Multi-laboratory Validation of a New Marine Biodegradation Screening Test for Chemical Persistence Assessment.

Current biodegradation screening tests are not specifically designed for persistence assessment of chemicals, often show high inter- and intra-test variability, and often give false negative biodegradation results. Based on previous studies and recommendations, an international ring test involving 13 laboratories validated a new test method for marine biodegradation with a focus on improving the reliability of screening to determine the environmental degradation potential of chemicals. The new method incorporated increased bacterial cell concentrations to better represent the microbial diversity; a chemical is likely to be exposed in the sampled environments and ran beyond 60 days, which is the half-life threshold for chemical persistence in the marine environment. The new test provided a more reliable and less variable characterization of the biodegradation behavior of five reference chemicals (sodium benzoate, triethanolamine, 4-nitrophenol, anionic polyacrylamide, and pentachlorophenol), with respect to REACH and OSPAR persistence thresholds, than the current OECD 306 test. The proposed new method provides a cost-effective screening test for non-persistence that could streamline chemical regulation and reduce the cost and animal welfare implications of further higher tier testing.

Elucidation of the RamA regulon in Klebsiella pneumoniae reveals a role in LPS regulation.

Klebsiella pneumoniae is a significant human pathogen, in part due to high rates of multidrug resistance. RamA is an intrinsic regulator in K. pneumoniae established to be important for the bacterial response to antimicrobial challenge; however, little is known about its possible wider regulatory role in this organism during infection. In this work, we demonstrate that RamA is a global transcriptional regulator that significantly perturbs the transcriptional landscape of K. pneumoniae, resulting in altered microbe-drug or microbe-host response. This is largely due to the direct regulation of 68 genes associated with a myriad of cellular functions. Importantly, RamA directly binds and activates the lpxC, lpxL-2 and lpxO genes associated with lipid A biosynthesis, thus resulting in modifications within the lipid A moiety of the lipopolysaccharide. RamA-mediated alterations decrease susceptibility to colistin E, polymyxin B and human cationic antimicrobial peptide LL-37. Increased RamA levels reduce K. pneumoniae adhesion and uptake into macrophages, which is supported by in vivo infection studies, that demonstrate increased systemic dissemination of ramA overexpressing K. pneumoniae. These data establish that RamA-mediated regulation directly perturbs microbial surface properties, including lipid A biosynthesis, which facilitate evasion from the innate host response. This highlights RamA as a global regulator that confers pathoadaptive phenotypes with implications for our understanding of the pathogenesis of Enterobacter, Salmonella and Citrobacter spp. that express orthologous RamA proteins.

Exposure of Salmonella enterica Serovar Typhimurium to Three Humectants Used in the Food Industry Induces Different Osmoadaptation Systems.

Common salt (NaCl) is frequently used by the food industry to add flavor and to act as a humectant in order to reduce the water content of a food product. The improved health awareness of consumers is leading to a demand for food products with reduced salt content; thus, manufacturers require alternative water activity-reducing agents which elicit the same general effects as NaCl. Two examples include KCl and glycerol. These agents lower the water activity of a food matrix and also contribute to limit the growth of the microbiota, including foodborne pathogens. Little is currently known about how foodborne pathogens respond to these water activity-lowering agents. Here we examined the response of Salmonella enterica serovar Typhimurium 4/74 to NaCl, KCl, and glycerol at three time points, using a constant water activity level, compared with the response of a control inoculum. All conditions induced the upregulation of gluconate metabolic genes after 6 h of exposure. Bacteria exposed to NaCl and KCl demonstrated the upregulation of the osmoprotective transporter mechanisms encoded by the proP, proU, and osmU (STM1491 to STM1494) genes. Glycerol exposure elicited the downregulation of these osmoadaptive mechanisms but stimulated an increase in lipopolysaccharide and membrane protein-associated genes after 1 h. The most extensive changes in gene expression occurred following exposure to KCl. Because many of these genes were of unknown function, further characterization may identify KCl-specific adaptive processes that are not stimulated by NaCl. This study shows that the response of S. Typhimurium to different humectants does not simply reflect reduced water activity and likely involves systems that are linked to specific humectants.

Elucidating the regulon of multidrug resistance regulator RarA in Klebsiella pneumoniae.

RarA is an AraC-type regulator in Klebsiella pneumoniae, which, when overexpressed, confers a low-level multidrug-resistant (MDR) phenotype linked to the upregulation of both the acrAB and oqxAB efflux genes. Increased rarA expression has also been shown to be integral in the development of tigecycline resistance in the absence of ramA in K. pneumoniae. Given its phenotypic role in MDR, microarray analyses were performed to determine the RarA regulon. Transcriptome analysis was undertaken using strains Ecl8ΔrarA/pACrarA-2 (rarA-expressing construct) and Ecl8ΔrarA/pACYC184 (vector-only control) using bespoke microarray slides consisting of probes derived from the genomic sequences of K. pneumoniae MGH 78578 (NC_009648.1) and Kp342 (NC_011283.1). Our results show that rarA overexpression resulted in the differential expression of 66 genes (42 upregulated and 24 downregulated). Under the COG (clusters of orthologous groups) functional classification, the majority of affected genes belonged to the category of cell envelope biogenesis and posttranslational modification, along with genes encoding the previously uncharacterized transport proteins (e.g., KPN_03141, sdaCB, and leuE) and the porin OmpF. However, genes associated with energy production and conversion and amino acid transport/metabolism (e.g., nuoA, narJ, and proWX) were found to be downregulated. Biolog phenotype analyses demonstrated that rarA overexpression confers enhanced growth of the overexpresser in the presence of several antibiotic classes (i.e., beta-lactams and fluoroquinolones), the antifungal/antiprotozoal compound clioquinol, disinfectants (8-hydroxyquinoline), protein synthesis inhibitors (i.e., minocycline and puromycin), membrane biogenesis agents (polymyxin B and amitriptyline), DNA synthesis (furaltadone), and the cytokinesis inhibitor (sanguinarine). Both our transcriptome and phenotypic microarray data support and extend the role of RarA in the MDR phenotype of K. pneumoniae.

ProP is required for the survival of desiccated Salmonella enterica serovar typhimurium cells on a stainless steel surface.

Consumers trust commercial food production to be safe, and it is important to strive to improve food safety at every level. Several outbreaks of food-borne disease have been caused by Salmonella strains associated with dried food. Currently we do not know the mechanisms used by Salmonella enterica serovar Typhimurium to survive in desiccated environments. The aim of this study was to discover the responses of S. Typhimurium ST4/74 at the transcriptional level to desiccation on a stainless steel surface and to subsequent rehydration. Bacterial cells were dried onto the same steel surfaces used during the production of dry foods, and RNA was recovered for transcriptomic analysis. Subsequently, dried cells were rehydrated and were again used for transcriptomic analysis. A total of 266 genes were differentially expressed under desiccation stress compared with a static broth culture. The osmoprotectant transporters proP, proU, and osmU (STM1491 to STM1494) were highly upregulated by drying. Deletion of any one of these transport systems resulted in a reduction in the long-term viability of S. Typhimurium on a stainless steel food contact surface. The proP gene was critical for survival; proP deletion mutants could not survive desiccation for long periods and were undetectable after 4 weeks. Following rehydration, 138 genes were differentially expressed, with upregulation observed for genes such as proP, proU, and the phosphate transport genes (pstACS). In time, this knowledge should prove valuable for understanding the underlying mechanisms involved in pathogen survival and should lead to improved methods for control to ensure the safety of intermediate- and low-moisture foods.

Epidemiology of Platelet Transfusions in Hospitalized Children: A Pediatric Hospital Information System Database Study.

OBJECTIVES: To describe the epidemiology and complications of platelet transfusions among hospitalized pediatric patients during 2010 to 2019. METHODS: We performed a retrospective cohort study of hospitalized children within the Pediatric Health Information System database. Pediatric encounters receiving at least one platelet transfusion during hospitalization from 2010 to 2019 were identified. Data regarding demographics, diagnoses, procedures required during hospitalization, complications, and outcomes were extracted for eligible encounters. RESULTS: Within the Pediatric Health Information System database, 6 284 264 hospitalizations occurred from 2010 to 2019. A total of 244 464 hospitalizations required at least one platelet transfusion, yielding a prevalence of 3.89% (95% confidence interval [CI], 3.87%-3.91%). Transfusion prevalence did not change significantly across the decade (P value = .152). Two-thirds of children receiving platelet transfusions were in their first 6 years of life, and the majority identified as male (55%). Recipients most commonly had diseases of the circulatory system (21%, 52 008 of 244 979), perinatal disorders (16%, 38 054 of 244 979), or diseases of the hematologic/immune systems (15%, 37 466 of 244 979). When adjusted for age, support by extracorporeal membrane oxygenation, mechanical ventilation, surgical intervention, and diagnostic category, the odds of thrombosis, infection, and mortality increased by 2% (odds ratio [OR], 1.02; 95% CI, 1.016-1.020), 3% (OR, 1.03; 95% CI, 1.028-1.033), and 7% (OR, 1.07; 95% CI, 1.067-1.071), respectively, with each additional transfusion. CONCLUSIONS: The prevalence of platelet transfusions among pediatric inpatients remained consistent across the decade. Our finding that increasing numbers of transfusions may be associated with elevated morbidity and mortality is consistent with other observation and experimental studies, highlighting the need to be thoughtful in weighing risks and benefits when prescribing repeated platelet transfusions to hospitalized children.

Corrigendum: Molecular Characterization of Salmonella Serovars Anatum and Ealing Associated with Two Historical Outbreaks, Linked to Contaminated Powdered Infant Formula.

[This corrects the article on p. 1664 in vol. 7, PMID: 27818652.].

Molecular Characterization of Salmonella Serovars Anatum and Ealing Associated with Two Historical Outbreaks, Linked to Contaminated Powdered Infant Formula.

Powdered infant formula (PIF) is not intended to be produced as a sterile product unless explicitly stated and on occasion may become contaminated during production with pathogens such as Salmonella enterica. This retrospective study focused on two historically reported salmonellosis outbreaks associated with PIF from the United Kingdom and France, in 1985 and 1996/1997. In this paper, the molecular characterization of the two outbreaks associated Salmonella serovars Anatum and Ealing is reported. Initially the isolates were analyzed using pulsed-field gel electrophoresis (PFGE), which revealed the clonal nature of the two outbreaks. Following from this two representative isolates, one from each serovar was selected for whole genome sequencing (WGS), wherein analysis focused on the Salmonella pathogenicity islands. Furthermore, the ability of these isolates to survive the host intercellular environment was determined using an ex vivo gentamicin protection assay. Results suggest a high level of genetic diversity that may have contributed to survival and virulence of isolates from these outbreaks.

Immune thrombocytopenic purpura in ulcerative colitis: a case report and systematic review.

Over 100 extraintestinal manifestations are reported in ulcerative colitis (UC). A commonly reported hematological manifestation is autoimmune hemolytic anemia. On rare occasions, immune thrombocytopenic purpura (ITP) has been reported with UC. The presence of thrombocytopenia can complicate the clinical scenario as the number of bloody bowel movements is an important indicator of disease activity in UC. A proposed theory for this association is antigenic mimicry between a platelet surface antigen and bacterial glycoprotein. We are reporting a case of UC and associated ITP managed successfully with anti-TNF therapy. We also performed a systemic review of case reports and a case series reporting this association.

Comparative analysis of Salmonella susceptibility and tolerance to the biocide chlorhexidine identifies a complex cellular defense network.

Chlorhexidine is one of the most widely used biocides in health and agricultural settings as well as in the modern food industry. It is a cationic biocide of the biguanide class. Details of its mechanism of action are largely unknown. The frequent use of chlorhexidine has been questioned recently, amidst concerns that an overuse of this compound may select for bacteria displaying an altered susceptibility to antimicrobials, including clinically important anti-bacterial agents. We generated a Salmonella enterica serovar Typhimurium isolate (ST24(CHX)) that exhibited a high-level tolerant phenotype to chlorhexidine, following several rounds of in vitro selection, using sub-lethal concentrations of the biocide. This mutant showed altered suceptibility to a panel of clinically important antimicrobial compounds. Here we describe a genomic, transcriptomic, proteomic, and phenotypic analysis of the chlorhexidine tolerant S. Typhimurium compared with its isogenic sensitive progenitor. Results from this study describe a chlorhexidine defense network that functions in both the reference chlorhexidine sensitive isolate and the tolerant mutant. The defense network involved multiple cell targets including those associated with the synthesis and modification of the cell wall, the SOS response, virulence, and a shift in cellular metabolism toward anoxic pathways, some of which were regulated by CreB and Fur. In addition, results indicated that chlorhexidine tolerance was associated with more extensive modifications of the same cellular processes involved in this proposed network, as well as a divergent defense response involving the up-regulation of additional targets such as the flagellar apparatus and an altered cellular phosphate metabolism. These data show that sub-lethal concentrations of chlorhexidine induce distinct changes in exposed Salmonella, and our findings provide insights into the mechanisms of action and tolerance to this biocidal agent.

Mechanisms of survival, responses and sources of Salmonella in low-moisture environments.

Some Enterobacteriaceae possess the ability to survive in low-moisture environments for extended periods of time. Many of the reported food-borne outbreaks associated with low-moisture foods involve Salmonella contamination. The control of Salmonella in low-moisture foods and their production environments represents a significant challenge for all food manufacturers. This review summarizes the current state of knowledge with respect to Salmonella survival in intermediate- and low-moisture food matrices and their production environments. The mechanisms utilized by this bacterium to ensure their survival in these dry conditions remain to be fully elucidated, however, in depth transcriptomic data is now beginning to emerge regarding this observation. Earlier research work described the effect(s) that low-moisture can exert on the long-term persistence and heat tolerance of Salmonella, however, data are also now available highlighting the potential cross-tolerance to other stressors including commonly used microbicidal agents. Sources and potential control measures to reduce the risk of contamination will be explored. By extending our understanding of these geno- and phenotypes, we may be able to exploit them to improve food safety and protect public health.

Phenotypic characterization of Salmonella isolated from food production environments associated with low-water activity foods.

Salmonella can survive for extended periods of time in low-moisture environments posing a challenge for modern food production. This dangerous pathogen must be controlled throughout the production chain with a minimal risk of dissemination. Limited information is currently available describing the behavior and characteristics of this important zoonotic foodborne bacterium in low-moisture food production environments and in food. In our study, the phenotypes related to low-moisture survival of 46 Salmonella isolates were examined. Most of the isolates in the collection could form biofilms under defined laboratory conditions, with 57% being positive for curli fimbriae production and 75% of the collection positive for cellulose production, which are both linked with stronger biofilm formation. Biocides in the factory environment to manage hygiene were found to be most effective against planktonic cells but less so when the same bacteria were surface dried or present as a biofilm. Cellulose-producing isolates were better survivors when exposed to a biocide compared with cellulose-negative isolates. Examination of Salmonella growth of these 18 serotypes in NaCl, KCl, and glycerol found that glycerol was the least inhibitory of these three humectants. We identified a significant correlation between the ability to survive in glycerol and the ability to survive in KCl and biofilm formation, which may be important for food safety and the protection of public health.