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S H Cohen

Publications and source records attributed to S H Cohen.

At least 19 recordsLinked to original sources
PubMed2002

One-step cloning and expression of Clostridium difficile toxin B gene (tcdB).

The toxin genes of Clostridium difficile have been previously cloned by reconstructing the entire gene in a series of steps in sequence using several cloned fragments. Amplification of a 7.9 kb fragment corresponding to the toxin B gene (tcdB) was obtained with EXPAND Long Template PCR system. The amplified fragment was inserted into the E. coli expression vector pBAD and cloned into competent E. coli TOP 10 cells. tcdB gene sequences representing the complete toxin gene were detected in 3/120 (2.5%) clones analyzed. Culture filtrates of 2/3 clones were found to have cytotoxic activity in human lung fibroblasts. The recombinant protein expressed in E. coli was identified as toxin B by Western immunoblot analysis using C. sordellii antitoxin. This rapid cloning method may be useful in determining the role that individual genes in the pathogenicity locus (PaLoc) play in the virulence of C. difficile. Our results also suggest that the activity of toxin B is independent of other genes in the PaLoc.

Bacterial Proteins↗
PubMed2001

Electroporation of DNA sequences from the pathogenicity locus (PaLoc) of toxigenic Clostridium difficile into a non-toxigenic strain.

Toxigenic Clostridium difficile is the etiologic agent of C. difficile-associated diarrhoea (CDAD), the most common cause of hospital-acquired infectious diarrhoea. The genes tcdA and tcdB, which encode for the toxin A and B proteins, are part of the pathogenicity locus (PaLoc) of toxigenic C. difficile. Genetic and virulence studies at the molecular level in C. difficile have been hindered by the lack of techniques for DNA manipulation in this species. We describe the electroporation of DNA fragments from a toxigenic isolate into a non-toxigenic strain of C. difficile. Using previously described methods of electroporation into Clostridium spp., the complete toxin B gene and polymerase chain reaction (PCR) fragments of the PaLoc were cloned and electroporated into a non-toxigenic strain of C. difficile. The resulting transformed clones were screened for the introduced gene fragments by PCR, which confirmed their presence. This is the first description of introduction of DNA into C. difficile by electroporation.

Bacterial Proteins↗
PubMed2001

Isolation of Clostridium innocuum from cases of recurrent diarrhea in patients with prior Clostridium difficile associated diarrhea.

Clostridium innocuum isolates resistant to vancomycin (MIC values of 16-24 microg/mL) were isolated from three patients with recurrent Clostridium difficile -associated diarrhea (CDAD). We discuss the clinical significance and problems associated with the identification and differentiation of these two clostridial species, which may result in misdiagnosis of patients.

Clostridioides difficile↗
PubMed2001

Resistance to moxifloxacin in toxigenic Clostridium difficile isolates is associated with mutations in gyrA.

Clostridium difficile is the etiological agent of antibiotic-associated colitis and the most common cause of hospital-acquired infectious diarrhea. Fluoroquinolones such as ciprofloxacin are associated with lower risks of C. difficile-associated diarrhea. In this study, we have analyzed 72 C. difficile isolates obtained from patients with different clinical courses of disease, such as toxic megacolon and relapses; the hospital environment; public places; and horses. They were investigated for their susceptibilities to moxifloxacin (MXF), metronidazole (MEO), and vancomycin (VAN). Mutants highly resistant to fluoroquinolones were selected in vitro by stepwise exposure to increasing concentrations of MXF. The resulting mutants were analyzed for the presence of mutations in the quinolone resistance-determining regions of DNA gyrase (gyrA), the production of toxins A and B, and the epidemiological relationship of these isolates. These factors were also investigated using PCR-based methods. All strains tested were susceptible to MEO and VAN. Twenty-six percent of the clinical isolates (19 of 72) were highly resistant to MXF (MIC > or = 16 microg/ml). Fourteen of these 19 strains contained nucleotide changes resulting in amino acid substitutions at position 83 in the gyrA protein. Resistant strains selected in vitro did not contain mutations at that position. These findings indicate that resistance to MXF in a majority of cases may be due to amino acid substitution in the gyrA gene.

Anti-Infective Agents↗
PubMed2001

Molecular typing methods for the epidemiological identification of Clostridium difficile strains.

Toxigenic Clostridium difficile is the etiologic agent of C. difficile-associated diarrhea (CDAD), the most common cause of nosocomial diarrhea. Cross-infection between patients and transmission through the environment and medical personnel are important factors in the acquisition of CDAD. In order to understand differences in epidemiology and pathogenesis, a number of typing schemes have been developed. We will review the typing methods used to study the epidemiology of C. difficile infections and how they have evolved from a phenotypic identification to state of the art molecular methods, detecting genetic polymorphisms among strains. These molecular methods include PCR-based methods (arbitrarily primed-PCR [AP-PCR] and PCR ribotyping), restriction endonuclease analysis (REA) and pulse field gel electrophoresis (PFGE). The application, usefulness and feasibility of these methods are compared and discussed. Finally, the role of genomics as a tool to investigate CDAD is introduced.

Bacterial Typing Techniques↗
PubMed2000

Genotyping of Bacteroides fragilis isolates from stool specimens by arbitrarily-primed-PCR.

In order to determine genetic relatedness of Bacteroides fragilis isolates from different clinical sources, arbitrarily primed polymerase chain reaction (PCR) (AP-PCR) was used to compare 17 strains isolated from patients with inflammatory bowel disease (IBD) and 20 strains isolated from foals with diarrhea. Three reference ATCC strains were also analyzed. Eighteen unique types were identified with a 22-mer arbitrary primer (ERIC-2) among the 20 patient isolates. Types 1 (enterotoxigenic) and 9 (nonenterotoxigenic), were each found in the stools of two patients. All other isolates showed a distinct and unique DNA banding pattern indicating a high degree of genotypic variability. Eleven types were identified among the foal isolates. Type 20, a nonenterotoxigenic type, was present in 30% of the foals. No correlation was found between the human and horse isolates. No clear relationship between a disease state (diarrhea or IBD) and specific types was observed. AP-PCR will be useful as a rapid method to determine genetic relatedness and in future epidemiologic studies of diarrheal diseases due to B. fragilis.

Animals↗
PubMed2000

Persistence of an endemic (toxigenic) isolate of Clostridium difficile in the environment of a general medicine ward.

The epidemiology of Clostridium difficile-associated diarrhea (CDAD) in an endemic setting was investigated by use of DNA typing methods to determine the strain identity of C. difficile isolates. Two predominant toxigenic clones were found in the environment and accounted for 29.8% (type 1) and 15.5% (type 2) of CDAD cases, respectively. In endemic settings, the environment and cross-transmission may play a role in acquisition of CDAD.

Clostridioides difficile↗
PubMed2000

Analysis of the pathogenicity locus in Clostridium difficile strains.

The genes for Clostridium difficile toxins A and B (tcdA and tcdB) are part of a 19.6-kb pathogenicity locus (PaLoc) that includes the genes tcdD, tcdE, and tcdC. To determine whether the C. difficile PaLoc is a stable and conserved genetic unit in toxigenic strains, a multiplex polymerase chain reaction was used to analyze 50 toxigenic, 39 nontoxigenic, and 2 toxin-defective isolates. The respective amplicons were identified for tcdA-E in the toxigenic isolates; these were absent in the nontoxigenic isolates. C. difficile P-829 lacked at least a fragment of tcdD, tcdB, tcdE, and tcdC, but tcdA was present. C. difficile 8864 had deletions in the tcdA and tcdC genes. These data suggest that the PaLoc is highly stable in toxigenic C. difficile, nontoxigenic isolates lack the unit, and isolates with a defective PaLoc can still cause clinical disease. Further studies are needed to define the role of individual genes in the pathogenesis of C. difficile-associated diarrhea.

Bacterial Proteins↗
PubMed2000

The emergency department presentation of patients with active pulmonary tuberculosis.

OBJECTIVE: To determine the clinical presentation of emergency department (ED) patients with active pulmonary tuberculosis (TB). METHODS: This was a retrospective medical record review of adult patients, identified through infection control records, diagnosed as having active pulmonary TB by sputum culture over a 30-month period at an urban teaching hospital. The ED visits by these patients from one year before to one year after the initial positive sputum culture were categorized as contagious or noncontagious, using defined clinical and radiographic criteria. The medical records of patients with contagious visits to the ED were reviewed to determine chief complaint, presence of TB risk factors and symptoms, and physical examination and chest radiograph findings. RESULTS: During the study period, 44 patients with active pulmonary TB made 66 contagious ED visits. Multiple contagious ED visits were made by 12 patients (27%; 95% CI = 15% to 43%). Chief complaints were pulmonary 33% (95% CI = 22% to 46%), medical but nonpulmonary 41% (95% CI = 29% to 54%), infectious but nonpulmonary 14% (95% CI = 6% to 24%), and traumatic/orthopedic 12% (95% CI = 5% to 22%). At least one TB risk factor was identified in 57 (86%; 95% CI% = 76 to 94%) patient visits and at least one TB symptom in 51 (77%; 95% CI = 65% to 87%) patient visits. Cough was present during only 64% (95% CI = 51% to 75%) of the patient visits and hemoptysis during 8% (95% CI = 3% to 17%). Risk factors and symptoms that, if present, were likely to be detected at triage were foreign birth, homelessness, HIV positivity, hemoptysis, and chest pain. CONCLUSIONS: Patients with active pulmonary TB may have multiple ED visits, and often have nonpulmonary complaints. Tuberculosis risk factors and symptoms are usually present in these patients but often missed at ED triage. The diversity of clinical presentations among ED patients with pulmonary TB will likely make it difficult to develop and implement high-yield triage screening criteria.

Adult↗
PubMed2000

Isolation and molecular characterization of Clostridium difficile strains from patients and the hospital environment in Belarus.

Toxigenic Clostridium difficile is the most common etiologic agent of hospital-acquired diarrhea in developed countries. The role of this pathogen in nosocomial diarrhea in Eastern Europe has not been clearly established. The goal of this study was to determine the prevalence of C. difficile in patients and the hospital environment in Belarus and to characterize these isolates as to the presence of toxin genes and their molecular type. C. difficile was isolated from 9 of 509 (1.8%) patients analyzed and recovered from 28 of 1,300 (2. 1%) environmental sites cultured. A multiplex PCR assay was used to analyze the pathogenicity locus (PaLoc) of all isolates, and strain identity was determined by an arbitrarily primed PCR (AP-PCR). The targeted sequences for all the genes in the PaLoc were amplified in all C. difficile strains examined. A predominantly homogeneous group of strains was found among these isolates, with five major AP-PCR groups being identified. Eighty-three percent of environmental isolates were classified into two groups, while patient isolates grouped into three AP-PCR types, two of which were also found in the hospital environment. Although no data on the role of C. difficile infection or epidemiology of C. difficile-associated diarrhea (CDAD) in this country exist, the isolation of toxigenic C. difficile from the hospital environment suggests that this pathogen may be responsible for cases of diarrhea of undiagnosed origin and validates our effort to further investigate the significance of CDAD in Eastern Europe.

Adolescent↗
PubMed1998

Isolation of a toxin B-deficient mutant strain of Clostridium difficile in a case of recurrent C. difficile-associated diarrhea.

Clostridium difficile-associated diarrhea (CDAD) recurs in approximately 15%-20% of patients after discontinuation of metronidazole or vancomycin therapy. Most recurrences are believed to be endogenous relapses due to the persistence of spores. However, there is evidence that reinfection with a different strain is a cause of recurrence. We report the case of a patient with a history of multiple episodes of C. difficile colitis. The patient, a 56-year-old female, has had 5 years of repeated recurrences, each shortly after discontinuing vancomycin therapy. During the course of these episodes, three isolates were cultured from her stools at different times. These isolates were analyzed for the presence of toxin A and B gene sequences and genotyped by means of arbitrarily primed polymerase chain reaction (AP-PCR). The original two isolates contained the toxin A and B genes, as determined by PCR, and were of the same AP-PCR type. During her last relapse, a C. difficile strain lacking at least a portion of the toxin B gene was isolated. AP-PCR analysis of this isolate showed a different DNA banding pattern from that of the previous isolates. A vancomycin susceptibility assay revealed a slight decrease in vancomycin activity as compared with that against the prior isolate. This case demonstrates two unique features: (1) recurrent infections can be due to reinfections and (2) toxin B mutants can possibly cause CDAD. This study also raises concerns about long-term vancomycin use and the development of resistance of C. difficile to vancomycin.

Bacterial Proteins↗
PubMed1998

Detection of Bacteroides fragilis enterotoxin gene by PCR.

Bacteroides fragilis constitutes about 1% of the bacterial flora in intestines of normal humans. Enterotoxigenic strains of B. fragilis have been associated with diarrheal diseases in humans and animals. The enterotoxin produced by these isolates induces fluid changes in ligated intestinal loops and an in vitro cytotoxic response in HT-29 cells. We developed a nested PCR to detect the enterotoxin gene of B. fragilis in stool specimens. After DNA extraction, a 367-bp fragment was amplified with two outer primers. The amplicon from this reaction was subjected to a second round of amplification with a set of internal primers. With these inner primers, a 290-bp DNA fragment was obtained which was confirmed as part of the B. fragilis enterotoxin gene by Southern blotting with a nonradioactive internal probe and a chemiluminescence system. By this approach, B. fragilis enterotoxin gene sequences were detected in eight known enterotoxigenic human isolates and nine enterotoxigenic horse isolates. No amplification products were obtained from DNA extracted from 28 nonenterotoxigenic B. fragilis isolates or B. distasonis, B. thetaiotaomicron, B. uniformis, B. ovatus, Escherichia coli, or Clostridium difficile. The sensitivity of this assay allowed us to detect as little as 1 pg of enterotoxin DNA sequences or 100 to 1,000 cells of enterotoxigenic B. fragilis/g of stool. Enterotoxin production of all isolates was confirmed in vitro in HT-29 cells. A 100% correlation was obtained between enterotoxin detection by cytotoxin assay and the nested PCR assay. This rapid and sensitive assay can be used to identify enterotoxigenic B. fragilis and may be used clinically to determine the role of B. fragilis in diarrheal diseases.

Animals↗
PubMed1997

Risk factors associated with isolation of Stenotrophomonas (Xanthomonas) maltophilia in clinical specimens.

OBJECTIVE: To determine risk factors for patients whose cultures grew Stenotrophomonas maltophilia. DESIGN: Retrospective case-control study of 60 patients with cultures positive for S maltophilia, matched by specimen site to 120 controls whose cultures grew other gram-negative aerobic bacteria. SETTING: University medical center. RESULTS: S maltophilia was identified from the following sites: respiratory (36), wound (13), urinary (6), blood (4), and cerebral spinal fluid (1). By univariate analysis, cases had a higher risk of exposure than controls for ampicillin (P < .001), gentamicin (P < .001), vancomycin (P = .001), metronidazole (P = .003), piperacillin (P = .007), cefotaxime (P = .014), ceftazidime (P = .017), ciprofloxacin (P = .030), tobramycin (P = .040), and chronic respiratory disease (P = .024). Length of time foreign objects were in place prior to positive culture differed significantly between cases and controls only for endotracheal tubes in patients with respiratory isolates (median number of days: 12.5 for cases, 5 for controls; P = .007). For patients with urinary tract infections, having a urinary catheter increased the odds of infection 10 times over controls. Exposures found by multivariate analysis to be significantly more prevalent in cases than controls included ampicillin, cefotaxime, erythromycin, gentamicin, metronidazole, piperacillin, tobramycin, chronic respiratory disease, and female gender. Odds ratios were > 1 indicating higher risk for cases, except for erythromycin, which had an odds ratio < 1. CONCLUSIONS: The primary risk factor associated with isolation of S maltophilia was antibiotic use. For patients with pulmonary infections, chronic respiratory disease and length of time an endotracheal tube was in place also contributed to the risk. This suggests that judicious use of antibiotics may prevent some cases of S maltophilia infection.

Adult↗
PubMed1997

Isolation of various genotypes of Clostridium difficile from patients and the environment in an oncology ward.

The epidemiology of Clostridium difficile-associated diarrhea (CDAD) is not well defined in nonepidemic situations because precise biotyping techniques have only recently become available. Arbitrarily primed polymerase chain reaction (AP-PCR) was used to determine strain identity of C. difficile isolates recovered on our oncology ward, at an incidence rate of 0.84%. Twenty-one strains of C. difficile, which were grouped into 18 different AP-PCR types, were isolated from patients' specimens. Forty-two C. difficile isolates recovered from the environment (33 toxigenic and 9 nontoxigenic) represented 9 different AP-PCR types. The most commonly found type, a toxigenic strain accounting for 29% of the environmental isolates, was widespread throughout the ward. None of the environmental types were found among the isolates from patients. Three patients' isolates were of the same AP-PCR type, and two of these patients had occupied neighboring rooms at the same time. The diversity of C. difficile isotypes suggests that endemic nosocomial CDAD is not necessarily clonally spread.

California↗
PubMed1995

The relationship between antecedent antibiotic use and resistance to extended-spectrum cephalosporins in group I beta-lactamase-producing organisms.

Gram-negative pathogens are increasingly resistant to extended-spectrum cephalosporins (ESCs). Using a prospective, case-controlled observational study, we examined the prevalence and the risk factors for development of resistance to ESCs in group I beta-lactamase-producing organisms. Of the 386 isolates of Enterobacter species, Pseudomonas aeruginosa, Citrobacter species, and Serratia marsescens from 340 consecutive patients, 70 (18.1%) were resistant to ESCs; the highest rates of resistance were found among Citrobacter freundii (40.9%), Enterobacter cloacae (31.1%), and Enterobacter aerogenes isolates (18.9%). Patients' prior antibiotic use and the mean number of antibiotics used were significantly greater in association with resistant vs. susceptible isolates. Resistance was associated with prior use of ceftizoxime or cefotaxime (P = .008), ceftazidime (P = .004), and piperacillin (P = .001). Other antibiotics were not associated with resistance. Resistance was less frequent in patients receiving ESCs and an aminoglycoside. We conclude that prior use of ESCs is associated with the isolation of resistant group I beta-lactamase-producing organisms. Concomitant use of an aminoglycoside may decrease this risk.

Adult↗