The presence of antibiotic resistance genes in coastal soil and sediment samples from the eastern seaboard of the USA.

Infections from antibiotic resistant microorganisms are considered to be one of the greatest global public health challenges that result in huge annual economic losses. While genes that impart resistance to antibiotics (AbR) existed long before the discovery and use of antibiotics, anthropogenic uses of antibiotics in agriculture, domesticated animals, and humans are known to influence the prevalence of these genes in pathogenic microorganisms. It is critical to understand the role that natural and anthropogenic processes have on the occurrence and distribution of antibiotic resistance in microbial populations to minimize health risks associated with exposures. As part of this research, 15 antibiotic resistance genes were analyzed in coastal sediments and soils along the eastern seaboard of the USA using presence/absence quantitative and digital polymerase chain reaction assays. Samples (53 soil and 192 sediment samples including 54 replicates) were collected from a variety of coastal settings where human and wildlife exposure is likely. At least one of the antibiotic resistance genes was detected in 76.4% of the samples. Samples that contained at least five or more antibiotic resistance genes (5.7%) where typically hydrologically down gradient of watersheds influenced by combined sewer outfalls (CSO). The most frequently detected antibiotic resistance target genes were found in 33.2%, 34.4%, and 42.2% of samples (target genes blaSHV, tetO, and aadA2, respectively). These data provide unique insight into potential exposure of AbR genes over a large geographical region of the eastern seaboard of the USA.

Molecular characterization of non-aureus Staphylococcus spp. from heifer intramammary infections and body sites.

The purpose of this study was to investigate non-aureus Staphylococcus spp. intramammary infections (IMI) in periparturient heifers and determine the relationship of precalving body site isolation with precalving IMI and postcalving IMI using molecular speciation and strain-typing methods. Primiparous heifers were enrolled at approximately 14 d before expected calving date. Precalving mammary quarter secretions and body site swabbing samples (teat skin, inguinal skin, muzzle, and perineum) were collected. Postcalving, mammary quarter milk samples were collected for culture and somatic cell counting. Precalving body site samples were cultured, and up to 10 staphylococcal colonies were saved for characterization. Staphylococcal isolates were speciated using matrix-assisted laser/desorption ionization time-of-flight mass spectrometry or sequencing of rpoB or tuf. Pulsed-field gel electrophoresis was used to strain type a subset of isolates. Overall, Staphylococcus chromogenes, Staphylococcus agnetis, and Staphylococcus simulans were the most common species identified in precalving mammary secretions, whereas S. chromogenes, Staphylococcus xylosus, and S. agnetis were the most common species found in postcalving milk samples. The most common species identified from body site samples were S. chromogenes, S. xylosus, and Staphylococcus haemolyticus. Mammary quarters that had a precalving mammary secretion that was culture positive for S. agnetis, S. chromogenes, or Staphylococcus devriesei had increased odds of having an IMI with the same species postcalving. A S. chromogenes IMI postcalving was associated with higher somatic cell count when compared with postcalving culture-negative quarters. Among heifers identified with a non-aureus Staphylococcus spp. IMI either precalving or postcalving, heifers that had S. agnetis or S. chromogenes isolated from their teat skin had increased odds of having the same species found in their precalving mammary secretions, and heifers with S. chromogenes, S. simulans, and S. xylosus isolated from their teat skin precalving were at increased odds of having an IMI with the same species postcalving. Overall, 44% of all heifers with a S. chromogenes IMI around the time of parturition had the same strain isolated from a body site. Based on pulsed-field gel electrophoresis, a high level of strain diversity was found.

Cross-sectional study to identify staphylococcal species isolated from teat and inguinal skin of different-aged dairy heifers.

The purpose of this study was to describe the prevalence and distribution of staphylococcal species on the teat and inguinal skin of dairy heifers across the various stages of the heifer life cycle. The cross-sectional study included 106 Holstein heifers with an age range of 0 d to 27 mo that were selected from 11 different groups, based on housing type and age, on a single dairy operation. A composite swabbing sample including all 4 teats and a second composite sample including both inguinal regions of each heifer were collected using gas-sterilized electrostatic dusters (Swiffers; Procter and Gamble, Cincinnati, OH). Swabbing samples were mixed with 10 mL of sterile saline, agitated, and cultured on mannitol salt agar plates. At 24 h, plates were read and up to 10 staphylococcal colonies were saved for further analysis. Staphylococcal isolates were speciated using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or PCR amplification and partial sequencing of rpoB or tuf. The prevalence of staphylococci was compared between the inguinal and teat regions using the chi-squared or Fisher's exact test, as applicable. Logistic regression models were used to investigate the relationship between a heifer's age (treated as a quantitative continuous variable) and the probability of isolating a given staphylococcal species from a given body site (inguinal region or teats). Overall, the most common species identified were Staphylococcus haemolyticus followed by Staphylococcus chromogenes, Staphylococcus xylosus, Staphylococcus devriesei, and Staphylococcus sciuri. Staphylococcus aureus was more prevalent on the teat than in the inguinal region, whereas Staphylococcus arlettae was more prevalent in the inguinal region than on the teat. All other staphylococcal species were as likely to be found on the teat skin as the inguinal region skin. Isolation from the inguinal and teat skin was associated with age for Staphylococcus agnetis, S. chromogenes, S. devriesei, Staphylococcus equorum, S. haemolyticus, Staphylococcus lentus, S. sciuri, Staphylococcus vitulinus, and S. xylosus. The probability of finding S. chromogenes and S. agnetis on the teat and inguinal region increased with age, whereas the probability of S. devriesei and S. haemolyticus decreased with age. This study provides further insight into the ecology of staphylococcal species involved in heifer mastitis.

The effect of sodium nitroprusside on psychotic symptoms and spatial working memory in patients with schizophrenia: a randomized, double-blind, placebo-controlled trial.

BACKGROUND: Sodium nitroprusside (SNP) has been reported to rapidly reduce psychotic symptoms in patients with schizophrenia. This has the potential to revolutionize treatment for schizophrenia. In this study, we tested the hypothesis that SNP leads to a reduction in psychotic symptoms and an improvement in spatial working memory (SWM) performance in patients with schizophrenia. METHOD: This was a single-centre, randomized, double-blind, placebo-controlled trial performed from 27 August 2014 to 10 February 2016 (clinicaltrials.gov identifier: NCT02176044). Twenty patients with schizophrenia aged 18-60 years with a diagnosis of schizophrenia or schizoaffective disorder were recruited from psychiatric outpatient clinics in the South London and Maudsley NHS Trust, London, UK. Baseline symptoms were measured using the Positive and Negative Syndrome Scale (PANSS) and the 18-item Brief Psychiatric Rating Scale (BPRS-18), and SWM was assessed using the CANTAB computerized test. Participants received either an infusion of SNP (0.5 µg/kg per min for 4 h) or placebo and were re-assessed for symptoms and SWM performance immediately after the infusion, and 4 weeks later. RESULTS: SNP did not lead to any reduction in psychotic symptoms or improvement in SWM performance compared to placebo. CONCLUSIONS: Although this study was negative, it is possible that the beneficial effects of SNP may occur in patients with a shorter history of illness, or with more acute exacerbation of symptoms.

Standard Establishment Through Scenarios (SETS): A new technique for occupational fitness standards.

BACKGROUND: An objective and scientific task analysis provides the basis for establishing legally defensible Physical Employment Standards (PES), based on common and essential occupational tasks. Infrequent performance of these tasks creates challenges when developing PES based on criterion, or content validity. OBJECTIVE: Develop a systematic approach using Subject Matter Experts (SME) to provide tasks with 1) an occupationally relevant scenario considered common to all personnel; 2) a minimum performance standard defined by time, distance, load or work. Examples provided here relate to the development of a new PES for the Canadian Armed Forces (CAF). METHODS: SME of various experience are selected based on their eligibility criteria. SME are required to define a reasonable scenario for each task from personal experience, provide occupational performance requirements of the scenario in sub-groups, and discuss and agree by consensus vote on the final standard based on the definition of essential. RESULTS: A common and essential task for the CAF is detailed as a case example of process application. Techniques to avoid common SME rating errors are discussed and advantages to the method described. CONCLUSION: The SETS method was developed as a systematic approach to setting occupational performance standards and qualifying information from SME.

Physiological employment standards I. Occupational fitness standards: objectively subjective?

This paper examines the processes involved in the establishment of a minimum occupational fitness standard, with particular reference to the interplay that inevitably occurs between objective measurements and subjective decisions. The areas considered include: the determination of the critical task on which to base a standard; establishing minimum acceptable performance and methods of best practice for the execution of these tasks; determining the physical demands of a task and a reasonable relative workload; producing the final standard. Finally, the impact of the subjective component of the development of an occupational fitness standard on its defensibility is discussed. It is concluded that all standards involve some subjective aspects; the extent of these could be reduced by further research. In the meantime, it would be prudent for those developing standards to detail the rationale, methods and evidence by which subjective decisions were reached, to provide an audit trail for subsequent investigation.

Subcutaneous botryomycosis due to Bibersteinia trehalosi in a Texas Longhorn steer.

A 3-year-old Texas Longhorn steer had a long history of progressive swelling of the soft tissues of the jaw and neck. At necropsy, multifocal to coalescing dermal and subcutaneous pyogranulomas were surrounded by fibrous tissue. Microscopically, the pyogranulomas contained aggregates of gram-negative coccobacilli surrounded by Splendore-Hoeppli material and were separated by bands of fibrovascular tissue (botryomycosis). Phylogenetic analysis of multilocus sequence-typing data revealed that the bacteria recovered in pure culture from swabs of submandibular tissue were most closely related to Bibersteinia [Pasteurella] trehalosi. The bacterial colonies were immunohistochemically reactive with a rabbit polyclonal anti-Pasteurella class C acid phosphatase antibody. Botryomycosis is a pyogranulomatous inflammation caused by a variety of nonbranching, nonfilamentous bacteria that elicit the formation of Splendore-Hoeppli material. This case of botryomycosis is unique for its association with Bibersteinia trehalosi.

Isolated and combined fractures of the diaphysis of the radius and ulna.

In summary, treatment of diaphyseal fractures requires accurate assessment of the injury to rule out concomitant ligamentous injury at the wrist or the elbow. Minimally displaced ulnar fractures can be managed with bracing. Displaced diaphyseal fractures of the radius and ulna should be plated, usually with 3.5-mm compression plates using AO technique. Complications may still occur but can be minimized if strict attention to technique is followed.

NadN and e (P4) are essential for utilization of NAD and nicotinamide mononucleotide but not nicotinamide riboside in Haemophilus influenzae.

Haemophilus influenzae has an absolute requirement for NAD (factor V) because it lacks almost all the biosynthetic enzymes necessary for the de novo synthesis of that cofactor. Factor V can be provided as either nicotinamide adenosine dinucleotide (NAD), nicotinamide mononucleotide (NMN), or nicotinamide riboside (NR) in vitro, but little is known about the source or the mechanism of uptake of these substrates in vivo. As shown by us earlier, at least two gene products are involved in the uptake of NAD, the outer membrane lipoprotein e (P4), which has phosphatase activity and is encoded by hel, and a periplasmic NAD nucleotidase, encoded by nadN. It has also been observed that the latter gene product is essential for H. influenzae growth on media supplemented with NAD. In this report, we describe the functions and substrates of these two proteins as they act together in an NAD utilization pathway. Data are provided which indicate that NadN harbors not only NAD pyrophosphatase but also NMN 5'-nucleotidase activity. The e (P4) protein is also shown to have NMN 5'-nucleotidase activity, recognizing NMN as a substrate and releasing NR as its product. Insertion mutants of nadN or deletion and site-directed mutants of hel had attenuated growth and a reduced uptake phenotype when NMN served as substrate. A hel and nadN double mutant was only able to grow in the presence of NR, whereas no uptake of NMN was observed.

Contribution of the DDDD motif of H. influenzae e (P4) to phosphomonoesterase activity and heme transport.

Haemophilus influenzae lipoprotein e (P4) is a member of the DDDD phosphohydrolase superfamily and mediates heme transport. Each of the aspartate residues of the signature motif is required for phosphomonoesterase activity, as none of the e (P4) single D mutants (D64A, D66A, D181N, and D185A) possessed detectable phosphomonoesterase activity. These results suggest that the signature motif is essential to the phosphomonoesterase activity of lipoprotein e (P4). When assessed for phosphomonoesterase-dependent heme transport activity in Escherichia coli hemA strains, plasmids containing D181N and D185A retained heme transport as indicated by aerobic growth while D64A and D66A did not. We conclude that phosphomonoesterase activity is not required for heme transport.

Purification and characterization of a recombinant Haemophilus influenzae outer membrane phosphomonoesterase e (P4).

Haemophilus influenzae is a common inhabitant of the upper respiratory tract and can cause serious infections of mucosal surfaces. Results from recent studies indicate that this pathogen possesses copious amounts of surface-localized phosphomonoesterase activity mediated by the bacterial lipoprotein e (P4). While the enzyme has previously been purified to apparent homogeneity, purification of large amounts of protein has been prevented by presence of N-terminal lipid modification. Recombinant DNA technology was employed to simultaneously replace the N-terminal lipid modification signal sequence with one for protein secretion without such modification and to place expression of the protein under the control of the T7-inducible promoter. Results from this work show that high levels of phosphomonoesterase activity were achieved after IPTG induction and purified to apparent homogeneity after two chromatography steps. Consistent with loss of the N-terminal lipid modification, the recombinant enzyme was easily extracted from the bacterial membrane and partitioned within the matrix of gel filtration chromatography resin while retaining a denatured molecular weight similar to that of wild-type e (P4). Results from physicochemical characterization suggest that the recombinant protein was similar to wild-type protein in SDS-PAGE-derived molecular weight, primary structure, substrate specificity, pH optimum, and sensitivity or resistance to various inhibitors. Acquisition of sufficient amounts of recombinant P4 was a prelude for studies to elucidate the structure and function of this unusual phosphomonoesterase.

Acid phosphatase activity as a measure of Haemophilus influenzae adherence to mucin.

Haemophilus influenzae is an important respiratory tract pathogen. Toward understanding the progression of H. influenzae from commensal to pathogen, we need to understand the steps of colonization and infection, processes which must involve overcoming the normal host mucociliary clearance mechanism. A reliable method for the screening and quantitation of mucin-H. influenzae binding to allow for the assessment of the physiological variables significant to H. influenzae-mucin interactions in the normal and diseased conditions, will provide insight on how to intervene to prevent, inhibit, or treat infection. The current methods for enumeration of mucin-bound H. influenzae are labor intensive and rely on viable organisms. In this report, we present a new detection method, which reduces the number of variables, processing steps, and time involved, providing an economical, rapid, and reliable means to screen for and quantitate mucin-bound H. influenzae. Organisms are applied to mucin-coated microtiter wells for a set time; nonadherent organisms are removed with gentle rinses; wells are incubated with the phosphomonoesterase substrate p-nitrophenyl phosphate; and the absorbance, reflecting phosphatase activity of the mucin-bound organisms, is read at 410 nm in a microtiter plate reader against enzymatic activity calibration curves. All nonencapsulated and encapsulated H. influenzae tested exhibited significant acid phosphate activity within 20 min, which provided linear relationships with the numbers of organisms present. H. influenzae mucin binding characteristics obtained by this method were generally comparable to published data, and ranged from 10(3) to 10(6) organisms per well, depending on both strain of organism and type of mucin employed. This convenient, rapid and economical mucin adherence assay, will enable more extensive and comprehensive studies of the interactions of H. influenzae adhesins and specific ligands on mucin macromolecules, as well as the nonspecific means by which mucins function in preventing bacterial infection.

Outer membrane lipoprotein e (P4) of Haemophilus influenzae is a novel phosphomonoesterase.

Haemophilus influenzae exists as a commensal of the upper respiratory tract of humans but also causes infections of contiguous structures. We describe the identification, localization, purification, and characterization of a novel, surface-localized phosphomonoesterase from a nontypeable H. influenzae strain, R2866. Sequences obtained from two CNBr-derived fragments of this protein matched lipoprotein e (P4) within the H. influenzae sequence database. Escherichia coli DH5alpha transformed with plasmids containing the H. influenzae hel gene, which encodes lipoprotein e (P4), produced high levels of a membrane-associated phosphomonoesterase. The isolated approximately 28-kDa enzyme was tartrate resistant and displayed narrow substrate specificity with the highest activity for arylphosphates, excluding 5-bromo-4-chloro-3-indolylphosphate. Optimum enzymatic activity was observed at pH 5.0 and only in the presence of divalent copper. The enzyme was inhibited by vanadate, molybdate, and EDTA but was resistant to inorganic phosphate. The association of phosphomonoesterase activity with a protein that has also been recognized as a heme transporter suggests a unique role for this unusual phosphohydrolase.

The respiratory burst-inhibiting acid phosphatase AcpA is not essential for the intramacrophage growth or virulence of Francisella novicida.

Acid phosphatases capable of inhibiting the respiratory burst of neutrophils have been identified in certain intracellular pathogens. Here we evaluate the role of AcpA, a respiratory burst-inhibiting acid phosphatase of Francisella, in the virulence and intracellular growth of this organism. An F. novicida acpA null mutant was created and found to exhibit wild-type growth kinetics in both cell-line and inflammatory mouse macrophages. The acpA mutant also shows wild-type replication in the spleens of experimentally infected mice. These data suggest that AcpA is not essential for the intracellular growth or virulence of F. novicida.

Internalization of Escherichia coli by human renal epithelial cells is associated with tyrosine phosphorylation of specific host cell proteins.

Human renal epithelial cells are capable of internalizing Escherichia coli regardless of whether the bacteria are isolated from individuals with pyelonephritis or from healthy volunteers. In this study, we investigated the role of host cell tyrosine kinase activity in internalization. We found that internalization of both fecal and pyelonephritis isolates is blocked by tyrosine kinase inhibitors. We found increased intensity of two tyrosine-phosphorylated proteins, with relative mobilities of approximately 123,000 and 110,000, in Western blots of extracts from human renal epithelial cells infected with E. coli. The increased intensity of these tyrosine-phosphorylated proteins was observed only in the Triton X-100-insoluble fraction, suggesting that these proteins could be associated with the cytoskeleton. Increased tyrosine phosphorylation of these proteins upon E. coli infection was observed in both transformed and primary human renal epithelial cells and in cells infected with several different strains of E. coli isolated from the feces of healthy individuals or from the blood or urine of patients with pyelonephritis. The increased tyrosine phosphorylation of these proteins required live bacteria and was blocked by tyrosine kinase inhibition but not by protein synthesis inhibitors or cytochalasin D. These experiments establish a strong link between E. coli internalization and host cell signaling through tyrosine kinases in human kidney cells and provide evidence that specific proteins are involved in these processes.

Characterization and sequencing of a respiratory burst-inhibiting acid phosphatase from Francisella tularensis.

Acid phosphatases (Acp) of intracellular pathogens have recently been implicated as virulence factors that enhance intracellular survival through suppression of the respiratory burst. We describe here the identification, purification, characterization, and sequencing of a novel burst-inhibiting acid phosphatase from the facultative intracellular bacterium, Francisella tularensis. Similar to other the burst-inhibiting Acps, F. tularensis Acp (AcpA) is tartrate-resistant and has broad substrate specificity. The AcpA enzyme is unique, however, in that it is easily released from the bacterial cell in soluble form, is a basic enzyme, suppresses the respiratory burst of not only fMet-Leu-Phe but also phorbol 12-myristate 13-acetate-stimulated neutrophils and does not fit into any of the three currently recognized classes of acid phosphatase. We also report the complete nucleotide sequence of the gene acpA, encoding AcpA, and the deduced primary structure of its encoded polypeptide. Comparative sequence analyses of AcpA is discussed. To our knowledge, this is the first report describing the cloning and sequencing of a burst-inhibiting acid phosphatase.

Autoantibody formation in burn patients after inhibition of suppressor T cell activity with polymyxin B.

After thermal injury, treatment with polymyxin B blocks suppressor T cell activity by uncoupling endotoxin-mediated T cell activation, but the effect on autoantibody formation is unknown. We examined the presence of antinuclear antibodies to native DNA; to soluble antigens Ro/SSA, La/SSB, Sm, nRNP; and to antiepithelial antibodies in 12 burn patients before and after treatment with polymyxin B and in 24 samples from control burn patients. Low titer antinuclear antibody activity was detected in 25% of pretreatment and 78% of posttreatment samples (p less than 0.01) and in 16.7% of control patients. One polymyxin B-treated patient had a significant antinuclear antibody titer both before and after treatment. Antiepithelial antibodies were detected in 16.7% of early polymyxin B-treated samples and 11.1% of late samples (p less than 0.05) but were also present in 20.8% of controls. Antibodies to native DNA, Ro/SSA, La/SSB, Sm, and nRNP were not detected in any sera.

Laser Doppler flowmetry evaluation of burn wound depth.

Hypothesizing that reduced blood flow to various levels in thermally damaged dermis is related to the depth of burn, we studied 59 burns in 41 patients, with an age range of 18 months to 67 years, with improved laser Doppler blood flow technology to determine burn depth on admission. Two hundred and sixty-eight laser Doppler measurements were made in 59 burn wounds in 41 patients. Of the 129 burn wounds with laser Doppler readings of less than 1.4 on admission, 127 were classified ultimately through clinical observation as either deep second- or third-degree burns and required excision and closure. Therefore predictive value of a positive result (i.e., laser Doppler value less than 1.4) on admission was 98.4% using laser Doppler measurements. Clinical estimation did not correlate as well with the depth of injury.

Neutrophil intracellular kill following thermal injury. Different bactericidal capability for patients' organisms and laboratory organisms.

Sixteen patients with septic complications of severe thermal injury were studied with respect to neutrophil intracellular-killing power against clinical isolates from the patients themselves and against other laboratory organisms. Simultaneous measurements of neutrophil chemotaxis, helper/suppressor lymphocyte ratios, and serum IgG concentrations were also carried out. Neutrophils from patients who survived had diminished intracellular-killing capacity for their own organisms, but normal capacity for killing laboratory organisms either matched or unmatched with the patients' own isolate's species. In these patients, the chemotactic index, the lymphocyte helper/suppressor ratio, and the serum IgG concentration remained within normal limits. Neutrophils from patients who died failed to kill their own, as well as laboratory, organisms. In these patients, the chemotactic index, lymphocyte helper/suppressor ratio, and IgG concentration were significantly diminished. The biological implications of these findings are noted.