Enrichment Broth Research Articles

Synthesis of nitrogen-doped carbon nanodots to destroy bacteria competing with Campylobacter jejuni in enrichment medium, and development of a monoclonal antibody to detect C. jejuni after enrichment

The objective of this study was to develop a method with improved sensitivity for Campylobacter jejuni detection in foods. Nitrogen-doped carbon nanodots (N-CNDs) were synthesized and added to an enrichment medium (Bolton broth) at a concentration of 10 mg/mL. A light-emitting diode (LED) at a wavelength of 425 nm was used to irradiate the N-CNDs-supplemented enrichment medium to induce an exothermic reaction for 1 h. Additionally, a monoclonal antibody specific to C. jejuni NCTC11168 was developed using hybridoma cells to aid detection. The C. jejuni detection capabilities of N-CNDs-supplemented enrichment medium and the conventional Bolton broth enrichment, were compared using duck samples. C. jejuni in the enrichment was detected with the monoclonal antibody based-indirect enzyme-linked immunosorbent assay (ID-ELISA). The N-CNDs-supplemented enrichment medium showed a better C. jejuni detection capability than the conventional Bolton broth enrichment. Additionally, data from ID-ELISA showed excellent detection efficiency and a shortened detection time in the N-CNDs-supplemented enrichment medium after LED irradiation at 425 nm. These results indicate that 1-h LED irradiation at 425 nm to Bolton broth supplemented with the N-CNDs increased the detection efficiency and shortened the detection time with the monoclonal antibody for C. jejuni in food.

Microbiological quality and safety of raw milk from direct sale in northern Germany

The microbiological status of raw milk sold from vending stations at farm level in northern Germany was determined in relation to hygiene quality parameters and the presence of foodborne pathogens. For this, one hundred and ten samples were examined. The total viable count (TVC) was higher than 5 log cfu mL−1 in 36.4% of the milk samples. Counts of Pseudomonas spp., Enterobacteriaceae, yeasts and moulds were detected at mean values of 3.9 ± 1.3 log cfu mL−1, 2.7 ± 1.2 log cfu mL−1, 3.1 ± 0.9 log cfu mL−1 and 1.5 ± 0.8 log cfu mL−1, respectively. Overall, only 43% of the raw milk samples were of satisfactory quality from a microbiological point of view. Campylobacter jejuni and Arcobacter butzleri were determined in the enrichment broth of three raw milk samples. A long-term monitoring of three farms revealed that individual differences in farm management practice influenced raw milk quality.

Wild horses and horse manure as reservoirs for Clostridiodes difficile in Australia

Background: Clostridiodes (Clostridium) difficile poses an ongoing threat as a cause of diarrhoea in both human and animal populations. Recent emergence of human disease caused by strains of C. difficile found in animals adds to mounting evidence that C. difficile infection (CDI) is a zoonosis. In Australia, horses represent a unique mix of companion and wild animals. While C. difficile is regularly isolated from domesticated horses, little is known about C. difficile in wild horses, known as brumbies in Australia. The aim of this study was to investigate the prevalence and characteristics of C. difficile in wild horse populations, and in publicly available horse manure to evaluate its potential as a source of C. difficile in the community. Methods and materials: One hundred and twenty-six wild horse faecal samples were collected from Western and South-Eastern Australia and 60 publicly available horse manure samples were collected from Western Australian metropolitan areas. Samples were cultured in enrichment broth followed by alcohol shock and subsequent plating onto differential agar. C. difficile isolates were characterised by ribotyping using capillary electrophoresis, and toxin profiling by PCR. Results: The overall C. difficile prevalence was 15.1% in wild horses and 31.7% in publicly available horse manure, a statistically significant difference (P = 0.048). Thirty-one ribotypes (RTs) were identified across all samples; the most prevalent were RTs 0.10 (a non-toxigenic strain), 0.54, 0.56 and 0.15. These RTs have been previously isolated from vegetables, livestock and humans in WA. Two additional noteworthy isolates included an esculin hydrolysis negative strain and a non-toxigenic RT078 strain. Conclusion: This study is the first known investigation into C. difficile in wild horse populations and the first to look specifically at C. difficile in horse manure available for public use. Prevalence of C. difficile was higher than previously reported in horses, and some unusual strains that require further investigation were isolated. The study provides preliminary information on the epidemiology of C. difficile in wild horses and support the hypothesis that horse manure represents a potential reservoir of C. difficile in the community.

Wild horses and horse manure as reservoirs for Clostridiodes difficile in Australia

Background: Clostridiodes (Clostridium) difficile poses an ongoing threat as a cause of diarrhoea in both human and animal populations. Recent emergence of human disease caused by strains of C. difficile found in animals adds to mounting evidence that C. difficile infection (CDI) is a zoonosis. In Australia, horses represent a unique mix of companion and wild animals. While C. difficile is regularly isolated from domesticated horses, little is known about C. difficile in wild horses, known as brumbies in Australia. The aim of this study was to investigate the prevalence and characteristics of C. difficile in wild horse populations, and in publicly available horse manure to evaluate its potential as a source of C. difficile in the community. Methods and materials: One hundred and twenty-six wild horse faecal samples were collected from Western and South-Eastern Australia and 60 publicly available horse manure samples were collected from Western Australian metropolitan areas. Samples were cultured in enrichment broth followed by alcohol shock and subsequent plating onto differential agar. C. difficile isolates were characterised by ribotyping using capillary electrophoresis, and toxin profiling by PCR. Results: The overall C. difficile prevalence was 15.1% in wild horses and 31.7% in publicly available horse manure, a statistically significant difference (P=0.048). Thirty-one ribotypes (RTs) were identified across all samples; the most prevalent were RTs 010 (a non-toxigenic strain), 054, 056 and 015. These RTs have been previously isolated from vegetables, livestock and humans in WA. Two additional noteworthy isolates included an esculin hydrolysis negative strain and a non-toxigenic RT078 strain. Conclusion: This study is the first known investigation into C. difficile in wild horse populations and the first to look specifically at C. difficile in horse manure available for public use. Prevalence of C. difficile was higher than previously reported in horses, and some unusual strains that require further investigation were isolated. The study provides preliminary information on the epidemiology of C. difficile in wild horses and support the hypothesis that horse manure represents a potential reservoir of C. difficile in the community.

Periodic screening of donor faeces with a quarantine period to prevent transmission of multidrug-resistant organisms during faecal microbiota transplantation: a retrospective cohort study

On June 13, 2019, the US Food and Drug Administration issued a warning after transfer of faeces containing an extended-spectrum β-lactamase (ESBL)-producing Escherichia coli by faecal microbiota transplantation led to bacteraemia in two immunocompromised patients. Consequently, we evaluated the effectiveness of the faeces donor-screening protocol of the Netherlands Donor Faeces Bank, which consists of screening of donors for multidrug-resistant organisms every 3 months, combined with additional screening on indication (eg, after travelling abroad) and application of a quarantine period for all faecal suspensions delivered within those 3 months. We did a retrospective cohort study of data collected between Jan 1, 2015, and Oct 14, 2019, on the multidrug-resistant organism testing results of donor faeces. Additionally, we tested previously quarantined faecal suspensions approved for faecal microbiota transplantation between Dec 12, 2016, and May 1, 2019, for the presence of multidrug-resistant organisms using both aselective and selective broth enrichment media. Whole-genome sequencing with core-genome multilocus sequence typing (cgMLST) was done on all multidrug-resistant isolates. Among initial screenings, six (9%) of 66 tested individuals were positive for multidrug-resistant organisms and 11 (17%) of 66 tested individuals were positive for multidrug-resistant organisms at any timepoint. Multidrug-resistant organisms were detected in four (25%) of 16 active donors, who had a median donation duration of 268 days (IQR 92 to 366). Among all screening results, 14 (74%) of 19 detected multidrug-resistant organisms were ESBL-producing E coli. 170 (49%) of 344 approved faecal suspensions had corresponding research faeces aliquots available and were tested (from 11 active donors with a median of eight [IQR five to 26] suspensions per donor). No multidrug-resistant organisms were detected in the 170 approved faecal suspensions (one-sided 95% CI 0 to 1·7). cgMLST revealed that all multidrug-resistant organisms were genetically different. Healthy faeces donors can become colonised with multidrug-resistant organisms during donation activities. Our screening protocol did not result in approval of multidrug-resistant organism-positive faecal suspensions for microbiota transplantation. None.

Isolation of clinically significant microorganisms from prosthetic joint tissue using BacT/ALERT paediatric blood culture bottles compared with solid culture media and enrichment broth

The diagnosis of prosthetic joint infections and isolation of causative microorganisms has been found to be challenging in microbiology laboratories due to low sensitivity of microbiological culture. The aim of this study was to compare the use of conventional culture methods with the use of both enrichment broth and BacT/ALERT paediatric blood culture bottles, for the diagnosis of prosthetic joint infections. A total of 121 specimens from 44 patients were processed using three methods of microbiological culture: solid media, enrichment broth and paediatric bottles. The paediatric bottle method had a significantly lower (p<0.0001) time to detection than the standard solid media method, and was significantly more sensitive than solid media when used independently (93.33%, CI 83.27-98.09, vs 60.00%, CI 45.43-73.33). The combination use of solid media with paediatric bottles was found to be superior to the conventional solid media method and combination use with enrichment broth.

The colonisation of Czech travellers and expatriates living in the Czech Republic by colistin-resistant Enterobacteriaceae and whole genome characterisation of E. coli isolates harbouring the mcr-1 genes on a plasmid or chromosome: A cross-sectional study

BackgroundTravellers were recognized as a risk cohort that can be colonized by mcr-1-mediated colistin-resistant Enterobacteriaceae. We aimed to investigate the carriage of mcr-mediated colistin resistance in Enterobacteriaceae in Czech travellers or expatriates residing temporarily in the Czech Republic. MethodsBetween August 2018 and September 2019, the stool samples were cultured in enrichment broth. The enriched cultures were tested for the presence of the mcr-1-8 genes and inoculated onto selective agar with colistin. Colistin-resistant Enterobacteriaceae were tested for the presence of the mcr-1-8 genes; the mcr-positive isolates were characterised by whole genome sequencing. ResultsFrom the 177 stool samples, 15 colistin-resistant Enterobacteriaceae isolates were cultured (7.9%); two of the E. coli isolates carried the mcr-1 gene (1.1%). In the E. coli multilocus sequence type (ST) 156, the mcr-1 gene was located in an ISApl1-mcr-1-orf-ISApl1 (Tn6330) and incorporated into the chromosome; in the E. coli ST23 isolate, the mcr-1 gene was harboured by the plasmid IncX4. Both of the mcr-1 positive E. coli isolates were multidrug-resistant and one isolate was an extended-spectrum β-lactamase producer (blaCTX-M-27). ConclusionPatients with an international travel history should be monitored for the carriage of the mcr-1 gene in order to prevent its dissemination into healthcare settings.

Prevalence, risk factors and molecular characteristics of Shiga toxin-producing Escherichia coli in beef abattoirs in Gauteng, South Africa

A cross-sectional study was conducted to determine the prevalence, risk factors, and molecular characteristics of Shiga toxin-producing Escherichia coli (STEC) in 12 beef abattoirs in Gauteng province, South Africa. The relationship between STEC contamination and aerobic bacterial counts on carcasses at various stages of processing was investigated. Multiplex PCR was used to detect virulence genes in broth-enriched samples, to determine O-serogroups in all samples positive for Shiga toxin-encoding genes (stx), and to characterize isolates of STEC. The overall prevalence of STEC determined by PCR in 419 selective enrichment broth samples was 35.1% (147/419), and was significantly higher (P = 0.037) in perineum hide swabs (50%) than in 24 h chilled carcass swabs (20%). The maximum total aerobic plate count (TAPC) was 3.8 log10 CFU/100 cm2 for carcass swabs, but was below the South African microbiological standard for meat export at all stages of carcass processing. There was no significant association between TAPC and STEC contamination. Serogroup O113 was the most prevalent serogroup (13.6%; 20/147) detected. Only 33 isolates, all non-O157 STEC, were recovered, amongst which six different genotype combinations were observed. Additionally, the clinically important serogroups O117, O8, and O2 were isolated. Multivariable logistic regression revealed that the odds of STEC contamination was lower in post-wash (OR = 0.42; 95% CI: 0.18–0.98; P = 0.045) and 24 h chilled (OR = 0.33; 95% CI: 0.12–0.91; P = 0.033) carcass swabs compared to pre- and post-evisceration swabs. It was concluded that non-O157 STEC serogroups more frequently colonize beef cattle slaughtered at abattoirs in our study area than O157 STEC, and therefore have a higher potential to enter the food chain during carcass processing, with food safety implications.

Direct Clostridioides difficile ribotyping from stool using capillary electrophoresis

Clostridioides difficile(C. difficile) genotyping is essential for surveillance of emerging strains, transmissions, and outbreak investigations, but culture is lengthy and may not be routinely performed, which necessitates culture-independent genotyping methods. We aimed to develop a direct from stool C. difficile PCR ribotyping algorithm using capillary electrophoresis. Ribotypes were generated directly from 66.8% of stools with 33.2% requiring broth enrichment. 16S and tcdB cycle thresholds (Ct) were significantly lower (P< 0.001) in directly ribotyped stools compared to enriched stools, and Ct correlated with direct ribotyping (area under the curve: 0.97 and 0.96, respectively). Direct and isolate ribotypes were 94.7% concordant. Mixed C. difficile ribotypes were presumptively identified in 14 (7.5%) samples with 12 (6.4%) mixtures confirmed. We have developed a rapid PCR ribotyping algorithm allowing for direct C. difficile genotyping from stool using capillary electrophoresis with occasional detection of mixed C. difficile populations in stool, which is a limitation of conventional isolate genotyping.

Evaluation of Contamination of Multiuse Suture Cassettes in an HQHVSN Environment.

Historically, it has been thought that suture cassettes become contaminated by bacteria through multiuse. However, High-Quality, High-Volume Spay/Neuter (HQHVSN) veterinarians have been using them for years without issue because of their significantly lower cost. The objective of this study was to determine if absorbable suture cassettes are contaminated through multiuse in an HQHVSN environment. A total of 101 suture samples from suture cassettes were collected from 25 HQHVSN clinics or shelters. The suture samples were placed in an enrichment broth tube and aerobic and anaerobic culture with microbe ID were performed. A total of 17/101 samples were positive for microbial growth, with 11/25 clinics having at least one positive sample. Based on these results, there is a significant risk to using suture cassettes that must be balanced against cost savings. Although HQHVSN veterinarians do not report an increase in infection using suture cassettes, based on the results of this study, there is likely contaminated suture being used during sterilization surgeries at these surgery sites.

AquaSparkTM - Rapid Environmental Monitoring of Listeria monocytogenes.

In order to prevent microbial contamination of food, monitoring of the production environment, together with the rapid detection of foodborne pathogens have proven to be of utmost importance for Food Safety. Environmental monitoring should detect harmful pathogens at the earliest point in time in order for the necessary interventions to be taken. However, current detection methods fall short with regards to speed, ease of use, and cost. This article aims to present the idea behind NEMIS Technologies, a startup company making use of the novel AquaSparkTM technology for the development of a new generation of bacterial detection methods. These methods utilize chemiluminescence in order to detect live target bacteria in a short period of time compared to that of conventional methods. We show that dry-stressed Listeria monocytogenes can be detected within 24 hours, using small-molecule chemiluminescent probes, together with a bacteria-specific proprietary enrichment broth containing a cocktail of bacteriophages, which enhance the specificity and sensitivity. This novel platform technology has the potential to extend beyond environmental monitoring towards food analyses as well as veterinary and human health.

Reducing time in detection of Listeria monocytogenes from food by MALDI-TOF mass spectrometry.

In this study, direct detection of L. monocytogenes from liquid culture and enrichment broths containing foods was investigated by using MALDI-TOF MS. For determining the sole effect of food constituents on detection and accuracy of identification in enrichment broths, sterile foods were used before the experiments with food. L. monocytogenes could be detected in BHI broth after 24h of incubation. Detection period was determined as 18h for 3 × 101cfu/mL initial bacterial count in BHI broth containing sterile food. The period extended in ONE broth containing sterile garnish, which was 24 and 30h for 3 × 101 and 1cfu/mL inoculum, respectively. It was found that identification times in UHT milk were longer than that of canned garnish. In the experiments performed with foods having a specific microbiota; White cheese, iceberg lettuce, parsley and watermelon were used. Although no reliable identification was obtained by using White cheese, iceberg lettuce and parsley, L. monocytogenes could be detected in 24h in the enrichment broth containing watermelon. Detection was achieved during a single step enrichment in a reduced time of 24h for even 1cfu/mL initial inoculum.

Variability in lag duration of Listeria monocytogenes strains in half Fraser enrichment broth after stress affects the detection efficacy using the ISO 11290-1 method

A collection of 23 Listeria monocytogenes strains of clinical and food origin was tested for their ability to recover and grow out in half Fraser enrichment broth following the ISO 11290-1:2017 protocol. Recovery of sub-lethally heat-injured cells in half Fraser broth was compared to reference cells with no stress pre-treatment. The enrichments were followed over time by plate counts and the growth parameters were estimated with the 3-phase model which described the data best. The reference cells without stress pre-treatment showed a short lag duration, which ranged from 1.4 to 2.7 h. However, significant variation in the ability to recover after 60 °C heat stress was observed among the tested strains and resulted in a lag duration from 4.7 to 15.8 h. A subset of strains was also exposed to low-temperature acid stress, and the lag duration showed to be also stress dependent. Scenario analyses and Monte Carlo simulations were carried out using the growth parameters obtained in the enrichments. This demonstrated that when starting with one cell, the detection threshold for efficient transfer of at least one cell to the secondary enrichment step, i.e. 2 log10 CFU/ml, was not reached by 11 of 23 strains tested (48%) after exposure to 60 °C heat stress. Increasing the incubation time from 24 to 26 h and the transfer volume from 0.1 to 1.0 ml can increase the average probability to transfer at least one cell to the secondary enrichment step from 79.9% to 99.0%. When optimizing enrichment procedures, it is crucial to take strain variability into account as this can have a significant impact on the detection efficacy.

Efficacy of a Sink Tailpiece Heating Device to Decrease Microbial Colonization of Sink Drains

Background: Many institutions have reported transmission of multidrug-resistant organisms to patients from colonized sinks. Prior data have shown that bacterial colonization of the sink drain, which can occur via biofilm from a colonized p-trap or via seeding from above, results in dispersion of bacteria in the area of the sink when water from the faucet comes in contact with the drain. Heat disruption of biofilm formation between the p-trap and sink drain is a potential strategy in preventing colonization of sink drains. Methods: In an academic center hospital, 54 tail-piece heaters were installed in 3 intensive care units and 2 acute-care units in an associated regional hospital. Half of the installed devices were sham (no heat). The devices were programmed to heat the tail piece to 72C for 1 hour every fourth hour. Rooms were randomized to heating or sham devices on a 1:1 basis within each unit. Sink drains and p-traps were sampled biweekly. Samples were assessed for semiquantitative growth of gram-negative bacteria on MacConkey agar, looking especially for Pseudomonas aeruginosa and Stenotrophomonas maltophilia. Carbapenemase-producing Enterobacterales (CPE) was detected by broth enrichment followed by growth on Colorex KPC agar. Frontline personnel were blinded to device assignment. Results: Linear mixed modeling revealed reduced risk of detectable gram-negative bacteria (OR, 0.16; 95% CI, 0.037–0.536) and Enterobacterales (OR, 0.17; 95% CI, 0.368–0.668) in sink drains with a heating device (Fig. 1), but no difference in risk of detectable P. aeruginosa or S. maltophilia (Table 1). We detected a trend toward reduction in CPE that did not reach statistical significance, and there was no difference in risk for detection of any bacteria in the p-trap between heating and sham devices. Audits of devices demonstrated that few reached the target heating temperature of 72C (median, 65.9C; range, 50.1–73.7C). Conclusions: Disruption of biofilm between the p-trap and the sink drain is a promising strategy for the prevention of sink-drain colonization with clinically important bacteria. The presence of a heating device was associated with reduced risk of detectable gram-negative organisms, specifically Enterobacterales, in sink drains. The limitations of this study included low overall rates of positivity for certain pathogens, including CPE, and suboptimal, inconsistent performance across heating devices. Further work with a larger sample size and more consistent heating devices is warranted, as are data regarding patient outcomes as a result of such interventions.Funding: NoneDisclosures: None

Value of Nontargeted Screening for Highly Resistant Microorganisms: The MOVE Study

Background: In the Erasmus MC University Medical Center, Rotterdam, the Netherlands, patients considered at risk for carrying highly resistant microorganisms (HRMO) are placed in isolation on admission, until tested negative for HRMO (ie, targeted screening). Patients without risk factors are not routinely screened (ie, nontargeted screening). However, nontargeted screening could identify patients colonized with HRMO missed by targeted screening. To determine the additional value of nontargeted screening, we compared the outcomes of the nontargeted screening approach with all available clinical cultures. Objective: We aim to identify patients colonized with HRMO, but missed by targeted screening, and to determine whether non-targeted screening has additional value. Methods: For the MOVE study, nontargeted admission and discharge cultures (nose and perianal) were obtained from randomly selected patients admitted to specific wards, regardless of HRMO risk factors. This study was part of a research initiative to identify the relation of a contaminated environment with the risk of becoming infected or colonized on a patient level. All bacteriological clinical samples positive for at least 1 HRMO from January 1, 2018, until August 31, 2019, were compared with the nontargeted screening samples. Samples were screened for methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) as well as highly resistant Pseudomonas aeruginosa, Acinetobacter baumannii, Enterococcus faecium, and Enterobacteriales. Broth enrichment was used for all cultures. Results: During the study period, 50,653 patients were admitted. 706 patients (1%) had a clinical sample positive for at least 1 HRMO during their hospital stay. 936 (1.8%) patients were included in the nontargeted screening for the MOVE study, and 40 patients were found to have at least 1 culture positive for HRMO (4.3%). Among these 40 patients, 28 were positive at admission and 12 were positive at discharge. Extended-spectrum β-lactamase (ESBL)–producing Enterobacteriales were most prevalent (n = 36, 90.0%) both at admission and discharge (n = 26 and n = 10, respectively). At admission, 1 patient was identified with MRSA and 1 patient was positive for vancomycin-resistant E. faecium (VRE). At discharge, 1 patient was identified with VRE and 1 had Verona Integron-encoded Metallo-β-lactamase (VIM)–positive P. aeruginosa. Conclusions: Our results show that the current targeted screening does not identify all HRMO carriers. Furthermore, patients who acquire an HRMO during admission are missed. The nontargeted screening identified 40 unknown carriers (4.3%). The limitations of the study are the restricted number of sample sites and the fact that we were unable to culture all patients. Therefore, it is likely that our study shows an underestimation of the true number of patients with HRMO.Funding: NoneDisclosures: None

New Approaches to Colonization Screening in Response to Emerging Antimicrobial Resistance

Background: The capacity to monitor the emergence of carbapenemase-producing organisms (CPO) is critical in limiting transmission. CPO-colonized patients can be identified by screening rectal specimens for carbapenemase genes and the Cepheid GeneXpert Carba-R (XCR), the only FDA-approved test, is limited to 5 carbapenemase genes and cannot identify the bacterial species. Objective: We describe the development and validation of culture-based methods for the detection of CPO in rectal cultures (RCs) and nonrectal cultures (NRCs) of tracheal aspirate and axilla-groin swabs. Methods: Colonization screening was performed at 3 US healthcare facilities; specimens of RC swabs and NRC ESwabs were collected. Each specimen was inoculated to a MacConkey broth enrichment tube for overnight incubation then were subcultured to MacConkey agar with meropenem and ertapenem 10 µg disks (BEMA) and CHROMagar KPC (KCHR) or CHROMagar Acinetobacter (ACHR). All media were evaluated for the presence of carbapenem-resistant organisms; suspect colonies were screened by real-time PCR for the most common carbapenemase genes. MALDI-TOF was performed for species identification. BEMA, a previously validated method, was the comparator for 52 RCs; clinical culture (CC) served as the comparator method for 66 NRCs. Select CPO-positive and -negative specimens underwent reproducibility testing. Results: Among 56 patients undergoing colonization screening, 12 (21%) carried a CPO. Only 1 patient had CPO solely from RC. Also, 6 patients had both CPO-positive RC and NRC, and 5 patients only had a CPO-positive NRC. Of the latter, 4 had a CPO-positive tracheal specimen, and 1 had a positive culture from both tracheal and axilla-groin specimens. Sensitivity of BEMA (70%) for NRC was lower than for KCHR (96%) and ACHR (88 %) for all specimens. All methods showed a specificity of 100% and reproducibility of 92%. The detected CPO included OXA-23–positive Acinetobacter baumannii, NDM-positive Escherichia coli, KPC-positive Pseudomonas aeruginosa and 4 genera of KPC-positive Enterobacteriaceae. Conclusions:The addition of nonrectal specimens and use of selective media contributed to increased sensitivity and enhanced identification of CPO-colonized patients. Positive cultures were equally distributed among the 3 specimen types. The addition of the nonrectal specimens resulted in the identification of more colonized patients. The culture-based method was successful in detecting an array of different CPOs and target genes, including genes not detected by the Carba-R assay (eg, blaOXA-23-like). Enhanced isolation and characterization of CPOs will be key in aiding epidemiologic investigations and strengthening targeted guidance for containment strategies.Funding: NoneDisclosures: We discuss the drug combination aztreonam-avibactam and acknowledge that this drug combination is not currently FDA approved.

High levels of toxigenic Clostridioides difficile contamination of hospital environments: a hidden threat in hospital-acquired infections in Kenya.

Introduction The contribution of Clostridioides difficile (formerly Clostridium difficile ) to the burden of hospital-associated infections (HAIs) remains undetermined in many African countries.Aim This study aimed to identify a sensitive and readily adaptable C. difficile detection assay and to evaluate the C. difficile HAI risk in Kenya.Methodology Sterile swabs in neutralizing buffer were used to sample equipment or surfaces that patients and clinical staff touched frequently. These swabs were either plated directly on chromogenic agar or cultured in an enrichment broth before plating. The swab suspensions, enrichment broth and plate cultures were screened by quantitative PCR (qPCR) to determine the most efficient detection method. The HAI risk was evaluated by testing the C. difficile -positive samples by qPCR for the A, B and binary toxins.Results C. difficile was detected on 4/57 (7.0 %) equipment and surfaces by direct culture. The additional enrichment step increased the detection rate 10-fold to 43/57 (75.4 %). In total, 51/57 (89.5 %) environmental samples were positive for C. difficile detected through either culture or qPCR. The genes encoding the primary toxins, tcdA and tcdB, were detected on six surfaces, while the genes encoding the binary toxins, cdtA and cdtB, were detected on 2/57 (3.5 %) and 3/57 (5.3 %) surfaces, respectively. Different C. difficile toxin gene profiles were detected: the tcdA+/tcdB− gene profile on 4/10 (40 %) high-touch surfaces, tcdA−/tcdB+ on 3/10 (30 %) surfaces, tcdA+/tcdB+/cdtA+/cdtB+ on 2/10 (20 %) surfaces and tcdA−/tcdB+/cdtB+ on one high-touch surface.Conclusion The widespread contamination of hospital environments by toxigenic C. difficile gives a strong indication of the high risk of C. difficile infections (CDIs). The two-step culture process described can easily be adapted for monitoring hospital environment contamination by C. difficile .

Application of Recombinase Polymerase Amplification with Lateral Flow for a Naked-Eye Detection of Listeria monocytogenes on Food Processing Surfaces.

The continuous contamination of foods with L. monocytogenes, highlights the need for additional controls in the food industry. The verification of food processing plants is key to avoid cross-contaminations, and to assure the safety of the food products. In this study, a new methodology for the detection of L. monocytogenes on food contact surfaces was developed and evaluated. It combines Recombinase Polymerase Amplification (RPA) with the lateral flow (LF) naked-eye detection. Different approaches for the recovery of the bacteria from the surface, the enrichment step and downstream analysis by RPA-LF were tested and optimized. The results were compared with a standard culture-based technique and qPCR analysis. Sampling procedure with sponges was more efficient for the recovery of the bacteria than a regular swab. A 24 h enrichment in ONE broth was needed for the most sensitive detection of the pathogen. By RPA-LF, it was possible to detect 1.1 pg/µL of pure L. monocytogenes DNA, and the complete methodology reached a LoD50 of 4.2 CFU/cm2 and LoD95 of 18.2 CFU/cm2. These results are comparable with the culture-based methodology and qPCR. The developed approach allows for a next-day detection without complex equipment and a naked-eye visualization of the results.

Improved culture enrichment broth for isolation of Arcobacter-like species from the marine environment

Arcobacter-like species are found associated with many matrices, including shellfish in marine environments. The culture media and conditions play a major role in the recovery of new Arcobacter-like species. This study was aimed to develop a culture media for isolation and enhanced growth of Arcobacter-like spp. from marine and shellfish matrices. For this purpose, 14 different Arcobacter-like spp. mostly isolated from shellfish, were grown in 24 different formulations of enrichment broths. The enrichment broths consisted of five main groups based on the organic contents (fresh oyster homogenate, lyophilized oyster either alone or in combination with other standard media), combined with artificial seawater (ASW) or 2.5% NaCl. Optical density (OD420nm) measurements after every 24 h were compared with the growth in control media (Arcobacter broth) in parallel. The mean and standard deviation were calculated for each species in each broth and statistical differences (p < 0.05) among broths were calculated by ANOVA. The results indicated that shellfish-associated Arcobacter-like species growth was significantly higher in Arcobacter broth + 50% ASW and the same media supplemented with lyophilized oysters. This is the first study to have used fresh or lyophilized oyster flesh in the enrichment broth for isolation of shellfish-associated Arcobacter-like spp.

Kars İlinde Gözlenen Atık Vakalarında Kampilobakteriozisin Patolojik, İmmunohistokimyasal, PCR ve Mikrobiyolojik Yöntemler ile Araştırılması

Campylobacteriosis is an infectious, zoonotic infection characterized by offspring and infertility, leading to economic losses in cattle and sheep breeding. In this study, we aimed to investigate the incidence of cattle and sheep abortion in Kars region in terms of Campylobacteriosis and evaluate the results by PCR, immunohistochemical, histopathological and microbiological methods. In this context, liver and lung tissue examples and abomasum contents of 444 abort cases brought from Kars Center and districts to Kafkas University Faculty of Veterinary Medicine Pathology Department between 2013-2019 years were examined. Tissue examples from animals were fixed in % 10 buffered formaldehyde solutions. After routine procedures, paraffin blocks were prepared and sections with a thickness of 5 μm were taken for Hematoxylin &amp;amp; Eosin staining and 4 μm were taken for immunohistochemical staining . Sections were examined under light microscope to determine histopathologic changes. Organs belonging to aborted fetuses and abomasum contents were inoculated into the Preston Campylobacter Enrichment Broth containing microbial study selective supplement, and then enriched by pre-enrichment and then passed through Preston Campylobacter Selective Agar. Cultures in which the culture was incubated after incubation were examined for colony morphology and microscopic appearance and Campylobacter spp. suspicious colonies were evaluated by biochemical tests. As a result of histopathologic studies, characteristically, 7 of 17 abortion cases with multifocal necrotic hepatitis pattern and yellow abomasum contents were blurred and clotted, PCR, immunhistochemical and microbiological methods detected as Campylobacter spp. positive towards the direction. As a result, we thought that Campylobacteriosis is an important place in the abortion cases from Kars region and should be taken into consideration in breeding.