Conjugative R plasmids in group C and G streptococci.

Two streptococcal isolates of groups C and G harbored conjugative R plasmids with molecular weights of 17 X 10(6) (pIP646) and 20 X 10(6) (pIP920). These plasmids carried genetic markers for resistance to macrolides and related drugs, as well as to chloramphenicol (pIP920), and have very similar HindIII restriction enzyme patterns.     

Conjugative transfer of multiple antibiotic resistance markers in Streptococcus pneumoniae.

Two antibiotic-resistant isolates of Streptococcus pneumoniae were investigated for conjugative transfer of their drug resistance markers into streptococcal (groups B and D) and pneumococcal (encapsulated and non-encapsulataed) recipients. Of these, 7 wild-type donor pneumococci transferred all their resistance markers (except Pc [penicillin], Su [sulfonamide], and Tp [trimethoprim]) into group D Streptococcus and non-encapsulated S. pneumoniae recipients at a low frequency (10(-5) to 10(-6)). The resistance markers transferred were Tc (tetracycline); Tc and Cm (chloramphenicol); Tc and MLS (macrolides, lincosamides, and streptogramin B); Tc, MLS, Km (kanamycin), and Cm. The transconjugants obtained retransferred their resistance markers into appropriate streptococcal or pneumococcal recipients or both. The resistance markers of streptococcal transconjugants could not be cured by chemical agents. All attempts to detect extra-chromosomal deoxyribonucleic acid from pneumococcal or streptococcal transconjugants were unsuccessful. The molecular weight of a streptococcal conjugative R plasmid (pIP501) was investigated after transfer into the non-encapsulated S. pneumoniae recipient and was found to be similar to that of the wild-type group B Streptococcus host (20 x 10(6)).     

Binding of collagen to group A, B, C, D and G streptococci

Abstract Binding of ¹²⁵I-labelled collagen type II to group A, B, C, D and G streptococci was studied. Strains of all five serogroups were found to bind. Binding to one high-binding strain (group G, strain 12127) was characterised. This was reversible, saturable with time and inhibited by unlabelled type II collagen, but not by other proteins such as fibronectin and ovalbumin. However, binding was inhibited by unlabelled type I, II and III collagens and gelatin, suggesting that a common structure of various collagens is involved in binding.     

Conjugational plasmid transfer from A, B and H streptococci to N streptococci

Plasmid-mediated resistance to erythromycin and chloramphenicol was successfully transferred from group A, B and H streptococci to group N streptococci by a process akin to conjugation. The results showed that plasmids from streptococcal groups other than N were able to replicate in lactic streptococci as well. The transfer experiments were carried out by using a membrane filter mating technique. Four of the five plasmids used (pSM15346, pSM10419, pIP501, and pEL1) were transferred at frequencies ranging from 10(-1) to 10(-8) transconjugants per donor colony-forming unit. The highest transfer frequencies were obtained when S. pyogenes strain 15346 (pSM15346) served as the donor strain. The identy of transconjugants was verified by testing for the presence of unselected markers of the recipient strains, and both transduction and transformation were ruled out as the mechanisms of transfer.     

Hyaluronic acid capsule: strategy for oxygen resistance in group A streptococci.

Unencapsulated variants of encapsulated, M-protein-positive group A streptococci are oxygen sensitive and secrete inhibitory concentrations of hydrogen peroxide when grown in aerated broth cultures. The organisms were equally sensitive to hydrogen peroxide, and neither exhibited catalase or peroxidase activity, suggesting that differences in oxygen sensitivity reflect dissimilarity in oxygen uptake. The encapsulated parental culture was found to grow in aggregates that take up oxygen more slowly than unencapsulated, oxygen-sensitive derivatives. Moreover, the latter grow in an unaggregated, homogenous suspension. The enzyme hyaluronidase was able to disrupt aggregates of the encapsulated strain increase the rate that these cells take up oxygen, and cause the accumulation of toxic concentrations of hydrogen peroxide earlier in their growth cycle. The evidence presented shows that the aggregation of streptococcal cells by their hyaluronic acid capsule provides this organism with a novel means to avoid self-destruction by oxygen metabolites--cells are shielded from oxygen. The reduced surface-to-volume ratio and limited diffusion of oxygen into the interior of aggregates are proposed as the protective mechanism.     

Improved lysis of group N streptococci for isolation and rapid characterization of plasmid deoxyribonucleic acid.

Procedures for effective cellular lysis and plasmid deoxyribonucleic acid (DNA) isolation from group N streptococci were developed. Cells were grown at 32 degrees C for 4 h in a modified Elliker broth containing 20 mM DL-threonine. After cellular digestion with 2 mg of lysozyme per ml for 7 min at 37 degrees C, 1% sodium dodecyl sulfate exposure resulted in complete and immediate lysis. Lactose (Lac) plasmid species in Streptococcus lactis C2 and S. cremoris B1 (30 and 37 megadaltons, respectively) were demonstrated upon examination of DNA from the cleared lysates by agarose gel electrophoresis. Increasing the lysozyme treatment to 20 min or more resulted in loss of the Lac plasmid, whereas other resident plasmids were unaffected and demonstrable in agarose gels. Diethylpyrocarbonate added before lysis prevented Lac plasmid loss in 20-min lysozyme-treated cells, but was not effective after 40 min of lysozyme treatment. The results suggested that endogenous nuclease activity during the lysozyme treatment period initiated Lac plasmid DNA loss. The development of an efficient lysis procedure for the group N streptococci allowed rapid identification and characterization of plasmid DNA by agarose gel electrophoresis. The plasmid composition of S. lactis C2 and S. cremoris B1, as determined by agarose gel electrophoresis, compared favorably to previous electron microscopic observations.     

Improved lysis of group N streptococci for isolation and rapid characterization of plasmid deoxyribonucleic acid.

Procedures for effective cellular lysis and plasmid deoxyribonucleic acid (DNA) isolation from group N streptococci were developed. Cells were grown at 32 degrees C for 4 h in a modified Elliker broth containing 20 mM DL-threonine. After cellular digestion with 2 mg of lysozyme per ml for 7 min at 37 degrees C, 1% sodium dodecyl sulfate exposure resulted in complete and immediate lysis. Lactose (Lac) plasmid species in Streptococcus lactis C2 and S. cremoris B1 (30 and 37 megadaltons, respectively) were demonstrated upon examination of DNA from the cleared lysates by agarose gel electrophoresis. Increasing the lysozyme treatment to 20 min or more resulted in loss of the Lac plasmid, whereas other resident plasmids were unaffected and demonstrable in agarose gels. Diethylpyrocarbonate added before lysis prevented Lac plasmid loss in 20-min lysozyme-treated cells, but was not effective after 40 min of lysozyme treatment. The results suggested that endogenous nuclease activity during the lysozyme treatment period initiated Lac plasmid DNA loss. The development of an efficient lysis procedure for the group N streptococci allowed rapid identification and characterization of plasmid DNA by agarose gel electrophoresis. The plasmid composition of S. lactis C2 and S. cremoris B1, as determined by agarose gel electrophoresis, compared favorably to previous electron microscopic observations.     

Solubilization of IgG-binding proteins from group A and G streptococci

The release of IgG-binding proteins from the cell surface of streptococcal strains AR-1 and G148 with various proteolytic enzymes, acid, alkali or SDS was investigated. The IgG-binding proteins were purified by affinity chromatography using IgG-Sepharose Fast Flow. After SDS-polyacrylamide gel electrophoresis and immuno-electroblotting the major proteins identified varied in relative molecular mass from 15,000 to 65,000 depending on the solubilizing agent used. The results showed that solubilization with trypsin gave the highest yield of IgG-binding proteins, that strain G148 yielded about twice the amount of protein as strain AR-1, and that elastase released an IgG-binding protein of high relative molecular mass of 65,000.     

Antibiotic sensitivity of beta-hemolytic Streptococcus group A on a new nutrient medium for the cultivation of streptococci

Sensitivity of 167 strains of beta-hemolytic streptococci of group A was studied with the method of serial dilutions on a solid agar medium for cultivation of streptococci. The medium was developed at the I. I. Mechnikov Research Institute of Vaccines and Sera. It does not require addition of blood or serum. The strains were found to be highly sensitive to penicillin, cephalothin and erythromycin. The number of the strains resistant to tetracycline, streptomycin, gentamycin, levomycetin (chloramphenicol) and ristomycin amounted to 51, 36, 23, 1.8 and 1.8 per cent, respectively. One of the strains (0.6 per cent) was resistant to lincomycin. Strains with multiple resistance were isolated. The necessity of regular control of distribution of antibiotic resistance among staphylococci is indicated.     

Involvement of locomotion of granulocytes in the phagocytosis of glass-adherent group B streptococci in the absence of opsonins

Abstract Phagocytosis of glass-adherent group B streptococci (GBS) by bovine polymorphonuclear leukocytes (PMN) was investigated by a fluorochrome microassay. Three out of the four tested strains were taken up in the absence of serum, but phagocytosis was increased when immobilized bacteria were pre-treated by either normal bovine serum (NBS, containing antibodies to GBS), precolostral calf serum (PCS, virtually devoid of antibodies), heat-inactivated PCS (H-PCS), or to a lesser extent by human serumalbumin (HSA) or gelatin. The fourth strain required opsonization by NBS to be ingested. The under-agarose PMN migration assay showed that HSA, PCS, H-PCS, and NBS had locomotion-promoting effects that ranked as for enhancement of phagocytosis. In addition, fixed antigen-antibody complexes inhibited both migration under agarose and surface phagocytosis. These findings suggested that when bacteria are sensitive to surface phagocytosis in the absence of opsonins, the enhancing effect of serum is essentially mediated through promotion of PMN locomotion and not through opsonin-enhanced ingestion.     

Transduction transmission of the extrachromosomal markers of antibiotic resistance in staphylococcal populations]

Transduction of extrachromosomal markers of resistance to penicillin and erythromycin in staphylococcal strains isolated from patients was studied. Two transduction methods were compared, i. e. transduction with a phage filtrate of the donor culture resistant to erythromycin and transduction on mixed cultivation of the donor and recipient. A higher transduction rate was observed with the latter method. Mixed cultivation of the donor cultures resistant to penicillin or erythromycin and the recipient strains of the wild type sensitive to these antibiotic resulted in transduction of the respective markers. Transductants which acquired prophage 6 simultaneously with marker Egg became donors of erythromycin resistance.