Number Of Intracellular Bacteria Research Articles

A 3-D airway epithelial cell and macrophage co-culture system to study Rhodococcus equi infection

We developed a 3-D equine bronchial epithelial cell (BEC) culture that fully differentiates into ciliary beating and mucus producing cells. Using this system, we evaluated how mucus affects the phagocytic activity of macrophages. Adult horse monocyte-derived macrophages were incubated with Rhodococcus equi for 4h either in the mucus layer of in vitro generated airway epithelium or on collagen coated membranes. Using light and electron microscopy, we noted that the number of macrophages with intracellular bacteria, and the number of intracellular bacteria per macrophage were lower in the presence of mucus. TNFα measurements revealed that the presence of BECs promoted TNFα production by R. equi-infected macrophages; a decrease in TLR-2 (involved in R. equi recognition) and an increase in EGF-R (involved in mucin production) mRNA expression were also noted. Interestingly, when foal macrophages were added to foal BECs, we made the opposite observation, i.e. many macrophages were loaded with R. equi. Our in vitro bronchial system shows great potential for the identification of mechanisms how BECs and mucus play a role in phagocyte activation and bacterial clearance. Further studies using this system will show whether the airway environment in the foal responds differently to R. equi infection.

Ipr1 Gene Mediates RAW 264.7 Macrophage Cell Line Resistance to Mycobacterium bovis

Tuberculosis caused by Mycobacterium bovis (M.bovis) seriously affects efficiency of animal production with impacts on public health as well. Effective programmes of prevention and eradication of M.bovis infection therefore are urgently needed. Intracellular pathogen resistance gene 1 (Ipr1) is well known to mediate innate immunity to Mycobacterium tuberculosis (MTB), but there are no reports as to whether Ipr1 can enhance the phagocytic ability of macrophage against M.bovis. In this investigation, RAW 264.7 macrophage was transduced with lentiviral vector carrying Ipr1 (named Lenti-Ipr1); transgenic cells were identified by RT-PCR and western blotting. Transgenic positive cells (R-Ipr1) were then infected with an M.bovis virulent strain, with non-transduced cells used as control. When cell proliferation, viability and apoptosis of the two groups were investigated, it was found that infected RAW 264.7 died by necrosis whereas R-Ipr1 underwent apoptosis. Furthermore, the numbers of intracellular bacteria in R-Ipr1 were lower than those in control cells (P < 0.05). To identify the role of Ipr1, we measured the genes of Casp3, Mcl-1 and NOS2A which associated with macrophage activation and apoptosis by real-time quantitative PCR. The results demonstrated that Ipr1 gene expression can enhance anti-M.bovis infection of macrophage. This establishes a basis for the future production of Ipr1-transgenic cattle to strengthen the tuberculosis resistance.

A New Twist in the Regulation of Legionella Replication through ASC and Caspase-1

A New Twist in the Regulation of Legionella Replication through ASC and Caspase-1

Intracellular Trafficking ofBordetella pertussisin Human Macrophages

Although Bordetella pertussis has been observed to survive inside macrophages, its ability to resist or evade degradation in phagolysosomes has not been defined. We here investigated the trafficking of B. pertussis upon entry into human macrophages. During the first hours following phagocytosis, a high percentage of bacteria were destroyed within acidic compartments positive for the lysosome-associated membrane proteins (LAMP). However, roughly one-fourth of the bacteria taken up evade this initial killing event, remaining in nonacidic compartments. Forty-eight hours after infection, the number of intracellular bacteria per cell increased, suggesting that B. pertussis is capable of replicating in this type of compartment. Viable bacteria accumulated within phagosomal compartments positive for the early endosomal marker Rab5 but not the late endosomal marker LAMP. Moreover, B. pertussis-containing phagosomes acquired exogenously added transferrin, indicating that intracellular bacteria have access to extracellular components and essential nutrients via the host cell recycling pathway. Overall, these results suggest that B. pertussis survives and eventually replicates in compartments with characteristics of early endosomes, potentially contributing to its extraordinary ability to persist within hosts and populations.

Induction of CXC Chemokine Messenger-RNA Expression in Chicken Oviduct Epithelial Cells by Salmonella enterica Serovar Enteritidis via the Type Three Secretion System–1

The messenger-RNA (mRNA) expression of selected cytokines and chemokines in primary chicken oviduct epithelial cells (COEC) was determined following in vitro infections with wild-type or type three secretion system (T3SS)-mutant Salmonella enterica serovar Enteritidis (SE) strains. All SE strains examined in this study elicited the expression of proinflammatory immune mediators including inducible nitric oxide synthase (iNOS), CXCLi1 (K60), CXCLi2 (IL-8), CCLi3 (K203), and CCLi4 (MIP-1beta). SE also triggered the expression of an anti-inflammatory cytokine, IL-10, but repressed TGF-beta3 transcription. Both T3SS-1 (sipA and sipB) and T3SS-2 (pipB and ssaV) mutants showed reduced capacity, compared to the wild-type SE, to stimulate iNOS mRNA expression in COEC. T3SS-1 (sipA and sipB) mutants were significantly impaired in their ability to induce the expression of CXCLi1 and CXCLi2. T3SS-2 mutants displayed a wild-type phenotype in terms of modulating the expression of chemokines and cytokines in COEC. The expression of iNOS, but not CXC chemokines, correlated with the number of intracellular bacteria in COEC. Genetic complementation of the sipA mutation restored a wild-type phenotype. Thus, SE induction of CXCLi1 and CXCLi2 was sipA-dependent. These results provide enhanced insights into the complex interplay between local host innate immune system and bacterial virulence factors.

Evidence that the p38 MAP kinase pathway is dysregulated in HLA–B27–expressing human monocytic cells: Correlation with HLA–B27 misfolding

To investigate the cause of the enhanced intracellular replication of Salmonella enteritidis in HLA-B27-transfected U937 human monocytic cells and the contribution of HLA-B27 heavy chain (HC) misfolding. U937 monocytic cell transfectants stably expressing pSV2neo resistant vector (mock), wild-type HLA-B27, or mutated HLA-B27 HCs with amino acid substitutions in the B pocket were differentiated, infected with S enteritidis, and treated with signaling pathway inhibitors or specific p38 small interfering RNA (siRNA). The numbers of living intracellular bacteria were determined with the colony-forming unit method. To visualize S enteritidis, the bacteria were transformed with green fluorescent protein, and studied by microscopy. Treatment with the p38 MAPK inhibitors or with p38 siRNA enhanced the replication of S enteritidis in U937 transfectants, whereas the other inhibitors had no effect. In mock-transfected cells and in cells expressing the mutated B27 HCs in which the misfolding had been corrected, p38 inhibitors impaired their ability to resist the replication of bacteria (mock, B27.A2B, B27.E45M, and B27.C67A). In contrast, the number of intracellular bacteria was not significantly increased in p38 inhibitor-treated cells expressing misfolded B27 HCs (B27g, B27cDNA, and B27.H9F). Our results show that p38 activity plays a crucial role in controlling intracellular S enteritidis in U937 cells. Enhanced replication of bacteria in B27-expressing cells requires that the HCs contain glutamic acid at position 45 and cysteine at position 67. Furthermore, in transfectants expressing misfolded B27 HCs, p38 inhibition had no significant effect on bacterial replication, suggesting that in these cells, the p38 pathway may not function properly.

A major role for intestinal epithelial nucleotide oligomerization domain 1 (NOD1) in eliciting host bactericidal immune responses to Campylobacter jejuni

Campylobacter jejuni is the foremost cause of bacterial-induced diarrhoeal disease worldwide. Although it is well established that C. jejuni infection of intestinal epithelia triggers host innate immune responses, the mechanism(s) involved remain poorly defined. Innate immunity can be initiated by families of structurally related pattern-recognition receptors (PRRs) that recognize specific microbial signature motifs. Here, we demonstrated maximal induction of epithelial innate responses during infection with live C. jejuni cells. In contrast when intestinal epithelial cells (IECs) were exposed to paraformaldehyde-fixed bacteria, host responses were minimal and a marked reduction in the number of intracellular bacteria was noted in parallel. These findings suggested a role for intracellular host-C. jejuni interactions in eliciting early innate immunity. We therefore investigated the potential involvement of a family of intracellular, cytoplasmic PRRs, the nucleotide-binding oligomerization domain (NOD) proteins in C. jejuni recognition. We identified NOD1, but not NOD2, as a major PRR for C. jejuni in IEC. We also found that targeting intestinal epithelial NOD1 with small interfering RNA resulted in an increase in number of intracellular C. jejuni, thus highlighting a critical role for NOD1-mediated antimicrobial defence mechanism(s) in combating this infection at the gastrointestinal mucosal surface.

Identification of intracellular oral species within human crevicular epithelial cells from subjects with chronic periodontitis by fluorescence in situ hybridization

Interactions between oral bacteria and gingival epithelial cells play an important role in the pathogenesis of periodontal diseases. This study used in situ hybridization with 16 rRNA probes and confocal microscopy to detect the periodontal pathogens Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Tannerella forsythia, and Treponema denticola within epithelial cells from periodontal pockets, gingival crevice, and buccal mucosa collected from subjects with chronic periodontitis (n = 14) and good periodontal health (n = 8). Each green fluorescent species-specific and universal probe was hybridized with all 58 epithelial samples from the 22 patients. The samples were observed by confocal microscopy to confirm the intracellular localization of oral species of bacteria. The mean frequency of detection and number of intracellular bacteria per epithelial cell were computed for each sample. The frequency of cells with internalized bacteria was higher in samples from the gingival crevice than in samples from the oral mucosa. Epithelial cells from all subjects harbored intracellular bacteria; however, patients with periodontitis presented significantly higher counts of bacteria per cell than periodontally healthy individuals (p < 0.05). Periodontal pathogens showed a trend to be detected in higher numbers in epithelial cells from periodontitis patients. In particular, T. forsythia and T. denticola were significantly more prevalent in periodontal pocket cells than healthy sulci and buccal cell samples in the periodontitis group (p < 0.05). Those findings indicate that crevicular and buccal cells present internalized bacteria, regardless of periodontal status. However, higher bacterial loads are detected in cells from subjects with periodontitis.

Porphyromonas gingivalis survives within KB cells and modulates inflammatory response

The purpose of the study was to investigate the intracellular survival of Porphyromonas gingivalis as a possible mechanism for maintaining periodontitis. P. gingivalis strains, the strain ATCC 33277 and seven clinical isolates, were co-cultured with KB cells. The number of intracellular bacteria was determined up to 3 days after infection. In addition, the numbers of KB cells per well, the concentrations of the cytokines interleukin-1beta (IL-1beta), IL-6, IL-8 and tumour necrosis factor-alpha (TNF-alpha) and the arginine-specific amidolytic activity were measured. The 16S rRNA of P. gingivalis and the mRNA expression of IL-1beta, IL-6, IL-8, TNF-alpha and rgpA were also determined. All the P. gingivalis strains studied were able to survive within KB cells. In contrast to the reduced values of colony-forming units at day 3, equal and higher levels of 16S rRNA were seen in comparison to day 0. Arginine-specific amidolytic activity declined in all samples during infection. Expression of mRNA for rgpA was not found after infection of KB cells by P. gingivalis strains. IL-8 was detectable in all samples 2 days after infection with P. gingivalis strains. Principal components analysis underlined a correlation between the arginine-specific amidolytic activity 1 h after infection and both the released IL-8 and the mRNA expression of IL-8. Associations were found between the cultivable numbers of intracellular P. gingivalis and the mRNAs of IL-1, IL-6 and TNF-alpha at the day of infection. The results indicate survival of P. gingivalis within epithelial cells, possibly in a non-cultivable stage. Invasion into cells modulates the virulence properties of P. gingivalis as well as the inflammatory response of the cells.

In VivoEfficacy of Phage Therapy forMycobacterium aviumInfection As Delivered by a Nonvirulent Mycobacterium

The emergence of mycobacteria resistant to currently available antimicrobial agents has become an important problem in modern medicine. Mycobacterium avium and M. tuberculosis are intracellular pathogens that replicate and survive within the mononuclear phagocytes. TM4 is a lytic mycobacteriophage that kills both extracellular M. avium and M. tuberculosis. When delivered by M. smegmatis transiently infected with TM4, it kills both M. avium and M. tuberculosis within RAW 264.7 macrophages. To evaluate the treatment of M. avium infection with phage in vivo, C57 BL/6 mice were infected with M. avium 109 and, 7 days later, treated either once or twice with TM4 phage (7.9 x 10(10) PFU/ml), M. smegmatis (4 x 10(8) cFU/ml), or M. smegmatis with TM4 phage delivered intravenously (i.v.). Treatment with TM4 phage alone or M. smegmatis without TM4 did not show a significant decrease in number of intracellular bacteria in the spleen compared with untreated control. In contrast, administration of M. smegmatis-TM4 resulted in a significant decrease in the number of M. avium in the spleen. However, 23% of bacteria recovered from treated mice were resistant to TM4. These in vivo studies confirmed the in vitro findings that an avirulent mycobacterium can be used as a carrier to deliver antimycobacterial phage intracellularly.

Encapsulated Streptococcus suis inhibits activation of signaling pathways involved in phagocytosis.

Streptococcus suis capsular type 2 is an important zoonotic agent of meningitis. Previous studies reported that, in contrast to nonencapsulated mutants, encapsulated S. suis is able to resist phagocytosis. However, the mechanisms by which S. suis avoids phagocytosis are unknown. To elucidate the signaling pathway(s) involved in S. suis antiphagocytosis, we compared the ability of an encapsulated strain and its nonencapsulated mutant to induce the activation of Akt and protein kinase C (PKC), which are downstream kinases of the phosphatidylinositol 3-kinase (PI-3K) pathway, known to be involved in the phagocytosis processes. The results demonstrated high levels of Akt and PKCalpha phosphorylation after infection of J774 macrophages with the nonencapsulated mutant, whereas the encapsulated strain showed reduced activation of PI-3K/Akt/PKCalpha signaling pathway, as well as several protein tyrosine events. These results correlated with the number of intracellular bacteria. Macrophages pretreated with specific PI-3K or PKC inhibitors showed reduced levels of Akt and PKCalpha phosphorylation, resulting in 50% reduction of phagocytosis. The role of phosphatases in the antiphagocytic mechanisms was evaluated by using phosphatase inhibitors, as well as SHP-1-deficient macrophages. Only in the absence of SHP-1 did the phagocytosis of encapsulated S. suis significantly increase, leading to Akt phosphorylation levels similar to those observed with the nonencapsulated strain, indicating activation of this important SH2 domain-containing tyrosine phosphatase by encapsulated S. suis. Finally, when purified S. suis capsular polysaccharide (CPS) was added to macrophages, no phosphorylation events were observed. In addition, CPS and encapsulated S. suis were able to inhibit the uptake of the nonencapsulated mutant. These results suggest the importance of CPS in the mechanisms, whereby S. suis downmodulates phagocytosis.

Tubular Glands of the Isthmus are the Predominant Colonization Site of Salmonella Enteritidis in the Upper Oviduct of Laying Hens

Salmonella enterica serovar Enteritidis is the serovar most frequently isolated from chicken eggs. Colonization of the upper oviduct of hens is believed to play an important role in egg contamination. The interaction of S. enteritidis with gland epithelial cells of the isthmus and the magnum was, therefore, studied in vitro and in vivo. In the first experiment, S. enteritidis bacteria were added to confluent monolayers of primary cultures of chicken tubular epithelial cells of the isthmus (ICTEC) or magnum (MCTEC). Intracellular bacteria in ICTEC and MCTEC were confirmed by a gentamicin protection assay. Internalization in the glandular cells was corroborated by confocal scanning microscopy. Although S. enteritidis was able to invade and proliferate intracellularly during 24 h in the cell culture of both segments, this was significantly more so in the ICTEC. In a second experiment, an in vivo loop model was developed for investigation of the invasiveness of S. enteritidis in the oviduct of laying hens. Loops in the isthmus and magnum of laying hens were made under anaesthesia. S. enteritidis was inoculated into each loop. After 1 h, tubular gland cells were isolated from the infected tissue under gentamicin. S. enteritidis invasiveness was measured as the ratio of isolated bacteria per isolated tubular gland cell. This ratio was higher (P < 0.01) in the isthmus (1.3 × 10-3) than in the magnum (5.3 × 10-5). In a third experiment, laying hens were intravenously infected with 5 × 107) cfu S. enteritidis bacteria. The number of intracellular bacteria was counted in the tubular gland cells of the isthmus and magnum after isolation under gentamicin. The ratio of isolated bacteria per isolated tubular gland cell was again significantly higher in the isthmus as compared with in the magnum. In all 3 assays, the tubular gland cells of the isthmus were more heavily invaded than those of the magnum.

Role of flagellum and chemotactic motility of Vibrio anguillarum for phagocytosis by and intracellular survival in fish macrophages

The role of the flagellum and chemotactic motility of Vibrio anguillarum for phagocytosis by and intracellular survival in fish macrophages was determined using a wild-type strain, a mutant without the flagellum, a mutant with a truncated flagellum and a non-chemotactic mutant. For all strains, the numbers of intracellular bacteria were relatively low and fell steadily during the observation period. The presence of a flagellum did not influence the uptake by the macrophages, but the smooth swimming phenotype of a non-chemotactic mutant increased its intracellular presence. We suggest that this is due to an increased collision between the mutant and the macrophage, due to a higher average speed of the non-chemotactic mutant.

Role of flagellum and chemotactic motility ofVibrio anguillarumfor phagocytosis by and intracellular survival in fish macrophages

The role of the flagellum and chemotactic motility of Vibrio anguillarum for phagocytosis by and intracellular survival in fish macrophages was determined using a wild-type strain, a mutant without the flagellum, a mutant with a truncated flagellum and a non-chemotactic mutant. For all strains, the numbers of intracellular bacteria were relatively low and fell steadily during the observation period. The presence of a flagellum did not influence the uptake by the macrophages, but the smooth swimming phenotype of a non-chemotactic mutant increased its intracellular presence. We suggest that this is due to an increased collision between the mutant and the macrophage, due to a higher average speed of the non-chemotactic mutant.

The importance of a beta-glucan receptor in the nonopsonic entry of nontypeable Haemophilus influenzae into human monocytic and epithelial cells.

Previous reports showed that nontypeable Haemophilus influenzae (NTHi) reside in macrophage-like cells in human adenoid tissue. This study investigated the ability of nonopsonized NTHi and encapsulated H. influenzae type b (Hib) to enter human monocytic and epithelial cells. The number of intracellular bacteria was determined by a viability assay and flow cytometry. To characterize the mechanisms responsible for the internalization of NTHi, different inhibitors of surface molecules, receptor turnover, and the cytoskeleton were used. Hib were found in monocytic cells at very low numbers (<100 bacteria/2x105 cells). In contrast, a great variation in intracellular numbers was detected between the different NTHi isolates (range, 0.0007%-0.28% of the inoculum for monocytes and 0.053%-3.5% for epithelial cells). NTHi entered human monocytic and epithelial cells via a receptor-mediated endocytosis involving mainly a beta-glucan receptor that could be blocked by laminarin.

Shigella infection in a SCID mouse-human intestinal xenograft model: role for neutrophils in containing bacterial dissemination in human intestine.

Shigellae infect human intestine and cause intense inflammation and destruction of colonic and rectal mucosa. To model the interactions of shigella with human intestine in vivo, we have studied shigella infection in human intestinal xenografts in severe combined immunodeficient mice (SCID-HU-INT mice). Inoculation of shigella into human intestinal xenografts caused severe inflammation and mucosal damage, which was apparent as soon as 4 h following infection. Shigella infection was associated with human intestinal production of interleukin-1B (IL-1B) and IL-8 and a marked neutrophil influx into the graft. Depletion of neutrophils from SCID-HU-INT mice reduced inflammation in the human intestinal xenograft in response to shigella infection but failed to significantly alter tissue damage. However, the number of intracellular bacteria was more than 20-fold higher in the human intestinal xenografts from neutrophil-depleted SCID-HU-INT mice. Infection of human intestinal xenografts with an attenuated vaccine strain of shigella (CVD1203) induced lower levels of IL-1B and IL-8 than wild-type shigella and caused only moderate damage to the intestinal permeability barrier. Our studies establish the SCID-HU-INT mouse as a viable model for studying the interactions between shigella and human intestine and indicate that neutrophils are important for controlling the invasion of human intestine by shigella.

Activity of moxifloxacin by itself and in combination with ethambutol, rifabutin, and azithromycin in vitro and in vivo against Mycobacterium avium.

Moxifloxacin activity against Mycobacterium avium complex (MAC) was evaluated in vitro against 25 strains. The MIC was determined to range from 0.125 to 2.0 microg/ml. In addition, U937 macrophage monolayers infected with MAC strain 101 (serovar 1) were treated with moxifloxacin (0.25 to 8 microg/ml) daily, and the number of intracellular bacteria was quantitated after 4 days. Moxifloxacin showed inhibitory activity at 0.5 microg/ml and higher. To assess the activity of moxifloxacin containing regimens in vivo, we infected C57BL bg(+)/bg(+) mice with 3 x 10(7) MAC strain 101 bacteria intravenously. One week later treatment was begun with the following: (i) moxifloxacin (50 mg/kg/day or 100 mg/kg/day), ethambutol (100 mg/kg/day), or a combination of moxifloxacin and ethambutol; or (ii) moxifloxacin (100 mg/kg/day), azithromycin (200 mg/kg/day), or rifabutin (40 mg/kg/day) as oral monotherapy; or (iii) all permutations of two-drug therapy or all three drugs in combination. All groups contained at least 14 animals, and the control group received the drug vehicle. After 4 weeks, quantitative blood cultures were obtained and the number of bacteria in liver and spleen was quantitated. Moxifloxacin, ethambutol, and azithromycin were active as single agents in liver, spleen, and blood. Rifabutin showed inhibitory activity only in the blood. Two-drug combinations containing azithromycin were no more active than azithromycin alone. Similarly, the three-drug combination was not more active than azithromycin alone in the spleen. Rifabutin did not add to the activity of any other single agent or two-drug combination. Moxifloxacin at both concentrations in combination with ethambutol was significantly more active than each drug alone.

Mefloquine is active in vitro and in vivo against Mycobacterium avium complex.

Despite the development of several agents, new classes of antimicrobials with activity against the Mycobacterium avium complex (MAC) are needed. Based on a broad screening of compounds, we found that mefloquine has MICs of 8 to 16 microg/ml by the BACTEC system and 16 microg/ml by broth microdilution for five MAC strains tested. An expansion of the screening with broth microdilution to 24 macrolide-susceptible strains and 6 macrolide-resistant strains determined that the MIC for all strains was 16 microg/ml. To determine the intracellular activity of mefloquine, U937 macrophage monolayers infected with MAC strain 101, 100, or 109 (serovars 1, 8, and 4) were treated with mefloquine daily, and the number of intracellular bacteria was quantitated after 4 days. Significant growth inhibition against the three MAC strains at concentrations greater than or equal to 10 microg/ml (P < 0.05) was obtained. Due to the encouraging anti-MAC activity, in vivo efficacy in beige mice infected with MAC 101 was evaluated. Animals were treated with 5, 10, 20, or 40 mg/kg of body weight daily, three times a week, twice a week, or once a week for 4 weeks, and bacteria were quantitated in blood, liver, and spleen. No toxicity was observed with any of the treatment regimens. Mefloquine had borderline bactericidal activity at a dosage of 40 mg/kg daily (100% inhibition compared with a 1-week control), and significant inhibition was obtained at dosages of 40 mg/kg three times a week, as well as 20 mg/kg daily. Mefloquine had no significant effect on bacteremia. A combination of mefloquine and ethambutol showed significantly more activity than did either drug alone in liver, spleen, and blood; the combination was also bactericidal against M. avium. Although safety is a potential concern, mefloquine and related compounds deserve further investigation as anti-MAC therapies.

Invasion and Persistence of Streptococcus dysgalactiae Within Bovine Mammary Epithelial Cells

Little is known about bacterial and host factors that contribute to the establishment and persistence of intramammary infection by Streptococcus dysgalactiae. Streptococcus dysgalactiae adheres to epithelial cells from the bovine mammary gland and to extracellular matrix proteins in vitro and invades mammary epithelial cell cultures, all of which can be potentially important pathogenic mechanisms. In this study, mechanisms involved in the invasion of Strep. dysgalactiae into epithelial cells from the bovine mammary gland were characterized. Studies were conducted to determine whether Strep. dysgalactiae invaded mammary epithelial cell cultures in a dose-dependent manner and whether mammary epithelial cells that harbored different numbers of Strep. dysgalactiae for varying times were damaged. Bacterial invasion increased as inoculum size increased; however, the number of intracellular bacteria was not proportional to the inoculum size, indicating that a finite number of organisms is capable of invading epithelial cells. No net increase in intracellular organisms was detected at any bacterial density evaluated; however, Strep. dysgalactiae remained viable throughout the evaluation. In addition, Strep. dysgalactiae did not appear to cause cell injury at any bacterial density or time point evaluated. These data suggest that Strep. dysgalactiae can survive within mammary epithelial cells for an extended time without losing viability or damaging the eukaryotic cell. This feature may be associated with the development of persistent infection and protection of organisms from antimicrobial drugs and host defense mechanisms and may provide a route for bacterial colonization of subepithelial tissues.

Killing mechanism of Listeria monocytogenes in activated macrophages as determined by an improved assay system

Exposure of Listeria monocytogenes to gentamicin 5 mg/L for 4 h resulted in the killing of most extracellular bacteria, but had no effect on the survival of bacteria inside macrophages. Higher concentrations of gentamicin caused a reduction in the number of intracellular bacteria. This effect was associated with cellular uptake of gentamicin, but was unaffected by activation of macrophages by interferon-gamma and lipopolysaccharide. In experiments in which exposure to gentamicin 5 mg/L for 4 h was used to kill extracellular bacteria, killing by activated macrophages was impaired when O2- production was inhibited by superoxide dismutase, but not when nitric oxide production was blocked by NG-monomethyl-L-arginine. These data suggest that the reactive oxygen intermediates are more important than nitric oxide in the killing of L. monocytogenes, at least in macrophages activated in vitro.