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ABSTRACTPrevention and management of Clostridium difficileinfection (CDI) can be improved by rapid and reliable diagnostics. The Vidas C. difficileglutamate dehydrogenase assay had performance comparable to that of the Quik Chek-60 assay (overall agreement, 95%) and a sensitivity of >93%; thus, it is suitable as the first test in two-stage algorithms for a CDI diagnosis.

ABSTRACTNo study to date has compared multilocus variable-number tandem-repeat analysis (MLVA) and whole-genome sequencing (WGS) in an investigation of the transmission of Clostridium difficileinfection. Isolates from 61 adults with ongoing and/or recurrent C. difficileinfections and 17 asymptomatic carriage episodes in children (201 samples), as well as from 61 suspected outbreaks affecting 2 to 41 patients in 31 hospitals in the United Kingdom (300 samples), underwent 7-locus MLVA and WGS in parallel. When the first and last samples from the same individual taken for a median (interquartile range [IQR]) of 63 days (43 to 105 days) apart were compared, the estimated rates of the evolution of single nucleotide variants (SNVs), summed tandem-repeat differences (STRDs), and locus variants (LVs) were 0.79 (95% confidence interval [CI], 0.00 to 1.75), 1.63 (95% CI, 0.00 to 3.59), and 1.21 (95% CI, 0.00 to 2.67)/called genome/year, respectively. Differences of >2 SNVs and >10 STRDs have been used to exclude direct case-to-case transmission. With the first serial sample per individual being used to assess discriminatory power, across all pairs of samples sharing a PCR ribotype, 192/283 (68%) differed by >10 STRDs and 217/283 (77%) by >2 SNVs. Among all pairs of cases from the same suspected outbreak, 1,190/1,488 (80%) pairs had concordant results using >2 SNVs and >10 STRDs to exclude transmission. For the discordant pairs, 229 (15%) had =2 SNVs but =10 STRDs, and 69 (5%) had =2 SNVs but =10 STRDs. Discordant pairs had higher numbers of LVs than concordant pairs, supporting the more diverse measure in each type of discordant pair. Conclusions on whether the potential outbreaks were confirmed were concordant in 58/61 (95%) investigations. Overall findings using MLVA and WGS were very similar despite the fact that they analyzed different parts of the bacterial genome. With improvements in WGS technology, it is likely that MLVA locus data will be available from WGS in the near future.

ABSTRACTIn the past decade, the incidence of Clostridium difficileinfections (CDI) with a more severe course has increased in Europe and North America. Assays that are capable of rapidly diagnosing CDI are essential. Two real-time PCRs (LUMC and LvI) targeting C. difficiletoxin genes (tcdB, and tcdAand tcdB, respectively) were compared with the BD GeneOhm PCR (targeting the tcdBgene), using cytotoxigenic culture as a gold standard. In addition, a real-time PCR targeting the tcdCframeshift mutation at position 117 (?117 PCR) was evaluated for detecting toxigenic C. difficileand the presence of PCR ribotype 027 in stool samples. In total, 526 diarrheal samples were prospectively collected and included in the study. Compared with those for cytotoxigenic culture, sensitivity, specificity, positive predicted value (PPV), and negative predicted value (NPV) were for PCR LUMC 96.0%, 88.0%, 66.0%, and 98.9%, for PCR LvI 100.0%, 89.4%, 69.7%, and 100.0%, for PCR ?117 98.0%, 90.7%, 71.9%, and 99.5%, and for PCR BD GeneOhm 88.3%, 96.9%, 86.5%, and 97.4%. Compared to those with feces samples cultured positive for C. difficiletype 027, the sensitivity, specificity, PPV, and NPV of the ?117 PCR were 95.2%, 96.2%, 87.0%, and 98.7%. We conclude that all real-time PCRs can be applied as a first screening test in an algorithm for diagnosing CDI. However, the low PPVs hinder the use of the assays as stand-alone tests. Furthermore, the ?117 PCR may provide valuable information for minimizing the spread of the epidemic C. difficilePCR ribotype 027.

ABSTRACTTotals of 102 and 56 Clostridium difficiletype 078 strains of human and porcine origins, respectively, from four European countries were investigated by an optimized multilocus variable-number tandem-repeat analysis (MLVA) and for tetracycline susceptibility. Eighty-five percent of all isolates were genetically related, irrespective of human or porcine origin. Human strains were significantly more resistant to tetracycline than porcine strains. All tetracycline-resistant strains contained the Tn916-like transposon harboring the tet(M) gene. We conclude that strains from human and porcine origins are genetically related, irrespective of the country of origin. This may reflect a lack of diversity and/or common source.

SUMMARYThe epidemiology of Clostridium difficileinfection (CDI) has changed dramatically during this millennium. Infection rates have increased markedly in most countries with detailed surveillance data. There have been clear changes in the clinical presentation, response to treatment, and outcome of CDI. These changes have been driven to a major degree by the emergence and epidemic spread of a novel strain, known as PCR ribotype 027 (sometimes referred to as BI/NAP1/027). We review the evidence for the changing epidemiology, clinical virulence and outcome of treatment of CDI, and the similarities and differences between data from various countries and continents. Community-acquired CDI has also emerged, although the evidence for this as a distinct new entity is less clear. There are new data on the etiology of and potential risk factors for CDI; controversial issues include specific antimicrobial agents, gastric acid suppressants, potential animal and food sources of C. difficile, and the effect of the use of alcohol-based hand hygiene agents.

ABSTRACTWe compared multilocus variable-number tandem-repeat analysis (MLVA) and macrorestriction endonuclease analysis using pulsed-field gel electrophoresis (PFGE) to determine their utility to identify clusters of Clostridium difficileinfection (CDI) among 91 isolates of PCR ribotype 027 (NAP1, for North American pulsed-field type 1) from nine hospitals (and 10 general practitioners associated with one institution) in England. We also examined whether mortality in CDI cases was associated with specific MLVA subtypes. PFGE discriminated between ribotype 027 strains at >98% similarity, identifying five pulsovars (I to V) of 1 to 53 isolates. MLVA was markedly more discriminatory, identifying 23 types of 1 to 15 isolates (>71% similarity). PFGE pulsovars I and IV contained 14 and 8 MLVA types, respectively. Twenty-one of twenty-three (91%) of MLVA types were specific to individual PFGE pulsovars. Four CDI clusters were identified in institution A by conventional epidemiological analysis. MLVA typing identified two enlarged and two additional clusters. Thirty of forty-four (68%) patients in institution A with CDI caused by ribotype 027 strains were assigned to seven distinct clusters by a combination of MLVA typing and epidemiological records. Of 33 patients, comprising 14 different MLVA types, nine (27%) died by day 30 (early deaths). Eight of nine (89%) were associated with PFGE type IV C. difficileribotype 027. Five of nine early deaths were associated with MLVA type 16, which was the dominant type in this cohort (10/33 cases); 4 other distinct MLVA types accounted for the other early deaths. MLVA was far superior to PFGE for analyzing clusters of CDI both within and between institutions. Further study is needed to examine whether subtypes of C. difficileribotype 027 affect outcome.

ABSTRACTWe prospectively studied the comparative epidemiology and risk factors for Clostridium difficile, Clostridium perfringens, and Staphylococcus aureusantibiotic-associated diarrhea (AAD). Four thousand six hundred fifty-nine inpatient fecal specimens (11 months) were tested for C. difficilecytotoxin, C. perfringensenterotoxin, and S. aureusby Vero cell assay, enzyme-linked immunosorbent assay, and growth on fresh blood agar, respectively. Two distinct age-, sex-, and location-matched control patient groups were used for multivariate logistic regression risk factor analyses: symptomatic patients who were AAD pathogen negative and asymptomatic patients with histories of recent antimicrobial therapy. All AAD pathogen isolates were DNA fingerprinted. In AAD cases, the prevalences of C. difficilecytotoxin, C. perfringensenterotoxin, and S. aureuswere 12.7%, 3.3%, and 0.2%, respectively (15.8% overall). Age of >70 years was a common risk factor. Other risk factors for infective AAD and C. difficileAAD included length of hospital stay and use of feeding tubes (length of stay odds ratios [OR], 1.017 and 1.012; feeding tube OR, 1.864 and 2.808). Female gender and use of antacids were significantly associated with increased risk of C. perfringensAAD (OR, 2.08 and 2.789, respectively), but unlike what was found for C. difficileAAD, specific antibiotic classes were not associated with increased risk. A limited number of genotypes caused the majority of C. difficileand C. perfringensAAD cases. Similar to what was found for C. difficileAAD, there was epidemiological evidence of C. perfringensAAD case clustering and reinfection due to different strains. C. difficileAAD was approximately 4 and 60 times more common than C. perfringensAAD and S. aureusAAD, respectively. Risk factors for these AAD pathogens differed, highlighting the need to define specific control measures. There is evidence of nosocomial transmission in cases of C. perfringensAAD.

We describe a new method for the measurement of antimicrobial concentrations in the biofilm associated with the endoluminal surface of intravascular catheters. We quantified endoluminal planktonic bacteria in haemodialysis catheters using the acridine orange method on catheter blood. After catheter removal, separate lumens were perfused in vitro with either vancomycin or linezolid to simulate in vivo antibiotic infusion. Biofilm was recovered using endoluminal brushes, weighed and assayed by fluoroimmunoassay for vancomycin and by bioassay for linezolid. Viable bacteria were counted by serial dilution and agar plating. Biofilm had measurable amounts of vancomycin in 11/11 catheter lumens post-infusion (0.3-18.2 mg biofilm per lumen, mean 6.8 mg; vancomycin concentration 0.2-89 mg/g biofilm, median 19 mg/g). By comparison, linezolid was detected in 4/11 catheter lumens post-infusion (0.5-18.1 mg biofilm per lumen, mean 5.9 mg; linezolid concentration 0.9-6.1 mg/g biofilm, median 1.5 mg/g). Percentage reductions in biofilm-associated bacterial counts post-antibiotic were 84-100%, median 95% (vancomycin) and 0-98%, median 91% (linezolid). We found a significant difference (P = 0.05; Wilcoxon rank sum test) in vancomycin concentrations in coagulase-negative staphylococcal biofilm (median 17.0 mg/g, mean 27.9 mg/g) compared with glycopeptide levels found in biofilm associated with other microorganisms (median 5.5 mg/g, mean 6.9 mg/g). Biofilm concentrations of vancomycin are generally higher than linezolid after antibiotic infusion, which can be explained partly by glycopeptide binding to glycocalyx. Neither antibiotic achieved consistent 100% kill of biofilm bacteria after single infusions, even when a very high concentration was present. The endoluminal brush technique can be used to measure antibiotic concentration in intravascular catheter-associated biofilm in situ. This approach can be exploited to measure biofilm antibiotic concentrations in vivo, without the need for catheter removal.

There is an increased interest in exploring antibacterial, biotherapy and immunological treatment options for Clostridium difficileinfection. The impetus has occurred from increased antibiotic use, the rising incidence of C. difficileinfection in the elderly and high rates of recurrence associated with metronidazole and vancomycin therapy. However, progress has been hampered by the varied aetiology of diarrhoea, spontaneous and unpredictable symptomatic resolution in some patients and inconsistencies in diagnosis. C. difficilemay be treated with vancomycin or metronidazole as the overall response and recurrence rates are not statistically significantly different. However, metronidazole is the antibiotic of first choice as it reduces the selective pressure for glycopeptide-resistant enterococci, promoted by the widespread use of vancomycin. Management of symptomatic recurrences is problematic; switching between vancomycin and metronidazole is not recommended as DNA fingerprinting studies demonstrate that relapses are often re-infections with different strains. Placebo-controlled, double-blind trials on Saccharomyces boulardiigiven prophylactically to patients receiving antibiotics reported a reduced incidence in antibiotic-associated diarrhoea. Further double-blind, placebo-controlled trials demonstrated that treatment with S. boulardiisignificantly reduced recurrent C. difficilediarrhoea. Case reports suggested that LactobacillusGG reduced recurrent C. difficileinfection and there is only one partially reported, prospective, randomized, placebo-controlled trial of LactobacillusGG in adults with C. difficileinfection. Prophylactic bovine immunoglobulin prevented diarrhoea in C. difficile-exposed hamsters and two patients with severe colitis unresponsive to antibiotics improved following intravenous immunoglobulin therapy. SYNSORB Cd®, a novel compound, is reported to reduce C. difficilediarrhoeal recurrence rates and is undergoing phase III study.

We compared the activities of ciprofloxacin and levofloxacin with those of the newer fluoroquinolones grepafloxacin, moxifloxacin, sparfloxacin and trovafloxacin against Clostridium difficile isolates. As there is good evidence of marked clonal spread of C. difficile, we studied both genotypically distinct (n = 26) and indistinguishable (n = 28) isolates as determined by random amplified polymorphic DNA and ribosomal spacer PCR fingerprinting. The indistinguishable strains examined represent the main UK epidemic C. difficile clone. For 17 of 54 strains (31%) we were unable to read MICs following inocula preparation using Mueller-Hinton broth. Using Schaedler's broth for inocula preparation 93% of strains had readable MICs, although geometric mean MICs were uniformly higher (2.5- to 5.4-fold) compared with results using Mueller-Hinton broth. Moxifloxacin and trovafloxacin, followed by grepafloxacin, were the most active fluoroquinolones tested and were 3- to 4-fold more active than older agents such as ciprofloxacin by both MIC methods. Unexpectedly, clonal C. difficile strains had markedly reduced susceptibility compared with the distinct strains to each of the fluoroquinolones tested. Clonal strains were more than seven-fold or 12- to 29-fold less susceptible (according to geometric mean MICs) than distinct strains to both moxifloxacin and trovafloxacin, depending on the MIC method used. It remains to be seen whether the enhanced activity of new fluoroquinolones such as moxifloxacin in comparison with other fluoroquinolones against C. difficile implies that these agents are unlikely to be associated with C. difficile infection. However, clinical use of new generation fluoroquinolones in elderly hospitalized patients where C. difficile is endemic requires further study, particularly given the reduced antibiotic susceptibility to all fluoroquinolones of the readily transmissible UK C. difficile clone.

Despite international protocols prohibiting the use of microbiological agents - viruses, bacteria, fungi and protozoa, as weapons of war, many countries have either retained or actively developed their capacity to produce such weapons. Biological warfare (BW) agents have long been studied, and actually used by armies for many centuries. One of the reasons for this is that BW agents have been considered an inexpensive way to kill or incapacitate one's enemies - the 'poor man's atomic bomb'. In view of recent international events, it is apposite to review the use of BW agents, with especial reference to bacteriological warfare - historical, bacterial types, delivery systems, detection, prevention, treatment and future developments utilising new microbiological techniques such as recombinant DNA technology.

The cell envelope protein profiles of Staphylococcus epidermidis cultured in used human peritoneal dialysate (HPD) differed markedly from those of cells cultured in nutrient broth. Compared with broth-grown cells, many cell wall proteins were repressed in HPD, although three proteins of 42, 48, and 54 kDa predominated and an iron-repressible 130-kDa protein was induced. Growth in HPD also resulted in expression of two cell membrane proteins of 32 and 36 kDa which were iron repressible. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis using monospecific polyclonal antisera raised against the 32- and 36-kDa proteins revealed considerable antigenic and molecular mass homology among 12 S. epidermidis isolates from patients with continuous ambulatory peritoneal dialysis-related peritonitis. The 32-kDa antiserum also cross-reacted with a 32-kDa S. aureus cell membrane protein. Immunoblots of S. epidermidis cell walls and membranes were also probed with normal human serum and serum and HPD from continuous ambulatory peritoneal dialysis patients. While the cell wall proteins of S. epidermidis appeared to be relatively poorly immunogenic, the 32- and 36-kDa membrane proteins reacted strongly with antibodies present in each of the body fluids evaluated. These results suggest that the highly conserved 32- and 36-kDa iron-repressible proteins are expressed during growth in vivo and may be involved in iron transport, since all 12 S. epidermidis strains examined also produced iron chelators.

Objective: To compare four vancomycin‐containing agar media for the isolation of glycopeptide‐resistant enterococci (GRE) from clinical fecal specimens: kanamycin‐aesculin‐azide (KAA) agar; bile‐aesculin‐polymixin (BAP) agar; aztreonam‐amphotericin blood (CBAA) agar; and neomycin blood (CBN) agar.

Used peritoneal dialysis fluid was collected from patients undergoing continuous ambulatory peritoneal dialysis, and its pH and composition were assessed after incubation in either air or air with 5% CO2. Precipitation of calcium, magnesium, phosphate, and proteins occurred in the dialysis fluid incubated in air at 37 degrees C and was associated with a mean pH increase of 1.23 U. Incubation of dialysis fluid in air with 5% CO2 prevented precipitation and maintained pCO2 and pH levels at those found physiologically. Coagulase-negative staphylococcal strains isolated from patients with peritonitis tended to grow less well in dialysis fluid incubated in air than in dialysis fluid incubated in the carbon dioxide-enriched atmosphere. MICs of cefuroxime, ciprofloxacin, and vancomycin for seven strains of coagulase-negative staphylococci in dialysis fluid were markedly affected by atmosphere type (16 of 21 MICs). Of these 16 atmosphere-dependent MICs, 14 were at least fourfold higher in air than in air with 5% CO2.

Coagulase-negative staphylococci obtained from patients with continuous ambulatory peritoneal dialysis-related peritonitis were cultured in nutrient broth in an atmosphere of air containing 5% carbon dioxide (reflecting the CO2 tension found in freshly used dialysate). Significant differences were observed between the surface chemistries of cells grown in the two atmospheres, as determined by X-ray photoelectron spectroscopy and changes in the cell wall protein profile. The growth atmosphere also influenced the adherence potential toward polystyrene and silicone in a proportion of strains examined. Thus, gaseous conditions can profoundly influence the nature of the staphylococcal surface, and this should be considered in any in vitro study of in vivo behavior.

Of 95 faecal specimens containing bacterial DNA amplified by PCR, 24% contained cultivable bacteria that were resistant to high-level ampicillin. When these samples were examined by PCR using primers to amplify the bla(TEM) gene, the number of positive samples identified increased significantly to 49 (52%). These results indicate that ampicillin resistance is common in the study population. Furthermore, the bla(TEM) gene is more common than indicated by the recovery of resistant bacteria in culture. This points to potential anomalies in the assessment of the prevalence of resistance when relying on recovery of resistant bacteria by culture.

ABSTRACTWe noted that some Clostridium difficileisolates are nonrecoverable after frozen storage and so used molecular typing analysis to characterize DNA from these strains. The recovery rate of C. difficilePCR ribotype 1 was statistically significantly greater than that of other strains. This observation has implications for C. difficileepidemiological studies.

Intravascular catheter-related infections (CRI) account for one third of nosocomial bloodstream infections in England. UK Department of Health guidelines state that antibiotic prophylaxis is not required during placement, or use of, central venous catheters, to prevent CRI. However, some clinicians continue to use antibiotics in an attempt to prevent CRI. We investigated the effect of extended routine perioperative antibiotic prophylaxis in cardiothoracic patients on rates of intravascular catheter (IVC) colonization and infection. Investigations were undertaken in patients undergoing uncomplicated cardiothoracic surgery during July 2001–February 2002. Patients who received three doses of cefuroxime as perioperative prophylaxis were compared with those who received extended cefuroxime prophylaxis until the IVC was removed. Patients were not randomized into groups, but received the different prophylaxis regimens according to the usual practice of the consultant cardiothoracic surgeon. A roll tip method was used to determine IVC colonization. Of 191 patients who fulfilled the inclusion criteria, 12 were excluded because data were incomplete. One hundred and forty-six patients received routine prophylaxis, and 33 prophylaxis until the IVC was removed. Twenty-three out of 146 (16%) IVCs in the ‘routine’ group and four out of 33 (12%) in the ‘extended’ group became colonized; no IVC-related bloodstream infections occurred during the survey. The duration of IVC placement and the types of operation performed in the two groups were not significantly different (<it>P</it> > 0.05). In routine cardiothoracic surgery patients, extending routine perioperative antibiotic prophylaxis until all IVCs have been removed does not influence rates of IVC colonization.