Clinical utility of multiplex PCR in the detection of pathogens from sterile body fluids.

Rapid identification of pathogens in normally sterile body fluid (NSBF) is essential for appropriate patient management, specifically antimicrobial therapy. Limited sensitivity and increased time to detection of traditional culture prompted us to evaluate additional testing to contribute to the diagnosis of infection. The purpose of this study was to evaluate the GenMark Dx ePlex Blood Culture Identification (BCID) Panels on positive body fluids inoculated into blood culture bottles for the detection of microorganisms. A total of 88 positive body fluids from blood culture bottles were analyzed using a Gram-Positive, Gram-Negative, and/or Fungal pathogen BCID Panel based on the Gram stain result. Each result was compared to routine culture performed from the positive bottle. When using culture as a reference standard, we found the ePlex multiplex panel performed with a positive percent agreement of 96.5% and a negative percent agreement of 99.8%. The use of multiplex PCR may be a useful supplement to routine culture for NSBF in blood culture bottles. IMPORTANCE: The identification of pathogens in normally sterile body fluid (NSBF) is performed using routine culture, the current gold standard. Limitations of this method include sensitivity and increased turnaround times which could potentially delay vital patient care, especially antimicrobial therapy. Adaptations of NSBF in blood culture bottles prompted us to consider the utility of additional methods to bridge the gap in diagnostic challenges for these life-threatening infections. Multiplex molecular panels have been manufactured for use with multiple specimen types including blood, cerebral spinal fluid, stool, and respiratory. Therefore, the purpose of this study was to evaluate the off-label use of ePlex Blood Culture Identification Panels on positive body fluids grown in blood culture bottles for the detection of microorganisms for research purposes.

Characterization of initial CLABSI culture results to support antimicrobial de-escalation in pediatric GI inpatients.

OBJECTIVES: Central Line-associated Bloodstream Infections (CLABSIs) pose a serious mortality and morbidity risk. An institutional protocol was developed for the evaluation and empirical antibiotic treatment of possible CLABSIs. The potential impact of de-escalating antimicrobial therapy based on initial Gram stain and molecular identification was assessed. METHODS: All positive blood cultures from patients admitted to the gastroenterology service at a large pediatric medical center were collected from 1/1/14 to 12/31/20. Cultures that were negative, repeated, or causative organisms that were unable to be identified with susceptibility data were excluded. Timepoints and organism(s) from each culture were recorded. Polymicrobial cultures were classified as containing only gram-positive organisms (polymicrobial GP), only gram-negative organisms (polymicrobial GN), or mixed spectrum. RESULTS: During the 6-year period, 361 positive blood cultures were included in the study. Single isolates were identified in 79.5% (287/361) of cultures. Polymicrobial cultures from confirmed central line source accounted for 15.0% (54/361), with 6.4% (23/361) Polymicrobial GP, 4.4% (16/361) Polymicrobial GN, and 4.2% (15/361) being mixed-spectrum cultures. Both organism types were detected on initial gram-stain in 40% (6/15) of the mixed-spectrum cultures, another 26.7% (4/15) had the opposite-spectrum organism identified within an average of <3 h and the remaining 33.3% (5/15) had the opposite-spectrum organism identified by culture growth. CONCLUSIONS: Polymicrobial mixed-spectrum cultures accounted for <5% of positive blood cultures and most isolates were identified within 3 h of first positivity. This may allow for further investigation of early de-escalation of therapy for this population and limit antimicrobial exposure.

AB569, a nontoxic chemical tandem that kills major human pathogenic bacteria.

Antibiotic-resistant superbug bacteria represent a global health problem with no imminent solutions. Here we demonstrate that the combination (termed AB569) of acidified nitrite (A-NO2-) and Na2-EDTA (disodium ethylenediaminetetraacetic acid) inhibited all Gram-negative and Gram-positive bacteria tested. AB569 was also efficacious at killing the model organism Pseudomonas aeruginosa in biofilms and in a murine chronic lung infection model. AB569 was not toxic to human cell lines at bactericidal concentrations using a basic viability assay. RNA-Seq analyses upon treatment of P. aeruginosa with AB569 revealed a catastrophic loss of the ability to support core pathways encompassing DNA, RNA, protein, ATP biosynthesis, and iron metabolism. Electrochemical analyses elucidated that AB569 produced more stable SNO proteins, potentially explaining one mechanism of bacterial killing. Our data implicate that AB569 is a safe and effective means to kill pathogenic bacteria, suggesting that simple strategies could be applied with highly advantageous therapeutic/toxicity index ratios to pathogens associated with a myriad of periepithelial infections and related disease scenarios.

Predicting Oral Beta-lactam susceptibilities against Streptococcus pneumoniae.

BACKGROUND: Oral beta-lactam antimicrobials are not routinely tested against Streptococcus pneumoniae due to presumed susceptibility based upon penicillin minimum inhibitory concentration (MIC) testing. Currently, Clinical and Laboratory Standards Institute provides comments to use penicillin MIC ≤0.06 to predict oral cephalosporin susceptibility. However, no guidance is provided when cefotaxime MIC is known, leading to uncertainty with interpretation. The purpose of this study was to evaluate cefotaxime and penicillin MICs and their respective correlation to oral beta-lactam categorical susceptibility patterns. METHODS: 249 S. pneumoniae isolates were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-ToF) and then tested by broth microdilution method to penicillin, cefotaxime, amoxicillin, cefdinir, cefpodoxime, and cefuroxime. RESULTS: Using Clinical and Laboratory Standards Institute (CLSI) non-meningitis breakpoints for cefotaxime, 240/249 isolates were classified as susceptible. Of the cefotaxime susceptible isolates, 23% of the isolates are misrepresented as cefdinir susceptible. Amoxicillin correlated well with penicillin MIC breakpoints with only 1 discordant isolate out of 249. CONCLUSION: The correlation between amoxicillin and penicillin creates a very reliable predictor to determine categorical susceptibility. However oral cephalosporins were not well predicted by either penicillin or cefotaxime leading to the possible risk of treatment failures. Caution should be used when transitioning to oral cephalosporins in cefotaxime susceptible isolates, especially with higher cefotaxime MICs.

Chlamydia Conjunctivitis in 2 Prepubertal Children: An Uncommon Presentation of Child Sexual Abuse.

Sexually transmitted infection as the result of child sexual abuse in prepubertal children is uncommon. Chlamydia trachomatis conjunctivitis is an even less common entity in prepubertal children outside the newborn period. This report details the presentation of 2 children with conjunctivitis who were subsequently diagnosed as having C. trachomatis conjunctivitis. One child was also diagnosed as having rectal and pharyngeal C. trachomatis infection, and the other also had genital C. trachomatis infection. Even with multisite C. trachomatis infection as an indication of sexual abuse, neither child gave a detailed disclosure of abuse to account for their infections. The absence of a clear disclosure is not uncommon. Previous literature reports that a disclosure in these circumstances occurs in less than half of cases. In this report, we review the recommendations for diagnosis of C. trachomatis using nucleic acid amplification testing and culture as well as treatment. Specific clinical features should alert the clinician to C. trachomatis conjunctivitis and lead to timely diagnosis and protection of the child from further sexual abuse.

Multicenter Clinical Validation of the Molecular BD Max Enteric Viral Panel for Detection of Enteric Pathogens.

The conventional methodology for gastrointestinal pathogen detection remains time-consuming, expensive, and of limited sensitivity. The objective of this study was to evaluate the performance of the BD Max enteric viral panel (Max EVP) assay for identification of viral pathogens in stool specimens from individuals with symptoms of acute gastroenteritis, enteritis, or colitis. Prospective and archival stool specimens from adult and pediatric patients with diarrhea were collected in Cary-Blair medium or unpreserved containers. The results for specimens tested by the Max EVP (on the BD Max platform) were compared to those obtained by the reference method (alternate PCR assays, followed by bidirectional sequencing). Positive percent agreement (PPA) and negative percent agreement (NPA) were calculated. A total of 2,239 specimens were collected, with 2,148 being included for analysis. In this population, 39.6% of specimens were from outpatients, 42.1% were from patients <21 years old, and 49.7% were from females. Prevalence rates for prospective specimens were 7.3%, 4.5%, 3.5%, 2.4%, and 1.2% for norovirus, sapovirus, astrovirus, rotavirus, and adenovirus, respectively. PPA was 92.8%, 84.9%, 93.0%, 100%, and 95.6%, for norovirus, sapovirus, astrovirus, rotavirus, and adenovirus, respectively. NPA was ≥99.4% for all targets. In conjunction with the clinical presentation, laboratory findings, and epidemiological information, the Max EVP assay is effective for the differential diagnosis of enteric disease caused by norovirus, sapovirus, astrovirus, rotavirus, and adenovirus. This assay can be used individually for patients at high risk for a viral enteropathogen (e.g., in outbreak settings) or as an adjunct to other enteric bacterial panels.

The gut-liver axis: impact of a mouse model of small-bowel bacterial overgrowth.

BACKGROUND: The mechanisms by which intestinal bacteria impact liver diseases remain poorly understood. The aim of this study was to develop a mouse model of small-bowel bacterial overgrowth and to determine its impact on hepatobiliary injury. MATERIALS AND METHODS: A jejunal self-filling blind loop (SFBL) was created in C57BL/6 mice. Three weeks after surgery, the mice were euthanized, and bacterial cultures of luminal content of the loop and extraintestinal tissues were performed. Liver and jejunum were collected for histological grading of inflammation and injury. Serum liver biochemistry assays were performed. Hepatobiliary transporter mRNA expression in liver was measured by quantitative real-time polymerase chain reaction. Bile and blood were collected for measurement of total bile acids, phospholipid, and cholesterol. Mice undergoing jejunal transection and reanastomosis and laparotomy only served as control groups. RESULTS: SFBL induced a dramatic increase in intraluminal bacterial counts, mesenteric lymph node bacterial translocation, and evidence of jejunal and hepatobiliary injury. Significant reductions in hepatic expression of hepatobiliary transporters involved in biliary canalicular export and basolateral uptake were observed in SFBL mice. SFBL resulted in a significant increase in biliary total bile acid concentration, decreases in bile phospholipid and cholesterol output, and an increase in the bile acid/phospholipid ratio. CONCLUSIONS: We have developed a reproducible mouse model of small-bowel bacterial overgrowth with evidence of liver inflammation, altered hepatobiliary transporter expression, and alterations in bile composition. This model may help to elucidate the mechanisms by which gut-derived bacterial factors impact the liver and contribute to the exacerbation of liver diseases and biliary injury.

Multisite Evaluation of the BD Max Extended Enteric Bacterial Panel for Detection of Yersinia enterocolitica, Enterotoxigenic Escherichia coli, Vibrio, and Plesiomonas shigelloides from Stool Specimens.

The purpose of this study was to perform a multisite evaluation to establish the performance characteristics of the BD Max extended enteric bacterial panel (xEBP) assay directly from unpreserved or Cary-Blair-preserved stool specimens for the detection of Yersinia enterocolitica, enterotoxigenic Escherichia coli (ETEC), Vibrio, and Plesiomonas shigelloides The study included prospective, retrospective, and prepared contrived specimens from 6 clinical sites. BD Max xEBP results were compared to the reference method, which included standard culture techniques coupled with alternate PCR and sequencing, except for ETEC, for which the reference method was two alternate PCRs and sequencing. Alternate PCR was also used to confirm the historical results for the retrospective specimens and for discrepant result analysis. A total of 2,410 unformed, deidentified stool specimens were collected. The prevalence in the prospective samples as defined by the reference method was 1.2% ETEC, 0.1% Vibrio, 0% Y. enterocolitica, and 0% P. shigelloides Compared to the reference method, the positive percent agreement (PPA) (95% confidence interval [CI]), negative percent agreement (NPA) (95% CI), and kappa coefficient (95% CI) for the BD Max xEBP assay for all specimens combined were as follows: ETEC, 97.6% (87.4 to 99.6), 99.8% (99.5 to 99.9), and 0.93 (0.87 to 0.99); Vibrio, 100% (96.4 to 100), 99.7% (99.4 to 99.8), and 0.96 (0.93 to 0.99); Y. enterocolitica, 99.0% (94.8 to 99.8), 99.9% (99.8 to 99.9), and 0.99 (0.98 to 1); P. shigelloides, 100% (96.4 to 100), 99.8% (99.5 to 99.9), and 0.98 (0.95 to 1), respectively. In this multicenter study, the BD Max xEBP showed a high correlation (kappa, 0.97; 95% CI, 0.95 to 0.98) with the conventional methods for the detection of ETEC, Vibrio, Y. enterocolitica, and P. shigelloides in stool specimens from patients suspected of acute gastroenteritis, enteritis, or colitis.

Utilizing BD MAX™ Enteric Bacterial Panel to Detect Stool Pathogens from Rectal Swabs.

BACKGROUND: The BD MAX™ Enteric Bacterial Panel (BDM-EBP) is designed and FDA-cleared to detect Salmonella, Shigella, Campylobacter, and Shiga toxin genes stx1/2 from stool samples. However, rectal swabs, which are not FDA-cleared for clinical testing with the BDM-EBP, are common specimens received from pediatric patients for enteric pathogen testing. The purpose of this study was to evaluate the ability of the BDM-EBP to detect stool pathogens from rectal swabs. METHODS: Routine cultures, Shiga toxin testing, and molecular testing with BDM-EBP were performed on 272 sequential rectal swabs collected from August 2015 to December 2015. Discrepant test results were resolved using Verigene® Enteric Pathogens Nucleic Acid Test (EP). 36 challenge samples (13 Salmonella spp., 3 Shigella spp., 10 Campylobacter spp., and 10 Shiga toxin positive Escherichia coli) were tested using reference strains (American Type Culture Collection) and previous patient isolates diluted to103-104 cfu/ml in saline then added to Sample Buffer Tube (SBT) with negative stool matrix delivered via a swab. Limit of detection testing was performed by serial 10 fold dilutions in saline then added to SBT with negative stool matrix provided via a swab. RESULTS: A total of 272 rectal swab specimens were evaluated and 89 were positive by culture and/or MAX EBP. All discrepant results were BDM-EBP positive and culture negative. 21 of 31 (68%) of the apparent false positive BDM-EBP discrepant results resolved as positive with Nanosphere's Verigene® EP. After resolution of the discordant results, the Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) are as follows for each target: Salmonella (n = 4) 100%, PPA and 100%, NPA; Shigella (n = 79) 100%, PPA and 95.3%, NPA; Campylobacter (n = 4) 100%, PPA and 99.6%, NPA; and Shiga toxin producing organisms (n = 2) 100%, PPA and 100%, NPA. 8.8% of the patient samples did not initially yield a result on the BDM-System. Upon repeat, half of the problematic samples resolved, and 4.4% of the total specimen tested did not yield a result. All organisms in the challenge samples were detected. Limits of detection for BDM-EBP testing of rectal swabs were as follows (in cfu/ml in SBT): Salmonella-1.44 × 102; Shigella-5.10 × 100; Campylobacter-1.51 × 101; and Shiga Toxin-1.13 ×103. CONCLUSION: Rectal swabs are acceptable samples for detecting Salmonella, Shigella, Campylobacter, and Shiga toxin using BDM-EBP.

Use of Shotgun Metagenome Sequencing To Detect Fecal Colonization with Multidrug-Resistant Bacteria in Children.

Prevention of multidrug-resistant (MDR) bacterial infections relies on accurate detection of these organisms. We investigated shotgun metagenome sequencing for the detection of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), and MDR Enterobacteriaceae Fecal metagenomes were analyzed from high-risk inpatients and compared to those of low-risk outpatients and controls with minimal risk for a MDR bacterial infection. Principal-component analysis clustered patient samples into distinct cohorts, confirming that the microbiome composition was significantly different between cohorts (P = 0.006). Microbial diversity and relative anaerobe abundance were preserved in outpatients compared to those in controls. Relative anaerobe abundance was significantly reduced in inpatients compared to that in outpatients (P = 0.006). Although the potential for MDR bacteria was increased in inpatients and outpatients compared to that in controls (P < 0.001), there was no difference between inpatients and outpatients. However, 9 (53%) inpatients had colonization with a MDR bacterium that was not identified by culture. Unlike culture, shotgun sequencing quantitatively characterizes the burdens of multiple MDR bacteria relative to all of the microbiota within the intestinal community. We propose consideration of key microbiome features, such as diversity and relative anaerobe abundance, in addition to the detection of MDR bacteria by shotgun metagenome sequencing as a novel method that might better identify patients who are at increased risk of a MDR infection.

Extraction of total nucleic acids from bacterial isolates using the bioMérieux NucliSENS easyMAG total nucleic acid extractor.

The BioMerieux NucliSENS easyMAG total nucleic acid extractor was evaluated for use on bacterial isolates in the clinical microbiology laboratory. Forty eight isolates were extracted, yielding quantifiable amounts of DNA for all isolates. The easyMAG is appropriate for DNA extraction from bacterial isolates and will be incorporated in the clinical laboratory.

Comparison of time-motion analysis of conventional stool culture and the BD MAX™ Enteric Bacterial Panel (EBP).

BACKGROUND: Conventional bacterial stool culture is one of the more time-consuming tests in a routine clinical microbiology laboratory. In addition, less than 5 % of stool cultures yield positive results. A molecular platform, the BD MAX™ System (BD Diagnostics, Sparks, MD) offers the potential for significantly more rapid results and less hands-on time. Time-motion analysis of the BD MAX Enteric Bacterial Panel (EBP) (BD Diagnostics, Quebec, Canada) on the BD MAX System was compared to conventional stool culture in the microbiology laboratory of a tertiary care pediatric hospital. METHODS: The process impact analysis of time-motion studies of conventional cultures were compared to those of EBP with 86 stool specimens. Sample flow, hands-on time, processing steps, and overall turnaround time were determined and analyzed. Data were obtained and analyzed from both standard operating procedures and direct observation. A regression analysis was performed to ensure consistency of measurements. Time and process measurements started when the specimens were logged into the accessioning area of the microbiology laboratory and were completed when actionable results were generated. RESULTS: With conventional culture, negative culture results were available from 41:14:27 (hours:minutes:seconds) to 54:17:19; with EBP, positive and negative results were available from 2:28:40 to 3:33:39. CONCLUSIONS: This study supports the suggestion that use of the EBP to detect commonly encountered stool pathogens can result in significant time savings and a shorter time-to-result for patients with acute bacterial diarrhea.

Stachybotrys eucylindrospora isolated from foreign material following a traumatic eye injury.

Stachybotrys eucylindrospora was characterised as a new species in 2007, and we present the first report of this organism isolated from foreign material recovered from a patient. It is probable that isolates of this species have been previously identified as either Stachybotrys chartarum or Stachybotrys cylindrospora.

Application of matrix-assisted laser desorption ionization-time of flight mass spectrometry for identification of the fastidious pediatric pathogens Aggregatibacter, Eikenella, Haemophilus, and Kingella.

The accuracy of matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS) in the identification of Haemophilus, Aggregatibacter, Cardiobacterium, Eikenella, and Kingella (HACEK) species was compared to that of phenotypic methods (Remel RapID and Vitek 2). Overall, Vitek MS correctly identified more isolates, incorrectly identified fewer isolates, and failed to identify fewer isolates than both phenotypic methods.

Multicenter clinical evaluation of the illumigene group A Streptococcus DNA amplification assay for detection of group A Streptococcus from pharyngeal swabs.

Acute pharyngitis is a nonspecific symptom that can result from a number of viral or bacterial infections. For most etiologies, symptoms are self-limited and resolve without lasting effects; however, pharyngitis resulting from infection with Streptococcus pyogenes (a group A Streptococcus [GAS]) can be associated with serious sequelae, including acute rheumatic fever and acute glomerulonephritis. Rapid accurate detection of GAS in pharyngeal specimens from individuals suffering from pharyngitis aids in the management and selection of antibiotic therapy for these patients. A total of 796 pharyngeal swabs were collected at three separate clinical centers. Each specimen was analyzed using the illumigene group A strep DNA amplification assay (Meridian Bioscience Inc., Cincinnati, OH). To confirm GAS identification, the results were compared to those from direct and extracted culture methods using Gram staining and a GAS-specific latex agglutination test. Discrepant results were resolved using an alternative nucleic acid amplification test. The prevalence of culture-detected GAS in this study was 12.8% (102/796 specimens). The illumigene assay detected GAS in 74/74 direct culture-positive specimens (100% sensitivity) and 100/102 extracted culture-positive specimens (98.0% sensitivity). GAS was detected by the illumigene assay in an additional 42 specimens that were direct culture negative (94.2% specificity) and 16 specimens that were extracted culture negative (97.7% specificity). Discrepant analysis using an alternative molecular assay detected GAS nucleic acid in 13/16 (81.3%) false-positive specimens and 1/2 false-negative specimens, resulting in a final sensitivity of 99.0% and a specificity of 99.6% for the detection of GAS in pharyngeal swabs using the illumigene assay.

Invasive Malassezia pachydermatis Infection in an 8-Year-Old Child on Lipid Parenteral Nutrition.

Invasive disease due to Malassezia pachydermatis infection is uncommon but increasingly recognized in children, especially neonates on parenteral nutrition or immunocompromised children. We describe a case of Malassezia pachydermatis fungemia in a demographically distinct patient and discuss the workup and current strategies for managing this infection in the setting of a central venous catheter.

Disinfection of Neonatal Resuscitation Equipment in Low-Resource Settings: The Importance, the Reality, and Considerations for the Future.

Preventable neonatal deaths due to prematurity, perinatal events, and infections are the leading causes of under-five mortality. The vast majority of these deaths are in resource-limited areas. Deaths due to infection have been associated with lack of access to clean water, overcrowded nurseries, and improper disinfection (reprocessing) of equipment, including vital resuscitation equipment. Reprocessing has recently come to heightened attention, with the COVID-19 pandemic bringing this issue to the forefront across all economic levels; however, it is particularly challenging in low-resource settings. In 2015, Eslami et al. published a letter to the editor in Resuscitation, highlighting concerns about the disinfection of equipment being used to resuscitate newborns in Kenya. To address the issue of improper disinfection, the global health nongovernment organization PATH gathered a group of experts and, due to lack of best-practice evidence, published guidelines with recommendations for reprocessing of neonatal resuscitation equipment in low-resource areas. The guidelines follow the gold-standard principle of high-level disinfection; however, there is ongoing concern that the complexity of the guideline would make feasibility and sustainability difficult in the settings for which it was designed. Observations from hospitals in Kenya and Malawi reinforce this concern. The purpose of this review is to discuss why proper disinfection of equipment is important, why this is challenging in low-resource settings, and suggestions for solutions to move forward.

AB569, a non-toxic combination of acidified nitrite and EDTA, is effective at killing the notorious Iraq/Afghanistan combat wound pathogens, multi-drug resistant Acinetobacter baumannii and Acinetobacter spp.

Multi-drug resistant (MDR) Acinetobacter baumannii (Ab) and Acinetobacter spp. present monumental global health challenges. These organisms represent model Gram-negative pathogens with known antibiotic resistance and biofilm-forming properties. Herein, a novel, nontoxic biocide, AB569, consisting of acidified nitrite (A-NO2-) and ethylenediaminetetraacetic acid (EDTA), demonstrated bactericidal activity against all Ab and Acinetobacter spp. strains, respectively. Average fractional inhibitory concentrations (FICs) of 0.25 mM EDTA plus 4 mM A-NO2- were observed across several clinical reference and multiple combat wound isolates from the Iraq/Afghanistan wars. Importantly, toxicity testing on human dermal fibroblasts (HDFa) revealed an upper toxicity limit of 3 mM EDTA plus 64 mM A-NO2-, and thus are in the therapeutic range for effective Ab and Acinetobacter spp. treatment. Following treatment of Ab strain ATCC 19606 with AB569, quantitative PCR analysis of selected genes products to be responsive to AB569 revealed up-regulation of iron regulated genes involved in siderophore production, siderophore biosynthesis non-ribosomal peptide synthetase module (SBNRPSM), and siderophore biosynthesis protein monooxygenase (SBPM) when compared to untreated organisms. Taken together, treating Ab infections with AB569 at inhibitory concentrations reveals the potential clinical application of preventing Ab from gaining an early growth advantage during infection followed by extensive bactericidal activity upon subsequent exposures.

Sodium nitrite-mediated killing of the major cystic fibrosis pathogens Pseudomonas aeruginosa, Staphylococcus aureus, and Burkholderia cepacia under anaerobic planktonic and biofilm conditions.

A hallmark of airways in patients with cystic fibrosis (CF) is highly refractory, chronic infections by several opportunistic bacterial pathogens. A recent study demonstrated that acidified sodium nitrite (A-NO(2)(-)) killed the highly refractory mucoid form of Pseudomonas aeruginosa, a pathogen that significantly compromises lung function in CF patients (S. S. Yoon et al., J. Clin. Invest. 116:436-446, 2006). Therefore, the microbicidal activity of A-NO(2)(-) (pH 6.5) against the following three major CF pathogens was assessed: P. aeruginosa (a mucoid, mucA22 mutant and a sequenced nonmucoid strain, PAO1), Staphylococcus aureus USA300 (methicillin resistant), and Burkholderia cepacia, a notoriously antibiotic-resistant organism. Under planktonic, anaerobic conditions, growth of all strains except for P. aeruginosa PAO1 was inhibited by 7.24 mM (512 µg ml(-1) NO(2)(-)). B. cepacia was particularly sensitive to low concentrations of A-NO(2)(-) (1.81 mM) under planktonic conditions. In antibiotic-resistant communities known as biofilms, which are reminiscent of end-stage CF airway disease, A-NO(2)(-) killed mucoid P. aeruginosa, S. aureus, and B. cepacia; 1 to 2 logs of cells were killed after a 2-day incubation with a single dose of ∼15 mM A-NO(2)(-). Animal toxicology and phase I human trials indicate that these bactericidal levels of A-NO(2)(-) can be easily attained by aerosolization. Thus, in summary, we demonstrate that A-NO(2)(-) is very effective at killing these important CF pathogens and could be effective in other infectious settings, particularly under anaerobic conditions where bacterial defenses against the reduction product of A-NO(2)(-), nitric oxide (NO), are dramatically reduced.

Trimethoprim in vitro antibacterial activity is not increased by adding sulfamethoxazole for pediatric Escherichia coli urinary tract infection.

PURPOSE: The combination of trimethoprim/sulfamethoxazole is often used to treat uncomplicated urinary tract infections in children. The rationale for combining trimethoprim and sulfamethoxazole is that they may act synergistically to increase antibacterial activity. However, approximately 3% of patients show allergic reactions to sulfamethoxazole, of which some are serious (liver failure and Stevens-Johnson syndrome). We determined whether adding sulfamethoxazole is necessary to increase in vitro antibacterial activity for pediatric urinary tract infection compared to that of trimethoprim alone. MATERIALS AND METHODS: We prospectively identified 1,298 children with urinary tract infection (greater than 100,000 cfu/ml Escherichia coli) from a total of 4 American regions. In vitro susceptibility of bacterial isolates to sulfamethoxazole, trimethoprim and trimethoprim/sulfamethoxazole was determined using disk diffusion. Ampicillin susceptibility was tested at 2 sites. At 1 site all uropathogens from consecutive urinary isolates were evaluated. RESULTS: E. coli susceptibility to trimethoprim was 70%, comparable to the 70% of trimethoprim/sulfamethoxazole (p = 0.9) and higher than the 56.9% of sulfamethoxazole (p <0.05). This susceptibility pattern was without regional differences. At 2 sites susceptibility to trimethoprim was significantly higher than to ampicillin. At 1 site the susceptibility of other uropathogens to trimethoprim and trimethoprim/sulfamethoxazole was similar to that of E. coli. CONCLUSIONS: In children with urinary tract infection in vitro susceptibility to trimethoprim was comparable to that to trimethoprim/sulfamethoxazole and significantly higher than to sulfamethoxazole. This finding was similar at all sites. Adding sulfamethoxazole appears unnecessary and may represent a risk to patients. Trimethoprim can be used as an alternative to trimethoprim/sulfamethoxazole based on in vitro antibacterial susceptibility. Routine trimethoprim/sulfamethoxazole use for urinary tract infection should be carefully reevaluated.