Biomedical subjects

P C Kohner

Publications and source records attributed to P C Kohner.

10 recordsLinked to original sources

PubMed2000

Comparison of protein A gene sequencing with pulsed-field gel electrophoresis and epidemiologic data for molecular typing of methicillin-resistant Staphylococcus aureus.

The epidemiologic relatedness of methicillin-resistant Staphylococcus aureus (MRSA) isolates is currently determined by analysis of chromosomal DNA restriction patterns by pulsed-field gel electrophoresis (PFGE). We have evaluated an alternative typing system (MicroSeq StaphTrack Kit; Perkin-Elmer Biosystems) based on the sequence analysis of the chromosomally encoded polymorphic repeat X region of the S. aureus protein A (spa) gene. A total of 69 clinical MRSA isolates were divided into 18 groups according to the number and nucleotide sequences of the spa repeats. Molecular typing results obtained both by spa sequencing and from the PFGE patterns were concordant except for one group, which contained 20 isolates recovered over a 2-year period from hospitalized patients at the Mayo Clinic. Although the spa typing patterns were indistinguishable for those isolates, PFGE analysis yielded seven related but distinguishable patterns. Further coagulase gene sequence analysis subtyped those 20 strains into four groups which followed distinct temporal and geographic distributions. During a 2-year epidemic period there were up to 7 fragment changes in PFGE patterns among epidemiologically related isolates, suggesting that PFGE may be unsuitable for long-term typing of strains involved in epidemics. Although more limited than PFGE in discriminatory power, spa sequencing analysis could be used as a screening method for typing of MRSA strains because of the shorter turnaround time, ease of use, and the inherent advantages of sequence analysis, storage, and sharing of information.

Animals↗

PubMed1998

Molecular, serological, and clinical features of 16 consecutive cases of invasive streptococcal disease. Southeastern Minnesota Streptococcal Working Group.

We performed a comprehensive analysis of the molecular, serological, and clinical features of 16 consecutive cases of invasive streptococcal disease (ISD). The majority of cases were linked to two group A streptococcus (GAS) clones closely related by pulsed-field gel electrophoresis (PFGE) and designated as PFGE-1 and PFGE-1.1. These clones, serotyped as M-3, T-3/B3264, carried an allelic variant of the gene that encodes pyrogenic exotoxin A (speA3) and the gene that encodes streptococcal superantigen (SSA) but different emm alleles that encode M-protein. The characteristics and clinical features of patients were similar to those described in previous reports, regardless of the responsible GAS clone. However, worse clinical outcomes (shock and death) were more frequent when patients infected with PFGE1/1.1 clones were considered as a group and compared with all other patients as a group. One striking feature in some patients with deep tissue infection was a lack of inflammatory cells despite the presence of numerous streptococci. An evaluation of PFGE profiles of GAS isolated elsewhere demonstrated that the PFGE-1 clone has caused invasive disease in other locations in the United States and in Japan.

Adult↗

PubMed1997

An outbreak of invasive group A streptococcal disease associated with high carriage rates of the invasive clone among school-aged children.

OBJECTIVES: To determine if a common strain of group A streptococcus was responsible for an outbreak of invasive streptococcal disease in southeastern Minnesota and to determine whether this strain was prevalent among residents of this area during the outbreak who either had streptococcal pharyngitis or were asymptomatic streptococcal carriers. DESIGN: Pharyngeal culture survey and case-contact evaluation. SETTING: Three adjacent counties in southeastern Minnesota defined as the outbreak area. Outbreak period, January 1 through March 31, 1995. PATIENTS: Seven patients with invasive streptococcal infection, 1249 patients (adults and children) with sore throat who resided in the outbreak area, children from an elementary school located in 1 community where the majority of invasive cases occurred, and 896 students from 3 schools located in Minnesota counties outside the outbreak area. MEASUREMENTS: Pulsed-field gel electrophoresis (DNA fingerprinting) of group A streptococcal isolates obtained from patients with invasive disease, throat swabs of patients with sore throat, and throat swabs of asymptomatic school-aged children. RESULTS: All patients with outbreak-associated invasive disease had group A streptococcal isolates that were indistinguishable by pulsed-field gel electrophoresis. Additional testing showed that these isolates carried significant virulence factors including pyrogenic exotoxin A and streptococcal superantigen. Five of these 7 patients with invasive disease had underlying medical conditions; 4 developed toxic shock syndrome and died (case fatality, 57%). The outbreak-associated group A streptococcal clone was found in 69 (26.5%) of the 260 patients with sore throat from whom group A streptococcus was isolated. The frequency of the outbreak clone among pharyngeal carriers from the 3 schools outside the outbreak area was significantly less (range, 0%-10%) than in children from the school in the outbreak area (78%; relative risk, 29; 95% confidence interval, 11.1-78.1; P<.001). Four of the 7 patients with outbreak-associated disease had contact with children who attended the school in the outbreak area. CONCLUSIONS: A single clone of group A streptococcus was responsible for 7 cases of invasive streptococcal disease during an outbreak in Minnesota and for a significant number of pharyngitis cases that also occurred during the outbreak. Invasive disease occurred most frequently in persons with underlying medical conditions. This outbreak was also associated with increased carriage rates of the invasive streptococcal clone among community school-aged children. Cases of invasive group A streptococcal infection may therefore reflect the tip of the iceberg with regard to the burden of colonization of a specific invasive streptococcal clone in a community.

Adult↗

PubMed1997

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.

Agar↗

PubMed1997

Comparison of agar dilution, broth microdilution, disk diffusion, E-test, and BACTEC radiometric methods for antimicrobial susceptibility testing of clinical isolates of the Nocardia asteroides complex.

An evaluation was undertaken to determine the optimal method for the in vitro susceptibility testing of 26 Nocardia asteroides complex isolates to the following antimicrobial agents: amikacin, ampicillin, amoxicillin-clavulanate, ceftriaxone, ciprofloxacin, erythromycin, imipenem, minocycline, and trimethoprim-sulfamethoxazole. Five testing methods were studied including the agar dilution, broth microdilution, and disk diffusion methods, the epsilometer test (E-test), and the BACTEC radiometric method. Results for each antimicrobial agent and each testing method were interpreted as indicating susceptibility, intermediate susceptibility, or resistance according to current guidelines of the National Committee for Clinical Laboratory Standards (NCCLS) for bacteria that grow aerobically and were then compared to a "gold standard" susceptibility test result. The gold standard result for each Nocardia isolate was established by a consensus of the results of the majority of testing methods used in the study. When the results were combined for all antimicrobial agents tested against all Nocardia isolates by all methods, the BACTEC radiometric method produced the highest level of agreement (97.9%) with the consensus results and had the fewest very major (n = 1), major (n = 2), and minor (n = 2) errors. In contrast, the results of the agar dilution method were in least agreement (93.2%) with the consensus results, and this method also produced the most very major (n = 8), major (n = 4), and, along with the disk diffusion method, minor (n = 6) errors. For all test methods, interpretive errors were most frequent when testing ampicillin or amoxicillin-clavulanate. Moreover, for all Nocardia nova isolates tested, ampicillin susceptibility results by any of the testing methods were not in agreement with the results of testing for beta-lactamase by the nitrocefin (Cefinase) disk method. We conclude that among the methods evaluated, the BACTEC radiometric method appeared to be the best for determining the in vitro susceptibilities of members of the N. asteroides complex to a panel of nine antimicrobial agents. However, none of the test methods, including the BACTEC method, accurately predicted the ampicillin resistance of the N. nova isolates tested, all of which produced beta-lactamase. Presuming that this beta-lactamase hydrolyzes ampicillin, this disparity may relate to the NCCLS breakpoints that were used, which may require modification for this antimicrobial agent when tested against N. nova isolates.

Drug Resistance, Microbial↗

PubMed1995

Comparison of in-house and commercially prepared haemophilus test media for disk diffusion testing of ampicillin against Haemophilus species.

The performance characteristics of two lots of Haemophilus Test Medium (HTM) prepared in-house at Mayo Clinic, one lot obtained from BBL (Cockeysville, Md.) and one lot obtained from Remel (Lenexa, Kans.), for disk diffusion susceptibility testing of ampicillin against 81 Haemophilus strains were assessed. Insufficient growth occurred in 2.5 to 13.6% of strains, depending on the HTM used, and was most frequent for beta-lactamase-negative, ampicillin-resistant strains when they were tested with one lot of HTM prepared in-house. Results for all 14 beta-lactamase-positive Haemophilus strains were in complete agreement for all lots of HTM when either 1990 or 1993 National Committee for Clinical Laboratory Standards (NCCLS) interpretive guidelines were followed. However, these beta-lactamase-positive Haemophilus strains could be as precisely, but more rapidly, identified by the cefinase disk method. Results for 56 beta-lactamase-negative, ampicillin-susceptible strains and 11 beta-lactamase-negative, ampicillin-resistant strains varied significantly among all HTM lots tested and as to whether 1990 or 1993 NCCLS interpretive guidelines were followed. We conclude that in our hands disk diffusion testing with HTM prepared in-house or obtained from two commercial sources and following either 1990 or 1993 NCCLS guidelines is an unacceptable method for determining the susceptibilities of Haemophilus strains to ampicillin.

Ampicillin↗

PubMed1994

Comparison of agar dilution, broth dilution, and disk diffusion testing of ampicillin against Haemophilus species by using in-house and commercially prepared media.

An evaluation to determine the optimal method for the in vitro susceptibility testing of Haemophilus strains to ampicillin was undertaken. In our hands, in-house-prepared Haemophilus Test Medium used by either the broth macrodilution or agar dilution method produced the most consistent results, especially for beta-lactamase-negative, ampicillin-resistant H. influenzae strains.

Agar↗

PubMed1978

Effect of cation content of agar on the activity of gentamicin, tobramycin, and amikacin against Pseudomonas aeruginosa.

Fifty-five strains of Pseudomonas aeruginosa were tested against arithmetic increments in concentrations of gentamicin, tobramycin, and amikacin in 14 different lots of Mueller-Hinton agar. The divalent cation content of each lot was determined by atomic absorption spectrometry. The relation between mean minimal inhibitory concentration (MIC) for strains within each lot and cation content was studied by stepwise regression. Among the cations, the content of Zn++ most highly correlated with the MIC of each aminoglycoside; however, Zn++ accounted for only 23%, 60%, and 47% of the variability in the mean MIC of gentamicin, tobramycin, and amikacin, respectively, against all strains. In two-cation models Zn++ with Ca++ or Cu++ was most highly correlated with the mean MICs of the three aminoglycosides against all strains. No divalent cation, either singly or in combination with one or two other cations, gave a good prediction of the MICs of the aminoglycosides in agar. Furthermore, there was variability in the cations that most highly correlated with the MICs for some strains. These observations support the concept that ionic strength, cations, and a variety of other as yet poorly defined components of media influence the activity of aminoglycosides against P. aeruginosa.

Agar↗

PubMed1976

Spurious ampicillin resistance by testing Haemophilus influenzae with agar containing supplement C.

Ampicillin resistance (minimal inhibitory concentration >/=10 mug/ml) in the absence of beta-lactamase activity by Haemophilus influenzae was noted in tests performed with Mueller-Hinton agar containing one lot of supplement C. All strains, except five with known resistance due to beta-lactamase activity, were inhibited by 0.6 mug or less of ampicillin per ml of chocolatized blood agar.

Ampicillin↗

PubMed1976

Analysis of certain variables in the agar dilution susceptibility test.

This study examined (i) the activity of gentamicin added to agar in ratios of 1:100 and 1:1,000 to produce a final concentration of 1 mug/ml, (ii) the uniformity of distribution of gentamicin in agar in relation to the amount of mixing, and (iii) the effect of agar depth on the activity of gentamicin against Pseudomonas aeruginosa. Although the ratio of antibiotic solution to agar had no significant effects on activity of the antibiotic, the amount of mixing did. Agar depth had no significant effect on the activity of gentamicin.

Agar↗