFB isolated from 36 salted fish pickled overnight. They were identified as six bacteria species: Vibrio alginolyticus, Vibrio rumoiensis, Staphylococcus saprophyticus, Staphylococcus xylosus, Lactococcus lactis, and Morganella morganii. The rate of confirmation of V. alginolyticus was highest (23 of 31), exceeding 200 mg/kg histamine. In particular, M. morganii produced a histamine amount that exceeded 2,000 mg/kg, although it was only one isolate in this study. In addition, five bacteria species of HFB were isolated from 34 dried salted fish. Among them, S. saprophyticus was dominant in the dried salted fish but produced histamines below 200 mg/kg. However, Enterobacter aerogenes from dried salted fish formed a histamine amount exceeding 200 mg/kg. The study showed that the dominant strain of HFB was different in two kinds of salted fish. Both kinds of salted fish contained HFB whose histamine-forming capacity exceeded 200 mg/kg. As a result, the safety of salted fish should be of concern, especially salted fish pickled overnight.

Inhibitory Effects of High-Hydrostatic-Pressure Processing on Growth and Histamine Formation of Histamine-Forming Bacteria in Yellowfin Tuna Meat during Storage.

Simple SummaryHigh-hydrostatic-pressure treatment effectively inhibited the growth and histamine formation of histamine-forming bacteria species in tuna meat during storage at low temperatures. Our findings can be used to enhance food safety and decrease the risk of histamine poisoning associated with consumption of tuna meat products.In the research, we evaluated the effects of high-pressure processing (HPP) on the growth and histamine formation of histamine-forming bacteria (HFB) in yellowfin tuna meat during storage. Tuna meat samples inoculated with the individual HFB species Morganella morganii and Photobacterium phosphoreum were subjected to HPP treatment at 250, 350, 450, and 550 MPa for 5 min, and the changes in bacterial count, total volatile basic nitrogen (TVBN) content, pH, and histamine content during storage at 4 and 15 °C were analyzed. The results indicate that the bacterial counts of the HFB species decreased significantly with increasing pressure, and HFB became undetectable in the samples treated at 450 and 550 MPa. At a storage temperature of 15 °C, the bacterial counts of both HFB species in the control group and samples subjected to HPP treatment at 250 and 350 MPa increased significantly with storage time. The bacterial counts of M. morganii in the samples stored at 4 °C decreased, whereas those of P. phosphoreum increased gradually owing to its psychrophilic nature. HPP treatment (>250 MPa) inhibited the increases in pH and TVBN content of the samples stored at 15 °C and delayed histamine formation in the samples during storage; these effects were more significant as the pressure during HPP treatment was increased.

Gamma-irradiation induced effects on histamine-forming bacteria isolated from the chilled mackerel fish

Gamma-irradiation induced effects on histamine-forming bacteria isolated from the chilled mackerel fish

Proofing banana blossom (Musa acuminata) water-soluble extract as histamine forming antibacteria

Banana blossom is a type of vegetable that can be used as a source of fiber. The previous studies showed that the addition of banana blossoms to the baby tuna shredded caused a decrease in the number of microbes compared to control. Histamine-forming bacteria in baby tuna originated from indigenous bacteria and contaminants such as Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, and Lactobacillus plantarum. This study was aimed to find out how much water-soluble extract of banana blossom anti-bacterial against histamine-forming bacteria. Testing of anti-bacterial ingredients in banana blossom Musa acuminata water-soluble extract with LC-HNMR, toxicity testing, and amino acid testing. The studies using the Kirby Bauer method and corroborated by SEM observations showed that water-soluble extract of banana blossom Musa acuminata had low anti-bacterial properties for Lactobacillus plantarum and Klebsiella pneumonia but not for Escherichia coli and Pseudomonas aeruginosa. Further testing of the anti-bacterial substance water-soluble extract of banana blossom was histidine, proline, isoleucine, threonine, and glutamic acid. This extract categorized as a non-toxic substance

DETERMINATION OF HISTAMINE-FORMING BACTERIA, TOTAL COLIFORM AND ESCHERICHIA COLI IN FERMENTED FOODS

Fermented fishery food products have been generally sold in the local market, Thailand. These products were frequently contaminated with various microorganisms due to raw material and unhygienic production process. Determination of microbial contamination of fermented food products helps to assure safety for consumers. Therefore, the aims of the study were to determine total bacteria, total coliform, Escherichia coli and histamine producing bacteria from fermented fishery food products commercially distributed in several regions of Chumphon and Surat Thani provinces. Twenty-two samples of fermented fishery food products were randomly collected for bacterial contamination assay. All samples revealed the levels of pH and salt content in the range of 3.67-7.46 and 8.46%-29.69%, respectively. The bacterial contamination of the samples was tested by total plate count method. Determination of coliform bacteria and E. coli was examined by multiple-tube fermentation technique. Total bacteria, total coliform and E. coli of all samples were in the range of 2.15-7.54 log CFU/g, < 3-460 MPN/g and < 3-93 MPN/g, respectively. In addition, thirteen histamine-forming bacteria were found on histamine-forming bacteria isolation agar after incubation at 37°C for 4 days. Five isolates of histamine-forming bacteria named M1, M3, M9, M11, and M13 were selected to identify by molecular techniques. M1 and M3 were obtained from shrimp paste (Kapi) and M11 was obtained from fermented fish (Pla Pang Dang). They were identified as Enterobacter sp. M9 and M13 isolated from fermented fish (Pla Som and Pla Ra) were identified as Citrobacter farmeri and Staphylococcus kloosii, respectively.

Survival and Histamine Production by Histamine-Forming Bacteria Exposed to Low Doses of Gamma Irradiation

Histamine poisoning occurs when fish containing high amount of histamine are consumed. Because histamine is thermally stable, control of histamine-forming bacteria in seafood is an appropriate strategy for preventing the formation of histamine. One prevention method is the use of gamma irradiation on the histamine formers. To understand the effect of gamma irradiation on the histamine-forming bacteria, laboratory isolates of the prolific histamine formers Morganella morganii, Klebsiella variicola, and Proteus vulgaris were exposed to various doses of gamma radiation in nutrient broth and tuna muscle spiked with histamine formers. None of the test bacteria survived in tuna muscle irradiated at 2.0 kGy. K. variicola was highly sensitive to gamma irradiation and was eliminated at a dose of 1.5 kGy. Histamine production also was reduced significantly as the radiation dose increased. These results suggest that gamma irradiation can effectively eliminate histamine-forming bacteria and reduce the threat of histamine poisoning from seafood.

High-Pressure Inactivation of Histamine-Forming Bacteria Morganella morganii and Photobacterium phosphoreum

The effects of high hydrostatic pressure (HHP) treatments on histamine-forming bacteria (HFB) Morganella morganii and Photobacterium phosphoreum in phosphate buffer and tuna meat slurry were investigated using viability counting and scanning electron microscopy. The first-order model fits the destruction kinetics of high pressure on M. morganii and P. phosphoreum during the pressure hold period. The D-values of M. morganii (200 to 600 MPa) and P. phosphoreum (100 to 400 MPa) in phosphate buffer ranged from 16.4 to 0.08 min and 26.4 to 0.19 min, respectively, whereas those in tuna meat slurry ranged from 51.0 to 0.09 min and 71.6 to 0.19 min, respectively. M. morganii had higher D-values than P. phosphoreum at the same pressure, indicating it was more resistant to HHP treatment. HFB had a higher D-value in tuna meat slurry compared with that in phosphate buffer, indicating that the HFB were more resistant to pressure in tuna meat slurry. The Zp values (pressure range that results in a 10-fold change in D-value) of M. morganii and P. phosphoreum were 162 and 140 MPa in phosphate buffer and 153 and 105 MPa in tuna meat slurry, respectively. Damage to the cell wall and cell membrane by HHP treatments can be observed by scanning electron microscopy. To our knowledge, this is the first report to demonstrate that HHP can be applied to inactivate the HFB M. morganii and P. phosphoreum by inducing morphological changes in the cells.

Inactivation and Damage of Histamine-Forming Bacteria by Treatment with High Hydrostatic Pressure.

The inactivation and damage of histamine-forming bacteria (HFB), Enterobacter aerogenes and Staphylococcus capitis, in a 0.1 M potassium phosphate buffer (pH 6.8) and marlin meat slurry by high hydrostatic pressure (HHP) treatments were studied using viability measurement and scanning electron microscopy (SEM). HHP treatments showed first order destruction kinetics to E. aerogenes and S. capitis during the pressure holding period. HFB in marlin meat slurry had higher D values and were more resistant to HHP treatments than in phosphate buffer. In phosphate buffer, E. aerogenes had higher D values than S. capitis at >380 MPa of pressure, whereas the reverse trend was noticed at lower pressures (<380 MPa). In marlin meat slurry, S. capitis had a higher D value than E. aerogenes at the same treatment pressure, indicating that S. capitis was more resistant to HHP treatment. To our knowledge, this is the first report to demonstrate that HHP can be used to inactivate HFB, E. aerogenes, and S. capitis, by causing disruption to bacterial cell membrane and cell wall as demonstrated by SEM micrographs.

Assessing the quality of fresh Whitemouth croaker (Micropogonias furnieri) meat based on micro-organism and histamine analysis using NGS, qPCR and HPLC-DAD.

Quality evaluation of fresh whitemouth croaker (Micropogonias furnieri) by histamine determination using the HPLC-DAD method and quantification of histamine-forming bacteria using NGS and qPCR. The histamine content of fresh whitemouth croaker was detected by high performance liquid chromatography with diode array detector with a concentration ranging from 258·52 to 604·62mgkg-1 being observed. The number of histidine decarboxylase (hdc gene) copies from Gram-negative bacteria and the bacteria Morganella morganii and Enterobacter aerogenes were quantified by quantitative polymerase chain reaction. All samples were positive, with copy numbers of the hdc gene ranging from 4·67 to 12·01 log10 per g. The microbial community was determined by sequencing the V4 region of the 16S rRNA gene using the Ion Torrent platform. The bioinformatics data generated by frog software showed that the phylum Proteobacteria was the most abundant, with the family Moraxellaceae being more prevalent in samples collected in the summer, whereas the Pseudomonadaceae was more present in the winter. All fish muscle samples analysed in this study presented histamine values higher than those allowed by CODEX Alimentarius. Additionally, a wide variety of spoilage micro-organisms capable of expressing the enzyme histidine decarboxylase were detected. Thus, improvements in handling and processing are required to minimize the prevalence of histamine-producing bacteria in fish. Global fish production in 2016 was 171 million tons, with the largest consumer being China, followed by Indonesia and the USA. In Brazil, 1·3 million tons of fish are consumed per year, with whitemouth croaker being the main fish landed. Notably, cases associated with histamine poisoning are quite common. According to the European Food Safety Authority and European Centre for Disease Prevention and Control, a total of 599 HFP outbreaks were identified in the European Union during the period 2010-2017. In the USA, there were 333 outbreaks with 1383 people involved between 1998 and 2008.

Growth Rate and Histamine Production of Citrobacter freundii CK01 in Various Incubation Temperatures

Improper handling and temperature fluctuations during postharvest of fish commonly allow the growth of histamine-forming bacteria (HFB) that may cause histamine poisoning. Citrobacter freundii CK01 is one of the HFB isolated from Skipjack landed on Sadeng, Yogyakarta. This study aimed to determine the temperature effect on the growth rate and histamine production by C. freundii CK01. Bacterial growth and histamine production was tested in tuna fish infusion broth (TFIB) at 5, 15, 30 and 40°C. The bacterial growth was tested using Total Plate Count method, while histamine was determined using Thin Layer Chromatography and densitometry method. The primary model for bacterial growth was plotted with incubation time using DMFit referred to Baranyi &amp; Roberts model. The secondary model was converted from growth rate and modeled using Ratkowsky Square Root Model. The equation for growth rate of C. freundii CK01 was μmax=[0.0105 (T + 13.886)]2 (Root Mean Square Error &lt;10%). Histamine production reached the highest concentration at 15°C in 96 hours up to 117,13 ppm. This study shows that temperature affected the growth rate and histamine production of C. freundii, indicating the importance of maintaining the low temperature stability during handling of skipjack.

Evaluation of modified Zobell marine agar for differential isolation of histamine-forming bacteria from fresh fish

A new series of pyrimidine (8, 14, 18 and 23) embellished analogues of 1,5-benzodiazepines were synthesized by the one-pot domino approach using the catalyst DABCO (1,4-diazabicyclo[2.2.2]octane). For each compound synthesized, anti-microbial efficacy was determined using broth microdilution assay and half maximal inhibitory concentration (IC50). Furthermore, FESEM (Field emission scanning electron microscope) studies were also carried out to observe the effect of the structure of test compounds on the morphology of both Gram-positive (S. aureus) and Gram-negative (E. coli) cell walls. The leakage of nucleotides and their integral components from compromised bacterial cells was assessed by plotting the optical density (OD) with respect to time of exposure at 320 nm. Anti-bacterial studies revealed that compound 23 was most active against targeted bacterial species. Results of the antibacterial study indicated that all the test compounds possess significant antibacterial potential against targeted bacterial strains. Amongst all, in the FE-SEM study, compound 23 caused marked alteration in bacterial cell morphology and resulted in maximum leakage of cell nucleotides in bacterial strains as compared to controls. Further efforts are required to establish their efficacy as antibacterial agents in clinical management.

Molecular Characterization of Histamine-Producing Psychrotrophic Bacteria Isolated from Red Octopus (Octopus maya) in Refrigerated Storage.

The present study aimed at determining the histamine production capacity of Gram (+) and Gram (−) bacteria isolated from Octopus maya, along with identifying the presence of amino acid decarboxylase genes. Of the total 80 psychrotrophic microorganisms, 32 strains were identified as histamine-forming bacteria. The recombinant DNA technique was used for genotypic identification of histidine (hdc), ornithine (odc), and lysine decarboxylases (ldc) genes. Thirty-two strains were able to produce 60–100 ppm in trypticase soy broth with 1.0% l-histidine after 6 h at 20 °C. NR6B showed 98% homology with Hafnia alvei. NR73 represented 18.8% of the total isolates and showed 98% homology with Enterobacter xianfengensis and Enterobacter cloacae. NR6A represented 6% of the total isolates, which were identified as Lactococcus sp. The hdc gen from NR6B showed 100% identity with hdc from Morganella morganii; ldc showed 97.7% identity with ldc from Citrobacter freundii. The Odc gene was detected only in NR73 and showed 100% identity with Enterobacter sp. All the isolated were identified as weak histamine–former. The ingestion of a food containing small amounts of histamine has little effect on humans; however, the formation of biogenic amines is often considered as an indicator of hygienic quality; this emphasizes the importance of improving good management practices and storage.

Occurrence of Histamine and Histamine-forming Bacteria in Philippine Traditional Dried-salted Fish Products

Occurrence of Histamine and Histamine-forming Bacteria in Philippine Traditional Dried-salted Fish Products

CHITOSAN INHIBITION FROM SHELL OF WASTE CRAB CULTIVATION “SOKA” (SCYLLA SP) AGAINST 4 HISTAMINE FORMING BACTERIA ISOLATE IN EUTHYNNUS AFFINIS

The purpose of this study was to determine the inhibition of chitosan from crab cultivation waste "soka" (Scylla sp) against histamine-forming bacteria in tuna (Euthynnus affinis). The formation of histamine on the cob caused by bacteria that can produce the enzyme histidine decarboxylase to transform histidine into histamine. Several studies have described the ability of chitosan to inhibit the activity of histamine-forming bacteria. In this research, three stages, the first characterization of chitosan from crab shell waste cultivation "soka". Second, isolation of bacteria producing histamine on the cob and a third is testing the activity of chitosan against bacteria producing histamine. Characteristics of chitosan from crab shell waste cultivation "soka" is the water content of 10.07 %, 0.20 % fat, 1.42 % ash content, protein content 3.18 % and 58.99 % degree of deacetylation. The result of the isolation of bacterial symbionts on the cob re-examined in the formation of histamine thus obtained 4 isolates that Serratia marcescens, Enterobacteriaceae sp, Enterobacter gergoviae and Citrobacter amalonaticus. Testing inhibition of chitosan against bacteria Serratia marcescens, Enterobacteriaceae sp, Enterobacter gergoviae and Citrobacter amalonaticus obtained a clear zone, respectively, are 0.54 ± 0.03 cm; 0.59 ± 0.01 cm; 0.41 ± 0.01 cm; and 0.40 ± 0.05 cm.

Histamine Formation in a Dry Salted Twaite Shad (Alosa fallax lacustris) Product

Landlocked shad is a freshwater clupeid fish ( Alosa fallax lacustris ) whose consumption is associated with the risk of scombrotoxin poisoning. Traditionally, fresh shad are subjected to an artisanal processing procedure, consisting of dry salting and maturation under pressure, to give a fish product named missoltino , which is stored in large metallic barrels and is sold to local consumers and restaurants. In recent years, the introduction of modern food packaging technologies has enabled this product to also be distributed in shops and supermarkets. Consequently, the determination of the safety of this product is an urgent issue. The aims of the present research were to measure histamine levels and histamine-forming bacteria in shad products collected at different phases of preparation and ripening, in order to minimize poison hazards, to provide technical information about risk, and to standardize the production process. One hundred twenty-six samples of shad (21 fresh fish and 105 dried) at different phases of preparation and ripening were collected from seven producers and were analyzed for chemical composition, histamine content, and microbiological properties. After 130 days of ripening, samples from three producers presented unacceptable amounts of histamine (>200 mg/kg), according to European Union legislation. A moderate negative correlation was found between histamine levels and salt content (r =-0.504, P < 0.01) and between histamine levels and water phase salt content (r =-0.415, P < 0.01). Several bacterial strains that were positive on Niven's medium were isolated during the early phases of production, whereas the extreme environment of salted shad at the end of ripening led to a drastic decrease of bacteria, but not of histamine. The most effective preventive measures for histamine formation and accumulation in salted shad were strictly related to fish handling and storage conditions during processing.

Prevalence and diversity of histamine-forming Lactobacillus parabuchneri strains in raw milk and cheese – A case study

In the present case study, we investigated the causes of consistently elevated histamine content in raw milk cheeses from a Swiss cheese dairy. Screenings of milk samples from 67 farmers revealed that 19.1% of the raw milk samples were contaminated with histamine-forming bacteria. Lactobacillus parabuchneri was detected in 97.4% of contaminated milk samples. The population density of L. parabuchneri was almost always below the limit of quantification of the qPCR method used. Genotyping of 469 isolates provided detailed insight into the diversity of L. parabuchneri in contaminated milks and cheeses. The results of the trace-back study clearly demonstrated that several milk suppliers were responsible for the histamine problem at the cheese dairy under study. Systematic controls of milking systems allowed persistent contamination sources of L. parabuchneri at the farm level to be identified and eliminated and thereby to reduce the percentage of contaminated milks to 8.5% within the study period.