Miscellaneous Gram-negative Bacilli Research Articles

Direct Disk Diffusion Susceptibility Testing for Gram-negative Bacteria from Blood Cultures: Diagnostic Accuracy and Impact on Antimicrobial Stewardship

BackgroundIn order to detect multidrug resistant (MDR) bacteria, our laboratory routinely performs direct susceptibility (DS) testing from positive blood cultures. We conducted a prospective study to determine the accuracy, reporting time (RT), and antimicrobial stewardship impact of DS testing for Gram-negative bacilli (GNB) positive blood cultures.MethodsFrom March – December 2016, first time positive blood cultures for GNB were included in the study. Broth from positive blood culture bottles was inoculated to standard media, as well as to Mueller–Hinton agar with cefoxitin (FOX), amoxicillin/clavulanic acid (AMC), ceftriaxone (CRO), ceftazidime (CAZ), ciprofloxacin (CIP) and meropenem (MEM) disks. The CRO and CAZ were adjacent to the AMC disk, which enabled detection of zone-enhancement with extended-spectrum β-lactamase (ESBL) producing organisms. CLSI breakpoints were used to guide interpretations of the DS results. Antibiotic therapy changes, made based on verbal reporting of DS results, were recorded. In order to determine RT, the following time points were recorded: blood culture positivity, reading of DS, and reporting of standardized susceptibilities (SS).ResultsThere were 105 unique, monomicrobial cultures consisting of: E. coli (N = 61), Klebsiella sp. (N = 15), Enterobacter sp. (N = 9), Proteus sp. (N = 5), Pseudomonas aeruginosa(N = 5), and 10 other miscellaneous GNB. RT was reduced from 38 to 22 hours, for SS and DS, respectively. For species with CLSI breakpoints (101 isolates), the major and minor errors for all antibiotics were 2% and 20%, respectively; 17% of isolates were DS-intermediate and SS-susceptible (minor error). CIP disk testing identified all resistant isolates correctly (N = 21), as did MEM (N = 7). Resistance to CRO/CAZ was correctly identified in 26/27 isolates. DS results changed antibiotic management for 23 patients. Antibiotics were narrowed for 7 patients, and treatment was expanded for 16 patients. For these patients, DS results were available 24 hours before SS.ConclusionDS testing is an accurate and rapid method to detect MDR GNB blood culture pathogens and facilitates the optimization of antimicrobial therapy. A relatively high rate of minor errors was detected due to DS disks testing in the intermediate zone for isolates ultimately identified as susceptible by SS.Disclosures All authors: No reported disclosures.

Biodegradative activities of some gram- negative bacilli isolated from kerosene treated soil grown with cowpea (Vigna unguiculata)

The biodegradative activities of three Gram negative bacilli (Aeromonas hydrophila, Vibro parahaemolyticus and Actinobacillus sp) isolated from soil contaminated with kerosene and planted with cowpea was investigated. The isolates were identified using MicrobactTM ID 24E system for the identification of Enterobacteriaceae and common miscellaneous Gram negative bacilli (MGNB). 2kg of soil collected from University of Ilorin was placed inside transparent, drilled buckets. Physicochemical properties of the soil were recorded. The soil was contaminated with kerosene at different concentrations of 0ml, 7ml, 14ml, 21ml, 56ml, 112ml, 168ml and 224ml. The setup was laid out in a randomized complete block design with three replicates. Cowpea seeds of the variety Samaru-40 were cultivated and observed for eight weeks. The results indicated that kerosene contamination of soils significantly affected the growth parameters: germination percentage, time of germination, rate of germination, plant height, number of leaves, leaf area and root nodules. A negative interaction existed between the level of the contaminant and the growth characteristics measured. Their biodegradative activities were studied and confirmed by the change in the Total Petroleum Hydrocarbon (TPH) using gravimetric method. The biodegradative abilities of the isolates were compared by measuring the optical densities, total viable count, pH and emulsification activity. The results showed that the organisms did better as a consortium rather than singly. Aeromonas hydrophila had the highest biodegradative activity followed by Vibro parahaemolyticus and then Actinobacillus sp. The study recommends the use of Aeromonas hydrophila and consortium for more effective biodegradation. Keywords: Bacterial Biodegradation, Soil, Cowpea, Kerosene, Total Petroleum Hydrocarbon

Control of Foodborne Pathogens during Sufu Fermentation and Aging

Control of the foodborne pathogens Escherichia coli O157:H7, Salmonella typhimurium, Staphylococcus aureus, and Listeria monocytogenes during sufu fermentation was evaluated. Before fermentation, pathogens were inoculated onto tofu (substrate for sufu) at 5 log cfu/g or 3 log cfu/g, and starter culture (Actinomucor elegans) was inoculated at 3 log cfu/g. After 2 days of fermentation at 30°C, the four pathogens reached 7 to 9 log cfu/g, and the mold count reached 6 to 7 log cfu/g. After fermentation, sufu samples were aged in a solution of 10% alcohol + 12% NaCl. After 1 month of aging, the total bacterial count was 6 to 7 log cfu/g, but all foodborne pathogens and mold were reduced to nondetectable levels. The total bacterial count decreased after aging for 2 months and 3 months, but the differences were not significant (P > 0.05) compared with the count after 1 month. Microorganism in experimental sufu from different aging periods and in commercial sufu were compared. A total of 270 isolates were purified and identified by the BBL Crystal Identification System. From the experimental sufu samples, 49 Bacillus spp. (20.4%), 167 Enterococcus spp. (69.6%), 6 Shewanella putrefaciens (2.4%), and 18 miscellaneous Gram-negative bacilli (7.5%) were identified. From commercial sufu samples, 17 Bacillus spp. (56.7%), 2 Enterococcus durans (6.7%), 5 miscellaneous Gram-negative bacilli (16.7%), 5 Corynbacterium aquaticum (16.7%), and 1 Shewanella putrefaciens (3.3%) were obtained. Although the longer aging period did not significantly decrease the total bacterial count, it may help in the development of sufu flavor. This study showed that sufu fermentation and aging can control common foodborne pathogens, so sufu is a safe product even though its preparation does not include pasteurization.

Control of Foodborne Pathogens during Sufu Fermentation and Aging

ABSTRACT: Referee: Dr. Catharina Y. W. Ang, National Center Toxicology Research, HFT-230, FDA, 3900 NCTR Road, Jefferson, AR 72079Control of the foodborne pathogens Escherichia coli O157:H7, Salmonella typhimurium, Staphylococcus aureus, and Listeria monocytogenes during sufu fermentation was evaluated. Before fermentation, pathogens were inoculated onto tofu (substrate for sufu) at 5 log cfu/g or 3 log cfu/g, and starter culture (Actinomucor elegans) was inoculated at 3 log cfu/g. After 2 days of fermentation at 30°C, the four pathogens reached 7 to 9 log cfu/g, and the mold count reached 6 to 7 log cfu/g. After fermentation, sufu samples were aged in a solution of 10% alcohol + 12% NaCl. After 1 month of aging, the total bacterial count was 6 to 7 log cfu/g, but all foodborne pathogens and mold were reduced to nondetectable levels. The total bacterial count decreased after aging for 2 months and 3 months, but the differences were not significant (P>0.05) compared with the count after 1 month. Microorganism in experimental sufu from different aging periods and in commercial sufu were compared. A total of 270 isolates were purified and identified by the BBL Crystal Identification System. From the experimental sufu samples, 49 Bacillus spp. (20.4%), 167 Enterococcus spp. (69.6%), 6 Shewanella putrefaciens (2.4%), and 18 miscellaneous Gram-negative bacilli (7.5%) were identified. From commercial sufu samples, 17 Bacillus. spp. (56.7%), 2 Enterococcus durans (6.7%), 5 miscellaneous Gram-negative bacilli (16.7%), 5 Corynbacterium aquaticum (16.7%), and 1 Shewanella putrefaciens (3.3%) were obtained. Although the longer aging period did not significantly decrease the total bacterial count, it may help in the development of sufu flavor. This study showed that sufu fermentation and aging can control common foodborne pathogens, so sufu is a safe product even though its preparation does not include pasteurization.

Microbial Identification by GLC

Laboratories offering comprehensive or specialized diagnostic microbiology services can complement other technology and enhance their capabilities for microbial identification by GLC. The main enticement of GLC is the versatility with which it can be applied. Except for the initial cost of capital equipment. expenses are modest. The specific applications employed by a given laboratory must be individualized, but GLC has considerable power as a tool in aiding the identification of nonfermentative and miscellaneous gram-negative bacilli, anaerobes, and mycobacteria. Applications that are more highly specialized—e.g., identification of un-usual or newly encountered pathogens, fastidious organisms, and atypical strains—are additional considerations for those laboratories engaged in such work.

Optimum use of selective plated media in primary processing of respiratory tract specimens from patients with cystic fibrosis.

A total of 258 respiratory tract specimens from patients with cystic fibrosis were inoculated onto nine different plated media, and the rates of recovery of potential pathogens were compared. Media included sheep blood agar, enriched chocolate agar, MacConkey agar for gram-negative bacilli, chocolate agar containing bacitracin for Haemophilus spp., bromcresol green agar for yeasts, cetrimide agar for Pseudomonas spp., sheep blood agar containing colistin and nalidixic acid for gram-positive cocci, mannitol salt agar for Staphylococcus aureus, and oxidation-fermentation agar containing 300 U of polymyxin B per ml and 2 U of bacitracin per ml (OF-PBL medium) for Pseudomonas cepacia. With two exceptions, all of these media proved useful in recovering potential pathogens from respiratory tract specimens from patients with cystic fibrosis. The two exceptions were cetrimide agar and colistin-nalidixic acid-supplemented sheep blood agar, which were found to be superfluous. In addition, the results of this study further delineated the prevalence of selected bacteria and fungi in respiratory tract secretions from patients with cystic fibrosis. In rank order of frequency of isolation, we recovered isolates of Pseudomonas aeruginosa, Haemophilus parainfluenzae, Candida albicans, S. aureus, Haemophilus influenzae, molds, members of the family Enterobacteriaceae, yeasts other than Candida albicans, miscellaneous gram-negative bacilli, beta-hemolytic streptococci, P. cepacia, and Streptococcus pneumoniae.

Critical assessment of blood culture techniques: analysis of recovery of obligate and facultative anaerobes, strict aerobic bacteria, and fungi in aerobic and anaerobic blood culture bottles.

Recent reports have documented a decrease in anaerobic bacteremias and have questioned the need for routine anaerobic blood cultures. At the same time, we and others have noted an increase in fungal bloodstream infections. In this two-part study, we first compared recoveries of obligate anaerobic bacteria with those of fungi over a 13-year period and then examined the recoveries of all bacteria and fungi in aerobic and anaerobic blood culture bottles during a 12-month period. During the 13-year period, the number of patients with anaerobic bacteremia remained relatively constant (average, 39 patients per year), while the incidence of fungemia steadily increased, from 12 patients in 1978 to 117 patients in 1990. Of the 1,090 anaerobic isolates, 55.1 and 90.2% were recovered in aerobic and anaerobic bottles, respectively, compared with 98.6 and 37.0% of the 2,582 fungi. During the 12-month period of evaluation, 2,980 bacteria and fungi were recovered in cultures collected from 1,555 patients. Overall, 21.1% more organisms were recovered in aerobic bottles than in anaerobic bottles, including significantly more Staphylococcus species; gram-positive aerobic bacilli; Escherichia, Enterobacter, Pseudomonas, Xanthomonas, and Acinetobacter species; miscellaneous gram-negative bacilli; and yeasts. Only anaerobic gram-negative bacilli and non-spore-forming gram-positive bacilli were isolated more commonly in anaerobic bottles. These data support the concepts that bacteremia caused by obligate anaerobic bacteria is decreasing relative to sepsis caused by other bacteria and fungi and that the routine use of unvented anaerobic blood culture bottles reduces the recovery of common aerobic bloodstream pathogens.

Efficacy of intravenous ofloxacin: a French multicentre trial in 185 patients

The efficacy of intravenous ofloxacin therapy (200 mg 12-hourly) followed, when appropriate, by oral administration of the same dose was evaluated in an open multicentre trial involving 185 patients in 31 French hospitals. Dosage adjustment was made for patients in renal failure. Infection was hospital-acquired in 35 cases, 53 patients required admission to an intensive care unit. The infections comprised septicaemia (n = 56), pneumonia (n = 18), bronchitis (n = 10), urinary tract (n = 78), female pelvis (n = 8), bone and joint (n = 5), skin and soft tissues (n = 10). The causative pathogens were: Staphylococcus spp. (n = 23), Streptococcus spp. (n = 11), Escherichia coli (n = 85), Haemophilus influenzae (n = 9), Klebsiella, Enterobacter or Serratia spp. (n = 21), Salmonella spp. (n = 22), Chlamydia spp. (n = 3), Legionella spp. (n = 1), Mycoplasma pneumoniae (n = 1) and miscellaneous Gram-negative bacilli (n = 17). All were ofloxacin-susceptible. Mean duration of therapy was 8.06 ( +/- 2.6) days for the i.v. and 14.8 ( +/- 14.39) days for the oral preparation. Clinical cure was achieved in 173 patients (93.5%). It is concluded that iv ofloxacin is an effective treatment for a range of infections due to susceptible organisms.

Sensitivity tests with cephalosporins.

The sensitivity of 127 strains of miscellaneous Gram-negative bacilli to various cephalosporins was determined by three methods. Disc tests with cephaloridine (CLOR), cephalothin (CTIN) and cephalexin (CLEX) made most strains of beta-lactamase-producing Escherichia coli appear sensitive. Eight cephalosporins were tested by the agar break-point method using concentrations of 16 and 4 mg/l for all except cefotaxime (CTAX), when 8 and 1 mg/l were used. In repeated tests there was considerable variation in the response of many strains to the lower concentration of CLOR, CTIN, CLEX and to a lesser extent, cefoxitin (CFOX) and cefuroxime (CROX), and beta-lactamase-producing E coli appeared as sensitive or moderately sensitive to all. Cephalosporins CLOR, CLEX, and CROX were then tested by inoculating 2 ml volumes of broth containing a 30 microgram disc; all the beta-lactamase-producing E coli grew in CLOR and CLEX but were inhibited by CROX.

A diagnostic schema for identifying enterobacteriaceae and miscellaneous gram-negative bacilli.

A diagnostic schema for identifying enterobacteriaceae and miscellaneous gram-negative bacilli.

LOCAL CHEMOPROPHYLAXIS FOR BURNS WITH GENTAMICIN AND OTHER AGENTS

In a series of controlled prophylactic trials of antibacterial creams on burns of less than 30%, a cream containing 0.5% silver nitrate had a significant prophylactic action against Pseudomonas aeruginosa when compared with an inactive control cream (containing penicillin), but no significantly greater prophylaxis was obtained against Ps. aeruginosa by the addition of chlorhexidine gluconate (0.2%) or of gentamicin sulphate (0.1%) to the silver-nitrate cream; gentamicin cream was as active in prophylaxis against Ps. aeruginosa as silver-nitrate/gentamicin cream. Prophylaxis against Proteus spp. followed a similar pattern, but the addition of gentamicin significantly added to the prophylactic value against Proteus spp. of the cream containing silver nitrate. Unlike these organisms, Staphylococcus aureus was not acquired less often by burns treated with silver-nitrate cream than by those treated with penicillin cream; significant prophylactic effects against Staph. aureus were, however, obtained by the addition of gentamicin and (to a smaller degree) of chlorhexidine to the silver-nitrate cream. Both silver nitrate and gentamicin seemed to have a prophylactic effect against miscellaneous gram-negative bacilli, but this did not quite reach statistical significance. The proportion of swabs that yielded no bacterial growth was significantly greater in the series taken from burns treated with silver-nitrate cream (26%) than from those treated with penicillin cream (4.4%), and in the series from burns treated with silver-nitrate/gentamicin and gentamicin creams (62% and 54% respectively) than in the series from burns treated with silver-nitrate cream. There was no significant emergence of gentamicin or silver-nitrate-resistant bacteria in burns during these trials.