The behaviour of Escherichia coli and Pseudomonas aeruginosa in bottled mineral water.

Microbial contamination of bottled water during the filling and capping procedure is a problem which should be avoided. The examination of the influence of carbon dioxide (CO2) on bacterial growth of Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa) in bottled mineral water was the aim of this study. Commercially available glass bottles with plastic screw caps filled with natural mineral water (without additional CO2 "still" (StMW) and with CO2 "sparkling" (SpMW) were obtained from a manufacturer in the province of Styria, Austria. The artificial contamination was performed in the lab by opening the bottle with subsequent addition of a bacterial solution with a defined number of bacteria. For each bacterial strain, 12 bottles were prepared. Samples (100 mL) were taken after a specific number of days, filtrated and placed on Endo Agar for cultivation. After incubation for 24 h bacterial colonies were counted. In this study CO2 addition to bottled water reduced colony forming units of the two investigated bacterial strains over time.

Background concentrations of airborne, culturable fungi and dust particles in urban, rural and mountain regions.

Geographic location and meteorological factors can affect the content of bioaerosol concentrations. This study was conducted to determine the natural background concentrations of culturable fungal spores and dust particles in three different geographical areas. Focus was given to the dominant airborne genera Cladosporium, Penicillium, Aspergillus and the species Aspergillus fumigatus. The influence of weather conditions on the microorganism concentrations in urban, rural and mountain regions were examined. Possible correlations between particle counts and culturable fungal spore concentrations were investigated. 125 measurements of the air were conducted using the air sampler MAS-100NT® and the particle counter Alphasense OPC-N3. The analyses of the collected samples were based on culture methods using different media. The highest median of fungal spore concentrations was detected in the urban region and was of 2.0 × 103 CFU/m3 for xerophilic fungi and 1.7 × 103 CFU/m3 for the genus Cladosporium. The concentrations of fine and coarse particles in rural and urban regions were the highest of 1.9 × 107 pa/m3 and 1.3 × 107 pa/m3, respectively. Little cloud cover and slight wind had a positive influence on the concentration of fungal spores. Furthermore, correlations were observed between air temperature and the concentrations of xerophilic fungi as well as the genera Cladosporium. In contrast, relative humidity correlated negatively with total fungi and Cladosporium and no correlation was found with the other fungi. For the region of Styria in summer and early autumn, the natural background concentration for xerophilic fungi ranged between 3.5 × 102 and 4.7 × 103 CFU/m3 air. No significant differences were detected between the fungal spore concentrations in urban, rural and mountainous regions. The data of this study could be used as a reference to compare the natural background concentrations of airborne culturable fungi in further studies concerning air quality assessment.

Dissolved Carbon Dioxide: The Lifespan of Staphylococcus aureus and Enterococcus faecalis in Bottled Carbonated Mineral Water.

During the process of mineral water production, many possible contamination settings can influence the quality of bottled water. Microbial contamination can originate from different sources, for example, the ambient air, the bottles, the caps, and from the bottling machine itself. The aim of this study was to investigate the influence of three different carbon dioxide (CO2) concentrations (3.0 g/L, 5.5 g/L, and 7.0 g/L; 20 bottles each) in bottled mineral water on the bacterial growth of Staphylococcus aureus (S. aureus) and Enterococcus faecalis (Ent. faecalis). The examined mineral water was artificially contaminated before capping the bottles inside the factory. After a specific number of days, water samples were taken from freshly opened bottles and after filtration (100 mL), filters were placed on Columbia Agar with 5% Sheep blood to cultivate S. aureus and Slanetz and Bartley Agar to cultivate Ent. faecalis. The respective colony-forming units (CFU) were counted after incubation times ranging from 24 to 120 h. Colony-forming units of S. aureus were not detectable after the 16th and 27th day, whereas Ent. faecalis was not cultivable after the 5th and 13th day when stored inside the bottles. The investigation of the bottles that were stored open for a certain amount of time with CO2 bubbling out showed only single colonies for S. aureus after the 5th day and no CFUs for Ent. faecalis after the 17th day. A reduction in the two investigated bacterial strains during storage in carbonated mineral water bottles means that a proper standardized disinfection and cleaning procedure, according to valid hygiene standards of industrial bottling machines, cannot be replaced by carbonation.

Corrigendum: Variation in accessory genes within the Klebsiella oxytoca species complex delineates monophyletic members and simplifies coherent genotyping.

[This corrects the article DOI: 10.3389/fmicb.2021.692453.].

High-Speed Dental Instruments: An Investigation of Protein-Contaminated Dental Handpieces with the Bicinchoninic Acid Assay in Dental Offices in Styria, Austria.

Due to permanent contact with bodily secretions such as blood and saliva, the dental workplace poses a high risk of infection for patients as well as for personnel. High-speed dental instruments are still considered one of the major hygienic risks, as the high-speed rotation of the attachments leads to the retraction of infectious material from patients' oral cavities. The aim of this study was to investigate the extent to which dental handpieces are contaminated after use. Spray-water samples were taken from different handpieces used in seven dental offices and protein concentrations were measured photometrically. In the first part of the study, samples were collected from each handpiece before and after the treatment of the patients. Additionally, the changes in protein concentration after consecutive treatments in which the same high-speed dental instrument was used were investigated. The results demonstrated measurable protein concentrations in 91.2% of a total of 398 samples, and 96.4% of the spray-water samples taken after treatment showed a discrepancy from the initial measured protein concentration. In 68.4% an increase in protein concentration was observed, whereas in 27.9% a decrease was measured. In conclusion, the internal contamination of high-speed dental instruments frequently occurs in daily usage and consequently may lead to the transmission of infectious agents by flushing the contaminated water out of the spray water tubes. Moreover, it must be pointed out that internal cleansing of handpieces is insufficient and that a final mechanical disinfection is indispensable.

Cefsulodin and Vancomycin: A Supplement for Chromogenic Coliform Agar for Detection of Escherichia coli and Coliform Bacteria from Different Water Sources.

Background microorganism growth on Chromogenic Coliform Agar (CCA) can be challenging. For this reason, a new alternative method with a Cefsulodin/Vancomycin (CV)-supplemented CCA should be developed in this study. CCA supplemented with CV was validated according to ÖNORM EN ISO 16140-4:2021 using water from natural sources in Styria, Austria. Results show that the alternative method using the supplemented CCA has similar values in relation to sensitivity (82.2%), specificity (98.6%) and higher selectivity (59%) compared to the reference method. Repeatability and reproducibility were acceptable for the alternative method and showed similar results with the reference method. The alternative method shows a very low false positive rate and a low false negative rate paired with good performance regarding the inclusion study. The exclusion study shows the advantage of our method by suppressing background microorganisms and facilitating the process of enumeration of Escherichia coli and other coliform bacteria on CCA plates. Aeromonas hydrophila and Pseudomonas aeruginosa growth was inhibited using the supplement. To conclude, the coliform CV selective supplement combined with CCA is an appropriate tool for coliform bacteria detection in water samples.

Concentrations of mesophilic bacteria in a poultry farm over two fattening periods focusing on the presence of staphylococci and enterococci.

The increasing animal numbers have a potential impact on the air quality of stables. The aim of this study was to assess the microbial load in the barn air from the day of entry of the chickens to the day of removal for slaughter. A total of 10 measurements in two fattening periods were conducted in a poultry farm with a capacity of 400 chickens in Styria, Austria. The samples were collected with an Air-Sampling Impinger for the investigation of mesophilic bacteria, staphylococci and enterococci. Chicken skin swab samples were collected to detect Staphylococcus aureus. The total colony forming units per cubic meter of mesophilic bacteria of the first measurement series of period I was 7.8 × 104 and increased to 1.4 × 108 at the end and at the fattening period II it increased from 2.5 × 105 to 4.2 × 107. In the measurement series of the fattening period I, the concentration of Staphylococcus spp. increased from 0 to 4.9 × 107 CFUs/m3 and from 0 to 2.1 × 107 CFUs/m3 in the fattening period II. Staphylococcus aureus could not be found on the chicken skin. An interesting finding was the increase of staphylococci while the intestinal enterococci were not detectable in the air of the barn toward the end of both fattening periods.

Variation in Accessory Genes Within the Klebsiella oxytoca Species Complex Delineates Monophyletic Members and Simplifies Coherent Genotyping.

Members of the Klebsiella oxytoca species complex (KoSC) are emerging human pathogens causing infections of increasing significance especially in healthcare settings. KoSC strains are affiliated with distinct phylogroups based on genetic variation at the beta-lactamase gene (bla OXY) and it has been proposed that each major phylogroup represents a unique species. However, since the typing methods applied in clinical settings cannot differentiate every species within the complex, existing clinical, epidemiological and DNA sequence data is frequently misclassified. Here we systematically examined the phylogenetic relationship of KoSC strains to evaluate robustness of existing typing methods and to provide a simple typing strategy for KoSC members that cannot be differentiated biochemically. Initial analysis of a collection of K. oxytoca, K. michiganensis, K. pasteurii, and K. grimontii strains of environmental origin showed robust correlation of core phylogeny and blaOXY grouping. Moreover, we identified species-specific accessory gene loci for these strains. Extension of species correlation using database entries initially failed. However, assessment of average nucleotide identities (ANI) and phylogenetic validations showed that nearly one third of isolates in public databases have been misidentified. Reclassification resulted in a robust reference strain set for reliable species identification of new isolates or for retyping of strains previously analyzed by multi-locus sequence typing (MLST). Finally, we show convergence of ANI, core gene phylogeny, and accessory gene content for available KoSC genomes. We conclude that also the monophyletic members K. oxytoca, K. michiganensis, K. pasteurii and K. grimontii can be simply differentiated by a PCR strategy targeting bla OXY and accessory genes defined here.

Multiresistant Bacteria Isolated from Intestinal Faeces of Farm Animals in Austria.

In recent years, antibiotic-resistant bacteria with an impact on human health, such as extended spectrum ß-lactamase (ESBL)-containing Enterobacteriaceae, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE), have become more common in food. This is due to the use of antibiotics in animal husbandry, which leads to the promotion of antibiotic resistance and thus also makes food a source of such resistant bacteria. Most studies dealing with this issue usually focus on the animals or processed food products to examine the antibiotic resistant bacteria. This study investigated the intestine as another main habitat besides the skin for multiresistant bacteria. For this purpose, faeces samples were taken directly from the intestines of swine (n = 71) and broiler (n = 100) during the slaughter process and analysed. All samples were from animals fed in Austria and slaughtered in Austrian slaughterhouses for food production. The samples were examined for the presence of ESBL-producing Enterobacteriaceae, MRSA, MRCoNS and VRE. The resistance genes of the isolated bacteria were detected and sequenced by PCR. Phenotypic ESBL-producing Escherichia coli could be isolated in 10% of broiler casings (10 out of 100) and 43.6% of swine casings (31 out of 71). In line with previous studies, the results of this study showed that CTX-M-1 was the dominant ESBL produced by E. coli from swine (n = 25, 83.3%) and SHV-12 from broilers (n = 13, 81.3%). Overall, the frequency of positive samples with multidrug-resistant bacteria was lower than in most comparable studies focusing on meat products.

Background Concentrations of Cultivable, Mesophilic Bacteria and Dust Particles in the Air in Urban, Rural and Mountain Regions.

Particulate air components can be of anthropogenic or natural origin. It is assumed that in different geographical areas varying concentrations of mesophilic bacteria are present in the ambient air. The aim of this study was to determine the background concentrations of airborne culturable mesophilic bacteria and particulate matter in the ambient air. Furthermore, the association between their concentrations and some environmental factors was analysed. In the period from July to October 2019, concentrations of mesophilic bacteria and dust particles were measured in urban, rural and mountain areas using the single-stage air sampler and the particle counter. The concentrations of bacteria and dust particles in the air were counted as number of Colony Forming Units per cubic metre (CFU/m3) and particles per cubic metre (pa/m3). Staphylococcus sp. were identified. The median values of the cultivated mesophilic bacteria at 30 °C and 37 °C were 7.1 × 102 CFU/m3 and 2.3 × 101 CFU/m3 in mountain regions, 1.3 × 102 CFU/m3 and 6.9 × 101 CFU/m3 in rural regions and 2.1 × 102 CFU/m3 and 6.5 × 101 CFU/m3 in urban regions. The median of Staphylococcus sp. was 2.5 × 100 CFU/m3 in alpine areas and 7.5 × 100 CFU/m3 in urban and rural areas. Higher bacterial concentrations were measured in sunshine and in windy weather. A relationship was observed between the concentrations of airborne mesophilic bacteria and the coarse particles in all three areas. The present study determined values between 5.0 × 100 and 4.6 × 102 CFU/m3 as natural background concentrations of airborne mesophilic bacteria and 1.2 × 107 pa/m3 and 6.5 × 104 pa/m3 for fine and coarse particles, respectively. These results can be proposed as baseline for the assessment of the emission sources of mesophilic bacteria for summer and early autumn.

Correction: Comparative study of impaction and sedimentation in an aerosol chamber using defined fungal spore and bacterial concentrations.

[This corrects the article DOI: 10.1371/journal.pone.0187039.].

Multiresistant Bacteria Isolated from Activated Sludge in Austria.

Wastewater contains different kinds of contaminants, including antibiotics and bacterial isolates with human-generated antibiotic resistances. In industrialized countries most of the wastewater is processed in wastewater treatment plants which do not only include commercial wastewater, but also wastewater from hospitals. Three multiresistant pathogens-extended spectrum ß-lactamase (ESBL)-harbouring Enterobacteriaceae (Gram negative bacilli), methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococci (VRE)-were chosen for screening in a state of the art wastewater treatment plant in Austria. Over an investigation period of six months all three multiresistant pathogens could be isolated from activated sludge. ESBL was the most common resistance mechanism, which was found in different species of Enterobacteriaceae, and in one Aeromonas spp. Sequencing of ESBL genes revealed the dominance of genes encoding members of CTX-M ß-lactamases family and a gene encoding for PER-1 ESBL was detected for the first time in Austria. MRSA and VRE could be isolated sporadically, including one EMRSA-15 isolate. Whereas ESBL is well documented as a surface water contaminant, reports of MRSA and VRE are rare. The results of this study show that these three multiresistant phenotypes were present in activated sludge, as well as species and genes which were not reported before in the region. The ESBL-harbouring Gram negative bacilli were most common.

Comparative study of impaction and sedimentation in an aerosol chamber using defined fungal spore and bacterial concentrations.

Biocontamination control is a very significant part of the manufacturing process of sterile drugs. Sterility is frequently monitored by active or passive air sampling measurements, but there are no specific rules as to how this is to be done. This study tested air sampling methods of active impaction and passive sedimentation under standardized conditions. Aspergillus niger (A. niger) and Staphylococcus aureus (S. aureus) were selected in this experiment to examine parallels, correlations and differences between the two methods. The results show that the number of colony forming units per plate (CFU/plate) was higher for A. niger in the active method, whereas for S. aureus it was higher in the sedimentation method. A high correlation coefficient was found between the impaction and sedimentation methods for A. niger. For S. aureus, depending on the culture media used and the time for passive air sampling, a larger number of CFU/plate was found than in active air sampling. This study concludes that active and passive air sampling can be used for monitoring the air in clean rooms. For fungal spore detection, the impaction is more efficient, as it is possible to sample a higher volume of air in a shorter period of time, whereas the optimal measurement methods for S. aureus depend on a number of factors.

Culturable fungi in potting soils and compost.

In the present study the spectrum and the incidence of fungi in potting soils and compost was investigated. Since soil is one of the most important biotopes for fungi, relatively high concentrations of fungal propagules are to be expected. For detection of fungi, samples of commercial soils, compost and soils from potted plants (both surface and sub-surface) were suspended and plated onto several mycological media. The resulting colonies were evaluated qualitatively and quantitatively. The results from the different sampling series vary, but concentrations on the surface of potted plants and in commercial soils are increased tenfold compared to compost and sub-surface soils. Median values range from 9.5 × 10(4) colony forming units (CFU)/g to 5.5 × 10(5) CFU/g. The spectrum of fungi also varies in the soils. However, all sampling series show high proportion of Aspergillus and Penicillium species, including potentially pathogenic species such as Aspergillus fumigatus. Cladosporium, a genus dominant in the ambient air, was found preferably in samples which were in contact with the air. The results show that potentially pathogenic fungi are present in soils. Immunocompromised individuals should avoid handling soils or potted plants in their immediate vicinity.

Multiresistant bacteria isolated from chicken meat in Austria.

Multidrug resistant bacteria (MDR bacteria), such as extended spectrum beta-lactamase (ESBL) Enterobacteriaceae, methicillin resistant Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococci (VRE), pose a challenge to the human health care system. In recent years, these MDR bacteria have been detected increasingly outside the hospital environment. Also the contamination of food with MDR bacteria, particularly of meat and meat products, is a concern. The aim of the study was to evaluate the occurrence of MDR bacteria in chicken meat on the Austrian market. For this study, 50 chicken meat samples were analysed. All samples originated from chickens slaughtered in Austrian slaughterhouses and were marked as produced in Austria. Samples were analysed for the presence of ESBL Enterobacteriaceae, methicillin resistant Staphylococci and VRE. Resistance genes of the isolated bacteria were characterised by PCR and sequencing. In the present study 26 ESBL producing E. coli, five mecA gene harbouring Staphylococci (but no MRSA), and four VRE were detected in chicken meat samples of Austrian origin. In 24 (48%) of the samples no ESBL Enterobacteriaceae, MRSA, methicillin resistant coagulase negative Staphylococcus (MRCNS) or VRE could be detected. None of the samples contained all three types of investigated multiresistant bacteria. In concordance to previous studies, CTX-M-1 and SHV-12 were the dominant ESBL genes.

Isolation and characterization of multidrug-resistant bacteria from minced meat in Austria.

INTRODUCTION: Resistant bacteria are a well-known public health problem. This study was conducted to investigate the prevalence and genetic characteristics of extended spectrum ß-lactamase (ESBL) producing enterobacteria, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) in mixed minced meat from pork and beef. METHODS: One hundred samples of mixed minced meat were collected from supermarkets (n = 70) and local butcher shops (n = 30) in the city of Graz (Austria). After enrichment and inoculation on selective media, bacteria were identified with MALDI-TOF MS or Vitek2 systems, tested for antibiotic resistance and further characterized with PCR and sequencing. RESULTS: In 20 of the 100 meat samples 24 ESBL positive Escherichia coli isolates were found. The most common ESBL among the isolates was CTX-M-1. Other detected bla genes contained CTX-M-14, CTX-M-32, SHV-12 and TEM-52 types. Nine samples were tested positive for MRSA and spa-typed. Detected spa-types were hospital-acquired t3928, as well as livestock-associated t011, t034 and t2241. No VRE were found. CONCLUSION: A contamination of meat with ESBL-producing E. coli and MRSA was confirmed in this study. The large diversity of ESBL producing E. coli could indicate a growing dissemination of ESBL genes in E. coli found in meat products from porcine and bovine origin.

Prevalence, antibiotic resistance patterns and molecular characterization of Escherichia coli from Austrian sandpits.

The aim was to determine the prevalence of E. coli and coliform bacteria in playground sand of all public children's sandpits in Graz (n = 45), Austria, and to assess the frequency of antimicrobial resistance in E. coli. Molecular characterization included the discrimination of O-serotypes and H-antigens and the determination of virulence and resistance genes, using a microarray technology. E. coli isolates were tested for susceptibility to a set of antibiotics by VITEK2 system and disk diffusion method. In total, 22 (49%) and 44 (98%) sandpits were positive for E. coli and coliform bacteria. Median concentrations of E. coli and coliform bacteria in the sand samples were: 2.6 × 10(4) CFU/100 g and 3.0 × 10(5) CFU/100 g. Resistance rates were: ampicillin, 12.5%; piperacillin, 10.4%; amoxicillin/clavulanic acid, 9.4%; cotrimoxazole, 6.3%; tetracycline, 6.3%; piperacillin/tazobactam, 5.2%. No ESBL- or carbapenemase-producing isolates were found. The most prevalent serogroups were O15, O6 and O4. Isolates harbored 0 up to 16 different virulence genes.

Synergism between pyronaridine and retinol in Plasmodium falciparum in vitro.

Pyronaridine, a naphthyridine derivative and Mannich base, is a highly active blood schizontocide and currently being explored as partner in artemisinin-based combination therapy (ACT). In this study, carried out 2008 in Mae Sot, Thailand, the activity of pyronaridine was found to be compromised, obviously as the result of mono-therapy in an adjacent area. The pyronaridine sensitivity and the interaction between pyronaridine and retinol at concentrations corresponding to the 50th, 65th and 80th percentile of the physiological retinol levels in healthy adults were assessed in 38 fresh isolates of P. falciparum. The mean IC50, IC90 and IC99 values for pyronaridine were 12.7, 201.4 and 3084.2 nM, those for pyronaridine + retinol "low" 1.2, 14.0 and 102.6 nM, those for pyronaridine + retinol "medium" 0.6, 7.4 and 54.8 nM, and those for pyronaridine + retinol "high" 0.9, 8.2 and 47.8 nM. There was significant evidence of strong synergism between pyronaridine and retinol against P. falciparum.

Synergism between quinine and retinol in fresh isolates of Plasmodium falciparum.

Following earlier reports of synergism between retinol and various antimalarial compounds, the pharmacodynamic interaction between retinol and quinine was investigated in 38 fresh isolates of Plasmodium falciparum. The study was carried out in western Thailand, an area with quinine-resistant P. falciparum. The combination of quinine with retinol in concentrations corresponding to the 50(th), 65(th) and 80(th) percentile of the physiological values in healthy subjects, significantly reduced the EC(50), EC(90), EC(99) and GMCOC for quinine. The FIC values at EC(90) and EC(99) indicate increasing synergism with rising EC and retinol concentration. The mean SFIC value dropped to a level as low as 0.2420, indicating strong synergism.