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PubMed2026

First characterization of Staphylococcus felis in diabetic foot osteomyelitis: from intracellular persistence to phage treatment.

Staphylococcus felis is a coagulase-negative Staphylococcus (CoNS) primarily associated with the feline microbiota and only rarely reported in human disease. Here, we report its implication in diabetic foot osteomyelitis, and provide the first comprehensive characterization of its pathogenic potential. Two isolates (NSF001 and NSF002), recovered 5 months apart from bone biopsies of the same patient, were analyzed for growth kinetics, biofilm formation, and intracellular persistence in macrophages and osteoblasts. Both isolates proliferated efficiently, produced robust biofilm, and persisted within host cells, most markedly in osteoblasts. In a zebrafish embryo infection model, both isolates caused significant mortality, confirming their pathogenic potential in vivo. Whole-genome sequencing revealed conserved virulence determinants, a narrow resistome, and strain-specific genomic variations affecting genes involved in virulence regulation, phage defense, and iron acquisition. The lytic phage SAVM02, previously characterized for activity against other Staphylococcus species, effectively inhibited S. felis growth in vitro and conferred protection in vivo against lethal infection. Notably, the two sequential isolates differed in their in vivo virulence and phage susceptibility, paralleling these within-host microevolutionary changes and illustrating bacterial adaptation during chronic infection. Altogether, this study establishes S. felis as a CoNS capable of intracellular persistence, biofilm formation, and in vivo virulence in chronic human infection. Our findings also highlight the therapeutic potential of lytic phages against virulent CoNS species and support further investigation of phage therapy for chronic staphylococcal infections.IMPORTANCECoagulase-negative staphylococci (CoNS) are increasingly recognized as genuine agents of chronic infection, yet the pathogenic capacity of most individual species remains undefined. Staphylococcus felis, a commensal of cats only exceptionally reported in humans, had never been implicated in a chronic human infection. Here, we describe two sequential S. felis isolates recovered from bone biopsies of a patient with diabetic foot osteomyelitis and show that this species combines biofilm formation, intracellular persistence in macrophages and osteoblasts, and lethality in a zebrafish embryo model. Whole-genome comparison of the two isolates uncovered microevolutionary changes, most notably in iron-acquisition and genome-defense loci, that paralleled differences in virulence and phage susceptibility. These findings extend the list of CoNS capable of causing invasive human disease and provide a rationale for lytic phage therapy against emerging, difficult-to-treat staphylococcal pathogens.

Staphylococcus felis↗

PubMed1979

Sequestration of staphylococci at an inaccessible focus.

The persistence of staphylococcal arthritis in a patient treated with nafcillin was attributed to intracellular sequestration of staphylococci. These intraphagocytic organisms were protected against bactericidal activity. The infection was cleared by rifampin--an agent which can penetrate leucocyte membranes. When clinical infections do not respond to seemingly adequate antimicrobial treatment, intracellular persistence of bacteria should be considered.

Adult↗

PubMed1979

Comparison of Brucella abortus and Brucella melitensis infections of mice and their effect on acquired cellular resistance.

By using mice infected with strains of Brucella abortus and Brucella melitensis we examined the histological responses to infection, the relationship of histology to persistence of organisms, and the relation of persistence of organisms to the acquisition of acquired cellular resistance (ACR). Infection with B. abortus resulted in well-formed granulomas in the livers, which persisted for more than 30 days. In contrast, infection with B. melitensis produced microabscesses in the livers which resolved before 30 days. The clearance of organisms from the tissues was also different. A total of 30 days after infection, large numbers of viable bacteria were recovered from the tissues of B. abortus-infected mice whereas bacteria were no longer recoverable from B. melitensis-infected animals. ACR to Listeria monocytogenes, another intracellular pathogen, persisted for more than 30 days in B. abortus-infected mice but waned rapidly in B. melitensis-infected animals. This disappearance of ACR due to B. melitensis paralleled the clearance of bacteria from the tissues.

Animals↗

PubMed1978

Immunoglobulin class distribution of measles virus antibodies in serum and spinal fluids of patients with subacute sclerosing panencephalitis.

Sera and cerebrospinal fluids (CSF) from patients with subacute sclerosing panencephalitis (SSPE) were tested by indirect immunofluorescence for the presence of measles virus specific antibodies of the various heavy chain classes. IgG and IgA antibodies were detected in the CSF while IgG, IgM, IgA, IgD and IgE measles virus antibodies were found in a significant number of the patient sera. Sera from SSPE patients had slightly elevated levels of IgG, IgM, IgD and IgE while IgA was decreased. The heterogeneous heavy chain class distribution of measles antibodies suggests the possibility that non-complement fixing antibodies serve as blocking antibodies which aid in the persistence of intracellular measles virus infection in patients with SSPE.

Antibodies, Viral↗

PubMed1979

Evolution of multiple genome mutations during long-term persistent infection by vesicular stomatitis virus.

Persistent infection of BHK21 cells was established with cloned vesicular somatitis virus plus purified Dl particles and maintained in vitro for over 5 years. After 1 year of persistence, the infectious virus RNA genome had evolved several oligonucleotide map changes, and numerous changes had accumulated by 3.5 years. Additional evolution occurred by the fourth year and continued until the fifth year. In contrast, repeated passage of virus in acute infections of several cell types in vitro or in vivo did not lead to detectable oligonucleotide map changes. The short Dl particle originally used to co-infect with infectious virus in establishing persistent infection has been displaced by an ever present and constantly changing population of other Dl particles of differing sizes and radically differing oligonucleotide maps. We conclude that the genomes of both infectious VSV and its Dl particles undergo continuous evolutionary change during years of persistence. In the infectious virus, these changes involve hundreds of mutations which are usually expressed as poorly replicating, temperature-sensitive, small plaque mutants. These are stable mutants which do not revert to wild-type when passaged repeatedly in acute infections at 37 or 33 degrees C. It appears that the sequestered intracellular environment of persistently infected cells favors rapid and continuous virus evolution.

Animals↗

PubMed1978

The interaction in vitro of Pneumocystis carinii with macrophages and L-cells.

A model was developed for studying the interaction between Pneumocystis, rat-derived cells, and humoral factors. Pneumocystis were obtained in large quantity by bronchial lavage of steroid-treated rats. The trophozoite was the predominant form obtained, and it could readily be recognized by phase contrast microscopy. Organisms maintained a typical morphology for at least 3 days in culture, and 10-20% took up radiolabeled nucleotides. Pneumocystis readily adhered to cell surfaces in a similar manner in alveolar macrophages from steroid-treated or normal rats, mouse peritoneal macrophages, and L-cells. Adherent organisms were not interiorized to a significant degree in the absence of antipneumocystis serum. After addition of rabbit antipneumocystis serum, rapid interiorization of organisms occurred from the surface of macrophages but not L-cells. Organisms appeared to be promptly destroyed within macrophages after interiorization. Persisting or multiplying intracellular forms were not seen. Antipneumocystis serum did not morphologically alter Pneumocystis. These observations suggest a role for antibody and mononuclear phagocytes during the immune response to Pneumocystis.

Animals↗

PubMed1979

A comparative study of tick-borne encephalitis virus RNA synthesis in acutely and persistently infected cells.

Rate zonal and buoyant density gradient centrifugation did not reveal any difference between tick-borne encephalitis virus virions released from acutely or persistently infected cells. All three RNA species characteristic for flavivirus replication were found both in acutely or persistently infected cells, but increased levels of intracellular 42S RNA polyadenylation was observed in persistently infected cells.

Animals↗

PubMed1976

Tissue pharmacokinetics, inhibition of DNA synthesis and tumor cell kill after high-dose methotrexate in murine tumor models.

In Sarcoma 180 and L1210 ascites tumor models, the initial rate of methotrexate accumulation in tumor cells in the peritoneal cavity and in small intestine (intracellularly) after s.c. doses up to 800 mg/kg, showed saturation kinetics. These results and the fact that initial uptake in these tissues within this dosage range was inhibited to the expected relative extent by the simultaneous administration of leucovorin suggest that carrier mediation and not passive diffusion is the major route of drug entry at these extremely high doses. Maximum accumulation of intracellular drug occurred within 2 hr and reached much higher levels in small intestine than in tumor cells at the higher dosages. At a 3-mg/kg dose of methotrexate s.c., intracellular exchangeable drug levels persisted more than four times longer in L1210 cells than in small intestine, but differences in persistence (L1210 cell versus gut) diminished markedly with increasing dosage. At 96 mg/kg, the difference in persistence was less than 2-fold. In small intestine and L1210 cells, theduration of inhibition of DNA synthesis at different dosages correlated with the extent to which exchangeable drug was retained. Toxic deaths occurred when inhibition in small intestine lasted longer than 25 to 30 hr. Recovery of synthesis in small intestine and L1210 cells occurred synchronously and only below dosages of 400 mg/kg. Within 24 hr after dosages of greater than 24 mg/kg, the rate of tumor cell loss increased to a point characterized by a single exponential (t1/2=8.5 hr). The total cell loss, but not the rate of cell loss, was dose dependent.

Animals↗

PubMed1979

Surface and cytoplasmic immunoglobulin distribution during treatment of a non-excretory myeloma.

A patient with non-excretory myeloma with severe clinical signs, has been assessed for cytoplasmic and surface immunoglobulins on peripheral mononuclear cells and bone marrow cells. A clearcut monoclonal pattern was found. During treatment with melphalan, methotrexate and prednisolone a clinical remission was obtained. The immunological studies were repeated during this remission and were in accordance with the clinical improvement, however the monoclonality persisted and indicated an intracellular dissocitaion of light and heavy chains. The patient died with broncho-pneumonia and marrow failure.

Bone Marrow↗

PubMed1979

The action of neurotensin on single myenteric neurones.

The action of neurotensin was studied on single myenteric neurones within ganglia of the myenteric plexus isolated from the guinea-pig ileum. Drugs were applied by adding them to the perfusing Krebs solution. Extracellular recording with glass suction electrodes indicated that neurotensin (100 pM-300 nM) caused a dose-dependent excitation of about 50% of myenteric neurones; the remaining neurones were unaffected. This effect persisted in calcium-free solutions. Intracellular recording showed that a similar proportion of Type 1 myenteric neurones were depolarized by neurotensin: this was associated with an increase in membrane resistance. Type 2 cells were either depolarized or hyperpolarized by neurotensin. The depolarization persisted in calcium-free solutions. The hyperpolarization disappeared in calcium-free solutions, suggesting either that the potential change itself is calcium-dependent or that it was due to release by neurotensin of a hyperpolarizing substance.

Animals↗

PubMed1975

Effects of metabolites present during growth of Tetrahymena pyriformis on the subsequent secretion of lysosomal hydrolases.

Tetrahymena were grown in proteose-peptone medium supplemented with glucose, mannose, fructose, galactose, acetate, succinate, or pyruvate and then washed and resuspended in a non-nutrient salt solution and the amounts of 7 acid hydrolases secreted into the medium in a one hour incubation were measured. Cells that had been grown in the presence of glucose secreted about half the amounts of acid phosphatase, beta-N-acetylglucosaminidase and acid protease as did control cells grown in unsupplemented medium. Pyruvate was about as effective as glucose and both were slightly more effective than acetate or fructose. Succinate had little effect. Similar experiments showed that alpha-mannosidase, beta-fucosidase, and beta-galactosidase are secreted into the salt solution and the secretion is reduced by prior growth of the cells in medium supplemented with glucose or mannose but not galactose. Except for alpha-mannosidase, these reductions in amounts of hydrolase secreted were not accompanied by appreciable changes in intracellular activity, and therefore demonstrate a persistent effect of growth in the presence of certain metabolites on the subsequent secretion of lysosomal hydrolases. Since the inhibition of subsequent secretion depended on both the individual metabolite and the particular hydrolase examined, it appears that the effect of metabolites is not limited to a general inhibition of secretion but may differentially alter some properties of lysosomal subpopulations. A preliminary characterization of the secreted acid protease of Tetrahymena suggests that there may be two acid proteases released, since up to 25% of the activity was not inhibited by high concentrations of pepstatin, leupeptin, or chymostatin.

Acetates↗

PubMed1977

Lanthanum probe studies of cellular pathophysiology induced by hypoxia in isolated cardiac muscle.

This study was undertaken to evaluate directly the relationship between evolution of irreversible myocardial injury induced by hypoxia in an isolated papillary muscle preparation and the development of pathophysiological alterations related to severely impaired membrane function. An ionic lanthanum probe technique was employed as a cytochemical marker to monitor the progression of cellular injury, and data from this cytologic technique were correlated with ultrastructure and measurements of contractile parameters in a total of 67 muscles subjected to control conditions or to graded intervals of hypoxia with or without reoxygenation. Marked depression of developed tension and rate of tension development occurred after 30 min of hypoxia. Contractile function showed significant recovery with reoxygenation after 1 h and 15 min of hypoxia but remained depressed when reoxygenation was provided after 2 or 3 h of hypoxia. Examination by transmission and analytical electron microscopy (energy dispersive X-ray microanalysis) revealed lanthanum deposition only in extracellular regions of control muscles and muscles subjected to 30 min of hypoxia. After hypoxic intervals of over 1 h, abnormal intracytoplasmic and intramitochondrial localization of lanthanum were detected. After 1 h and 15 min of hypoxia, abnormal intracellular lanthanum accumulation was associated with only minimal ultrastructural evidence of injury; muscle provided reoxygenation after 1 h and 15 min of hypoxia showed improved ultrastructure and did not exhibit intracellular lanthanum deposits upon exposure to lanthanum during the reoxygenation period. After 2 to 3 h of hypoxia, abnormal intracellular lanthanum accumulation was associated with ultrastructural evidence of severe muscle injury which persisted after reoxygenation. Thus, the data support the conclusion that cellular and membrane alterations responsible for abnormal intracellular lanthanum deposition precede the development of irreversible injury but evolve at a transitional stage in the progression from reversible to irreversible injury induced by hypoxia in isolated feline papillary muscles.

Animals↗

PubMed1975

A model for the quantitative study of Arthus (immunologic) hypersensitivity in rats.

A model of reverse passive Arthus (RPA) reaction in the pleural cavity of rats is described. The time course of development of exudate and migration of cells has been examined. It has been found to be complement dependent and dominated by polymorphonuclear cells. The reaction reaches a peak around 6 hours after challenge. Cyclic AMP levels have been measured both intracellularly and extracellularly and have been found to persist at high levels after the waning of the reaction.

Animals↗

PubMed1976

[Iodine-induced hypothyroidism (author's transl)].

Excessive administration of iodine over a long period may cause hypothyroidism in suitably disposed individuals and provoke the formation of a goiter. The underlying reason for this is the pathological lack of adaptation of the thyroid gland to high doses of iodine with persisting inhibition of hormone production through excess of intracellular iodine (Wolff-Chaikoff effect). Asthmatic patients--as in the case described - are usually affected, when they have taken iodine-containing expectorants over a long time. Provocation of an iodine-induced hypothyroidism by the recently required iodine prophylaxis of endemic goiter is not to be expected because the quantities of iodine lie within the physiological range.

Asthma↗

PubMed1977

Use of polarized-light microscopy in histological diagnostics of early stages of ischaemic and metabolic myocardial lesions.

Animal experiments have proved that the earliest morphological manifestations of acute lesions of myocardial cells (at the optical microscopic level) are myofibrillar changes detectable by polarized-light microscopy. As basic types of myocardial cell lesions there can be distinguished segmental and subsegmental contractures, intracellular myocytolysis, and primary granular disintegration. These changes persist in animal myocardium for many hours and thus can be recognized at autopsy. The application of polarized-light microscopy makes possible a diagnosis of early stages of myocardial infarction, associated with lesions of myocardial cells appearing at the periphery of the ischaemic zone already within the first minutes, revelation of focal metabolic changes leading to acute heart failure, and diagnosis of ventricular fibrillation, characterized by subsegmental contractures of the subepicardial and subendocardial layers of the myocardium.

Animals↗

PubMed1977

Interdependence of ion transport and the action of quabain in heart muscle.

1 The influence of ouabain (0.4 muM) on contractile force and cellular Na and K concentrations was investigated in isolated left atria of the guinea-pig at rest and at different beat frequencies. Simultaneously the binding of ouabain to the tissue was determined.2 Strict dependence of rates of onset of positive iontropic action and of binding of ouabain on beat frequency are limited to conditions where no alterations of cellular Na and K concentrations occur. A correlation was observed between sodium flux per unit time and the development of positive inotropism and binding to the receptors of ouabain.3 Ouabain exerts its positive inotropic effect without affecting the intracellular Na and K concentrations in spite of the fact that under these conditions even the majority of binding sites, i.e. Na-K-adenosine triphosphatases (Na-K-ATPases), are occupied by the drug. The positive inotropic effect may be explained by a ouabain-induced conformational alteration of the Na-K-ATPase which leads to structural alterations of the plasmalemma connected with an increased availability of coupling calcium.4 Increasing the frequency of stimulation over a critical value, which appears to be determined by an overloading of the Na pump, induces a decrease in contractile force, cellular accumulation of Na and loss of K, and eventually contracture.5 The rate of binding of ouabain appears to depend on the actual concentration of particular conformations of the Na-K-ATPase with high affinity for ouabain. These conformations transiently occur during a pumping cycle and their concentration may therefore be dependent on the frequency of cycling which in turn is determined by the frequency of contraction.6 Ouabain can easily be washed out from the tissue irrespective of the condition of the muscle. If, however, the intracellular Na and K homeostasis is impaired, the inhibition of the pump persists even if ouabain is released from the binding sites upon wash-out. It is suggested that the inhibition of the pump is maintained by an increased intracellular Ca ion concentration and a depletion of ATP.7 A kinetic model is proposed for the interaction between cardiac glycosides and the Na-K-ATPase in intact heart muscle cells.

Animals↗

PubMed1978

Immune response to persistent mycobacterial infection in mice.

Mycobacterium marinum has been recommended as a possible model of M. leprae for use in laboratory studies of antileprosy immunity. M. marinum introduced into the footpads of normal mice underwent a steady decline in viability, with less than 1% survival after a 30-day period. Small numbers of viable bacilli were recovered from the footpads of these mice up to 12 months later. Similarly, mice infected with M. simiae exhibited bacterial populations that persisted for up to 18 months with little change in viability. Injection of M. simiae into the footpads was followed by an extensive redistribution of the organisms in the tissues. Eventally, bacterial counts for footpads and draining lymph nodes stabilized, with small numbers of bacilli still present in the footpads 18 months later. Persistent growth, with little sign of any immune response, was also observed in mice infected with several strains of M. avium, as well as with one strain of M. intracellulare. Other strains of M. intracellulare, as well as M. vaccae and M. nonchromogenicum, failed to establish persistent infections in normal mice, regardless of whether they were introduced by an intravenous or subcutaneous (footpad) route. The relevance of these findings is discussed in relation to antileprosy immunity in experimental animals and in humans.

Animals↗

PubMed1975

Intracellular (muscle-fiber) habitat of Ancylostoma caninum in some mammalian hosts.

The persistence and precise location of Ancylostoma caninum larvae in tissues of vertebrate paratenic hosts and the nature of host responses were studied in mouse, cat, and monkey. Mice were infected percutaneously and examined at various intervals up to 260 days after exposure. Long-persisting larvae were found only in the muscles. Histologic sections revealed that within 4 hr after exposure some larvae had migrated through the skin and had entered individual fibers of the underlying muscles. After the 1st day nearly all larvae found in muscles were within fibers. Granuloma formation and encapsulation were not observed, suggesting that inside the fibers the larvae produced no direct inflammatory reaction. Only diffuse infiltration of inflammatory cells was observed in muscles and this appeared to be in response to destruction of muscle fibers. Larvae were similarly located in muscles of a cat and a rhesus monkey examined 16 and 17 days, respectively, after cutaneous exposure. The histologic changes observed in muscle fibers invaded by A. caninum larvae are similar to those observed in early Trichinella spiralis infections. The larvae of A. canninum lying coiled within the muscle fibers also bear superficial resemblance to the larvae of T. spiralis. A. caninum larvae were also recovered by tissue digestion from muscles of naturally infected dogs, which suggests that larvae reside in this location in the bitch prior to transfer to the neonate via the milk.

Abdomen↗