Elevated lactoferrin is associated with moderate to severe Clostridium difficile disease, stool toxin, and 027 infection.

We evaluated blood and fecal biomarkers as indicators of severity in symptomatic patients with confirmed Clostridium difficile infection (CDI). Recruitment included patients with CDI based on clinical symptoms and supporting laboratory findings. Disease severity was defined by physician's assessment and blood and fecal biomarkers were measured. Toxigenic culture done using spore enrichment and toxin B detected by tissue culture were done as confirmatory tests. Polymerase chain reaction (PCR) ribotyping was performed on each isolate. There were 98 patients recruited, with 85 (87%) confirmed cases of toxigenic CDI (21 severe, 57 moderate, and seven mild), of which 68 (80%) were also stool toxin-positive. Elevated lactoferrin (p = 0.01), increased white blood cell (WBC) count (p = 0.08), and low serum albumin (p = 0.03) were all associated with the more severe cases of CDI. Ribotype 027 infection accounted for 71% of severe cases (p < 0.01) and patients with stool toxin had significantly higher lactoferrin levels and WBC counts (p < 0.05). Our findings show that elevated fecal lactoferrin, along with increased WBC count and low serum albumin, were associated with more severe CDI. In addition, patients infected with ribotype 027 and those with stool toxin had significantly higher fecal lactoferrin and WBC counts.

Persistently positive culture results in a patient with community-acquired pneumonia due to Legionella pneumophila.

We describe a patient with community-acquired pneumonia due to Legionella pneumophila serogroup 6. This patient was found to have bronchoalveolar carcinoma of the lung by means of cytologic testing in 1 of 2 bronchoalveolar lavage samples, but no lesions were visible on bronchoscopy. Despite intravenous administration of azithromycin to the patient, repeat culture and polymerase chain reaction showed persistence of Legionella; the isolates remained susceptible to azithromycin. The patient did not respond to 14 doses of daily intravenously administered azithromycin. The poor outcome may have been partially due to the suspected underlying lung malignancy, as shown by cytologic examination, and by a delay in seeking medical attention.

Fluoroquinolone-resistant Moraxella catarrhalis in a patient with pneumonia: report from the SENTRY Antimicrobial Surveillance Program (1998).

Fluoroquinolone resistance in Moraxella catarrhalis isolates has been quite rare. This report presents a case history of a 22-year-old man with compromised immune status and severe pneumonia caused by M. catarrhalis. The organism was markedly resistant (MICs, 1.5- > 32 micrograms/mL) to several marketed fluoroquinolones including the agent (levofloxacin) used for concurrent and prior therapy. The emergence of this problematic strain seems related to chronic exposure of the patient to compounds in the class and poor patient compliance to prescribed medications. The strain was not clonally related to other M. catarrhalis strains isolated in the same hospital during early 1998. This second documented case of a fluoroquinolone-resistant M. catarrhalis clinical isolate presents a warning that resistances can emerge in at-risk patients, and that surveillance systems (SENTRY) will be necessary to monitor for unusual organisms and spread of resistance phenotypes among commonly isolated respiratory tract pathogens.

Group A streptococcal necrotizing fasciitis. Diagnosing and treating the "flesh-eating bacteria syndrome".

Over the past decade the incidence of necrotizing fasciitis due to group A streptococci has increased. Appropriate management of this life-threatening infection requires rapid recognition, immediate antibiotic therapy, and expeditious surgical debridement or excision.

Spread of serious disease-producing M3 clones of group A streptococcus among family members and health care workers.

Streptococcus pyogenes causes a variety of diseases ranging from mild pharyngitis to severe toxic shock syndrome (TSS) and acute rheumatic fever. Since 1987 there has been a resurgence of severe group A streptococcus infections including TSS, necrotizing fasciitis, and myositis. Using molecular and serotyping procedures, we recently studied two clusters of group A streptococcus disease that occurred within separate family units. The first cluster involved two family members (one with TSS and one with necrotizing fasciitis) and three health care workers who attended one of the index patients. The second cluster included a mother (with necrotizing fasciitis of the hand) and her three children. Group A streptococci isolated from individuals within both cluster groups were serotype M3;T3/13/B3264, and pulsed field gel electrophoresis revealed that all isolates except one had identical fingerprints of Sma I-digested chromosomal DNA. The findings demonstrate the potential for spread of serious group A streptococcus disease among individuals and the need for barrier protection when health care workers are exposed to secretions from infected individuals.

Development of a rapid enzyme immunoassay for Clostridium difficile toxin A and its use in the diagnosis of C. difficile-associated disease.

A rapid (2.5 h) direct enzyme immunoassay (EIA) for Clostridium difficile toxin A was developed for clinical use. Specimen centrifugation and filtration were not required. The EIA detected toxin A levels in patient stool as low as 20 pg (2 ng/ml of stool). The test was 5,000 times more sensitive for toxin A than it was for toxin B and did not react with a panel of other bacterial species with the exception of one highly toxigenic strain of Clostridium sordellii. The EIA was compared with the cytotoxin assay, culture of toxigenic C. difficile (toxigenic culture), and latex agglutination by using 313 fresh stool specimens submitted from patients with suspected C. difficile-associated disease. Results read visually and with a plate reader were similar. Sixty-two specimens were positive by one or more tests, but only 22 (35%) were positive by all four laboratory methods. The EIA was 84.1% sensitive and 98.9% specific when it was compared with the cytotoxin assay. The use of toxigenic culture to referee discrepant results (EIA versus cytotoxin assay) showed the EIA sensitivity and specificity to be 95.1 and 99.3%, respectively, with respect to other laboratory methods. Patient charts were reviewed for antibiotic-associated diarrhea on 108 specimens, including all those that were positive by at least one test method. Of 34 patients determined to have C. difficile-associated disease, 29 (85.3%) were positive by EIA, 32 (94.1%) were positive by the cytotoxin assay, 27 (79.4%) were positive by toxigenic culture, and 20 (58.8%) were positive by latex agglutination. Seven patients with antibiotic-associated diarrhea had a positive latex result, but results were negative by EIA, the cytotoxin assay, and toxigenic culture. The EIA demonstrated high specificity and good sensitivity for C. difficile-associated disease cases. The test can be used alone or in combination with the cytotoxin assay or toxigenic culture to provide rapid and sensitive results.

Evaluation of four newer antimicrobial agents in the Avantage susceptibility test system.

Antimicrobial elution disks containing amoxicillin-clavulanic acid (Augmentin), cefotetan, ciprofloxacin, or norfloxacin were tested in the Avantage automated susceptibility test system. Performance was compared against an agar diffusion procedure in a three-site collaborative study. Results of 1,500 comparison with amoxicillin-clavulanic acid showed a full accord (agreement of both systems) of 93.6% and an essential accord (agreement excluding minor discrepancies) of 97.6%. Results for cefotetan showed a full accord of 95.1% and an essential accord of 98.3% by the two methods. Results for both ciprofloxacin and norfloxacin were in full accord for more than 98% of tests with gram-negative bacilli and staphylococci, but tests with enterococci gave 38 and 26.1% minor discrepancies (the result of one method was resistant or susceptible and the result of the other method was intermediate), respectively. The results indicated that the Avantage test system is accurate and reliable and provides appropriate determination of bacterial susceptibility with the four antibiotics tested.

Wide variability in Pseudomonas aeruginosa aminoglycoside results among seven susceptibility testing procedures.

Seven commonly used antimicrobial susceptibility testing methods were used to test the susceptibility of 150 isolates of Pseudomonas aeruginosa against gentamicin, tobramycin, amikacin, carbenicillin, and piperacillin. Results were compared with respect to the susceptibility characteristics of the population of isolates as defined by each method. Conventional methods included agar disk diffusion and agar dilution, carried out in accordance with current recommendations of the National Committee for Clinical Laboratory Standards, as well as broth microdilution testing with cation-supplemented Mueller-Hinton broth (CSMHB). Methods in which instrumentation was used for result determination included the Autobac I, Avantage, Sensititre Autoreader (using a breakpoint panel at 18 h of incubation), and Vitek (AMS-240, using the GNS susceptibility card). When necessary for comparison, MIC data were converted to categorical interpretations (susceptible, intermediate, and resistant). With respect to gentamicin, no significant differences were noted among the results of disk diffusion, broth microdilution, Sensititre Auto breakpoint, or Vitek methods which characterized 60 to 67% of isolates as susceptible, 16 to 22% as intermediate, and 13 to 17% as resistant. In contrast, agar dilution, Autobac, and Avantage, although yielding gentamicin results similar to those of one another, were each significantly different in result reporting from the other four methods above for gentamicin results, and they characterized the Pseudomonas population largely as susceptible (88 to 97%), with 0 to 6% intermediate and only 3% to 6% resistant. More isolates were characterized as being resistant to gentamicin in the Avantage test if an assay broth supplemented with increased amounts of calcium was used. Cation impregnation of Autobac disks did not appreciably change Autobac results. The geometric mean MIC of gentamicin was 4 micrograms/ml lower in the agar dilution method than in the CSMHB microdilution method, despite monitoring of the agar for cation content through performance disk diffusion testing with P. aeruginosa ATCC 27853. Tobramycin activity was greater than gentamicin activity, and susceptibility to tobramycin ranged from 89 to 97%, with few statistically significant differences noted among the seven methods studied. Differences in MIC distribution and geometric mean MIC between agar dilution and CSMHB microdilution testing were minimal and suggested less of a cation influence on tobramycin than gentamicin results. Although amikacin was also more active than gentamicin (83 to 99% of isolates were susceptible), differences in the amikacin results among methods tended to reflect the same trends in reporting as seen with gentamicin testing, with the exception that results of Avantage testing were similar to those of disk diffusion, CSMHB microdilution, Sensititre, and Vitek. A difference in geometric mean MIC of 5 micrograms/ml between CSMHB testing and agar dilution testing suggested the influence of divalent cations on amikacin results. Few highly significant differences were noted among methods when isolates were tested against carbenicillin and piperacillin, except that Avantage piperacillin results (66% susceptible) and Autobac piperacillin results (98% susceptible) were noticeably different from the percent piperacillin susceptibility (range, 85 to 92%) measured by the other methods. Method-dependent variability among aminoglycoside susceptibility results, particularly when testing gentamicin, prevents meaningful comparison of Pseudomonas susceptibility trends among hospitals when different methods are used and promotes confusion and frustration among clinical microbiologists and clinicians owing to the uncertainties of clinical meaning of these data.

Use of the RIM Escherichia coli Kit for rapid identification of Escherichia coli.

The Rapid Identification Method (RIM) Escherichia coli Kit (Austin Biologicals Laboratories Inc., Austin, TX) was evaluated as a means to rapidly differentiate at reduced cost clinical isolates of Escherichia coli from other members of the Enterobacteriaceae. A group of 408 gram-negative rods that were isolated on primary isolation plates were tested with the use of both the kit and complete bacterial identification. The RIM E. coli Kit identified 92.5% (272 of 294) of the E. coli isolates. Only 1 of 114 non-E. coli isolates was misidentified as E. coli (positive predictive value equal to 99.6). Although the RIM E. coli Kit was sufficiently rapid (less than one hour), any supply cost savings would depend upon the proportion of true E. coli in the group of organisms tested. Savings derived from the lower cost of using the RIM E. coli Kit would be reduced by the extra cost of screening non-E. coli isolates, which would then have to be completely identified.

Evaluation of the Spot-CAMP test for the rapid presumptive identification of group B streptococci.

A rapid Spot-CAMP test was evaluated for its ability to accurately identify colonies of Streptococcus agalactiae (Lancefield Group B) growing on primary sheep blood agar plates. The test uses a beta-lysin-containing filtrate, which is prepared from a broth culture of Staphylococcus aureus. A drop of beta-lysin filtrate is applied adjacent to a suspected group B Streptococcus (GBS) colony and the plate is incubated and then examined for a zone of synergistic hemolysis. The Spot-CAMP test demonstrated 100% correlation with both a Standard CAMP procedure and Lancefield serogrouping. The rapid Spot-CAMP test was easy to perform and inexpensive, and could presumptively identify within 30 minutes colonies of GBS growing on primary isolation plates.

Direct identification and susceptibility testing of gram-negative bacilli from BACTEC bottles by use of the MS-2 system with updated bacterial identification software.

The Abbott MS-2 system (Abbott Laboratories, Diagnostic Division, Irving, Tex.), equipped with updated bacterial identification software (version 03.02), was used to perform both direct identification and susceptibility tests on gram-negative bacilli from positive BACTEC blood culture bottles. Ninety-eight of 101 Enterobacteriaceae strains, one strain of Acinetobacter calcoaceticus, and two strains of Pseudomonas aeruginosa were correctly identified by following a direct inoculation procedure. One Enterobacter sakazakii isolate was misidentified as Enterobacter cloacae. Ninety-seven percent of 936 direct susceptibility results were identical to the results obtained by using the standard MS-2 susceptibility procedure. Only five tests yielded a sensitive-direct and a resistant-standard MS-2 susceptibility result.

Evaluation of the updated MS-2 Bacterial Identification system in comparison with the API 20E system.

The recently updated MS-2 Bacterial Identification system software (Abbott Laboratories, Diagnostic Division, Irving, Tex.) was compared with the original MS-2 Bacterial Identification software and the API 20E, using 968 strains of Enterobacteriaceae. The updated MS-2 software correctly identified 94.4% of the isolates tested. API 20E and the original MS-2 software correctly identified 91 and 85.3% of the strains, respectively. MS-2 responses were considered to be equivocal (needing additional tests for verification) if the percent likelihood values were less than 80%. The percentage of equivocal responses was reduced from 6.5% with the original software to 2.2% with the updated software, and the percentage of incorrect identifications was reduced from 8.2 to 3.4% with the original and updated software, respectively. Organisms belonging to 25 taxonomic groups were tested. Direct comparison of the two MS-2 programs showed that the updated MS-2 software increased the identification accuracy of Salmonella spp., Enterobacter cloacae, Providencia stuartii, Escherichia coli, Shigella spp., Klebsiella pneumoniae, Serratia marcescens, Proteus mirabilis, and Acinetobacter calcoaceticus. A decrease in accuracy was seen with Citrobacter freundii, Hafnia alvei, Enterobacter agglomerans, and Yersinia pseudotuberculosis when the updated software was used. The remaining 12 taxonomic groups were not affected by the software changes. The updated MS-2 software appears to significantly improve the identification accuracy of the MS-2 Bacterial Identification system.

Enhanced detection of bacteremia with a new BACTEC resin blood culture medium.

In this multicenter study, 621 sets of blood culture specimens were drawn from 280 patients who were suspected of being septic and who were receiving antimicrobial therapy. Equal volumes of each specimen were inoculated into BACTEC 6B and 16B media. The 16B medium contained adsorbent and cationic resins for neutralizing the effects of the drugs. Of the 621 sets drawn, there were 72 positive cultures in 16B and 52 positive cultures in 6B. In 23 cases the organism was detected only in the 16B medium, and in 3 cases the organism was detected in 6B only. The remaining 49 positives were detected in both culture bottles. In 13 of these 49 cultures, detection in 16B was made between 1 and 5 days earlier than in 6B, whereas 3 of 49 specimens were detected 1 day earlier in 6B; the remaining 33 cultures became positive at approximately the same time in both media. There were a total of 43 patients with positive cultures in this study. Of these patients, 28 had sepsis detected in both the 16B and 6B media. The 6B medium alone detected an additional three cases of sepsis, and the 16B resin medium alone identified 12 additional cases. Supplementary culturing of samples from patients receiving antimicrobial therapy significantly increased the number of positive cultures and positive patients, as well as significantly shortening the time to positivity in these cultures.

In vitro activity of netilmicin, gentamicin, tobramycin and amikacin against glucose fermenting and nonfermenting bacteria.

The in vitro activity of netilmicin was compared to gentamicin, tobramycin and amikacin against 461 strains of glucose fermenting and nonfermenting bacteria. The minimum inhibitory concentrations of netilmicin, gentamicin and tobramycin against the majority of Enterobacteriaceae, Staphylococcus and Streptococcus were quite similar. Gentamicin, however, was approximately fourfold more active against strains of S. marcescens. Amikacin was the most effect antibiotic against strains of fermenting and nonfermenting bacilli resistant to at least one aminoglycoside. Many gentamicin-resistant species of nonfermenting bacilli, however, remain highly resistant to all four aminoglycosides tested.

In vitro activity of rosamicin and erythromycin against a group of nonfermenting gram-negative bacilli.

The in vitro inhibitory activity of rosamicin and erythromycin against 283 strains of nonfermenting, gram-negative bacilli was determined by using a broth dilution procedure. Rosamicin demonstrated greater activity than erythromycin against most strains tested. A number of species demonstrated significantly lower minimum inhibitory concentrations to rosamicin and would fall within the therapeutic range of the drug based on current pharmacological data.

A quantitative ultrastructural and chemical investigation of the accumulation of iodophilic polysaccharide in two cariogenic strains of Streptococcus mutans.

The amount of intracellular, iodophilic, glycogen-like polysaccharide (IPS) present in cells of two strains of Streptococcus mutans at various stages of growth in a chemically defined medium was determined by quantitative electron microscopy. The results obtained were then compared with the chemically determined, iodophilic polysaccharide content of cultures. The ultrastructural method used determined the fraction of area of central longitudinal sections of individual cells occupied by stained granules, and was therefore capable of determining amounts of polysaccharide in cells starved of glucose. Although the results of the two methods showed very good quantitative correlation, the ultrastructural method allowed study of glycogen synthesis on a cellular basis, and detected some heterogeneity in amounts of IPS stored by individual cells. The ultrastructural method also permits the detection of much smaller amounts of stored IPS than does the chemical method.

Unbalanced growth and macromolecular synthesis in Streptococcus mutans FA-1.

The continued synthesis of deoxyribonucleic acid, protein, cell wall peptidoglycan and intracellular iodophilic polysaccharide (IPS) by Streptococcus mutans strain FA-1 after several treatments intended to inhibit protein synthesis was studied. Exponential-phase cultures were: (i) simultaneously deprived of two required amino acids (cystine and leucine) that are not present in the cell wall peptidoglycan of this species; (ii) depreived of required amino acids (lysine or glutamate plus glutamine ) that are present in both peptidoglycan and protein; or (iii) treated with tetracycline. Each of these three types of treatment was accompanied by a different pattern of unbalanced growth. The patterns of unbalanced growth that accompanied treatments (i) or (ii) differed substantially from the patterns observed previously for Streptococcus faecalis ATCC 9790, a noncariogenic organism that does not contain IPS. In contrast to S. faecalis 9790, S. mutans FA-1 failed to accumulate peptidoglycan and thicken its wall when deprived of non-wall amino acids. Instead, S. mutans FA-1 continued to accumulate IPS to levels substantially higher than those found in exponential-phase cells. Again, in contrast to S. faecalis, S. mutans FA-1 failed to autolyze upon deprivation of essential precursors of wall peptidoglycan. Under conditions of lysine of glutamate/glutamine deprivation, S. mutans FA-1 continued to accumulate IPS to very high levels. Treatment with tetracycline did result in peptidoglycan accumulation and wall thickening in a manner very similar to that observed previously for inhibition of protein synthesis in S. faecalis. Realtively little IPS synthesis continued after tetracycline treatment. Accumulation of IPS appeared to occur when both ribonucleic acid and peptidoglycan synthesis were severely inhibited. The observations are discussed in terms of the survival of cariogenic organisms in the oral environment.

Measurement of intracellular iodophilic polysaccharide in two cariogenic strains of Streptococcus mutans by cytochemical and chemical methods.

Quantitative cytological and chemical methods have been developed to study the intracellular iodophilic polysaccharide (IPS) content of two strains of Streptococcus mutans. The cytological method uses a periodic acid-chlorite treatment of thin sections to increase the affinity of IPS for uranyl and lead salts. This results in the IPS appearing as individual electron-dense granules which can be counted for quantitative studies. As a basis for these quantitative studies, IPS was measured chemically by dissolving whole cells with hot KOH and quantitating spectrophotometrically the amount of iodine-polysaccharide complex formed.