Enrichment Broth Research Articles

Development of an Effective Two-Step Enrichment Process to Enhance Bax System Detection of Healthy and Injured Salmonella Enteritidis in Liquid Whole Egg and Egg Yolk

The BAX system for pathogen detection has been highly accurate in a variety of food products. However, false-negative results have been reported for the detection of pathogens in liquid egg products because of failed pathogen resuscitation and the existence of inhibitory components. In this study, a short-time enrichment step was used to simultaneously resuscitate the target cells to the detection level and to dilute the inhibitory components to reduce detection interference. The MP medium (BAX system) enabled faster multiplication of healthy Salmonella cells than did buffered peptone water (BPW) in tested liquid whole egg and egg yolk. However, MP failed to resuscitate heat-injured cells even after 24 h of incubation. Therefore, MP was replaced with BPW as the enrichment broth for the BAX system. However, the use of BPW for a one-step enrichment was not effective for removal of PCR inhibitors in egg yolk, and unstable detection results were obtained. To improve detection accuracy, a second step of enrichment with brain heart infusion was added. This two-step enrichment process shortened the enrichment time to 14 h and greatly increased the number of samples in which the pathogen was detected during the same enrichment time, especially in the liquid egg yolk samples. The validation study revealed 100% diagnostic accuracy of the two-step enrichment process plus the BAX system. These results indicate that a two-step enrichment process added to the BAX system can improve the detection of pathogenic Salmonella in liquid egg products.

Selenite Cystine Agar for Enumeration of Inoculated Salmonella Serovars Recovered from Stressful Conditions During Antimicrobial Validation Studies.

Process validation studies often require the inoculation of select foodborne pathogens into targeted foods to determine the lethality of the process or antimicrobial ingredients, and quantitative recovery of surviving inoculum bacteria helps to make those assessments. Such processes introduce various stressors on the inoculated challenge microorganisms whereby traditional selective media are too harsh to enumerate the remaining viable and injured population quantitatively. Innate antibiotic resistance of challenge organisms has often been used to establish simple selective media (i.e., Tryptic Soy Agar/TSA + antibiotics) for recovering inoculated strains, but sometimes antibiotic resistant background microorganisms are higher than desired. Salmonella Thompson 120, Salmonella Heidelberg F5038BG1, Salmonella Hadar MF60404, Salmonella Enteritidis H3527, and Salmonella Typhimurium H3380 were characterized for antibiotic resistance and acid adaptation in Tryptic Soy Broth containing 0%, 0.25%, or 1.0% glucose. Sodium pyruvate was evaluated for recovery after stress but no enhancing effect was observed, possibly because the strains were acid-adapted. Selenite Cystine Broth, traditionally used as a selective enrichment broth, was used as the basis for Selenite Cystine Agar (SCA) in combination with three antibiotics to which our Salmonella are resistant. Serovars of Salmonella, both individually and in mixtures, were enumerated on TSA, SCA, Xylose Lysine Desoxycholate (XLD), and Hektoen Enteric (HE) selective agars (all containing the same antibiotics) after conditions of nutrient starvation, desiccation, acid stress, and thermal stress. The data show that quantitative enumeration of our Salmonella serovars on SCA was not significantly different (p > 0.05) than those achieved on TSA for all tested stress categories. Levels of Salmonella enumerated on XLD and/or HE were significantly different (p < 0.05) than on TSA and SCA and often more than 1–2-log lower, consistent with the inhibition of injured cells. These data confirm that SCA (+ antibiotics) is a suitable selective medium for enumeration of these acid-adapted Salmonella serovars as challenge organisms recovered from various conditions of stress.

Inoculation of cattle manure with microbial agents increases efficiency and promotes maturity in composting.

In this study, fresh cattle manure was mixed with rice straw at a ratio of 10:1 in fresh weight and then composted in a self-built, aerated static composting box, whose dimension was 1m × 0.8m × 0.8m with a volume of approximately 0.6m3. To deal with the inconvenient and time-consuming problem of multiple stage inoculation, a single, one-time inoculation agent containing diverse microorganisms that are active at both the initial heating and thermophilic phases was developed. A total of 12 from 42 strains isolated from the none-inoculated Experiment 1 composting system were selected as microorganismal agents in Experiment 2 according to their species, prevalence and cultural temperature. 200mL of each microorganism enrichment broth was mixed to the inoculation group at the beginning of composting. A total of 2400mL of sterilize distilled water was added to the control group. The parameters of temperature, moisture, pH, C/N ratio, organic matter degradation, and germination index were investigated for both inoculation and control composting groups. Results showed that inoculation did not significantly shorten composting time. However, the pile temperature was increased with the maximum temperatures of 64.6°C and 60.3°C for the inoculation and control groups, respectively. The degradation of organic matter was accelerated (P < 0.05), and significantly higher GI value (P < 0.05) indicated that the maturity was promoted by the inoculation microorganism. This suggests that the final composting product would provide value as alternative source of nutrients for plants. Conclusively, we suggested a multiple microorganism inoculation method to increase the efficiency and promote maturity in cattle manure composting.

Criterium pencil mines as an alternative to platinum rods (Inoclic) in achievement of the antibiogram in agar medium for countries with limited resources?

Experimental study evaluating the comparability of the results between Criterium and Inoclic mines (by counting bacterial cells on agar after 5 successive dilutions of reason 10 from a bacterial suspension obtained after piercing through a colony; by measuring the inhibition diameters of 4 ATCC reference bacterial strains on an antibiogram in an agar medium) and evaluating the sterility of the criterium mines by culturing them on enriched broth (heart - brain type). 42 bacterial strains were used for bacterial cell counting. The results were of the same order of magnitude (107 CFU/mL) between Inoclic and criterium mines, for all strains and at all dilutions. The antibiotic susceptibility tests performed for the 4 reference strains by the Inoclics and criterium mines all complied (100%) with the expected limits for determining their sensitivity profile to the antibiotics tested. Compared to the bacterial growth inhibition diameters on antibiotic susceptibility tests, no intra-operator variability was observed, while significant inter-operator variability (both with Inoclic and 0.5 mm criterium mines) was observed with some strains and for inhibition diameters greater than 10 mm. The enriched broth cultures (BCC) and their subculture carried out on 10 criterium mines from 5 different batches were negative. Criterium mines seem to be a serious and less expensive alternative to Inoclic for the realization of antibiotic susceptibility testing in our resource-limited countries.

Expedient management of Listeria monocytogenes endovascular graft infection in an immunosuppressed patient

Introduction Listeria monocytogenes is a rare cause of infection following endovascular aneurysm repair (EVAR); there remains lack of consensus on the optimal management strategy, in particular the need for life-long suppression. Despite this organism’s increased pathogenicity amongst immunosuppressed hosts, to date no EVAR infections have been described in this cohort. Here we describe the first case ofListeria monocytogenesEVAR infection in an immunocompromised host. Case description A 75-year-old gentleman presented with a 5-day history of back pain, fever and dysuria despite 3 days of oral co-amoxiclav for presumed urinary tract infection. There was no recent gastrointestinal upset. Admission blood and urine cultures were negative. Past medical history included on-going methotrexate therapy for rheumatoid arthritis and EVAR of the infra-renal aorta in 2013. A CT abdomen, performed to exclude intra-abdominal pathology, revealed an enlarged aneurysmal sac and fat stranding, secondary to inflammation. A CT-guided sample of aneurysmal fluid was obtained before commencing empirical piperacillin-tazobactam and vancomycin; Listeria monocytogeneswas isolated from subculture after 5 days broth enrichment. The EVAR was removed and replaced 7 days after admission and antimicrobials rationalised to intravenous amoxicillin, ciprofloxacin and metronidazole. The patient completed 6 weeks intravenous therapy, then commenced lifelong suppressive therapy with oral co-trimoxazole 960mg OD. Discussion Listeria monocytogenesEVAR infection has been described in only 8 patients; this is the first in an immunosuppressed patient. This case adds to the literature by outlining a putative management strategy, involving explanation and life-long antimicrobials, for immunocompromised patients with Listeria monocytogenes EVAR infection.

Prevalence and characterisation of methicillin-resistant staphylococci from bovine bulk tank milk in England and Wales

ObjectivesTo investigate the prevalence and characteristics of methicillin-resistant staphylococci on dairy farms in England and Wales including zoonotic MRSA. MethodsBulk tank milk was sampled from 363 dairy farms in 2015-2016 and methicillin-resistant staphylococci were isolated by salt broth enrichment and plating on MRSA Brilliance selective agar. Isolates were characterised through antimicrobial susceptibility testing and whole-genome sequencing. ResultsMethicillin-resistant staphylococci were isolated from ∼5% of dairy farms and belonged to six different species, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lentus, Staphylococcus saprophyticus, Staphylococcus fleurettii and Staphylococcus sciuri. Whole-genome sequencing revealed a large variety of antimicrobial resistance genes and SCCmec elements were present, including mecA and mecC alleles. Potentially zoonotic methicillin-resistance S. aureus were found at a low prevalence (0.83% of sampled dairy farms). Whole-genome sequencing also provided evidence for the mobility of a primordial mec gene complex, independently of a SCCmec element, which appears to have been acquired by S. saprophyticus from S. fleurettii. ConclusionsThese data give new insight into the epidemiology of veterinary methicillin-resistant staphylococci to inform future surveillance and zoonotic risk evaluation. Our data indicate that MRSA has likely decreased in prevalence since earlier survey work in England and Wales during 2011-12 and highlights the diversity of methicillin resistance and other resistance determinants among bovine-associated staphylococci with implications for veterinary and human medicine.

Detection of multiple naturally occurring Salmonella serotypes from commercial broiler carcasses with conventional methods

AbstractMany laboratories sampling foods for Salmonella are interested only in presence or absence of Salmonella, so only one colony may be selected. The objectives of this study were to use two selective enrichment broths and two selective agar plating media for Salmonella recovery from naturally contaminated broiler carcass rinsates and evaluate these media combinations on Salmonella serotypes recovered from each carcass. Broiler carcasses (n = 52) from a commercial processing plant prior to chilling were rinsed with buffered peptone water and after incubation subcultured to gram‐negative and tetrathionate, and after inoculation to Rappaport Vassiliadis broth which was incubated and then streaked onto plates of Brilliant Green Sulfa and Xylose‐Lysine‐Tergitol‐4 agar. On 11/49 positive carcasses, both plating media yielded the same serotypes; for the other 38 positive samples different serotypes were found on the different plating media. Enrichment and plating media combinations influence the serotypes recovered and demonstrates bias even in a limited study such as this.

Bayesian estimation of diagnostic accuracy of fecal culture and PCR-based tests for the detection of Salmonella enterica in California cull dairy cattle.

Epidemiological studies of low prevalence disease problems are often hindered by the high cost of diagnostic testing. The objective of this study was to evaluate PCR screening of both individual and pooled fecal samples from culled dairy cows for the invA gene of Salmonella followed by culture to determine if the sensitivity and specificity were comparable to the results from traditional culture methods applied to individual samples. Cows from six different dairies were sampled in all four seasons. A total of 240 individual cow fecal samples, 24 fecal pools and 24 pools of 24-hour tetrathionate enrichment broth were tested. Diagnostic sensitivity of PCR screening followed by culture of PCR positive or indeterminate samples (i.e PCR-CUL method) was lower than that of culture (CUL) when applied to individual fecal samples (94.8%, 99.5%), however the specificity was comparable (99.6% and 97.7% respectively). For pools of five fecal samples and pools of five, 24 h tetrathionate broth samples, the specificity of both tests were comparable (∼98%); however, their sensitivity was only comparable in pooled fecal samples (∼93%) but greater for culture compared to PCR-CUL in pooled broth samples (∼99% versus ∼93%). Compared to culture results from testing of individual fecal samples, testing pooled fecal samples by culture had a relative sensitivity of 74% and relative specificity of 96%, testing pooled fecal samples by PCR-CUL resulted in relative sensitivity of 90% and relative specificity of 96%. Testing of pooled 24-hour enrichment broth by PCR-CUL increased the relative sensitivity and specificity to 100%. PCR testing followed by culture of positive or indeterminate samples is a time saving alternative to traditional methods. In addition, pooling of samples may be a useful method for decreasing cost if study aims can accommodate a moderate loss of relative sensitivity.

Development of An Innovative and Quick Method for the Isolation of Clostridium botulinum Strains Involved in Avian Botulism Outbreaks.

Avian botulism is a serious neuroparalytic disease mainly caused by a type C/D botulinum neurotoxin produced by Clostridium botulinum group III, one of the entwined bacterial species from the Clostridium novyi sensu lato genospecies. Its isolation is very challenging due to the absence of selective media and the instability of the phage carrying the gene encoding for the neurotoxin. The present study describes the development of an original method for isolating C. botulinum group III strains. Briefly, this method consists of streaking the InstaGene matrix extraction pellet on Egg Yolk Agar plates and then collecting the colonies with lipase and lecithinase activities. Using this approach, it was possible to isolate 21 C. novyi sensu lato strains from 22 enrichment broths of avian livers, including 14 toxic strains. This method was successfully used to re-isolate type C, D, C/D, and D/C strains from liver samples spiked with five spores per gram. This method is cheap, user-friendly, and reliable. It can be used to quickly isolate toxic strains involved in avian botulism with a 64% success rate and C. novyi sensu lato with a 95% rate. This opens up new perspectives for C. botulinum genomic research, which will shed light on the epidemiology of avian botulism.

Isolation and characterization of Escherichia coli serotype O157:H7 and other verotoxin-producing E. coli in healthy Indian cattle.

Background and Aim:Cattle are the main reservoir of Escherichia coli O157:H7 and other verotoxigenic E. coli (VTEC); therefore, there is an increased risk of infection to humans by either direct or indirect mode of transmissions. However, the prevalence of E. coli O157:H7 in the healthy cattle population of India is yet to be ascertained. This study aimed to screen the dairy cattle in and around Pune, Maharashtra, India, for verotoxin-producing E. coli O157:H7.Materials and Methods:A total of 257 rectal swabs were collected from 15 different organized and unorganized dairy farms of Pune during the period, January-March 2015. The screening involved enrichment in EC broth followed by differential identification on MacConkey sorbitol agar. The presumptive positive isolates were further confirmed by multiplex polymerase chain reaction (PCR) using primers specific to rfbE (O157), fliC (H7), VT1 (MK1), and VT2 (MK2). Vero-toxicity and antibiotic sensitivity were examined in PCR confirmed isolates.Results:Out of the 257 samples analyzed, 1.9% (2/105) were positive for O157:H7 and 39% (41/105) were positive for VTEC. Two PCR confirmed positive O157:H7 strains and two randomly selected PCR-positive VT strains exhibited in vitro cytopathic effect on Vero cells on day-7 post-inoculation. Antibiotic sensitivity profiling of O157:H7 strains exhibited resistance against penicillin G, kanamycin, ampicillin, tetracycline, gentamycin, cefotaxime, streptomycin, and piperacillin.Conclusion:These findings reveal the presence of pathogenic E. coli O157:H7 in the healthy cattle of Pune; in a situation, wherein regular surveillance for O157:H7 is not a norm. Therefore, the findings presented herein warrant routine surveillance and public awareness to prevent the transfer of such pathogens and manage health risks to the public.

Modification and Matrix Extension of the Bio-Rad iQ-Check E. coli O157:H7, STEC VirX, and STEC SerO Test Kits for the Detection of Shiga Toxin-Producing Escherichia coli (STEC) and Escherichia coli O157 From a Single Enrichment.

The iQ-Check Real-Time PCR kits use PCR technology based on gene amplification and detection by a real-time PCR thermalcycler for the detection of target analytes in select food matrices. The iQ-Check E. coli O157:H7 [Performance Tested MethodSM (PTM) 020801] and STEC VirX and STEC SerO (combined PTM 121203) methods were previously validated for different matrices under different enrichment schemes. To modify the current iQ-Check E. coli O157:H7 Kit for the detection of Escherichia coli O157:H7 from 25 to 375 g for raw ground beef (17% fat), raw beef trim, and fresh spinach. In addition, a matrix extension was validated for iQ-Check E. coli O157:H7 for raw chicken breast without skin (25 g), raw chicken thigh with skin (25 g), mechanically separated chicken (25 g), and raw ground pork (25 g). The study also included the modification of the iQ-Check STEC VirX and SerO Kits for the detection of non-O157 Shiga toxin-producing E. coli (STEC) for raw ground beef (375 g), raw beef trim (375 g), and fresh spinach (375 g) from STEC Enrichment Broth to buffered peptone water (BPW). All tests were carried out at 8-22 h (10-22 h for fresh spinach). Ground beef, beef trim, and spinach were co-inoculated with E. coli O157:H7, non-O157 STECs, and Salmonella spp. and analyzed for E. coli O157:H7 and non-O157 STECs after an 8-22 h enrichment in BPW for the beef matrices and after a 10-22 h enrichment in BPW for spinach. The chicken matrices were inoculated with E. coli O157:H7 only and analyzed after an 8-22 h enrichment in BPW. The iQ-Check Free DNA Removal Solution workflow was utilized for all matrices. Confirmations at the 22 h time point and method comparisons were conducted with the appropriate reference method as outlined in the U.S. Food and Drug Administration Bacteriological Analytical Manual Chapter 4A or the U.S. Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Guidebook Chapters 5.09 and 5B.05. For the iQ-Check STEC VirX and STEC SerO Kits, inclusivity and exclusivity were also performed. The two inclusivity and exclusivity evaluations indicated that the test methods can accurately detect the target analytes and correctly excluded nontarget organisms after 8 h of enrichment. In the method comparison study, the iQ-Check E. coli O157:H7 and STEC VirX and STEC SerO test kits demonstrated no statistically significant differences between candidate and reference method results or between presumptive and confirmed results for all food matrices analyzed and the two time points (8 or 10 and 22 h). Both time points produced the same results, with no discrepancies. The iQ-Check real-time PCR kits are effective methods for the detection of E. coli O157 and non-O157 STECs (both the virulence factors and the O groups) from raw ground beef, raw beef trim, and fresh spinach in 375 g samples enriched in BPW for 8-22 h (10-22 h for fresh spinach). In addition, the iQ-Check E. coli O157 Kit is effective in detecting E. coli O157 in 25 g samples of raw chicken breast without skin, raw chicken thigh with skin, mechanically separated chicken, and raw ground pork. The iQ-Check test kits allow the end user to pair enrichments for multiple target analytes, allowing the user to prepare a single enrichment and perform a single DNA extraction. The Free DNA Removal Solution removes free DNA from samples prior to PCR analysis, protecting DNA from intact and living cells. The method modifications were granted based on the data collected.

Optimization of Salmonella detection in garlic, onion, cinnamon, red chili pepper powders and green tea

Salmonella is the causative agent of many outbreaks related to spice consumption. However, because of the antimicrobial properties of various spices which hinders recovery and detection, Salmonella detection in spices remains a challenge. The objective of this study was to optimize an enrichment broth for Salmonella growth in different spices and tea, in order to maintain an adequate pH and decrease the antimicrobial effects of spices during Salmonella enrichment and subsequent detection. Salmonella contaminated spice and tea dried samples were prepared and the detection of Salmonella was assessed using the developed broth and automated DNA extraction and RT-PCR. Double strength Buffered Peptone Water (BPW) was used to maintain pH, and L-cysteine and DL-serine were added to the broth to reduce the effects of antimicrobial compounds in spices. The modified enrichment broth allowed the growth of Salmonella from each spice sample. Sample to broth ratios varied from 1:9 (garlic powder, chili peppers and tea), to 1:20 (cinnamon). The pH value of each enrichment varied but remained above 4.8. The addition of L-cysteine (30 mmol/L) allowed Salmonella recovery and growth in garlic and onion samples and the addition of DL-serine (11.23 mmol/L) allowed the recovery and growth in cinnamon. The results indicated that Salmonella detection was achieved in <24 h in the modified (BPW + L-cysteine and DL-serine) enrichment broth followed by detection by RT-PCR. This protocol could allow for a more rapid, robust, and sensitive enrichment method for Salmonella in spices.

Assessing the intestinal carriage rates of vancomycin-resistant enterococci (VRE) at a tertiary care hospital in Hungary

Excessive use of antibiotics contributes to the selection of resistant bacteria and intestinal colonization with multiresistant pathogens poses a risk factor for subsequent infections. The present study assessed vancomycin-resistant enterococci (VRE) carriage rates in patients admitted to our tertiary care hospital. Stool samples sent for routine culturing were screened with vancomycin containing solid or broth enrichment media. VRE isolates were identified with matrix-assisted laser desorption/ionization-time of flight mass spectrometry and antibiotic susceptibilities were tested by E-test. Vancomycin resistance genes were detected by polymerase chain reaction. Medical records of carriers were examined for suspected risk factors for colonization. Altogether 3025 stool specimens were analyzed. Solid media identified a VRE carriage rate of 2.2% while broth enrichment detected 5.8%. Seventy percent of the isolates were Enterococcus faecium. VanB genotype was detected in 38.2%, VanA in 37.3%, VanC1 in 22.6%, and VanC2 in 1.9%. All VRE were sensitive to linezolid, daptomycin, and tigecycline. Collective risk factors for carriage were diabetes, normal flora absence, Clostridioides difficile positivity, longer hospital stay, and advanced age. 78.5% of the carriers received antibiotic therapy which was metronidazole in most cases (47.3%). We recommend regular screening of risk groups such as patients with diabetes, history of recent hospitalization, or former C. difficile infection as an imperative step for preventing VRE dissemination.

Identification of Campylobacter species in Jahrom slaughterhouse chickens in 2016-17

A Introduction: Campylobacteria is one of the most common groups of bacteria which is transmitted between humans and animals. Among the animal source foods, the presence of campylobacter species in poultry meat has been extensively reported and the consumption of undercooked meat is the most important cause of campylobacter transmission to humans. The aim of this study was to identify Campylobacter spp.in Jahrom slaughterhouse chickens in2016-17. Material&Methods: In this descriptive cross-sectional study, 328 samples of poultry carcasses were randomly selected from slaughterhouses of Jahrom during spring and winter of 2016-2017.Campy-Thio transient medium were used for transferring samples. Exeter Broth enrichment medium and Agar Skirrow selective medium and specific Bacteriological tests were used to identify campylobacter.Data were analyzed by SPSS software version21in descriptive statistics level and using frequency, percentage and chi-square test at the significant level of 0.05. Results: Out of 328 samples, contamination with Campylobacter species was showed in 217(66.2%) ones, 146 (67.3%) and 71 (32.7%) of the samples were infected with Campylobacter jejuni and other Campylobacter species, respectively. It is necessary to mention that 118samples (72%) were contaminated in spring and 99 ones (60.4%) in winter. Also, Frequency of Campylobacter species in liver was79.3%, gizzard 69.5%, heart 61% and meat of poultry55%. Conclusion: Due to the contamination of poultry carcasses (66.2%) especially the liver, which is a potential source of Campylobacter species infection and one of the major health hazards in human society, quality control, and the significant association between infection frequency and season Notification about not consuming certain parts of chicken and training proper methods of consumption and full baking of poultry meat and precautions during the different seasons seems essential.

Austria-wide survey on resistant, potentially pathogenic bacteria at Austrian bathing sites, 2017

Summary There is growing concern about human-induced antibiotic resistance and on the occurrence of antibiotic-resistant, potentially pathogenic bacteria in the environment. The aim of this study was to investigate the incidence of resistant, clinically relevant bacteria at bathing sites. In total, 27 of 263 bathing sites authorized under the EU Bathing Water Directive (3 per Austrian state) were sampled during the summer of 2017. Samples were tested for antibiotic-resistant bacteria by enrichment in thioglycollate broth and cultivation on chromogenic media. The screening for potentially pathogenic antibiotic-resistant bacteria was negative in 23 of the 27 samples. Antibiotic-resistant bacteria were detected from 4 of the 27 bathing sites: one Pseudomonas aeruginosa and three resistant Enterobacteriaceae (piperacillin/tazobactam-resistant Enterobacter cloacae with high-level expression of AmpC beta-lactamase, carbapenem-resistant Enterobacter mori, extended-spectrum beta-lactamase-producing Escherichia coli). Despite the occurrence of resistant bacteria, we consider the public health risk at Austrian bathing sites as low.

Optimal detection of extended-spectrum β-lactamase producers, carbapenemase producers, polymyxin-resistant Enterobacterales, and vancomycin-resistant enterococci from stools

This study compared the impact of different approaches, namely, nonenrichment, nonselective enrichment, and selective (antibiotic-containing) enrichment steps, for detecting extended-spectrum β-lactamase producing Enterobacterales (ESBL-E), carbapenemase-producing Enterobacterales (CPE), polymyxin-resistant Enterobacterales (PMR-E), and vancomycin-resistant enterococci (VRE) from spiked stools. The use of a nonselective 18-h enrichment broth culture significantly improved the recovery rate of all types of resistant bacteria after their plating onto selective media. In addition, the detection of ESBL-E, CPE, PMR-E, and VRE was further improved when using an enrichment step using antibiotic-supplemented broths respectively supplemented with cefotaxime (0.1 μg/mL), ertapenem (0.1 μg/mL), colistin (0.5 μg/mL), and vancomycin (1 μg/mL). Therefore, we showed here that a screening strategy based on a selective broth enrichment step significantly contributes to an increased rate of detection of multidrug-resistant bacteria, which may be crucial in term of improvement of infection control.

2427. A Laboratory Simulation and Field Study of Simplified Qualitative and Quantitative Environmental Cultures for Clostridioides difficile

BackgroundReliable, simple methods to assess environmental contamination with Clostridioides difficile are needed for studies of terminal disinfection effectiveness and transmission of C. difficile from fomites. We compared two novel sampling methods for assessment of C. difficile contamination: (1) isopropanol wipe sampling with broth enrichment (IW), where alcohol wipes are used to sample surfaces followed by immersion in CDBB-TC broth and (2) ethanol-shock hand imprint sampling (HI), where surfaces are sampled by gloved hands moistened with ethanol hand sanitizer followed by imprinting hands on selective agar (CDBA-TC). Both methods allow for sampling of complex surfaces common in hospital environments.MethodsWe performed a laboratory simulation study using six 10-fold dilutions (101–106) of C. difficile spores from two strains sampled from fiberglass surfaces by IW and HI. We then performed a field study of 10 sites in 11 hospital rooms of patients with C. difficile infection (CDI) diagnosed < 48 hours prior to sampling. Results for the HI and IW method were quantitative and qualitative, respectively. We compared room contamination prevalence (total CFU per room by HI and sites positive/total sites per room) to the cycle crossing threshold (CT) from the diagnostic C. difficile PCR test (Cepheid) from the room occupant.ResultsThe IW laboratory limit of detection was 101 spores compared with 104 spores for HI. In the field study, IW and HI detected C. difficile contamination in 9/11 (82%) rooms at 22/110 (20%) sites and 5/11 (45%) rooms at 22/110 (20%) sites, respectively (Table 1). Six rooms with no detection by the HI method had a lower median number of sites positive per room compared with the 5 rooms with ≥1 cfu (1 vs 6, P < 0.05) (Table 1). The most commonly contaminated sites were linens (53%), call bells (43%), toilets (40%), blood pressure cuffs (40%), over-bed tables (38%), and bedrails (30%) (Table 2). Higher tcdB CT was associated with a lower total room contamination prevalence (Figures 1 and 2).ConclusionOur data suggest that the IW method is more sensitive for detection of C. difficile in the hospital environment, but the HI method offers quantitation. The prevalence of hospital room contamination for CDI patients appears to correspond with their toxin burden as estimated by PCR. Disclosures All authors: No reported disclosures.

534. Active Screening for Carbapenemase Producing Enterobacteriaceae: Yield and Cost Considerations

BackgroundDuring 2016, our hospital experienced an outbreak with carbapenemase-producing Enterobacteriaceae (CPE) in our solid-organ transplant (SOT) population. Since this outbreak and until now, our hospital has implemented active CPE screening of patients admitted to SOT units and point prevalence surveillances in any unit with a known CPE patient. The present study evaluates the yield of these screening tests and their cost since implementation.MethodsThis retrospective cohort was performed in a 600-bed hospital in Milwaukee, WI. CPE screening tests were retrieved from the clinical microbiology laboratory dataset from January 2016 to April 2019. CPE tests are performed on rectal swabs or stool samples using the CDC broth enrichment method followed by MIC confirmation using Etest. CPE patients were placed on enhanced precautions (gowns, gloves, booties) and were cohorted geographically and to 1:1 nursing and nurse aid staff.ResultsA total of 6,684 samples belonging to 3,383 patients were processed (1.9 samples/patient). Two hundred thirty (3.44%) had carbapenem-resistant Enterobacteriaceae, although only 33 isolates (0.49%) were confirmed as either KPC (n = 31) or NDM (n = 2) positive. Out of the 3,383 patients tested, 121 were identified as carriers of carbapenem-resistant isolates but only 11 (0.32%) were CPE (KPC = 11; NDM = 2). The incidence of new CPE patients during 2016 was 0.82% but decreased to 0.28% and 0.33% in 2017 and 2018, respectively. The units with the highest number of CPE patients were the transplant intensive care unit (n = 6) and the step-down SOT unit (n = 3). Negative cultures were quoted at $8.49 per sample but culture plates with colonies increased the cost per test to $28.44. The total cost for all the 6,684 screening tests was calculated at $61,335. The cost of CPE screening per positive CPE patient identified comes up to $5,575 (not including RN collection time).ConclusionIn an institution with staff and CPE patient cohorting, active screening of CPE positive patients was relatively expensive given our low -level of transmission. In the near future, we plan to stop staff and patient cohorting due to the high stress that these interventions place on our hospital staff. This might ensue in increase transmission, which will be detected by CPE screening tests.Disclosures All authors: No reported disclosures.

Application of gyrB targeted SYBR green based qPCR assay for the specific and rapid detection of Vibrio vulnificus in seafood

A SYBR green based qPCR assay targeting a unique region of gyrB was developed for the detection of Vibrio vulnificus. The specificity of the assay was studied using V. vulnificus and other bacterial strains belonging to Vibrio and non-Vibrio species. The assay unambiguously distinguished V.vulnificus with a sensitivity of 101 CFU/mL in pure culture while 102CFU/g was detected in clam meat homogenate with an efficiency of ≥98%.The utility of the qPCR assay was validated with naturally incurred seafood samples, where 24 out of 59(40.67%) seafood samples tested positive for V. vulnificus after 6–8 h enrichment in APW-P broth. In contrast, conventional PCR could detect only 11 samples (18.64%). Our results showed that qPCR assay developed in this study could be used as a rapid method for screening seafood samples for the presence of V. vulnificus, as the assay can be completed within 9–12 h including the enrichment of seafood in APW-P broth. The gyrB targeted qPCR developed in this study can provide excellent results on the presence and load of V. vulnificus in naturally contaminated samples quickly and efficiently; thus it could find application as a routine test in the seafood industry for the analysis V. vulnificus.

SKRINING BAKTERI BERPOTENSI PENDEGRADASI POLIETILEN OXO-DEGRADABLE DARI TANAH GAMBUT DI SEKITAR TPA KUALA DUA RASAU JAYA

The use of polyoethylene degradable polyethylene in Indonesia, especially in West Kalimantan, is high enough to potentially cause environmental pollution. One way to resolve pollution caused by polyethylene oxo-degradable plastic waste is to utilize isolates from the peat soil around the Kuala Dua Final Disposal Site, Rasau Jaya, which is able to degrade oxo-degradable polyethylene. This research aims to determine the bacterial genera that can to be expected to degrade Oxo-Degradable Polyethylene. Samples were taken from peat soil with the incubation method of oxo-degradable polyethylene for 2 weeks, 4 weeks and 6 weeks. Isolation use the pour plate method with Enrichment Broth Media and Enrichment Agar Media. The isolation results obtained 9 (nine) bacterial isolates which are distinguished based on incubation time and morphological characters.