Will Antigen Testing Remain Relevant in the Point-of-Care Testing Environment?

Before the molecular age, cell culture was the gold standard for confirmatory diagnosis of viral and atypical infectious diseases. Typical cell culture methodologies are costly, require days (or weeks) for results, and require significant technical expertise. As a result, cell culture is impractical for timely diagnostic testing in most of the health care environments. Traditional bacterial culture methods, also have disadvantages due to the need for incubation, subsequent identification of pathogens, and significant technical expertise. This article discusses the general considerations of antigen and molecular assays and the merits and factors to consider when implementing diagnostic assays for several common pathogens.

Evaluation of cerebrospinal fluid white blood cell count criteria for use of the BioFire® FilmArray® Meningitis/Encephalitis Panel in immunocompromised and nonimmunocompromised patients.

We implemented the BioFire® FilmArray® Meningitis/Encephalitis Panel (MEP) with guidance for use based on patient age, cerebrospinal fluid (CSF) white blood cell (WBC) count and immune system status. MEPs results over 2 years (1/1/2017 to 12/31/18) were reviewed and clinical significance of positive MEP results in patients with CSF WBC ≤ 10 evaluated. Overall, 12% (51/453) of MEPs were positive with 4/184 (2%) positive in nonimmunocompromised (non-IC) with ≤ 10 CSF WBCs. Among positive results in non-IC patient with ≤10 CSF WBCs, none were judged clinically significant. Four of 6 results in immunocompromised patients with ≤10 CSF WBCs were clinically significant. Redundant testing was common and guideline adherence could have safely decreased MEPs use 41% saving >$56,000. Guideline adherence was poor and MEP use can be safely avoided in non-IC adults with <10 CSF WBC, but clinically significant results did occur in IC patients with low CSF WBC. Clinical decision support could reduce unneeded testing and result in significant cost savings.

Hypervirulent Streptococcus agalactiae septicemia in twin ex-premature infants transmitted by breast milk: report of source detection and isolate characterization using commonly available molecular diagnostic methods.

BACKGROUND: Group B Streptococcus (GBS) infections caused by Streptococcus agalactiae is a leading cause of meningitis and sepsis in neonates, with early-onset GBS symptoms emerging during the first week of life and late-onset occurring thereafter. Perinatal transmission of GBS to the neonate through the birth canal is the main factor associated with early-onset neonate infections, while less is understood about the source of late-onset infections. METHODS: In this report we describe a case of twin ex-premature infants who presented one month after birth with GBS septicemia. The mother had been appropriately screened at gestational age 35-37 weeks and laboratory methods failed to detect GBS colonization by culture or clinical molecular methods. In attempts to identify and isolate the source of GBS infection, additional surveillance swabs were collected from the mother at the time of neonate admission. Culture and a commercially available, FDA-cleared molecular PCR assay were performed. RESULTS: No GBS was detected from swabs collected from the perianal, thigh/groin or axillary areas. However, expressed breast milk and swabs from the breastmilk pump were positive by both methods. Since simultaneous culture and molecular methods which used breastmilk as a source were performed, investigators ascertained the limit of detection for GBS in breastmilk. The limit of detection was determined to be tenfold lower than that of LIM-broth enriched cultures-the FDA-approved source. Subsequent whole genome sequencing (WGS) analysis of isolates recovered from breastmilk and blood cultures from the infants demonstrated all strains were related and characterized as ST-452. Both infants responded very well to treatment and continued to have no related events or concerns at the two-year follow up appointment. CONCLUSIONS: Strain type 452 (capsular type IV) has recently emerged as a hypervirulent strain and has previously been documented as causing GBS infections in elderly populations. Antibiotic therapy resolved both mother and infant infections. Subsequent testing for the presence of GBS in breastmilk samples also showed an absence of bacteria. This is the first report of infant twins late-onset GBS infections caused by the hypervirulent S. agalactiae ST-452 with breastmilk as the source.

First Records of Established Populations of Ixodes scapularis (Acari: Ixodidae) Collected From Three Nebraska Counties.

Reported cases of Lyme disease in Nebraska have been assumed to be imported from other endemic areas. Previous surveillance efforts provided no evidence of established populations as only individual specimens of Ixodes scapularis (Say) had been collected. In the winter of 2018, adult I. scapularis were found on a dog at Two Rivers State Recreation Area, Douglas County, prompting tick collection at the site and nearby natural areas. In May 2019, all life stages of host-seeking I. scapularis were collected using dragging and flagging techniques in sites located near the Platte River in Douglas, Sarpy, and Saunders counties. This is the first documentation of established populations of I. scapularis in Nebraska.

One Size Fits All? Application of Susceptible-Dose-Dependent Breakpoints to Pediatric Patients and Laboratory Reporting.

The Clinical and Laboratory Standards Institute antimicrobial and antifungal standards define a susceptible-dose-dependent (SDD) category for certain organisms and drug combinations. Reporting MICs within the SDD category suggests that treatment success is likely with increased drug exposure. These breakpoints are based on pharmacokinetic, pharmacodynamic, and clinical outcome data from adults and not pediatric patients. This commentary aims to discuss the implications of reporting SDD interpretations for pediatric patients and recommends laboratory reporting comments.

Mumps outbreak and MMR IgG surveillance as a predictor for immunity in military trainees.

In 2017, a mumps outbreak occurred in a barrack holding 249 service members. Suspected cases were evaluated with a combination of mumps IgG, IgM, viral culture, PCR and sequencing. Seven cases were diagnosed in febrile patients presenting with parotitis or orchitis. Mumps infection was confirmed by IgM or positive PCR with 5/7 cases having notable IgG levels before infection. Sequencing confirmed mumps genotype G strain. Serum from all 249 service members collected prior to the outbreak was withdrawn from the Department of Defense (DoD) Serum Repository and the IgG values of measles, mumps and rubella determined with 20.2%, 12.3% and 9.7% service members being seronegative, respectively. No specific IgG seronegativity combination predicted IgG marker levels to another virus within the same vaccine. This paper provides additional evidence that mumps serology is not a reliable surrogate for mumps immunity and that we need better laboratory correlates to confirm immunity.

Multicenter investigation of hemostatic dysfunction in 15 dogs with acute pancreatitis.

OBJECTIVE: To characterize hemostatic profiles in dogs with acute pancreatitis. DESIGN: Prospective and observational study. SETTING: Tertiary referral centers. ANIMALS: Fifteen client-owned dogs with acute pancreatitis enrolled between December 1, 2011 and June 1, 2012. MATERIALS AND METHODS: Blood samples were collected on admission for measurement of platelet count, PCV, thromboelastography (TEG), antithrombin, prothrombin time, activated partial thromboplastin time, D-dimer, von Willebrand factor, and fibrinogen values, which were compared to reference intervals derived from healthy dogs. The Wilcoxon rank-sum test was used to test for differences in continuous variables between study subjects and reference intervals. MEASUREMENTS AND MAIN RESULTS: Dogs with acute pancreatitis were globally hypercoagulable using TEG when compared with reference intervals. Dogs with acute pancreatitis had significantly higher D-dimers (1,144 µg/L vs 251 µg/L [6264.5 vs 1374.5 nmol/L]; P = 0.001), fibrinogen (837 vs 232 mg/dL [8.37 vs 2.32 g/L]; P < 0.001), and von Willebrand factor (92.9% vs 65.1%; P = 0.02) as well as significantly lower antithrombin (85.7% vs 120%; P < 0.001) and prothrombin time values (3.8 vs 7.6 sec; P < 0.001) than reference intervals. CONCLUSIONS: Laboratory evidence of hypercoagulability was present in dogs with acute pancreatitis. TEG may be useful in dogs with acute pancreatitis for monitoring response to therapy and guiding therapeutic interventions.

A Tertiary Care Center's Experience with Novel Molecular Meningitis/Encephalitis Diagnostics and Implementation with Antimicrobial Stewardship.

Background: Novel molecular techniques, such as the Biofire FilmArray Meningitis/Encephalitis (ME) panel, are increasingly used to improve pathogen detection and time to detection (TtD). The Brooke Army Medical Center antibiotic stewardship program evaluated the impact of the ME panel on empiric antimicrobial usage. Methods: Negative ME panels were analyzed for days of therapy (DOT). The ME panel became available at Brooke Army Medical Center on January 1, 2016 and a retrospective chart review was performed on all hospitalized patients tested by ME panel through April 30, 2016. Demographic data, cerebral spinal fluid (CSF) leukocyte count, immunocompromised status, and intensive care unit admission status were collected. TtD by ME panel and CSF culture were compared and DOT for common antimicrobials were quantified. Positive ME panels were analyzed for same demographic data, diagnoses, and microbiologic workup including CSF cultures and send out polymerase chain reactions. Results: Of the 77 ME panels performed during the study period, 54 (70%) were conducted on inpatients and included in the analysis. The majority of patients were males (n = 29, 54%) and the median age was 24 yr (interquartile range [IQR] 45; range 1 d to 83 yr). A total of eight (15%) patients were immunocompromised and 17 (31%) required intensive care unit level of care. The median TtD with the ME panel and CSF culture was 2.75 (IQR 2.16, 3.64) and 68.5 (IQR 63.87, 78.37) h, respectively. For negative ME panels, the overall median DOT for antimicrobials was 3 (IQR 1.5, 4.0) d, whereas the median DOT for individual agents was 2 (IQR 1.0, 4.0) d for vancomycin (n = 15), 1.5 (IQR 1.0, 2.25) d for ceftriaxone (n = 16), 3 (IQR 3.0, 4.0) d for ampicillin (n = 15), 3.5 (IQR 2.75, 4.0) d for gentamicin (n = 8), 3.5 (IQR 2.25, 4.0) d for cefotaxime (n = 6), and 5 (IQR 3.0, 5.5) d for acyclovir (n = 7); the median CSF leukocyte is of 2 cells/mm3 (IQR 1.0, 7.5). DOT excluded cases of positive ME panels: human herpes virus-6 (n = 2), herpes simplex virus-2 (n = 3), enterovirus (n = 1), and Streptococcus pneumoniae (n = 1). Of these, there were two discordance diagnoses between ME panel and convention microbiologic methods. S. pneumonia was detected on the ME panel and not on the CSF culture. One bone marrow transplant recipient had symptoms of encephalitis caused by human herpes virus-6 detected only by the ME panel, the send out human herpes virus-6 polymerase chain reaction was negative. Conclusion: The ME panel appears to improve diagnostic yield in our facility, and there is potential for improvement in decreasing empiric antimicrobial usage, particularly in patients with a negative ME panel and absence of CSF pleocytosis. This demonstrates the need for antibiotic stewardship program involvement to assist in implementation of rapid diagnostic tests through methods such as education, clinical guidelines, and prospective audit and feedback to improve meningitis and encephalitis management.

Evaluation of red blood cell distribution width as a prognostic indicator in cats with acquired heart disease, with and without congestive heart failure.

Objectives Congestive heart failure secondary to cardiomyopathy is a common manifestation of cardiac disease in cats, carrying a variable prognosis. The objective of this retrospective study was to evaluate the relationship between red blood cell distribution width (RDW) and survival time in feline patients with acquired heart disease with and without congestive heart failure (CHF). Methods Three hundred and forty-nine client-owned cats with echocardiograms and complete blood count, including RDW measurement, performed between March 2006 and December 2011, were included in the study. Patient characteristics, including signalment, hematocrit, RDW, echocardiographic parameters and survival, were recorded. Comparisons between RDW in cats with asymptomatic acquired heart disease and those with CHF were made. Survival was documented and compared at 30 days and 6 months. Results CHF was present in 80 cats and absent in 269 cats. Cats with CHF had an increase in mortality compared with cats without CHF at 30 days and 6 months ( P = 0.007 and P = 0.04, respectively). RDW was not significantly associated with survival in cats with or without CHF at 30 days or 6 months. A significant difference was found between median RDW values in cats with CHF vs cats without CHF (16.3% vs 15.8%; P = 0.02). The median RDW value was significantly higher in cats with unclassified cardiomyopathy compared with cats with other types of cardiomyopathy (16.3% vs 15.8%; P = 0.03). Conclusions and relevance Single RDW values did not predict mortality in cats with acquired heart disease but may be useful in determining if cats have decompensated heart disease and CHF. Human studies indicate that incremental increases in serial RDW measurements are associated with decreased survival; serial RDW measurements in cats may be an area of future study.

Retrospective evaluation of the incidence and prognostic significance of spontaneous echocardiographic contrast in relation to cardiac disease and congestive heart failure in cats: 725 cases (2006-2011).

OBJECTIVE: To determine whether the presence of spontaneous echocardiographic contrast (SEC) in cats with cardiomyopathy is associated with increased mortality. To establish whether specific types of cardiomyopathy are more often associated with SEC in an attempt to provide a risk-stratification scheme for cats with increased risk of thromboembolic events. DESIGN: Retrospective study 2006-2011. SETTING: Tertiary referral and teaching hospital. ANIMALS: Seven hundred twenty-five client-owned cats undergoing echocardiographic evaluation. MEASUREMENTS AND MAIN RESULTS: Patient characteristics, including age, breed, clinical signs, type of cardiovascular disease, presence of SEC, and survival time were recorded. Thyroxine, HCT, and blood pressure were recorded when available. Among cats diagnosed with cardiac abnormalities based on echocardiographic findings, those with SEC were at significantly increased risk of death as compared to those without SEC. Cats with dilated cardiomyopathy, unclassified cardiomyopathy, and hypertrophic cardiomyopathy were significantly more likely to have SEC compared to cats with other types of cardiac disease. CONCLUSIONS: Cats with cardiomyopathy and SEC have an increased risk of death compared to cats without SEC, although other previously identified factors such as the presence of congestive heart failure and increased left atrium to aorta ratio remain important determinants of mortality. Cats with hypertrophic cardiomyopathy, unclassified cardiomyopathy, and dilated cardiomyopathy may benefit from anticoagulant therapy due to the increased risk of SEC in these subpopulations.

From the Battlefield to the Bedside: Supporting Warfighter and Civilian Health With the "ART" of Whole Genome Sequencing for Antibiotic Resistance and Outbreak Investigations.

Awareness, responsiveness, and throughput characterize an approach for enhancing the clinical impact of whole genome sequencing for austere environments and for large geographically dispersed health systems. This Department of Defense approach is informing interagency efforts linking antibiograms of multidrug-resistant organisms to their genome sequences in a public database.

Isolation of Rapidly Growing Nontuberculous Mycobacteria in Wounds Following Combat-Related Injury.

OBJECTIVES: Rapidly growing nontuberculous mycobacteria (RGNTM) have yet to be described in combat-related injuries. This study investigates the epidemiology, clinical findings, treatment, and outcomes of RGNTM infections among combat casualties wounded in Afghanistan from 2010 to 2012. METHODS: Patients with RGNTM were identified from the Department of Defense Trauma Registry through the Trauma Infectious Disease Outcomes Study. Trauma history, surgical management, and clinical data were collected. Six isolates from patients requiring antimycobacterial therapy were sequenced. RESULTS: Seventeen cases were identified. Six cases, predominantly associated with Mycobacterium abscessus, required aggressive debridement and a median of 180 days of multidrug antimycobacterial therapy that included clofazimine. M. abscessus isolates expressed the erythromycin resistance methylase (erm(41)) gene for inducible macrolide resistance, yet there were no clinical treatment failures when macrolides were utilized in combination therapy. No clonal similarity between M. abscessus isolates was found. Eleven cases had positive wound cultures, but did not require antimycobacterial therapy. The median duration of time of injury to first detection of a RGNTM was 57 days. CONCLUSIONS: This represents the first report of RGNTM infections in war-wounded patients. RGNTM should be recognized as potential pathogens in grossly infected combat wounds. Surgical debridement and multidrug antimycobacterial therapy, when clinically indicated, was associated with satisfactory clinical outcomes.

The Challenges of Implementing Next Generation Sequencing Across a Large Healthcare System, and the Molecular Epidemiology and Antibiotic Susceptibilities of Carbapenemase-Producing Bacteria in the Healthcare System of the U.S. Department of Defense.

OBJECTIVE: We sought to: 1) provide an overview of the genomic epidemiology of an extensive collection of carbapenemase-producing bacteria (CPB) collected in the U.S. Department of Defense health system; 2) increase awareness of the public availability of the sequences, isolates, and customized antimicrobial resistance database of that system; and 3) illustrate challenges and offer mitigations for implementing next generation sequencing (NGS) across large health systems. DESIGN: Prospective surveillance and system-wide implementation of NGS. SETTING: 288-hospital healthcare network. METHODS: All phenotypically carbapenem resistant bacteria underwent CarbaNP® testing and PCR, followed by NGS. Commercial (Newbler and Geneious), on-line (ResFinder), and open-source software (Btrim, FLASh, Bowtie2, an Samtools) were used for assembly, SNP detection and clustering. Laboratory capacity, throughput, and response time were assessed. RESULTS: From 2009 through 2015, 27,000 multidrug-resistant Gram-negative isolates were submitted. 225 contained carbapenemase-encoding genes (most commonly blaKPC, blaNDM, and blaOXA23). These were found in 15 species from 146 inpatients in 19 facilities. Genetically related CPB were found in more than one hospital. Other clusters or outbreaks were not clonal and involved genetically related plasmids, while some involved several unrelated plasmids. Relatedness depended on the clustering algorithm used. Transmission patterns of plasmids and other mobile genetic elements could not be determined without ultra-long read, single-molecule real-time sequencing. 80% of carbapenem-resistant phenotypes retained susceptibility to aminoglycosides, and 70% retained susceptibility to fluoroquinolones. However, among the CPB-confirmed genotypes, fewer than 25% retained susceptibility to aminoglycosides or fluoroquinolones. CONCLUSION: Although NGS is increasingly acclaimed to revolutionize clinical practice, resource-constrained environments, large or geographically dispersed healthcare networks, and military or government-funded public health laboratories are likely to encounter constraints and challenges as they implement NGS across their health systems. These include lack of standardized definitions and quality control metrics, limitations of short-read sequencing, insufficient bandwidth, and the current limited availability of very expensive and scarcely available sequencing platforms. Possible solutions and mitigations are also proposed.

Administration of fenoldopam in critically ill small animal patients with acute kidney injury: 28 dogs and 34 cats (2008-2012).

OBJECTIVE: To describe the clinical features and outcomes of critically ill dogs and cats with acute kidney injury (AKI) receiving fenoldopam infusions compared to patients with AKI that did not receive fenoldopam. DESIGN: Retrospective clinical study from May 1, 2008 until June 1, 2012. SETTING: Private emergency and specialty referral hospital. ANIMALS: Client-owned dogs (28) and cats (34) with AKI that received fenoldopam compared with similar patients with AKI (30 dogs and 30 cats) that did not. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The medical records of 62 critically ill dogs and cats with AKI that received fenoldopam were reviewed. Presenting clinical signs, physical examination findings, and primary and secondary disease processes were identified in all patients. The mean number of days on fenoldopam was 1.5 days (range 0.3-4.0 days) for dogs and 1.9 days (range 1.0-4.0 days) for cats. Eleven of 28 (39%) dogs survived to discharge and 13 of 34 (38%) of the cats survived to discharge. Of the animals in the group receiving fenoldopam that died, the majority (84%) were euthanized. Potential adverse reactions were evaluated, with hypotension being the most commonly encountered adverse effect (7% of fenoldopam group [FG] dogs and 23% of FG cats). When compared with patients with AKI that did not receive fenoldopam, no significant differences were found between the groups with regards to survival, length of hospital stay, adverse effects, or changes in creatinine, BUN, or sodium concentrations except that patients receiving fenoldopam were significantly more likely to have received other renally active medications. CONCLUSIONS: In this study of patients with AKI, fenoldopam administration at 0.8 µg/kg/min in dogs and 0.5 µg/kg/min in cats appeared relatively safe but was not associated with improvement in survival to discharge, length of hospital stay, or improvement in renal biochemical parameters when compared to patients with AKI not receiving fenoldopam.

Carbapenem-resistant Enterobacteriaceae and the correlation between carbapenem and fluoroquinolone usage and resistance in the US military health system.

Whether carbapenem or fluoroquinolone usage is correlated with carbapenem-resistant Enterobacteriaceae (CRE) has not been investigated at the level of an entire US nationwide managed health care system. We analyzed 75 million person-years of surveillance and 1,969,315 cultures from all 266 hospitals in the geographically dispersed US military health system. Incidences of CRE remained under 1 case per 100,000 person-years. Incidences of CRE increased relative to 2005 baseline levels in 3 of 7 subsequent years, then decreased in 2012 (P<0.05). Incident proportions of carbapenem resistance (CR) differed significantly among years, geographical regions, and bacterial species. Although use and resistance strongly correlated (R>0.80) for several "drug-bug" combinations, none were significant at the national or facility level. One exception was that inpatient consumption of fluoroquinolones was significantly correlated (P=0.0007) with CR in Escherichia coli when data from the major referral centers of the Southern and Northern regions were combined.

IS5 element integration, a novel mechanism for rapid in vivo emergence of tigecycline nonsusceptibility in Klebsiella pneumoniae.

Tigecycline nonsusceptibility is concerning because tigecycline is increasingly relied upon to treat carbapenem- or colistin-resistant organisms. In Enterobacteriaceae, tigecycline nonsusceptibility is mediated by the AcrAB-TolC efflux pump, among others, and pump activity is often a downstream effect of mutations in their transcriptional regulators, cognate repressor genes, or noncoding regions, as demonstrated in Enterobacteriaceae and Acinetobacter isolates. Here, we report the emergence of tigecycline nonsusceptibility in a longitudinal series of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Klebsiella pneumoniae isolates collected during tigecycline therapy and the elucidation of its resistance mechanisms. Clinical isolates were recovered prior to and during tigecycline therapy of a 2.5-month-old Honduran neonate. Antimicrobial susceptibility tests to tigecycline determined that the MIC increased from 1 to 4 µg/ml prior to the completion of tigecycline therapy. Unlike other studies, we did not find increased expression of ramA, ramR, oqxA, acrB, marA, or rarA genes by reverse transcription-quantitative PCR (qRT-PCR). Whole-genome sequencing revealed an IS5 insertion element in nonsusceptible isolates 85 bp upstream of a putative efflux pump operon, here named kpgABC, previously unknown to be involved in resistance. Introduction of the kpgABC genes in a non-kpgABC background increased the MIC of tigecycline 4-fold and is independent of a functional AcrAB-TolC pump. This is the first report to propose a function for kpgABC and identify an insertion element whose presence correlated with the in vivo development of tigecycline nonsusceptibility in K. pneumoniae.

Physiological and transcriptional responses to osmotic stress of two Pseudomonas syringae strains that differ in epiphytic fitness and osmotolerance.

The foliar pathogen Pseudomonas syringae is a useful model for understanding the role of stress adaptation in leaf colonization. We investigated the mechanistic basis of differences in the osmotolerance of two P. syringae strains, B728a and DC3000. Consistent with its higher survival rates following inoculation onto leaves, B728a exhibited superior osmotolerance over DC3000 and higher rates of uptake of plant-derived osmoprotective compounds. A global transcriptome analysis of B728a and DC3000 following an osmotic upshift demonstrated markedly distinct responses between the strains; B728a showed primarily upregulation of genes, including components of the type VI secretion system (T6SS) and alginate biosynthetic pathways, whereas DC3000 showed no change or repression of orthologous genes, including downregulation of the T3SS. DC3000 uniquely exhibited improved growth upon deletion of the biosynthetic genes for the compatible solute N-acetylglutaminylglutamine amide (NAGGN) in a minimal medium, due possibly to NAGGN synthesis depleting the cellular glutamine pool. Both strains showed osmoreduction of glnA1 expression, suggesting that decreased glutamine synthetase activity contributes to glutamate accumulation as a compatible solute, and both strains showed osmoinduction of 5 of 12 predicted hydrophilins. Collectively, our results demonstrate that the superior epiphytic competence of B728a is consistent with its strong osmotolerance, a proactive response to an osmotic upshift, osmoinduction of alginate synthesis and the T6SS, and resiliency of the T3SS to water limitation, suggesting sustained T3SS expression under the water-limited conditions encountered during leaf colonization.

Compartment syndrome: pathophysiology, clinical presentations, treatment, and prevention in human and veterinary medicine.

OBJECTIVE: To review the human and veterinary literature pertaining to all forms of compartment syndrome (CS). DATA SOURCES: Data sources included scientific reviews and original research publications from the human and veterinary literature. HUMAN DATA SYNTHESIS: While CS affecting the extremities has been recognized in people for decades, other forms of CS in the abdominal and thoracic cavities are recently gaining more attention. The role of CS in critically ill people is a rapidly growing area of interest. More research on prevention and treatment of CS is being conducted in people because some studies have found mortality rates as high as 80% for those suffering from these conditions. VETERINARY DATA SYNTHESIS: While a significant amount of experimental studies of CS have been performed on small animals, there is a marked lack of primary veterinary studies. The majority of the veterinary literature includes case reports and series, and many of these studies were published over a decade ago. However, the increased recognition of CS in people has sparked an interest in veterinary critical care medicine and this has been demonstrated by the recent increased evaluation of compartment pressures in veterinary patients. CONCLUSIONS: CS is a complex clinical condition where increased pressure within a compartment can cause significant adverse effects within the compartment as well as throughout the body. Systemic inflammatory responses and local ischemia-reperfusion elements can contribute to the detrimental effects seen in CS. This cascade of events results in increased mortality rates and contributes to the development of CS elsewhere. A better understanding of CS will help veterinarians improve patient care and outcome. Future studies on incidence, prevention, and treatment of CSs in the critical care patient are needed in veterinary medicine.

Cell-cell and cell-surface interactions mediated by cellulose and a novel exopolysaccharide contribute to Pseudomonas putida biofilm formation and fitness under water-limiting conditions.

The composition of the exopolysaccharide matrix of Pseudomonas putida mt2 biofilms is relatively undefined as well as the contributions of each polymer to ecological fitness. Here, we describe the role of two putative exopolysaccharide gene clusters, putida exopolysaccharide A (pea) and bacterial cellulose (bcs) in biofilm formation and stability, rhizosphere colonization and matrix hydration under water-limiting conditions. Our findings suggest that pea is involved in the production of a novel glucose, galactose, and mannose-rich polymer that contributes to cell-cell interactions necessary for pellicle and biofilm formation and stability. In contrast, Bcs plays a minor role in biofilm formation and stability, although it does contribute to rhizosphere colonization based on a competition assay. We show that pea expression is highly induced transiently under water-limiting conditions but only slightly by high osmolarity, as determined by qRT-PCR. In contrast, both forms of water stress highly induced bcs expression. Cells deficient in making one or more exopolysaccharide experienced greater dehydration-mediated cell-envelope stress, leading to increased alginate promoter activity. However, this did not lead to increased exopolysaccharide production, except in bcs or pea mutants unable to produce alginate, indicating that P. putida compensates by producing, presumably more Pea or Bcs exopolysaccharides, to facilitate biofilm hydration. Collectively, the data suggest that Pea and Bcs contribute to biofilm formation and in turn their presence contributes to fitness under water-limiting conditions, but not to the extent of alginate.

Transient alginate gene expression by Pseudomonas putida biofilm residents under water-limiting conditions reflects adaptation to the local environment.

Under water-limiting conditions Pseudomonas putida produces the exopolysaccharide alginate, which influences biofilm development and facilitates maintaining a hydrated microenvironment. Since alginate is a minor biofilm matrix component it is important to determine whether alginate production occurs by all or a subset of residents, and when and to what extent cells contribute to alginate production. To address these questions we employed stable and unstable fluorescent reporters to measure alginate biosynthesis (algD) operon expression and metabolic activity in vivo quantitatively by flow cytometry and visually by microscopy. Here we report that during growth under water-limiting conditions and when biofilms become dehydrated most residents transiently express the alginate biosynthesis genes leading to distinct spatial patterns as the biofilm ages. Transient alginate gene expression was not a consequence of decreased metabolic activity, since metabolic reporters were still expressed, nor was it likely due to transient cytosolic availability of the alternative sigma factor AlgT, based on qRT-PCR. Our findings also indicate that one or more biofilm attribute, other than alginate, provides protection from desiccation stress. Collectively, our findings suggest that differentiated cells dedicated to alginate production are not part of the P. putida biofilm lifestyle under water-limiting conditions. Alternatively, P. putida biofilm cells may be responding to their own local environment, producing alginate because of the fitness advantage it confers under those particular conditions.