Colistin-nalidixic Acid Agar Research Articles

Processing of Positive Blood Cultures Using a Total Laboratory Automation System: Workflow and Clinical Validation

Abstract Automated systems for culture-based microbiology are in the early phase of implementation in clinical laboratories. Here, our objective was to evaluate the performance of the BD Kiestra Total Laboratory Automation (TLA) System for inoculation, incubation, and imaging of positive blood culture broth specimens. To optimize parameters for clinical testing, 56 clinical specimens were processed using both TLA and manual standard-of-care (SOC) methods. For TLA processing, 3 mL positive blood culture broth (35 VersaTREK: 19 aerobic, 16 anaerobic; 21 BD BACTEC: 15 aerobic, 6 anaerobic) was transferred to a no-additive vacutainer using a safety adapter and syringe. This aliquot was then placed on TLA for fully automated processing: 10 µL was inoculated to blood, chocolate, and MacConkey agar (Remel) and, for anaerobic bottles only, Brucella blood agar (Hardy Diagnostics). Kiestra cross-streak pattern 5 was optimal for obtaining isolated colonies and was superior to quadrant-streaking methods. Additional media types were added based on Gram stain results of the positive blood specimen: CandiSelect (Bio-Rad) and Sabouraud Dextrose (Remel) were added if yeast were identified, and Colistin Nalidixic Acid agar (Remel) for Gram stains with mixed Gram-positive and Gram-negative morphology. Plates were imaged at 6, 8, 10, 12, 18, 38, and 62 hours. Anaerobic media were placed by the system in a media stacker, incubated off-line, and then imaged on the TLA at 24, 48, and 72 hours. SOC cultures were evaluated after overnight incubation and on days 2 and 3. Organism identification was performed using MALDI-TOF MS (Bruker). Based on our evaluation, optimal parameters for clinical implementation of TLA were identified. Microbial growth was scant at 6 hours of incubation, but by 8 hours, small discrete colonies were observed for most aerobes. Thus, imaging parameters selected for routine clinical testing were 8, 18, and 38 hours for aerobic media and one image taken at 38 hours for anaerobic media. TLA and SOC culture results had 96% agreement (29 Gram positive, 12 Gram negative, 5 mixed, 6 yeast, 1 no growth). Two specimens with a second low-abundance bacterial population were observed on TLA that were not observed with SOC. Reproducibility of TLA was tested by processing 6 positive samples in triplicate and found to be 100%. There was no carryover or contamination noted between specimens processed simultaneously on TLA. To evaluate if implementation of TLA impacted epidemiology of positive blood cultures, we compared the 10 most common organisms recovered pre- and post-TLA implementation (August-November 2017 compared to August-November 2018). We found these organisms were not significantly impacted by TLA processing. In conclusion, automated processing of positive blood cultures improves laboratory workflows and facilitates faster workup at 8 hours of incubation. These results demonstrate that automation is a viable avenue for the processing of positive blood cultures.

Effects of Solid-Medium Type on Routine Identification of Bacterial Isolates by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a rapid method for the identification of bacteria. Factors that may alter protein profiles, including growth conditions and presence of exogenous substances, could hinder identification. Bacterial isolates identified by conventional methods were grown on various media and identified using the MALDI Biotyper (Bruker Daltonics, Billerica, MA) using a direct smear method and an acid extraction method. Specimens included 23 Pseudomonas isolates grown on blood agar, Pseudocel (CET), and MacConkey agar (MAC); 20 Staphylococcus isolates grown on blood agar, colistin-nalidixic acid agar (CNA), and mannitol salt agar (MSA); and 25 enteric isolates grown on blood agar, xylose lysine deoxycholate agar (XLD), Hektoen enteric agar (HE), salmonella-shigella agar (SS), and MAC. For Pseudomonas spp., the identification rate to genus using the direct method was 83% from blood, 78% from MAC, and 94% from CET. For Staphylococcus isolates, the identification rate to genus using the direct method was 95% from blood, 75% from CNA, and 95% from MSA. For enteric isolates, the identification rate to genus using the direct method was 100% from blood, 100% from MAC, 100% from XLD, 92% from HE, and 87% from SS. Extraction enhanced identification rates. The direct method of MALDI-TOF analysis of bacteria from selective and differential media yields identifications of varied confidence. Notably, Staphylococci spp. from CNA exhibit low identification rates. Extraction enhances identification rates and is recommended for colonies from this medium.

Comparison of selective and nonselective enrichment broth media for the detection of vaginal and anorectal colonization with group B streptococcus

The use of a selective enrichment broth medium has been widely recommended to optimize the recovery of group B streptococci (GBS) from genital and anorectal samples. Because selective antibiotic-containing versions of broth media are significantly more expensive than their nonselective parent formulations, we sought to examine whether the use of the nonselective Todd-Hewitt broth (THB) could accomplish comparable recovery of GBS to the recommended, selective version of this medium (Lim broth). During the study, vaginal and anorectal swab samples submitted to our laboratory for GBS culture were all inoculated onto Columbia colistin–nalidixic acid agar (CNA) and into either THB or Lim broth (alternated on a weekly basis). During the 45-week study period, 1200 samples (600 in each study arm) were evaluated. GBS were recovered from 164 samples (27.3%) in the Lim arm of the study, of which 60 (36.7%) were positive only on subculture of broths. In the THB study arm, 161 samples (26.8%) yielded GBS, of which 58 (36.0%) were positive only in broths. This study conclusively demonstrates that a nonselective enrichment broth media provides comparable sensitivity to the recommended selective broth for detection of GBS colonization during pregnancy.

An evidential example of airborne bacteria in a crowded, underground public concourse in Tokyo

Abstract We examined airborne bacteria in an underground concourse in Tokyo and investigated conditions that influenced bacterial counts. Airborne bacteria were collected by using an impactor sampler. Colonies on plate count agar (PCA) and Columbia colistin–nalidixic acid agar with 5% sheep blood (CNA agar) were enumerated. The range, geometric mean, and 95% CI of the bacterial counts (CFU m−3) on PCA and CNA agar were 150–1380, 456, 382–550 and 50–990, 237, 182–309, respectively. Bacterial counts on PCA significantly correlated with number of the pedestrians (r=0.89), relative humidity (r=0.70) and airborne dust (PM5.0) (r=0.73). Results of a multiple regression indicated independent positive association between the number of pedestrians and bacterial counts on PCA (p

Corynebacterium nigricans sp. nov.: proposed name for a black-pigmented Corynebacterium species recovered from the human female urogenital tract.

Six independent isolates of an unusual black-pigmented Corynebacterium species (strains CN-1, CN-2, CN-3415, W70124, 91-0032, and 92-0360) were recovered from the human female urogenital tract. Four of the six source patients had complications of pregnancy, including spontaneous abortion, preterm labor, and low amniotic fluid volume at the time of the pathogen isolation. One isolate was recovered from a vaginal ulcer. All six strains yielded black-pigmented colonies on sheep blood agar, chocolate agar, and colistin-nalidixic acid agar after 24 to 48 h of incubation at 35 degrees C. The dry, adherent colonies pitted the agar surface. The cells were coccobacillary to rod-shaped, catalase positive, nonmotile, and nonlipophilic. Only five of six isolates were available for characterization. Biochemical and chemotaxonomic studies revealed that the strains belong to the genus Corynebacterium but differ from known corynebacterial species. Comparative 16S rRNA gene sequence analysis showed that the strains are closely related and form a new subline within the genus Corynebacterium. We propose the name Corynebacterium nigricans sp. nov. for this group of coryneforms. The type strain of Corynebacterium nigricans is CN-1. It is deposited in the American Type Culture Collection (assigned strain number ATCC 700975) and in the Institute Pasteur collection (assigned strain number CIP 107346).

Occurrence of the vancomycin-resistant genes vanA, vanB, vanC1, vanC2 and vanC3 in Enterococcus strains isolated from poultry and pork

It is suspected that the use of avoparcin as a feeding antibiotic for the fat stock contributes to development of cross-resistance against vancomycin and teicoplanin. After isolating enterococci strains from poultry and pork meat by cultivation on citrate azide Tween carbonate agar (CATC) and screening the vancomycin resistance on Columbia colistin nalidixic acid agar (CNA, supplemented with 5% sheepblood and 5 mg vancomycin/l) the polymerase chain reaction (PCR) was used for the detection of the vancomycin resistance genes vanA (‘high level’), vanB (‘moderate high level’), vanC1, vanC2 and vanC3 (‘low level’). Out of 1643 E.-isolates from 115 poultry and 50 pork samples, 420 isolates could be identified as vancomycin resistant, 202 isolates of which carry the vanA, one isolate both the vanA and the vanC1, 38 isolates the vanC1, 14 isolates the vanC2, nine isolates both the vanC1 and the vanC3 gene and 156 isolates carry no gene. The vanB gene was not found in these isolates. Comparing vanA-positive food isolates with those from different human sources by means of the pulsed field gel electrophoresis (PFGE) it could clearly be demonstrated that they do not show homological fingerprints according to the source of origin. It is therefore unlikely that there is a close genetic relationship between isolates from animal foodstuff and humans.

Screening Pregnant Women for Group B Streptococcal Colonization

The recovery rates of group B streptococcus (GBS) from anorectal swabs (RS) and vaginal swabs (VS) that were enriched were compared to the routine method to determine the optimal procedure. Separate RS and VS were collected from women attending antenatal clinics. RS and VS were placed in 2 ml enrichment and selective broth. Swabs were inoculated onto colistin/nalidixic acid agar (CNA) upon arrival in the laboratory and onto 5% sheep blood agar (SBA) and CNA after 24 h enrichment. The routine method consisted of a VS sent in transport medium and inoculated in the laboratory onto SBA (no enrichment). The overall GBS colonization rate was 24% (64/264). Of the 64 GBS carriers, 77% were colonized in the vagina and 89% were colonized in the anorectum. The anorectum was the only site of colonization in 24% of the women, whereas the vagina was the only site of colonization in 11% of cases. Enrichment increased the detection of GBS from both RS (55 versus 42; P < 0.025) and from VS (49 versus 27; P < 0.001). Of the 64 cases, enriched RS detected 86%, enriched VS detected 77% and the standard VS detected only 41%. Enriched RS and enriched VS collectively detected 99% of cases. SBA was better than CNA for subculture of the enrichment broth because of a higher recovery rate (98–100% versus 80–82%; P < 0.01) and the fact that the hemolysis on SBA made it easier to differentiate GBS from enterococci. The data confirm that optimal screening of pregnant women for GBS should include a combined RS/VS swab placed in enrichment broth that is then subcultured onto SBA after 24 h incubation.

Screening pregnant women for group B streptococcal colonization.

The recovery rates of group B streptococcus (GBS) from anorectal swabs (RS) and vaginal swabs (VS) that were enriched were compared to the routine method to determine the optimal procedure. Separate RS and VS were collected from women attending antenatal clinics. RS and VS were placed in 2 ml enrichment and selective broth. Swabs were inoculated onto colistin/nalidixic acid agar (CNA) upon arrival in the laboratory and onto 5% sheep blood agar (SBA) and CNA after 24 h enrichment. The routine method consisted of a VS sent in transport medium and inoculated in the laboratory onto SBA (no enrichment). The overall GBS colonization rate was 24% (64/264). Of the 64 GBS carriers, 77% were colonized in the vagina and 89% were colonized in the anorectum. The anorectum was the only site of colonization in 24% of the women, whereas the vagina was the only site of colonization in 11% of cases. Enrichment increased the detection of GBS from both RS (55 versus 42; P < 0.025) and from VS (49 versus 27; P < 0.001). Of the 64 cases, enriched RS detected 86%, enriched VS detected 77% and the standard VS detected only 41%. Enriched RS and enriched VS collectively detected 99% of cases. SBA was better than CNA for subculture of the enrichment broth because of a higher recovery rate (98-100% versus 80-82%; P < 0.01) and the fact that the hemolysis on SBA made it easier to differentiate GBS from enterococci. The data confirm that optimal screening of pregnant women for GBS should include a combined RS/VS swab placed in enrichment broth that is then subcultured onto SBA after 24 h incubation.

Oral staphylococcus in older subjects with rheumatoid arthritis.

To determine if Staphylococcus aureus and Staphylococcus epidermidis, etiologic bacterial agents to late prosthetic joint infections (LPJI), are more prevalent in the oral flora of older individuals with rheumatoid arthritis (RA) than in an age and gender-matched nonarthritic control population (NA). Cultures were obtained from the nares, oropharynx, saliva, tongue, and gingival crevice, and the results were compared between older patients with RA and controls. University of Michigan Medical Center, Ann Arbor, VA Medical Center, and University of Michigan School of Dentistry. A total of 111 community-dwelling subjects with a diagnosis of RA and 83 gender-matched control subjects. Colistin nalidixic acid agar plates with 5% sheep's blood were inoculated and incubated. Isolates were speciated using the API Staph Trac micro method and catalase and coagulase tests. Individuals with RA had a higher prevalence of S. aureus isolated from the oral cavity. However, only the oropharynx and tongue revealed higher rates; all other sites were insignificant. The presence of oral S. aureus was associated with xerostomia. Staphylococcus epidermidis was not detected from any of the oral sites sampled. Sixty-two percent (10/16) of the S. aureus isolates from the RA subjects were resistant to penicillin and ampicillin, whereas none were resistant to a cephalosporin. These findings suggest that rheumatoid arthritis may be a risk factor for LPJI in older prosthetic joint patients undergoing invasive dental procedure in the posterior oral cavity. This increased risk is caused, in part, by a higher prevalence of S. aureus in the posterior oral cavity. The prevalence and the antibiotic resistance of S. aureus must be considered when determining the need for chemoprophylaxis.

Performance of a new DNA probe for the detection of group B streptococcal colonization of the genital tract

To test the performance characteristics of a new DNA probe designed for the rapid identification of heavy colonization of the genital tract with group B streptococci. Vaginal and combined vaginal-perianal samples were collected from 193 pregnant women and cultured on colistin-nalidixic acid agar plates. Bacterial growth was classified semiquantitatively. Specimens were also tested by a new DNA probe in two formats: a direct assay performed on the swabs soon after collection and an assay performed after the swabs were incubated for 24 hours in an enriched culture medium. The agar cultures were positive in 36 of 193 patients (18.6%, 95% confidence interval 13.2–24). Nineteen women were lightly colonized, and 17 were heavily colonized (at least 10 4 colonies/mL). The combined vaginal-perianal swabs yielded positive results more often than the vaginal swabs alone (26 versus 16, χ 2 = 24, P < .01). In its direct form, the assay had only 8.3% sensitivity in identifying colonized women. In heavily colonized women, the sensitivity of the assay increased slightly to 12%. After a 16–24-hour incubation, the sensitivity of the assay was 81%. The direct assay is insufficiently sensitive for clinical use. The delayed assay offers no advantage over standard cultures.

Recovery of vancomycin-resistant gram-positive cocci from children

A cross-sectional survey of vancomycin-resistant gram-positive cocci (VRGPC) in the feces of children was initiated after several bacteremic infections with these organisms occurred at our hospital. A selective medium consisting of colistin-nalidixic acid agar, 5% sheep blood, vancomycin (5 mg/liter), and amphotericin B (8 mg/liter) was developed to isolate VRGPC. A single stool specimen submitted to the clinical microbiology laboratory from each of 48 patients was inoculated onto the medium. Plates were incubated at 35 degrees C with 5% carbon dioxide and examined at 24, 48, and 72 h. Susceptibilities were determined by broth microdilution. A total of 14 isolates from 11 of 48 (22%) children were recovered. The density of growth ranged from a single colony to 2+. The VRGPC were identified as Leuconostoc lactis (n = 2), Lactobacillus confusus (n = 4), Enterococcus species (n = 5), and Lactococcus lactis (n = 3). One strain of Lactobacillus confusus was recovered from both the stool and the blood of one of these patients. The MICs of vancomycin were 4 micrograms/ml for one of the isolates, 8 micrograms/ml for four of the isolates, and more than 16 micrograms/ml for the remaining eight isolates. All isolates were susceptible to both penicillin and ampicillin. Only 1 of the 11 children had received prior treatment with vancomycin. We conclude that low concentrations of VRGPC may be common in the gastrointestinal tracts of children.

Growth of coagulase-negative staphylococci on colistin-nalidixic acid agar and susceptibility to polymyxins.

Colistin-nalidixic acid agar, although recently recommended as a replacement for blood agar for primary plating of urine specimens ( Fung et al., J. Clin. Microbiol. 16:632-636, 1982), has also been reported to suppress the growth of some strains of staphylococci that are susceptible to colistin (polymyxin E). The susceptibility of 11 species of staphylococci to polymyxins was determined, and the ability of these species to grow on colistin-nalidixic acid agar was examined. Although the MICs for most of the strains tested were 8 micrograms/ml or less, only a few coagulase-negative staphylococci grew on or were inhibited by colistin-nalidixic acid agar. This descrepancy was explained by the antagonistic effects that medium components, such as physiological concentrations of magnesium and calcium and 5% sheep blood, had on the activity of polymyxin. Colistin-nalidixic acid agar is still recommended for routine urine processing; however, the poor growth of 13% of the Staphylococcus saprophyticus strains tested suggests that blood agar should be included in the primary plating battery of urine specimens obtained from female outpatients.

Primary culture media for routine urine processing.

It has been recommended that routine microbiological processing of urine specimens include quantitative plating onto blood agar medium along with a selective and differential agar such as MacConkey agar for gram-negative organisms. Few data have been published to justify this combination. To evaluate the validity of this recommendation 2,553 midstream, clean-voided urine samples were quantitatively plated onto blood agar, MacConkey agar, and colistin-nalidixic acid agar, which is a selective medium for gram-positive organisms. The amounts of growth on each of the three media were compared. Results indicated that the best medium combination was colistin-nalidixic acid agar and MacConkey agar. The use of colistin-nalidixic acid agar instead of blood agar increased the detection of significant growth of enterococci, lactobacilli, and Torulopsis glabrata.

Growth of Thermoactinomyces

<h3>To the Editor.—</h3> In response to inquiries concerning the finding of<i>Thermoactinomyces</i>in environmental samples (236:344, 1976), I wish to answer two questions that were frequently asked. To grow these bacteria, use tryptic soy agar or Columbia colistin nalidixic acid agar medium. After spreading the sample on the surface of the agar, slip the Petri dish into a plastic bag with a tightly closed top to prevent drying of the agar. Incubate at least one day at 55°C. Colonies of Thermoactinomyces are chalky white and are easily recognized. A Gram stain will show refractile, round endospores when viewed microscopically at a magnification of 1,000. For a recent clinical report on hypersensitivity pneumonitis caused by Thermoactinomyces, I recommend the report by Fink and colleagues in the Annals of Internal Medicine (84:406-413, 1976).