Mechanisms of erythromycin resistance of Campylobacter spp. isolated from food, animals and humans

Macrolides are regarded as drugs of choice for treatment of human campylobacteriosis. The use of antimicrobials for this purpose as well as in food animal production could result in macrolide resistance in Campylobacter species. Campylobacter isolates exhibit two different phenotypes with regard to erythromycin resistance: high-level resistance (HLR) and low-level resistance (LLR). Thirty-six food/animal and human isolates of Campylobacter jejuni and C. coli were examined for their mechanisms of resistance to erythromycin. The data presented here confirm the previous findings that the A2075G mutation in the 23S rRNA gene is the most frequently reported mechanism of high-level erythromycin resistance in Campylobacter isolates. The efflux pump inhibitor PAbetaN increased susceptibility to erythromycin for at least 16-32-fold in all examined HLR isolates, suggesting that the efflux mechanism acts in synergy with the 23S rRNA mutation to confer high-level erythromycin resistance. This was also confirmed in the isolates with sequence variation in the efflux pump cmeB gene. Additionally, the PAbetaN restored the susceptibility of LLR strains to the level of minimal inhibitory concentrations (MICs) of the susceptible strains and also reduced the MICs of the susceptible C. jejuni and C. coli isolates. The data suggest that active efflux contributes to the intrinsic resistance to erythromycin in Campylobacter and also contribute to high-level resistance.     

A Review of Hop Resistance in Beer Spoilage Lactic Acid Bacteria

Hop bitter acids play a major role in enhancing the microbiological stability of beer. However, beer spoilage lactic acid bacteria (LAB) are able to grow in beer by exhibiting strong hop resistance. Recently two hop resistance genes, horA and horC, have been identified in beer spoilage Lactobacillus brevis ABBC45. The horA gene was shown to encode an ATP dependent multidrug transporter that extrudes hop bitter acids out of bacterial cells. In contrast, the product of the horC gene confers hop resistance by presumably acting as a proton motive force (PMF)‐dependent multidrug transporter. Strikingly, the homologs of horA and horC genes were found to be widely and almost exclusively distributed in various species of beer spoilage LAB strains, indicating these two hop resistance genes are excellent species‐independent genetic markers for differentiating the beer spoilage ability of LAB. Furthermore the nucleotide sequence analysis of horA and horC homologs revealed that both genes are essentially identical among distinct beer spoilage species, indicating horA and horC have been acquired by beer spoilage LAB through horizontal gene transfer. Taken collectively, these insights provide a basis for applying horA and horC to the species‐independent determination of beer spoilage LAB, including yet uncharacterized species. In addition to the hop resistance mechanisms mediated by multidrug transporters, proton translocating ATPase and the ATP production system were shown to contribute to the hop resistance mechanisms in beer spoilage LAB by generating PMF and ATP that are necessary for survival in beer.     

Reversible and irreversible pollutant-induced bacterial cellular stress effects measured by ethidium bromide uptake and efflux

Chemical pollution is known to affect microbial community composition but it is poorly understood how toxic compounds influence physiology of single cells that may lay at the basis of loss of reproductive fitness. Here we analyze physiological disturbances of a variety of chemical pollutants at single cell level using the bacterium Pseudomonas fluorescens in an oligotrophic growth assay. As a proxy for physiological disturbance we measured changes in geometric mean ethidium bromide (EB) fluorescence intensities in subpopulations of live and dividing cells exposed or not exposed to different dosages of tetradecane, 4-chlorophenol, 2-chlorobiphenyl, naphthalene, benzene, mercury chloride, or water-dissolved oil fractions. Because ethidium bromide efflux is an energy-dependent process any disturbance in cellular energy generation is visible as an increased cytoplasmic fluorescence. Interestingly, all pollutants even at the lowest dosage of 1 nmol/mL culture produced significantly increased ethidium bromide fluorescence compared to nonexposed controls. Ethidium bromide fluorescence intensities increased upon pollutant exposure dosage up to a saturation level, and were weakly (r(2) = 0.3905) inversely correlated to the proportion of live cells at that time point in culture. Temporal increase in EB fluorescence of growing cells is indicative for toxic but reversible effects. Cells displaying high continued EB fluorescence levels experience constant and permanent damage, and no longer contribute to population growth. The procedure developed here using bacterial ethidium bromide efflux pump activity may be a useful complement to screen sublethal toxicity effects of chemicals.     

Occurrence of and a possible mechanism for resistance to a quaternary ammonium compound in Listeria monocytogenes

In a study of 200 Listeria monocytogenes isolates, 10% were determined to be resistant to benzalkonium chloride (BC). Serial subcultivation of initially BC sensitive (BC(S)) and BC resistant (BC(R)) isolates in sublethal concentrations of BC resulted in enhanced and approximately equal resistance of all strains to the compound. Fifty per cent of the BC(R) isolates showed resistance to ethidium bromide (EB) as well. A proton motive force (pmf)-dependent efflux of EB was demonstrated in BC(R) isolates, and in originally sensitive strains adapted to grow in BC. This efflux was not found in BC(S) strains. The result indicate that BC can induce a broad resistance mechanism based on a pmf-driven efflux pump. There was no indication that this type of resistance was related to resistance to antibiotics.     

沙门菌传代中(氟)喹诺酮类抗生素筛选株药敏性及相关基因突变和表达

目的研究在不同浓度(氟)喹诺酮类抗生素存在条件下,沙门菌在传代时得到的筛选株中与耐药性相关部分基因的变异和表达水平变化规律及其与耐药性间的关联性。方法使用含有一定浓度(氟)喹诺酮类抗生素的肉汤培养基对沙门菌进行培养,在含有相同浓度同类抗生素的平板上划线筛选突变株,微量肉汤稀释法测定筛选株的药敏性,聚合酶链式反应(PCR)结合DNA测序确定喹诺酮耐药决定区(quinolone resistance-determining region,QRDR)基因突变,实时荧光定量PCR(real-time qPCR)法检测外排泵AcrAB-TolC编码基因表达水平。结果沙门菌(ATCC 14028s)在含有不同代、不同浓度(氟)喹诺酮类抗生素的LB培养基中培养、筛选后,筛选株对抗生素耐受性均有不同程度增加。萘啶酮酸第5～7代筛选株gyrA突变,发生Asp87Tyr变异;环丙沙星第4～7代筛选株gyrA突变,发生Asp87Asn变异;加替沙星、左氧氟沙星和德拉沙星第1～7代筛选株gyrA中均未检出核苷酸突变。随着抗生素浓度增加,各抗生素相应筛选株中外排泵AcrAB-TolC编码基因表达水平较原始菌株增加,差异有统计学意义(P0.05),第7代菌株acrAB-tolC表达量间差异无统计学意义(P0.05)。(氟)喹诺酮类抗生素对不同代筛选株的最小抑菌浓度(MIC)与其对沙门菌(ATCC 14028s)的MIC值比值、gyrA突变、acrAB-tolC表达水平与抗生素作用浓度和筛选代数间呈正相关。结论在(氟)喹诺酮类抗生素作用下,沙门菌可通过QRDR基因突变及增加acrAB-tolC表达适应抗生素胁迫环境;新一代(氟)喹诺酮类抗生素作用于沙门菌时,菌株QRDR靶位点突变概率降低;多次重复作用后,菌株acrAB-tolC表达量虽然增加,但表达量间差异无统计学意义(P0.05),避免了因突变导致耐药性的进一步增强。     

Efflux pump activity in fluoroquinolone and tetracycline resistant Salmonella and E. coli implicated in reduced susceptibility to household antimicrobial cleaning agents

It has been shown that the inappropriate use of antimicrobial household agents selects for organisms with resistance mechanisms (e.g. efflux pumps), which could lead to the development of antibiotic resistance. The reverse hypothesis, that antibiotic-resistant organisms become tolerant to other antibacterial agents (e.g. disinfectants) due to the action of efflux pumps, has however not been extensively examined. The objective of this study was to establish whether there is a link between antibiotic resistance in potential gastrointestinal pathogens and reduced sensitivity of these organisms to commonly used household antimicrobial agents. In this study, tetracycline and ofloxacin sensitive and resistant Escherichia coli (9 strains) and Salmonella spp. (8 strains) were isolated from poultry and clinical samples. In order to assess whether these bacteria had active efflux pumps, ethidium bromide accumulation assays were performed. Extrusion of the active components of three commercial household agents (triclosan, sodium salicylate, and ortho-phenylphenol) by efflux pumps was tested using spectrophotometric accumulation assays. In order to simulate the kitchen environment, in-use disinfectant testing using the commercial household agents was performed to determine changes in their efficacy due to antibiotic resistance. Active efflux pump activity and extrusion of all three active ingredients was observed only in the antibiotic resistant organisms. The antibiotic sensitive bacteria were also more susceptible than the resistant isolates to the household antimicrobial agents at concentrations below that recommended by the manufacturer. These resistant bacteria could potentially be selected for and result in hard to treat infections.     

Isolation and Identification of Potential Beer‐Spoilage Pediococcus inopinatus and Beer‐Spoilage Lactobacillus backi Strains Carrying the horA and horC Gene Clusters

Four beer‐spoilage strains, LA20, LA21, LA22 and LA23, were isolated from brewery environments. Based on the 16S rRNA gene sequence, LA20 was identified as Pediococcus inopinatus and the remaining three were identified as Lactobacillus backi. The homologs of horA and horC, the hop resistance genes originally identified in L. brevis ABBC45, were detected simultaneously in LA22 and LA23, while only a horA homolog and a horC homolog were found in LA20 and LA21 respectively. The 5.6 kb DNA regions containing the horA homolog in LA20 and LA22 were almost 99% identical with the corresponding region of ABBC45. Similarly the 8.2 kb regions containing the horC homolog in LA21 and LA22 were more than 99% identical with that of ABBC45. Interestingly the horA‐containing 5.6 kb regions in LA20 and LA22 were found to be completely identical despite the distinct genus status. Coupled with the fact that LA20 and LA22 were isolated from the same sampling site, these results, taken collectively, reinforce our hypothesis that horA and horC genes were acquired by beer‐spoilage species through horizontal gene transfer and confirm the usefulness of horA and horC as genetic markers for the species‐independent determination of beer‐spoilage ability in lactic acid bacteria.     

CaALK8, an alkane assimilating cytochrome P450, confers multidrug resistance when expressed in a hypersensitive strain of Candida albicans

We report the isolation of a novel C. albicans gene designated CaALK8, by its ability to complement drug hypersensitivity of a pdr5 (ABC: ATP-binding cassette drug extrusion pump) null mutant of S. cerevisiae (JG436). CaALK8 in JG436 conferred resistance to drugs such as cycloheximide (CYH), fluconazole (FCZ), O-phenanthroline (PHE) and 4-nitroquinoline oxide (NQO). The gene was so designated because its sequence was identical to a partial sequence entry named as ALK8 in the Candida database (http://alces.med.umn.edu/candida.html). CaALK8 encodes for a putative 515 amino acid protein highly homologous to alkane-inducible cytochromes P450 (CYP52 gene family) of C. maltosa and C. tropicalis. The ability of CaALK8 to confer drug resistance was also established by its expression in another drug-hypersensitive strain of S. cerevisiae (AD 1234568), which was deleted in seven ABC efflux pumps. The homozygous disruption of CaALK8 in a wild-type C. albicans strain (CAI4) did not result in altered drug susceptibilities. The overexpression of CaALK8 in CAI4 resulted in only FCZ resistance. However, a distinct MDR phenotype was evident when CaALK8 was overexpressed in a drug-hypersensitive C. albicans strain disrupted in both CDR1 and CDR2 (ABC drug extrusion pumps of C. albicans). Alk8p, similar to other Alk proteins from C. maltosa and C. tropicalis, could hydroxylate alkanes and fatty acids. In this study we demonstrate that several drugs could compete with the hydroxylation activity by directly interacting with CaAlk8p. Taken together, our results suggest that a member of the CYP52 gene family could mediate MDR in C. albicans, although it does not seem to be involved in the development of azole resistance in clinical isolates. The nucleotide sequence reported in this paper has been submitted to GenBank under Accession No. Y14766.     

Lipid composition and cell surface hydrophobicity of Candida albicans influence the efficacy of fluconazole–gentamicin treatment

Adherence of the fungus, Candida albicans, to biotic (e.g. human tissues) and abiotic (e.g. catheters) surfaces can lead to emergence of opportunistic infections in humans. The process of adhesion and further biofilm development depends, in part, on cell surface hydrophobicity (CSH). In this study, we compared the resistance of C. albicans strains with different CSH to the most commonly prescribed antifungal drug, fluconazole, and the newly described synergistic combination, fluconazole and gentamicin. The hydrophobic strain was more resistant to fluconazole due to, among others, overexpression of the ERG11 gene encoding the fluconazole target protein (CYP51A1, Erg11p), which leads to overproduction of ergosterol in this strain. Additionally, the hydrophobic strain displayed high efflux activity of the multidrug resistance Cdr1 pump due to high expression of the CDR1 gene. On the other hand, the hydrophobic C. albicans strain was more susceptible to fluconazole–gentamicin combination because of its different effect on lipid content in the two strains. The combination resulted in ergosterol depletion with subsequent Cdr1p mislocalization and loss of activity in the hydrophobic strain. We propose that C. albicans strains with different CSH may possess altered lipid metabolism and consequently may differ in their response to treatment.     

Study on ATP Production of Lactic Acid Bacteria in Beer and Development of a Rapid Pre‐Screening Method for Beer‐Spoilage Bacteria

Three beer‐spoilage strains of lactic acid bacteria (LAB), Lactobacillus brevis ABBC45, L. lindneri DSM 20690^T and L. para‐collinoides DSM 15502^T, exhibited strong ATP‐yielding ability in beer. To investigate energy sources, these beer‐spoilage strains were inoculated into beer. After the growth of the strains in beer, utilized components were determined by high performance liquid chromatography (HPLC). As a result, it was shown that citrate, pyruvate, malate and arginine were consumed by beer‐spoilage LAB strains examined in this study. The four components induced considerable ATP production even in the presence of hop compounds, accounting for the ATP‐yielding ability of the beer‐spoilage LAB strains observed in beer. We have further examined the ATP‐yielding ability of other strains of bacteria in beer. Beer‐spoilage bacteria, including Pectinatus frisingensis and P. cerevisiiphilus, showed strong ATP‐yielding abilities, whereas species frequently isolated from brewery environments exhibited low ATP‐yielding abilities. Although some of the nonspoilage LAB strains produced substantial amount of ATP in beer, the measurement of ATP‐yielding ability was considered to be useful as a rapid pre‐screening method for potential beer‐spoilage bacteria isolated from brewery environments.     

副溶血弧菌耐药诱导株的生物特性与多药外排泵表达

本研究将初始菌株副溶血弧菌F7经过环丙沙星连续体外诱导得到的实验室诱导株H128、H256、H512作为研究对象,研究了菌株F7、H128、H256、H512的药敏性、泳动能力、生长曲线、生物膜形成能力等生物学特性,分析了多药外排泵基因vme B、vme D、vpo C、vmr A的相对表达量。结果表明,诱导菌株不仅对诱导抗生素环丙沙星的耐药性明显提高,还对多种非诱导抗生素呈现交叉耐药。与初始菌株F7相比,诱导菌株H128、H256、H512泳动能力下降,泳动圈直径分别为4.83 mm、4.57 mm、5.13 mm,生物膜形成能力增强,吸光值分别为4.35 nm、4.35 nm、4.29 nm。诱导菌株H128、H256、H512均检测到外排泵基因vme D的相对表达量均高于F7,分别上调了1.20、3.55、2.02倍,仅在H256中检测到vme B、vpo C、vmr A基因表达量上调,分别上调了1.47、2.90、1.71倍,说明RND型外排系统Vme CD-Vpo C是与副溶血弧菌对环丙沙星的耐药性以及交叉耐药相关的主要外排系统。本研究为耐药性副溶血弧菌的风险评估提供参考依据,为解决副溶血弧菌耐药问题提供新的思路。     

Prevalence of the Campylobacter multi-drug efflux pump (CmeABC) in Campylobacter spp. Isolated from freshly processed Turkeys

The prevalence of the Campylobacter multi-drug efflux pump (CmeABC) was evaluated in Campylobacter isolates recovered from freshly processed turkeys at two Midwestern processing plants. A total of 94 Campylobacter isolates recovered from processed turkeys were examined using polymerase chain reaction (PCR) to determine the presence of the multi-drug efflux pump genes cmeA, cmeB, and cmeC. Results from this study found that 51% of all isolates tested were positive for CmeABC. 46.6% of these positive isolates were from plant A and 55.1% from plant B. Differences were observed in the prevalence of individual genes found among Campylobacter isolates from each plant. Additional analysis found that among the isolates positive for CmeABC, 85.5% were identified as C. jejuni and 14.5% identified as C. coli. There was a relatively high occurrence of the Campylobacter multi-drug efflux pump genes in Campylobacter spp. recovered from processed turkeys, however, the presence of the genes could not be significantly linked to antimicrobial resistance observed in the test strains and suggests that the CmeABC genes are only one factor associated with antimicrobial resistance in Campylobacter spp.     

Adaptive tolerance to phenolic biocides in bacteria from organic foods: Effects on antimicrobial susceptibility and tolerance to physical stresses

The aim of the present study was to analyze the effects of step-wise exposure of biocide-sensitive bacteria from organic foods to phenolic biocides triclosan (TC) and hexachlorophene [2,2′-methylenebis(3,4,6-trichlorophenol)] (CF). The analysis included changes in the tolerance to the biocide itself, the tolerance to other biocides, and cross-resistance to clinically important antibiotics. The involvement of efflux mechanisms was also studied as well as the possible implication of modifications in cytoplasmic membrane fluidity in the resistance mechanisms. The influence of biocide tolerance on growth capacity of the adapted strains and on subsequent resistance to other physical stresses has also been analyzed. Repeated exposure of bacteria from organic foods to phenolic biocides resulted in most cases in partially increased tolerance to the same biocide, to dissimilar biocides and other antimicrobial compounds. Nine TC-adapted strains and six CF-adapted strains were able to develop high levels of biocide tolerance, and these were stable in the absence of biocide selective pressure. Most strains adapted to TC and one CF-adapted strain showed significantly higher anisotropy values than their corresponding wildtype strains, suggesting that changes in membrane fluidity could be involved in biocide adaptation. Exposure to gradually increasing concentrations of CF induced a decrease in heat tolerance. Biocide adaptation had no significant effects of gastric acid or bile resistance, suggesting that biocide adaptation should not influence survival in the gastrointestinal tract.     

Characterization of the quinolone resistance mechanism in foodborne Salmonella isolates with high nalidixic acid resistance

Sixteen Salmonella strains resistant to nalidixic acid isolated from kimbab, the most popular ready-to-eat (RTE) food in Korea, and chicken meat were selected for this study. The resistant strains were shown to have high minimal inhibitory concentrations (MICs) against nalidixic acid (512 ~ 4096 μg/mL). Among them, 4 Salmonella enterica serovar Haardt isolates showed multi-drug resistance (MDR) patterns with reduced susceptibility to fluoroquinolone (0.5 μg/mL of ciprofloxacin MICs). The mechanisms of quinolone resistance in the nalidixic acid resistant strains were characterized by PCR and sequence analysis. The presence of plasmid-mediated quinolone resistance (PMQR) genes and amino acid changes in the quinolone resistance determining region (QRDR) were investigated by PCR-based detection and sequencing, and the efflux pump inhibition test was also done using phe-arg-β-naphthylamide (PAβN). Although PMQR genes were not detected in any of the tested strains, the QRDR mutations were found in this study: single mutation in gyrA (Asp87Tyr, Asp87Gly, and Asp87Asn), double mutations in gyrA (Ser83Thr) and parC (Thr57Ser), and single mutation in parC (Thr57Ser). MICs of nalidixic acid were reduced by 2- to 32-folds by the efflux pump inhibitor, PAβN. Pulsed-field gel electrophoresis (PFGE) was carried out to confirm the epidemiological relationship between the nalidixic acid resistant strains. The PFGE patterns were classified into 6 groups at cutoff level of 70 ~ 100% correlation on the dendrogram. Some strains of serotype Haardt and Enteritidis showed several values of genomic identity in accordance with strains, sources, and isolation year. We suggest that point mutation on QRDR and efflux pump systems involved in antimicrobials had independent effects on drug-resistance regardless of bacterial genomic variation.     

Key physiological differences in Candida albicans CDR1 induction by steroid hormones and antifungal drugs

The Candida albicans CDR1 gene, encoding an ABC transporter that functions as an efflux pump, is thought to be involved in pathogenic adaptation and uses mammalian hormones and other environmental cues to regulate its activity. Exposure of several clinical isolates of C. albicans to 1 x 10(-8) M 17beta-oestradiol increased CDR1 expression and the isolates showed a positive correlation between oestrogen induction of CDR1 and growth in the presence of oestrogen. A reporter strain carrying the GFP gene under the control of the CDR1 promoter was used to analyse the effect of steroid hormones and antifungal drugs on CDR1 expression by flow cytometry. We found that among the many hormones tested, only oestradiol and progesterone induce CDR1 expression. CDR1 induction requires hormone concentrations greater than 10(-8) M, a threshold reached in vivo only by progesterone. Using the GFP-reporter strain, we show CDR1 induction by female but not male human serum and demonstrate that exposure of C. albicans to physiological concentrations of progesterone measurably increases resistance to fluconazole, miconazole and 5-fluorouracil. Simultaneous exposure of C. albicans to hormones and antifungal drugs provided evidence that both agents induce CDR1 expression via different mechanisms with different saturation points.     

Characterization of a wild strain of Alicyclobacillus acidoterrestris: heat resistance and implications for tomato juice

This article reports the characterization of a wild strain of Alicyclobacillus acidoterrestris and describes the implications of the heat resistance of this microorganism in tomato juice. The strain (labeled as A. acidoterrestrisγ4) showed pH and temperature ranges for growth typical of the species (3.0 to 6.0 for the pH and 35 to 60 °C for the temperature); heat resistance in tomato juice was as follows: D(T) values of 40.65, 9.47, and 1.5 min (at 85, 90, and 95 °C, respectively) and z-value of 7 °C. A treatment at 70 °C for 15 min was found to be optimal for spore activation, whereas Malt Extract Agar, acidified to pH 4.5, showed good results for spore recovery. Concerning the implications of heat resistance of A. acidoterrestris on tomato juice, high temperatures required for spore inactivation determined a general decrease of the antioxidant activity (increase of the redox potential and reduction of the chain-breaking activity), but not the formation of brown compounds (namely, hydroxymethylfurfural), thus suggesting an effect on the secondary antioxidants (carotenoids and ascorbic acid) rather than on lycopene. PRACTICAL APPLICATION: Alicyclobacillus acidoterrestris is an emerging spore-forming microorganism, capable of causing spoilage in tomato juice. Due to their high thermal resistance, spores could be used as targets for the optimization of heat processing; this article reports on the assessment of thermal resistance of a wild strain of A. acidoterrestris, then focusing on the effect of the thermal treatment necessary to inactivate spores on the quality of tomato juice.     

Nucleotide Sequence Identities of horA Homologues and Adjacent DNA Regions Identified in Three Species of Beer‐Spoilage Lactic Acid Bacteria

The horA homologues and adjacent DNA regions identified in beer‐spoilage Lactobacillus lindneri DSM 20690^Tand L. paracollinoides DSM15502^Twere examined and compared with the corresponding DNA region of beer‐spoilage L. brevis ABBC45, a strain in which the hop‐resistance gene horA was originally identified. The PCR analysis suggests ORFB1‐B5 regions surrounding horA are conserved in all of the strains. The nucleotide sequence comparison of the conserved DNA regions revealed extremely high levels of identities among the three beer‐spoilage strains that are not typical for distinct species. The PCR methods using primers specific to the adjacent ORFs were found to be able to differentiate beer‐spoilage Lactobacillus strains from non‐spoilers, indicating these ORFs are also useful genetic markers for determining the beer‐spoilage ability of lactobacilli. The presence or absence of the adjacent ORFs in 92 bacterial strains was completely identical with that of horA homologues, indicating the ORFB1‐B5 regions are generally conserved in beer‐spoilage Lactobacillus strains. Taken together, these results suggest the ORFB1‐B5 regions have been acquired by beer‐spoilage lactobacilli through horizontal gene transfer and provide a theoretical basis for applying a trans‐species genetic marker such as horA to deal with unencountered species of beer‐spoilage lactobacilli.     

Recombinant lamb chymosin as an alternative coagulating enzyme in cheese production

Recombinant lamb chymosin (RLC) was prepared and tested for its potential use in cheese production. The milk clotting activity and proteolytic activity of RLC were evaluated in comparison with commercial recombinant calf chymosin (RCC), cow rennet (CR), and microbial coagulant (MC). RLC, RCC, and MC showed similar responses to pH, with a sharp increase of the coagulation time at pH 6.6 to 6.8 and decrease of curd firmness at the pH 6.5 to 6.6. In the case of CR, we observed two clear increases in the coagulation time and decreases in the curd firmness, at pH 6.4 to 6.5 and 6.6 to 6.8. Optimal clotting activity was obtained for RLC at 40 degrees C, for both CR and RCC at 45 degrees C, and for MC at 60 degrees C. The temperature instability of RLC at temperatures above 45 degrees C could constitute a benefit in making hard cheese varieties. The additon of CaCl2 to milk resulted in enhanced clotting activity of all coagulants, most prominently for CR. The proteolytic activity of RLC was significantly lower from that of CR but not significantly different from the activity of RCC. The lower proteolytic activity in the cheese made with RLC did not have negative effect on organoleptic properties. The overall quality of the cheese made with RLC was at least comparable to that of the cheese made with RCC, and both cheeses were better scored than the cheese made with CR.