Performance of eight methods, including two new rapid methods, for detection of oxacillin resistance in a challenge set of Staphylococcus aureus organisms.

Using a set of 55 Staphylococcus aureus challenge organisms, we evaluated six routine methods (broth microdilution, disk diffusion, oxacillin agar screen, MicroScan conventional panels, MicroScan rapid panels, and Vitek cards) currently used in many clinical laboratories and two new rapid methods, Velogene and the MRSA-Screen, that require less than a day to determine the susceptibility of S. aureus to oxacillin. The methods were evaluated by using the presence of the mecA gene, as detected by PCR, as the "gold standard." The strains included 19 mecA-positive heterogeneously resistant strains of expression class 1 or 2 (demonstrating oxacillin MICs of 4 to >16 microg/ml) and 36 mecA-negative strains. The oxacillin MICs of the latter strains were 0.25 to 4 microg/ml when tested by broth microdilution with 2% NaCl-supplemented cation-adjusted Mueller-Hinton broth as specified by the NCCLS. However, when tested by agar dilution with 4% salt (the conditions used in the oxacillin agar screen method), the oxacillin MICs of 16 of the mecA-negative strains increased to 4 to 8 microg/ml. On initial testing, the percentages of correct results (% sensitivity/% specificity) were as follows: broth microdilution, 100/100; Velogene, 100/100; Vitek, 95/97; oxacillin agar screen, 90/92; disk diffusion, 100/89; MicroScan rapid panels, 90/86; MRSA-Screen, 90/100; and MicroScan conventional, 74/97. The MRSA-Screen sensitivity improved to 100% if agglutination reactions were read at 15 min. Repeat testing improved the performance of some but not all of the systems.

Analysis of Clostridium difficile isolates from nosocomial outbreaks at three hospitals in diverse areas of Japan.

Clostridium difficile isolates recovered from patients with C. difficile-associated diarrhea (CDAD) at three hospitals located in diverse areas of Japan were analyzed by three typing systems, PCR ribotyping, pulsed-field gel electrophoresis (PFGE), and Western immunoblotting. At the three hospitals examined, a single PCR ribotype strain (type smz) was predominant and accounted for 22 (65%) of 34, 18 (64%) of 28, and 11 (44%) of 25 isolates, respectively. All of the 51 isolates that represented PCR ribotype smz were nontypeable by PFGE because of DNA degradation. Since the type smz strain did not react with any of the antisera against 10 different serogroups (A, B, C, D, F, G, H, I, K, and X), we prepared a new antiserum against a type smz isolate. All 51 type smz isolates presented identical banding patterns, reacting with the newly prepared antiserum (designated subserogroup JP-0 of serogroup JP). These results were compared with those of a strain from a hospital outbreak that occurred in New York, which has been identified as type J9 by restriction enzyme analysis and type 01/A by arbitrarily primed PCR but was nontypeable by PFGE because of DNA degradation. This strain was reported to be epidemic at multiple hospitals in the United States. The J9 strain represented a PCR ribotype pattern different from that of a type smz strain and was typed as subserogroup G-1 of serogroup G by immunoblot analysis. A single outbreak type causing nosocomial CDAD in Japan was found to be different from the strain causing multiple outbreaks in the United States, even though the outbreak strains from the two countries were nontypeable by PFGE because of DNA degradation.

A 5' nuclease PCR (TaqMan) high-throughput assay for detection of the mecA gene in staphylococci.

In an effort to find a rapid, efficient, and reliable method of screening large numbers of bacterial isolates for specific antimicrobial resistance genes, we compared conventional PCR results to the results generated using the TaqMan 5' nuclease PCR kit in conjunction with an ABI Prism 7700 Sequence Detector for detecting the mecA gene in various species of staphylococci. DNA was extracted using two techniques. The first used a high-salt extraction method suitable for conventional PCR but resulted in a 7.2% rate of PCR inhibition with the TaqMan technique. PCR inhibition could be overcome by diluting samples 1:5 prior to testing. The second method used the Qiagen QIAamp Tissue Kit; no instances of PCR inhibition were encountered with this method. A total of 197 (96%) of the 206 samples with no inhibition showed agreement between the two methods. Eight of the nine disagreements were likely the result of low-level DNA cross contamination caused by frequent specimen handling. Target DNA in all eight of these samples was first detected in the initial tests only after >30 PCR cycles, and all were negative upon repeat testing even after 40 PCR cycles using freshly extracted DNA. Among those positive samples in agreement, target DNA was invariably detected before 30 PCR cycles. The TaqMan assay eliminated the need to load, run, stain, and read agarose gels and provided the advantage of instant detection of PCR product by laser-activated fluorescence. Thus, final results were obtained 2 h after PCR was initiated, as opposed to a requirement of 2 days to examine 96 samples by agarose gel electrophoresis.

Epidemics of diarrhea caused by a clindamycin-resistant strain of Clostridium difficile in four hospitals.

BACKGROUND: Large outbreaks of diarrhea caused by a newly recognized strain of Clostridium difficile occurred in four hospitals located in different parts of the United States between 1989 and 1992. Since frequent use of clindamycin was associated with the outbreak in one of the hospitals, we examined the resistance genes of the epidemic-strain isolates and studied the role of clindamycin use in these outbreaks. METHODS: Case-control studies were performed at three of the four hospitals to assess the relation of the use of clindamycin to C. difficile-associated diarrhea. All isolates of the epidemic strain and representative isolates of other strains identified during each outbreak were tested for susceptibility to clindamycin. Chromosomal DNA from these representative isolates was also analyzed by dot blot hybridization and amplification with the polymerase chain reaction (PCR) with the use of probes and primers from a previously described determinant of erythromycin resistance - the erythromycin ribosomal methylase B (ermB) gene - found in C. perfringens and C. difficile. RESULTS: In a stratified analysis of the case-control studies with pooling of the results according to the Mantel-Haenszel method, we found that the use of clindamycin was significantly increased among patients with diarrhea due to the epidemic strain of C. difficile, as compared with patients whose diarrhea was due to nonepidemic strains (pooled odds ratio, 4.35; 95 percent confidence interval, 2.02 to 9.38; P<0.001). Exposure to other types of antibiotics or hospitalization in a surgical ward was not significantly associated with the risk of C. difficile-associated diarrhea due to the epidemic strain. All epidemic-strain isolates were highly resistant to clindamycin (minimal inhibitory concentration, >256 microg per milliliter). DNA hybridization and PCR analysis showed that all these isolates had an ermB gene, which encodes a 23S ribosomal RNA methylase that mediates resistance to macrolide, lincosamide, and streptogramin antibiotics. Only 15 percent of the nonepidemic strains were resistant to clindamycin. CONCLUSIONS: A strain of C. difficile that is highly resistant to clindamycin was responsible for large outbreaks of diarrhea in four hospitals in different states. The use of clindamycin is a specific risk factor for diarrhea due to this strain. Resistance to clindamycin further increases the risk of C. difficile-associated diarrhea, an established complication of antimicrobial use.

Comparison of restriction enzyme analysis, arbitrarily primed PCR, and protein profile analysis typing for epidemiologic investigation of an ongoing Clostridium difficile outbreak.

During an outbreak of diarrhea in a general hospital in 1992, 166 Clostridium difficile isolates from 102 patients were typed by restriction enzyme analysis (REA), arbitrarily primed PCR (AP-PCR), and protein profile analysis (PP) techniques. A total of 18 types and 5 subtypes were identified by REA, 32 types were identified by AP-PCR, and 9 types were identified by PP. Analysis of the data indicated the presence of a predominant strain among 76, 75, and 84% of the isolates by REA, AP-PCR, and PP, respectively. Subsequently, 45 C. difficile isolates which had been collected in 1990 from 33 patients in the same hospital following a significant increase in the number of cases of diarrhea caused by C. difficile were studied by REA, AP-PCR, and PP typing techniques. Thirteen types and one subtype were identified by REA, 12 types were identified by AP-PCR, and 5 types were identified by PP. As with the isolates from 1992, a dominant strain was identified. This strain was represented by 53, 64, and 70% of the total number of isolates when the strains were typed by REA, AP-PCR, and PP, respectively. Every isolate (210 of 211) from both 1990 and 1992 that was available for typing was typeable by all three methods. Furthermore, the same dominant strain was identified in both 1990 and 1992 by each method. This study demonstrates that each of the three typing methods can be useful in epidemiologic investigations of C. difficile outbreaks and that one strain can be dominant in an institution over a number of years.

Risk factors for early recurrent Clostridium difficile-associated diarrhea.

Recurrence is a common sequela of Clostridium difficile-associated diarrhea (CDD) and may increase morbidity, costs, and treatment-related antimicrobial resistance. Because recurrent CDD (RCDD) frequently occurs very soon after an initial episode, our goal was to determine the risk factors for early RCDD (occurring < or = 45 days after the initial episode). We conducted a case-control study, comparing 13 patients with early RCDD (case patients) with 46 patients who had only one CDD episode (control patients) at Centre Hospitalier Angrignon (Québec) during January 1993 through November 1994. Risk factors for early RCDD included a history of chronic renal insufficiency, a white blood cell count of > or = 15 x 10(3)/mm3, and community-acquired diarrhea with the first CDD episode. For seven of eight case patients, C. difficile strains from the first and second CDD episodes were identical, suggesting that relapse is more common than reinfection. These results suggest that treatments should be directed at preventing relapses in patients at high risk for early RCDD.

Multicenter typing comparison of sporadic and outbreak Clostridium difficile isolates from geographically diverse hospitals.

In a collaborative study by three laboratories, arbitrarily primed polymerase chain reaction (AP-PCR), HindIII restriction enzyme analysis (REA), and pulsed-field gel electrophoresis (PFGE) using SmaI were compared for typing of Clostridium difficile. The study included 30 isolates from nosocomial outbreaks in six geographically disparate hospitals and 15 isolates from sporadic cases of C. difficile diarrhea. REA distinguished a total of 23 types representing 10 groups; AP-PCR performed at Deaconess Hospital resolved 19 types; AP-PCR performed at the Centers for Disease Control resolved 15 types. Thirty isolates exhibited degradation of larger sized fragments during processing and therefore were nontypeable by PFGE; among the remaining 15 isolates, PFGE resolved 11 types. Outbreak isolates in five different hospitals represented REA group J and constituted a single AP-PCR strain. In summary, nosocomial outbreaks of C. difficile diarrhea in five hospitals were associated with a single genetic lineage as resolved by multiple strain typing systems.

Further observations on ghee as a risk factor for neonatal tetanus.

BACKGROUND: Previous case-control studies of neonatal tetanus (NNT) in the North West Frontier Province of Pakistan indicated that clarified butter (ghee) applied to the umbilical wound of newborns was a significant risk factor for NNT. However, the mechanisms underlying the risk remained undisclosed. METHODS: A hospital-based case-control study was undertaken to evaluate further ghee and other factors possibly associated with risk of NNT. Mothers of several recent ghee-associated cases were visited in their homes, asked to simulate the procedures used in preparing the ghee, and samples of ghee were collected for culture. RESULTS: Topical application of ghee to the umbilical wound was again shown to pose a significant risk for NNT. In-use contamination of ghee was documented as mothers repeatedly heated and manipulated samples of ghee set aside in special containers for this purpose. Ghee was usually applied to the umbilical wound of the baby several times each day for the first few days of life. Mothers of cases were again confirmed to be substantially more likely to report prior NNT cases than mothers of controls. CONCLUSIONS: Educational interventions to reduce umbilical ghee use or to wash hands before each manipulation might reduce the risk of NNT in babies exposed to ghee who are born to non-immunized mothers. Increased efforts to immunize women of childbearing age with tetanus toxoid are also needed, with special priority for mothers known to have been associated with a previous NNT case. Topical antibiotics should be further evaluated for protective effects in non-immunized mothers.

PIP: Previous investigations in Pakistan revealed that the application of ghee (clarified butter) to umbilical wounds is a risk factor for neonatal tetanus (NNT), but the underlying mechanisms remained unknown because multiple cultures of ghee obtained from relevant households failed to culture Clostridium tetani. This study used a case-control approach to continue the evaluation of the risk of ghee applications through within-household observations of patterns of use of ghee and further microbiological tests. 100 physician-diagnosed cases who were hospitalized with NNT from September 1990 to January 1991 were compared with 300 controls matched as nearly as possible in age and sex. Data collected through questionnaires were submitted to descriptive analyses, matched analysis with single variables, stratified analysis, and other tests of statistical significance. Conditional logistic regression and Pearson correlation coefficients were also assessed. The only significant factors discovered were delivery by an academically trained attendant (which had a protective effect against NNT) and use of ghee on the umbilical wound (which was a risk factor for NNT). The household investigations revealed that the ghee for use on the newborn is kept in a separate container than that for general household use. The newborn's ghee is reheated and manipulated frequently by the mother. 25% of the samples from the secondary pots of ghee were contaminated. Heating is likely to activate rather than kill the spore-bearing bacteria which causes NNT. The influence of maternal practices is also seen in the fact that the incidence of NNT among previous births was statistically higher for mothers of cases than for mothers of controls. Since use of ghee is unlikely to be abandoned for sociocultural reasons, the added use of topical antibiotics should be evaluated for their impact on the risk factor posed by ghee. Also, special priority should be paid to the immunization of mothers of NNT cases.

Use of arbitrary primer PCR to type Clostridium difficile and comparison of results with those by immunoblot typing.

An arbitrarily primed PCR (AP-PCR) assay was used to type Clostridium difficile isolates from a hospital outbreak of antibiotic-associated diarrhea. Forty-one isolates were separated into nine groups, with 66% falling into one group; no other group contained more than 10%. Comparison of AP-PCR grouping with that when the immunoblot technique was used showed agreement for 33 of 34 isolates typed by both techniques, and AP-PCR grouped seven isolates that were not typeable by immunoblotting.

Typing of Clostridium difficile by western immunoblotting with 10 different antisera.

Western blotting (immunoblotting) with antisera against each of 10 reference serogroups was evaluated as a means of typing Clostridium difficile. A total of 164 clinical isolates of C. difficile were tested. Variations in band profiles in each serogroup were used to type isolates into subserogroups. This technique was useful for an epidemiological investigation.

Detection of toxigenic Clostridium difficile in stool specimens by the polymerase chain reaction.

Polymerase chain reaction (PCR) amplification of a segment of the toxin A gene was used to detect toxigenic Clostridium difficile directly from stool specimens of patients with antibiotic-associated diarrhea. Although PCR-inhibitory substances were recognized in DNA prepared from stool specimens, the inhibitory substances were eliminated by using an ion-exchange column after phenol-chloroform extraction. Eventually, 39 stool specimens were evaluated by PCR. PCR results for detection of toxigenic C. difficile were in complete agreement with cell culture assay results; all 12 PCR-positive stool specimens were positive by cytotoxin assay, and all 27 PCR-negative specimens were negative by cytotoxin assay. Toxigenic C. difficile was cultured from all PCR-positive specimens. These results suggest that PCR amplification may be an effective method for laboratory diagnosis of C. difficile-associated diarrhea and colitis.

Radiation sterilization of surgical instruments with a consideration of metal shielding on sterilization efficiency.

The feasibility of the use of radiation for sterilization of surgical instruments was evaluated. Two aspects were considered: radiation biology of relevant microorganisms, that is, bacterial spores and viruses, and shielding and radiation protection by the metal of the instruments. After proper cleaning and hot water machine washing, surgical instruments carry few, if any contaminants; however, subsequent handling increases the contamination load. Although large instruments may attenuate as much as 30% of the incident radiation, spores dried on the metal are sensitized to irradiation by some 40%. A dose of 25 kGy (2.5 Mrad) is adequate to inactivate a potential contamination load of approximately 10(7) bacterial spores or approximately 10(4) viruses. Therefore, 25 kGy will provide a high sterility assurance level, and can be recommended with a considerable degree of confidence for hospital-based sterilization of surgical instruments.

Comparison of Schaedler agar and trypticase soy-yeast extract agar for the cultivation of anaerobic bacteria.

Schaedler agar (SA) and Trypticase soy-yeast extract agar (TSYEA), both supplemented with rabbit blood (5%, v/v) and menadione (0.5 mg/liter), were compared with respect to quantitative recovery, quality of growth, and rapidity of growth of selected anaerobic bacteria. The media were stored for 2 to 4 days prior to use in an anaerobic glove box, where all subsequent bacteriological procedures were performed. After 24 hr of incubation, colonies of Clostridium cadaveris (C. capitovale), C. haemolyticum, C. novyi A, and C. perfringens were larger on SA than on TSYEA, and the appearance of C. novyi B colonies on SA at 24 hr antedated their appearance on TSYEA. Quantitative recovery of C. novyi B was improved on SA; recovery of the other clostridia tested was comparable on the two media (inconclusive results were obtained with C. novyi A). Rough colonial types of some of the clostridia emerged on SA. No appreciable differences in results with the two media were noted for Bacteroides fragilis, B. melaninogenicus, or Fusobacterium fusiforme.