Bacterial Recoveries Research Articles

Persistence of Bacillus thuringiensis and Bacillus pumilus potential biological control agents of the coffee berry borer under field conditions of Puerto Rico

Field trials were conducted in two years to evaluate persistence of B. thuringiensis and Bacillus pumilus under field conditions. Strains of B. thuringiensis (3971) and B. pumilus (4185) were isolated from coffee agro-ecosystem in Puerto Rico. Field trials consisted in plots of nine coffee trees arranged in a randomized complete block design, sprayed with Bt-3971 and Bp-4185 at 108, 109, or 1010 colony forming units CFU ml-1 or non-inoculated. In year 1, B. thuringiensis and B. pumilus survived up to six months after inoculation. Monitoring carried out during year 2, re-confirmed the survival of Bt-3971 and Bp-4185 inoculated in coffee trees under field conditions. Overall, Bt-3971 had significantly more recovery rates than Bp-4185. However, the higher bacterial recoveries were 1.24x1013 CFU ml-1 in coffee trees treated with Bt-3971 and 2.48x1013 CFU ml-1 in coffee trees treated with Bp-4185 in year 1 and year 2, respectively, both sprayed with 1010 spores ml-1. B. thuringiensis (3971) and B. pumilus (4185) isolated from coffee fields of Puerto Rico are well adapted to the environmental conditions of the island, and are valuable biological control agents against coffee berry borer.

Impact of biofilm on bacterial transport and deposition in porous media

Laboratory scale experiments were conducted to obtain insights into factors that influence bacterial transport and deposition in porous media. According to colloidal filtration theory, the removal efficiency of a filter medium is characterized by two main factors: collision efficiency and sticking efficiency. In the case of bacterial transport in porous media, bacteria attached to a solid surface can establish a thin layer of biofilm by excreting extracellular polymeric substances which can significantly influence both of these factors in a porous medium, and thus, affect the overall removal efficiency of the filter medium. However, such polymeric interactions in bacterial adhesion are not well understood and a method to calculate polymeric interactions is not yet available. Here, to determine how the migration of bacteria flowing within a porous medium is affected by the presence of surface-associated extracellular polymeric substances previously produced and deposited by the same bacterial species, a commonly used colloidal filtration model was applied to study transport and deposition of Pseudomonas fluorescens in small-scale columns packed with clean and biofilm coated glass beads. Bacterial recoveries were monitored in column effluents and used to quantify biofilm interactions and sticking efficiencies of the biofilm coated packed-beds. The results indicated that, under identical hydraulic conditions, the sticking efficiencies in packed-beds were improved consistently by 36% when covered by biofilm.

In-vitro experimental evaluation of skin-to-surface recovery of four bacterial species by antibacterial and non-antibacterial medical examination gloves

BackgroundThe number of bacteria recovered from a stainless steel coupon after touching a pigskin substrate with an examination glove coated on its outside with polyhexanide (PHMB), as compared to the number of bacteria recovered in the same manner with non-coated control gloves was evaluated.MethodsSuspensions containing 1 × 109 colony-forming units of 4 clinically relevant bacterial species (Enterococcus faecium ATCC #51559; Escherichia coli ATCC #25922; Klebsiella pneumoniae ATCC #4352; and Staphylococcus aureus ATCC #33591) were used to contaminate Gamma-irradiated pigskin substrates. Bacterial recoveries from the pigskin substrate, stainless steel coupons, and each glove swatch were performed. A difference in the bacterial recovery from the stainless steel coupons after touching with coated and uncoated control gloves was measured.ResultsFor E. faecium, the coated glove showed a reduction of 4.63 log10 cfu recovery, when compared to control gloves. For E. coli, the coated glove showed 5.48 log10 cfu, for K. pneumoniae 5.03 log10 cfu, and for S. aureus 5.72 log10 cfu recovery, when compared to the non-coated control glove.ConclusionAn in-vitro experiment designed to mimic cross-contamination of clinically relevant bacteria in a simulated healthcare setting following glove contact with a contaminated biological surface and cross-transfer to a stainless steel surface has demonstrated that an examination glove coated on its outside surface with PHMB was able to reduce bacterial recovery from a contaminated surface by > 4 log10 cfu, compared to a control non-coated examination glove. These elaborated results may encourage further clinical investigation on the clinical impact of an antibacterial examination glove.

Bacterial attachment to iron-impregnated granular activated carbon

Bacterial attachment to iron-impregnated granular activated carbon (Fe-GAC) was investigated in this study using Enterococcus faecalis ATCC 10100 and charcoal-based GAC. Two sets of column experiments were performed under different ionic strengths and pH conditions. Breakthrough curves of bacteria were obtained by monitoring effluent. Mass recoveries and attachment rate coefficients were quantified from these curves. In addition, characteristics of Fe-GAC were analyzed using field emission scanning electron microscopy (FESEM) and X-ray spectrometry (EDS). Results show that Fe-GAC was characterized by mosaic-like deposition layers of iron oxides with about 2 μm in thickness. Color mapping with FESEM visualized the spatial distribution of carbon (yellow-green) and iron (red) on Fe-GAC. EDS indicates that iron was distinctly found from Fe-GAC at three peak positions. Results also reveal that bacterial attachment to Fe-GAC was affected by ionic strength and pH. Bacterial mass recoveries decreased from 62.9 to 41.7% with increasing ionic strength from 1 to 50 mM. This indicates that bacterial attachment to the surfaces of Fe-GAC was enhanced with increasing ionic strength. With increasing pH from 6.46 to 9.19, mass recoveries increased from 50.5 to 84.2%, indicating that bacterial attachment to Fe-GAC was reduced with increasing pH. This study demonstrates that iron oxides offer favorable attachment sites for bacteria on the surfaces of Fe-GAC and further improves the knowledge of bacterial removal in Fe-GAC.

Straining phenomena in bacteria transport through natural porous media

Transport of bacteria through natural porous media is an issue of increasing concern arising in several very important environmental processes. These include the percolation of bacteria from fecal waste to drinking water reservoirs, thus leading to a risk for human health, or the bioremediation of contaminated soils in which the bacteria are expected to travel long distances underground in order to reach contaminated areas and degrade chemicals originating from accidental spills. An understanding of bacterial retention and transport mechanisms in porous media would be of great help in the development of models able to predict the distance covered by bacterial suspensions in these situations. Experiments were carried out preparing columns filled of soil and sand, introducing bacteria culture (Escherichia coli, Pseudomona putida, and Listeria innocua) solutions by the top of the column. Breakthrough curves were obtained to see the transport of the bacteria in the column. The transport of different bacteria in the two soils aimed at establishing the relative importance of straining in different conditions. This has enabled us to obtain certain parameters, such as the sticking coefficients derived from the filtration theory or bacterial recoveries after multi-step elution, which aid our understanding of how bacteria are retained by mechanisms different to those usually included in the physico-chemical filtration theory. Several indicators may be used to determine the degree of relevance of straining as a mechanism acting during bacterial transport through porous media. Usually, in natural media, neither straining nor physico-chemical filtration is the sole mechanism contributing to bacterial retention. The retention of bacteria by straining mechanisms can be assessed by means of elution profiles under varying conditions. The inversion of flow in our experiments gave rise to secondary elution peaks, probably originating from bacteria retained in narrow pores According to experimental observations, straining was shown to contribute highly to bacterial retention in all the soils tested, in particular in the soils with a broader grain size distribution and more irregular shape. In both media, an increase in ionic strength did not lead to significant differences in bacterial retention, possibly due to the lack of relevance of ionic repulsion as a barrier to physico-chemical attachment of particles The study of bacteria transport in natural soil is an important step in the development of decontamination processes. The importance of the straining in the transport process has been revealed in the work carried out in this paper.

Rotavirus detection in environmental water samples by tangential flow ultrafiltration and RT-nested PCR

Environmental monitoring was conducted in Otranto (Italy), from January 2006 to April 2007, to monitor the circulation of rotaviruses in various water matrices (raw and treated sewage, surface waters and seawater) and to identify any correlation with the traditional bacteriological indices (faecal coliforms). The viruses were detected using tangential flow ultrafiltration and reverse transcriptase-nested polymerase chain reaction, whilst detection of feaecal coliform was performed according to standard methods. The results showed widespread viral contamination, particularly in spring, of the matrices tested, with the exception of seawater, which at all times tested negative for the presence of rotaviruses. The typing of the rotavirus strains identified the circulation in the studied area of only two genotypes: G1 (22%) and G2 (78%). The bacterial recoveries confirmed the presence of faecal pollution indicators in all examined samples, sometimes with high values. A very weak correlation was found between the presence of faecal coliforms and the circulation of rotaviruses in the environment. The presence of rotaviruses in the environmental water samples may constitute a potential health risk for the local population.

Transport and retention of Escherichia coli in a mixture of quartz, Al‐coated and Fe‐coated sands

AbstractTransport and retention of Escherichia coli through the mixture of quartz, Al‐coated and Fe‐coated sands was examined using column experiments to investigate the effect of geochemical heterogeneity on bacteria transport. The first set of the experiments was performed in quartz, Al‐coated and Fe‐coated sand mixtures (coated sand: 0, 5, 10, 25, 50, 100%) to examine the influence of positively‐charged sand grains on bacteria transport. The second experiments were carried out to observe the impact of pH (range 6·74–8·21) on bacteria transport in the mixture of quartz 50% and Fe‐coated sand 50%. The third experiments were conducted to analyse the effect of ionic strength (0, 50, 100, 200 mM) on bacteria transport in the mixture of quartz 50% and Al‐coated sand 50%. The first experiments show that bacterial mass recoveries were in the range of 3·6–43·4%, decreasing nonlinearly as the content of Al‐ and Fe‐coated sands increased. In the second experiments, the bacterial mass recoveries were in the range of 35·5–79·2%, increasing linearly as the solution pH increased. In the third experiments, the mass recovery was 3·4% at 0 mM. As the ionic strength increased to 50mM, the mass recovery decreased to 0%. When the ionic strength increased further to 100 and 200 mM, no bacterial mass was recovered as in the case of 50 mM. It indicates that in the mixed medium of quartz 50% and Al‐coated sand 50% both positive (increment of bacterial adhesion) and negative (decrement) effects of ionic strength may be counterbalanced, minimizing the impact of ionic strength on the bacterial adhesion. This study helps to understand the role of metal oxides and solution chemistry in the transport of bacteria in geochemically heterogeneous media Copyright © 2008 John Wiley & Sons, Ltd.

Determination of bacterial mass recovery in iron-coated sand: Influence of ionic strength

Column experiments were performed in this study to investigate the influence of ionic strength on the mass recovery of Escherichia coli in iron-coated sand. The first set of the experiments was performed in the coated sand under various NaCl concentrations. The second experiments were carried out in the coated sand under various NaCl concentrations with a fixed phosphate concentration. Bacterial mass recoveries were quantified from breakthrough curves. The mass recoveries were compared with those obtained from the experiments in quartz sand under the same ionic strength/composition. Experimental results show that the mass recovery in quartz sand decreased from 76.7 to 9.2% with increasing effective ionic strength (I e ) from 0 to 149.4 mM using NaCl. In the coated sand, however, the mass recovery remained constant in the range between 2.7 and 3.7% even though I e increased in the same range. This indicates that bacterial adhesion to the coated sand may not be affected by ionic strength in the presence of NaCl. Results also illustrate that the mass recovery in quartz sand decreased from 64.7 to 13.3% with increasing I e from 0.97 to 149.6 mM using NaCl under a fixed phosphate concentration (0.97 mM as I e ). In the coated sand, the mass recovery increased sharply to 58.5% in 0.97 mM phosphate concentration compared to the case in deionized water (3.0%). This indicates that in the coated sand bacterial mass recovery can increase due to the presence of phosphate. In addition, the mass recovery in the coated sand decreased from 58.5 to 6.7% with increasing I e from 0.97 to 149.6 mM using NaCl under a fixed phosphate concentration (0.97 mM as I e ). This demonstrates that bacterial adhesion to the coated sand may be influenced by ionic strength in the presence of phosphate.

Bacteria transport through goethite-coated sand: Effects of solution pH and coated sand content

This study investigated the transport of bacteria through goethite-coated sand, focusing on the effects of solution pH and coated sand content on the transport of Escherichia coli ATCC 11105. The first set of column experiments was performed in columns (length 30 cm, diameter 5 cm) packed with quartz sand coated with goethite in solution having a pH in the range of 6–9. The second was carried out in columns (length 30 cm, diameter 2.5 cm) with varying coated sand contents ranging from 0 to 100%. Results indicate that the bacteria transport in the coated sand was influenced by solution pH. Around pH 6 and 7, bacterial mass recoveries were low at 2.4–6.7% while they were high at 76.3–81.6% around pH 8 and 9. Around pH 8, the positively charged coated sand may convert to being negatively charged, causing an electrostatically repulsive interaction between the coated sand and bacteria, thus effecting a sharp change in the mass recovery. Results also reveal that the mass recovery decreased from 76.7 to 2.7% as the coated sand content increased from 0 to 100%, showing the nonlinear dependency of mass recovery on the content of coated sand. This study demonstrates the importance of the solution pH and coated sand content in the adhesion of bacteria to goethite-coated sand and furthermore contributes to the knowledge of bacterial removal in positively charged porous media.

Patterns of maternal-offspring behaviour of dairy sheep and potential association with mammary health

Behaviour during lactation and its relationship with mammary health was investigated in 12 dairy ewes (six with single lambs and six with twins) for 42 d after lambing. Behavioural recordings were made. Samples of teat duct material and of mammary secretion were collected for bacteriological and cytological examination. Ewes performed similar patterns of behaviour as those described in the literature for meat breeds, but with some differences. No negative maternal behaviours were observed; ewes showed reduced “Grooming” and “Sniffing” immediately post-partum. Of 480 duct or secretion samples, 39 and 3, respectively (P < 0.001), yielded bacteria. There was no difference in the frequency of bacterial isolation from ewes suckling singles or twins: 46 and 54% of isolates, respectively (P = 0.637). There was greater frequency of bacterial isolation during the first stage of lactation (day 0–day 3), 0.125, compared with the second (day 4– day 11), 0.083, the third (day 12–day 30), 0.063, and the fourth (day 31–day 42), 0.069 (P < 0.001). There was also greater frequency of positive California Mastitis Test scores during the first stage of lactation, 0.917, compared with that in stages 2 (0.550), 3 (0.140) and 4 (0.181) (P < 0.001). There was decreasing duration of “Sucking attempt” and “Successful suck” throughout lactation (P < 0.05). There was increasing frequency of “Hindering sucking” and of “Head up posture” from first to subsequent stages of lactation (P = 0.038 and P = 0.002), whilst “Hindering sucking” coincided with end of “Sucking bouts” of lambs. It is concluded that termination of lamb’s sucking activity by the ewe when her mammary glands have been emptied may prevent damage to the teat and likely development of mastitis. Furthermore, frequent and longer sucking events by lambs during early lactation, may contribute to increased bacterial recoveries from mammary glands of ewes. Key words: Sheep, maternal behaviour, mastitis, sucking, post-partum period, lactation, mammary infection

Immunogenic and Protective Potential of Mutans Streptococcal Glucosyltransferase Peptide Constructs Selected by Major Histocompatibility Complex Class II Allele Binding

Mutans streptococcal glucosyltransferases (GTF) have been demonstrated to be effective components of dental caries vaccines. We had previously selected peptide subunits of GTF for vaccine development based on putative functional significance and conservation of GTF primary structure among enzyme isoforms. In this study, 20 20-mer linear GTF peptides were synthesized, 17 identified on the basis of the highest potential major histocompatibility complex (MHC) class II-binding activity using computer-generated algorithms (Epimatrix and ProPred) and 3 with previously demonstrated functional significance. The immunoreactivities of these peptides were explored with rodent systems. Sera from GTF-immunized rats, assessed for binding to linear peptides by enzyme-linked immunosorbent assay, demonstrated immunoglobulin G antibody reactivity with peptides 6 and 11 and a T-cell proliferation response to peptides 6, 9, 11, and 16. Multiple antigenic peptide (MAP) constructs were synthesized from promising linear sequences. Rats that were immunized with MAP 7, 11, or 16, respectively, responded well to the immunizing MAP. Most importantly, a robust immune response (antibody and T-cell proliferation) was observed to native GTF following MAP 11 (amino acids 847 to 866; VVINNDKFVSWGITDFEM) immunization. This response inhibited GTF enzyme function. Two dental caries pathogenesis experiments were performed wherein rats were immunized with MAP constructs 11, 16, and/or 11 plus 16, followed by infection with cariogenic Streptococcus sobrinus. In both experiments cariogenic bacterial recoveries were reduced relative to total streptococci in the MAP 11- and MAP 11 plus 16-immunized groups, and the extent of dental caries was also significantly reduced in these groups. Thus, we have identified a peptide with projected avid MHC-binding activity that elicited immunoreactivity with native GTF and demonstrated protection against dental caries infection after immunization, implying that this peptide may be important in a subunit dental caries vaccine.

Experimental Comparison of Excision and Swabbing Microbiological Sampling Methods for Carcasses

Bovine sides, ovine carcasses, and porcine carcasses were individually inoculated by dipping in various suspensions of a marker organism (Escherichia coli K-12 or Pseudomonas fluorescens), alone or in combination with two meat-derived bacterial strains, and were sampled by two standard methods: cotton wet-dry swabbing and excision. The samples were examined for bacterial counts on plate count agar (PCA plate counts) and on violet red brilliant green agar (VRBGA plate counts) by standard International Organization for Standardization methods. Average bacterial recoveries by swabbing, expressed as a percentage of the appropriate recoveries achieved by excision, varied widely (2 to 100%). Several factors that potentially contributed to relatively low and highly variable bacterial recoveries obtained by swabbing were investigated in separate experiments. Neither the difference in size of the swabbed area (10, 50, or 100 cm2 on beef carcasses) nor the difference in time of swabbing (20 or 60 min after inoculation of pig carcasses) had a significant effect on the swabbing recoveries of the marker organism used. In an experiment with swabs preinoculated with the marker organism and then used for carcass swabbing, on average, 12% of total bacterial load was transferred inversely (i.e., from the swab to the carcass during the standard swabbing procedure). In another experiment, on average, 14% of total bacterial load was not released from the swab into the diluent during standard swab homogenization. Use of custom-made swabs with abrasive butts, around which metal pieces of pan scourers were wound, markedly increased PCA plate count recoveries from noninoculated lamb carcasses at commercial abattoirs compared with cotton swabs. In spite of the observed inferiority of the cotton wet-dry swabbing method compared with the excision method for bacterial recovery, the former is clearly preferred by the meat industry because it does not damage the carcass. Therefore, further large-scale evaluation of the two carcass sampling methods has been undertaken under commercial conditions and reported separately.

The lantibiotic lacticin 3147 produced in a milk-based medium improves the efficacy of a bismuth-based teat seal in cattle deliberately infected with Staphylococcus aureus

A preparation of the bacteriocin lacticin 3147 (prepared from a demineralized whey protein fermentation liquor) was combined as a powder with a bismuth-based intramammary teat seal and evaluated for its potential as an antimicrobial in non-lactating cows. The lacticin/teat seal formulation enabled significant bacteriocin release from the seal without the requirement for a surfactant. Studies in vivo in lactating cows demonstrated that this formulation was effective in reducing bacterial recoveries (approximately 20-fold) from teats deliberately inoculated with Staphylococcus aureus after infusion. Moreover, this formulation also significantly reduced the numbers of Staph. aureus recovered from teats that were exposed to the challenge bacterium before the infusion of the teat seal preparation. The powdered preparation of lacticin 3147 did, however, cause some teat irritation as evidenced by associated rises in somatic cell count (SCC). However, this effect was short-lived and when the mean SCC readings pre-infusion and the final two readings post-infusion were compared, there was no significant difference in the immunological acceptance between treatments.

Direct detection of bacterial pathogens in representative dairy products using a combined bacterial concentration-PCR approach

To develop a simple, rapid method to concentrate and purify bacteria and their nucleic acids from complex dairy food matrices in preparation for direct pathogen detection using polymerase chain reaction (PCR). Plain non-fat yogurt and cheddar cheese were each seeded with Listeria monocytogenes or Salmonella enterica serovar. Enteritidis in the range of 10(1)-10(6) CFU per 11-g sample. Samples were then processed for bacterial concentration using high-speed centrifugation (9700 g) followed by DNA extraction, PCR amplification, and amplicon confirmation by hybridization. Bacterial recoveries after centrifugation ranged from 53 to >100% and 71 to >100% for serovar. Enteritidis and L. monocytogenes, respectively, in the non-fat yogurt samples; and from 77 to >100% and 69 to >100% for serovar. Enteritidis and L. monocytogenes, respectively, in the cheddar cheese samples. There were no significant differences in recovery efficiency at different inocula levels, and losses to discarded supernatants were always <5%, regardless of dairy product or pathogen. When followed by pathogen detection using PCR and confirmation by amplicon hybridization, detection limits of 10(3) and 10(1) CFU per 11-g sample were achieved for L. monocytogenes and serovar. Enteritidis, respectively, in both product types and without prior cultural enrichment. This study represents progress toward the rapid and efficient direct detection of pathogens from complex food matrices at detection limits approaching those that might be anticipated in naturally contaminated products.

Investigating Infectious Keratitis

In a recently published study, Kaye et al. (2) compared two approaches to evaluating bacterial keratitis: the conventional method, in which samples are obtained by taking several corneal scrapes and placing them on different media and slides (direct method, as described by Kaye et al.), and a proposed method (indirect), in which only one scrape is placed into a tube containing brain heart broth for ulterior processing at the laboratory. Their experiments with in vitro suspensions of ocular pathogens and with enucleated pig eyes inoculated with the same organisms showed that both methods led to similar bacterial recoveries. However, the authors apparently underestimated the fact that, with a low inoculum, a variation in the results was found by both techniques due to sampling problems, as bacteria were recovered in some cases but not in others. Even more important are results obtained with real corneal specimens. Kaye et al. found no differences in the percentages of positive results obtained by both techniques. However, it seems that they overlooked the fact that around 20 to 25% of diagnoses would be missed if only one of the methods was used (the numbers of positive cultures were 16, 15, and 20 for the indirect method, the direct method, and both methods, respectively). Indeed, this rate of missed diagnoses can also be attributed to sampling, and it argues in favor of making several corneal scrapes, since sampling enhances microbial detection in infectious keratitis. This fact is remarkable, and it is in concordance with my experience of attending patients from all across Argentina in a private clinic of Buenos Aires (Centro Oftalmologico “Dr. Brunzini”) that specializes in ocular infection. At this center, both ophthalmologists and microbiologists work in a close fashion with suffering patients. Samples are obtained by taking multiple corneal scrapes with a Kimura spatula and are directly seeded onto several media (chocolate and blood agar, brain heart broth, Sabouraud agar, nonnutrient agar for acanthamoebas, and occasionally Lowenstein-Jensen medium). In addition, at a minimum, two to three smears are made for stains (Gram, calcofluor white, and occasionally Ziehl-Neelsen or Giemsa). An enormous variety of etiologic agents must be investigated, and neither clinical signs nor risk factors, although helpful, can replace a definitive diagnosis (1, 3). An etiologic agent was identified in 178 out of 253 patients (70.4%); 71.4% of the agents were bacterial (including mycobacterial), 17.1% were fungal, and 11.5% were Acanthamoeba spp. (F. Nicola, M. Maltagliatti, R. Brunzini, and M. Brunzini, J. Argent. Microbiol., abstr. 070, 2003). Similarly to Kaye et al. (2), Nicola et al. found that 55% of patients had received an antibiotic treatment before being evaluated in our center, and this clearly affected the recovery of an etiologic agent (45% versus 80%, with and without previous treatment, respectively [P < 0.01]). Moreover, in some cases, the pathogens were detected in only one of the inoculated media, and it was not unusual that very few colonies (fewer than five) grew on agar plates. It could be inferred then that bacterial load in naturally infected corneas is usually low, and in addition, significant variations in microorganism distribution in corneal abscesses occur. Finally, it is believed that expertise in both taking representative samples by firm corneal scraping and microbiologic processing of the samples, in a direct and rapid manner, is invaluable for a good diagnostic performance. Although this approach may be difficult to implement in all ophthalmologic settings, the recommendation is to make a special effort. If no laboratory-skilled assistant can be present at the time of sampling, basic microbiologic training should be given to the ophthalmologist, and fresh media should be provided as required. Data showing a rate of definitive diagnosis (70.4%) that is among the highest of those published (1, 2, 3) are the basis for this encouragement.

Magnetized Carbonyl Iron and Insoluble Zirconium Hydroxide Mixture Facilitates Bacterial Concentration and Separation from Nonfat Dry Milk

A mixture of magnetized carbonyl iron and insoluble zirconium hydroxide was investigated for its ability to concentrate various foodborne pathogens from 25-ml samples of reconstituted nonfat dry milk. Each sample was artificially contaminated with 103 to 106 CFU/25 ml of representative foodborne pathogens (Salmonella enterica serovar Enteritidis, Listeria monocytogenes, and Bacillus cereus spores) and processed for bacterial concentration with high-speed centrifugation for the primary concentration followed by a secondary concentration step involving the carbonyl iron–zirconium hydroxide mixture. Bacterial recoveries, as evaluated on the basis of loss to discarded supernatants, exceeded 75% for all organisms at all inoculum levels and were usually >90%. Recovery was confirmed by direct plating of the immobilized pellet, for which the values were similar albeit more varied. Additional experiments confirmed that the magnetized carbonyl iron–insoluble zirconium hydroxide mixture was relatively nontoxic to both Salmonella Enteritidis and L. monocytogenes. Overall, the entire concentration scheme resulted in a 25-fold reduction in sample volume with the recovery of viable bacterial cells. This novel compound shows promise for facilitating inexpensive, rapid, and effective bacterial concentration in food systems.

Immobilization with metal hydroxides as a means to concentrate food-borne bacteria for detection by cultural and molecular methods.

The application of nucleic acid amplification methods to the detection of food-borne pathogens could be facilitated by concentrating the organisms from the food matrix before detection. This study evaluated the utility of metal hydroxide immobilization for the concentration of bacterial cells from dairy foods prior to detection by cultural and molecular methods. Using reconstituted nonfat dry milk (NFDM) as a model, two food-borne pathogens (Listeria monocytogenes and Salmonella enterica serovar Enteritidis) were concentrated from 25-ml samples by the sequential steps of clarification and high-speed centrifugation (designated primary concentration) and immobilization with zirconium hydroxide and low-speed centrifugation (designated secondary concentration). Sample volume reduction after immobilization with zirconium hydroxide was 50-fold, with total bacterial recoveries ranging from 78 to 96% of input for serovar Enteritidis and 65 to 96% of input for L. monocytogenes. Immobilized bacteria remained viable and could be enumerated by standard cultural procedures. When followed by RNA extraction and subsequent detection by reverse transcription (RT)-PCR, detection limits of 10(1) to 10(2) CFU/25 ml of reconstituted NFDM were achieved for both organisms. The bacterial-immobilization step was relatively nonspecific, resulting in recovery of >50% of the input cells when evaluated on a panel of representative bacterial strains of significance to foods. The method could be adapted to more complex dairy products, such as whole milk and ice cream, for which bacterial recoveries after immobilization ranged from 64 to >100%, with subsequent RT-PCR detection limits of >/=10(2) CFU/ml for whole milk and >/=10(1) CFU for ice cream for both serovar Enteritidis and L. monocytogenes. The bacterial-immobilization method is easy, rapid, and inexpensive and may have applications for the concentration of a wide variety of food-borne bacteria prior to detection by both conventional and alternative methods.

Sampling Performance of Impactors for Bacterial Bioaerosols

Impactor is more commonly used than impinger and filter for bioaerosol sampling because of direct collection without post-collection sample processing. In this study, sampling bacterial performance of microbial impactors with different sampling velocities and cut-point diameters was assessed. By the evaluated impactors, the recoveries of hardy bacterial spores was observed to be much higher than those of sensitive gram-negative bacteria. In addition, it was demonstrated that sampling biological recoveries of Andersen 1-STG sampler were much higher than those of MAS-100 and Burkard samplers. These might be related to lower cut-point diameter of Andersen 1-STG sampler with higher collection efficiency. Moreover, a sampling time of < 40 min was not indicated to significantly influence bacterial recoveries of the evaluated impactors.

Sterilization of infected root-canal dentine by topical application of a mixture of ciprofloxacin, metronidazole and minocycline in situ.

The aim of this study was to observe the potential of a mixture of ciprofloxacin, metronidazole and minocycline to kill bacteria in the deep layers of root canal dentine in situ. After the crowns of extracted teeth had been removed, the drug combination (0.5 mg of each drug), or sterile saline, as the control, was placed in the root canals which had been previously irrigated ultrasonically with G4M EDTA. The penetration and bactericidal efficacy were estimated by various procedures as follows. (1) A cell suspension of E. coli was placed into small cavities prepared parallel to the root canals on the cut planes of nine single-rooted teeth. The teeth were then entirely covered with blue inlay wax. At time 0, and at 5h, 24h and 48h after the drug combination had been applied, cells of E. coli were recovered from the cavities by washing the cavities several times with sterile saline solution, and were cultured on the surfaces of heart-infusion (HI) agar plates. Total colony-forming units were tuen counted. Bacterial recoveries decreased with time, and no bacteria were recovered 48 h after application of the drug combination, while bacteria survived in all cases with the controls, (2) After the drug combination or sterile saline had been placed into and sealed in the root canal with blue inlay wax, the teeth were placed into HI agar plates where cells of E. coli had been inoculated. After culturing, a clear zone caused by the inhibition of bacterial growth was observed around the teeth, but not in the control experiment. (3) After sampling infected root dentine of 12 freshly extracted teeth as positive controls, the drug combination (0.5 mg each) was placed in the root canals. No bacteria were recovered from the infected dentine of the root canal wall 24 h after application of the drug combination, except in one case in which a few bacteria were recovered. On the basis of these results, penetration through dentine and antibacterial efficacy of the drug combination can be expected against bacteria infecting the dentine of the root canal wall in situ when the drugs were placed in root canals which had been irrigated ultrasonically.

Simultaneous Caries Induction and Calculus Formation in Rats

Weanling specific pathogen-free Osborne-Mendel rats were fed a high-calcium, high-phosphorus diet with various levels of sucrose and inoculated with Streptococcus sobrinus strain 6715-13WT and Actinomyces viscosus strain OMZ-105 in order to determine whether calculus and caries could develop simultaneously. Rats consumed diets designated RC-16-5, RC-16-25, or RC-16-50 which partially replaced the corn starch component with progressively higher levels of sucrose, thus, to 5, 25, or 50% sucrose. In general, bacterial recoveries of A. viscosus declined with higher sucrose content of the diet, but a pattern of recovery for S. sobrinus was less clear with respect to dietary sucrose. S. sobrinus, however, was recovered at higher percentages from the tooth surface flora at the later two of three sampling dates. Most calculus--identified by the brittle quality, staining characteristics, and apatitic x-ray diffraction patterns of tooth surface deposits--was formed on the maxillary molars, and most carious lesions occurred on mandibular molars. While there was minimal association of the calculus score with the amount of sucrose in the diet, calculus scores increased greatly from 23 to 43 days after infectious challenge. Caries scores, of both fissure and smooth surfaces, by contrast, increased in a dose-response fashion with increasing dietary sucrose and with time. It is thus possible to induce calculus formation and caries simultaneously in specific pathogen-free Osborne-Mendel rats consuming a high-calcium and -phosphorus diet conducive to calculus formation and containing sucrose.(ABSTRACT TRUNCATED AT 250 WORDS)