Bioterrorism: implications for the clinical microbiologist.

The specter of bioterrorism has captured the attention of government and military officials, scientists, and the general public. Compared to other sectors of the population, clinical microbiologists are more directly impacted by concerns about bioterrorism. This review focuses on the role envisioned for clinical laboratories in response to a bioterrorist event. The microbiology and clinical aspects of the biological agents thought to be the most likely tools of bioterrorists are presented. The historical background of the problem of bioterrorism and an overview of current U.S. preparedness planning, with an emphasis on the roles of health care professionals, are also included.

The influence of sulbactam on the in vitro activity of mezlocillin, piperacillin and cefotaxime.

In a multicentre study, the in-vitro activity of mezlocillin (MEZ, Chemical Abstract Service [CAS] 51481-65-3), piperacillin (PIP, CAS 61477-96-1) and cefotaxime (CTX, CAS 63527-52-6) against mezlocillin-resistant organisms was determined alone and in combination with the beta-lactamase inhibitor sulbactam (SBT, CAS 68373-14-8). A total of 870 strains were investigated (481 Enterobacteriaceae, 57 Pseudomonas aeruginosa, 41 Acinetobacter spp., 194 Bacteroides fragilis and 97 Staphylococcus spp.). MIC values were determined using the agar dilution test (aerobic organisms) or the microbroth dilution test (Bacteroides spp.) in accordance with Deutsche Industrie für Normung 58 940. SBT was added in fixed concentrations of 5 mg/l and 10 mg/l. For all combinations with SBT investigated, the geometric mean of the MIC and the MIC(50) and MIC(90) values were reduced as compared with the antibiotic alone (without SBT). Consequently, the proportion of sensitive strains was appreciably increased, for example in the Enterobacteriaceae: MEZ 1%, MEZ + 10 mg/l SBT 53%; PIP 4%, PIP + 10 mg/l SBT 54%; CTX 52%, CTX + 10 mg/l SBT 68%. The effect of SBT was especially pronounced on Bacteroides spp. For this organism, the proportion of sensitive strain rose from 2% to 97% (MEZ), 6% to 95% (PIP) and from 7% to 98% (CTX). The results show that adding SBT appreciably enhances the activity of MEZ, PIP and CTX against resistant strains of microorganism, and extends the activity spectrum to include anaerobic organisms. Thus the availability of SBT as a single-agent preparation for use in combination with various beta-lacta antibiotics represents a worthwhile enlargement of the therapeutic armamentarium for treating bacterial infections.

Diagnosis and detection of drug-resistant strains of M. tuberculosis.

AIDS: Factors contributing to the recent upswing in tuberculosis (TB) cases in the United States include the shifting of management of TB cases from specialized hospital units to lesser-equipped outpatient settings; reduced medical school teaching of TB-related issues; large-scale immigration from regions endemic for TB; and the homelessness and AIDS cases that have created a pocket of disease in high-risk populations. At least 33 percent of current cases can be attributed to new rather than reactivated infection, and many of these are caused by the ever-increasing number of drug-resistant strains. Multidrug-resistant TB has a higher overall fatality rate and disproportionately affects immunocompromised and HIV-infected individuals. Clearly, expedient diagnosis is important in controlling the spread of tuberculosis. Sputum samples, evaluated first by direct microscopic evaluation (smear), are visualized with either the easily detected acid-fast fluorochrome dye auramine O, or the more specific Ziehl-Neelsen stain. Specimens are cultured on either solid media (Lowenstein-Jensen slant), or are grown in a liquid medium, such as the BACTEC automated radiometric system. Next, biochemical or nucleic acid probe testing is used to identify various strains. Isolates are tested for resistance to commonly used antituberculosis drugs, often by using the new method of susceptibility testing in liquid broth rather than the traditional agar dilution method. Clinical laboratories await FDA approval of two new methods employing hybridization with rRNA genes or polymerase chain reaction (PCR) that will further speed up diagnosis of TB.^ieng

Double-blind, comparative study of rufloxacin once daily versus amoxicillin three times a day in treatment of outpatients with exacerbations of chronic bronchitis.

In a double-blind, randomized, multicenter study, the efficacy and safety of two dosage schedules of rufloxacin once daily were compared with those of amoxicillin three times a day in the treatment of 192 outpatients with exacerbations of chronic bronchitis. Rufloxacin was given as a single oral dose of 400 mg on day 1 and single daily doses of 200 mg on the subsequent 9 days (n = 64) or as 300 mg on day 1 and then 150 mg daily for 9 days (n = 63); amoxicillin was given as 500 mg orally three times a day for 10 days (n = 65). Clinical and bacteriological assessments were carried out before treatment, between study days 3 and 5, and at days 1 and 8 after treatment. Pretreatment cultures were positive for 139 patients, the most frequently isolated pathogens being Streptococcus pneumoniae, Moraxella catarrhalis, and Haemophilus influenzae. Clinical success rates were comparable in the three groups (94, 95, and 98%, respectively), as were bacteriological success rates at the end of treatment (93, 95, and 91%, respectively) and at follow-up (88, 95, and 98%, respectively). The power to detect a significant 15% difference in cure rates was 74.9%. Follow-up bacteriological failures from pneumococcal infection were 18% in both rufloxacin groups combined and 5% in the amoxicillin group. The 200-mg dose regimen achieved average steady-state concentrations in plasma higher than did the 150-mg dose regimen (3.75 versus 2.72 micrograms/ml). Adverse events occurred in 11 and 13 patients, respectively, on rufloxacin and 8 on amoxicillin. This study shows that rufloxacin once daily ay be a possible option for the treatment of acute exacerbations of chronic bronchitis. The 200-mg daily oral dose preceeded by a loading dose of 400 mg displays a better pharmacokinetic profile than the lower dose.

[The effect of sulbactam on the in vitro activity of mezlocillin, piperacillin and cefotaxime]. / Zum Einfluss von Sulbactam auf die In-vitro-Aktivität von Mezlocillin, Piperacillin und Cefotaxim.

The in vitro activity of mezlocillin (MZL, CAS 51481-65-3), piperacillin (PIP, CAS 61477-96-1) and cefotaxim (CTX, CAS 63527-52-6) alone and in combination with sulbactam (SBT; CAS 68373-14-8) against mezlocillin-resistant pathogens was determined in a multicenter study. A total of 870 strains were investigated (481 Enterobacteriaceae, 57 Pseudomonas aeruginos, 41 Acinetobaster spp., 194 Bacteroides fragilis, and 97 Staphylococcus spp.). Determinations of MIC were performed according to DIN-guidelines (agar-dilution method for aerobes and microbroth-dilution method for anaerobes). Sulbactam was added in fixed concentrations of 5 mg/l and 10 mg/l. In all sulbactam-combinations examined mean MIC as well as MIC50 and MIC90 were reduced compared to the respective values for the antibiotics alone. Consequently, percentages of susceptible strains increased significantly: i.e. for Enterobacteriaceae: MZL 1% vs. MZL + 10 mg/l SBT 53%; PIP 4% vs. PIP + 10 mg/l SBT 54%; CTX 52% vs. CTX + 10 mg/l SBT 68%. The effect of sulbactam was most pronounced in Bacteroides spp. with an increase in susceptible strains from 2% to 97% for MZL, from 6% to 95% for PIP and from 7% to 98% for CTX. The results indicate that by adding sulbactam the in vitro activity of mezlocillin, piperacillin and cefotaxim against resistant pathogens is augmented significantly. In addition, the spectrum of antibacterial activity is extended to anaerobic pathogens such as Bacteroides spp. The availability of sulbactam as a monosubstance for combination with various beta-lactam-antibiotics thus represents a useful improvement of therapeutic options in bacterial infections.

In vitro activity of the combination trimethoprim plus sulfamethoxazole compared with that of other chemotherapeutic agents.

For many years now, the combination trimethoprim plus sulfamethoxazole (active ingredients of Bactrim) has proved to be an effective chemotherapeutic agent with a broad spectrum of antibacterial activity against both gram-positive and gram-negative organisms, including beta-hemolytic streptococci, staphylococci, pneumococci, Haemophilus influenza, Escherichia coli, Proteus mirabilis, Proteus vulgaris, Klebsiella, Enterobacter and Citrobacter. In this comparative study, the antibacterial activity of the combination against 2,229 gram-negative and 1,338 gram-positive strains encountered in domiciliary practice was tested and compared with that of other commonly used antimicrobials. Minimum inhibitory concentrations (MICs) were determined in microtitre plates using serial dilutions. With regard to the gram-negative strains, trimethoprim + sulfamethoxazole, with an MIC90 of less than 2 mg/l for most isolates, was the most active substance apart from norfloxacin. The combination also had a powerful inhibitory effect on gram-positive strains, the MIC90 being between 2 and 4 mg/l for all strains except Staphylococcus epidermidis.

Effectiveness and tolerance of pre- and postexposure treatment with purified inactivated rabies vaccine prepared on Vero cell line.

The results are reported of a field trial which was designated to demonstrate the inocuity and efficacy of the purified inactivated rabies vaccine (PVRV), produced on Vero cells by the Institut Mérieux, Lyon, France in pre- and postexposure treatment in man. Four sex and age matched groups of veterinary students and medical personnel received the vaccine. The vaccine was given according to WHO recommendations for pre- and postexposure regimens. The 82 volunteers were divided into four groups and vaccinated as follows. Group IA consisted of 27 individuals, receiving injections on days 0, 7 and 21. Group IB consisted of 29 individuals, injected on days 0, 28 and 56. Group II consisted of 16 individuals, receiving a postexposure schedule of vaccinations on days 0, 3, 7, 14, 28 and 90. Group III consisted of 10 subjects, all of whom had been previously immunized with various antirabies vaccines and received one single booster inoculation of this vaccine. Serum samples were taken on the days of vaccination and 14 days later in all groups and in addition on day 70 in group IA. Neutralizing antibodies against the rabies virus were determined in the rapid fluorescent focus inhibition test (RFFIT). In conclusion it can be stated that, regardless of the schedule of prophylactic immunization, three 0.5 ml inoculations of PVRV result in very high titres of virus neutralizing antibodies and a single vaccination in previously immunized individuals is sufficient to raise the amount of antibodies to a high level. Due to the high purity of the product the vaccine is very well tolerated by the vaccinees.

Sensitivity of clinical isolates from German hospitals to amoxicillin/clavulanic acid (Augmentin) compared with other antibiotics.

17,244 pathogens isolated from clinical specimens of 24 hospitals in the Moers area (North-Rhine Westphalia, FRG) were tested in regard to their susceptibility to Augmentin (amoxicillin and clavulanic acid). For this purpose, minimal inhibitory concentrations were determined by use of microbroth dilution technique. 80% of Gram-negative, 98% of Gram-positive and 97% of anaerobic isolates were susceptible to Augmentin (breakpoint 4 mg/l amoxicillin in the presence of 2.5 mg/l clavulanic acid). In a second part of the study the susceptibility to Augmentin of 4.137 Gram-negative and 10.958 Gram-positive pathogens was compared to their sensitivity against benzylpenicillin, flucloxacillin, mezlocillin, erythromycin, clindamycin, fusidic acid, ampicillin, cefaclor and doxycyclin.

Antibacterial effect of imipenem in vitro against important aerobic and anaerobic strains isolated from clinical specimens.

Imipenem is a thienamycin antibiotic of the first generation with broad antibacterial activity. It covers all gram-positive organisms (including Streptococcus faecalis) and gram-negative bacteria (including Pseudomonas aeruginosa and Serratia spp.) as well as Bacteroides fragilis and other Bacteroides species. In this comparative study the antimicrobic effect against 1020 gram-negative, 927 gram-positive and 352 anaerobic strains from fresh clinical isolates was tested and compared with that of other frequently used antibiotics. The minimum inhibitory concentrations (MIC) were determined by means of a serial dilution test with micro standard plates. Within the group of gram-negative strains, imipenem was the most active antibiotic with a MIC90 of less than or equal to 0.25 mg/l for most isolates. Imipenem shows a broad spectrum of activity against gram-negative pathogenic bacteria including Escherichia coli, Klebsiella spp., Proteus spp, Enterobacter spp., Citrobacter spp. and Serratia spp., and also covers resistant strains of Pseudomonas aeruginosa, Acinetobacter spp. and Alcaligenes faecalis. Imipenem also shows high inhibiting activity against gram-positive strains and anaerobic pathogens.

Comparative in vitro activity of ciprofloxacin against aerobic and anaerobic bacteria from clinical isolates.

1-Cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-piperazin-1- ylquinoline-3-carboxylic acid (ciprofloxacin, Bay o 9867, Ciprobay) is a broad spectrum antibiotic of the 4-quinolone group. It possesses a bactericidal effect attributable to the property of DNA-gyrase inhibition. The antimicrobial action comprises all grampositive strains (including Streptococcus faecalis) and gramnegative strains (including Pseudomonas aeruginosa and Serratia spp.), as well as Bacteroides fragilis and other Bacteroides species. In this comparative study the antimicrobial effect of ciprofloxacin was tested against 665 gramnegative, 412 grampositive and 274 anaerobic strains from fresh clinical isolates and compared with that of other frequently used antibiotics. The minimum inhibitory concentrations (MIC) were determined by means of a serial dilution test with micro standard plates. Within the group of gramnegative strains, ciprofloxacin was the most active antibiotic with an MIC90 of 0.12 mg/l to 0.5 mg/l for most isolates. Ciprofloxacin shows a broad spectrum of activity against gramnegative pathogenic bacteria including Escherichia coli, Klebsiella spp., Citrobacter spp., Enterobacter spp., Serratia spp. and Acinetobacter spp., and also covers resistant strains of Pseudomonas aeruginosa and Alcaligenes faecalis. Ciprofloxacin also shows a high inhibiting activity against grampositive strains (Staphylococci, Enterococci) and anaerobic pathogens.

[Comparative in vitro study of the antimicrobial activity of ceftazidime against clinical isolates]. / Vergleichende In vitro-Studie zur antimikrobiellen Aktivität von Ceftazidim gegen klinische Isolate.

The antimicrobial activity of ceftazidime was tested against 1482 gram-negative and 1216 gram-positive strains isolated from fresh clinical specimens and compared with generally used antibiotics including other third generation cephalosporins and broad spectrum penicillins. Minimal inhibitory concentrations were determined in a broth dilution test on microtiter plates. In the group of the gram-negative bacteria ceftazidime was the most active of the antimicrobial agents tested with an MIC of 0.5 mg/l (MIC90) for most of the isolates. Ceftazidime exhibited a broad spectrum of activity against gram-negative pathogenic bacteria including Enterobacteriaceae (Escherichia coli, Klebsiella spp., Proteus spp. Enterobacter spp., Citrobacter spp., Serratia spp.) including frequently resistant strains of Pseudomonas aeruginosa, Acinetobacter spp. and Alcaligenes faecalis. The activity against P. aeruginosa is the most remarkable property of the agent. Ceftazidime is less effective against gram-positive compared to gram-negative bacteria. No inhibitory action can be observed against Streptococcus faecalis.

A survey of temocillin sensitivity of strains resistant to newer beta-lactam antibiotics.

The susceptibility of a total of 2014 Gram-negative clinical isolates (except Pseudomonas aeruginosa) to a number of antibiotics, including temocillin, was investigated in 2 geographically distinct areas. Overall, more strains were sensitive to temocillin than to mezlocillin, piperacillin, amoxycillin plus clavulanic acid, cefotaxime, cephazolin, latamoxef (moxalactam), cefoxitin, or netilmicin. Susceptibility to temocillin of multiple-resistant strains was studied.

In-vitro activity of augmentin against clinically important gram-positive and gram-negative bacteria in comparison with other antibiotics.

The susceptibility to Augmentin of a total of 1,417 bacterial isolates was investigated. Augmentin is a new formulation of the broad-spectrum beta-lactam-antibiotic amoxicillin together with the beta-lactamase-inhibitor clavulanic acid. It was demonstrated that 88% of all isolates tested were sensitive to Augmentin, 9% were resistant. 88% of all Pseudomonas aeruginosa strains fell in the "resistant" category. Only 1/71 anaerobes and 15/286 staphylococci were classified as resistant to Augmentin.

Rabies post-exposure treatment and side-effects in man using HDC (MRC 5) vaccine.

126 patients underwent a post-exposure antirabies treatment using the beta-propiolactone inactivated (HDC) MRC-5 vaccine. The immunization schedule consisted of single doses of 1 ml of vaccine administered on days 0, 3, 7, 14, 30 and 90. Within the post-exposure group several patients suffered wounds from known rabid animals. Serum samples were collected on days 0, 14, 30, 90 and 104. Neutralizing antibodies against the rabies virus were determined in the rapid-fluorescent-focus-inhibition (RFFI) test. The rise in neutralizing antibodies was steeper than that in the prophylactic group whereas the mean titer at day 30 did not significantly exceed the values obtained with the preventive immunization scheme. A 1 ml booster injection on day 90 resulted in a three-fold increase of antibody titer within two weeks. Although no major side-effects were observed, an analysis of questionnaires filled out by the patients is presented.

Human rabies immunoprophylaxis using HDC (MRC-5) vaccine.

In a field trial 181 high risk persons of both sexes between ages of 2--71 years were prophylactically vaccinated against rabies with the beta-propiolactone inactivated rabies-vaccine (hdc) mrc-5. 1 ml of vaccine was administered intramuscularly on days 0, 7 and 21. Serum samples were collected on days 0, 7, 21 and 35. Neutralizing antibody titers against the rabies virus were determined in the rapid fluorescent focus inhibition test, 7 days after the first injection measurable titers of antibodies were found in all sera with one exception. On day 21 all persons showed a 100% seroconversion with a mean titer of 1:1000 (8,3 IU/ml). On day 35 the final mean titer measured 1:2400 (20 IU/ml). Major side effects during the prophylactic vaccination were not observed.

Ultrastructural studies of cell-virus interaction in reptilian cell lines. IV. Effects of chloramphenicol and ethidium bromide on VSW cell mitochondria and associated virions.

Structural effects of chloramphenicol (CAP) and ethidium bromide (EB) on VSW cell mitochondria and intramitochondrial virions (IMV) have been studied on a comparative basis by thin-section electron microscopy. CAP-treated cells show a wide variety of mitochondrial alterations, frequently involving swelling of the organelle and loss of cristae orientation. IMV are generally severely disrupted, particularly in peripheral regions. In such configurations, strand-like material radiates in a spokelike fashion from the shell zone to adjacent cristae-matrix area. EB-treated cells also display considerable mitochondrial distortion evidenced primarily by the formation of small, localized multimembrane regions. IMV exposed to EB, however, are less structurally damaged than CAP-treated ones. The relative incidence of IMV production is enhanced approximately fourfold in EB-treated cells compared to CAP-treated ones, suggesting that virion synthesis may be under nuclear, rather than mitochondrial, control.

Studies of mitochondria and mitochondrial DNA extracted from organelles harboring an intramitochondrial virus.

(1) Two viper cells lines were investigated, one which harbors IMV in the mitochondria (VSW cells) and one without detectable IMV (VH3 cells). (2) The size of closed circular mtDNA molecules from both VSW and VH3 cells was found to be significantly greater (5.4 to 5.6 micron) than the contour lengths of typical mammalian cells (4.8 to 5.2 micron). (3) A small percentage of mini-circles ranging in size from 0.1 to 0.6 micron was observed to band with closed circular mtDNA from both cell lines. Minicircles were especially abundant in VH3 cells. (4) MtDNA from VSW cells contained 34.1% dimers plus oligomers (10.2% oligomers), whereas VH3 cells had only 14.8% dimeric and oligomeric forms (5.4% oligomers). (5) Treatment of VSW cells with 1 microng/ml ethidium bromide for 48 hours resulted in an increased incidence of IMV (IMV in 15% of mitochondrial sections) as compared with untreated VSW cells (IMV in 3% of mitochondrial sections).

Establishment and characterization of ethidium bromide resistance in simian virus 40-transformed hamster cells. Effects on mitochondria in vivo.

This study describes the isolation and subsequent characterization of four mammalian cell lines resistant to ethidium bromide (EB). Treatment of the simian virus 40- (SV40) transformed hamster cell line F5-1 first led to the establishment of the F2 cell line, which is resistant to 2 microg EB/ml. At this concentration cytochromes c and b are present in almost normal or only slightly diminished amounts, whereas cytochromes a + a(3) show an obvious decrease. The mitochondria of the F2 cell show a normal ultrastructure, not distinct from the parental cell line F5-1, and contain closed circular DNA. The sensitive parental F5-1 cells, however, when exposed to the same dye concentration exhibit the typical EB-induced ultrastructural changes in the mitochondria, and no more component I mitochondrial DNA can be demonstrated. 1 yr after establishment we derived from the F2 cell three more cell lines, resistant against 4, 8, and 16 microg of EB/ml. These cell lines, termed F4, F8, and F16, respectively, also revealed relatively intact-appearing mitochondria, although distinguishable from F5-1 and F2 mitochondria by a more condensed or unorthodox cristae conformation. F4, F8, and F16 cell lines contained closed circular mitochondrial DNA in the same position as that of the parental F5-1 cells, when analyzed in an isopycnic CsCl-EB gradient. A small shoulder at the lower density side of the DNA I peaks was observed. The newly acquired drug resistance of the F cells is hereditarily transmitted to the progeny cells and retained even after a period of growth in EB-free medium.