Characterization of a toxin A-negative, toxin B-positive strain of Clostridium difficile responsible for a nosocomial outbreak of Clostridium difficile-associated diarrhea

Abstract

Clostridium difficile-associated diarrhea (CAD) is a very common nosocomial infection that contributes significantly to patient morbidity and mortality as well as to the cost of hospitalization. Previously, strains of toxin A-negative, toxin B-positive C. difficile were not thought to be associated with clinically significant disease. This study reports the characterization of a toxin A-negative, toxin B-positive strain of C. difficile that was responsible for a recently described nosocomial outbreak of CAD. Analysis of the seven patient isolates from the outbreak by pulsed-field gel electrophoresis indicated that this outbreak was due to transmission of a single strain of C. difficile. Our characterization of this strain (HSC98) has demonstrated that the toxin A gene lacks 1.8 kb from the carboxy repetitive oligopeptide (CROP) region but apparently has no other major deletions from other regions of the toxin A or toxin B gene. The remaining 1.3-kb fragment of the toxin A CROP region from strain HSC98 showed 98% sequence homology with strain 1470, previously reported by M. Weidmann in 1997 (GenBank accession number Y12616), suggesting that HSC98 is toxinotype VIII. The HSC98 strain infecting patients involved in this outbreak produced the full spectrum of clinical illness usually associated with C. difficile-associated disease. This pathogenic spectrum was manifest despite the inability of this strain to alter tight junctions as determined by using in vitro tissue culture testing, which suggested that no functional toxin A was produced by this strain.

FIG. 1

Diagnostic tests performed on samples from patients involved in the outbreak. Each solid horizontal line represents the time line for the length of hospitalization for one of the 16 patients involved in the initial outbreak. Gaps in the line indicate when the patient was discharged and then was readmitted. Arrows indicate that the patient was still in the hospital beyond this date. There were four wards involved in this outbreak; ward 1 (), ward 2 (), and ward 4 () were general medical wards, and ward 3 () was an oncology ward. The EIA tests used detected toxin A alone (a solid circle represents a positive test and an open circle represents a negative test) or toxin A and toxin B (a solid diamond represents a positive test and an open diamond represents a negative test). The cytotoxin test using HFF-cell culture was also performed (a solid square represents a positive test and an open square represents a negative test).

FIG. 2

PFGE of C. difficile outbreak isolates. Isolates of C. difficile were detected in 7 of the 16 patients involved in the initial outbreak. PFGE was done using SmaI as the restriction enzyme by the method described in Materials and Methods. Lanes 1 and 12, lambda ladder size markers; lanes 2 to 8, isolates from patients 1, 11, 11, 2, 7, 6, and 9, respectively. The two isolates from patient 11 had different colony morphologies. Lanes 9 to 11, C. difficile isolates that were toxin A positive and toxin B positive that were not part of the outbreak.

FIG. 3

Restriction endonuclease digestion patterns for the A3′ amplicon of the CROP region of the toxin A gene for HSC98. The PCR amplicon product A3′ (lane 2) obtained from HSC98 using the A3 primer set (product was 1,250 bp) was exposed to SpeI (lane 3) and EcoI (lane 4) restriction endonucleases to determine restriction fragment length patterns. The A3′ amplicon (lane 2) had no restriction site for EcoRI (lane 4) and had one restriction site for SpeI (lane 3). A 500-bp DNA ladder is shown in lanes 1 and 6, and a 100-bp ladder is shown in lane 5.

FIG. 4

Effect of HSC98 culture supernatant fluid on CaCo-2 tight junctions. Filter-sterilized culture medium from dialysis membrane culture (as described in Materials and Methods) was used to inoculate the apical side of the CaCo-2 monolayer. C. difficile strains 10463 (toxin A positive and toxin B positive) (■), 8864 (toxin A negative and toxin B positive) (⧫), and HSC98 (toxin A negative and toxin B positive) (▴) were tested. Each point plotted represents the average of nine electrical resistance readings consisting of triplicate readings from each of three separate inserts.

FIG. 5

Comparison of CPE on HFF cells elicited by C. difficile HSC98 and C. difficile 10463. Culture supernatant from the dialysis culture method described in Materials and Methods was filter sterilized for C. difficile strains HSC98 and 10463. HSC98 supernatant was inoculated in the absence (A) or presence (B) of C. sordellii antitoxin, and similarly, strain 10463 was inoculated in the absence (C) or presence (D) of C. difficile antitoxin. CPE was produced by both HSC98 and 10463, which was neutralized by C. difficile antitoxin (B and D); however, the CPE produced by HSC98 showed rounding with no spindle formation, whereas strain 10463 produced rounded cells that had spindles (arrows).