Phages of Lactobacillus casei/paracasei: response to environmental factors and interaction with collection and commercial strains

AIM: To investigate the influence of several environmental factors on the viability and cell-adsorption for two Lactobacillus casei/paracasei bacteriophages (PL-1 and J-1). METHODS AND RESULTS: Both phages showed a remarkably high specificity of species, sharing similar host spectra. Two phages and four sensitive strains were used to conform five phage/strain systems. Each showed a particular behaviour (burst size: ranging from 32 to 160 PFU/infective centre; burst time: 120-240 min and latent time: 5-90 min). For both phages, the viability was not significantly affected from pH 4 to 11 (room temperature) and from pH 5 to 10 (37 degrees C). Adsorption rates were not influenced by calcium ions, but decreased after the thermal inactivation of cells. Adsorption rates were high between 0 and 50 degrees C with maximum values at 30 degrees C and pH 6. System PL-1/Lact. paracasei A showed noticeable differences in comparison with the others, being times required to reach 90% of adsorption of 4 h and lower than 45 min, respectively. CONCLUSIONS: The data obtained in this work demonstrated that environmental parameters can influence the viability and cell adsorption rates of Lact. casei/paracasei phages. The extent of this influence was phage dependent. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes to the enlargement of the currently scarce knowledge of phages of probiotic bacteria.     

Integration of the group c phage JCL1032 of Lactobacillus delbrueckii subsp. lactis and complex phage resistance of the host

AIMS: Sequences related to Lactobacillus delbrueckii phage JCL1032 genome integration, the maintenance of lysogeny and putative immunity genes were characterized. Phenotypic changes of the JCL1032 lysogens were investigated. METHODS AND RESULTS: Integration of JCL1032 DNA into the host genome and the location of phage and bacterial attachment sites were studied by standard molecular methods. The frequency of lysogenization was 10(-7), and stable lysogeny was an even rarer phenomenon. JCL1032 integrates its genome into two distinct host genes of unknown functions. According to EOP (efficiency of plating) and adsorption tests JCL1032 lysogens showed resistance against several virulent and temperate Lactobacillus phages at different steps of phage infection. CONCLUSIONS: Temperate JCL1032 integrates its genome into bacterial DNA with exceptionally low frequency. JCL1032 lysogens express a complex phage resistance against several Lact. delbrueckii phages. An antagonistic arms race between the temperate phage and its host is proposed. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that the genome integration of a group c Lact. delbrueckii phage has been described. The characterized lysogens could facilitate studies on Lact. delbrueckii phage receptors and phage resistance mechanisms. The genetic information gained from this study benefits the development of integration vectors and phage resistant starters.     

Production, purification and characterization of chitosanase produced by Gongronella sp. JG

AIMS: To optimize the production condition of chitosanases of Gongronella sp. JG and to characterize the major chitosanase. METHODS AND RESULTS: In the optimized medium and culturing condition, strain JG produced 800 micromol min(-1) l(-1) chitosanase activity at 72 h. The major chitosanase - csn1 was purified through three chromatography steps: CM (carboxymethyl)-Sepharose fast flow (FF), Sephacryl S200, SP (sulfopropyl)-Sepharose FF. The molecular weight and the pI value of csn1 were about 90,000 Da and 5 x 8, respectively. Its specific activity was 82 micromol min(-1) mg(-1). The optimal reaction pH for csn1 was between 4 x 6 and 4 x 8. The optimal reaction temperature was 50 degrees C. The half-life of csn1 at 50 degrees C was estimated to be about 65 min. Mn(2+) was a strong stimulator of csn1 activity, both at 1 and 10 mmol l(-1). csn1 showed its highest activity with chitosan of 85% degree of deacetylation, but did not hydrolyse colloidal chitin and carboxylmethyl cellulose. In 20 mmol l(-1) sodium acetate buffer (pH 4 x 8) and at 50 degrees C, the K(m) of csn1 was calculated to be 4 x 5 mg ml(-1). CONCLUSIONS: The production condition of chitosanases by Gongronella JG was optimized and the major chitosanase, csn1, was characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: The present work for the first time reported the production, purification and characterization of chitosanases produced by fungus of Gongronella sp. These results provided us more information on fungal chitosanases.     

Effect of freezing modes on the survival of Escherichia coli bacteriophages

The object of this work was to study the effect of freezing down to--196 degrees C at different cooling and warming rates on the survival of T3, T4 and phiX174 phages. Phage particles survived when T3 phage was frozen at a rate of 20-400 degrees/min and phiX174 phage at a rate of 20-45 degrees/min. The survival rate of T4 phage was highest when it was frozen at a rate of 45 degrees/min. The survival of the phages depended also on the regime of warming. The susceptibility of the phages to freezing correlated with their sensitivity to osmotic shock in NaCl and sucrose solutions.     

Milk contamination and resistance to processing conditions determine the fate of Lactococcus lactis bacteriophages in dairies

Milk contamination by phages, the susceptibility of the phages to pasteurization, and the high levels of resistance to phage infection of starter strains condition the evolution dynamics of phage populations in dairy environments. Approximately 10% (83 of 900) of raw milk samples contained phages of the quasi-species c2 (72%), 936 (24%), and P335 (4%). However, 936 phages were isolated from 20 of 24 (85%) whey samples, while c2 was detected in only one (4%) of these samples. This switch may have been due to the higher susceptibility of c2 to pasteurization (936-like phages were found to be approximately 35 times more resistant than c2 strains to treatment of contaminated milk in a plate heat exchanger at 72 degrees C for 15 s). The restriction patterns of 936-like phages isolated from milk and whey were different, indicating that survival to pasteurization does not result in direct contamination of the dairy environment. The main alternative source of phages (commercial bacterial starters) does not appear to significantly contribute to phage contamination. Twenty-four strains isolated from nine starter formulations were generally resistant to phage infection, and very small progeny were generated upon induction of the lytic cycle of resident prophages. Thus, we postulate that a continuous supply of contaminated milk, followed by pasteurization, creates a factory environment rich in diverse 936 phage strains. This equilibrium would be broken if a particular starter strain turned out to be susceptible to infection by one of these 936-like phages, which, as a consequence, became prevalent.     

Characterization of three Lactobacillus delbrueckii subsp. bulgaricus phages and the physicochemical analysis of phage adsorption

AIMS: Three indigenous Lactobacillus delbrueckii subsp. bulgaricus bacteriophages and their adsorption process were characterized. METHODS AND RESULTS: Phages belonged to Bradley's group B or the Siphoviridae family (morphotype B1). They showed low burst size and short latent periods. A remarkably high sensitivity to pH was also demonstrated. Indigenous phage genomes were linear and double-stranded DNA molecules of approx. 31-34 kbp, with distinctive restriction patterns. Only one phage genome appeared to contain cohesive ends. Calcium ions did not influence phage adsorption, but it was necessary to accelerate cell lysis and improve plaque formation. The adsorption kinetics were similar on viable and nonviable cells, and the adsorption rates were high between 0 and 50 degrees C. SDS and proteinase K treatments did not influence the phage adsorption but mutanolysin and TCA reduced it appreciably. No significant inhibitory effect on phage adsorption was observed for the saccharides tested. This study also revealed the irreversibility of phage adsorption to their hosts. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The study increases the knowledge on phages of thermophilic lactic acid bacteria.     

Diversity among Lactobacillus paracasei phages isolated from a probiotic dairy product plant

Aims:  To evaluate the phage diversity in the environment of a dairy industry which manufactures a product fermented with a probiotic strain of Lactobacillus paracasei .
Methods and Results:  Twenty-two Lact. paracasei phages were isolated from an industrial plant that manufactures a probiotic dairy product. Among them, six phages were selected based on restriction profiles, and two phages because of their notable thermal resistance during sample processing. Their morphology, host range, calcium dependency and thermal resistance were investigated. All phages belonged to the Siphoviridae family (B1 morphotype), were specific for Lact. casei and paracasei strains showing identical host spectrum, and only one phage was independent of calcium for completing its lytic cycle. Some of the phages showed an extraordinary thermal resistance and were protected by a commercial medium and milk.
Conclusions:  Phage diversity in a probiotic product manufacture was generated to a similar or greater extent than during traditional yogurt or cheese making.
Significance and Impact of the Study:  This work emphasizes probiotic phage infections as a new ecological situation beyond yogurt or cheese manufactures, where the balanced coexistence between phages and strains should be directed toward a favourable state, thus achieving a successful fermentation.     

Physicochemical characterization of phage adsorption to Lactobacillus helveticus ATCC 15807 cells

Characterization of the adsorption process by the phages hv and ATCC 15807-B1 to Lactobacillus helveticus ATCC 15807 was carried out. For this purpose, the influence of Ca²⁺ ions, temperature and physiological cell state were studied. The ability of several saccharides and related compounds to inactivate the phages hv and ATCC 15807-B1 was determined to investigate their potential role as phage receptors. Furthermore, several chemical treatments on the sensitive strain cells were carried out to study their influence on phage adsorption. Cell lysis and plaque formation were independent of Ca²⁺ ions for phage hv, but the cation was indispensable for completion of the lytic cycle of phage ATCC 15807-B1. However, for this phage, Ca²⁺ was not necessary for the adsorption process. The adsorption rates were almost normal for both phages within the temperature range examined (0 – 50 °C) and the adsorption kinetics were practically identical on viable and non-viable cells. The saccharides and related compounds used did not produce inactivation of the phages, suggesting that they were not essential components of phage receptor structures. Lactobacillus helveticus ATCC 15807 cells treated with SDS 1%, SDS 0·5% -EDTA 50 mmol l⁻¹ or NaOH 50 mmol l⁻¹ exhibited reduced adsorption of the phages, indicating possible damage or extraction of receptors from the cell wall. Phage adsorption presents an extremely attractive target for interfering in the lytic cycle of phages.     

The influence of dissolved CO(2) concentration on the death kinetics of Saccharomyces cerevisiae

AIMS: The effects of temperature and concentration of dissolved CO(2) on the inactivation of Saccharomyces cerevisiae were investigated using a plug-flow system. METHODS AND RESULTS: Several combinations of pressure (4, 6, 8, 10 mega-Pa (MPa)) and temperature (30, 34, 36, 38 degrees C) were used. The D-values obtained were 0.14 min at 8 MPa and 38 degrees C, and 0.15 min at 10 MPa and 36 degrees C. The log D-values were related linearly to the treatment temperature and to the dissolved CO(2) concentration. The thermal resistance constant (zCO(2)(T)) was 9.5 degrees C in the media, including significant levels of CO(2), and the CO(2) resistance constant was z(temp.)(gamma)=7.2 gamma. CONCLUSION: This work has shown that inactivation followed first-order death kinetics, and the effects of temperature and CO(2) concentration were consistent through the critical temperature and pressure of CO(2). Therefore, it is feasible to estimate D-values at any temperature and any CO(2) concentration. SIGNIFICANCE AND IMPACT OF THE STUDY: Non-thermal inactivation of micro-organisms in acidic beverages could be realized by the present technique.     

Isolation and characterization of a Lactobacillus fermentum temperate bacteriophage from Chinese yogurt

AIMS: The aim of this study was to investigate the properties of temperate bacteriophage of Lactobacillus fermentum, based on its morphology, restriction patterns, protein profile and the impact on the growth of host strain. METHODS AND RESULTS: With Mitomycin C, seven temperate phages were induced from Lactobacilli derived from Chinese yogurt. The temperate phages induced belong to the most common Bradley's group B, having hexagonal head and long, noncontractile tail. They were furthermore confirmed to be the same bacteriophage by identical restriction patterns. SDS-PAGE profile showed that the phage studied had one major structure protein about 31.9 kDa. The presence of the prophage influenced the cell shape and colony size of its lysogenic strain. CONCLUSIONS: The phage obtained had similar, but not complete identical properties with other L. fermentum phages reported. It influenced the growth behaviour of its lysogenic strain. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides some information about bacteriophages occurring in the Chinese yoghurt manufacture and contributes to our knowledge on the bacteriophage diversity in the dairy industry.     

Evaluation of the international phage typing set and some experimental phages for typing of Listeria monocytogenes from poultry in Spain

AIMS: The validity of the international phage set and 13 experimental phages for subtyping Listeria monocytogenes strains isolated from poultry in Spain was investigated. METHODS AND RESULTS: Ninety-six L. monocytogenes strains (52 from serogroup 1/2 and 44 from serogroup 4) were phage-typed using the international phage set, 10 experimental phages for typing serogroup 1/2 strains (seven isolated in France: 1313, 9425, 1807, 351, 881, 717 and 586-, and three from Denmark: 5775, 12682 and 6223-) and three experimental phages isolated in France for typing serogroup 4 strains (2425 A, 4286 and 197). Percentages of serogroup 1/2, serogroup 4 and total phage-typeable strains were 57.7%, 52.3% and 55.2%, respectively. Important differences in the behaviour of the phages tested were found. The typeability rate, the specificity index and the percentage of strong reactions were greater in the phages of international set than in the experimental phages. The number of phage typeable strains and the number of phage types (42) were not modified by the use of experimental phages. CONCLUSIONS: The phage set used was not effective for typing L. monocytogenes strains from poultry in Spain, because a low typeability rate was found. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggest the importance of the availability of new phages specific to a geographical area in order to improve the typeability of the system.     

Mode of antimicrobial action of vanillin against Escherichia coli, Lactobacillus plantarum and Listeria innocua

AIMS: To investigate the mode of action of vanillin, the principle flavour component of vanilla, with regard to its antimicrobial activity against Escherichia coli, Lactobacillus plantarum and Listeria innocua. METHODS AND RESULTS: In laboratory media, MICs of 15, 75 and 35 mmol l(-1) vanillin were established for E. coli, Lact. plantarum and L. innocua, respectively. The observed inhibition was found to be bacteriostatic. Exposure to 10-40 mmol l(-1) vanillin inhibited respiration of E. coli and L. innocua. Addition of 50-70 mmol l(-1) vanillin to bacterial cell suspensions of the three organisms led to an increase in the uptake of the nucleic acid stain propidium iodide; however a significant proportion of cells still remained unstained indicating their cytoplasmic membranes were largely intact. Exposure to 50 mmol l(-1) vanillin completely dissipated potassium ion gradients in cultures of Lact. plantarum within 40 min, while partial potassium gradients remained in cultures of E. coli and L. innocua. Furthermore, the addition of 100 mmol l(-1) vanillin to cultures of Lact. plantarum resulted in the loss of pH homeostasis. However, intracellular ATP pools were largely unaffected in E. coli and L. innocua cultures upon exposure to 50 mmol l(-1) vanillin, while ATP production was stimulated in Lact. plantarum cultures. In contrast to the more potent activity of carvacrol, a well studied phenolic flavour compound, the extent of membrane damage caused by vanillin is less severe. CONCLUSIONS: Vanillin is primarily a membrane-active compound, resulting in the dissipation of ion gradients and the inhibition of respiration, the extent to which is species-specific. These effects initially do not halt the production of ATP. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding the mode of action of natural antimicrobials may facilitate their application as natural food preservatives, particularly for their potential use in preservation systems employing multiple hurdles.     

The newly isolated lytic bacteriophages st104a and st104b are highly virulent against Salmonella enterica

AIMS: To screen Irish faecal samples from a variety of sources with a view to isolating novel anti-Salmonella phages and to subsequently evaluate their lytic capability. METHODS AND RESULTS: Two novel anti-Salmonella phages st104a and st104b were isolated from a screening programme based on their lytic capability. The phages produced significantly larger plaques (2 mm) on the chosen indicator Salmonella enterica strain, DPC6046, when compared with the well-known control phage, Felix 01 (0.5 mm). Both phages st104a and st104b were found to have a broad host range within the Salm. enterica species. During in vitro trials, both phages (st104a and st104b) reduced Salm. enterica numbers more than 99% within 1 h. In vivo studies, involving the addition of the phage to porcine gastric juice (pH 2.5) demonstrated that phage st104a and phage Felix 01 were capable of surviving (10 and 30% survival respectively) the acidic conditions, unlike st104b, which was undetectable after 2 h exposure. CONCLUSIONS: Two novel lytic anti-Salmonella phages were isolated and characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: With the exception of phage Felix 01, there has been relatively little phage therapy work performed using lytic Salmonella phage. In this study, the lytic phages st104a and st104b were isolated as a result of a faecal screening programme. Subsequently, phage st104a was found to have potential for biocontrol of Salm. enterica numbers if administered orally to pigs given their survival in porcine gastric juice, whereas, phage st104b may have potential in reducing cell numbers if applied by alternative approaches.     

Milk Contamination and Resistance to Processing Conditions Determine the Fate of Lactococcus lactis Bacteriophages in Dairies

Milk contamination by phages, the susceptibility of the phages to pasteurization, and the high levels of resistance to phage infection of starter strains condition the evolution dynamics of phage populations in dairy environments. Approximately 10% (83 of 900) of raw milk samples contained phages of the quasi-species c2 (72%), 936 (24%), and P335 (4%). However, 936 phages were isolated from 20 of 24 (85%) whey samples, while c2 was detected in only one (4%) of these samples. This switch may have been due to the higher susceptibility of c2 to pasteurization (936-like phages were found to be approximately 35 times more resistant than c2 strains to treatment of contaminated milk in a plate heat exchanger at 72°C for 15 s). The restriction patterns of 936-like phages isolated from milk and whey were different, indicating that survival to pasteurization does not result in direct contamination of the dairy environment. The main alternative source of phages (commercial bacterial starters) does not appear to significantly contribute to phage contamination. Twenty-four strains isolated from nine starter formulations were generally resistant to phage infection, and very small progeny were generated upon induction of the lytic cycle of resident prophages. Thus, we postulate that a continuous supply of contaminated milk, followed by pasteurization, creates a factory environment rich in diverse 936 phage strains. This equilibrium would be broken if a particular starter strain turned out to be susceptible to infection by one of these 936-like phages, which, as a consequence, became prevalent.     

Comparative Study of 35 Bacteriophages of Lactobacillus helveticus: Morphology and Host Range

This survey included 23 phages isolated from cheese whey and 12 temperate phages induced with mitomycin from their lysogenic host strains. All of the phages had an isometric head and a tail with a contractile sheath. In addition, short-tailed (160-nm-long) and long-tailed (260-nm-long) phages were distinguished. Short-tailed phages were by far the most widespread in French cheese factories (32 of the 35 phages studied). The study of phage relationships enabled two large groups of strains to be distinguished: those not or slightly sensitive to phages and those very sensitive to phages. There was an obvious relationship in the first group between phage sensitivity (or resistance) and the geographic origin of the strains. The second group contained primarily strains from large international collections and those isolated from commercial starters. The relationships among short-tailed phages, either temperate or isolated as lytic, suggest that lysogenic strains could be the major source of phages in French cheese factories.     

Inactivation of Escherichia coli by O- water

AIMS: To clarify the effects of O(-) (atomic oxygen radical anion) water on the viability and morphological alteration of Escherichia coli. METHODS AND RESULTS: O(-) water (OW) was prepared by bubbling of O(-)/argon (Ar) flux into deionized water. O(-) and hydrogen peroxide (H(2)O(2)) in the resultant OW were analysed by electron paramagnetic resonance and ultraviolet (UV) absorption spectroscopy. The population of E. coli treated by a typical OW of pH 4.30 +/- 0.20 [(2.5 +/- 0.8) x 10(-3) mmol l(-1) O(-); 0.5 +/- 0.2 mmol l(-1) H(2)O(2)) was reduced by more than 3 log CFU ml(-1) within 60 min at 30 degrees C. Through scanning electron microscopy observation, the OW-treated cells appeared dramatically collapsed. The release of nucleic acid induced by OW was identified by UV absorption spectroscopy. CONCLUSIONS: O(-) water can result in inactivation of E. coli, nucleic acid release and cellular damage under the controlled laboratory conditions in excess of 15-30 min. Reactive oxygen species may play an important role in the inactivation process. SIGNIFICANCE AND IMPACT OF THE STUDY: This study first revealed that OW could inactivate E. coli, which may be potentially useful in developing a novel approach for the microbial decontamination of food, water or heat-sensitive material.     

Transduction of penicillinase production and other antibiotic-resistance markers in Staphylococcus epidermidis.

Transduction of resistance from a multiply antibiotic-resistant strain of Staphylococcus epidermidis sub-group II was studied using the typing phage 108. The effect of increasing doses of ultraviolet radiation on the transducing phage was used to indicate the chromosomal or plasmid nature of the genes. Tetracycline and chloramphenicol resistance behaved as plasmid genes and streptomycin resistance as a chromosomal marker. It was also possible to transduce penicillin resistance (Pc) due to penicillinase production (bla+) using a low level of benzylpenicillin (0.03 microgram ml-1) for recovery. Approximately 10(-5) transductant colonies per phage input were obtained and ultraviolet kinetics indicated that Pc was plasmid carried. Pc transductants fell into two categories. In one group PC was stable as in the donor strain and transductants had the same phage sensitivity as the recipient. In the other, Pc was unstable at 37 degrees C and the instability was enhanced by growth at approximately 43.5 degrees C; these transductants also gained genes for restriction and modification of certain phages. Transductants that subsequently lost bla+ also lost the restriction and modification characters.     

The isolation and characterization of bacteriophages infecting obligately thermophilic strains of Bacillus

Twenty-four thermophilic bacteriophages have been isolated from diverse sources such as compost, soil, silage and rotting straw. Although considerable individual host specificity was observed, the phages were able to infect most of the major taxonomic groups of Bacillus thermophiles. The phages varied considerably in morphology and size; the phage heads were either cylindrical or polyhedral with tails varying in length between 15 and 500 nm. Most of the phages were stable at 50 degrees C for 4-5 h but at 70 degrees C the plaque-forming units decreased by between 10(2)- and 10(7)-fold in 2 h. The DNA of morphologically similar phages was examined by restriction enzyme analysis, and some differences in the DNA fragment patterns were found. Efficiency of plating data indicated that 'B. caldotenax' has a restriction and modification system. These phages may be valuable for the study of the genetics of thermophilic bacilli: transduction of 'B. caldotenax' and 'B. caldovelox' by phage JS017 has been observed.     

Isolation and characterization of large lactococcal phage resistance plasmids by pulsed-field gel electrophoresis

Abstract Five phage-resistant Lactococcus lactis strains were able to transfer by conjugation the lactose-fermenting ability (Lac⁺) to a plasmid-free Lac⁻ L. lactis strain. In each case, some Lac⁺ transconjugants were phage-resistant and contained one or two additional plasmids of high molecular mass, as demonstrated by pulsed-field gel electrophoresis. Plasmids pPF144 (144 kb), pPF107 (107 kb), pPF118 (118 kb), pPF72 (72 kb) and pPF66 (66 kb) were characterized: they are conjugative (Tra⁺), they confer a phage-resistant phenotype and they bear lactose-fermenting ability (Lactose plasmid) except for the last two. Plasmids pPF144, pPF107 and pPF118 resulted probably from a cointegrate formation between the Lactose plasmid and another plasmid of the donor strain, whereas pPF72, pPF66 and the Lactose plasmid were distinct in the corresponding transconjugants. Plasmids pPF72 and pPF66 produced a bacteriocin. At 30°C, the phage resistance conferred by the plasmids was complete against small isometric-headed phage and partial against prolate-headed phage, except for pPF107 whose phage resistance mechanism was totally effective against both types of phages, but was completely inactivated at 40°C. Restriction maps of four of the plasmids were constructed using pulsed-field gel electrophoresis.     

Isolation and phenotypic characterization of Lactobacillus casei and Lactobacillus paracasei bacteriophage-resistant mutants

Aims: To isolate and characterize bacterial strains derived from Lactobacillus casei and Lactobacillus paracasei strains and resistant to phage MLC‐A. Methods and Results: Two of nine assayed strains rendered resistant mutants with recovery efficiencies of 83% (Lact. paracasei ATCC 27092) and 100% (Lact. casei ATCC 27139). DNA similarity coefficients (RAPD–PCR) confirmed that no significant genetic changes occurred while obtaining resistant mutants. Neither parent nor mutant strains spontaneously released phages. Phage‐resistant mutants were tested against phages PL‐1, J‐1, A2 and MLC‐A8. Lactobacillus casei ATCC 27092 mutants showed, overall, lower phage resistance than Lact. paracasei ATCC 27092 ones, but still higher than that of the parent strain. Lactobacillus paracasei ATCC 27092 mutants moderately adsorbed phage MLC‐A only in calcium presence, although their parent strain successfully did it with or without calcium. Adsorption rates for Lact. casei ATCC 27139 and its mutants were highly influenced by calcium. Again, phage adsorption was higher on the original strain. Conclusions: Several isolates derived from two Lact. casei and Lact. paracasei strains showed resistance to phage MLC‐A but also to other Lact. casei and Lact. paracasei phages. Significance and Impact of the Study: This study highlights isolation of spontaneous bacteriophage‐resistant mutants from Lact. casei and Lact. paracasei as a good choice for use in industrial rotation schemes.