Viable Bacterial Numbers Research Articles

Lag Plot: A Novel Method to Determine Viable Bacterial Cell Number via a Single OD Measurement.

Bactericidal activity is a valuable parameter which is considered and measured for antimicrobial compounds. The available standard protocol to evaluate bactericidal activity is based on the direct colony count. Colony counting requires serial dilution, plating, overnight incubation, and direct counting, which is time-and labor-intensive. In regard to eliminate direct plate count, novel techniques were developed based on the real-time growth monitoring which can come with some limitations and drawbacks. These drawbacks encourage us to develop a novel technique with simple procedure to determine viable bacterial cell count. In this procedure, real-time growth monitoring is not required. In fact, after an incubation time, the number of viable bacteria can be determined with a single OD measurement and through an equation. In this regard, four standard bacterial strains, including Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), and Bacillus subtilis (ATCC 23857), were cultured with descending inoculum densities (1.5 × 107 to 15CFU/mL) and growth curves were drawn. As expected, growth in the samples with lower inoculum densities recorded with longer lag phase. Also, a direct relation was observed between the recorded turbidities and initial cell counts. A logarithmic curve (lag plot) was obtained by plotting the OD, after 12-18h incubation, against initial cell counts. In all examined strains, R2 was calculated in the range of 0.96-0.99 which is acceptable value for coefficient of determination. Equation corresponding to the lag plot was obtained and called lag equation. This equation is applicable to calculate the number of viable cells in unknown samples simply by inoculation, incubation, and single OD measurement. Developed technique can be introduced as a simple substitution for labor- and time-intensive direct colony counting.

PSV-12 Evaluating the effect of lyoprotectants in preserving community structure and bacterial viability in the lyophilized fecal community of pigs

Abstract Fecal microbiota transplantation (FMT) stands as a potential method for enhancing pig health. FMT has progressed from the basic delivery of fresh stool, to lyophilized preparations which is beneficial for sample storage, but can damage bacterial cells. This has necessitated the need for lyoprotectants to preserve bacterial viability. However, the ideal lyoprotectant for retaining community structure and viability remains unclear. Furthermore, DNA-based microbial profiling methods such as 16S rRNA and metagenomics sequencing do not distinguish live and dead bacteria, prompting the need for other methods of distinguishing live and dead members of the bacterial community after lyophilization. PMA-seq (propidium monoazide treatment followed by 16S rRNA gene amplicon sequencing) has been shown to differentiate between live and dead bacteria in microbial communities. To refine FMT in pigs, we sought to ascertain the impact of four lyoprotectants on bacterial viability and fecal microbiome structure. Fecal samples from six pigs were treated with mannitol, maltodextrin, trehalose, maltodextrin-trehalose mix (2.5% each) as lyoprotectants or left untreated. Lyophilized fecal samples underwent PMA treatment prior to 16S rRNA sequencing to capture the active community, and total and viable bacterial cells number were quantified. One-way ANOVA was used to analyze total and viable bacterial cells, and richness, while PERMANOVA was used to compare beta diversity. Results revealed no initial differences in total, viable bacteria, or community structure (P > 0.05). However, untreated, and mannitol-treated groups showed significantly reduced total and viable bacterial cells post-lyophilization (P < 0.01), unlike the other lyoprotectants (P > 0.05). Mannitol and the untreated group also displayed decreased richness and beta diversity shifts (P < 0.05). Overall, lyophilization with mannitol or no lyoprotectant notably impacted pig fecal microbiota community structure, while maltodextrin, trehalose, and maltodextrin-trehalose effectively preserved bacterial viability and structure.

Isolation and identification of blueberry-derived lactic acid bacteria and their probiotic, antioxidant, and fermentation properties

In this study, high-throughput sequencing was used to analyze the microbial diversity and relative abundance of the blueberry epidermis and found that lactic acid bacteria (LAB) represent a relatively abundant group. Putative LAB strains were isolated and purified from the blueberry epidermis by traditional biological methods. Thirty LAB strains were identified by genotype identification, physiological and biochemical test, and 16 S rDNA sequence analysis revealed 100% sequence identity between 8 isolated strains and the representative LAB in the blueberry epidermis. Of the 8 strains, ZYN-0417 (Lactobacillus rhamnosus) and ZYN-0221 (Lactobacillus plantarum) showed strong tolerance in the probiotic test. Under gastric acid conditions with a pH of 2.0, the respective survival rate reached 65.71% and 68.85%. The respective survival rate reached 96.45% and 98.37% in 1.8% bile salt. After incubation in vitro gastrointestinal fluid for 240 min, the respective survival rate reached 91.00% and 86.26%. The antioxidant capacity test showed that the DPPH scavenging rate of ZYN-0417 and ZYN-0221 reached 85.45% and 87.45%, with ABTS free radical scavenging rate of 76.03% and 66.17%, OH scavenging rate of 57.30% and 46.93%, and Fe2+ chelating ability of 46.34% and 43.70%, respectively. Fermentation study showed that ZYN-0417 and ZYN-0221 were capable of strong acid production with respective pH values and total acid content of 3.64 and 3.63, 14.88 mg⋅L−1 and 14.85 mg⋅L−1 after 36 h of fermentation. Moreover, ZYN-0417 and ZYN-0221 showed strong extracellular protease activity (82.99 U⋅mL−1 and 95.17 U⋅mL−1, respectively). Blueberry juice fermentation showed robust growth of ZYN-0417 and ZYN-0221 and the viable bacterial number of LAB reached 13–14 log CUF⋅mL−1 at the end of fermentation. In summary, blueberry-derived LAB have good probiotic and antioxidant properties, and strong fermentation properties in low acid environments, thus showing the potential of their use for the lactobacillus fermentation of fruit and vegetables.

Do the Culturable Microbial Groups Present in Cutaway Bogs Change According to Temporal Variation? Pilot Study Based on the Midlands in the Republic of Ireland

Cutaway peatlands in the midlands of the Republic of Ireland are rarely the focus of scientific studies. The soil quality and related microenvironment have been severely impacted by peat extraction. Returning them to a ‘near-natural state’ would require greater insights into this ecological niche. The current research took the initiative to study the microbiology of vast cutaway sites in the midlands of Ireland. Peat was collected over two seasons in January, February and April. Homogenised peat was aseptically cultured on a range of specific and non-specific culture media. Microbial enumeration, Gram staining and other microscopic observations of morphologically distinct microorganisms were performed. The total viable bacterial and fungal numbers were highest in February (1.33 × 105 CFU ml−1 and 5.93 × 106 CFU ml−1, respectively) and were lowest in April (1.14 × 103 CFU ml−1 and 5.57 × 106 CFU ml−1). Penicillium spp. and Trichoderma spp. were common in all the sites. The highest values of phosphate solubilisation index were recorded in peat collected in April (SI = 3.167 & 3.000). Overall, there is a statistically significant difference (p ≤ 0.0001) among the microbial numbers across the three months. This variation could be due to the temperature and pH differences across peat soil.

Antimicrobial Effect of Polyhexanide on Denture Base and Soft Lining Materials

Objective: Polyhexanide (PHMB; polyhexamethylene biguanide) is a well-known antiseptic agent; however, no data exist for its applicationon denture base and lining materials. In the present in vitro study, the aim was to compare viable bacterial or fungal cell numbers and theiradhesion on different types of denture base and lining materials in diverse concentrations of PHMB.Methods: Light-activated urethane dimethacrylate (UDMA), heat-polymerized polymethyl methacrylate (PMMA), heat-polymerizedpolydimethylsiloxane, and autopolymerizing polyethylmethacrylate discs were prepared for each group (n = 10). 1×108 CFU/mL of all the testedspecies were appended separately to discs, and they were immersed into different PHMB suspensions (0.5%, 1%, 2%, and 5%) for 10 minutes.The antimicrobial activity and number of adherent species on the surface were evaluated.Results: In the PMMA group, all studied species except C. albicans, L. acidophilus, and S. aureus were decreased in various concentrations (p <0.05), and all studied species presented a significant decrease in every concentration of PHMB in the UDMA group (p < 0.01) in comparison to thecontrol. N. sicca, K. pneumoniae, S. pyogenes, S. sanguis, C. pseudotuberculosis, and S. aureus (p < 0.05) were reduced in the heat-polymerizedpolydimethylsiloxane group, while all tested species except B. subtilis were decreased in the autopolymerizing polyethylmethacrylate group incomparison to the control (p < 0.01). Among all tested materials and species, no significant difference was detected in adherent cell number(p > 0.05).Conclusion: PHMB suspension, in various concentrations, can reduce some species of bacterial and yeast cells.

Smart Chimeric Lysin ClyC Loaded Alginate Hydrogel Reduces Staphylococcus aureus Induced Bone Infection

Staphylococcus aureus (S. aureus) is the most common cause of hospital and community-acquired infections. The current clinical treatment is limited by the emergence of drug-resistant strains. We previously developed a chimeric ClyC that effectively inhibited S. aureus strains. Nonetheless, an efficient delivery system to provide sustained release of ClyC to infected site is needed. Thus, we engineered a chimeric ClyC loaded alginate hydrogel (ClyC-AH) to improve the therapeutic outcomes against S. aureus. ClyC-AH retained the stability and activity of ClyC while providing a sustained release of ClyC and a continuous antibacterial effect against S. aureus. Compared to ClyC alone, the use of ClyC-AH was relatively safe, as there was no significant cytotoxicity to BHK-21 cells at a ClyC concentration≤250 μg/ml. Furthermore, in a S. aureus infected mouse model of osteomyelitis, ClyC-AH reduced bacterial burden in the femur and surrounding tissues, with a reduction of 2 log10 (CFU/ml) in viable bacterial number. Based on these results, hydrogel-delivered chimeric lysin ClyC provides a promising future in the S.aureus targeting therapy.

A Highly Active Chimeric Lysin with a Calcium-Enhanced Bactericidal Activity against Staphylococcus aureus In Vitro and In Vivo.

Lysins, including chimeric lysins, have recently been explored as novel promising alternatives to failing antibiotics in treating multi-drug resistant (MDR) pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). Herein, by fusing the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) catalytic domain from the Ply187 lysin with the non-SH3b cell-wall binding domain from the LysSA97 lysin, a new chimeric lysin ClyC was constructed with Ca2+-enhanced bactericidal activity against all S. aureus strains tested, including MRSA. Notably, treating S. aureus with 50 μg/mL of ClyC in the presence of 100 μM Ca2+ lead to a reduction of 9 Log10 (CFU/mL) in viable bacterial number, which was the first time to observe a lysin showing such a high activity. In addition, the effective concentration of ClyC could be decreased dramatically from 12 to 1 μg/mL by combination with 0.3 μg/mL of penicillin G. In a mouse model of S. aureus bacteremia, a single intraperitoneal administration of 0.1 mg/mouse of ClyC significantly improved the survival rates and reduced 2 Log10 (CFU/mL) of the bacterial burdens in the organs of the infected mice. ClyC was also found stable after lyophilization without cryoprotectants. Based on the above observations, ClyC could be a promising candidate against S. aureus infections.

Natural photosensitizers from Tripterygium wilfordii and their antimicrobial photodynamic therapeutic effects in a Caenorhabditis elegans model

Tripterygium wilfordii Hook. f. is a traditional medicinal plant and has long been used in East Asia to treat many diseases. However, the extract and active components have never been investigated as potential photosensitizers for photodynamic treatment to kill pathogenic microorganisms. Here, the antimicrobial photodynamic treatment (APDT) effects of the extract, fractions, and compounds of T. wilfordii were evaluated in vitro and in vivo. Ethanolic extract (TWE) and the photosensitizer-enriched fraction (TW-F5) were prepared from dried T. wilfordii. Six active compounds were isolated from TW-F5 by semipreparative high-performance liquid chromatography, and their chemical structures were characterized through spectroscopic and spectrometric analysis. The singlet oxygen from extracts, fractions, and compounds was measured by using the imidazole-N,N-dimethyl-4-nitrosoaniline method. These extracts, fractions, and compounds were used as photosensitizers for the inactivation of bacteria and fungi by red light at 660 nm. The in vitro APDT effects were also evaluated in the model animal Caenorhabditis elegans. APDT with TWE showed effective antimicrobial activity against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), and Candida albicans. TW-F5, consisting of six pheophorbide compounds, also showed strong APDT activity. The photosensitizers were taken up into the bacterial cells and induced intracellular ROS production by APDT. TWE and TW-F5 also induced a strong APDT effect in vitro against skin pathogens, including Staphylococcus epidermidis and Streptococcus pyogenes. We evaluated the APDT effects of TWE and TW-F5 in C. elegans infected with various pathogens and found that PDT effectively controlled pathogenic bacteria without strong side effects. APDT reversed the growth retardation of worms induced by pathogen infection and decreased the viable pathogenic bacterial numbers associated with C. elegans. Finally, APDT with TWE increased the survivability of C. elegans infected with S. pyogenes. In summary, TWE and TW-F5 were found to be effective antimicrobial photosensitizers in PDT.

Evaluation of Novel Virulent Phages Infecting the Aeromonas Hydrophila and Escherichia coli Isolated from Sewage Water Samples

MULTIDRUG-resistant bacteria are now emerging for almost all the present-day antibiotics. Aeromonas hydrophila D2007 and Escherichia coli W102 were isolated from fresh food and drinking water samples and they were resistant to 57.14% and 85.71% of tested common antibiotics respectively. Three bacteriophages (phages) were isolated from sewage samples. Morphological examinations suggested that phage ΦAHP7, which infects A. hydrophila D2007, belongs to the Myoviridae family and other phages ΦECP8 and ΦECP9 capable of lysing E. coil W102 belongs to Siphoviridae and Podoviridae families, respectively. For the three phages, the optimal multiplicity of infection (MOI) was calculated to be 0.001. Phages were characterized by determining their host range and stability in pHs, temperatures, and salinity. The latent periods of phages ΦAHP7, ΦECP8, and ΦECP9 were 10, 20 and 10min with average burst sizes of 53.5±0.5, 26.5±0.5 and 67.5±0.5 phages per infected cell, respectively. The three phages gradually reduced OD600 and are able to stop the growth of A. hydrophila D2007 and E. coli W102 in vitro at a low MOI of 0.001. Phages ΦAHP7, ΦECP8, and ΦECP9 treatments achieved 1.55, 1.68 and 2.28 log CFU/g (P< 0.01) reduction of viable bacterial number in red cabbage and 1.48, 1.38 and 1.68 log CFU/g (P< 0.01) reduction in tomato after 30 min at room temperature (28°C) respectively. Applications of lytic ΦAHP7, ΦECP8, and ΦECP9 bacteriophages lead to a rapid reduction of A. hydrophila D2007 and E. coli W102 counts in fresh food for human consumption.

Antibacterial activity and mechanism of Litsea cubeba essential oil against food contamination by Escherichia coli and Salmonella enterica

AbstractLitsea cubeba essential oil (LC‐EO) has been reported as antibacterial agents, but there are few studies about its possible antibacterial mechanism. The antibacterial activities and the underlying mechanisms of LC‐EO against Escherichia coli O157: H7 and Salmonella enterica were investigated. The results showed that the LC‐EO was more effective against gram‐negative bacteria. The inhibition zone for E. coli O157: H7 and S. enterica were 3.1 ± 0.8 and 4.5 ± 0.6 mm, respectively. The minimum inhibitory concentration of LC‐EO against both bacteria was 0.9 μg/ml, while the minimum bactericidal concentrations were 4.5 and 9 μg/ml. Gas chromatography–mass spectrometry analysis confirmed that citral (86.8%) was the main component of LC‐EO. The results of a time–kill analysis illustrated that treatment with LC‐EO led to a rapid decrease in viable bacterial cell number. The release of electrolytes and nucleic acids from the bacterial cells increased with the dose of LC‐EO. Propidium iodide uptake revealed that LC‐EO caused cell membrane damage. Scanning electron and transmission electron microscopy showed that LC‐EO caused damage to the cell walls and membranes, resulting in cell deformation, atrophy, and large central voids. Thus, LC‐EO may provide the basis for the development of new natural food preservatives.

Effects of Osmotic Stress and Sodium Hypochlorite on Endodontic Microbiota: An In-Vitro Study.

Objective:To assess the effect of osmotic stress on various bacteria in a planktonic milieu and the effect of exposure to sodium hypochlorite (NaOCl) on the microbial cells previously subjected to osmotic stress.Methods:Enterococcus faecalis, Streptococcus sanguinis, Fusobacterium nucleatum, Porphyromonas gingivalis and Prevotella intermedia were suspended as follows: Iso-osmotic group 0.9% NaCl; Hypo-osmotic group “ultrapure water”; Hyper-osmotic group 9% NaCl solution for 120 hours before exposure to 0.0001% NaOCl for 10 minutes. Quantitative analyses of viable cells were performed at 0 and 120 hours and after exposure to NaOCl to obtain colony forming units (CFU/mL). A linear mixed-effects model was used to find the association between mean CFU/mL (logarithmic transformation) and the interaction of solution Group and Time (P<0.001).Results:F. nucleatum, P. gingivalis and P. intermedia did not survive after 24 hours in any of the solutions and were excluded from further testing. For S. sanguinis there were significant differences at each time interval, when holding solution group constant. After 120 hours, the Hyper-osmotic group presented with the highest CFU/mL and was significantly different to the Iso-osmotic group (P<0.001). For E. Faecalis, there was a significant difference for each pairwise comparison of time (P<0.001) in mean CFU/mL between 0 hours and 120 hours for the Iso-osmotic and Hyper-osmotic groups. At 120 hours, no significant differences were found between the three groups. Significant differences were also found between 0 hours and Post-NaOCl administration, and between 120 hours and Post-NaOCl administration for all three groups (P<0.001). Exposure to NaOCl after hypo-osmotic stress was associated with significantly less CFU/mL for S. sanguinis compared to hyperosmosis and iso-osmosis (P<0.001) and for E. Faecalis only compared to hyperosmosis (P<0.001).Conclusion:S. sanguinis and E. faecalis were able to withstand osmotic stress for 120 hours. Hypo-osmotic stress before contact with NaOCl was associated with lower viable bacterial numbers, when compared to the other media for the above species. Hyper-osmotic stress was associated with higher viable bacterial numbers after NaOCl exposure for E. faecalis.

Viable and Total Bacterial Populations Undergo Equipment- and Time-Dependent Shifts during Milk Processing.

We set out to identify the viable and total bacterial content in milk as it passes through a large-scale, dairy product manufacturing plant for pasteurization, concentration, separation, blending, and storage prior to cheese manufacture. A total of 142 milk samples were collected from up to 10 pieces of equipment for a period spanning 21 h on two collection dates in the spring and late summer of 2014. Bacterial composition in the milk was determined by 16S rRNA marker gene, high-throughput DNA sequencing. Milk samples from the late summer were paired such that half were treated with propidium monoazide (PMA) to enrich for viable cells prior to quantification by PCR and identification by DNA sequence analysis. Streptococcus had the highest median relative abundance across all sampling sites within the facility on both sampling dates. The proportions of Anoxybacillus, Thermus, Lactococcus, Lactobacillus, Micrococcaceae, and Pseudomonas were also elevated in some samples. Viable cells detected by PMA treatment showed that Turicibacter was enriched after high-temperature short-time pasteurization, whereas proportions of Staphylococcus were significantly reduced. Using clean-in-place (CIP) times as a reference point, Bacillus, Pseudomonas, and Anoxybacillus were found in high relative proportions in several recently cleaned silos (<19 h since CIP). At later times (>19 h after CIP), 10 of 11 silos containing elevated viable cell numbers were enriched in Acinetobacter and/or Lactococcus These results show the tremendous point-to-point and sample-dependent variations in bacterial composition in milk during processing.IMPORTANCE Milk undergoes sustained contact with the built environment during processing into finished dairy products. This contact has the potential to influence the introduction, viability, and growth of microorganisms within the milk. Currently, the population dynamics of bacteria in milk undergoing processing are not well understood. Therefore, we measured for total and viable bacterial composition and cell numbers in milk over time and at different processing points in a cheese manufacturing facility in California. Our results provide new perspectives on the dramatic variations in microbial populations in milk during processing even over short amounts of time. Although some of the changes in the milk microbiota were predictable (e.g., reduced viable cell numbers after pasteurization), other findings could not be easily foreseen based on knowledge of bacteria contained in raw milk or when the equipment was last cleaned. This information is important for predicting and controlling microbial spoilage contaminants in dairy products.

Stability of probiotics with antibiotics via gastric tube by simple suspension method: An in vitro study

Data on the stability of probiotics with antibiotics delivered via gastric tube using the simple suspension method (SSM) are limited. Therefore, we investigated bacterial survivability in probiotics treated with antibiotics prepared by the SSM in vitro. Probiotics and antibiotics were suspended in 20 mL of sterilized hot water (55 °C) and then 1-mL of the suspensions were taken each at 10, 60, 120, 180 and 360 min. Thereafter, the samples were inoculated on 3 media and cultured at 37 °C for 24 h. Survival of probiotic strains was measured in colony-forming units. The growth of Clostridium butyricum did not change without antibiotics at all experimental times, but in the case of Enterococcus faecium tended to increase. On the other hand, the viable bacterial number of C. butyricum was decreased significantly by treatment with cefdinir, tosufloxacin, clarithromycin, or azithromycin, but was not altered by levofloxacin, minocycline, or vancomycin. The viable bacterial number of E. faecium was significantly decreased by treatment with tosufloxacin, levofloxacin, minocycline, vancomycin, or azithromycin, and was significantly increased by clarithromycin. In conclusion, our results suggest that the efficacy of probiotic therapies might be reduced by the SSM when specific antibiotics are used. Moreover, antibiotics might inhibit probiotic growth, although some probiotics are spore-forming and have high minimum inhibitory concentrations. Additionally, early administration of non-spore-forming bacteria might be desirable. Therefore, when patients are administered therapy combining probiotics and antibiotics by the SSM, we should consider the characteristics of the probiotics and the administration times.

Rapid Quantification of the Total Viable Bacterial Population on Human Hands Using Flow Cytometry with SYBR® Green I.

Monitoring the efficiency of hand washing typically relies on determining the presence or absence of targeted organisms on the hands, not necessarily determining if the method is working. The results focus on the organism studied and do not give a true reflection of the efficiency of the hygiene intervention. To obtain a better picture, this study tested flow cytometry with SYBR® Green I to quantify bacterial populations on the hands of participants in a healthcare training clinic at a University in South Africa. Participants "washed" both hands in a buffer solution in a sterile bag, and the total and viable bacterial populations numbers were determined using flow cytometry and compared with standard culture-based methods, testing for total coliforms, E. coli and heterotrophic organisms (IDEXX Colilert®-18 Quanti-Trays™ and SimPlates®) and fastidious organisms with hemolytic activity (Blood Agar). Compared to the culture-based method, flow cytometry rapidly distinguished total, viable, and dead bacterial populations in all samples. As expected, the culture-based methods gave lower bacterial counts and were limited by the detection limit of each test, requiring further testing with additional costs. Although the exact bacterial species in the population could not be identified with flow cytometry, viable counts from flow cytometric analysis were two times more precise than the data obtained with any of the culture-based methods tested. Flow cytometry was found to be suitable for immediate, accurate, and automatic detection of bacteria, and because it describes the viable bacterial population, it is ideal to monitor hand hygiene interventions. © 2019 International Clinical Cytometry Society.

In vitro effect of diode laser against biofilm of Aggregatibacter actinomycetemcomitans.

Objective:The main objective is to investigate the antibacterial effect of diode laser against Aggregatibacter actinomycetemcomitans biofilm.Materials and Methods:Biofilms of A. actinomycetemcomitans plus Streptococcus sanguinis grown on bovine root surfaces were treated with an 810-nm diode laser using a noncontact pulsed mode with a pulse interval and pulse length of 20 ms. Four protocols, that is, one episode of 1.5 or 2.5 W for 30 s and three episodes of 1.5 or 2.5 W for 30 s were tested. No treatment and 0.2% chlorhexidine treatment served as negative and positive controls, respectively. Viable bacterial number was determined by colony counting.Results:Treatment with chlorhexidine and all laser protocols except that using single episode of 1.5 W reduced the number of A. actinomycetemcomitans in either single-species or dual-species biofilm compared to negative control. A higher percentage of A. actinomycetemcomitans reduction was demonstrated after increasing the power output or repeating the irradiation.Conclusions:The laser protocols used in this study could reduce the number of viable bacteria but not eradicate A. actinomycetemcomitans biofilm.

Evaluation of Bacteriophage Anti-Biofilm Activity for Potential Control of Orthopedic Implant-Related Infections Caused by Staphylococcus aureus.

Despite significant advancements in surgical protocols and biomaterials for orthopedics, peri-prosthetic joint infection (PJI) remains a leading cause of implant failure. Staphylococcus aureus nasal colonization is an established risk factor for PJI, with methicillin-sensitive S. aureus a leading cause of orthopedic implant-related infections. The purpose of these in vitro studies was to investigate the antibacterial activity of a tailored bacteriophage cocktail against planktonic and biofilm-associated S. aureus. The S. aureus strains (n = 30) were screened for their susceptibility to a library of S. aureus-specific bacteriophage (n = 31). Five bacteriophage preparations that demonstrated bactericidal activity against >90% of S. aureus strains tested were combined as a StaPhage cocktail and assessed for their antibacterial activity toward planktonic and biofilm-associated S. aureus, with biofilms established on three-dimensional-printed porous titanium scaffolds. StaPhage treatment immediately after bacterial inoculation inhibited growth of S. aureus by >98% in eight hour cultures when multiplicity of infection of phages to bacteria was greater than 1:1 (p < 0.01). Viable bacterial numbers within biofilms on titanium surfaces were significantly reduced (6.8 log10 to 6.2 log10 colony forming units [CFU]; p < 0.01) after exposure to the StaPhage cocktail, in vitro. No significant reduction was observed in biofilms exposed to 100 times the minimal inhibitory concentration of cefazolin (log10 6.81 CFU). Combined, these data demonstrate the in vitro efficacy of S. aureus-specific bacteriophage cocktails against S. aureus growing on porous titanium and warrant further in vivo studies in a clinically relevant animal model to evaluate the potential application of bacteriophage in the management of PJI caused by S. aureus.

Effects of monochloramine and hydrogen peroxide on the bacterial community shifts in biologically treated wastewater

Amending feed water with biocide is one of the strategy conventionally used to control biofouling in membrane-based water treatment systems. In this study, the impacts of two biocides, monochloramine (MCA) and hydrogen peroxide (H2O2), on the bacterial community in wastewater samples were investigated at equivalent biocidal efficiency levels. Viable bacterial numbers were determined before and after treatment for 10 min and 60 min using both culture-dependent heterotrophic plate count (HPC) and culture-independent propidium monoazide (PMA)-droplet digital PCR (ddPCR). Shifts of the live bacterial diversity were studied using high-throughput sequencing of 16S rRNA genes and followed by bioinformatics analysis. At the genus level, MCA treatment increased the relative abundance of Mycobacterium, Pseudomonas, Sphingomonas, Clostridium, Streptococcus, Undibacterium, Chryseobacterium and Cloacibacterium, while decreasing Arcobacter, Nitrospira and Sphingobium. H2O2 treatment increased the relative abundance of Anaerolinea and Filimonas, and diminished Denitratisoma and Thauera. The findings of this study suggest a combination of different types of biocide may be the most efficient strategy for biofouling mitigation and increasing membrane treatment efficiency.

Within-host spatiotemporal dynamics of systemic Salmonella infection during and after antimicrobial treatment.

ObjectivesWe determined the interactions between efficacy of antibiotic treatment, pathogen growth rates and between-organ spread during systemic Salmonella infections.MethodsWe infected mice with isogenic molecularly tagged subpopulations of either a fast-growing WT or a slow-growing ΔaroC Salmonella strain. We monitored viable bacterial numbers and fluctuations in the proportions of each bacterial subpopulation in spleen, liver, blood and mesenteric lymph nodes (MLNs) before, during and after the cessation of treatment with ampicillin and ciprofloxacin.ResultsBoth antimicrobials induced a reduction in viable bacterial numbers in the spleen, liver and blood. This reduction was biphasic in infections with fast-growing bacteria, with a rapid initial reduction followed by a phase of lower effect. Conversely, a slow and gradual reduction of the bacterial load was seen in infections with the slow-growing strain, indicating a positive correlation between bacterial net growth rates and the efficacy of ampicillin and ciprofloxacin. The viable numbers of either bacterial strain remained constant in MLNs throughout the treatment with a relapse of the infection with WT bacteria occurring after cessation of the treatment. The frequency of each tagged bacterial subpopulation was similar in the spleen and liver, but different from that of the MLNs before, during and after treatment.ConclusionsIn Salmonella infections, bacterial growth rates correlate with treatment efficacy. MLNs are a site with a bacterial population structure different to those of the spleen and liver and where the total viable bacterial load remains largely unaffected by antimicrobials, but can resume growth after cessation of treatment.

Alloiococcus otitidis Forms Multispecies Biofilm with Haemophilus influenzae: Effects on Antibiotic Susceptibility and Growth in Adverse Conditions.

Otitis media with effusion (OME) is a biofilm driven disease and commonly accepted otopathogens, such as Haemophilus influenzae, Streptococcus pneumonia, and Moraxella catarrhalis, have been demonstrated to form polymicrobial biofilms within the middle ear cleft. However, Alloiococcus otitidis (A. otitidis), which is one of the most commonly found bacteria within middle ear aspirates of children with OME, has not been described to form biofilms. The aim of this study was to investigate whether A. otitidis can form biofilms and investigate the impact on antibiotic susceptibility and survivability in polymicrobial biofilms with H. influenzae in vitro. The ability of A. otitidis to form single-species and polymicrobial biofilms with H. influenzae was explored. Clinical and commercial strains of A. otitidis and H. influenzae were incubated in brain heart infusion with and without supplementation. Biofilm was imaged using confocal laser scanning microscopy and scanning electron microscopy. Quantification of biofilm biomass and viable bacterial number was assessed using crystal violet assays and viable cell counting in both optimal growth conditions and in adverse growth conditions (depleted media and sub-optimal growth temperature). Antimicrobial susceptibility and changes in antibiotic resistance of single-species and multi-species co-culture were assessed using a microdilution method to assess minimal bactericidal concentration and E-test for amoxicillin and ciprofloxacin. A. otitidis formed single-species and polymicrobial biofilms with H. influenzae. Additionally, whilst strain dependent, combinations of polymicrobial biofilms decreased antimicrobial susceptibility, albeit a small magnitude, in both planktonic and polymicrobial biofilms. Moreover, A. otitidis promoted H. influenzae survival by increasing biofilm production in depleted media and at suboptimal growth temperature. Our findings suggest that A. otitidis may play an indirect pathogenic role in otitis media by altering H. influenzae antibiotic susceptibility and enhancing growth under adverse conditions.

Alcohol fixation of bacteria to surgical instruments increases cleaning difficulty and may contribute to sterilization inefficacy

Alcohol is frequently inappropriately used on surgical instruments to reduce bacterial contamination, but fixes protein to stainless steel. Here we compare the effect of air drying, prolonged soaking in water, and alcohol treatment on cleaning difficulty of contaminated forceps. Haltsted-mosquito forceps were contaminated with only Staphylococcus aureus. Instruments were air-dried for 10 (control), 75, or 240 minutes, soaked in water, or air dried then treated with ethanol or isopropanol for 10 seconds. All instruments were prewashed for 15 minutes. Forceps contaminated with blood and S aureus or Pseudomonas aeruginosa were dried and then sprayed or wiped with ethanol, and prewashed. Bacterial viability and soiling were determined by standard plate culture and crystal violet staining, respectively. Soaking or spraying instruments with alcohol significantly reduced viable bacterial numbers, but significantly increased soil attached to forceps, as did air drying. Wiping instruments with alcohol had little effect on bacterial viability, but increased cleaning difficulty. Soaking in water for 75 or 240 minutes increased cleaning difficulty perhaps due to bacterial attachment to forceps. Treating contaminated instruments with alcohol, allowing them to dry, or allowing them to soak in water for prolonged periods increases cleaning difficulty and should be discouraged.