Antibiotic-free Controls Research Articles

The impact of physiological state and environmental stress on bacterial load estimation methodologies for Mycobacterium tuberculosis

When processed in solid or liquid medium, tuberculosis patient samples yield different proportions of a heterogenous bacterial community over the duration of treatment. We aimed to derive a relationship between methodologies for bacterial load determination and assess the effect of the growth phase of the parent culture and its exposure to stress on the results. Mycobacterium tuberculosis H37Rv was grown with and without antibiotic (isoniazid or rifampicin) and sampled on day 0, 3, 11 and 21 of growth in broth culture. The bacterial load was estimated by colony counts and the BD BACTEC MGIT system. Linear and nonlinear mixed-effects models were used to describe the relationship between time-to-positivity (TTP) and time-to-growth (TTG) versus colony forming units (CFU), and growth units (GU) versus incubation time in MGIT. For samples with the same CFU, antibiotic-treated and stationary phase cells had a shorter TTP than antibiotic-free controls and early-logarithmic phase cells, respectively. Similarly, stationary phase samples reached higher GUs and had shorter TTG than early-log phase ones. This suggests that there is a population of bacterial cells that can be differentially recovered in liquid medium, giving us insight into the physiological states of the original culture, aiding the interpretation of clinical trial outputs.

Erythromycin and florfenicol treatment of rainbow trout Oncorhynchus mykiss (Walbaum) experimentally infected with Flavobacterium psychrophilum.

Flavobacterium psychrophilum is responsible for significant economic losses in rainbow trout aquaculture. Antimicrobial treatment remains the primary means of control; however, there are limited choices available for use. The objectives of the study were therefore to determine the minimum inhibitory concentrations for erythromycin and florfenicol in selected F.psychrophilum isolates and to evaluate their clinical treatment efficacy in experimentally infected rainbow trout. All isolates tested had moderate susceptibility to florfenicol and erythromycin except one isolate, which had low susceptibility to erythromycin. Two isolates (one with moderate and one with low susceptibility to erythromycin) were used in an experimental infection trial. Rainbow trout juveniles were injected intraperitoneally with 108 cfu/fish and after mortality had begun, fish were given erythromycin- and florfenicol-medicated feed at a rate of 75mg kg- 1 day- 1 and 10mg kg- 1 day- 1 fish body weight, respectively, for 10 consecutive days. The splenic F.psychrophilum load was determined using an rpoC quantitative PCR throughout the 30-day trial. Relative to antibiotic-free controls, erythromycin treatment significantly (p<0.05) reduced mortality of rainbow trout juveniles infected with FPG101, even when treatment was initiated after clinical signs developed.

Effect of sub-MIC values of metronidazole, ciprofloxacin, and imipenem on the growth and toxin production in Clostridioides difficile.

This study intends to investigate the effect of sub-minimum inhibitory concentration (sub-MIC) of metronidazole, ciprofloxacin, and imipenem on the growth and toxin production in Clostridioides difficile. C. difficile is the most common causative agent of hospital-acquired diarrhea. Toxin production in C. difficile appears to be a critical process for induction of the disease. Several factors such as antibiotics can facilitate growth and toxin production in C. difficile. Five C. difficile strains were grown with and without sub-MIC concentrations of metronidazole, ciprofloxacin, and imipenem (0.5x MIC). The bacterial growth was measured by density at OD620 nm in 0, 4, 8, 12 and 24 h post inoculation. Toxin production was detected using ELISA in culture supernatants as well as in cell pellet. The five strains showed minor growth variations in the presence and absence of antibiotic sub-MIC values, except for metronidazole, in which the sub-MIC concentration reduced the growth rate of the resistant isolate in comparison with the control without antibiotic. There were no significant variations in the levels of toxin production with the sub-MIC values of antibiotics examined in comparison with antibiotic-free controls. However, the amount of toxin production in the culture supernatant was greater than in the cell pellet. The findings of this study suggested that sub-MIC concentrations of antibiotics may have minor effects on bacterial growth and toxin production of C. difficile. Taken together, these findings suggest that presence of antimicrobial agents increased expression levels of certain genes, particularly virulence genes, which may help C. difficile to survive.

Effects of vancomycin versus nafcillin in enhancing killing of methicillin-susceptible Staphylococcus aureus causing bacteremia by human cathelicidin LL-37.

Recent studies have demonstrated that anti-staphylococcal beta-lactam antibiotics, like nafcillin, render methicillin-resistant Staphylococcus aureus (MRSA) more susceptible to killing by innate host defense peptides (HDPs), such as cathelicidin LL-37. We compared the effects of growth in 1/4 minimum inhibitory concentration (MIC) of nafcillin or vancomycin on the LL-37 killing of 92 methicillin-susceptible S. aureus (MSSA) isolates. For three randomly selected strains among these, we examined the effects of nafcillin, vancomycin, daptomycin, or linezolid on LL-37 killing and autolysis. Growth in the presence of subinhibitory nafcillin significantly enhanced LL-37 killing of MSSA compared to vancomycin and antibiotic-free controls. Nafcillin also reduced MSSA production of the golden staphylococcal pigment staphyloxanthin in 39% of pigmented strains vs. 14% for vancomycin. Among the antibiotics tested, only nafcillin resulted in significantly increased MSSA autolysis. These studies point to additional mechanisms of anti-staphylococcal activity of nafcillin beyond direct bactericidal activity, properties that vancomycin and other antibiotic classes do not exhibit. The ability of nafcillin to enhance sensitivity to innate HDPs may contribute to its superior effectiveness against MSSA, as suggested by studies comparing clinical outcomes to vancomycin treatment.

Key Roles of Human Polymorphonuclear Cells and Ciprofloxacin in Lactobacillus Species Infection Control.

Lactobacilli have the potential to act as reservoirs of antibiotic resistance genes similar to those found in human pathogens, with the risk of transferring these genes to many pathogenic bacteria. In this study, we investigated the role of human polymorphonuclear cells (PMNs) against Lactobacillus spp. both resistant and susceptible to ciprofloxacin (a fluoroquinolone) and the effect of ciprofloxacin on the interaction between PMNs and three Lactobacillus spp. with different patterns of susceptibility to this drug. Hence, the primary functions of PMNs, such as phagocytosis and bacterial intracellular killing, against lactobacilli were investigated. The rate of PMN phagocytosis was high for ciprofloxacin-sensitive and ciprofloxacin-resistant strains. The patterns of intracellular killing of ciprofloxacin-sensitive and ciprofloxacin-resistant strains by PMNs underline that PMNs alone were able to kill lactobacilli. The addition of ciprofloxacin to PMNs did not result in a significant increase in the bacterial uptake by phagocytes. On the contrary, ciprofloxacin had a marked effect on PMN intracellular killing, resulting in increased numbers of killed ciprofloxacin-sensitive bacteria in comparison with antibiotic-free controls. Our data show that by itself, the profile of antibiotic resistance does not constitute an intrinsic factor of greater or lesser pathogenicity toward the host. The ability of PMNs to kill a diverse array of bacterial pathogens is essential for human innate host defense, primarily in immunocompromised patients.

The importance of growth kinetic analysis in determining bacterial susceptibility against antibiotics and silver nanoparticles.

Routine antibiotics susceptibility testing still relies on standardized cultivation-based analyses, including measurement of inhibition zones in conventional agar diffusion tests and endpoint turbidity-based measurements. Here, we demonstrate that common off-line monitoring and endpoint determination after 18–24 h could be insufficient for reliable growth-dependent evaluation of antibiotic susceptibility. Different minimal inhibitory concentrations were obtained in 20- and 48 h microdilution plate tests using an Enterococcus faecium clinical isolate (strain UKI-MB07) as a model organism. Hence, we used an on-line kinetic assay for simultaneous cultivation and time-resolved growth analysis in a 96-well format instead of off-line susceptibility testing. Growth of the Enterococcus test organism was delayed up to 30 h in the presence of 0.25 μg mL-1 of vancomycin and 8 μg mL-1 of fosfomycin, after which pronounced growth was observed. Despite the delayed onset of growth, treatment with fosfomycin, daptomycin, fusidic acid, cefoxitin, or gentamicin resulted in higher maximum growth rates and/or higher final optical density values compared with antibiotic-free controls, indicating that growth stimulation and hormetic effects may occur with extended exposure to sublethal antibiotic concentrations. Whereas neither maximum growth rate nor final cell density correlated with antibiotic concentration, the lag phase duration for some antibiotics was a more meaningful indicator of dose-dependent growth inhibition. Our results also reveal that non-temporal growth profiles are only of limited value for cultivation-based antimicrobial silver nanoparticle susceptibility testing. The exposure to Ag(0) nanoparticles led to plasma membrane damage in a concentration-dependent manner and induced oxidative stress in Enterococcus faecium UKI-MB07, as shown by intracellular ROS accumulation.

The significant contribution of fungi to soil N2O production across diverse ecosystems

Sporadic observations from pure culture study and direct soil measurement have indicated that fungi can substantially contribute to soil N2O production. Yet, it is still uncertain whether this fungal significance is a more general ecological phenomenon. In this study, relative contributions of fungi and bacteria to soil N2O production were examined in five ecosystems, including conventional farming (CON), integrated crop and livestock system (ICL), organic farming (ORG), plantation forestry (PF), and abandoned agriculture field subjected to natural succession (SUCC). Soil N2O production was measured at 90% water-filled pore space from antibiotic-free controls and soils amended with streptomycin, cycloheximide, or both. Streptomycin and cycloheximide additions significantly reduced soil N2O fluxes from the five systems, ranging from 31% to 54% and 40% to 51%, respectively. Fungi contributed more to soil N2O fluxes than bacteria in PF, whereas fungi and bacteria made comparable contributions in other four systems. Furthermore, soil pH was correlated positively with the percentage of bacterial contribution to soil N2O flux, but negatively with the percentage of fungal contribution to soil N2O flux as well as the ratio of fungal-to-bacterial contributions. Our results showed that fungi could potentially contribute to soil N2O production in diverse agroecosystems and their contribution might be more pronounced in the acidic plantation forestry.

Sub-inhibitory concentrations of some antibiotics can drive diversification of Pseudomonas aeruginosa populations in artificial sputum medium.

BackgroundPseudomonas aeruginosa populations within the cystic fibrosis lung exhibit extensive phenotypic and genetic diversification. The resultant population diversity is thought to be crucial to the persistence of infection and may underpin the progression of disease. However, because cystic fibrosis lungs represent ecologically complex and hostile environments, the selective forces driving this diversification in vivo remain unclear. We took an experimental evolution approach to test the hypothesis that sub-inhibitory antibiotics can drive diversification of P. aeruginosa populations. Replicate populations of P. aeruginosa LESB58 were cultured for seven days in artificial sputum medium with and without sub-inhibitory concentrations of various clinically relevant antibiotics. We then characterised diversification with respect to 13 phenotypic and genotypic characteristics.ResultsWe observed that higher population diversity evolved in the presence of azithromycin, ceftazidime or colistin relative to antibiotic-free controls. Divergence occurred due to alterations in antimicrobial susceptibility profiles following exposure to azithromycin, ceftazidime and colistin. Alterations in colony morphology and pyocyanin production were observed following exposure to ceftazidime and colistin only. Diversification was not observed in the presence of meropenem.ConclusionsOur study indicates that certain antibiotics can promote population diversification when present in sub-inhibitory concentrations. Hence, the choice of antibiotic may have previously unforeseen implications for the development of P. aeruginosa infections in the lungs of cystic fibrosis patients.

In vitro interference of tigecycline at subinhibitory concentrations on biofilm development by Enterococcus faecalis

Since biofilm formation is the hallmark of Enterococcus faecalis isolates, the aim of this study was to quantify biofilm formation in the presence of subinhibitory concentrations of tigecycline. Interference of tigecycline on biofilm formation was spectrophotometrically quantified using 20 biofilm-producing E. faecalis isolates with tigecycline MICs of 0.12 (8 strains) or 0.25 mg/L (12 strains). Biofilm production was measured in antibiotic-free tryptic soy broth supplemented with 1% glucose and compared with biofilm production in the same medium with tigecycline at subinhibitory concentrations (0.25× or 0.5× MIC, similar to trough concentrations in serum or concentrations in the colon after a standard dose) by reading the optical density at 450 nm (OD(450)) after staining with Crystal Violet. In the presence of subinhibitory tigecycline concentrations, pooled OD(450) values for the 20 strains [median (IQR)] were significantly lower than those for controls: 0.468 (0.379-0.516) for antibiotic-free controls versus 0.295 (0.200-0.395) for 0.25× MIC tigecycline (P < 0.001) and 0.287 (0.245-0.479) for 0.5× MIC tigecycline (P < 0.001), with significant differences between pooled OD(450) values obtained with each concentration of tigecycline (P = 0.022). In 17 out of 20 (85%) strains the OD(450) obtained with 0.25× MIC tigecycline was significantly (P < 0.05) lower than the basal OD(450), while this occurred in 12 out of 20 (60%) strains with 0.5× MIC. In vitro tigecycline subinhibitory concentrations were able to interfere with biofilm formation by E. faecalis.

Comparison of the bactericidal activity of various fluoroquinolones against Mycobacterium tuberculosis in an in vitro experimental model

To compare the bactericidal activity of various fluoroquinolones against Mycobacterium tuberculosis in the latent and exponential growth phases. Ciprofloxacin, levofloxacin and moxifloxacin were tested against 16 M. tuberculosis clinical isolates (4 resistant and 12 susceptible to fluoroquinolones) from Elche, Spain, isolated between 1992 and 2009. To study bactericidal activity, an inoculum of approximately 10(5) cfu of each isolate was cultured in Middlebrook 7H9 broth. The broth was previously acidified to pH 4.6 to obtain the microorganism in the stationary phase. Cultures with different concentrations (0.1 to 50 mg/L) of antibiotic and antibiotic-free controls were incubated for 48 h then plated onto Middlebrook 7H11 to detect bacterial killing. In all stages of the process the M. tuberculosis strain ATCC 41323 was included as a quality control to ensure reproducible results. Moxifloxacin and levofloxacin were found to exhibit bactericidal activity at lower concentrations and against more strains in both the latent and the exponential growth phases compared with ciprofloxacin. The bactericidal activity of moxifloxacin was greater than that of levofloxacin against microorganisms in the exponential growth phase, but the opposite was true in the latent phase. Our data confirm the usefulness of moxifloxacin in the treatment of tuberculosis and suggest that levofloxacin may be used as an alternative drug in the treatment of latent tuberculosis when it is not possible to use isoniazid. Based on the results presented, ciprofloxacin appears to be a poor choice.

Antibiotic Susceptibility of Stenotrophomonas maltophilia in the Presence of Lactoferrin

Stenotrophomonas maltophilia is resistant to most antibiotics and infects the respiratory tract of cystic fibrosis (CF) patients (5). CF sputum contains a high concentration of lactoferrin (0.9 mg/ml) (9). Lactoferrin damages outer membranes of gram-negative bacteria (6), which might explain why MICs for Pseudomonas aeruginosa of rifampin and chloramphenicol were reduced in the presence of 0.9 mg/ml of human lactoferrin (7). Colistin (8) and other agents (4) that increase outer membrane permeability of gram-negative bacteria have enhanced the susceptibility of S. maltophilia to rifampin. As potentiation of antibiotic activity by lactoferrin may influence treatment of CF infections, we investigated the effect of lactoferrin (0.9 mg/ml) on susceptibilities of CF S. maltophilia isolates to drugs used to treat S. maltophilia infections (ceftazidime, gentamicin, trimethoprim, and rifampin). Lactoferrin enhanced the sensitivity of P. aeruginosa (7) to chloramphenicol, so we looked for similar effects with S. maltophilia. Clinical isolates of S. maltophilia were from Booth Hall Hospital, Manchester, United Kingdom. Human recombinant lactoferrin was from Agennix Inc., Houston, Tex.; rifampin and chloramphenicol were from Mast Laboratories, United Kingdom; gentamicin sulfate from Sigma; ceftazidime pentahydrate was from GlaxoSmithKline; and trimethoprim was from APS-Berk, Sussex, United Kingdom. Determination of MICs with and without 0.9 mg/ml lactoferrin was by broth microdilution (7) including antibiotic-free controls with and without lactoferrin. Bacterial suspensions were diluted to a final concentration of 105 CFU/ml. The minimum bactericidal concentration (MBC) was the concentration that led to 99.9% killing. For each MIC and MBC, eight replicate tests were performed, and results are presented as medians. Analysis was by Mann-Whitney U test. Rifampin MICs (Table ​(Table1)1) for all isolates were lower (2- to 16-fold) with lactoferrin than without, while the rifampin MBCs were lowered 2- to 4-fold in the presence of lactoferrin (P 256 μg/ml), and those for the remaining strains were unchanged on addition of lactoferrin. All isolates were resistant to trimethoprim (MICs, 64 to 256 μg/ml). TABLE 1. Median MICs and MBCs (μg/ml) of rifampin and gentamicin for clinical isolates (eight replicates per isolate) of S. maltophilia with (+LF) and without (−LF) lactoferrin (0.9 mg/ml) Rifampin MBCs for S. maltophilia were lower with lactoferrin, as were MICs which fell from levels that are not clinically obtainable to 12 μg/ml or below (levels that have been recorded in the sputum of some rifampin-treated patients) (1). This finding supports use of rifampin in treatment of S. maltophilia infections of CF patients. Our findings with rifampin concur with reports of similar effects with other gram-negative bacteria, such as Burkholderia cepacia (2) and mucoid P. aeruginosa isolates from CF patients (2, 7), Escherichia coli (3, 6), and Salmonella enterica serovar Typhimurium (10). In contrast to S. enterica serovar Typhimurium (10) and P. aeruginosa (7), susceptibility of S. maltophilia to chloramphenicol was not enhanced by lactoferrin. These and previous findings (2, 7) suggest that for CF respiratory infections, it may be more appropriate to test antibiotic sensitivities in the presence of lactoferrin.

Effects of fluoroquinolones on the migration of human phagocytes through Chlamydia pneumoniae-infected and tumor necrosis factor alpha-stimulated endothelial cells.

The anti-inflammatory activities of three quinolones, levofloxacin, moxifloxacin, and gatifloxacin, were investigated with an in vitro model of transendothelial migration (TEM). Human umbilical vein endothelial cells (HUVEC) were seeded in Transwell inserts, treated with serial dilutions of antibiotics, infected with Chlamydia pneumoniae, or stimulated with tumor necrosis factor alpha (TNF-alpha). Neutrophils or monocytes were also preincubated with serial dilutions of each antibiotic. TEM was assessed by light microscopic examination of the underside of the polycarbonate membrane, and levels of interleukin-8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) were measured by enzyme-linked immunosorbent assay. In HUVEC infected with C. pneumoniae or stimulated with TNF-alpha, all fluoroquinolones significantly decreased neutrophil and monocyte TEM, compared to antibiotic-free controls. Moxifloxacin and gatifloxacin produced a significant decrease in IL-8 in C. pneumoniae-infected and TNF-alpha-stimulated HUVEC; however, moxifloxacin was the only fluoroquinolone that produced a significant decrease in MCP-1 levels under both conditions. Results from this study indicate similarities in the anti-inflammatory activities of these fluoroquinolones, although no statistically significant decrease in chemokine secretion was observed when levofloxacin was used. Mechanisms of neutrophil and monocyte TEM inhibition by fluoroquinolone antibiotics are unknown but may be partially due to inhibition of IL-8 and MCP-1 production, respectively.

Effects of sub-MIC concentrations of antibiotics on growth of and toxin production by Clostridium difficile.

Effects on growth and toxin A production of sub-MIC concentrations of six different antibiotics were investigated in three strains of Clostridium difficile: reference strain NCTC 11223, a fully sequenced strain (630) and a locally endemic isolate (strain 338a). The antibiotics chosen for investigation were the agents used to treat C. difficile-associated disease (CDAD), i.e. vancomycin and metronidazole, and four antibiotics that are commonly involved in precipitating CDAD (amoxycillin, clindamycin, cefoxitin and ceftriaxone). Strains were cultured in sublethal concentrations of antibiotics (1/2, 1/4 and 1/8 MIC) over 104 h and growth and toxin A production were measured three times a day. Effects varied between strain and antibiotic. The most common effect on growth of the strains was to increase the initial lag period by approximately 4 h, compared with antibiotic-free controls; however, strain NCTC 11223, which has high-level clindamycin resistance (> or = 512 microg ml(-1)), showed no lag whatsoever in comparison with the controls when grown in this antibiotic. The most common effect on production of toxin A was in the onset of toxin elaboration. Normally, toxins began to appear at low levels in the early stationary phase, before accumulating to high levels by the start of decline. In the presence of sub-MIC antibiotics, this onset appeared before that of the antibiotic-free controls. This effect was seen with metronidazole, amoxycillin and clindamycin, rarely with vancomycin and never with cefoxitin. These results suggest a very complex, strain-dependent relationship between the effects of growth and toxin production.

Comparison of the effects of kanamycin and geneticin on regeneration of papaya from root tissue

Kanamycin and geneticin are commonly used for the selection of neomycin phosphotransferase II (npt II) transformed plants. Since papaya tissue is sensitive to both antibiotics, it is difficult to explore their effects on the regeneration process solely based on using non-transformed tissues. Adventitious roots derived from npt II-transgenic and non-transgenic papaya shoots in vitro were used as explants in this investigation. The effects of kanamycin and geneticin on callus formation, embryogenesis, and conversion of somatic embryos to shoots were compared. Callus growth derived from npt II-transformed root explants was apparently enhanced on kanmycin within 50–200 mg l−1 or on geneticin within 12.5–50 mg l−1 as compared to those on antibiotic-free controls. The percentages of npt II-transformed somatic embryo-forming callus were not significantly different (16.3–18.3%) on geneticin less than 6.25 mg l−1 and only slightly reduced (11.2–15.7%) on geneticin within 12.5–50 mg l−1, whereas, formation of somatic embryos was strongly suppressed on kanamycin media. Conversion rates of npt II-transformed somatic embryos to shoots were not significantly different among all kanamycin or geneticin treatments. Percentages of the callus derived from non-transformed root explants were greatly reduced on the medium containing more than 25 mg l−1 kanamycin or geneticin, and no somatic embryos formed from untransformed callus on any kanamycin or geneticin media. Our results indicated that somatic embryogenesis of callus derived from npt II-transformed root explants of papaya was strongly inhibited by kanamycin. Thus, to regenerate npt II-transformed cells from papaya root tissue, we recommend using the lower concentration geneticin (12.5–25 mg l−1) to avoid the adverse effects of kanamycin on embryogenesis.

Effect of macrolide antibiotics on human endothelial cells activated by Chlamydia pneumoniae infection and tumor necrosis factor-alpha.

This study investigated the potential anti-inflammatory activity of 3 macrolide antibiotics, clarithromycin, roxithromycin, and azithromycin, in an in vitro model of transendothelial migration (TEM). Human umbilical vein endothelial cells (HUVECs) were seeded in Transwell inserts, treated with serial dilutions of the antibiotics, and infected with Chlamydia pneumoniae or stimulated with tumor necrosis factor (TNF)-alpha. In HUVECs infected with C. pneumoniae or stimulated with TNF-alpha, both azithromycin and roxithromycin caused significant decreases in neutrophil and monocyte TEM, compared with antibiotic-free controls. Clarithromycin had no detectable effect in either group. Azithromycin caused significant decreases in interleukin (IL)-8 and monocyte chemotactic protein (MCP)-1, whereas roxithromycin significantly decreased IL-8. This study indicates heterogeneity in the anti-inflammatory activity of these antibiotics. Mechanisms of monocyte and neutrophil TEM inhibition by azithromycin and roxithromycin are unclear but may be partially due to inhibition of IL-8 and MCP-1 production.

Effects of ampicillin, ceftriaxone, chloramphenicol, pefloxacin and trimethoprim-sulphamethoxazole on Salmonella typhi within human monocyte-derived macrophages

The killing effect of various antimicrobial agents used in the therapy of Salmonella typhi infection was tested against Salm. typhi strain Ty2 after phagocytosis by human monocyte-derived macrophages. The macrophages, cultured in 96-well microtitre plates, were infected for 1 h at 37 degrees C by opsonized Salm. typhi Ty2 at a bacteria-cell ratio of 9:1. When added to the infected macrophage monolayers, at one and ten times the MIC, ampicillin, ceftriaxone and pefloxacin appeared to be highly bactericidal (less than 0.25 log10 cfu/well after 20 h, against 4 log10 cfu/well in antibiotic-free controls). Trimethoprim-sulphamethoxazole was bactericidal at ten times the MIC, but not at the MIC. Chloramphenicol was mostly bacteriostatic at the concentrations tested. As a control, gentamicin (10 mg/l) did not exhibit any significant antibacterial effect, indicating that most or all the bacteria recovered from lysed cells were intracellular. Other controls for phagocytosis were also performed with heat-killed Candida albicans. Our results seem to correlate with the known clinical effect of some antimicrobials in human Salm. typhi infection. The in-vitro assay described here may be useful for assessing the activity of antimicrobial agents against Salm. typhi infection.

Reverse inoculum effect in bactericidal activity and other variables affecting killing of group B streptococci by penicillin

Variables of the effect of penicillin G on the numbers of viable group B streptococci in broth cultures were studied. One-fourth of the MIC was the lowest concentration that reduced the viable count compared with antibiotic-free controls. The rate of killing increased with the concentration of penicillin up to 4 X MIC, but no further. During the first 2 or 3 h, the bactericidal activity was more rapid than later on. The MIC and supraoptimal concentrations of penicillin killed an inoculum of 10(6) organisms more rapidly than an inoculum of 10(4) organisms. The MIC was not inoculum dependent. The reverse inoculum effect was revealed by the killing curves but not by the MBC. There were reproducible differences among strains as to the rate of killing by penicillin; these did not correlate with the rate of multiplication, which also varied among strains. Among the 11 strains tested, there were no tolerant ones.