Survey of antimicrobial resistance in lactic streptococci

A total of 26 strains of Streptococcus cremoris and 12 strains of Streptococcus lactis were challenged with 18 antimicrobial agents and with nisin in the Bauer-Kirby disk susceptibility test. All strains were susceptible to ampicillin, bacitracin, cephalothin, chloramphenicol, chlortetracycline, erythromycin, penicillin G, tetracycline, and vancomycin. All strains were resistant to trimethoprim, and almost all strains were resistant to sulfathiazole. Variability in resistance to gentamicin, kanamycin, lincomycin, nafcillin, neomycin, nisin, rifampin, and streptomycin was observed. MICs of these substances for the less susceptible strains were determined, and high-level resistance factors could not be detected, except in the case of nisin. S. lactis ATCC 7962 was resistant to at least 40-fold-higher concentrations of nisin (greater than 64 micrograms/ml) than most other strains tested. This strain was a potent nisin producer.     

Inhibition of tyrosine-sensitive 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase by glyphosate in Candida maltosa

Abstract A gene block controlling sucrose-fermenting ability, nisin resistance and nisin production was found to be transmissible by a conjugation-like process. The 'pSN' (sucrose nisin) plasmid was transferred from 8 different nisin-producing donor strains into MG1614, a plasmid-free derivative of Streptococcus lactis 712. In the new host low yields of a plasmid of approx. 30 MDa were isolated but its authenticity as a pSN plasmid has not yet been established. Possibilities for increased nisin yield by genetic manipulation in S. lactis 712 must exist.     

Use of the method of protoplast fusion in the selection of a nisin producer

Experimental data on selection of Streptococcus lactis producing the polypeptide antibiotic nisin with the method of protoplast fusing, one of the modern methods of cell engineering are presented. Four strains of Streptococcus lactis differing in their nisin-producing levels and difficult for protoplasting were used in the study. It was shown possible to transfer them to the protoplast form when respective conditions for their preliminary cultivation and regeneration are provided. Distinctive features of these strains with respect to the antibiotic resistance, sugar fermentation and growth component requirements were revealed. The protoplast fusing yielded hybrids differing from the parent strains by a number of phenotypical features and nisin-synthesizing activity.     

Conjugal transfer of nisin plasmid genes from Streptococcus lactis 7962 to Leuconostoc dextranicum 181.

Acriflavine-generated mutants of Streptococcus lactis 7962 with various combinations of plasmid molecular masses were screened for nisin production. Nisin was produced by both the wild type and mutants that contained a 17.5-megadalton plasmid, which was obscured by chromosomal fragments. No nisin was produced by plasmid-free mutants. Sucrose fermentation and nisin production were simultaneously expressed. A transconjugant obtained from nisin-producing donor S. lactis 7962 and recipient Leuconostoc dextranicum 181 was a "supernisin" producer. The L. dextranicum Nis+ transconjugant was resistant to S. lactis 7962 phage and vancomycin (greater than 1,000 micrograms/ml), and it contained an extra 17.5-megadalton plasmid.     

Conjugal transfer of nisin plasmid genes from Streptococcus lactis 7962 to Leuconostoc dextranicum 181

Acriflavine-generated mutants of Streptococcus lactis 7962 with various combinations of plasmid molecular masses were screened for nisin production. Nisin was produced by both the wild type and mutants that contained a 17.5-megadalton plasmid, which was obscured by chromosomal fragments. No nisin was produced by plasmid-free mutants. Sucrose fermentation and nisin production were simultaneously expressed. A transconjugant obtained from nisin-producing donor S. lactis 7962 and recipient Leuconostoc dextranicum 181 was a "supernisin" producer. The L. dextranicum Nis+ transconjugant was resistant to S. lactis 7962 phage and vancomycin (greater than 1,000 micrograms/ml), and it contained an extra 17.5-megadalton plasmid.     

Molecular and genetic characterization of a novel nisin variant produced by Streptococcus uberis

Streptococcus uberis is one of the principal causative agents of bovine mastitis. In this study, we report that S. uberis strain 42 produces a lantibiotic, nisin U, which is 78% identical (82% similar) to nisin A from Lactococcus lactis. The 15.6-kb nisin U locus comprises 11 open reading frames, similar in putative functionality but differing in arrangement from that of the nisin A biosynthetic cluster. The nisin U producer strain exhibits specific resistance (immunity) to nisin U and cross-resistance to nisin A, a finding consistent with the 55% sequence similarity of their respective immunity peptides. Homologues of the nisin U structural gene were identified in several additional S. uberis strains, and in each case cross-protective immunity was expressed to nisin A and to the other producers of nisin U and its variants. To our knowledge, this is the first report both of characterization of a bacteriocin by S. uberis, as well as of a member of the nisin family of peptides in a species other than L. lactis.     

Lysogeny in Lactic Streptococci Producing and Not Producing Nisin

Eighty-seven strains of lactic streptococci (46 of Streptococcus lactis, 24 of S. diacetilactis, and 17 of S. cremoris) were tested for lysogeny; 12 S. lactis strains produced nisin. Lysogeny was found in five S. lactis strains (two of them were nisin producers) and in two S. diacetilactis strains. Four S. lactis and two S. diacetilactis lysogens liberated phages both spontaneously and after ultraviolet treatment, and one S. lactis strain liberated phages spontaneously only. No lysogens were found among the S. cremoris strains tested. An initial characterization of the lysogens and their phages was made. The lytic spectrum of some of the examined phages was very narrow (homospecific), whereas that of others was wide, including strains of the three investigated species.     

Plasmid-encoded copper resistance in Lactococcus lactis

A 54-kb plasmid (pND306) from Lactococcus lactis subsp. lactis 1252D encoded resistance to both Cu and Sn . The copper resistance determinant was subcloned on a 12.8-kb PvuII DNA fragment and mapped using a number of restriction endonucleases. Six other copper resistant lactococcal strains were also identified and all contained multiple plasmids. Plasmids in five of these strains showed strong hybridization with a probe made using the 12.8-kb DNA fragment, however no chromosomal homologs were detected. The copper resistance determinant was further isolated as a 10.6-kb SphI fragment and used to construct pND968 that expresses resistance to both copper and nisin.     

一个含有乳链菌肽抗性基因的乳酸乳球菌质粒pTS50的鉴定

在添加乳链菌肽、乳糖及溴甲酚紫的M1 7选择培养基上 ,从 1 97个新鲜牛奶样品中筛选到 3株乳链菌肽抗性菌株 ,PCR扩增证实它们都含有乳链菌肽抗性基因。菌种生理生化特性鉴定及特异性 1 6SrDNAPCR扩增产物的序列测定结果表明这 3株菌都属于乳酸乳球菌乳酸亚种。质粒转化实验发现乳酸乳球菌乳酸亚种TS 1 640中的乳链菌肽抗性基因位于一个约47kb的大质粒pTS50上。BamHI、EcoRI、HindⅢ、NcoI、PstⅠ酶切分析和Southern杂交 ,进一步将乳链菌肽抗性基因定位于pTS50的一个约 1 9kbEcoRI酶切片段中     

Conjugative 40-megadalton plasmid in Streptococcus lactis subsp. diacetylactis DRC3 is associated with resistance to nisin and bacteriophage.

Streptococcus lactis subsp. diacetylactis DRC3 was examined for plasmid DNA and found to contain a previously unreported plasmid of 40 X 10(6) daltons. This plasmid, designated pNP40, was conjugally transferred to a plasmid-cured derivative of S. lactis C2. Transconjugants containing pNP40 acquired resistance to nisin produced by strains of S. lactis and to commercially available nisin when assay plates were incubated at 21, 32, and 37 degrees C. In addition, c2 phage growth was completely restricted in transconjugants containing pNP40 at 21 and 32 degrees C, but not at 37 degrees C. This result suggests that pNP40 may be coding for a temperature-sensitive enzyme that restricts phage growth at 21 and 32 degrees C, but not at 37 degrees C. Eight consecutive transfers of a transconjugant containing pNP40 in Elliker broth at 37 degrees C resulted in 100% loss of resistance to c2 phage when colonies were tested at 32 degrees C. These phage-sensitive isolates had lost pNP40 and had also become sensitive to nisin. This result suggests that pNP40 may also be thermosensitive in its replication. The finding of a phage resistance determinant located on a conjugative plasmid should prove useful in constructing phage-resistant variants for dairy fermentation processes.     

Conjugative 40-megadalton plasmid in Streptococcus lactis subsp. diacetylactis DRC3 is associated with resistance to nisin and bacteriophage

Streptococcus lactis subsp. diacetylactis DRC3 was examined for plasmid DNA and found to contain a previously unreported plasmid of 40 X 10(6) daltons. This plasmid, designated pNP40, was conjugally transferred to a plasmid-cured derivative of S. lactis C2. Transconjugants containing pNP40 acquired resistance to nisin produced by strains of S. lactis and to commercially available nisin when assay plates were incubated at 21, 32, and 37 degrees C. In addition, c2 phage growth was completely restricted in transconjugants containing pNP40 at 21 and 32 degrees C, but not at 37 degrees C. This result suggests that pNP40 may be coding for a temperature-sensitive enzyme that restricts phage growth at 21 and 32 degrees C, but not at 37 degrees C. Eight consecutive transfers of a transconjugant containing pNP40 in Elliker broth at 37 degrees C resulted in 100% loss of resistance to c2 phage when colonies were tested at 32 degrees C. These phage-sensitive isolates had lost pNP40 and had also become sensitive to nisin. This result suggests that pNP40 may also be thermosensitive in its replication. The finding of a phage resistance determinant located on a conjugative plasmid should prove useful in constructing phage-resistant variants for dairy fermentation processes.     

Analysis of the genetic determinant for production of the peptide antibiotic nisin

The structural gene for the precursor of the peptide antibiotic nisin was isolated and characterized. As with other lanthionine-containing antibiotics, nisin is synthesized as a pre-propeptide which undergoes post-translational modification to generate the mature antibiotic. The sequence data obtained agreed with those of precursor nisin genes isolated by other workers from different Lactococcus lactis strains. Analysis of regions flanking the precursor nisin gene revealed the presence of a downstream open reading frame that may be involved in maturation of the precursor molecule. Nucleotide sequences characteristic of an IS element were located upstream of the nisin determinant. This element, termed IS904, is present in multiple copies in the genome of L. lactis. The nisin determinant of L. lactis is a component of a large transmissible gene block that also encodes nisin resistance and sucrose-metabolizing genes. Gene probe experiments indicated that the nisin/sucrose gene block was located in the chromosome. Furthermore, the copy of IS904 identified adjacent to the precursor nisin gene lies at, or very close to, one end of this transmissible DNA segment and may play a role in mediating its transfer between strains.     

Cloning of nisin resistance determinant and replication origin on 7.6-kilobase EcoRI fragment of pNP40 from Streptococcus lactis subsp. diacetylactis DRC3.

The nisin resistance determinant and an origin of replication on pNP40, a plasmid of about 60 kilobases that is present in Streptococcus lactis subsp. diacetylactis DRC3, was cloned on a 7.6-kilobase EcoRI fragment. When self-ligated, this fragment existed as an independent replicon (pFM011) and contained a 2.6-kilobase EcoRI-XbaI fragment encoding nisin resistance.     

Cloning of nisin resistance determinant and replication origin on 7.6-kilobase EcoRI fragment of pNP40 from Streptococcus lactis subsp. diacetylactis DRC3

The nisin resistance determinant and an origin of replication on pNP40, a plasmid of about 60 kilobases that is present in Streptococcus lactis subsp. diacetylactis DRC3, was cloned on a 7.6-kilobase EcoRI fragment. When self-ligated, this fragment existed as an independent replicon (pFM011) and contained a 2.6-kilobase EcoRI-XbaI fragment encoding nisin resistance.     

Nisin resistance of Streptococcus bovis

The growth of Streptococcus bovis JB1 was initially inhibited by nisin (1 microM), and nisin caused a more than 3-log decrease in viability. However, some of the cells survived, and these nisin-resistant cells grew as rapidly as untreated ones. To see if the nisin resistance was merely a selection, nisin-sensitive cells were obtained from agar plates lacking nisin. Results indicated that virtually any nisin-sensitive cell could become nisin-resistant if the ratio of nisin to cells was not too high and the incubation period was long enough. Isolates obtained from the rumen were initially nisin sensitive, but they also developed nisin resistance. Nisin-resistant cultures remained nisin resistant even if nisin was not present, but competition studies indicated that nisin-sensitive cells could eventually displace the resistant ones if nisin was not present. Nisin-sensitive, glucose-energized cells lost virtually all of their intracellular potassium if 1 microM nisin was added, but resistant cells retained potassium even after addition of 10 microM nisin. Nisin-resistant cells were less hydrophobic and more lysozyme-resistant than nisin-sensitive cells. Because the nisin-resistant cells bound less cytochrome c, it appeared that nisin was being excluded by a net positive (i.e., less negative) charge. Nisin-resistant cells had more lipoteichoic acid than nisin-sensitive cells, and deesterified lipoteichoic acids from nisin-resistant cells migrated more slowly through a polyacrylamide gel than those from nisin-sensitive cells. These results indicated that lipoteichoic acids could be modified to increase the resistance of S. bovis to nisin. S. bovis JB1 cultures were still sensitive to monensin, tetracycline, vancomycin, and bacitracin, but ampicillin resistance was 1,000-fold greater.     

Nisin Resistance in Clostridium botulinum Spores and Vegetative Cells

The frequencies at which vegetative cells and spores of Clostridium botulinum strains 56A, 62A, 17409A, 25763A, 213B, B-aphis, and 169B formed colonies on agar media containing 0, 10(sup2), 10(sup3), and 10(sup4) IU of nisin per ml at 30(deg)C were determined. Strain 56A had the highest frequencies of nisin resistance, while strains 62A, 169B, and B-aphis had the lowest. For most strains, spores were more resistant than vegetative cells. One exposure to nisin was sufficient to generate stable nisin-resistant isolates in some strains. Stepwise exposure to increasing concentrations of nisin generated stable resistant isolates from all strains. Spores produced from nisin-resistant isolates maintained their nisin resistance. The frequency of spontaneous nisin resistance was reduced considerably by lowering the pH of the media and adding 3% NaCl. Nisin-resistant isolates of strains 56A and 169B also had increased resistance to pediocin PA1, bavaricin MN, plantaricin BN, and leuconocin S.     

Properties of nisin Z and distribution of its gene, nisZ, in Lactococcus lactis.

Two natural variants of the lantibiotic nisin that are produced by Lactococcus lactis are known. They have a similar structure but differ in a single amino acid residue at position 27; histidine in nisin A and asparagine in nisin Z (J.W.M. Mulders, I.J. Boerrigter, H.S. Rollema, R.J. Siezen, and W.M. de Vos, Eur. J. Biochem, 201:581-584, 1991). The nisin variants were purified to apparent homogeneity, and their biological activities were compared. Identical MICs of nisin A and nisin Z were found with all tested indicator strains of six different species of gram-positive bacteria. However, at concentrations above the MICs, with nisin Z the inhibition zones obtained in agar diffusion assays were invariably larger than those obtained with nisin A. This was observed with all tested indicator strains. These results suggest that nisin Z has better diffusion properties than nisin A in agar. The distribution of the nisin variants in various lactococcal strains was determined by amplification of the nisin structural gene by polymerase chain reaction followed by direct sequencing of the amplification product. In this way, it was established that the nisZ gene for nisin Z production is widely distributed, having been found in 14 of the 26 L. lactis strains analyzed.     

Streptococcus cremoris M12R transconjugants carrying the conjugal plasmid pTR2030 are insensitive to attack by lytic bacteriophages

Conjugal transfer of lactose-fermenting ability (Lac+), nisin resistance (Nisr), and phage resistance (Hsp+) was demonstrated in matings between Streptococcus lactis ME2 (donor) and Streptococcus cremoris M43a (recipient), a derivative of M12R. Transconjugants were detected by transfer of Lac+ and were found to exhibit Nisr and harbor a 40-megadalton plasmid (pTR1040). Fifty-six percent of Lac+ transconjugants were resistant to the S. cremoris M12R lytic phage. Efficiency of plaquing for phage m12r . M12 on a phage-resistant transconjugant, T2r-M43a, was less than 4.3 X 10(-10). Five additional phages which were virulent for S. cremoris M12R and isolated from industrial sources failed to plaque on S. cremoris T2r-M43a. Mating experiments with T2r-M43a revealed that phage resistance was accompanied by high-frequency conjugation ability (Tra+) and the appearance of both pTR1040 and pTR2030 encoding Lac+ Nisr and Tra+ Hsp+, respectively, in transconjugants of S. lactis LM2302. Phage-sensitive Lac+ transconjugants of S. cremoris M43a (T2s-M43a) showed no conjugal ability. These observations confirmed that pTR2030 was present and responsible for the phage resistance and conjugal ability exhibited by the S. cremoris transconjugant T2r-M43a. Unlike the S. lactis LM2302 transconjugant carrying pTR2030, resistance of T2r-M43a to phage was not affected at high temperatures (35 to 40 degrees C) or destabilized in repeated transfers through a starter culture activity test. These results demonstrated that phage resistance conferred by pTR2030 in the S. cremoris transconjugant was effective against industrially significant phages under fermentation conditions normally encountered during cheese manufacture.     

Streptococcus cremoris M12R transconjugants carrying the conjugal plasmid pTR2030 are insensitive to attack by lytic bacteriophages.

Conjugal transfer of lactose-fermenting ability (Lac+), nisin resistance (Nisr), and phage resistance (Hsp+) was demonstrated in matings between Streptococcus lactis ME2 (donor) and Streptococcus cremoris M43a (recipient), a derivative of M12R. Transconjugants were detected by transfer of Lac+ and were found to exhibit Nisr and harbor a 40-megadalton plasmid (pTR1040). Fifty-six percent of Lac+ transconjugants were resistant to the S. cremoris M12R lytic phage. Efficiency of plaquing for phage m12r . M12 on a phage-resistant transconjugant, T2r-M43a, was less than 4.3 X 10(-10). Five additional phages which were virulent for S. cremoris M12R and isolated from industrial sources failed to plaque on S. cremoris T2r-M43a. Mating experiments with T2r-M43a revealed that phage resistance was accompanied by high-frequency conjugation ability (Tra+) and the appearance of both pTR1040 and pTR2030 encoding Lac+ Nisr and Tra+ Hsp+, respectively, in transconjugants of S. lactis LM2302. Phage-sensitive Lac+ transconjugants of S. cremoris M43a (T2s-M43a) showed no conjugal ability. These observations confirmed that pTR2030 was present and responsible for the phage resistance and conjugal ability exhibited by the S. cremoris transconjugant T2r-M43a. Unlike the S. lactis LM2302 transconjugant carrying pTR2030, resistance of T2r-M43a to phage was not affected at high temperatures (35 to 40 degrees C) or destabilized in repeated transfers through a starter culture activity test. These results demonstrated that phage resistance conferred by pTR2030 in the S. cremoris transconjugant was effective against industrially significant phages under fermentation conditions normally encountered during cheese manufacture.     

Partial characterization of the genetic basis for sucrose metabolism and nisin production in Streptococcus lactis.

We attempted to identify the genetic loci for sucrose-fermenting ability (Suc+), nisin-producing ability (Nip+), and nisin resistance (Nisr) in certain strains of Streptococcus lactis. To obtain genetic evidence linking the Suc+ Nip+ Nisr phenotype to a distinct plasmid, both conjugal transfer and transformation were attempted. A conjugation procedure modified to protect the recipients against the inhibitory action of nisin allowed the conjugal transfer of the Suc+ Nip+ Nisr marker from three Suc+ Nip+ Nisr donors to various recipients. The frequency of transfer ranged from 1.7 x 10(-4) to 5.6 x 10(-8) per input donor, depending on the mating pair. However, no additional plasmid DNA was apparent in these transconjugants. Transformation of S. lactis LM0230 to the Suc+ Nip+ Nisr phenotype by using the plasmid pool of S. lactis ATCC 11454 was not achieved, even though other plasmids present in the pool were successfully transferred. However, two results imply the involvement of plasmid DNA in coding for the Suc+ Nip+ Nisr phenotype. The Suc+ Nip+ Nisr marker was capable of conjugal transfer to a recipient deficient in host-mediated homologous recombination (Rec-), and the Suc+ Nip+ Nisr marker exhibited bilateral plasmid incompatibility with a number of lactose plasmids found in S. lactis. Although our results indicate that the Suc+ Nip+ Nisr phenotype is plasmid encoded, no physical evidence linking this phenotype to a distinct plasmid was obtained.