Presence of UDP-N-acetylmuramyl-hexapeptides and -heptapeptides in enterococci and staphylococci after treatment with ramoplanin, tunicamycin, or vancomycin

Analyses of the peptidoglycan nucleotide precursor contents of enterococci and staphylococci treated with ramoplanin, tunicamycin, or vancomycin were carried out by high-pressure liquid chromatography coupled with mass spectrometry (MS). In all cases, a sharp increase in the UDP-N-actetylmuramoyl-pentapeptide or -pentadepsipeptide pool was observed. Concomitantly, new peptidoglycan nucleotide peptides of higher molecular masses with hexa- or heptapeptide moieties were identified: UDP-MurNAc-pentapeptide-Asp or pentadepsipeptide-Asp in enterococci and UDP-MurNAc-pentapeptide-Gly or -Ala and UDP-MurNAc-pentapeptide-Gly-Gly or -Ala-Gly in staphylococci. These new compounds are derivatives of normal UDP-MurNAc-pentapeptide or -pentadepsipeptide precursors with the extra amino acid(s) linked to the lysine epsilon-amino group as established by various analytical procedures (MS, MS-MS fragmentation, chemical analysis, and digestion with R39 D,D carboxypeptidase). Except for tunicamycin-treated cells, it was not possible to ascertain whether these unusual nucleotides were formed by direct addition of the amino acids to UDP-MurNAc-pentapeptide (or -pentadepsipeptide) or whether they arose by reverse reactions from lipid I intermediates to which the amino acids had been added.     

Modification of vancomycin nephrotoxicity by other antibiotics in rats

Vancomycin (VCM) was intravenously administered to rats for 14 days at doses of 150 mg/kg/day and 250 mg/kg/day alone or in combination with 1,000 mg/kg/day of latamoxef (LMOX), flomoxef (FMOX) or cefpirome (CPR) or 250 mg/kg/day of fosfomycin (FOM), and the influences of combined antibiotics on the VCM-induced renal damage were studied. The renal impairment caused by VCM alone was, morphologically, demonstrated mainly as regeneration of tubular epithelium: slight regeneration was observed in a half of rats administered 150 mg/kg/day and slight to extensive regeneration in all the rats administered 250 mg/kg/day. Clinical examinations found apparent increases in urinary LDH and MDH activities in rats administered 250 mg/kg/day, thus showing a good correlation with renal pathological changes. In addition, increase in kidney weight and increase in urinary NAG activity were noted, while changes in plasma urea-N and creatinine were mild, and gamma-GTP activity and protein in urine could not be used as a parameter of the renal impairment. The slight renal impairment as noted in rats administered VCM 150 mg/kg/day alone was not observed at all when LMOX or FMOX was administered concomitantly, and less pronounced even when FOM was administered concomitantly. When CPR was administered concomitantly, the changes were the same as those observed with VCM alone. The renal impairment in rats administered VCM 250 mg/kg/day was apparently less severe when combined with LMOX, FMOX and FOM than that in rats administered VCM alone, and this was supported by apparent reduction of clinical examination values as the parameter of VCM-induced nephrotoxicity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterogeneity of staphylococci and other bacteria isolated from six-week-old broiler chickens

In broiler operations, various health problems develop during the final 2 wk of the growing period, resulting in increased mortality and condemnation losses. At this stage, sickly birds were found to be systemically infected by various bacteria regardless of varied clinical signs, and the purpose of this study was to carry out thorough microbiological investigations on this problem. Thirty-one 6-wk-old broilers showing signs of illness were obtained from three farms, and bacterial isolations were carried out from the blood, liver, and hock joint. Bacteria were isolated from 87, 90, and 71% of the blood, liver, and hock joint samples, respectively. Mean bacterial counts in log10 of the blood (per milliliter) and liver (per gram) were 2.15 and 2.93, respectively. Among 132 bacterial isolates, major species were Staphylococcus (60%), Corynebacterium (18%), Escherichia coli (5%), and Stomatococcus (4%). Among 79 Staphylococcus isolates, 77 were coagulase-negative. Major species of staphylococci were S. lentus (19%), S. simulans (18%), S. cohnii (13%), S. gallinarum (10%), and S. captis (7%). In addition, six species of gram-positive and five species of gram-negative organisms were isolated. Thus, the apparent systemic infections were not caused by predominant pathogenic bacterial species, and adequately described as mixed infections. There were some significant relationships between isolated bacterial species and sampling sites, suggesting that certain organisms were abundant in the environment of a particular poultry house. These results indicate that systemic infections in market age broilers are caused by mixed bacterial species and suggest that they are caused by suppressed host antibacterial systems rather than pathogenic factors of microorganisms.     

Yellow staphylococci with borderline resistance to methicillin

Over the last four years there has been an increase in the incidence of borderlineresistant Staphylococcus aureus isolated from bacteriological samples at the Ullev?l University Hospital, Department of Medical Microbiology. Several severe infections caused by these bacteria have been noticed in the Department of Infectious Diseases at Ullev?l University Hospital. From December 1994 to April 1997, 24 patients suffering from this type of S. aureus infection were examined with regard to clinical and microbiological outcome. 15 of the patients had hospital-acquired infections, and all except one had acquired the infection in Norway. 13 of the patients had at least one predisposing factor, 50% had received antibiotics (mainly cefalosporins) beforehand. Three of the 24 patients died from the infection. We discuss etiology, identification of groups at risk and management of the infection.     

Susceptibilities of penicillin- and erythromycin-susceptible and -resistant pneumococci to HMR 3647 (RU 66647), a new ketolide, compared with susceptibilities to 17 other agents

Susceptibility of 230 penicillin- and erythromycin-susceptible and -resistant pneumococci to HMR 3647 (RU 66647), a new ketolide, was tested by agar dilution, and results were compared with those of erythromycin, azithromycin, clarithromycin, roxithromycin, rokitamycin, clindamycin, pristinamycin, ciprofloxacin, sparfloxacin, trimethoprim-sulfamethoxazole, doxycycline, chloramphenicol, cefuroxime, ceftriaxone, imipenem, and vancomycin. HMR 3647 was very active against all strains tested, with MICs at which 90% of the strains were inhibited (MIC90s) of 0.03 microg/ml for erythromycin-susceptible strains (MICs, < or =0.25 microg/ml) and 0.25 microg/ml for erythromycin-resistant strains (MICs, > or =1.0 microg/ml). All other macrolides yielded MIC90s of 0.03 to 0.25 and >64.0 microg/ml for erythromycin-susceptible and -resistant strains, respectively. The MICs of clindamycin for 51 of 100 (51%) erythromycin-resistant strains were < or =0.125 microg/ml. The MICs of pristinamycin for all strains were < or =1.0 microg/ml. The MIC90s of ciprofloxacin and sparfloxacin were 4.0 and 0.5 microg/ml, respectively, and were unaffected by penicillin or erythromycin susceptibility. Vancomycin and imipenem inhibited all strains at < or =1.0 microg/ml. The MICs of cefuroxime and cefotaxime rose with those of penicillin G. The MICs of trimethoprim-sulfamethoxazole, doxycycline, and chloramphenicol were variable but were generally higher in penicillin- and erythromycin-resistant strains. HMR 3647 had the best kill kinetics of all macrolides tested against 11 erythromycin-susceptible and -resistant strains, with uniform bactericidal activity (99.9% killing) after 24 h at two times the MIC and 99% killing of all strains at two times the MIC after 12 h for all strains. Pristinamycin showed more rapid killing at 2 to 6 h, with 99.9% killing of 10 of 11 strains after 24 h at two times the MIC. Other macrolides showed significant activity, relative to the MIC, against erythromycin-susceptible strains only.     

Comparison of agar dilution, broth microdilution, E-test, disk diffusion, and automated Vitek methods for testing susceptibilities of Enterococcus spp. to vancomycin

An evaluation was undertaken to determine the optimal method for testing the susceptibilities of 100 clinical isolates and two reference strains of Enterococcus spp. to vancomycin in vitro. Six testing methods were studied by using the following media and incubation times: agar screen with the Synergy Quad Plate (Remel, Lenexa, Kans.), an in-house-prepared brain heart infusion (BHI) agar plate, and an in-house-prepared Mueller-Hinton (MH) agar plate, all incubated for 24 or 48 h; broth microdilution (Sensititre Just One Strip; AccuMed International, Inc., West Lake, Ohio) with BHI or cation-adjusted MH broth incubated for 24 or 48 h; agar dilution with BHI or MH agar incubated for 24 or 48 h; epsilometer test (E test; AB BioDisk, Solna, Sweden) with BHI or MH agar incubated for 24 or 48 h; disk diffusion with BHI or MH agar incubated for 24 or 48 h; and the automated Vitek method with the gram-positive susceptibility Staphylococcus aureus card and R02.03 software (bioMerieux, Inc., Hazelwood, Mo.). Growth failures occurred with MH media (n = 6) but not with BHI media. One growth failure occurred with the Vitek method. Results for each testing method for each Enterococcus strain were interpreted as susceptible, intermediate, or resistant according to current National Committee for Clinical Laboratory Standards (NCCLS) criteria and compared to the vancomycin resistance genotype (i.e., vanA, vanB, vanC-1, or vanC-2/3). For all methods, extension of the incubation time from 24 h to 48 h either produced no difference in the results or gave poorer results. The following methods produced no very major or major interpretive errors: broth microdilution with BHI media incubated for 24 h, agar dilution with BHI media incubated for 24 or 48 h, and E test with BHI media incubated for 24 or 48 h. Unacceptable frequencies of very major errors (> 1%) occurred with all methods for which MH media were used. Minor interpretive errors were frequent with all methods. These minor interpretive errors also occurred most frequently with Enterococcus strains with vanC genes, which encoded low-level vancomycin resistance (MIC < or = 8 microg/ml), as opposed to Enterococcus strains which possessed vanA or vanB genes, which encoded higher-level vancomycin resistance (MIC > or = 64 microg/ml). Modification of NCCLS breakpoints, especially for motile Enterococcus spp. (E. casseliflavus, E. flavescens, and E. gallinarum), may resolve this problem; however, in the current study, one E. faecalis strain and one E. faecium strain carried only the vanC gene. The agar screen method may also require reformulation. The current agar screen plate contains 6 microg of vancomycin per ml, which may not detect all low-level resistance associated with vanC genotypes. Nevertheless, the clinical significance of this low-level vancomycin resistance remains unknown.     

Comparison of vancomycin and teicoplanin for prophylaxis of sepsis with coagulase negative staphylococci (CONS) in very low birth weight (VLBW) infants

The hourly flight periodicity of adults of the biting midge Culicoides impunctatus was sampled at a site in Western Scotland, using suction traps over 18 days in July/August 1994. In addition, meteorological conditions were logged continuously. Female but not male C. impunctatus had a bimodal pattern of activity, with peaks at dawn and dusk. The dawn peak (05.00-07.00 hours) was most distinct. Correlation analysis revealed significantly positive relationships between catches of female midges and both relative humidity and rainfall, and negative relationships with wind velocity. The calculation of partial correlation coefficients reinforced the influence of relative humidity on female activity, and highlighted a further positive relationship with air temperature. Male C.impunctatus activity was negatively correlated with air temperature, although the total male catch was relatively small (15% of total trap catches) and further data would be required to confirm this result. Overall, the results help to clarify previous confusion as to whether C.impunctatus has a circadian rhythm of activity, with the data matching closely predictions of a bimodal pattern. Clearly, this pattern will be damped by meteorological conditions, which may vary greatly on a local scale.     

Postoperative mediastinitis due to methicillin resistant Staphylococcus epidermidis with low sensitivity to vancomycin

BACKGROUND: Vancomycin is the drug of choice for methicillin-resistant Staphylococcus. Antibiotherapy failure is rarely clinically related to Staphylococcus with vancomycin low susceptibility. CASE REPORT: A surgical cure of an aortic stenosis in a neonate was complicated by a Staphylococcus mediastinitis. After initiation of antibiotherapy with vancomycin and rifampin and surgical debridement, there was a rapid improvement. Few days later, failure of therapy was obvious. Despite continuous infusion of vancomycin, with a serum level of 29 mg/L, blood cultures were positive again to Staphylococcus. There was no endocarditis or inadequate surgical drainage. Susceptibility of the Staphylococcus was tested, looking for a tolerant strain. The vancomycin minimum bactericidal concentration was 30 mg/L (above usual value 2 to 8 mg/L), while the minimum inhibitory concentration was 3.75 mg/L. A higher dosage of vancomycin associated with fusidic acid was rapidly efficient, and total recovery was achieved. CONCLUSION: In case of failure of vancomycin therapy, despite correct serum levels, the susceptibility of the Staphylococcus strain has to be determined. A low susceptibility strain prescribes more prolonged combination of two antibiotics.     

In vivo activities of ceftriaxone and vancomycin against Borrelia spp. in the mouse brain and other sites

Borrelia burgdorferi, the agent of Lyme disease, and B. turicatae, a neurotropic agent of relapsing fever, are susceptible to vancomycin in vitro, with an MIC of 0.5 microgram/ml. To determine the activity of vancomycin in vivo, particularly in the brain, we infected adult immunocompetent BALB/c and immunodeficient CB-17 scid mice with B. burgdorferi or B. turicatae. The mice were then treated with vancomycin, ceftriaxone as a positive control, or normal saline as a negative control. The effectiveness of treatment was assessed by cultures of blood and brain and other tissues. Ceftriaxone at a dose of 25 mg/kg of body weight administered every 12 h for 7 to 10 days eliminated cultivable B. burgdorferi or B. turicatae from all BALB/c or scid mice in the study. Vancomycin at 30 mg/kg administered every 12 h was effective in eliminating infection from immunodeficient mice if treatment was started within 3 days of the onset of infection. If treatment with vancomycin was delayed for 7 days or more, vancomycin failed to eradicate infection with B. burgdorferi or B. turicatae from immunodeficient mice. The failure of vancomycin in eradicating established infections in immunodeficient mice was associated with the persistence of viable spirochetes in the brain during antibiotic treatment.     

Influence of zinc on Pseudomonas aeruginosa susceptibilities to imipenem

Serial dilution susceptibility testing of imipenem against 59 clinical isolates of Pseudomonas aeruginosa, conducted simultaneously on single lots of Difco and BBL Mueller-Hinton agar (MHA), resulted in MICs for 90% of strains tested of 8 and 16 micrograms/ml, respectively. MICs for Escherichia coli, Klebsiella pneumoniae, and Pseudomonas spp. were also higher on BBL MHA. Quantification of the cation content of the two MHAs by atomic absorption spectroscopy demonstrated that the zinc concentration in BBL MHA was 15 times greater than that measured in Difco MHA (2.61 and 0.17 micrograms/ml, respectively). Concentrations of calcium, magnesium, iron, manganese, and copper in the two agars were similar. Addition of zinc to Difco MHA resulted in increases in MICs of imipenem for P. aeruginosa but not in the MICs of ceftazidime or cefpirome for P. aeruginosa (P < 0.01). A lesser zinc effect was seen on the activity of imipenem against E. coli, K. pneumoniae, and Pseudomonas spp. The activities of ceftazidime and cefpirome were similar on both MHAs when tested against all gram-negative organisms in this study. Thus, the effect of zinc in MHA was clearly demonstrated by a significant increase in the MICs of imipenem for P. aeruginosa, and, to a lesser extent, for other gram-negative bacilli.     

In vitro susceptibilities of 27 rickettsiae to 13 antimicrobials

The MICs of 13 antibiotics (doxycycline, thiamphenicol, rifampin, amoxicillin, gentamicin, co-trimoxazole, ciprofloxacin, pefloxacin, ofloxacin, erythromycin, josamycin, clarithromycin, and pristinamycin) were determined for 27 available rickettsial species or strains. We used two in vitro cell culture methods described previously: the plaque assay and the microplaque colorimetric assay. Our results confirm the susceptibilities of rickettsiae to doxycycline, thiamphenicol, and fluoroquinolones. Beta-lactams, aminoglycosides, and cotrimoxazole were not active. Typhus group rickettsiae were susceptible to all macrolides tested, whereas the spotted fever group rickettsiae, R. bellii, and R. canada were more resistant, with josamycin, a safe alternative for the treatment of Mediterranean spotted fever, being the most effective compound. Strain Bar 29, R. massiliae, R. montana, R. aeschlimannii, and R. rhipicephali, which are members of the same phylogenetic subgroup, were more resistant to rifampin than the other rickettsiae tested. Heterogeneity in susceptibility to rifampin, which we report for the first time, may explain in vivo discrepancies in the effectiveness of this antibiotic for the treatment of rickettsial diseases. We hypothesize that rifampin resistance and erythromycin susceptibility may reflect a divergence during the evolution of rickettsiae.     

In vivo and in vitro cellular response to Schistosoma japonicum eggs in hosts with differing susceptibilities

A comparative study of the cellular response to Schistosoma japonicum eggs was conducted in order to explore its significance, using hosts with differing susceptibilities to the parasite. In experimentally induced, synchronized hepatic granuloma formation, animal species formed each characteristic feature of the granulomas, and the magnitude of tissue reaction was significantly larger in highly susceptible hosts, such as mice and hamsters, while less susceptible hosts, such as rats and quails, formed smaller granulomas. Confluent neutrophils were seen within the tissue lesions for mice and hamsters, while rats and quails showed obviously scanty neutrophils. Guinea pigs failed to develop any granulomas. When splenic cells and bone marrow cells were used for in vitro granuloma formation, bone marrow cells showed markedly higher reactions than splenic cells from naive or sensitized animals and the reactivities of bone marrow cells from susceptible hosts, mice and hamsters, were clearly higher than those from rats, indicating similar results to those of in vivo granuloma formation. This study indicates that the in vivo and in vitro cellular response to S. japonicum eggs varies greatly according to the host's susceptibility, independent of whether the host is a naive or sensitized animal. Our results seem to support the concept that the parasites exploit the host immune system in order to complete their life-span.     

Interactions of vancomycin resistant enterococci with biomaterial surfaces

Many cytokines transmit signals to the cell interior through activation of receptor-associated, Janus family protein tyrosine kinases (Jak PTKs). The interleukin-2 receptor (IL-2R) is associated with the Jak1 and Jak3 PTKs, and ligand-induced activation of these PTKs is essential for lymphocyte proliferation. Here, the nonreceptor PTK, Pyk2, was found to be activated following IL-2 stimulation in a Jak-dependent manner. Furthermore, physical association was detected between endogenous Pyk2 and Jak3, and a dominant interfering mutant of Pyk2 inhibited IL-2-induced cell proliferation without affecting Stat5 activation. Collectively, these results suggest that Pyk2 is a newly identified component of the Jak-mediated IL-2 signaling pathway.     

Synthesis and pharmacological activity of deltorphin and dermorphin-related glycopeptides

The solid phase procedure, based on the Fmoc chemistry, was used to prepare some opioid deltorphin (H-Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2, DEL C) and dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2, DER) analogues in which a D-glucopyranosyl moiety is beta-O-glycosidically linked to a Thr4 or Thr7 side chain. Their activities were determined in binding studies based on displacement of mu- and delta-receptor selective radiolabels from rat brain membrane synaptosomes, in guinea pig ileum and rabbit jejenum bioassays, and, in vivo, by a mouse tail-flick test after intracerebroventricular (icv) and subcutaneous (sc) administrations. The glyco analogues modified at position 4 displayed low opioid properties, while Thr7-glycosylated peptides retained high delta- or mu-selectivity and remarkable activity in vivo. In particular, as systemic antinociceptive agents, the latter glucoside-bearing compounds were more potent than the parent unglycosylated peptide counterparts, showing a high blood to brain rate of influx which may be due to the glucose transporter GLUT-1.     

Sensitivity to antibiotics in coagulase-negative staphylococci isolated from patients with central venous catheters

We examined 25 coagulase-negative staphylococci isolated from children, of whom 17 with leukaemia and 8 with terminal renal failure. Strain identification performed by api Staph system revealed the presence of S. epidermidis in 21 children, S. hominis in 3 patients and S. haemolyticus in 1 patient. By diffusion method we examined the activity of penicillin, methicillin, cephalexin, cephtriaxon, lincomycin, erythromycin, vancomycin, co-trimoxasol, gentamicin, amikacin, chloramphenicol, rifampicin and fusidic acid. MICs of seven antibiotics were obtained by agar dilution method. MIC50 and MIC90 were as follows: /ml Methicillin 3.13 mg/ml and 50 mg/ml, lincomycin 100 mg/ml and 100 mg/ml, gentamicin 25 mg/ml and 100 mg/ml, chloramphenicol 6.25 mg/ml and 50 mg/ml, amikacin 1.56 mg/ml and 100 mg/ml, rifampicin 0.09 mg/ml and 12.5 mg/ml, fusidic acid 6.25 mg/ml and 12.5 mg/ml, vancomycin 1.56 mg/ml and 3.13 mg/ml. These data show that the examined strains are highly resistant to numerous antibiotics. Thirty six percent of all strains were resistant to methicillin, 88% to lincomycin, 60% to gentamicin, 52% to chloramphenicol, 24% to amikacin, 52% to rifampicin and 56% to fuscidic acid. All the examined strains were sensitive to vancomycin.