Chromogenic Agar Plates Research Articles

Rapid counting of coliforms and Escherichia coli by deep learning‐based classifier

AbstractTo ensure that food has been handled hygienically, manufacturers routinely examine the numbers of indicator bacteria, such as coliforms and Escherichia coli. Using the deep‐learning algorithm YOLO, we developed a classifier that automatically counts the number of coliforms (red colonies) and E. coli (blue colonies) on a chromogenic agar plate. Using Citrobacter freundii IAM 12471T and E. coli NBRC 3301, we trained our YOLO‐based classifier with images of Petri dishes grown with each strain alone (10 images) and/or with a mixture of both strains (5 images). When the performance of the classifier was evaluated using 83 images, the accuracy rates for coliforms and E. coli reached 99.4% and 99.5%, respectively. We then investigated whether this classifier could detect other, non‐trained coliform species (22 species) and E. coli strains (13 strains). The accuracy rates for coliforms and E. coli were 98.7% (90 Petri dishes) and 94.1% (46 Petri dishes), respectively. Furthermore, we verified the practical feasibility of the developed classifier using 38 meats (chicken, pork, and beef). The accuracy rates for coliforms and E. coli in meat isolates were 98.8% (80 Petri dishes) and 93.8% (35 Petri dishes), respectively. The time required to count coliforms/E. coli on a single plate was ~70 ms. This novel method should enable users to rapidly quantify coliforms/E. coli without relying on a human inspector's color vision, leading to improved assurance of food safety.

Prevalence of Klebsiella pneumoniae and Acinetobacter baumannii in Urine Samples of Pregnant Women in South-East, Nigeria

Background: Pregnancy induces so many changes in the woman’s body that likely predisposes them to Klebsiella pneumoniae and Acinetobacter baumannii which causes Urinary Tract Infections (UTIs). Aims: To determine the prevalence of Klebsiella pneumoniae and Acinetobacter baumannii in urine samples of pregnant women attending ante-natal clinics at three health institutions within the study area. Study Design: This study was a descriptive cross-sectional study. Place and Duration of Study: Department of Microbiology Federal University Teaching Hospital, Owerri, Teaching Hospital Orlu and General Hospital Okigwe, Imo State between October 2021 and February 2022. Methodology: Urine samples were collected aseptically from 408 pregnant women between 18-45 years and analyzed by culturing onto the surface of freshly prepared MacConkey and Chromogenic agar plates for the isolation of Klebsiella pneumoniae and Acinetobacter baumannii respectively and confirmed by appropriate biochemical tests. Results: From the results, there was a prevalence rate of 145/408 (35.54%) for both isolates with Klebsiella pneumoniae being more dominant with a prevalence rate of 96/408 (23.53%) as against Acinetobacter baumannii that was 49/408 (12.01%). The highest prevalence of the isolates was seen in the Owerri zone with a prevalence rate of 37 (9.07%) for Klebsiella pneumoniae and 23 (5.64%) for Acinetobacter baumannii, respectively. The highest age-specific prevalence of the isolates within the three zones was observed in the 24–29-year-olds with a prevalence rate of 42 (10.29%) in the Owerri zone. In contrast, the prevalence of both organisms was higher in pregnant women in their third trimester of pregnancy with a prevalence rate of 72 (17.65%) and in multigravida women with a prevalence rate of 217 (53.2%). Conclusion: The results from this study showed that these isolates are becoming more predominant in causing both symptomatic and asymptomatic urinary tract infections in pregnant women, hence regular check-ups and proper treatments of urinary tract infections in pregnant women should be encouraged.

Clinical and Microbiological Spectrum of Invasive Trichosporonosis at a Tertiary Care Institute: A Retrospective Observational Study

Introduction: Invasive trichosporonosis was considered rare in the past decades, but it has emerged as an opportunistic pathogen causing invasive infections in immunocompromised patients. It can colonise various parts of the body, and it is important to differentiate between colonisation and infection. Moreover, knowledge of the epidemiology and risk factors associated with the disease is limited. Its inherent resistance to echinocandins poses a therapeutic challenge in treating patients. If not treated appropriately, it may lead to disseminated infections. Aim: To understand the clinical and microbiological spectrum of infections caused by Trichosporon spp. Materials and Methods: This retrospective observational study was conducted in the Department of Microbiology at Nizam’s Institute of Medical Sciences, Hyderabad, Telangana, India for a peroid of one year period from January 2019 to December 2019. The study analysed demographic data such as age, sex, risk factors, clinical features, microbiological diagnosis, treatment, and patient outcomes related to invasive trichosporonosis from the case records. The final sample size was 14 cases. Samples were processed for both aerobic and fungal cultures. The samples were inoculated into Chromogenic agar plates (bioMeriux), 5% sheep blood agar plates (bioMeriux), and Sabouraud dextrose agar. The plates were incubated at 37°C for 48 hours. Culture identification was performed using the Vitek® 2 compact system with the yeast panel YST, and antifungal susceptibility was determined by Broth Microdilution (BMD). Descriptive statistics were used for analysis, and categorical data were described as frequencies with percentages. The data was entered into a Microsoft Excel sheet and analysed using Statistical Packages for the Social Sciences (SPSS) Version 20.0. Results: During the study period, there were 14 cases of invasive trichosporonosis. The predominant age group was 60-70 years. Risk factors included the administration of broad-spectrum antibiotics in 13 (92.8%) of patients, followed by prolonged Intensive Care Unit (ICU) stay in 8 (57.1%) of the patients. Trichosporon spp. were isolated from urine in 10 (71.4%) cases, blood in 2 (14.2%) cases, and tissue in 2 (14.2%) cases. Trichosporon asahii was the predominant species isolated in 12 (85.7%) patients. All isolates were sensitive to voriconazole and amphotericin B. Mortality was reported in 5 (35.7%) of the patients. Conclusion: In present study, Trichosporon spp. was predominantly isolated from urine in the majority of patients. The pathogen was isolated from patients with various risk factors. Hence, proper identification of the pathogen, understanding its clinical significance, and determining antifungal susceptibility would aid in the appropriate management of patients.

Shiga toxin-producing Escherichia coli (STEC) stool multiplex PCR can replace culture for clinical diagnosis and follow-up.

Shiga toxin (stx)-producing Escherichia coli (STEC) causes potentially severe gastrointestinal infections. Due to its public health importance, control measures are required, and carriers may need to refrain from work or daycare when the risk of spread to vulnerable people is high. We evaluated the use of direct stool multiplex PCR compared to culture for primary STEC diagnostics and for follow-up in order to update the national guidelines for STEC monitoring. We analyzed primary and follow-up samples of 236 STEC PCR-positive cases at HUSLAB, Helsinki, Finland in 2016-2017, altogether 858 samples. All STEC PCR-positive samples were inoculated on non-selective chromogenic agar plates. Culture positivity was confirmed from culture sweeps by PCR. 211 (89%) of the cases were culture positive in their primary sample. Of all primary and follow-up samples, 499 were PCR positive and of these 450 (90%) were culture positive. PCR-negative follow-up samples were available from 125 cases. Of these, 88 cases were followed for at least three consecutive PCR-negative samples. Two cases (2%) had culture-positive sample(s) after two consecutive PCR-negative samples. The median time for STEC clearance was 22-23 days. The laboratory-developed multiplex PCR test used in this study is a reliable method for STEC diagnostics and follow-up in a clinical laboratory. When non-selective methodology is used, the majority of PCR-positive samples (90%) are also culture positive. Furthermore, only two cases (2%) in our material had two consecutive PCR-negative samples followed by positive samples. Consequently, to demonstrate the clearance from STEC infection, we consider two PCR-negative follow-up samples sufficient. The Finnish national guidelines for STEC monitoring have been updated accordingly.

Multicentre evaluation of a selective isolation protocol for detection of mcr-positive E. coli and Salmonella spp. in food-producing animals and meat.

This study was conducted to evaluate the performance of a screening protocol to detect and isolate mcr‐positive Escherichia coli and Salmonella spp. from animal caecal content and meat samples. We used a multicentre approach involving 12 laboratories from nine European countries. All participants applied the same methodology combining a multiplex PCR performed on DNA extracted from a pre‐enrichment step, followed by a selective culture step on three commercially available chromogenic agar plates. The test panel was composed of two negative samples and four samples artificially contaminated with E. coli and Salmonella spp. respectively harbouring mcr‐1 or mcr‐3 and mcr‐4 or mcr‐5 genes. PCR screening resulted in a specificity of 100% and a sensitivity of 83%. Sensitivity of each agar medium to detect mcr‐positive colistin‐resistant E. coli or Salmonella spp. strains was 86% for CHROMID® Colistin R, 75% for CHROMagarTM COL‐APSE and 70% for COLISTIGRAM. This combined method was effective to detect and isolate most of the E. coli or Salmonella spp. strains harbouring different mcr genes from food‐producing animals and food products and might thus be used as a harmonized protocol for the screening of mcr genes in food‐producing animals and food products in Europe.

Deep Learning-Enabled Detection and Classification of Bacterial Colonies Using a Thin-Film Transistor (TFT) Image Sensor

Early detection and identification of pathogenic bacteria such as Escherichia coli (E. coli) is an essential task for public health. The conventional culture-based methods for bacterial colony detection usually take >24 hours to get the final read-out. Here, we demonstrate a bacterial colony-forming-unit (CFU) detection system exploiting a thin-film-transistor (TFT)-based image sensor array that saves ~12 hours compared to the Environmental Protection Agency (EPA)-approved methods. To demonstrate the efficacy of this CFU detection system, a lensfree imaging modality was built using the TFT image sensor with a sample field-of-view of ~10 cm^2. Time-lapse images of bacterial colonies cultured on chromogenic agar plates were automatically collected at 5-minute intervals. Two deep neural networks were used to detect and count the growing colonies and identify their species. When blindly tested with 265 colonies of E. coli and other coliform bacteria (i.e., Citrobacter and Klebsiella pneumoniae), our system reached an average CFU detection rate of 97.3% at 9 hours of incubation and an average recovery rate of 91.6% at ~12 hours. This TFT-based sensor can be applied to various microbiological detection methods. Due to the large scalability, ultra-large field-of-view, and low cost of the TFT-based image sensors, this platform can be integrated with each agar plate to be tested and disposed of after the automated CFU count. The imaging field-of-view of this platform can be cost-effectively increased to >100 cm^2 to provide a massive throughput for CFU detection using, e.g., roll-to-roll manufacturing of TFTs as used in the flexible display industry.

Utility of InTray COLOREX Screen agar and InTray COLOREX ESBL agar for urine culture in the Lao PDR.

BackgroundThere is a need for simple microbiology diagnostics to enable antimicrobial resistance surveillance in low- and middle-income countries.ObjectivesTo investigate the field utility of InTray COLOREX plates for urine culture and ESBL detection.MethodsClinical urine samples from Mahosot Hospital, Vientiane, Lao PDR were inoculated onto chromogenic media and InTray COLOREX Screen plates between June and August 2020. Urine and isolates from other clinical specimens were inoculated onto COLOREX ESBL plates. A simulated field study investigating the field utility of the InTray COLOREX plates was also completed.ResultsIn total, 355 urine samples were inoculated onto standard chromogenic agar and InTray COLOREX Screen plates, and 154 urine samples and 54 isolates from other clinical specimens on the COLOREX ESBL plates. Growth was similar for the two methods (COLOREX Screen 41%, standard method 38%) with 20% discordant results, mainly due to differences in colony counts or colonial appearance. Contamination occurred in 13% of samples, with the COLOREX Screen plates showing increased contamination rates, potentially due to condensation. ESBL producers were confirmed from 80% of isolates from the COLOREX ESBL plates, and direct plating provided rapid detection of presumptive ESBL producers. Burkholderia pseudomallei also grew well on the ESBL plates, a relevant finding in this melioidosis-endemic area.ConclusionsThe InTray COLOREX Screen and ESBL plates were simple to use and interpret, permitting rapid detection of uropathogens and ESBLs, and have the potential for easy transport and storage from field sites and use in laboratories with low capacity.

Diagnostic Performance of Various Methodologies for Group B Streptococcus Screening in Pregnant Woman in China.

Maternal vaginal/rectal colonization of group B streptococcus (GBS) is a main risk for neonatal invasive infection. Efficient determination of GBS colonization in pregnant women is crucial. This study aimed to investigate the prevalence of GBS carriage and evaluate the diagnostic performance of six methodologies for GBS screening conducted in China, including blood agar plate, liquid chromogenic medium, and loop-mediated isothermal amplification (LAMP) without pre-enrichment, chromogenic agar plate with pre-enrichment, and GBS antigen detection without and with pre-enrichment in comparison with the standard reference method (Lim broth-enriched subculture with plating on 5% sheep blood agar). Vaginal/rectal swabs were collected from 1,281 pregnant women at 35–37 weeks of gestation. Of them, 309 were taken in triplicate, one for Lim broth-enriched subculture, one for blood agar plate, and the third for GBS antigen detection (Reagent W); 177 were acquired in duplicate, one for Lim broth-enriched subculture and the other for GBS antigen detection (Reagent H); 502 were obtained in duplicate, one for Lim broth-enriched subculture and the other for liquid chromogenic medium; 158 were collected in duplicate, one for Lim broth-enriched subculture and the other for LAMP; and 135 were inoculated in Lim broth-enriched for GBS antigen detection (Reagent W) and subculture with chromogenic agar plate and 5% blood agar plate. The overall prevalence of GBS carriage was 10.1% (130/1,281, 95% CI: 8.5–12.1%) according to the standard reference method. Compared with the standard reference method, the LAMP had excellent performance of sensitivity (100%, 95%CI: 83.4–100%), specificity (94%, 95%CI: 88.1–97.1%), and Yoden index (0.940); as well as the blood agar plate with sensitivity (81.5%, 95%CI: 61.3–93.0%), specificity (100%, 95%CI: 98.3–100.0%), and Yoden index (0.815). The other four methods were not sufficient to reach the threshold in terms of sensitivity or specificity compared to the standard reference method. Furthermore, for LAMP, results can be obtained within 0.5–1 h, while for blood agar plate, which needed 24–48 h, and further identification was required. Our data suggested that the performance of LAMP was highly comparable to the standard Lim broth-enriched subculture and LAMP is considered as an alternative for fast and accurate GBS screening.

Verification of UriSed 3 PRO automated urine microscope in regional laboratory environment

Background and aimsTen UriSed 3 PRO automated microscopes (77 Elektronika, Hungary) were verified for nine HUSLAB laboratories with 160 000 annual urine samples. Materials and methodsParticle counting of the primary UriSed 3 PRO instrument (77 Elektronika, Hungary) was verified against reference visual microscopy with 463 urine specimens, and against urine culture on chromogenic agar plates with parallel 396 specimens. Nine secondary instruments were compared pairwise with the primary instrument. ResultsRelative imprecisions compared to Poisson distribution, R(CV), were estimated to be 1.0 for white blood cell (WBC) and 1.5 for red blood cell (RBC) counts, respectively. Spearman’s correlations against visual microscopy were rS = 0.94 for WBC, rS = 0.87 for RBC, and rS = 0.82 for squamous epithelial cell (SEC) counts. Agreement with visual microscopy (Cohen’s weighted kappa) was 0.94 for WBC, 0.89 for RBC, 0.88 for SEC, 0.59 for combined casts, and 0.49 for non-squamous epithelial cells (NEC). Bacteria were detected with a sensitivity of 90% and specificity of 39 against culture at 107 CFB/L (104 CFU/mL). Created flagging limits allowed automated reporting for 70–75% of patient results. ConclusionsUriSed 3 PRO instruments were adopted into routine use after acceptance of the verification.

Growth Inhibition of Common Enteric Pathogens in the Intestine of Broilers by Microbially Produced Dextran and Levan Exopolysaccharides.

Antibiotics are generally applied for treatment or as subtherapeutic agents to overcome diseases caused by pathogenic bacteria including Escherichia coli, Salmonella and Enterococcus species in poultry. However, due to their possible adverse effects on animal health and to maintain food safety, probiotics, prebiotics, and synbiotics have been proposed as alternatives to antibiotic growth promoters (AGPs) in poultry production. In this study, the effects of prebiotics on the augmentation of broiler's indigenous gut microbiology were studied. Day old 180 broilers chicks were divided into four treatment groups: G, L, C1, and C2. The groups G and L were fed with basal diet containing 3% dextran and 3% levan, respectively. Control groups were fed with basal diets without antibiotic (C1) and with antibiotics (C2). The experimental groups showed decreased mortality as compared to control groups. After 35days, the chickens were euthanized and intestinal fluid was analyzed for enteric pathogens on chromogenic agar plates and by 16S rRNA gene sequencing. Inhibition of the growth of E. coli and Enterococcus was observed in groups G and L, respectively, whereas Salmonella was only present in group C1. Also, high populations of lactic acid bacteria were detected in the intestine of prebiotic fed birds as compared to controls. These results depict that dextran and levan have the potential to replace the use of antibiotics in poultry feed for inhibiting the growth of common enteric pathogens. To the best of our knowledge, this is the first study where effects of dextran and levan on intestinal microbiota of broilers have been reported.

Performance of the Check-Direct ESBL Screen for BD MAXTM for detection of asymptomatic faecal carriage of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae

ObjectivesAccurte diagnostic methods are crucial for the detection of extended-spectrum β-lactamase-producing Enterobacterales (ESBL-E). Besides culture-based gold-standard methods, new molecular gene detection tests are reaching the market. The aim of this study was to investigate the performance of the direct quantitative PCR (qPCR)-based methods Check-Direct ESBL and CPE Screen for BD MAXTM in relation to traditional culture-based methods for detection of ESBL-E faecal carriage. MethodsFaecal samples were collected from healthy adult volunteers. Samples were cultured on chromogenic ESBL agar plates and were screened for ESBL-producing Escherichia coli and Klebsiella pneumoniae. Confirmed ESBL- and AmpC-producing isolates were further analysed using whole-genome sequencing. In addition, faecal samples were analysed using Check-Direct ESBL and CPE Screen for BD MAXTM and the results were compared with the gold-standard culture-based method. ResultsOf 176 faecal samples examined, 11 (6.3%) grew ESBL-producing E. coli or K. pneumoniae isolates. Among 173 analysed samples, Check-Direct ESBL Screen for BD MAXTM detected 22 (12.7%) ESBL-positive samples. No carbapenemase-producing isolates were detected. Two culture-positive samples remained negative with Check-Direct ESBL Screen for BD MAXTM. Culture-negative but qPCR-positive discrepancy was observed in 12 samples (6.9%). Altogether, concordant results were obtained for 158 samples (91.3%; 9 positive and 149 negative). ConclusionCheck-Direct ESBL Screen for BD MAXTM is a fast screening method for ESBL carriage. However, several discrepant results were observed, which hinders interpretation. More clinical samples should be tested in combination with culture to evaluate the true benefits of this method.

Rapid Methods for Detection of MRSA in Clinical Specimens.

Traditional antimicrobial susceptibility test methods for detection of S. aureus resistant to oxacillin (MRSA) such as disk diffusion, broth microdilution, and oxacillin screen plate require 18-24 h of incubation after having the organism growing in pure culture. Rapid and accurate identification of MRSA isolates is essential not only for patient care, but also for effective infection control programs to limit the spread of MRSA. In the last few years, several commercial rapid tests for detection of MRSA directly from nasal and wound swabs, as well as from positive blood cultures, have been developed for use in clinical laboratories. Chromogenic agar plates and real-time PCR and other molecular tests are gaining popularity as MRSA screening tests because they have the advantage of a lower turnaround time than that of traditional culture and susceptibility testing and they are capable of detecting MRSA directly from nasal and wound swabs, allowing rapid identification of colonized or infected patients. In addition, molecular methods able to detect and differentiate S. aureus and MRSA (SA/MRSA) directly from blood cultures are becoming a useful tool for rapid detection of bacteremia caused by MSSA and MRSA. This review focuses on the procedures for performing testing using rapid methods currently available for detection of MRSA directly from clinical specimens.

Characterisation of carbapenem-resistant Gram-negative organisms from clinical specimens in Yola, Nigeria.

This study aimed to identify carbapenem-resistant Gram-negative bacteria from clinical specimens of patients in Yola, Nigeria. Routine clinical specimens were screened for the presence of carbapenem-resistant Gram-negative bacteria using chromogenic agar plates. Susceptibility of all presumptive isolates to carbapenems was tested by MIC and disk diffusion methods. Real-time PCR was used to test for the presence of carbapenemase genes. Screening of 1741 clinical specimens yielded 119 (6.8%) presumptive carbapenem-resistant Gram-negative bacteria. Antimicrobial susceptibility testing confirmed carbapenem resistance in 105 of these isolates. New Delhi metallo-β-lactamase (blaNDM) gene was detected in 26 isolates and Verona integron-encoded metallo-β-lactamase (blaVIM) gene was detected in four. The mechanism of resistance could not be identified in approximately two thirds of the carbapenem-resistant isolates. While blaNDM and blaVIM accounted for 28.6% of the resistance seen, further molecular-based studies are needed to characterise the other mechanisms of carbapenem resistance in these isolates.

Prevalence of nasal carriage of methicillin-resistant Staphylococcus aureus in patients at hospital admission in The Netherlands, 2010–2017: an observational study

ObjectivesWe determined the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) nasal carriage upon hospital admission, among patients who were screened preoperatively for nasal S. aureus carriage between 2010 and 2017. We also aimed to evaluate the prevalence of MRSA carriers without the standard risk factors. MethodsWe conducted an observational study to determine the prevalence of MRSA nasal carriage among patients who were screened preoperatively for nasal S. aureus carriage between 2010 and 2017. Samples of cardiothoracic patients were tested by polymerase chain reaction (PCR), other samples were cultured using chromogenic agar plates. A Poisson regression model with robust error variance was used to assess whether there was a trend in the prevalence of MRSA over time. ResultsIn total, 31 093 nasal swabs were obtained from 25 660 patients. Three-hundred and seventy-five swabs (1.2%) had an invalid result. Therefore, 30 718 swabs (98.8%) were included in our analysis. Overall, S. aureus was detected in 7981/30 718 patients (26.0% 95% CI 25.5–26.5%) of whom 41 were MRSA (0.13% 95% CI 0.10–0.18%). The MRSA prevalence varied from 0.03% to 0.17% over the years without evidence of a changing trend over time (p = 0.40). Results of the questionnaire revealed that 30 of the 41 patients (73.2%) had no known risk factors for MRSA carriage (0.10%; 95% CI 0.07–0.14%). ConclusionOur study revealed a sustained low prevalence of MRSA carriage upon hospital admission over 7 years. This supports the effectiveness of the Dutch Search and Destroy policy, in combination with a restrictive antibiotic prescription policy.

Persistence and Diffusion of mecC-Positive CC130 MRSA Isolates in Dairy Farms in Meurthe-et-Moselle County (France).

Background: Methicillin resistance in Staphylococcus aureus (MRSA) is classically conferred by the acquisition of the mecA gene encoding an additional penicillin binding protein with low affinity for beta-lactams. A mecA variant, named mecC, was described in 2011. MRSA isolates harboring mecC of both animal and human origin have since been collected in different European countries. In France, animal cases were reported in 4 dairy farms between 2008 and 2013 in the Meurthe-et-Moselle county, all located in a 30 km perimeter, suggesting a possible dissemination of mecC-positive MRSA strains. We performed a prospective study to evaluate the local epidemiology of such strains in terms of (i) dissemination among animals, humans and in the environment, and (ii) persistence in Meurthe-et-Moselle dairy cattle farms.Methods: The 4 French dairy farms with previous reports of mecC-positive MRSA strains and 14 farms in the same perimeter were included in this study. In each farm, nasal swabs, rectal swabs and milk samples were collected from 10 randomly selected cows, as well as nasal samples from family pets, volunteer farmers and veterinarians. One farm (E0), in which mecC-MRSA isolates were detected, was selected to study more deeply the dissemination of mecC-positive strains within the farm. After pre-enrichment of swabs and milk, they were subcultured on MSSA/MRSA chromogenic selective agar plates. S. aureus colonies were tested with a multiplex PCR to detect the mecA and mecC genes. The mecC-positive strains were characterized using DNA microarray.Results: mecC-positive strains were recovered in four farms, corresponding to the ones with previous reports of mecC-positive MRSA strains, and originated only from dairy cow samples. The screening in the E0 farm showed that 22% of the dairy cows carried mecC-positive MRSA. Three strains were also isolated from the environmental samples. All mecC-positive strains belonged to the clonal complex CC130 and harbored the same spa-type t1736.Conclusion: This study found that mecC-positive MRSA isolates are able to persist within the same farms for several years after being introduced in this setting and are able to widely disseminate but only among dairy cows suggesting that milking machines might be a key player.

Limitations of a chromogenic agar plate for the identifying bacteria isolated from equine endometritis samples

The use of commercial chromogenic agar plates for the rapid, easy and correct identification of equine endometritis-causing bacteria has been proposed. Preliminary tests in our lab revealed undescribed limitations. Therefore, we tested the ability of the Brilliance UTI agar, a commercially available chromogenic agar, to accurately identify bacteria causing equine endometritis. To 1) investigate whether bacteria present in the equine uterus are able to grow on this chromogenic agar plate, 2) determine whether these bacteria belong to the genera for which these agar plates were originally designed and 3) consider whether these bacterial genera can be correctly identified, based only on the colour appearance. In vitro experiments. Macroscopic growth and colour appearance on the Brilliance UTI agar of 58 bacterial isolates, from previously collected equine uterine samples, were evaluated. Isolates were tentatively identified at the genus level using the manufacturer's guidelines and results were compared with MALDI-TOF MS as a "gold standard". The study revealed that 1) 77% (N = 45/58) of the bacterial isolates grew well on this chromogenic agar, 2) 83% of the investigated isolates (N = 48/58) belonged to one of the genera for which guidelines for identification were provided by the manufacturer and 3) only 50% of the isolates (N = 29/58) were correctly identified at the genus level, based only on colour appearance. The current study uses purified bacterial isolates to inoculate the chromogenic agar plates, instead of fresh uterine samples. Bacteria were identified to the genus level using MALDI-TOF MS. This study shows that identification at the genus level based only on colour appearance, without additional tests or the simultaneous use of other media, is not reliable based on the existing identification guidelines.

Efficient processing of MRSA screening specimens by a modified inoculation protocol.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of healthcare-associated infections and mortality, and therefore constitutes a serious cost factor in public health. Culture-based MRSA screening is a crucial part of MRSA-infection prevention and control strategies in the hospital setting. Manual inoculation of screening swabs onto culture plates still constitutes the major part of the technicians' workload in laboratories. We present a modified inoculation protocol that comprises direct inoculation of specimen onto a chromogenic MRSA-selective agar plate without further streaking for isolation. This study aims to evaluate the impact of this inoculation protocol on technicians' workload and the downstream workflow in our laboratory. Batches of 50 specimens were processed by different technicians and the hands-on time was compared between the standard and modified inoculation protocol. To assess the impact on downstream processing, a retrospective analysis of the rate of subcultures and turnaround time (TAT) of specimens yielding putative MRSA colonies from 9months before (n = 1548) and after (n = 1267) the protocol change was carried out based on laboratory information system (LIS) data. The implementation of the modified protocol significantly reduced technicians' hands-on time needed for inoculation by 26.5% without altering the overall turnaround time of surveillance cultures or causing higher costs for extra plates needed for subcultures. Our modified inoculation protocol offers a cost-effective and easy to implement procedure for MRSA surveillance cultures which significantly decreases technicians' workload and does not impede the downstream workflow. It therefore increases the capacity of laboratory technicians' to execute more demanding tasks.

Molecular Characterization and Antimicrobial Resistance Pattern of Escherichia coli Recovered from Wastewater Treatment Plants in Eastern Cape South Africa.

Wastewater treatment plants (WWTPs) are designed to eliminate organic matter and pathogens but most WWTPs discharges antimicrobial resistance pathogens into aquatic milieu. The study aimed to examine the antibiotics resistant patterns and the presence of some resistance genes among E. coli isolates from WWTPs effluents. Water were collected from WWTPs final effluents, filtered through nitrocellulose membrane and the filter papers were placed on chromogenic agar plates, incubated for 24 h at 37 °C. Presumptive E. coli isolates (173) were obtained from the culture method. From the presumptive E. coli isolates screened by polymerase chain reaction (PCR), 111 isolates were positive and the positive isolates were further screened for six diarrheagenic E. coli pathotypes (EPEC, ETEC, EHEC, DAEC, EIEC, and EAEC) and from the pathotypes screened, nine isolates harboured daaE gene. The phenotypic susceptibility patterns of the 111 isolates to 12 antibiotics were determined by Kirby-Bauer disk diffusion technique. All the isolates were resistant to erythromycin and clindamycin. From the resistance genes screened, 31 isolates harboured mcr-1 gene and nine isolates harboured ermA gene. The study reveals that water samples recovered from the final effluents of WWTPs may likely be one of the major sources of antibiotic-resistant in Escherichia coli.

Prevalence of Nasal Carriage of Methicillin-Resistant <i>Staphylococcus Aureus</i> in Patients at Hospital Admission in the Netherlands, 2010 - 2017: An Observational Study

Background: The MRSA Search and Destroy strategy relies on active screening of high-risk groups. However, a substantial proportion of MRSA carriers found do not belong to the known risk groups, pointing to a reservoir of MRSA carriers in the community. Methods: We conducted an observational study to determine the prevalence of MRSA nasal carriage upon hospital admission, among patients who were screened preoperatively for nasal S. aureus carriage between 2010 and 2017. Samples of cardiothoracic patients were tested by PCR, other samples were cultured using chromogenic agar plates. A Poisson regression model with robust error variance was used to assess whether there was a trend in the prevalence of MRSA over time. Findings: In total, 31,093 nasal swabs were obtained from 25,660 patients. Three hundred seventy-five swabs (1.2%) had an invalid result . Therefore, 30,718 swabs (98.8%) were included in our analysis. Overall, S. aureus was detected in 7,981/30,718 patients (26,0% 95%CI 25.5 - 26.5%) of whom 41 were MRSA (0.13% 95%CI 0.10 - 0.18%). The MRSA prevalence varied from 0.03% to 0.17% over the years without evidence of a changing trend over time (p=0.40). Results of the questionnaire revealed that 30 of the 41 patients (73.2%) had no known risk factors for MRSA carriage (0.10%; 95% CI 0.07 - 0.14%). Interpretation: Our study revealed a sustained low prevalence of MRSA carriage upon hospital admission over 7 years. This supports the effectiveness of the Dutch Search and Destroy policy, in combination with a restrictive antibiotic prescription policy. Funding: None. Declaration of Interest: We declare no competing interests. Ethical Approval: The data used in this study were part of routine practices in The Netherlands and do not require approval from an ethics committee. The data were anonymized and analysed according to the local regulations and laws that apply for medical information.

Comparison of Different Phenotypic Approaches To Screen and Detect mecC -Harboring Methicillin-Resistant Staphylococcus aureus

Similar to mecA, mecC confers resistance against beta-lactams, leading to the phenotype of methicillin-resistant Staphylococcus aureus (MRSA). However, mecC-harboring MRSA strains pose special difficulties in their detection. The aim of this study was to assess and compare different phenotypic systems for screening, identification, and susceptibility testing of mecC-positive MRSA isolates. A well-characterized collection of mecC-positive S. aureus isolates (n = 111) was used for evaluation. Routinely used approaches were studied to determine their suitability to correctly identify mecC-harboring MRSA, including three (semi)automated antimicrobial susceptibility testing (AST) systems and five selective chromogenic agar plates. Additionally, a cefoxitin disk diffusion test and an oxacillin broth microdilution assay were examined. All mecC-harboring MRSA isolates were able to grow on all chromogenic MRSA screening plates tested. Detection of these isolates in AST systems based on cefoxitin and/or oxacillin testing yielded overall positive agreements with the mecC genotype of 97.3% (MicroScan WalkAway; Siemens), 91.9% (Vitek 2; bioMérieux), and 64.9% (Phoenix, BD). The phenotypic resistance pattern most frequently observed by AST devices was "cefoxitin resistance/oxacillin susceptibility," ranging from 54.1% (Phoenix) and 83.8% (Vitek 2) to 92.8% (WalkAway). The cefoxitin disk diffusion and oxacillin broth microdilution assays categorized 100% and 61.3% of isolates to be MRSA, respectively. The chromogenic media tested confirmed their suitability to reliably screen for mecC-harboring MRSA. The AST systems showed false-negative results with varying numbers, misidentifying mecC-harboring MRSA as methicillin-susceptible S. aureus This study underlines cefoxitin's status as the superior surrogate mecC-positive MRSA marker.