Chemical and biochemical studies for the differentiation of coagulase-positive staphylococci.

The cell wall composition, the configuration of lactic acid produced from glucose under anaerobic conditions, the occurrence of fructose-1,6-diphosphate (FDP) activated L-lactate dehydrogenase (L-LDH), and the esterase pattern were determined from more than 80 strains of coagulase-positive staphylococci isolated from man and animal. Strains isolated from man, swine, bovines and hares form a rather homogeneous group. They exhibit a similar cell wall composition, produce predominantly D,L-lactate and have a characteristic and simple esterase pattern. Coagulase-positive staphylococci isolated from dogs, horses, minks and pigeons are quite distinct from typical Staphylococcus aureus strains. They exhibit a different cell wall composition, produce only L-lactate, possess an L-LDH which is specifically activated by FDP, and have a quite complex esterase pattern.     

Antibiotic sensitivity of the microflora isolated in ENT diseases]

Sensitivity of beta-hemolytic streptococci of group A, Streptococcus viridans, Staphylococcus aureus, epidermal staphylococci, pneumococci, Proteus and Ps. aeruginosa isolated in 1975-1978 from patients with tonsillitis, otitis, sinusitis and other otorhinolaryngological diseases was studied with respect to 19 antibiotics. Data on comparison of the antibiotic sensitivity of the microflora isolated from the patients with otorhinolaryngological diseases in 1964-1974 and 1975-1978 are presented. It was shown that beta-hemolytic streptococci were highly sensitive to all the antibiotics tested except tetracycline. Among Streptococcus viridans the strains resistant to many antibiotics were more frequent than among beta-hemolytic streptococci. Most of the Staphylococcus aureus strains were sensitive to gentamycin, cephaloridin, oxacillin and resistant to the other antibiotics. The epidermal staphylococci were characterized by approximately the same antibiotic sensitivity as Staphylococcus aureus. Resistance of the predominating majority of the Pneumococcus strains to tetracycline was noted. Proteus and Ps. aeruginosa were resistant to all the antibiotics except aminoglycosides. The microflora isolated from the cases with otorhinolaryngological diseases in 1975-1978 were mainly characterized by lower antibiotic sensitivity than that isolated from the cases with the same diseases in 1964-1974. It is possible to suppose that the microorganisms isolated from the patients with otorhinolaringological diseases had no significant differences with respect to their antibiotic sensitivity from those isolated from the patients with other pathological processes.     

Bacteriostatic activity of serum against staphylococci

Cybulska, Janina (State Institute of Hygiene, Warsaw, Poland), and J. Jeljaszewicz. Bacteriostatic activity of serum against staphylococci. J. Bacteriol. 91:953-962. 1966.-Antistaphylococcal activity of normal serum against strains exhibiting various patterns of coagulase, clumping-factor, and staphylokinase production is not connected with the presence of these factors. Purified coagulase does not influence this property of serum. Coagulase-negative strains with clumping-factor activity grow in normal serum as typical pathogenic staphylococci. Serum bacteriostatic activity against staphylococci may be reversed by several nonspecific factors, such as sterile broth, supernatant fluids of coagulase-negative strains, and ammonium sulfate precipitates of culture supernatant fluids of various staphylococci. Immune sera with a high agglutinating titer for staphylococcal cells do not prevent growth of serum-resistant strains; serum-susceptible strains are inhibited as in normal serum control. Activation or blocking of the serum fibrinolytic system does not influence serum bacteriostatic activity. The growth rate of serum-resistant strains is identical in serum and in Todd-Hewitt broth; serum-susceptible strains are inhibited to the inoculum level, but decreases and increases in viable count are noted during a 24-hr observation period. Observations made with sera of 10 animal species clearly demonstrated differences in serum bacteriostatic activity, mouse serum being completely noninhibitory and cat serum only weakly inhibitory. The technique of quantitative determination of serum susceptibility of staphylococci is described, and the importance of serum antistaphylococcal activity in vitro is discussed. Experimental staphylococcal infection produced in rabbits by intravenous injection of different Staphylococcus aureus strains did not result in significant changes in serum antistaphylococcal activity. The technique of experimental infection used caused chronic infection, with a peak on the 14th day; this was proved by means of a newly developed 5'-nucleotidase test. At the same time, sera of infected animals exhibited slight inhibitory properties, which returned to initial values 1 week later. Infection was produced by strains recognized as nonpathogenic and was inhibited in vitro by sera from both normal and infected rabbits. It is concluded that antistaphylococcal activity of serum should be considered as an "in vitro" phenomenon, which seems to have no importance in defense mechanisms of rabbits infected intravenously with staphylococci.     

Antibiotic sensitivity of the causative agents of suppurative surgical infection]

Sensitivity of 1492 strains of the causative agents of the surgical purulent infections, i. e. pathogenic Staphylococcus, Proteus, Ps. aeruginosa and E. coli to benzylpenicillin, streptomycin, levomycetin, tetracycline, erythromycin, oleandomycin, monomycin, kanamycin, novobiocin, ampicillin, oxacillin, ceporin, gentamicin and rifampicin was studied. Gentamicin was most active against all the bacterial species tested. The staphylococci were in addition sensitive in a high percentage of the cases to rifampicin, novobiocin, ceporin, monomycin and kanamycin. The isolates of E. coli were in addition sensitive to ceporin, monomycin and kanamycin. Sensitivity of the strains of Ps. aeruginosa and Proteus was low to all of the antibiotics except gentamycin. Most of the strains of the causative agents of the surgical purulent infections were multiresistant to 4 antibiotics. The number of the staphylococcal strains sensitive to benzylpenicillin, streptomycin and levomycetin increased in 1976 as compared to 1975 on the background of a limited use of these antibiotics in clinics.     

The effect of staphylococci on the manifestation of opsonic cooperation in the complement system

The influence of the cultures of 14 Staphylococcus aureus and Staphylococcus epidermidis strains on the capacity of factor C3b of the complement for mediating the adhesive reaction of human neutrophils was studied. In experiments with 5 out of 11 S. aureus strains the essential weakening of reactions was registered, while one of the strains considerably enhanced reactions. Out of 3 S. epidermidis strains, the weakening of C3b-dependent reaction was noted in one case. The activity of the cultures did not correlate with the gelatinase properties of staphylococci and was absent in all gelatinase-positive Pseudomonas aeruginosa strains. The model worked out by the authors may be used for studying the influence of bacterial metabolites and biologically active substances on the effector properties of factor C3b of the complement.     

Use of ampicillin trihydrate in transplantation surgery]

Ampicillin trihydrate was used for the treatment of 29 patients with purulent inflammatory processes, such as peritonitis, suppurating operative wound, urinary tract infection after the kidney allotransplantation. The antibacterial activity of ampicillin was preliminarily tested on 517 microbial strains, i.e. staphylococci, streptococci, E. coli, Proteus and Ps. aeruginosa isolated from the surgical patients. The strains of penicillin sensitive staphylococci, streptococci and E. coli were most sensitive to the drug effect, the MIC ranging from 0.03 to 16 gamma/ml. It was shown that the blood retention time of the antibiotic was much more prolonged in the patients with a decreased excretion function of the kidneys. The treatment was performed under control of the clinical, bacteriological and biochemical parameters. The drug was used in a dose of 0.5 g 6--8 times a day for 5 to 15 days. A satisfactory therapeutic effect was registered in 73 per cent of the cases.     

Transferable amikacin and cefamandole resistance: Pseudomonas maltophilia and Acinetobacter strains as possible reservoirs of R plasmids

Three strains belonging to gramnegative non-fermenting rods, i.e. a Pseudomonas maltophilia strain and two strains of Acinetobacter, were tested, as representatives of different types of nosocomial strains, for transferability of their multiple drug resistance. As all of them posed difficulties in demonstrating the transferability of their resistance by conventional methods, a three-step procedure was developed that includes a transfer to rifampicin-resistant P. aeruginosa recipients, then to susceptible P. aeruginosa intermediate strains, and, finally, from these strains to rifampicin-resistant Enterobacteriaceae. In three strains studied three genetically different types of R plasmids have been demonstrated. P. maltophilia transferred Amikacin resistance, as well as resistance to other antibiotics, to P. aeruginosa and then to Enterobacteria. In contrast, an Amikacin-resistant Acinetobacter with quite identical multiple drug resistance spectrum transferred its resistance to P. aeruginosa only, but not to Enterobacteria. Finally, another Acinetobacter strain, resistant to Gentamicin but susceptible to Amikacin transferred this resistance directly to Enterobacteria (and, separately, to P. aeruginosa, too). All three strains transferred Cefamandole resistance together with other resistances. Non-fermenting rods, thus, might be a source of transmissible resistance to reserve antibiotics as Amikacin, and advanced-type Cephalosporins.     

Phenotypic and genotypic identification of staphylococci isolated from wild small mammals

229 strains of Staphylococcus isolated from the intestines of wild small mammals (Insectivora and rodents) were analysed by phenotypic tests and 16S-23S rDNA intergenic spacer PCR amplification (ITS-PCR). Based on the results of three methods (phenotypical tests, API numerical profiles and ITS-PCR) 65.5% of all staphylococcal strains could be identified. The remaining strains were compared by cluster analysis with reference and identified strains. It is quite possible that, a part of unidentified strains represent hitherto undescribed species, all the more as their ITS types were not found among other members of the genus Staphylococcus. Moreover, the results show that small mammals provides a suitable new habitat for certain staphylococci.     

The reciprocal effect of the causative agents in a mixed infection in burn injury

In vitro experiments on the joint cultivation of Pseudomonas aeruginosa, Escherichia coli, staphylococci and in vivo experiments carried out on mice with the experimental mixed infection of a burn injury revealed the pronounced antagonistic action of P. aeruginosa with respect to staphylococci and E. coli. Under the same conditions, in the joint cultivation of P. aeruginosa and fungi of the genus Candida and in the mixed infection of a burn wound caused by the same microorganisms the mutual stimulation of multiplication and a considerable aggravation of the clinical course of a burn wound were observed. The mutual influence of associates in the mixed infection of a burn injury is manifested by an increase in the number of an antibiotic-resistance microorganisms in their populations and, so far as the macroorganism is concerned, in the aggravation of the course of the infectious process and the formation of the pronounced state of immunodeficiency.     

Antagonistic action of Pseudomonas aeruginosa against Staphylococci, coliform bacilli and various types of Proteus

Antagonistic activity of 2 fresh isolates and 3 collection strains of Pseudomonas aeruginosa against 177 microbial strains was determined with the method of late antagonism. Among the microbial strains there were 56 staphylococcal strains isolated from patents and carriers. 38 nontypable colon bacilli isolated from healthy persons, 59 enteropathogenic colon bacilli of various serogroups, 12 strains of Proteus and 12 colon bacilli, carriers of multiple drug resistance factors (R factors). All the cultures were sensitive to the antagonistic action of 5 or at least 3 strains of Pseudomonas used in the study. The most active antagonists were the fresh isolates of Pseudomonas as compared to the collection strains. Among the staphylococci S. aureus proved to be the most resistant to the antagonistic action of Pseudomonas as compared to S. epidermidis, the same as the strains isolated from carriers as compared to the strains isolated from patients. As for the enteric bacilli the most resistant were the strains of Proteus. Acquiring of transmissive R factors by the colon bacilli markedly increased their sensitivity to the antagonistic action of Pseudomonas.     

Antibacterial activity of rifampicin against the causative agents of suppurative, septic diseases]

The antibacterial activity of rifampicin was studied in comparison with other antibiotics with respect to clinical strains isolated from cases with various purulent inflammatory processes caused by Staphylococcus, E. coli, Ps. aeruginose, Proteus. The aim of the study was to define the role of rifampicin in the treatment of the above infections. No rifampicin resistant strains were found among staphylococci belonging to the phenotype carrying the determinants of resistance to 2-8 antibiotics. Rifampicin was less active against gramnegative organisms. High heterogeneity of the microbial population of rifampicin was shown with respect to all microbial strains tested. The rate of the spontaneous mutants was high. The average rate of the mutants was 1-7.7-10-8. The studies on the dynamics of the rifampicin resistance increase in the strains of Staphylococci, E. Coli, Ps. aeruginosa and Proteus showed that the resistance increased after 1-2 passages, which means that one-stage mutation was characteristic rifampicin.     

Slide Agglutination Method for the Serological Identification of Neisseria gonorrhoeae with Anti-Gonococcal Antibodies Adsorbed to Protein A-Containing Staphylococci

A rapid slide agglutination test has been developed for the identification of Neisseria gonorrhoeae that are primarily detected as oxidase-positive colonies in gonococcal cultures. The technique is based on the specific nonimmune reactivity between the Fc portion of immunoglobulin (Ig)G and staphylococcal protein A. IgG molecules adsorbed to stabilized staphylococci will thereby become oriented with their antigen-reactive sites that are directed outwards. Protein A-containing staphylococci with unabsorbed anti-gonococcal antibodies gave positive co-agglutination reactions with gonococci but also with meningococci, some Moraxella, Haemophilus, and Pseudomonas strains. These crossreactions were eliminated by absorption of the anti-gonococcal antiserum with meningococcal and Moraxella organisms prior to the coating of reagent staphylococci. In the routine culture diagnosis of N. gonorrhoeae the use of specific gonococcal reagent staphylococci gave concordant results with fermentation procedures and immunofluorescent techniques.     

Crude petroleum-oil biodegradation efficiency of Bacillus subtilis and Pseudomonas aeruginosa strains isolated from a petroleum-oil contaminated soil from North-East India

The efficiency of Bacillus subtilis DM-04 and Pseudomonas aeruginosa M and NM strains isolated from a petroleum contaminated soil sample from North-East India was compared for the biodegradation of crude petroleum-oil hydrocarbons in soil and shake flask study. These bacterial strains could utilize crude petroleum-oil hydrocarbons as sole source of carbon and energy. Bioaugmentation of TPH contaminated microcosm with P. aeruginosa M and NM consortia and B. subtilis strain showed a significant reduction of TPH levels in treated soil as compared to control soil at the end of experiment (120 d). P. aeruginosa strains were more efficient than B. subtilis strain in reducing the TPH content from the medium. The plate count technique indicated expressive growth and biosurfactant production by exogenously seeded bacteria in crude petroleum-oil rich soil. The results showed that B. subtilis DM-04 and P. aeruginosa M and NM strains could be effective for in situ bioremediation.     

Combined Staphylococcus-Proteus-Pseudomonas vaccine. II. The toxicity of the vaccine in "chronic" experiments and its ability to protect animals against Proteus and Staphylococcus infections

The combined preparation consisting of the antigenic complexes of staphylococci (1 part), Proteus (1 part) and P. aeruginosa parts) was capable of protecting mice from infection with staphylococci, Proteus and P. aeruginosa and prolonging the survival time of rabbits under the conditions of the development of staphylococcal sepsis. The staphylococcal component of the combined preparation possessed adjuvant activity, increasing the immunogenicity of Proteus antigen. The combined vaccine enhanced a short-time increase in the nonspecific resistance of the animals. The moderate toxicity of the preparation permits making multiple injections of the preparation to mice without inhibiting the weight gain of the animals.     

Structure of the microflora of suppurative wounds and its sensitivity to antibiotics]

Clinico-bacteriological examination of patients with purulent infections showed that Staphylococcus was the predominating microflora in the wounds. Simultaneously an increasing role of gram-negative conditionally pathogenic bacteria was shown. Multiple drug resistance was found in the organisms tested. The highest sensitivity levels were observed to gentamicin, kanamycin, tetracycline, levomycetin. It was shown by means of special typing methods that staphylococci of phage group III and Ps. Aeruginosa of serotype II predominated in the infected wounds. When the pathological material contained the antibiotic resistant cultures of Ps. aeruginosa, Proteus, Klebsiella and toxigenic strains of Staphylococcus, a tendency for prolongation of the suppurative process was observed.     

Chemical and biochemical studies for the differentiation of coagulase-positive staphylococci

The cell wall composition, the configuration of lactic acid produced from glucose under anaerobic conditions, the occurrence of fructose-1,6-diphosphate (FDP) activatedl-lactate dehydrogenase (l-LDH), and the esterase pattern were determined from more than 80 strains of coagulase-positive staphylococci isolated from man and animal. Strains isolated from man, swine, bovines and hares form a rather homogencous group. They exhibit a similar cell wall composition, produce predominantlyd,l-lactate and have a characteristic and simple esterase pattern. Coagulasepositive staphylococci isolated from dogs, horses, minks and pigeons are quite distinct from typicalStaphylococcus aureus strains. They exhibit a different cell wall composition, produce onlyl-lactate, possess anl-LDH which is specifically activated by FDP, and have a quite complex esterase pattern.List of Abbreviations BBP bromphenol blue - FDP fructose-1,6-diphosphate - d-LDH d-lactate dehydrogenase - l-LDH l-lactate dehydrogenase - NAD nicotinamide adenine dinucleotide     

Safety-related properties of staphylococci isolated from food and food environments

Aims: To test some safety‐related properties within 321 staphylococci strains isolated from food and food environments. Methods and Results: The isolates were identified as Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus pasteuri, Staphylococcus sciuri, Staphylococcus warneri and Staphylococcus xylosus. Decarboxylase activity was quite common for the various Staphylococcus spp., and tyrosine was the most frequently decarboxylated amino acid. The frequency of antibiotic resistance was highest in Staph. pasteuri and Staph. xylosus. Several of the isolates were tolerant to QAC compounds, and in some cases, QAC tolerance was present in antibiotic‐resistant strains. Most of the strains displayed moderate to high adhesion rates to stainless steel and Teflon^®. The strains that readily formed biofilms belonged to the species Staph. aureus, Staph. epidermidis and Staph. pasteuri. Conclusions: An high incidence of some safety hazards was found within the staphylococcal strains of food origin tested in this study. In particular, amino acid decarboxylase activity and biofilm‐forming ability were common within strains, and antibiotic resistance and tolerance to QAC‐based compounds occurred frequently as well. These characteristics are an important safety concern for food industry. Significance and Impact of the Study: This work gives a first picture of safety hazards within staphylococcal species isolated from food environments. The presence of disinfectant‐resistant staphylococci is a concern because resistance can be genetically transferred between the various Staphylococcus species. This could lead an increase and spread of resistant enterotoxic staphylococci and/or pathogenic staphylococci.     

Lysozyme Production as an Aid for Identification of Potentially Pathogenic Strains of Staphylococci

Lysozyme production is a frequent property of potentially pathogenic staphylococci. In the present study, 1,186 strains of human origin, 85 strains of animal origin, and 156 strains of Staphylococcus albus (epidermidis) were tested. Of 1,114 coagulase-positive strains of human and animal origin, 1,098 were lysozyme-positive (98.5%). On the other hand, of 157 coagulase-negative strains which, based on further investigations, belong to the potentially pathogenic staphylococci, all were lysozyme-positive. All of the 156 strains (100%) belonging to the species S. albus (epidermidis) were lysozyme-negative. We conclude that lysozyme production is a better index of potentially pathogenic staphylococci than the measurement of free coagulase, especially in cases of strains of animal origin. It is possible that lysozyme production allows a differentiation between pathogenic and nonpathogenic coagulase-negative staphylococci.     

Determination of toxigenicity of Escherichia coli and Vibrio cholerae strains by radioimmunologic method

A solid phase variant of radioimmunoassay has been elaborated for screening toxin-producing strains of E. coli and V. cholerae grown on agar plates. The method is based on the ability of cholera-like toxins to be absorbed on nitrocellulose filters and their further identification with the use of homologous sera and [125I]-A protein from staphylococci. Sensitivity of the method reaches 20 pg. The proposed technique permits identification of intracellular enterotoxin and is aimed at a massive screening of E. coli strains, NAG-vibrios and V. cholerae strains for toxin production.     

Rifampicin in the treatment of infections of non-tuberculous etiology

Clinical efficacy of rifampicin, a semisynthetic broad spectrum antibiotic was estimated in 247 patients with purulent inflammations. It was shown advisable to use rifampicin intravenously in treatment of severe bronchopulmonary pathology, disorders of the bile excretion system, osteomyelitis, severe wound infections and in prophylaxis of postoperative purulent complications in cardiovascular surgery and other cases. High rifampicin sensitivity of staphylococci and streptococci belonging to various species was revealed. Rifampicin was found to be less active against gramnegative pathogens. The isolation frequency of rifampicin sensitive strains of E. coli, Proteus spp., Klebsiella spp. and P. aeruginosa amounted to 88.4, 52.1, 58.8 and 49.3 per cent respectively.