Richard B. Thomson

The Roles of Bacteria and TLR4 in Rat and Murine Models of Necrotizing Enterocolitis

Bacteria are thought to contribute to the pathogenesis of necrotizing enterocolitis (NEC), but it is unknown whether their interaction with the epithelium can participate in the initiation of mucosal injury or they can act only following translocation across a damaged intestinal barrier. Our aims were to determine whether bacteria and intestinal epithelial TLR4 play roles in a well-established neonatal rat model and a novel neonatal murine model of NEC. Neonatal rats, C57BL/6J, C3HeB/FeJ (TLR4 wild type), and C3H/HeJ (TLR4 mutant) mice were delivered by Cesarean section and were subjected to formula feeding and cold asphyxia stress or were delivered naturally and were mother-fed. NEC incidence was evaluated by histological scoring, and gene expression was quantified using quantitative real-time PCR from cDNA generated from intestinal total RNA or from RNA obtained by laser capture microdissection. Spontaneous feeding catheter colonization or supplementation of cultured bacterial isolates to formula increased the incidence of experimental NEC. During the first 72 h of life, i.e., the time frame of NEC development in this model, intestinal TLR4 mRNA gradually decreases in mother-fed but increases in formula feeding and cold asphyxia stress, correlating with induced inducible NO synthase. TLR4, inducible NO synthase, and inflammatory cytokine induction occurred in the intestinal epithelium but not in the submucosa. NEC incidence was diminished in C3H/HeJ mice, compared with C3HeB/FeJ mice. In summary, bacteria and TLR4 play significant roles in experimental NEC, likely via an interaction of intraluminal bacteria and aberrantly overexpressed TLR4 in enterocytes.

Bifidobacterial supplementation reduces the incidence of necrotizing enterocolitis in a neonatal rat model

BACKGROUND & AIMSnNeonatal necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease of premature infants partly caused by intestinal bacterial proliferation. Because bifidobacteria are thought to reduce the risk for intestinal disturbances associated with pathogenic bacterial colonization, we hypothesized that exogenous bifidobacterial supplementation to newborn rats would result in intestinal colonization and a reduction in the incidence of neonatal NEC.nnnMETHODSnNewborn rat pups were given Bifidobacterium infantis (10(9) organisms per animal daily), Escherichia coli, or saline control and exposed to the NEC protocol consisting of formula feeding (Esbilac; 200 cal. kg(-1). day(-1)) and asphyxia (100% N(2) for 50 seconds followed by cold exposure for 10 minutes). Outcome measures included stool and intestinal microbiological evaluation, gross and histological evidence of NEC, plasma endotoxin concentration, intestinal phospholipase A(2) expression, and estimation of intestinal mucosal permeability.nnnRESULTSnBifidobacterial supplementation resulted in intestinal colonization by 24 hours and appearance in stool samples by 48 hours. Bifidobacteria-supplemented animals had a significant reduction in the incidence of NEC compared with controls and E. coli-treated animals (NEC, 7/24 B. infantis vs. 19/27 control vs. 16/23 E. coli; P < 0.01). Plasma endotoxin and intestinal phospholipase A(2) expression were lower in bifidobacteria-treated pups than in controls, supporting the role of bacterial translocation and activation of the inflammatory cascade in the pathophysiology of NEC.nnnCONCLUSIONSnIntestinal bifidobacterial colonization reduces the risk of NEC in newborn rats.

Detection of Toxigenic Clostridium difficile in Stool Samples by Real-Time Polymerase Chain Reaction for the Diagnosis of C. difficile-Associated Diarrhea

BACKGROUNDnClostridium difficile-associated diarrhea (CDAD) is the major cause of health care-associated infectious diarrhea. Current laboratory testing lacks a single assay that is sensitive, specific, and rapid. The purpose of this work was to design and validate a sensitive and specific real-time polymerase chain reaction (PCR) diagnostic test for CDAD.nnnMETHODSnThis observational validation study of a new real-time PCR assay occurred from July 2004 through April 2006 and involved the testing of 1368 stool samples. As the final validation portion of the investigation, 350 inpatients were prospectively interviewed for clinical findings for 365 episodes of diarrheal illness. Test results and clinical criteria were used to assess the performance of 4 assays.nnnRESULTSnUsing clinical criteria requiring at least 3 loose stools in 1 day as part of the reference standard for a positive test result supporting CDAD, the sensitivity, specificity, and positive and negative predictive values were 73.3%, 97.6%, 73.3%, and 97.6%, respectively, for enzyme immunoassay; 93.3%, 97.4%, 75.7%, and 99.4%, respectively, for real-time PCR; 76.7%, 97.1%, 69.7%, and 97.9%, respectively, for cell culture cytotoxin assay; and 100.0%, 95.9%, 68.2%, and 100.0%, respectively, for anaerobic culture (for toxigenic C. difficile strains). The real-time PCR and anaerobic culture assays were significantly more sensitive than the enzyme immunoassay (P<.01 to P<.05).nnnCONCLUSIONSnWith an assay turnaround time of <4 h, real-time PCR is a more sensitive and equally rapid test, compared with enzyme immunoassay, and is a feasible laboratory option to replace enzyme immunoassay for toxigenic C. difficile detection in clinical practice, as well as for use during the development of new therapeutic agents.

Role of Clinical Microbiology Laboratories in the Management and Control of Infectious Diseases and the Delivery of Health Care

Modern medicine has led to dramatic changes in infectious diseases practice. Vaccination and antibiotic therapy have benefited millions of persons. However, constrained resources now threaten our ability to adequately manage threats of infectious diseases by placing clinical microbiology services and expertise distant from the patient and their infectious diseases physician. Continuing in such a direction threatens quality of laboratory results, timeliness of diagnosis, appropriateness of treatment, effective communication, reduction of health care-associated infections, advances in infectious diseases practice, and training of future practitioners. Microbiology laboratories are the first lines of defense for detection of new antibiotic resistance, outbreaks of foodborne infection, and a possible bioterrorism event. Maintaining high-quality clinical microbiology laboratories on the site of the institution that they serve is the current best approach for managing todays problems of emerging infectious diseases and antimicrobial agent resistance by providing good patient care outcomes that actually save money.

Topical Therapy for Methicillin-Resistant Staphylococcus aureus Colonization Impact on Infection Risk

OBJECTIVEnWe evaluated the usefulness of topical decolonization therapy for reducing the risk of methicillin-resistant Staphylococcus aureus (MRSA) infection among MRSA-colonized inpatients.nnnDESIGNnRetrospective cohort study.nnnSETTING AND INTERVENTIONnThree hospitals with universal surveillance for MRSA; at their physicians discretion, colonized patients could be treated with a 5-day course of nasal mupirocin calcium 2%, twice daily, plus chlorhexidine gluconate 4% every second day.nnnPATIENTS AND METHODSnMRSA carriers were later retested for colonization (407 subjects; study 1) or followed up for development of MRSA infection (933 subjects; study 2). Multivariable methods were used to determine the impact of decolonization therapy on the risks of sustained colonization (in study 1) and MRSA infection (in study 2).nnnRESULTSnIndependent risk factors for sustained colonization included residence in a long-term care facility (odds ratio [OR], 1.8 [95% confidence interval [CI], 1.1-3.2]) and a pressure ulcer (OR, 2.3 [95% CI, 1.2-4.4]). Mupirocin at any dose decreased this risk, particularly during the 30-60-day period after therapy; mupirocin resistance increased this risk (OR, 4.1 [95% CI, 1.6-10.7]). Over a median follow-up duration of 269 days, 69 (7.4%) of 933 patients developed infection. Independent risk factors for infection were length of stay (hazard ratio [HR], 1.2 per 5 additional days [95% CI, 1.0-1.4]), chronic lung disease (HR, 1.7 [95% CI, 1.0-2.8]), and receipt of non-MRSA-active systemic antimicrobial agents (HR, 1.8 [95% CI, 1.1-3.1]). Receipt of mupirocin did not affect the risk of infection, although there was a trend toward delayed infection among patients receiving mupirocin (median time to infection, 50 vs 15.5 days; P=.06).nnnCONCLUSIONSnMupirocin-based decolonization therapy temporarily reduced the risk of continued colonization but did not decrease the risk of subsequent infection.

Direct Detection of Staphylococcus aureus from Adult and Neonate Nasal Swab Specimens Using Real-Time Polymerase Chain Reaction

Nasal carriage of Staphylococcus aureus is considered a source of subsequent infection in health care settings. Utilization of real-time polymerase chain reaction (PCR) for detection of S. aureus has the potential to dramatically affect infection control practice by rapidly identifying S. aureus-colonized patients. We developed and validated the use of real-time PCR for detection of S. aureus colonization in two patient populations. Paired nasal swabs were collected from 299 neonates and from 151 adult patients at Evanston Hospital. One swab was used for culture and the other placed into a bacterial lysis solution containing achromopeptidase. The DNA liberated was used as the template for real-time PCR with primers for the femA gene. SYBR Green was used for amplicon detection. In the neonatal population the sensitivity, specificity, predictive value positive and predictive value negative for culture and PCR was 92% versus 96%, 100% versus 100%, 100% versus 100%, and 98% versus 99%, respectively. In the adults the results were 90% versus 100%, 100% versus 98%, 100% versus 96%, and 95% versus 100%, respectively. Real-time PCR was able to detect S. aureus in 2 hours compared to 1 to 4 days for culture and provided sensitivity equal to or greater than culture.

Real-Time PCR Can Rapidly Detect Methicillin-Susceptible and Methicillin-Resistant Staphylococcus aureus Directly From Positive Blood Culture Bottles

We developed, validated, and implemented real-time polymerase chain reaction (PCR) detection of the femA gene for Staphylococcus aureus and the mecA gene for methicillin resistance directly from BACTEC (Becton Dickinson, Sparks, MD) blood culture bottles showing gram-positive cocci in clusters. For the 332 positive blood cultures tested, the assay had 100% sensitivity and specificity for identifying methicillin-susceptible (n=28) and methicillin-resistant (n=28) S aureus, and overall was 98% sensitive and 94% specific, with 3 uninterpretable test results when identification of coagulase-negative staphylococci was included. PCR detection yields rapid (2-3 hours) results and accurate identification of S aureus directly from signal-positive blood culture bottle samples.

Neonatal Sepsis Caused by Streptococcus bovis Variant (Biotype II/2): Report of a Case and Review

ABSTRACT Streptococcus bovis is an uncommon cause of infection in neonates. However, S. bovis is capable of causing fulminant neonatal sepsis or meningitis that is indistinguishable clinically from that caused by group B streptococcus. S. bovis and S. bovis variant (sometimes referred to as S. bovis biotypes I and II, respectively) are phenotypically similar but may be differentiated by expanded testing. In adults, specific associations between disease states and different biotypes of S. bovis are apparent. No data exist on possible differences or clinical relevance of neonatal infection caused by different biotypes or newer species of S. bovis. We report a 3-day-old neonate with bacteremia and meningitis caused by S. bovis variant (S. bovis biotype II/2) and review the literature.

Microbiologic Surveillance Using Nasal Cultures Alone Is Sufficient for Detection of Methicillin-Resistant Staphylococcus aureus Isolates in Neonates

ABSTRACT During an outbreak of methicillin-resistant Staphylococcus aureus (MRSA) in the neonatal intensive care units at two hospitals, we assessed several sites for detection of MRSA colonization. Nasal cultures found 32 of 33 MRSA-colonized patients (97%). Rectal cultures detected 29% of 24 MRSA-colonized patients identified by paired rectal and nasal samples and axillary samples found 22% of 9 MRSA-colonized patients identified by axillary samples paired with nasal swabs. There were no positive umbilical samples.

Left-sided endocarditis caused by Pseudomonas aeruginosa: successful treatment with meropenem and tobramycin

Medical treatment alone is rarely successful in left-sided infective endocarditis caused by Pseudomonas aeruginosa. We report the cure of such a case with high-dose meropenem in combination with tobramycin.