Direct detection of lactococcal bacteriophages in cheese whey using DNA probes

Abstract We have designed a new method for the rapid detection of lactococcal phage directly in milk samples. The method was based on a dot blot analysis and did not require a biological assay with sensitive indicator strains. Culture media or whey permeate samples containing phage were spotted directly onto a nylon membrane and the phage were lysed in situ prior to hybridization. For skim milk, whole milk and whey samples, the samples were first treated with 50 mM EDTA, 20% Triton X-100 and heated at 60°C for 5 min, prior to spotting on the membrane. The detection limit was approximately between 10⁵ and 10⁷ pfu/spot. A large number of samples could be processed simultaneously and the results were obtained within 24 h.     

Characterization and classification of virulent lactococcal bacteriophages isolated from a Cheddar cheese plant

C.N. CASEY, E. MORGAN, C. DALY AND G.F. FITZGERALD, 1993. Twenty-two bacteriophages, isolated from cheese-vat whey samples over a period of 4 years, were found to be active against one or more of four different strains of Lactococcus lactis subsp. cremoris used in a defined strain starter system in an Irish Cheddar cheese factory. All the phages were small isometric-headed with non-contractile tails, a baseplate and a collar; they had genome sizes of approximately 30 kb, and belonged to a single DNA hybridization group. All 22 phages could be classified into four distinct groups based on restriction analysis which overlapped perfectly with those based on host range. Each group of phage examined showed cross-reactive host ranges. None of the phage DNAs hybridized to the chromosomes of any of the seven cultures used in the factory during the four cheesemaking seasons, and neither could phages be induced from any of the strains by mitomycin-C or ultraviolet light treatment.     

Identification of the receptor-binding protein in 936-species lactococcal bacteriophages

The aim of this work was to identify genes responsible for host recognition in the lactococcal phages sk1 and bIL170 belonging to species 936. These phages have a high level of DNA identity but different host ranges. Bioinformatic analysis indicated that homologous genes, orf18 in sk1 and orf20 in bIL170, could be the receptor-binding protein (RBP) genes, since the resulting proteins were unrelated in the C-terminal part and showed homology to different groups of proteins hypothetically involved in host recognition. Consequently, chimeric bIL170 phages carrying orf18 from sk1 were generated. The recombinant phages were able to form plaques on the sk1 host Lactococcus lactis MG1614, and recombination was verified by PCR analysis directly with the plaques. A polyclonal antiserum raised against the C-terminal part of phage sk1 ORF18 was used in immunogold electron microscopy to demonstrate that ORF18 is located at the tip of the tail. Sequence analysis of corresponding proteins from other lactococcal phages belonging to species 936 showed that the N-terminal parts of the RBPs were very similar, while the C-terminal parts varied, suggesting that the C-terminal part plays a role in receptor binding. The phages investigated could be grouped into sk1-like phages (p2, fd13, jj50, and phi 7) and bIL170-like phages (P008, P113G, P272, and bIL66) on the basis of the homology of their RBPs to the C-terminal part of ORF18 in sk1 and ORF20 in bIL170, respectively. Interestingly, sk1-like phages bind to and infect a defined group of L. lactis subsp. cremoris strains, while bIL170-like phages bind to and infect a defined group of L. lactis subsp. lactis strains.     

Taxonomic differentiation of 101 lactococcal bacteriophages and characterization of bacteriophages with unusually large genomes

Sixty-three virulent bacteriophages of Lactococcus lactis were differentiated by DNA-DNA hybridization. The results, including those of a previous classification of 38 phages of the same bacterial species (P. Relano, M. Mata, M. Bonneau, and P. Ritzenthaler, J. Gen. Microbiol. 133:3053-3063, 1987) show that 48% of the phages analyzed belong to a unique DNA homology group (group III). Phages of this most abundant group had small isometric heads. Group I comprised 29% of the phages analyzed and was characterized by a small phage genome (19 to 22 kilobases) and a particular morphology with a prolate head. Like group III, this group contained representative phages of other classifications. Group II (21%) included virulent and temperate phages with small isometric heads. Two large isometric-headed phages, phi 109 and phi 111, were not related to the three DNA homology groups I, II, and III. The genome of phi 111 was unusually large (134 kilobases) and revealed partial DNA homology with another large isometric phage, 1289, described by Jarvis (type e) (A. W. Jarvis, Appl. Environ. Microbiol. 47:343-349, 1984). The protein compositions of phi 111 and 1289 were similar (three common major proteins of 21, 28, and 32 kilodaltons).     

Multiplex PCR for detection and identification of lactococcal bacteriophages

Three genetically distinct groups of Lactococcus lactis phages are encountered in dairy plants worldwide, namely, the 936, c2, and P335 species. The multiplex PCR method was adapted to detect, in a single reaction, the presence of these species in whey samples or in phage lysates. Three sets of primers, one for each species, were designed based on conserved regions of their genomes. The c2-specific primers were constructed using the major capsid protein gene (mcp) as the target. The mcp sequences for three phages (eb1, Q38, and Q44) were determined and compared with the two available in the databases, those for phages c2 and bIL67. An 86.4% identity was found over the five mcp genes. The gene of the only major structural protein (msp) was selected as a target for the detection of 936-related phages. The msp sequences for three phages (p2, Q7, and Q11) were also established and matched with the available data on phages sk1, bIL170, and F4-1. The comparison of the six msp genes revealed an 82. 2% identity. A high genomic diversity was observed among structural proteins of the P335-like phages suggesting that the classification of lactococcal phages within this species should be revised. Nevertheless, we have identified a common genomic region in 10 P335-like phages isolated from six countries. This region corresponded to orfF17-orf18 of phage r1t and orf20-orf21 of Tuc2009 and was sequenced for three additional P335 phages (Q30, P270, and ul40). An identity of 93.4% within a 739-bp region of the five phages was found. The detection limit of the multiplex PCR method in whey was 10(4) to 10(7) PFU/ml and was 10(3) to 10(5) PFU/ml with an additional phage concentration step. The method can also be used to detect phage DNA in whey powders and may also detect prophage or defective phage in the bacterial genome.     

An improved plaque assay for poor plaque-producing temperate lactococcal bacteriophages

This report summarizes the results from an effort to optimize the double-agar plate assay for visualization of the plaques made by six temperate bacteriophages induced from industrial strains of Lactococcus lactis . Among the several parameters found to influence the plaque assay, the effect of incorporating glycine into the growth medium was most striking, resulting in extensive increase in the plaque size of all of the 13 phage–host pairs tested. Notable effects on the plaque size of other factors such as the procedure for sterilization of the agar medium, the volume and softness of the top and bottom layers, and the number and growth stage of the bacterial cells added to the lawn, were also observed. By exploiting these findings in an optimized procedure for plaque assaying, several indicator strains were identified which were unable to support the development of plaques on standard double-agar plates. Since bacterial hosts usually are identified by their ability to support the development of plaques, this observation suggests that the severe difficulty experienced in identifying lactococcal starter strains that are sensitive towards a temperate phage, partly is a problem of methodology.     

Occurrence of Propionibacterium freudenreichii bacteriophages in swiss cheese.

We isolated bacteriophages active against Propionibacterium freudenreichii from 16 of 32 swiss cheese samples. Bacteriophage concentrations ranged from 14 to 7 x 10(5) PFU/g, depending on the sample and the sensitive strain used for detection. Only a few strains, 8 of the 44 strains of P. freudenreichii in our collection, were sensitive. We observed that multiplication of bacteriophages occurred in the cheese loaf during multiplication of propionibacteria in a warm curing room, but it seems that these bacteriophages have no adverse effect on the development of the propionic flora. We also found that sensitive cells, originating from either the starter or the cheese-making milk, were present at a high level (10(9) CFU/g) in the cheese.     

Detection of lactococcal 936-species bacteriophages in whey by magnetic capture hybridization PCR targeting a variable region of receptor-binding protein genes

AIMS: To develop PCR assays able to distinguish between groups within lactococcal 936-species bacteriophages, as defined by their different receptor-binding protein (RBP) genes. METHODS AND RESULTS: DNA sequences of RBP genes from 17 lactococcal bacteriophages of the 936-species were compared, and six phage groups were identified. For each phage group a specific primer pair targeting a variable region of the RBP genes was designed. In nine of 20 whey samples, from dairies with recorded phage problems, between one and six phage groups were identified by conventional PCR. The sensitivity and specificity of the method was improved by magnetic capture hybridization (MCH)-PCR using a capture probe targeting an 80-bp highly conserved region just upstream from the RBP gene in all the investigated phages. The MCH-PCR was performed on 100 microl whey samples and the detection limit of the assay was 10(2)-10(3) PFU ml(-1) as opposed to the detection limit of 10(4) PFU ml(-1) for conventional PCR performed on 1-microl whey samples. CONCLUSIONS: In this study, PCR assays have been developed to detect six different types of RBP genes in lactococcal 936-species bacteriophages. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCR assays have practical applications at cheese plants for detection of 936-species phages with different RBP and thereby potentially with different host ranges. This knowledge will make it possible to improve starter culture rotation systems in the dairy industry.     

Proteolytic enzyme activities in Cheddar cheese juice made using lactococcal starters of differing autolytic properties

AIMS: To determine proteolytic enzyme activities released in Cheddar cheese juice manufactured using lactococcal starter strains of differing autolytic properties. METHODS AND RESULTS: The activities of residual chymosin, cell envelope proteinase and a range of intracellular proteolytic enzymes were determined during the first 70 days of ripening when starter lactococci predominate the microbial flora. In general, in cell free extracts (CFE) of the strains, the majority of proteolytic activities was highest for Lactococcus lactis HP, intermediate for L. lactis AM2 and lowest for L. lactis 303. However, in cheese juice, as ripening progressed, released proteolytic activities were highest for the highly autolytic strain L. lactis AM2, intermediate for L. lactis 303 and lowest for L. lactis HP. CONCLUSIONS: These results indicate that strain related differences in autolysis influence proteolytic enzyme activities released into Cheddar cheese during ripening. No correlation was found between proteolytic potential of the starter strains measured in CFE prior to cheese manufacture and levels of activities released in cheese juice. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings further support the importance of autolysis of lactococcal starters in determining the levels of proteolytic activities present in cheese during initial stages of ripening.     

Metabolism of airborne methyl salicylate in adjacent plants

ABSTRACT

Salicylic acid (SA) and methyl salicylate (MeSA) are synthesized in many plants and are crucial components that establish their disease responses. The metabolism of airborne MeSA to SA has been previously reported. In this report, it was found that SA glucose ester (SAGE), ether (SAG), and salicyloyl-L-aspartic acid (SA-Asp) are metabolites of airborne MeSA. Furthermore, it was found that airborne MeSA was able to increase the endogenous amount of rosmarinic acid in Perilla frutescens, which is known as one of the functional components that contributes to the maintenance of human health.     

Exposure to mutagenic airborne particulate in a rubber manufacturing plant

Epidemiological studies conducted in the 1980s revealed that people working in the rubber manufacturing industry had an increased risk of cancer. Even now, workers employed in rubber processing are still at risk despite the measures adopted to improve their working conditions. The aim of the study was to evaluate the presence of a genotoxic risk in a rubber industry and to verify whether or not it was possible to locate the most dangerous position among the different rubber-working processes. The mutagenic activity of airborne particulate was evaluated in samples collected in the mixing department of a rubber manufacturing plant. Ambient air samples were taken over 3-h period in two stable positions near the mixing (Banbury mixer) and calendering areas. Personal air samples were taken over 2-h period during a normal workday from five workers employed in different rubber processing operations (mixing, weighing, calendering, compounding and extruding). The mutagenic activity of the air samples was determined by plate incorporation assay using Salmonella typhimurium strains (TA 98, TA 98NR, TA 100, YG 1021) with and without metabolic activation. Polycyclic aromatic hydrocarbon (PAH) concentrations were determined by high-performance liquid chromatography (HPLC); the presence of other presumable contaminants were carried out by gas chromatography-mass spectrometry (GC-MS). The results showed substantial direct and indirect frameshift mutagenicity in both ambient and personal samples. No mutagenic activity was present in S. typhimurium TA 100, except in the personal sample from a worker employed on the Banbury mixer. HPLC analysis revealed very low concentrations of PAHs. GC-MS analysis showed the presence of compounds such as azulene derivative, 1,2-dihydro-2,2,4-trimethylquinoline, N-methyl N-phenylbenzenamine, diphenylamine, bis(2-ethylhexyl)phthalate and bis(methyl-propyl)phthalate. We conclude that the high levels of mutagenic activity in ambiental and personal samples indicate the presence of substances with high genotoxic potency; no substantial differences were seen among the several rubber processing operations. PAHs were not involved in indoor pollution. GC-MS analysis revealed the presence of compounds which may be produced by high temperatures to which the raw materials are subjected during rubber manufacturing processes. These substances are potential carcinogen though their mutagen properties have not been clearly determined.     

Detection and enumeration of airborne biocontaminants

The sampling and analysis of airborne microorganisms has received attention in recent years owing to concerns with mold contamination in indoor environments and the threat of bioterrorism. Traditionally, the detection and enumeration of airborne microorganisms has been conducted using light microscopy and/or culture-based methods; however, these analyses are time-consuming, laborious, subjective and lack sensitivity and specificity. The use of molecular methods, such as quantitative polymerase chain reaction amplification, can enhance monitoring strategies by increasing sensitivity and specificity, while decreasing the time required for analysis.     

Ragweed plants and airborne pollen in the Baltic states

Presence of pollen in the atmosphere was determined inTartu in 1989–1997, in Kuressaare in 1996–1997 and iniauliai in 1997. Ragweed airpollen was found in latesummer in three years. The distribution, abundance andflowering of local ragweed plants were estimated. Itappeared that the number of local sources did notexceed twenty per country per year. Majority of thesesources consisted of a small number of pollinating A. artemisiifolia individuals. The largest localsources, consisting of several hundreds ofindividuals, were exclusively rare. Transport ofragweed pollen by air masses from distant sources wasstudied. It was found that incursions of ragweedpollen were determined by air fluxes originating fromthe Ukraine as well as from the southeastern andsouthern regions of the European part of Russia. Theseincursions occurred in extensive high-pressure areaswhose centre was located in the central region of theEuropean part of Russia with a periphery extending tothe Baltic countries.     

Restriction/Modification systems and restriction endonucleases are more effective on lactococcal bacteriophages that have emerged recently in the dairy industry

Recently, eight lytic small isometric-headed bacteriophages were isolated from cheese-manufacturing plants throughout North America. The eight phages were different, but all propagated on one strain, Lactococcus lactis NCK203. On the basis of DNA homology, they were classified in the P335 species. Digestion of their genomes in vitro with restriction enzymes resulted in an unusually high number of type II endonuclease sites compared with the more common lytic phages of the 936 (small isometric-headed) and c2 (prolate-headed) species. In vivo, the P335 phages were more sensitive to four distinct lactococcal restriction and modification (R/M) systems than phages belonging to the 936 and c2 species. A significant correlation was found between the number of restriction sites for endonucleases (purified from other bacterial genera) and the relative susceptibility of phages to lactococcal R/M systems. Comparisons among these three phage species indicate that the P335 species may have emerged most recently in the dairy industry.     

Identification of the lower baseplate protein as the antireceptor of the temperate lactococcal bacteriophages TP901-1 and Tuc2009

The first step in the infection process of tailed phages is recognition and binding to the host receptor. This interaction is mediated by the phage antireceptor located in the distal tail structure. The temperate Lactococcus lactis phage TP901-1 belongs to the P335 species of the Siphoviridae family, which also includes the related phage Tuc2009. The distal tail structure of TP901-1 is well characterized and contains a double-disk baseplate and a central tail fiber. The structural tail proteins of TP901-1 and Tuc2009 are highly similar, but the phages have different host ranges and must therefore encode different antireceptors. In order to identify the antireceptors of TP901-1 and Tuc2009, a chimeric phage was generated in which the gene encoding the TP901-1 lower baseplate protein (bppL(TP901-1)) was exchanged with the analogous gene (orf53(2009)) of phage Tuc2009. The chimeric phage (TP901-1C) infected the Tuc2009 host strain efficiently and thus displayed an altered host range compared to TP901-1. Genomic analysis and sequencing verified that TP901-1C is a TP901-1 derivative containing the orf53(2009) gene in exchange for bppL(TP901-1); however, a new sequence in the late promoter region was also discovered. Protein analysis confirmed that TP901-1C contains ORF53(2009) and not the lower baseplate protein BppL(TP901-1), and it was concluded that BppL(TP901-1) and ORF53(2009) constitute antireceptor proteins of TP901-1 and Tuc2009, respectively. Electron micrographs revealed altered baseplate morphology of TP901-1C compared to that of the parental phage.     

Classification of virulent bacteriophages of Streptococcus salivarius subsp. thermophilus isolated from yoghurt and Swiss-type cheese

Summary Twelve isometric-headed bacteriophages virulent against Streptococcus salivarius subsp. thermophilus were differentiated into three subgroups by analysis of the phage genomes and the structural proteins. Subgroup I is composed of two phages (P6 and P8) with a genome size of 41.2 and 44.2 kb pairs, respectively, complete DNA homology, and identical protein composition (main proteins of sizes 39.8, 24.0, 14.8 kilodaltons in sodium dodecyl sulphate-polyacrylamide gel electrophoresis). One phage (a10/J9) with low DNA homology to the other phages was classified into subgroup II. Subgroup III consists of nine phages with a genome size of 33.8 to 36.7 kb pairs and two major structural proteins (30.9 and 24.0 kilodaltons, or 30.9 and 26.3 kilodaltons). In general, phages with different host spectra revealed different restriction enzyme patterns, and DNA homologies of various degrees were detected among all phages tested.     

Study of Enterobacteriaceae throughout the manufacturing and ripening of hard goats' cheese

The evolution of the counts and the species of Enterobacteriaceae as well as some physico-chemical parameters (pH, α_w and NaCl and moisture contents) during manufacturing and ripening of a hard Spanish goats' cheese of the Armada-Sobado variety were studied. Enterobacteriaceae (mean log counts 4.45 g^-1 in milk) increased 0.71–2.18 log units in curd and afterwards decreased until they disappeared after 2–4 weeks of ripening. This premature disappearance seems to be due to the decrease in α_w values and in moisture contents. However, the low pH values, reached from the beginning of the ripening process, could also contribute to this phenomenon.
The most abundant species in milk was Serratia liquefaciens (57.5% of isolates), followed by Morganella morganii (27.5%), Hafnia alvei (5%), Klebsiella oxytoca (5%) and Yersinia enterocolitica (5%). Yersinia enterocolitica was not subsequently isolated from either curd or in cheese. Hafnia alvei numbers increased in curd and in 1-week-old cheese where this micro-organism was the most abundant (47.5% and 75% of the isolates respectively). Escherichia coli , which was not isolated from milk, curd or 1-week-old cheese, was the predominant organism in 2-week-old cheese (57.8% of isolates). This confirms the finding of other authors who have shown that it is one of the most resistant species in ripening cheeses.