A Note on the Use of the Spiral Plater to Study the In Vitro Effects of Amoxycillin, Spectinomycin and Chloramphenicol on a Porcine Escherichia coli

The use of the Spiral Plater to count bacteria during a minimum inhibitory concentration determination is described for amoxycillin, spectinomycin and chloramphenicol against a porcine Escherichia coli (Abbotstown strain). Chloramphenicol was bacteriostatic. Spectinomycin showed slow bactericidal activity. Amoxycillin was rapidly bactericidal.     

Comparative Characteristics of Human and Porcine Staphylococci and Their Differentiation in Burn Xenografting Procedures

Staphylococcus epidermidis from porcine skin differed from human cutaneous S. epidermidis in that the former strains were principally of the Baird-Parker biotype III group. The porcine-type strains were more proteolytic on casein and gelatin than were human strains, which were primarily of biotype II. Porcine strains were also elastolytic. Using supernatant fluids of broth cultures, the biotype II strains, but not the type III strains, were lipolytic in action on triolein. Both types of staphylococci were similar in enzymatic activities on Tween 80, egg yolk, and tributyrin. Elastase activity was not found in broth supernatant fluid of these bacteria. The porcine strains were retarded or inhibited from growing in media at pH 5.5. Action on casein agar followed by demonstration of elastase activity were used as markers to detect the porcine S. epidermidis strains in xenografts and on human burn wound grafting sites.     

Coagulase-Negative,Novobiocin-Resistant Staphylococci on the Skin of Animals and Man,on Meat and in Milk

The occurrence of coagulase-negative, novobiocin-resistant staphylococci, i.e. Staphylococcus cohnii, Staphylococcus saprophyticus, Staphylococcus sciuri and Staphylococcus xylosus, on the skin of animals and man has been studied. On cultures from cats, cows, dogs, guinea pigs, mice, rabbits and sheep studied, such organisms were predominant among the coagulase-negative staphylococci. From the skin of the hands of 21 of 38 persons whose professions brought them into contact with animals, e.g. inséminât ors, slaughterhouse workers and veterinarians, coagulase-negative, novobiocin-resistant staphylococci were isolated. This finding contrasted with that regarding 50 persons lacking such contacts, of whom only 1 harboured such bacteria. S. saprophyticus was isolated only from those slaughterers presenting with wounds on their hands. Coagulase-negative, novobiocin-resistant staphylococci were also isolated from every second specimen collected from the surface of meat at a slaughterhouse. No difference in the culture results could be demonstrated from specimens collected before and after cutting-up of the carcasses. Of 26 strains of coagulase-negative, DNase-negative staphylococci isolated from milk with pathological CMT, all but 5 were novobiocin-resistant. Fifteen were classified as S. xylosus, 4 as S. sciuri and 1 as S. cohnii. Of another 15 DNase-positive strains, 3 were resistant to novobiocin. Finally, clinical infections with coagulase-negative, novobiocin-resistant staphylococci in man, e.g. urinary tract infections caused by S. saprophyticus, are considered in relation to possible contagious reservoirs and modes of spread.     

Mammalian PGRPs: novel antibacterial proteins

Peptidoglycan recognition proteins (PGRPs) are innate immunity molecules conserved from insects to mammals. Insects have up to 19 PGRPs, which activate Toll or Imd signal transduction pathways or induce proteolytic cascades that generate antimicrobial products, induce phagocytosis, hydrolyse peptidoglycan, and protect insects against infections. Mammals have four PGRPs, which were hypothesized to function as signal-transducing pattern recognition receptors. However, all mammalian PGRPs are secreted, usually as disulphide-linked homo- and heterodimers. One mammalian PGRP, PGLYRP-2, is an N-acetylmuramoyl-L-alanine amidase that hydrolyses bacterial peptidoglycan and reduces its proinflammatory activity. PGLYRP-2 is secreted from liver into blood, and is also induced by bacteria in epithelial cells. The three remaining mammalian PGRPs are bactericidal or bacteriostatic proteins. PGLYRP-1 is expressed primarily in the granules of polymorphonuclear leucocytes (PMNs) , and PGLYRP-3 and PGLYRP-4 are expressed in the skin, eyes, salivary glands, throat, tongue, esophagus, stomach and intestine, and protect the host against infections. They kill bacteria by interacting with their cell wall peptidoglycan, rather than permeabilizing their membranes. These PGRPs therefore are a new class of bactericidal and bacteriostatic proteins that have different structure, mechanism of action, and expression pattern from currently known vertebrate antimicrobial peptides. Direct bactericidal activity of these PGRPs either evolved in vertebrates or mammals, or it is yet to be discovered in insects.     

Bactericidal and bacteriostatic antigonococcal activities produced by urogenital staphylococci

We have overcome some of the difficulties in obtaining soluble antigonococcal activity produced by staphylococci by using a very sensitive detection method. This method is based on the light absorbance determinations of liquid cultures of the gonococcus incubated for 6 h in the presence of serial dilutions of the inhibitor as compared to the absorbance of uninhibited control cultures. Antigonococcal activity was detected in the liquid phase prepared from semisolid agar cultures of all twenty two staphylococcal isolates tested. Sixteen supernatants from liquid cultures were also found to be active. The antigonococcal activity detected was differentiated by colony forming units counts into two types, bacteriostatic and bactericidal. After 6 h of incubation of the gonococcus in the presence of five arbitrary units (AU)/ml of the bactericidal activity produced by one of the strains of staphylococci, isolate 37, the loss of viability was over 99.9%, while 10 AU/ml of the bacteriostatic activity produced by isolate 66 did not cause any loss of viability of the gonococcus.     

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.     

Bacteriostatic and bactericidal action of bile acids and antibiotics on staphylococci

It was found in vitro that desoxycholic, cholic, glycocholic and choleinic acids inhibited the growth and development of staphylococci. The staphylococci isolated from bile were more resistant to the bacteriostatic and bactericidal effect of bile acids than the microorganisms isolated from other sources not containing cholates. Under the effect of these substances the activity of some antibiotics especially those from the group of aminoglycosides markedly increased.     

In vitro measurement of the adherence of Staphylococcus epidermidis to plastic by using cellular urease as a marker.

A rapid and sensitive in vitro assay was developed to quantitatively assess the adherence of Staphylococcus epidermidis to a hydrophobic plastic surface. The assay is based upon the detection of cell-associated urease activity as a marker of bacteria remaining adherent to the polystyrene microwells of flat-bottomed, 96-well tissue culture plates. Using ATCC 35984, a slime-producing strain of S. epidermidis, the assay could detect as few as 3 x 10(3) bacteria and was linear to 3.5 x 10(7) bacteria. The adherence of both slime-positive and slime-negative coagulase-negative staphylococci could be evaluated by using this method. This assay could be used to examine factors which influence the adherence of individual S. epidermidis strains to hydrophobic surfaces and to develop agents or coating materials which suppress the adherence of coagulase-negative staphylococci to biomedical implants.     

In vitro measurement of the adherence of Staphylococcus epidermidis to plastic by using cellular urease as a marker

A rapid and sensitive in vitro assay was developed to quantitatively assess the adherence of Staphylococcus epidermidis to a hydrophobic plastic surface. The assay is based upon the detection of cell-associated urease activity as a marker of bacteria remaining adherent to the polystyrene microwells of flat-bottomed, 96-well tissue culture plates. Using ATCC 35984, a slime-producing strain of S. epidermidis, the assay could detect as few as 3 x 10(3) bacteria and was linear to 3.5 x 10(7) bacteria. The adherence of both slime-positive and slime-negative coagulase-negative staphylococci could be evaluated by using this method. This assay could be used to examine factors which influence the adherence of individual S. epidermidis strains to hydrophobic surfaces and to develop agents or coating materials which suppress the adherence of coagulase-negative staphylococci to biomedical implants.     

Antibiotic resistance of staphylococci isolated from outpatients

The aim of the study was to determine susceptibility of 587 strains of S. aureus and 85 strains of coagulase-negative staphylococci isolated from outpatients in Poznań to co-trimoxazole, amoxycillin/clavulanic acid, erythromycin, gentamycin, doxycycline, ampicillin, oxacillin, cephradine, clindamycin and neomycin. Also methicillin-resistant strains were determined as well as strains ability to produce beta-lactamases. Susceptibility testing and examination of methicillin-resistant strains were performed by the disc diffusion techniques according to recommendation of NCCLS. Methicillin-resistant strains were additionally examined to their sensitivity to vankomycin and teicoplanin. beta-lactamase production was detected using nitrocefin impregnated discs and iodometric method. Amoxacillin/clavulanic acid, gentamycin, co-trimoxazole, cephradin, oxacillin and clindamycin occurred to be very active against both, S. aureus and coagulase-negative staphylococci. 84.7% to 100% of examined strains were sensitive to these drugs. Doxycyclin, erythromycin and ampicillin were less effective. Nine strains (1.5%) of 587 strains of S. aureus as well as 7 strains (8.7%) of coagulase-negative staphylococci were methicillin-resistant. All of methicillin-resistant strains were sensitive to vancomycin and teicoplanin. More than 75% of S. aureus and close to 50% of coagulase-negative staphylococci were able to produce beta-lactamases.     

Susceptibility of Coagulase-negative Staphylococci to Lysostaphin and Other Antibiotics

In general, coagulase-negative staphylococci were found to be relatively less susceptible to the lytic action of lysostaphin than coagulase-positive staphylococci. To achieve, arbitrarily, a lysis greater than 75%, it was necessary to use an increased concentration of enzyme or a longer incubation period than that usually required with coagulase-positive strains. For the most part, the cultures studied were sensitive to oxacillin, cloxacillin, dicloxacillin, nafcillin, ancillin, cephalothin, cephaloridine, fusidic acid, lincomycin, novobiocin, and neomycin [median minimal inhibitory concentrations (MIC) of 1.56 mug/ml or less]. Some degree of resistance (median MIC values of 12.5 mug/ml or greater) to benzylpenicillin, ampicillin, methicillin, tetracycline, chloretetracycline, erythromycin, ristocetin, and lysostaphin was found. Ten methicillin-resistant, coagulase-negative staphylococal strains were found to be cross-resistant to all nine of the penicillins tested, but much less resistant to the two cephalosporin analogues. In several instances, some of these strains seemed to be more sensitive to benzylpenicillin and to certain of the semisynthetic penicillins than to methicillin. Of the 18 antibiotics tested with the viable plate count method, the methicillin-resistant strains were found to be the most sensitive to lincomycin and novobiocin.     

The antibacterial activity against MRSA strains and other bacteria of a <500Da fraction from maggot excretions/secretions of Lucilia sericata (Diptera: Calliphoridae)

The application of Lucilia sericata larvae to chronic, infected wounds results in the rapid elimination of infecting microorganisms, including MRSA. Previously, we demonstrated in vitro antibacterial activity of native excretions/secretions (nES) from L. sericata and partially purified two low mass antibacterial compounds with masses of 0.5-10kDa and <500Da. The present study reports the antibacterial effects of the <500Da fraction (ES<500) on the growth and morphology of a range of bacteria, including 12 MRSA strains. Distinct morphological changes were observed in Bacillus cereus and Escherichia coli following exposure to ES<500. Flow cytometry and confocal microscopy analyses, in conjunction with turbidometric and CFU assays, revealed bacteriostatic activity of nES against S. aureus and E. coli. ES<500 also demonstrated bacteriostatic activity against S. aureus, however, bactericidal activity and the induction of a viable but non-culturable state were observed with ES<500-treated E. coli.     

Antagonistic action of corynebacteria and bacilli of a skin ecotype on staphylococci

582 strain of corynebacteria and 235 cultures of bacilli isolated from the skin surface of mammary glands of 120 nursing women were studied for their antagonistic action on staphylococci. It is established that bacilli, especially Bacillus subtilis, B. firmus and B. alvei exert more pronounced inhibitory effect on staphylococci. Representatives of the family Corynebacteriaceae possess a weak antagonistic action both on coagulase-positive and on coagulase-negative species. The ability of coryneform bacteria to retain the colonization resistance is supposed to be due to other factors. Antagonistic properties of B. subtilis are shown expedient to be used for elimination of hospital strains of Staphylococcus aureus from the skin of mammary glands.     

Bactericidal Activity and Synergy Studies of Peptide AP-CECT7121 Against Multi-resistant Bacteria Isolated from Human and Animal Soft Tissue Infections

AP-CECT7121 is an antimicrobial peptide, produced by Enterococcus faecalis CECT7121, with bactericidal activity against Gram-positive bacteria. The aim of this study was to evaluate the bactericidal activity of AP-CECT7121, alone and with gentamicin, against multi-resistant bacteria isolated from human and animals with soft tissue infections. During the period 2014–2015, bacterial strains producing human and animal soft tissue infections were studied. Samples from patients attended at a general hospital and cattle from four dairies in the Province of Buenos Aires (Argentina) were included. Twenty-two methicillin-resistant Staphylococcus aureus (11, human blood samples; 11, cow milk) and five vancomycin-resistant Ent. faecium strains isolated from four mastitic dairy cows were tested. AP-CECT7121 (12 mg/L) potency was assessed by time-kill curves alone or with sub-inhibitory concentrations of gentamicin. All staphylococcal strains were susceptible to gentamicin; enterococci did not show high-level gentamicin resistance. Colony counts were carried out at 0, 2, 4, 8, and 24 h of incubation. AP-CECT7121 showed bactericidal activity against all the enterococcal strains. In addition, AP-CECT7121 had a bactericidal effect on most staphylococci (16/22). Early AP-CECT7121/gentamicin synergy (4–8 h) for all staphylococci was detected. At 24 h, synergy (19/22) and indifference (3/22) were observed. Synergy with gentamicin was detected for staphylococci. AP-CECT7121 constitutes an attractive candidate for its use as a natural therapeutic tool for the treatment of infections produced by multi-resistant Staph. aureus and vancomycin-resistant Ent. faecium isolated from humans and animals.     

Bactericidal activity of the membrane fraction isolated from phagocytes of mice and its stimulation by melittin

The membrane fraction prepared from mouse peritoneal exudate cells was incubated with mycobacteria, staphylococci, or E. coli in acetate buffer of pH 5.6 to follow the fate of viable bacilli. The membrane fraction exhibited bactericidal effect on mycobacteria and staphylococci, but not on E. coli. The activity to kill mycobacteria, as well as the endogenous phospholipase A2 activity, of the membrane fraction was markedly enhanced by melittin, a basic peptide from bee venom, and inhibited by indomethacin and EDTA. The role of the enzyme activity in the bactericidal activity was discussed.     

Rifampicin for lepromatous leprosy: nine years' experience.

Over 100 patients with lepromatous leprosy were treated with rifampicin in a series of pilot, uncontrolled, and controlled trials in 1968-77. The rapid bactericidal effect of rifampicin on Mycobacterium leprae was confirmed. Clinical improvement became apparent sometimes as early as 14 days after the start of treatment. Nevertheless, a few persisting viable M leprae were detected as long as five years after the start of treatment with rifampicin either by itself or in combination with the bacteriostatic drug thiambutosine. Treatment with rifampicin and dapsone for six months reduced the number of persisting leprosy bacteria more than treatment with dapsone alone. Although rifampicin proved more effective than dapsone, it is unlikely that used by itself if can significantly shorten the length of treatment in lepromatous leprosy. Therefore initial intensive combined treatment with two or more bactericidal drugs (including rifampicin) warrants further investigation in both untreated leprosy and lepromatous leprosy resistant to dapsone.     

鼠李糖乳杆菌PUM1749高盐和胆盐耐受能力与抑菌活性研究

以结肠癌术后患者粪便中分离出的鼠李糖乳杆菌PUM1749作为研究对象,通过检测其耐高盐、耐胆盐能力和抑菌活性,评价其益生特性。

以鼠李糖乳杆菌GG为对照,将其和鼠李糖乳杆菌PUM1749分别培养于含不同浓度NaCl和胆盐的MRS肉汤中,采用滴种法进行活菌计数,采用双层琼脂夹心法结合滴种法检测两株菌对8种指示菌的抑菌效果。

在不同浓度NaCl溶液培养24 h后,2株鼠李糖乳杆菌活菌数均在10⁸ CFU/mL数量级以上。当浓度低于2 g/100 mL时,2株菌活菌数随NaCl浓度增加而减少,之后随着NaCl浓度的提高,PUM1749株活菌数均高于GG株;在不同浓度胆盐肉汤中培养4 h后,2株菌生长均被不同程度抑制,活菌数显著下降,PUM1749株在胆盐浓度为0.1 g/100 mL时生长被完全抑制,但继续提高胆盐浓度后,PUM1749株活菌数反而明显优于GG株;2株菌对8种指示菌均具有较好的抑菌效果,且对革兰阳性菌和革兰阴性菌的抑菌效果无明显差别。

鼠李糖乳杆菌PUM1749在不同高盐和胆盐浓度条件下的耐受能力和抑菌活性均强于或持平鼠李糖乳杆菌GG。鼠李糖乳杆菌PUM1749具有良好的益生特性。

     

Inactivation of gram-negative bacteria by lysozyme, denatured lysozyme, and lysozyme-derived peptides under high hydrostatic pressure

We have studied the inactivation of six gram-negative bacteria (Escherichia coli, Pseudomonas fluorescens, Salmonella enterica serovar Typhimurium, Salmonella enteritidis, Shigella sonnei, and Shigella flexneri) by high hydrostatic pressure treatment in the presence of hen egg-white lysozyme, partially or completely denatured lysozyme, or a synthetic cationic peptide derived from either hen egg white or coliphage T4 lysozyme. None of these compounds had a bactericidal or bacteriostatic effect on any of the tested bacteria at atmospheric pressure. Under high pressure, all bacteria except both Salmonella species showed higher inactivation in the presence of 100 microg of lysozyme/ml than without this additive, indicating that pressure sensitized the bacteria to lysozyme. This extra inactivation by lysozyme was accompanied by the formation of spheroplasts. Complete knockout of the muramidase enzymatic activity of lysozyme by heat treatment fully eliminated its bactericidal effect under pressure, but partially denatured lysozyme was still active against some bacteria. Contrary to some recent reports, these results indicate that enzymatic activity is indispensable for the antimicrobial activity of lysozyme. However, partial heat denaturation extended the activity spectrum of lysozyme under pressure to serovar Typhimurium, suggesting enhanced uptake of partially denatured lysozyme through the serovar Typhimurium outer membrane. All test bacteria were sensitized by high pressure to a peptide corresponding to amino acid residues 96 to 116 of hen egg white, and all except E. coli and P. fluorescens were sensitized by high pressure to a peptide corresponding to amino acid residues 143 to 155 of T4 lysozyme. Since they are not enzymatically active, these peptides probably have a different mechanism of action than all lysozyme polypeptides.     

Effect of molybdate ions on methanation of simulated and natural waste-waters

Summary Sulphate in concentrations of 500 and 1000 mg SO₄-S/l did not inhibit methanation of synthetic waste-water (acetate + methanol + glucose) by sludge from a digester treating neutral spent sulphite process effluents. The role of sulphate reducers in the conversion of those substrates was minor although sulphate-reducing bacteria were present with a viable count similar to that of methane-producing bacteria in the sludge. Neutral spent sulphite liquor was partially converted to methane (40% of chemical oxygen demand) under these conditions.Molybdate (20 mM) inhibited methanation of both synthetic waste-water and neutral spent sulphite liquor. Acetate accumulated in glucose plus molybdate media. Molybdate had a direct inhibitory effect on enriched acetoclastic methane-producing bacteria. Molybdate was bacteriocidic to sulphate-reducing bacteria and bacteriostatic to methane-producing bacteria.     

Biomaterial-associated staphylococcal peritoneal infections in a neutropaenic mouse model

Abstract Adhesion of staphylococcal cells to polyethylene with end point-attached heparin was quantified by bioluminescence. Staphylococcus epidermidis 3380 and the slime-producing S. epidermidis RP12 adhered to the highest extent, and S. lugdunensis 2342 to the least extent. Preincubation of the polymer with dialysis fluid reduced adhesion of S. epidermidis 3380 and RP12 but enhanced that of S. aureus , and preadsorption of the surface with fibronectin decreased subsequent adhesion of S. epidermidis and S. haemolyticus strains. When staphylococci were grown in the presence of a biomaterial their ability to activate peritoneal cells was decreased. The bactericidal activity was impaired, whereas ingestion of opsonized coagulase-negative staphylococci (CNS) strains was unaffected. With S. epidermidis RP12 the presence of biomaterial did not influence either phagocytosis or bactericidal effect of peritoneal cells. After intra-peritoneal challenge with staphylococcal strains, the organ uptake of S. aureus Cowan 1 was increased in normal mice whereas immunosuppressed mice died. CNS strains increased mainly in the peritoneal cavity of immunosuppressed mice. The uptake of bacteria in liver and kidneys was increased with S. epidermidis 3380, S. lugdunensis 2343 and S. schleiferi 667-88. Generally, CNS strains persisted in the peritoneal cavity of both normal and immunosuppressed mice. These data indicate that host defense mechanisms, mainly polymorphonuclear neutrophils, fail to eliminate CNS infections in the peritoneum, and that initial adhesion to an implanted biomaterial may be of lesser importance in the peritoneal cavity than in e.g. catheter-associated infections. There are strain-specific virulence factors of bacteria, and slime producing strains evade the host defense mechanisms more efficiently than non-slime producing strains.