Assessment of Ceftazidime-Avibactam 30/20-µg Disk, Etest versus Broth Microdilution Results When Tested against Enterobacterales Clinical Isolates.

The objective of this research was to evaluate the correlation between inhibitory zones and MIC when testing ceftazidime-avibactam using disk diffusion, Etest, and broth microdilution method established by the Clinical and Laboratory Standards Institute (CLSI). Four-hundred and 58 isolates of Enterobacterales isolated from 54 medical centers from the China Antimicrobial Surveillance Network (CHINET) in 2016 to 2020 were collected. Antimicrobial susceptibility testing using broth microdilution, Etest, and disk diffusion were performed according to the CLSI. Of the 458 Enterobacterales, 17.2% (79/458) and 82.8%(379/458) were resistant or susceptible to ceftazidime-avibactam by broth microdilution, respectively. Compared with the broth microdilution method, the categorical agreement (CA) and essential agreement (EA) of the Etest were 99.6% (456/458) and 94.8% (434/458), respectively; the major error (ME) and very major error (VME) were both 0.2% (1/458). For disk diffusion, the CA and VME were 99.8% (457/458) and 0.2% (1/458), respectively. For Escherichia coli, the CA and EA of the Etest were 100% and 97.1% (135/139), respectively. The CA of the disk diffusion was 100%. For Klebsiella pneumoniae, the CA and EA of the Etest were 99.3% (288/290) and 93.4% (271/290), respectively, the ME and VME were both 0.3% (1/290). The CA and VME of disk diffusion were 99.7% (289/290) and 0.3% (1/290), respectively. For other Enterobacterales, the CA and EA of the Etest were 100% and 96.6% (28/29), respectively. The CA of the disk diffusion was 100%. Ceftazidime-avibactam disk diffusion (30/20-µg disks) and Etest demonstrated good performance for ceftazidime-avibactam susceptibility testing against Enterobacterales clinical isolates. IMPORTANCE Multidrug-resistant Gram-negative bacteria, especially for extended-spectrum ß-lactamases-producing and carbapenemase-producing Enterobacterales, are disseminating rapidly around the world. Treatment options for these infections are limited, which prompt the development of novel or combinational therapies to combat the infections caused by multidrug-resistant pathogens. The newly available ß-lactam combination agent ceftazidime-avibactam has been demonstrated good in vitro and in vivo activity against ESBL, AmpC, KPC-2, or OXA-48-like-producing isolates and has shown promise in treating carbapenem-resistant Enterobacterales infections. Concerningly, there are few available automated systems for ceftazidime-avibactam susceptibility testing, and the broth microdilution method is hard to perform in most routine laboratories. Therefore, we urgently need an economical and practical method for the accurate detection of ceftazidime-avibactam activity against Gram-negative bacilli. Here, we evaluate the performance of the disk diffusion and Etest compared with the reference broth microdilution method against Enterobacterales clinical strains.

An optimized algorithm with improved turnaround time for detection of carbapenemase-producing Enterobacterales using the NG Test CARBA 5 in a routine laboratory.

Introduction. Rapid and reliable detection of carbapenemase-producing Enterobacterales (CPE) from surveillance cultures is critical in supporting a good infection control programme. We implemented a new algorithm for CPE detection incorporating the NG Test CARBA 5 in January 2019.Aim. Our goals were to compare turnaround time (TAT), costs and staff requirements between the old and new algorithm, and to evaluate the performance of the CARBA 5 test directly on colonies grown on CARBA Smart agar.Methodology. We analysed and compared the TAT of CPE surveillance cultures processed using the old and new CPE screening algorithm. The total actual reagent costs and staff requirements for the new CPE algorithm were compared with the estimated costs and staff requirements of the old CPE algorithm.Results. Of 197 isolates included in the evaluation of the new algorithm, 64 were positive for carbapenemases by both CARBA 5 and Xpert Carba-R assay. Of the 133 that were negative, two were found to harbour NDM and IMI genotypes. Significant improvements in TAT were achieved with 88.7 % of cultures with CPE, reported on the same day as growth was observed on CARBA Smart agar compared to none in the old algorithm. The new algorithm incurred lower costs and, based on our workload, the new algorithm is estimated to save 28.9 man-hours annually.Conclusion. CARBA 5 performs well on colonies growing on CARBA Smart agar and significant improvements in TAT can be achieved without incurring additional costs or staff requirements.

Prospective evaluation of rapid antimicrobial susceptibility testing by disk diffusion on Mueller-Hinton rapid-SIR directly on blood cultures.

BACKGROUND: With the worldwide spread of antibiotic resistance, delivering antibiotic susceptibility test (AST) results in a timely manner represents a major challenge. In cases of sepsis, rapid AST may facilitate early optimization of empiric antibiotic therapy. Disc diffusion is a well-standardized AST method, however 16 to 24 h are required to achieve an overall AST profile according to antimicrobial societies. METHODS: In this prospective pilot study, we evaluated the performance of Mueller-Hinton-Rapid-SIR (MHR-SIR) agar after 6-8 h of incubation in comparison with standard MH agar after 16 h of incubation directly on positive blood cultures caused by Enterobacteriaceae and Staphylococcus aureus from routine clinical microbiology. A total of 133 positive blood samples including 110 Enterobacteriaceae (83%) and 23 Staphylococcus aureus (17%) were tested in parallel by two direct AST methods, each using EUCAST breakpoints. For each combination bacterium and antibiotic, we compared the categorical agreement and the correlation between the diameters obtained by MHR-SIR and by standard MH. RESULTS: Our results showed 97.7% categorical agreement for Enterobacteriaceae, with 1.4% minor errors, 0.4% major errors and 0.5% very major errors. For S. aureus, we observed 97.8% categorical agreement, 1.9% minor errors, 0.3% major errors and no very major errors. CONCLUSION: Our results showed excellent categorical agreement and correlations between diameters for MHR-SIR and standard MH methods. MHRSIR can predict the result of overall AST profile within 6-8 h with reliable results. AST is obtained on the same day the blood culture becomes positive, with a very moderate cost.

Antimicrobial Activity of the Phenolic Compounds of Prunus mume against Enterobacteria.

Mume fruit, the Japanese apricot (Prunus mume SIEB. et ZUCC.), is popular in Japan and is mostly consumed in the pickled form called umeboshi. This fruit is known to have anti-microbial properties, but the principal constituents responsible for the antimicrobial properties have not yet been elucidated. We investigated the antimicrobial activities of the phenolic compounds in P. mume against enterobacteria. In this study, growth inhibitory activities were measured as an index of the antibacterial activities. The phenolic compounds were prepared from a byproduct of umeboshi called umesu or umezu (often translated as "mume vinegar"). Umesu or umezu phenolics (UP) contain approximately 20% phenolic compounds with p-coumaric acid as a standard and do not contain citric acid. We observed the inhibitory effects of UP against the growth of some enterobacteria, at a relatively high concentration (1250-5000 µg/mL). Alkali hydrolysates of UP (AHUP) exhibited similar antibacterial activities, but at much lower concentrations of 37.5-300 µg/mL. Since AHUP comprises hydroxycinnamic acids such as caffeic acid, p-coumaric acid, and ferulic acid, the antibacterial activities of each of these acids were examined. Our study shows that the phenolic compounds in P. mume other than citric acid contribute to its antimicrobial activity against enterobacteria in the digestive tract.

Testing commercial biopreservative against spoilage microorganisms in MAP packed Ricotta fresca cheese.

Ricotta fresca cheese is susceptible to secondary contamination and is able to support the growth of pathogens or spoilage psychotrophic bacteria during storage. The aim of the present study was to evaluate which among three commercial biopreservatives was suitable to be used to control the growth of spoilage microorganisms in sheep's milk MAP ricotta fresca cheese. 144 Ricotta fresca cheese samples were inoculated either with the bioprotective culture Lyofast FPR 2 (including Enterococcus faecium, Lactobacillus plantarum e Lactobacillus rhamnosus) or Lyofast CNBAL (Carnobacterium spp) or the fermentate MicroGARD 430. Not inoculated control and experimental ricotta were MAP packed (30% CO2 and 70% N2) and stored at 4 °C. Triplicate samples were analyzed after 5 h and 7, 14 and 21 days after inoculation for total bacterial count, mesophilic lactic acid bacteria, Enterobacteriaceae, Pseudomonas spp, Listeria monocytogenes, moulds and yeasts. Among the tested biopreservatives only Carnobacterium spp was able to control Pseudomonas spp and Enterobacteriaceae. The maximum reduction in the concentration of Pseudomonas spp and Enterobacteriaceae was respectively 1.93 and 2.66 log10 cfu/g, observed 14 days after production. Therefore, Carnobacterium spp was selected as the culture of choice to conduct a challenge study against Pseudomonas spp.

Genotoxicity assessment of pulp and paper mill effluent before and after bacterial degradation using Allium cepa test.

A lignin peroxidases-producing Serratia liquefaciens was used for bioremediation of pulp and paper (P&P) mill effluent. The treatment led to reduction of chemical oxygen demand (COD), colour, lignin and phenolic content by 84%, 72%, 61% and 95%, respectively. The effluent detoxification was studied by genotoxicity assays using Allium cepa L. (onion) root tip cells. Genotoxicity studies included measuring mitotic index (MI), chromosomal aberrations (CA) and nuclear abnormalities (NA) in root tip cells following treatment with 25, 50, 75 and 100% (v/v) of effluent. The root tip cells grown in untreated effluent showed a significant decrease in MI from 69% (control) to 32%, 27%, 22% and 11% at 25%, 50%, 75% and 100% effluent concentration, respectively. This indicated that the untreated effluent was highly cytotoxic in nature. Further, root tip cells, when treated with different concentrations of effluent showed various CA and NA including c-mitosis, stickiness, chromosome loss, chromosome break, anaphase bridge, multipolar anaphase, vagrant chromosomes, micronucleated and binucleated cells. The MI observed in root tip cells grown in bacterial treated effluents at similar concentrations (25, 50, 75 and 100% v/v) showed an increase of 33%, 36%, 42% and 66%. CA showed a substantial decrease and in some instances, complete absence of CA was also observed. The findings suggest that S. liquefaciens culture could be a potential bacterial culture for bioremediation of P&P mill effluent, as it is effective in substantial lowering of pollutants load as well as reduces the cytotoxic and genotoxic effects of effluent.

High Occurrence Rate and Contamination Level of Bacillus cereus in Organic Vegetables on Sale in Retail Markets.

Organic foods have risen in popularity recently. However, the increased risk of bacterial contamination of organic foods has not been fully evaluated. In this study, 100 samples each of organic and conventional fresh vegetables (55 lettuce samples and 45 sprout samples) sold in South Korea were analyzed for aerobic bacteria, coliforms, Escherichia coli, and Bacillus cereus. Although the aerobic bacteria and coliform counts were not significantly different between the two farming types (p > 0.05), the occurrence rate of B. cereus was higher in organically cultivated vegetables compared with those grown conventionally (70% vs. 30%, respectively). The mean contamination level of B. cereus-positive organic samples was also significantly higher (1.86 log colony-forming unit [CFU]/g vs. 0.69 log CFU/g, respectively) (p < 0.05). In addition, six samples of organic vegetables were found to be contaminated with B. cereus at over 4 log CFU/g categorized as unsatisfactory according to Health Protection Agency guideline. The relatively higher occurrence rate of B. cereus in organic vegetables emphasizes the importance of implementing control measures in organic vegetable production and postharvest processing to reduce the risk of food poisoning.

Population Pharmacokinetic Assessment and Pharmacodynamic Implications of Pediatric Cefepime Dosing for Susceptible-Dose-Dependent Organisms.

The Clinical and Laboratory Standards Institute (CLSI) revised cefepime (CFP) breakpoints forEnterobacteriaceaein 2014, and MICs of 4 and 8 µg/ml were reclassified as susceptible-dose dependent (SDD). Pediatric dosing to provide therapeutic concentrations against SDD organisms has not been defined. CFP pharmacokinetics (PK) data from published pediatric studies were analyzed. Population PK parameters were determined using NONMEM, and Monte Carlo simulation was performed to determine an appropriate CFP dosage regimen for SDD organisms in children. A total of 664 CFP plasma concentrations from 91 neonates, infants, and children were included in this analysis. The median patient age was 1.0 month (interquartile range [IQR], 0.2 to 11.2 months). Serum creatinine (SCR) and postmenstrual age (PMA) were covariates in the final PK model. Simulations indicated that CFP dosing at 50 mg/kg every 8 h (q8h) (as 0.5-h intravenous [i.v.] infusions) will maintain free-CFP concentrations in serum of >4 and 8 µg/ml for >60% of the dose interval in 87.1% and 68.6% of pediatric patients (age, ≥30 days), respectively, and extending the i.v. infusion duration to 3 h results in 92.3% of patients with free-CFP levels above 8 µg/ml for >60% of the dose interval. CFP clearance (CL) is significantly correlated with PMA and SCR. A dose of 50 mg/kg of CFP every 8 to 12 h does not achieve adequate serum exposure for older children with serious infections caused by Gram-negative bacilli with a MIC of 8 µg/ml. Prolonged i.v. infusions may be useful for this population.

Population Pharmacokinetics and Pharmacodynamics of Extended-Infusion Piperacillin and Tazobactam in Critically Ill Children.

The study objective was to evaluate the population pharmacokinetics and pharmacodynamics of extended-infusion piperacillin-tazobactam in children hospitalized in an intensive care unit. Seventy-two serum samples were collected at steady state from 12 patients who received piperacillin-tazobactam at 100/12.5 mg/kg of body weight every 8 h infused over 4 h. Population pharmacokinetic analyses were performed using NONMEM, and Monte Carlo simulations were performed to estimate the piperacillin pharmacokinetic profiles for dosing regimens of 80 to 100 mg/kg of the piperacillin component given every 6 to 8 h and infused over 0.5, 3, or 4 h. The probability of target attainment (PTA) for a cumulative percentage of the dosing interval that the drug concentration exceeds the MIC under steady-state pharmacokinetic conditions (TMIC) of ≥50% was calculated at MICs ranging from 0.25 to 64 mg/liter. The mean ± standard deviation (SD) age, weight, and estimated glomerular filtration rate were 5 ± 3 years, 17 ± 6.2 kg, and 118 ± 41 ml/min/1.73 m(2), respectively. A one-compartment model with zero-order input and first-order elimination best fit the pharmacokinetic data for both drugs. Weight was significantly associated with piperacillin clearance, and weight and sex were significantly associated with tazobactam clearance. Pharmacokinetic parameters (mean ± SD) for piperacillin and tazobactam were as follows: clearance, 0.22 ± 0.07 and 0.19 ± 0.07 liter/h/kg, respectively; volume of distribution, 0.43 ± 0.16 and 0.37 ± 0.14 liter/kg, respectively. All extended-infusion regimens achieved PTAs of >90% at MICs of ≤16 mg/liter. Only the 3-h infusion regimens given every 6 h achieved PTAs of >90% at an MIC of 32 mg/liter. For susceptible bacterial pathogens, piperacillin-tazobactam doses of ≥80/10 mg/kg given every 8 h and infused over 4 h achieve adequate pharmacodynamic exposures in critically ill children.

Microbiological quality of raw and processed wild and cultured edible snails.

BACKGROUND: An increasing interest in snail farming in Greece and other European countries has been observed. Despite the fact that edible snails have been involved with problems of Salmonella spp. contamination, there are to our knowledge only limited studies regarding microbiological safety and hygiene of such products. Enumeration of microbial populations and presence/absence of Salmonella spp. in snail meat and intestines of wild Cornu aspersum, Helix lucorum and cultured Cornu aspersum snails from indoor/outdoor type farms was conducted. Furthermore, snail-processing steps were simulated in the laboratory and the population reduction in snail meat was determined. RESULTS: Microbial populations were higher in intestines than snail meat in almost all cases. Escherichia coli/coliforms and Enterococcus spp. populations were lower in the intestines and snail meat of cultured C. aspersum. Salmonella spp. were detected in the intestines and snail meat of wild snails only. The high levels of bacterial populations were considerably reduced after the appropriate processing. CONCLUSION: The lower populations of E. coli/coliforms, Enterococcus spp. and especially the absence of Salmonella spp. in cultured snails show that the controlled conditions decrease the possibility of pathogen presence and contribute to food safety and public health.

Analyzing indicator microorganisms, antibiotic resistant Escherichia coli, and regrowth potential of foodborne pathogens in various organic fertilizers.

This study analyzed various organic fertilizers for indicator microorganisms, pathogens, and antibiotic-resistant Escherichia coli, and evaluated the growth potential of E. coli O157:H7 and Salmonella in fertilizers. A microbiological survey was conducted on 103 organic fertilizers from across the United States. Moisture content ranged from approximately 1% to 86.4%, and the average pH was 7.77. The total aerobic mesophiles ranged from approximately 3 to 9 log colony-forming units (CFU)/g. Enterobacteriaceae populations were in the range of <1 to approximately 7 log CFU/g, while coliform levels varied from <1 to approximately 6 log CFU/g. Thirty samples (29%) were positive for E. coli, with levels reaching approximately 6 log CFU/g. There were no confirmed positives for E. coli O157:H7, Salmonella, or Listeria monocytogenes. The majority of E. coli isolates (n=73), confirmed by glutamate decarboxylase (gad) PCR, were from group B1 (48%) and group A (32%). Resistance to 16 antibiotics was examined for 73 E. coli isolates, with 11 isolates having resistance to at least one antibiotic, 5 isolates to ≥ 2 antibiotics, and 2 isolates to ≥ 10 antibiotics. In the presence of high levels of background aerobic mesophiles, Salmonella and E. coli O157:H7 grew approximately 1 log CFU/g within 1 day of incubation in plant-based compost and fish emulsion-based compost, respectively. With low levels of background aerobic mesophiles, Salmonella grew approximately 2.6, 3.0, 3.0, and 3.2 log CFU/g in blood, bone, and feather meals and the mixed-source fertilizer, respectively, whereas E. coli O157:H7 grew approximately 4.6, 4.0, 4.0, and 4.8 log CFU/g, respectively. Our results revealed that the microbiological quality of organic fertilizers varies greatly, with some fertilizers containing antibiotic resistant E. coli and a few supporting the growth of foodborne pathogens after reintroduction into the fertilizer.

Modelling of biological Cr(VI) removal in draw-fill reactors using microorganisms in suspended and attached growth systems.

The kinetics of hexavalent chromium bio-reduction in draw-fill suspended and attached growth reactors was examined using sugar as substrate and indigenous microorganisms from the industrial sludge of the Hellenic Aerospace Industry. Initially, experiments in suspended growth batch reactors for Cr (VI) concentrations of 1.4-110 mg/l were carried out, to extensively study the behaviour of a mixed culture. The maximum Cr(VI) reduction rate of 2 mg/l h was achieved for initial concentration 12.85 mg/l with biomass production rate 4.1 mg biomass/l h. Analysis of the microbial structure in the batch reactor culture indicated that the dominant bacterial communities were constituted by bacterial members of Raoultella sp., Citrobacter sp., Klebsiella sp., Salmonella sp., Achromobacter sp. and Kerstersia sp. while the dominant fungal strain was that of Pichia jadinii. Experiments using the same mixed culture were also carried out in packed-bed reactors with plastic support media. High removal rates were achieved (2.0 mg/l h) even in high initial concentrations (109 mg/l). A combination of the model of Tsao and Hanson for growth enhancement and that of Aiba and Shoda for growth inhibition was used in order to describe and predict the process of Cr(VI) bio-reduction in suspended growth and packed-bed reactors. Kinetic constants of the equation obtained from both batch (or draw-fill) culture experiments. In the draw-fill experiments at the packed-bed reactor, hexavalent chromium inhibitory effects were minimized increasing the inhibitory constant value K(i)' at 148.5 mg/l, compared to suspended growth experiments which was K(i) = 8.219 mg/l. The model adequately predicts hexavalent chromium reduction in both batch reactors for all initial concentrations tested.

Use of meropenem Adatabs dissolved in MacConkey agar for screening NDM-1 positive Enterobacteriaceae in faecal surveillance cultures.

AIMS: Carbapenem resistance due to metallo-ß-lactamases (MBLs) in Enterobacteriaceae has gained much prominence in recent months. The emergence and subsequent spread of New Delhi metallo-ß-lactamase-1 (NDM-1) constitutes a potential threat in terms of the management of affected patients and institutional infection control efforts. We evaluated an in-house prepared meropenem impregnated MacConkey agar versus CHROMagar KPC. METHODS: The lowest limit of detection (LLD) was compared for nine clinical isolates of NDM-1 producing Enterobacteriaceae on the above mentioned agar media. RESULTS: LLD was comparable for all the nine clinical isolates on both media. The cost of the antibiotic impregnated MacConkey agar was considerably lower (US$0.39) as compared to the commercial media, while its performance remained unaltered for a period of at least 8 weeks of storage. CONCLUSION: The in-house medium proved to be a suitable and cheap alternative to the CHROMagar KPC.

Reducing SO2 in fresh pork burgers by adding chitosan.

The use of 0.02 or 0.05% chitosan is proposed to reduce from 450 to 150 mg kg⁻¹ the SO2 required to preserve pork burgers aerobically packed and stored at 2 °C for up to 21 days under retail display conditions. The effects of chitosan and/or sulfite addition and the storage time were determined in fresh (color deterioration, lipid oxidation, pH, total viable counts, Escherichia coli and coliforms, Salmonella, appearance and odor) and cooked (appearance, odor, flavor and texture) burgers. The addition of either 0.02 or 0.05% chitosan was not detected by sensory analysis, and extended the shelf life of low-SO2 burgers from 7 to 14 days. Chitosan enhanced the preservative effects of sulfite at a low dose, acting on the main causes of meat deterioration (bacterial spoilage, color stability and lipid oxidation), and provided good sensory properties to fresh and cooked pork burgers.

Quantity of the tetracycline resistance gene tet(M) differs substantially between meat at slaughterhouses and at retail.

UNLABELLED: Concentrations of the tetracycline resistance gene tet(M) per square centimeter were assessed in meat from the slaughterhouse (n = 100) and from retail (n = 100) by real-time quantitative PCR. The study revealed a substantial contamination of retail meat with the tetracycline resistance gene tet(M), with a mean of 4.34 log copies per cm² fasces in chicken and 5.58 log copies per cm² fasces in pork. Quantitative resistance gene analysis provides an interesting tool for risk assessment and is becoming increasingly important. For both chicken and pork, tet(M) concentrations were significantly higher in meat at retail, compared to meat at slaughter. Cultural investigations revealed substantial differences in the prevalence of listeria and enterococci, and of E. coli and coliforms, between meat at slaughter (n = 500) and at retail (n = 500). However, the differences in the prevalence of 2 investigated groups of potential tet(M)-carriers (enterococci, listeria) could not sufficiently explain the differences in tet(M) concentrations, since increasing concentrations of tet(M) were accompanied by decreasing prevalences of these potential tet(M)-carriers. The percentage of tetracycline susceptible indicator bacteria (E. faecalis, E. coli) did not differ between meat at slaughter and meat at retail. Higher concentrations of tet(M) at retail might correlate with the proliferation of other genera than enterococci and listeria, but there is also a reason to discuss whether secondary contaminants might carry tet(M) more often than the primary flora of meat. PRACTICAL APPLICATION: We successfully applied the direct quantitative monitoring of resistance genes in meat, which generally might aid as a useful and rapid additional tool for risk assessment. We know that bacteria provide a large pool of resistance genes, which are widely shared between each other-the larger the pool is, the more genes might be exchanged. Thus, in terms of resistance gene monitoring, we should sometimes overcome the restricted view on single bacteria and look at the gene pool, instead.

Hot water surface pasteurisation of lamb carcasses: microbial effects and cost-benefit considerations.

Although hot water pasteurisation of carcasses is accepted as a general intervention in USA, this is not the case in Europe. The aims of this study were (i) to evaluate the microbiological effects of hot water pasteurisation of lamb carcasses, both after slaughtering and dressing and following subsequent chilling and storage; (ii) to discuss hot water pasteurisation from a public health and cost-benefit perspective; (iii) to discuss the benefits of hot water pasteurisation compared with use of separate meat processing streams for high-risk carcasses; (iv) to evaluate the use of recycled hot water in a hygienic context and in relation to EU regulations; and (v) to consider the technological and sensory aspects of hot water pasteurisation of lamb carcasses. Samples were collected from 420 naturally contaminated lamb carcasses, with 50% of the carcasses (n=210) subject to hot water pasteurisation at 82 °C for 8s immediately after slaughter. Surface swab samples from 4500 cm² areas on carcasses were collected at slaughter, after chilling for 24 h, and after chilling for five days. The microbial analyses included Escherichia coli, Enterobacteriaceae, Bacillus cereus, Clostridium perfringens and aerobic plate count (APC). A resuscitation step using Tryptone Soya Agar was included in the microbiological analyses. Hot water pasteurisation significantly reduced the levels of E. coli, Enterobacteriaceae, B. cereus and APC (all P<0.001). E. coli colony forming unit (CFU) reduction was 99.5%, corresponding to a reduction of 1.85 log CFU per carcass. E. coli was isolated from 66% of control carcasses and from 26% of pasteurised carcasses. After 24h storage, the reduction in E. coli was increased to 2.02 log, and after five days E. coli could not be isolated from the pasteurised carcasses. These results suggest that surface pasteurisation could be an important and efficient procedure (critical control point) for reducing generic E. coli and thereby shiga toxin-producing E. coli on carcasses, and thus the risk for disease among consumers. The recycled water had acceptable physical and chemical parameters and no spore-forming bacteria were detected. Although some carcass discolouration was observed, after 24h the colour was acceptable. Our data provide relevant input for some of the data gaps regarding hot water pasteurisation and indicate that replacing the expensive system of separate processing of high-risk carcasses with hot water surface pasteurisation should be considered as a serious option.

A new cost-effective, selective and differential medium for the isolation of Cronobacter spp.

The objective of the present study was to develop a new selective, differential and cost-effective medium (Kim and Rhee - KR-medium) for the isolation of Cronobacter spp. In this new medium, which contained salicin as a differential agent, Cronobacter spp. generated typical colonies with characteristic violet-colored centers surrounded by a transparent to opalescent border, and the growth of other microorganisms (40 strains) was inhibited or produced visually distinguishable colonies. Using healthy and heat- and desiccation-injured cells, the quantity of nutrients was adjusted to determine the optimal recovery rate, selectivity, differentiation and cost-effectiveness. Peptone and salicin concentrations were established as 10 and 8 g/L, respectively. The KR medium was then validated using salicin fermenting organisms, including Cronobacter spp. (52 strains), Enterobacter cloacae (50 strains) and Klebsiella pneumonia (10 strains) isolated from clinical and food specimens. All strains of Cronobacter spp. produced typical colonies and other salicin fermenting organisms were easily distinguishable from Cronobacter spp. with the exception of 2 E. cloacae strains. The verification of KR medium was carried out in powdered infant formula artificially inoculated with healthy, heat-injured, and desiccation-injured Cronobacter spp. and the expected typical colonies were appeared. The KR medium had a high specificity (98%) and sensitivity (100%), with no false-negative results. Moreover, we show that the cost of the KR medium is much lower than that of other selective and differential media. The use of the KR medium for the selective isolation of Cronobacter spp. in laboratories and food industry settings may therefore lessen the financial burden of Cronobacter spp. detection.

Spatial assessment of Langat River water quality using chemometrics.

The present study deals with the assessment of Langat River water quality with some chemometrics approaches such as cluster and discriminant analysis coupled with an artificial neural network (ANN). The data used in this study were collected from seven monitoring stations under the river water quality monitoring program by the Department of Environment (DOE) from 1995 to 2002. Twenty three physico-chemical parameters were involved in this analysis. Cluster analysis successfully clustered the Langat River into three major clusters, namely high, moderate and less pollution regions. Discriminant analysis identified seven of the most significant parameters which contribute to the high variation of Langat River water quality, namely dissolved oxygen, biological oxygen demand, pH, ammoniacal nitrogen, chlorine, E. coli, and coliform. Discriminant analysis also plays an important role as an input selection parameter for an ANN of spatial prediction (pollution regions). The ANN showed better prediction performance in discriminating the regional area with an excellent percentage of correct classification compared to discriminant analysis. Multivariate analysis, coupled with ANN, is proposed, which could help in decision making and problem solving in the local environment.

Assessment of antimicrobial resistance transfer between lactic acid bacteria and potential foodborne pathogens using in vitro methods and mating in a food matrix.

The transferability of antimicrobial resistance from lactic acid bacteria (LAB) to potential pathogenic strains was studied using in vitro methods and mating in a food matrix. Five LAB donors containing either erythromycin or tetracycline resistance markers on transferable elements were conjugally mated with LAB (Enterococcus faecalis, Lactococcus lactis) and pathogenic strains (Listeria spp., Salmonella ssp., Staphylococcus aureus, and Escherichia coli). In vitro transfer experiments were carried out with the donors and recipients using both the filter and plate mating methods. The food matrix consisted of fermented whole milk (fermented with the LAB donors) with the pathogenic recipients added as contaminants during the production process. All transconjugants were confirmed by phenotypic and molecular methods. Erythromycin resistance transfer from LAB strains to Listeria spp. was observed using both in vitro mating methods at high transfer frequencies of up to 5.1 x 10(-4) transconjugants per recipient. Also, high frequency transfer (ranging from 2.7 x 10(-8) up to 1.1 x 10(-3) transconjugants per recipient) of both erythromycin and tetracycline-resistance was observed between LAB species using in vitro methods. No resistance transfer was observed to Salmonella spp., Staphylococcus aureus, and E. coli. The only conjugal transfer observed in the fermented milk matrix was for tetracycline resistance between two LAB strains (at a transfer frequency of 2.6 x 10(-7) transconjugants per recipients). This study demonstrates the transfer of antimicrobial resistance from LAB to Listeria spp. using in vitro methods and also the transfer of resistance between LAB species in a food matrix. It highlights the involvement of LAB as a potential source of resistance determinants that may be disseminated between LAB and pathogenic strains including Listeria spp. Furthermore, it indicates that food matrices such as fermented milks may provide a suitable environment to support gene exchange.

Microbial assessment in school foodservices and recommendations for food safety improvement.

This study evaluated microbial food safety in school foodservices. Five school foodservices were randomly selected, and samples from water, cooking utensils, tableware, foodservice surroundings, and linen were collected in summer and winter (N=420). Tap and drinking water samples were collected, samples of food contact surfaces were collected by swab-kit, and samples for foodservice workers' hands and gloves were prepared by glove juice method. Aerobic plate count (APC) and coliform bacterial populations were enumerated on plate count agar (PCA) and desoxycholate lactose agar, respectively. The presence of Escherichia coli, Salmonella, Listeria monocytogenes, and Staphylococcus aureus was also examined by biochemical identification tests. In addition, PCA agar for APCs and Baird-Parker agar for S. aureus were used to enumerate airborne microorganisms. Higher APCs (< 0 to 5.1 log CFU/mL) than acceptable level were generally observed in water samples, while low coliform counts were found in the samples. High APCs were enumerated in cooking utensils, foodservice workers, tableware, and food-service surroundings, and coliforms were also found in the samples for both seasons. The presence of Salmonella was found from only 10% of plastic glove samples (summer), and the presence of L. monocytogenes was not observed in all samples. S. aureus was detected in some of water, cooking utensils, tableware, employees, and foodservice surroundings, and E. coli was observed in cooking utensils (10% to 20%; summer). No obvious airborne bacteria were detected. These results showed that sanitation practice in school foodservices should be improved, and the results may be useful in microbial assessment of school foodservices.