Protective effects of C5a blockade in sepsis.

Sepsis in humans is a difficult condition to treat and is often associated with a high mortality rate. In this study, we induced sepsis in rats using cecal ligation and puncture (CLP). In rats depleted of the complement factor C3, CLP led to very short survival times (about 4 days). Of the rats that underwent CLP ('CLP rats') that were C3-intact and treated with preimmune IgG, most (92%) were dead by 7 days. Blood neutrophils from these rats contained on their surfaces the powerful complement activation product C5a. This group had high levels of bacteremia, and their blood neutrophils when stimulated in vitro had greatly reduced production of H2O2, which is known to be essential for the bactericidal function of neutrophils. In contrast, when companion CLP rats were treated with IgG antibody against C5a, survival rates were significantly improved, levels of bacteremia were considerably reduced, and the H2O2 response of blood neutrophils was preserved. Bacterial colony-forming units in spleen and liver were very high in CLP rats treated with preimmune IgG and very low in CLP rats treated with IgG antibody against C5a, similar to values obtained in rats that underwent 'sham' operations (without CLP). These data indicate that sepsis causes an excessive production of C5a, which compromises the bactericidal function of neutrophils. Thus, C5a may be a useful target for the treatment of sepsis.

Permeability of thermally damaged skin: I. Immediate influences of 60 degrees C scalding on hairless mouse skin.

Freshly sacrificed hairless mice were burned dorsally by direct contact with 60 degrees C water for periods ranging from 15 seconds to 8 min. Wounds ranging in degree from superficial epidermis damage to injury penetrating well into subcutaneous musculature were inflicted. Burned skin sections and reference abdominal skin sections were excised, placed in diffusion cells and investigated with regards to their permeabilities to water, methanol, ethanol, n-butanol and n-octanol. The data were couched in terms of ratios of permeability coefficients of burned skin to normal skin (scalding coefficients) for the same animal. Scalding increased permeability of skin to all compounds studied but the effects leveled out by 60 seconds. Protracted scalding was without great effect despite progressively increased depth of damage to the tissue as noted in histological sections. The degree of lost barrier competency attributable to 60 degrees C scalding was not marked for any compound but was definitely different for different alkanols. An approximately 3-fold permeability increase was noted with n-butanol, the most affected compound. The data demonstrate that near instantaneous alterations in permeability of skin accompany scalding, that decreased barrier competency does not correlate with the severity of a burn as measured in depth of the burn, and that thermal alteration of permeabilities is dependent on the physicochemical characteristics of the permeants.

Hydration and percutaneous absorption: I. Influence of hydration on alkanol permeation through hairless mouse skin.

A method to study the influence of hydration on skin permeability where the skin is immersed in saline for up to 30 hr and under circumstances where a steady state rate of permeation can be established in several minutes is indicated. These circumstances allow multiple, sequential runs over a period where the permeability coefficients of some chemicals are gradually changing. It has been found that the permeabilities of water, methanol and ethanol are little affected by such hydration. However, there is a doubling of the permeability coefficients of butanol and hexanol during the first 10 hr of immersion. More hydrophobic alkanols seem to be less sensitive to the protracted aqueous conditioning. In general the results indicate that there are complex molecular structure-permeability relationships operating in skin. More specifically, the hydration effects are insightful with respect to developing barrier models for skin as they are further indications that different parallel diffusional paths are followed by polar and semi- and nonpolar species.

Frequency of antibiotic resistance among group B Streptococcus isolated from healthy college students.

We report resistant rates to erythromycin and clindamycin among Streptococcus agalactiae (group B Streptococcus) isolated from a random sample of healthy male and nonpregnant female college students. Observed resistance rates were twice as high as those reported among pregnant women from the same geographic area 2 years prior.

Ureaplasma urealyticum and chronic lung disease in very low birth weight infants during the exogenous surfactant era.

BACKGROUND: An association between recovery of Ureaplasma urealyticum from the respiratory tract of very low birth weight (VLBW) infants (< or =1500 g) and later chronic lung disease (CLD) was reported by several authors before the routine use of exogenous surfactant (SURF). We sought to assess whether this relation persists in the era of routine SURF. METHODS: We prospectively studied a cohort of 105 VLBW infants who required mechanical ventilation at < 12 h of age. Tracheal aspirates for U. urealyticum culture were obtained before administration of SURF or antibiotics. Clinicians were unaware of U. urealyticum status. Chest radiographs at 28 days were reviewed by a single pediatric radiologist, blinded to U. urealyticum status. Sample size was predetermined to detect a 30% increase in CLD among those with U. urealyticum recovery from tracheal culture (U. urealyticum-positive) with alpha <0.05 and beta <0.20. RESULTS: Of the study infants 22 were U. urealyticum-positive and 83 were U. urealyticum-negative. No differences were found between the groups for birth weight, gestational age, gender, inborn, antenatal or postnatal steroid use, SURF therapy, non-U. urealyticum infection, necrotizing enterocolitis, patent ductus arteriosus, intraventricular hemorrhage or cystic periventricular leukomalacia. At 28 days U. urealyticum-positive patients were significantly more likely to have CLD than U. urealyticum-negative [15 of 22 (68%) vs. 30 of 83 (36%); P < 0.02]. The U. urealyticum-positive patients also required significantly longer courses of supplemental oxygen and mechanical ventilation. No significant differences were found for CLD at 36 weeks postconception or duration of hospitalization, although type II error could not be excluded for these secondary endpoints. CONCLUSIONS: Respiratory U. urealyticum at or shortly after birth remains associated with CLD at 28 days despite routine use of SURF. Controlled trials of anti-Ureaplasma therapy in U. urealyticum-positive VLBWs as soon after birth as possible may determine whether CLD, duration of respiratory support and attendant costs can be decreased.

Evaluation of the new DrySlide beta-lactamase test.

Four hundred thirteen recent clinical isolates of Staphylococcus, Haemophilus, Moraxella, and Neisseria species were tested for beta-lactamase production using the Difco DrySlide beta-lactamase test. These results were compared with those of the BBL Cefinase reagent. Of the 413 isolates tested, 258 (62.5%) were beta-lactamase positive. There was 99.8% agreement between the two test methods; only one isolate of Staphylococcus aureus was DrySlide-negative and Cefinase-positive. The DrySlide beta-lactamase reagent is an accurate and convenient format for the evaluation of beta-lactamase activity in the clinical laboratory.

Treatment of established prosthetic vascular graft infection with antibiotics preferentially concentrated in leukocytes.

The efficacy of treating established vascular graft infections with rifampin and clindamycin (preferentially concentrated in leukocytes) and cefazolin (not concentrated in leukocytes) was studied in a canine model. Infrarenal aortic, 6 mm by 6 cm knitted Dacron double velour grafts were implanted and infected with 10(8) colony-forming units (CFU) of coagulase-positive Staphyloccus aureus organisms injected intravenously immediately after graft placement. Antibiotic therapy was instituted at 3 months postimplantation. Three groups were studied: (I) untreated controls (n = 3); (II) therapy with intravenous cefazolin 15 mg/kg/8 hr for 28 days (n = 7); and (III) combined therapy with intravenous rifampin 13 mg/kg/24 hr and intravenous clindamycin 13 mg/kg/8 hr for 28 days (n = 7). Grafts were removed for quantitative bacteriologic studies after the 28-day course of therapy. Two group I control grafts remained patent with 6.4 X 10(6) and 8.1 X 10(3) CFU S. aureus/gm of graft. The third control graft was thrombosed. Two group II animals demonstrated 1.6 X 10(7) and 2.3 X 10(5) CFU S. aureus organisms/gram of graft, respectively; the remaining five group II grafts were free of organisms. All group III grafts were sterile--a significant difference (p less than 0.05) from group I grafts. In this experimental model, established prosthetic graft infections were eradicated by intensive treatment with antibiotics preferentially concentrated in leukocytes.

Frequent resistance of clinical group B streptococci isolates to clindamycin and erythromycin.

OBJECTIVE: To determine both the frequency of reported penicillin allergy in parturients and the frequency of resistance in vitro of clinical isolates of group B streptococci to clindamycin and erythromycin. METHODS: One hundred clinical isolates of group B streptococci were tested to determine the frequency of resistance to clindamycin, erythromycin, penicillin G, vancomycin, and cefazolin. The frequency of beta-lactam allergy and reported allergic reaction also were recorded for all consecutive laboring women during the 4-month study. RESULTS: The frequency of group B streptococcal resistance to clindamycin was 15% and to erythromycin was 16%. No isolates were resistant to penicillin G, vancomycin, or cefazolin. Twelve percent of the 963 women who delivered during the study reported a penicillin allergy, but only 30% of those could describe their allergic reaction. CONCLUSION: In vitro resistance of group B streptococci to clindamycin and erythromycin occurred frequently in this population. Whereas the importance of this finding in vivo is uncertain, it raises concern about the possibility of inadequate prophylaxis using currently recommended alternatives in penicillin-allergic patients. Artful questioning of women reporting penicillin allergy may lessen the likelihood of using these less desirable agents in the setting of intrapartum antimicrobial prophylaxis.

Comparison of susceptibility to beta-lactam antimicrobial agents among bacteria isolated from intensive care units.

Evaluating microbial isolation and susceptibility patterns from institutional settings is a well-established component of ongoing infection control activities. At the University of Michigan Hospitals, susceptibility profiles for selected bacteria tested from 1984 to 1989 were analyzed for major changes in the percentage of organisms susceptible to beta-lactam antimicrobials. Data on bacteria isolated from respiratory specimens obtained from 1433 patients in intensive care units (ICUs) during a 10-month interval were compared with like data obtained from 750 non-ICU patients. Antimicrobial agents studied were chosen based on hospital formulary availability and prevailing usage in the institution. Susceptible and moderately susceptible categories were combined for purposes of reporting, since empiric therapeutic doses would cover strains having both susceptibility levels. Antimicrobic susceptibilities were compared and differences analyzed among the ICUs. Major shifts in susceptibility were noted during the 5-year period. The incidence and susceptibility profiles of the microorganisms varied considerably between ICU and non-ICU patients. Pseudomonas aeruginosa isolates from individual ICUs showed large variations in prevailing susceptibilities, with the burn unit harboring the most resistant strains. However, the neurological, surgical, and critical care medicine units also showed large numbers of antimicrobial-resistant pseudomonads. Among Enterobacter cloacae isolates, only imipenem showed a high level of activity against both ICU and total hospital isolates. When examined by individual ICU, however, imipenem resistance was seen in the general medicine and burn units. The burn and pediatric ICUs showed increased rates of recovery of beta-lactam-resistant E. cloacae isolates, although significantly high resistance rates were seen throughout all ICUs. The surgical ICU was noted to have an abnormally high incidence of lower respiratory infections caused by P. aeruginosa. The antibiogram indicated that one possible epidemic strain was involved. However, when the isolates were subjected to fatty-acid profiling by gas-liquid chromatography, it was found that cross-contamination with five discernible strains had occurred among the ten patients tested. These preliminary data suggest that resistant pseudomonads can be harbored and spread within an ICU, and that the ICU can act as a reservoir of resistance that is spread to a "step-down" unit.

A five-year multicenter study of the susceptibility of the Bacteroides fragilis group isolates to cephalosporins, cephamins, penicillins, clindamycin, and metronidazole in the United States.

Over 2800 clinical strains of the Bacteroides fragilis group were collected during a 5-year period from ten geographically separate sites and tested for their susceptibility to various antimicrobial agents using a broth microdilution method. Among the cephalosporins, ceftizoxime was the most active (13% resistance) and importantly exhibited relatively equal activity against both B. fragilis species and non-B. fragilis species. Cefotaxime exhibited similar activity with an overall resistance rate of 18%. Both ceftriaxone and cefoperazone were appreciably less active cephalosporins especially against non-B. fragilis species. With regard to cephamycins, cefoxitin (MIC90, 32 micrograms/ml) was more active than cefotetan (MIC90, > or = 256 micrograms/ml) and cefmetazole (MIC90, 64 micrograms/ml). Non-B. fragilis species were highly resistant to cefotetan and cefmetazole. Imipenem was highly active against all strains with the exception of four strains of B. fragilis. Ampicillin-sulbactam, amoxicillin-clavulanate, piperacillin-tazobactam, and cefoperazone-sulbactam were all highly active with resistance rates < 2%. No resistance was detected to metronidazole, whereas 14% of isolates were resistant to clindamycin. When compared with other studies, these findings underscore the wide variability in susceptibility patterns reported nationwide and the need to continue monitoring these patterns to aid in choosing the most active compounds for therapy.

Detection and characterization of atypical mycobacteria by the polymerase chain reaction.

The purpose of this study was to develop a simple protocol of nested reamplification polymerase chain reaction (PCR) to detect and characterize diverse mycobacterial species. DNA extracted from 126 pure mycobacterial cultures isolated from clinical specimens was amplified by nested PCR with use of a novel set of oligonucleotide primers specific for the 65-kDa antigen gene of mycobacteria. The PCR products were each digested with three restriction enzymes and electrophoresed on an agarose gel. The observed DNA fragment sizes of the different species with each enzyme were compiled into a simple algorithm. This method can rapidly detect and characterize a wide variety of mycobacterial species, including the most common pathogens Mycobacterium tuberculosis, Mycobacterium avium-intracellulare, and Mycobacterium kansasii, without hybridization to labeled probes. The application of this method to surgical pathology was demonstrated by amplification and identification of atypical mycobacteria, including M. kansasii and Mycobacterium leprae, in formalin-fixed paraffin-embedded tissue. This protocol broadens the diagnostic potential of PCR for rapidly diagnosing mycobacterial infection in clinical samples, particularly in paraffin-embedded tissue sections.

Pulsed-field gel electrophoresis genomic fingerprinting of hospital Escherichia coli bacteraemia isolates.

Pulsed-field gel electrophoresis (PFGE), because of the increased sensitivity it affords over other methods of bacterial genotyping, represents a potentially powerful tool for the characterisation of isolates from hospital infections. Genomic fingerprinting by PFGE was applied to all clinical isolates of Escherichia coli obtained from blood during a 6-month period (78 isolates, 58 patients) at the University of Michigan Medical Center. The rare-restriction patterns of these isolates, in contrast to those of isolates from the E. coli reference collection (ECOR), were not randomly distributed through the E. coli species. Four related clusters, which represented c. 21% of the blood isolates, were identified. Two of these genotypic clusters were also clustered temporally, their members all being isolated within the same 2-week period, while the other two clusters spanned the study period. These observations indicate in-hospital endemic vectors or the occurrence of specialised E. coli lineages that are capable of invading the bloodstream and exploiting in-hospital vectors, or both.

Antimicrobial susceptibility patterns of the Bacteroides fragilis group in the United States, 1989.

A national survey of Bacteroides fragilis group was continued in 1989 for the ninth consecutive year. Seven hundred thirty-nine isolates of B fragilis group from eight centers were tested for susceptibility to 14 antimicrobials. Sulbactam and clavulanic acid, beta-lactamase inhibitors, were tested at a constant concentration of 8 micrograms/ml and 2 micrograms/ml, respectively. Sulbactam was also tested in a fixed ratio of 1:2. Imipenem, ampicillin+sulbactam, and ticarcillin+clavulanic acid had resistance of less than 1% at breakpoints of 8 micrograms/ml, 16 micrograms/ml, and 64 micrograms/ml, respectively. At 32 micrograms/ml, resistance to cefoxitin, cefotetan, ceftizoxime, and ceftriaxone were 4%, 25%, 26%, and 46%, respectively. Clindamycin resistance was 10% at a breakpoint of 4 micrograms/ml. No isolates were resistant to chloramphenicol or metronidazole. Resistance for five B fragilis species to cefoxitin, ceftizoxime, and cefotetan varied greatly among both species and participating institutions. The addition of a beta-lactamase inhibitor increased the potency of the beta-lactam drugs tested as combinations. This finding suggests that beta-lactamase production is the major resistance factor in members of the B fragilis group.

Bacteriology of pustules occurring during treatment of psoriasis.

Pustular lesions developed in 16% of our psoriatic inpatients treated with a modified Goeckerman regimen. The role of bacterial infection in the development of these lesions remains controversial. Representative lesions were cultured, and the recovered organisms were quantitated. We were not able to culture a significant number of bacteria from these lesions. Pustules were prone to develop during the summer months in hirsute patients, in patients with hyperhidrosis, and in patients in whom topical treatment of a more occlusive nature was applied. The male-female ratio was 4.8:1. Topical antibiotic treatment of these pustules in psoriatic patients appeared to be unwarranted since the lesions resolved when topical therapy was discontinued. These pustules appeared to be distinct from pustular psoriasis.

Permeation of skin and eschar by antiseptics I: baseline studies with phenol.

To assess how the permeability of phenol is altered by thermal injury, it was first necessary to have baselines of comparison on normal skin. Using in vitro diffusion cells and the skin of the hairless mouse, [14C]phenol was applied to skin in an aqueous medium with a reference copermeating species, [3H]methanol, and 37 degrees permeability coefficients of the pair were evaluated as functions of animal age, skin hydration, stripping of the skin, dermis isolation, and phenol concentration. Age proved to be of little consequence to permeability over a wide age range. Prolonged aqueous soaking of the skins was also without much effect. Stripping of the skin and isolating the dermis by soaking techniques allowed assessment of individual skin strata diffusion resistances. When applied to skin in trace radiochemical concentrations, phenol behaved diffusionally as an alkanol with a chain length of six. But at concentrations greater than 2% w/v, phenol facilitated the permeation rates of itself and methanol; the effect was markedly concentration sensitive and only fractionally reversible. Concentration studies using silicone rubber membranes proved that the effects on the skin were the results of destroyed barrier integrity. At 6% phenol concentration there was an essentially instantaneous, 10-fold increase in the phenol permeability coefficient, raising it to two-thirds that observed with fully stripped skin. Overall, the data suggest that the stratum corneum is proportionally impaired as the phenol concentration is increased.

Permeation of skin and eschar by antiseptics II: influence of controlled burns on the permeation of phenol.

The safe antiseptic use of phenol over the burn-traumatized surface depends on knowledge of how the systemic accumulation of phenol is affected by burn processes. To gain insight into the underlying permeation phenomenon, the diffusion of phenol and a reference cosolute, methanol, through both scalded and branded dorsal skin sections of the hairless mouse was studied as a function of burn temperature using in vitro diffusion cells. Temperatures up to 100 and 150 degrees were used for scalding and branding, respectively, using a 60-sec; exposure time. Permeability coefficients of the traumatized skins were assessed at 37 degrees and compared with control values. Coefficients of both permeating species were not increased significantly by burn temperatures up to 70 degrees applied either by scalding or branding, however, at higher temperatures exaggerated increases in permeation rates were noted. A limiting increase of approximately 7 times the control value was noted for phenol irrespective of the burn method. Permeability of methanol was altered even more dramatically and at 100 degrees by scalding and 150 degrees by branding was over 50 times the control rate. At 80 and 100 degrees for methanol and at 80 degrees for phenol, scalding produced larger increases in the permeability coefficients than branding. Since contact for 1 min at 60 degrees is capable of producing a full-thickness burn injury, it is clear that eschar permeability to phenol immediately postburn is not related to the clinical degree of burning, but is a function of the thermal intensity (hotness) of the burn stimulus. Full-thickness wounds can be expected to have highly variable rates of systemic absorption as a direct consequence of the wide-ranging permeability possible for such burns, with the risks of topical application varying accordingly.

Hydration and percutaneous absorption III: Influences of stripping and scalding on hydration alteration of the permeability of hairless mouse skin to water and n-alkanols.

The influence of hydration on the permeability of stripped and scalded skins of hairless mice was investigated in vitro using water and n-alkanols as test permeants. Irrespective of pretreatment, the permeation rates of water, methanol, and ethanol were unaffected by aqueous immersion of skin sections in a diffusion cell, consistent with earlier data on unprocessed skins. The permeation rates of butanol and hexanol also were insensitive to hydration, differing from earlier studies on normal, intact skin in which both solutes' rates doubled after 10 hr of soaking. Following both pretreatments, the permeability of octanol declined over the first 5-10 hr of maceration, but remained invariant thereafter. The decline was most pronounced for the scalded skins. With untreated skin, octanol permeability initially increased and then declined before assuming a constant value. This study indicates that the barrier properties of the epidermis and dermis are not particularly sensitive to extended hydration except in the case of octanol. Scalding at 60 degrees for 60 sec rapidly hydrates the skin, altering tissue permeability to about the same extent as a 10-hr (or longer) immersion in water at 37 degrees. Octanol's unique hydration profile is explained by locating the origin of permeability decline in tissue beneath the horny exterior of the skin.