Survival of Campylobacter jejuni during in vitro culture with mixed bovine ruminal microorganisms in the presence of methanogen inhibitors.

Foodborne pathogen Campylobacter jejuni has been associated with ruminants. The objectives of this experiment were to determine C. jejuni survivability in mixed in vitro rumen microbial populations and the impact on methane production with or without methane inhibitors 2-bromosulfonate (BES) and/or sodium nitrate. When inoculated into rumen microbial populations without or with 0.5 mM BES, 5.0 mM nitrate or their combination, C. jejuni viability decreased from 4.7 ± 0.1 log10 colony forming units (CFU)/mL after 24 h. Loss of C. jejuni viability was greater (P < 0.05) when incubated under 100% CO2 compared to 50% H2:50% CO2, decreasing 1.46 versus 1.15 log units, respectively. C. jejuni viability was also decreased (P < 0.05) by more than 0.43 log units by the anti-methanogen treatments. Rumen microbial populations produced less methane (P = 0.05) when incubated with than without C. jejuni regardless of whether under 100% CO2 or 50% H2:50% CO2. For either gas phase, nitrate was decreased (13.2 versus 37.9%) by the anti-methanogen treatments versus controls although not always significant. C. jejuni-inoculated populations metabolized 16.4% more (P < 0.05) nitrate under H2:CO2 versus 100% CO2. Apparently, C. jejuni can compete for H2 with methanogens but has limited survivability under rumen conditions.

Gene expression in Lucilia sericata (Diptera: Calliphoridae) larvae exposed to Pseudomonas aeruginosa and Acinetobacter baumannii identifies shared and microbe-specific induction of immune genes.

Antibiotic resistance is a continuing challenge in medicine. There are various strategies for expanding antibiotic therapeutic repertoires, including the use of blow flies. Their larvae exhibit strong antibiotic and antibiofilm properties that alter microbiome communities. One species, Lucilia sericata, is used to treat problematic wounds due to its debridement capabilities and its excretions and secretions that kill some pathogenic bacteria. There is much to be learned about how L. sericata interacts with microbiomes at the molecular level. To address this deficiency, gene expression was assessed after feeding exposure (1 h or 4 h) to two clinically problematic pathogens: Pseudomonas aeruginosa and Acinetobacter baumannii. The results identified immunity-related genes that were differentially expressed when exposed to these pathogens, as well as non-immune genes possibly involved in gut responses to bacterial infection. There was a greater response to P. aeruginosa that increased over time, while few genes responded to A. baumannii exposure, and expression was not time-dependent. The response to feeding on pathogens indicates a few common responses and features distinct to each pathogen, which is useful in improving the wound debridement therapy and helps to develop biomimetic alternatives.

Interactions of organic acids with vancomycin-resistant Enterococcus faecium isolated from community wastewater in Texas.

AIMS: Investigate the interactions of organic acids (OAs), acetic, butyric, citric, formic, lactic and propionic acid against 50 Gram-positive vancomycin-resistant Enterococcus faecium (VRE) strains to determine whether pH, undissociated or dissociated acid forms correlate with bacterial inhibition. METHODS AND RESULTS: Concentrations of undissociated and dissociated OAs at the molar minimum inhibitory concentrations (MICM s) of the VRE were calculated using the Henderson-Hasselbalch equation. The pH at the MICM s of all VRE strains against acetic, butyric, formic and propionic acids was similar, 4·66 ± 0·07, but there was a 1·1 pH unit difference for all six OAs. Inhibition of VRE by all six OAs did not appear to be solely dependent on pH or on the undissociated OA species. The inhibition of VRE by all six dissociated acids was within Δ = 3·1 mmol l-1 . CONCLUSIONS: Vancomycin-resistant Enterococcus faecium inhibition correlated with the dissociated OA species. A small decrease in the concentration of the dissociated OAs from optimum may result in allowing VRE strains to escape disinfection. SIGNIFICANCE AND IMPACT OF THE STUDY: When an OA is used to disinfect VRE strains, the concentration of the dissociated OA should be carefully controlled. A concentration of at least 20 mmol l-1 dissociated OA should be maintained when disinfecting VRE.

Characterization of antibiotic and disinfectant susceptibility profiles among Pseudomonas aeruginosa veterinary isolates recovered during 1994-2003.

AIMS: To evaluate susceptibility of Pseudomonas aeruginosa veterinary isolates to antibiotics and disinfectants. METHODS AND RESULTS: Pseudomonas aeruginosa isolates collected from dogs (n = 155) and other animals (n = 20) from sixteen states during 1994-2003 were tested for susceptibility. Most isolates were resistant to twenty-one antimicrobials tested, and the highest prevalence of resistance was to ß-lactams (93.8%) and sulphonamides (93.5%). Fluoroquinolone resistance did not increase from 1994 to 2003. Ciprofloxacin and enrofloxacin had a 5 and 16% prevalence of resistance, respectively, while sarafloxacin and nalidixic acid had a prevalence of resistance of 97 and 98%, respectively. Strains were pan-resistant to triclosan and chlorhexidine, were highly resistant to benzalkonium chloride and demonstrated high susceptibility to other disinfectants. Didecyldimethylammonium chloride was the most active ammonium chloride. Inducible resistance was observed to cetyl ammonium halides, chlorhexidine and benzyl ammonium chlorides, which formulate disinfectants used in veterinary clinics and dairies. Organic acid inhibition was associated with the dissociated acid species. CONCLUSIONS: Dissociated organic acids appear able to inhibit Ps. aeruginosa, and rates of fluoroquinolone resistance merit sustained companion animal isolate surveillance. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of Ps. aeruginosa susceptibility to 24 disinfectants and illustrates the high resistance of Ps. aeruginosa to both antibiotics and disinfectants.

Transient gut retention and persistence of Salmonella through metamorphosis in the lesser mealworm, Alphitobius diaperinus (Coleoptera: Tenebrionidae).

AIMS: This study was undertaken to determine the retention of Salmonella through Alphitobius diaperinus metamorphosis and its contribution, through defecation, to external contamination. METHODS AND RESULTS: Insects were exposed to a tagged Salmonella enterica and evaluated for external elimination. (i) Each day for 3 weeks, a filter collected frass from a restrained insect for analysis. (ii) Exposed larvae in a closed container were followed through pupation, and newly emerged adults were examined for their retention of marker bacteria. CONCLUSIONS: Exposed adults and larvae produced Salmonella-positive frass for an average of 8 days, ranging from 6 to 11 days and 6 to 12 days, respectively. Nineteen per cent of the larvae carried Salmonella through metamorphosis and eclosion, with 5% of the pupal exuviae being positive as well. SIGNIFICANCE AND IMPACT OF THE STUDY: Many sources of foodborne pathogens within the poultry production facilities, including reservoir populations, currently go unrecognized. This diminishes the ability of producers to mitigate the transfer of pathogens between animals, humans and the environment. Poultry management standards accept the reutilization of litter. Alphitobius diaperinus survive between flock rotations on the reutilized litter, and it was demonstrated in this study that the Salmonella they carry can survive with them.

Planktonic and biofilm communities from 7-day-old chicken cecal microflora cultures: characterization and resistance to Salmonella colonization.

Information implicating bacterial biofilms as contributory factors in the development of environmental bacterial resistance has been increasing. There is a lack of information regarding the role of biofilms within the microbial ecology of the gastrointestinal tract of food animals. This work used a continuous-flow chemostat model derived from the ceca of 7-day-old chicks to characterize these communities and their ability to neutralize invasion by Salmonella enterica serovar Typhimurium. We characterized and compared the biofilm and planktonic communities within these microcosms using automated ribotyping and the Analytical Profile Index biotyping system. Eleven species from eight different genera were identified from six culture systems. Klebsiella pneumoniae was isolated from all planktonic communities and four of the biofilm communities. Three of the communities resisted colonization by Salmonella enterica serovar Typhimurium, two communities suppressed growth, and one community succumbed to colonization. In cultures that resisted colonization, no Salmonella could be isolated from the biofilm; in cultures that succumbed to colonization, Salmonella was consistently found within the biofilms. This study was one of a series that provided a molecular-based characterization of both the biofilm and planktonic communities from continuous-flow culture systems derived from the cecal microflora of chicks, ranging in age from day-of-hatch to 14 days old. The one common factor relating to successful colonization of the culture was the presence of Salmonella within the biofilm. The capacity to sequester the introduced Salmonella into the biofilm appears to be a contributing factor to the inability of these cultures to withstand colonization by the Salmonella.

The horizontal transfer of Salmonella between the lesser mealworm (Alphitobius diaperinus) and poultry manure.

There is need to determine the nature of enduring reservoirs of Salmonella contributing to perpetual contamination within poultry flocks. The dispersal of Salmonella between birds, litter and the lesser mealworm has been established, but the extent that these act as critical components in the epidemiology of Salmonella infection during broiler grow-out and flock rotation has not been delineated; in particular, the level of participation by the lesser mealworm beetles (LMB) as agents of retention and dispersal. This study defines this route of transmission and provides empirical data on bacterial loads that facilitate Salmonella transfer. Results showed differential Salmonella transfer dependent on bacterial concentration. At 103  cfu/ml, only a small, but not significant, amount of Salmonella was transferred, from the LMB to the manure and back to uninfected LMB; while from 105 to 107  cfu/ml, a significant acquisition and transfer occurred both internally and externally to the LMB over 4 and 24 hr exposures. These data will be used in correlation with facility management practices to develop intervention strategies to mitigate the establishment and spreading of reservoir Salmonella populations contributing to pre-harvest contamination of poultry flocks.

Relationships between exposure, cell loss and proliferation, and manifestation of Hprt mutant T cells following treatment of preweanling, weanling, and adult male mice with N-ethyl-N-nitrosourea.

Experiments were performed to characterize the age-related patterns of appearance and frequency of hypoxanthine-guanine phosphoribosyl transferase (Hprt) mutant T lymphocytes in thymus and spleen following exposure of preweanling (12-day-old), weanling (22-day-old), and young adult (8-week-old) male B6C3F1 mice to ethylnitrosourea (ENU). Mice were given single i.p. injections of 0 or 40 mg ENU/kg and then groups of animals were necropsied from 2 h to 116 days after treatment to examine the relationships between exposure, cell loss and proliferation, and the frequency of Hprt mutant T cells in thymus and spleen. Hprt mutant frequency (Mf) data for thymus of ENU-exposed (0, 11.7, 35, 58, or 72 mg/kg, or five weekly doses of 1.7 mg/kg i.p.) male C57BL/6 mice (12- or 62-week-old), obtained during an earlier study of spleen cells [I.M. Jones, K. Burkhart-Schultz, C.L. Strout, T.L. Crippen, Factors that affect the frequency of thioguanine-resistant lymphocytes in mice following exposure to ethylnitrosourea, Environ. Mutagen, 9 (1987) 317-329.], were compared to results in B6C3F1 mice. Isolated T cells were cultured in the presence of mitogen, growth factor, and 6-thioguanine to detect Hprt mutants. The time required to achieve maximum Mfs in thymus was uniformly found at 2 weeks after ENU treatment, while the times needed to reach peak values in spleen were proportional to animal age at treatment. These data indicate that age-related differences in the appearance of Hprt mutant cells in spleen are largely defined by the physiologically based, age-dependent trafficking of mutant cells from or through the thymus. Three modes of handling the resulting Hprt Mf data were evaluated: (i) comparing the Mfs at a single time point, (ii) comparing the maximum Mfs observed, and (iii) comparing the change in Mfs over time (or the mutant T cell 'manifestation' curves in treated vs. control mice) in each age group post-exposure. Measuring the Mfs in spleen at multiple time points after cessation of exposure and integrating the frequency of mutants as a function of time appeared to be the superior method for comparing mutagenic responses in different age groups. Some of the underlying assumptions of this approach, as well as its strengths and weaknesses, are discussed.

Cytokine-induced neutrophil chemoattractant production by primary rat alveolar type II cells.

This study was designed to determine the production of the chemokine cytokine-induced neutrophil chemoattractant (CINC) by primary rat alveolar type II (ATII) cells upon stimulation with exogenous and endogenous proinflammatory factors. Cultures of primary rat ATII cells were exposed to lipopolysaccharide (LPS), interleukin-1 beta (IL-1 beta) or tumor necrosis factor-alpha (TNF alpha) over a 16 hour period and the production of CINC both apically and basolaterally was measured by ELISA. Compared to unstimulated (UNS) cultures, LPS, IL-1 beta and TNF alpha were found to significantly increase the level of CINC detected in culture by two, four and sixteen hours post stimulation, respectively. ATII cells also demonstrated a polar secretion of CINC. The accumulation of CINC basolaterally was significantly more than apically; 133%, 45%, 117% and 123% for UNS, IL-1 beta, LPS and TNF alpha respectively. We demonstrated that primary rat ATII cells may participate in the chemokine network during inflammation by the production of CINC upon stimulation with endogenous and exogenous factors.

Chicken mim-1 protein, P33, is a heterophil chemotactic factor present in Salmonella enteritidis immune lymphokine.

Lymphokine (ILK) secreted from concanavalin A-stimulated T cells from Salmonella Enteritidis-immune chickens is an undefined mixture of proteins that confers protection against Salmonella infectivity when administered to day-old chicks. It has previously been shown that polyclonal antibodies raised against human granulocyte colony-stimulating factor (GCSF) can neutralize the heterophil activation that is responsible for ILK's protective effect. Western blot analysis of ILK probed with anti-GCSF antibodies detects a prominent protein of mass 33 kDa. We have sequenced the first 20 amino acids of this protein and found it to be identical to residues 24 to 43 of P33, a 326-amino acid protein of unknown function encoded by the chicken mim-1 gene. The primary structure of P33 consists of two 140-residue imperfect repeats that are each homologous to a mammalian neutrophil chemotactic factor termed leukocyte cell-derived chemotaxin 2 (LECT2). We have expressed mim-1 in Escherichia coli and demonstrated in vitro that recombinant P33 is chemotactic for heterophils, the avian equivalent of mammalian neutrophils. We have also constructed a derivative of P33 that consists of residues 33 to 165 (P33[33-165]), the first repeat sequence of P33 that is homologous to LECT2. P33(33-165) is chemotactic for heterophils both in vitro and in vivo, inducing an influx of heterophils into the peritoneum in a response similar to that observed with ILK. These results suggest that P33 functions as a chemotactic factor in chickens and that it plays an active role in ILK-mediated protection against Salmonella infection.

Cell proliferation in the bone marrow, thymus and spleen of mice studied by continuous, in vivo bromodeoxycytidine labelling and flow cytometric analysis.

We have applied the technique of labelling dividing cells with bromodeoxyuridine (BrdUrd) in combination with in vivo continuous labelling, propidium iodide (PI) staining for DNA content, and flow cytometric analysis, for the determination of cell proliferation in bone marrow, thymus and spleen of mice. The percentage of BrdUrd labelled cells increased as a function of exposure time in a tissue specific manner for each of the three tissues. Thymus and bone marrow had cell populations which exhibited different kinetics for the accumulation of label: (1) those that cycled and became labelled within 2-3 days (88% in 2 days for bone marrow, 84% in 3 days for thymus); (2) those that cycled during the remainder of the 6 day infusion period (11% of bone marrow and 13% of thymus cells); and (3) those that did not cycle during the 6 day period studied (less than 2% of bone marrow and 3% of thymus cells). In contrast, the spleen exhibited a slower, constant accumulation of labelled cells. After six days of infusion a large proportion of spleen cells (50%) had not become labelled. These results suggest that a larger proportion of spleen cells are long lived than indicated by other methods. We also have found the period of labelling with BrdUrd extended several days beyond the period of infusion. This method will be very useful in studying perturbations of cell populations induced in mice exposed to toxic agents.

Cloned mouse lymphocytes permit analysis of somatic mutations that occur in vivo.

As part of our mouse model of somatic mutation, we have begun to characterize spontaneously occurring hypoxanthine phosphoribosyltransferase (HPRT) -deficient mouse lymphocytes. Lymphocytes were cloned by in vitro exposure of spleen cells from male C57B1/6 mice to the mitogen concanavalin A, conditioned medium containing lymphocyte growth factors, and thioguanine (TG), in a limiting dilution assay. The 17 TG-resistant clones recovered were all highly deficient in HPRT activity and were found by analysis of surface antigens to be representative of the major subclasses of T lymphocytes. Southern analysis of lymphocyte genomic DNA detected alterations of the hprt gene in 12/17 of the HPRT-deficient lymphocyte clones. Of these 12, 2/17 were lacking the entire hprt locus, 7/17 lacked part of the locus, and 3/17 had other, unidentified alterations.

Regulation of cytokine-induced neutrophil chemoattractant in rat bone marrow-derived macrophages by inflammatory mediators.

During the course of inflammation, macrophages are highly influenced by their local environment and changes in the cytokine milieu. Exposure of macrophages to various factors during different phases of the inflammatory response may have a strong influence on the pattern of gene expression, which a macrophage exhibits. We examined how these mediators affect the regulation of the expression and production of cytokine-induced neutrophil chemoattractant (CINC). Our study demonstrates that CINC can be induced in bone marrow-derived macrophages by lipopolysaccharide, interleukin-1 beta, tumor necrosis factor-alpha (TNFalpha), and interferon-gamma/TNF alpha. These mediators are factors which a macrophage would be expected to encounter early in an inflammatory process. In contrast, transforming growth factor-beta (TGFbeta), which is expressed late in the inflammatory process during mesenchymal cell proliferation and tissue repair, did not induce detectable amounts of CINC and functioned to suppress CINC production stimulated by early inflammatory mediators. Suppression of CINC production occurred whether TGF beta was added simultaneously, 12 or 24 h prior to the stimulus.

Differential regulation of the expression of cytokine-induced neutrophil chemoattractant by mouse macrophages.

The production of cytokine-induced neutrophil chemoattractant (CINC) by functionally diverse mouse bone-marrow-derived macrophages was determined. Studies showed that beta1,3-glucan, IL-beta, TNFalpha and IFNgamma/TNFalpha induced expression and production of CINC in macrophages while neither IFNgamma nor TGFbeta alone induced detectable CINC expression. Pretreatment or simultaneous treatment of macrophages with TGFbeta resulted in suppression of CINC protein production. These studies demonstrate that IFNgamma and TNFalpha, found early during the inflammatory response, induce production of CINC, as well as induce macrophages into a cytocidal state that are capable of killing transformed cells, parasites and bacteria, and recruiting neutrophils. In contrast, TGFbeta, found during reparative stages of the inflammatory response, suppressed production of CINC, while inducing the development of inflammatory macrophages that are capable of producing lysosomal enzymes, enhanced endocytosis and ingestion of particulate matter and function to scavenge debris, debride tissue and stimulate repair.

Factors that affect the frequency of thioguanine-resistant lymphocytes in mice following exposure to ethylnitrosourea.

The frequency of thioguanine(TG)-resistant lymphocytes in mice treated with ethylnitrosourea (ENU) was followed for a period of 51 wk using our clonogenic assay [Jones et al, 1985a,b]. The effects of dose (0-58 mg/kg), time since treatment (2-51 wk), dose rate (5 weekly X 11.7 mg/kg versus 1 X 58 mg/kg), and age at time of treatment (3 vs 15 mo) on the frequency of TG-resistant, concanavalin A-responsive spleen cells were evaluated. The frequencies of TG-resistant spleen cells were generally dose responsive for 51 wk after exposure to ENU. They also were dependent upon the time that had elapsed since treatment with ENU, increasing to maximal values at 10 wk as previously reported [Jones et al, 1985a], and holding essentially stable at values of approximately 20% of the maximum frequency from week 15 until at least week 40 for the 3-month-old mice. Fractionation of 58 mg ENU/kg into 5 weekly doses did not affect the frequency of ENU-induced TG-resistant cells detected in the spleen but did increase the rate of appearance in the spleen, and the efficiency of induction by the unit dose, of TG-resistant cells. The mice exposed to ENU at 15 mo of age appeared to have a 4-fold reduction in the rate of increase in frequency of ENU-induced TG-resistant spleen cells. One set of control mice was found to have a 10-fold elevated frequency of TG-resistant cells in both the spleen and thymus, indicating that mutations can occur in stem cells of untreated animals.