Chromogenic Medium Research Articles

High Frequency of Antibiotic Resistance Genes (ARGs) in the Lerma River Basin, Mexico.

The spread of beta-lactamase-producing bacteria is of great concern and the environment has been found to be a main source of contamination. Herein, it was proposed to determine the frequency of antimicrobial-resistant-Gram-negative bacteria throughout the Lerma River basin using phenotypic and molecular methods. Resistant bacteria were isolated with chromogenic media and antimicrobial susceptibility tests were used to characterize their resistance. ARGs for beta-lactams, aminoglycosides, and quinolones were detected by PCR. Species were identified by Sanger sequencing the 16S rRNA gene and the representative genomes of MDR strains were sequenced by NGS. A high variation in the number of isolates was observed in the 20 sampled sites, while observing a low diversity among the resistant bacteria. Of the 12 identified bacterial groups, C. freundii, E. coli, and S. marcescens were more predominant. A high frequency of resistance to beta-lactams, quinolones, and aminoglycosides was evidenced, where the blaCTX,qnrB, qnrS y, and aac(6')lb-cr genes were the most prevalent. C. freundii showed the highest frequency of MDR strains. Whole genome sequencing revealed that S. marcescens and K. pneumoniae showed a high number of shared virulence and antimicrobial resistance genes, while E. coli showed the highest number of unique genes. The contamination of the Lerma River with MDR strains carrying various ARGs should raise awareness among environmental authorities to assess the risks and regulations regarding the optimal hygienic and sanitary conditions for this important river that supports economic activities in the different communities in Mexico.

Validation of the AquaCHROM™ ECC for the Detection and Enumeration of Coliforms and Escherichia coli in Water Samples: AOAC Performance Tested MethodSM 072202.

The AquaCHROM™ ECC method from CHROMagar is intended for the detection and enumeration of Escherichia coli and coliform bacteria in 100 mL water samples after 18-24 h of incubation at 35-37°C. To validate the AquaCHROM ECC method for qualitative and quantitative detection of E. coli and coliforms with different water matrixes. Inclusivity/exclusivity studies were conducted. AquaCHROM ECC was compared to U.S. cultural reference methods in unpaired matrix studies for detection of E. coli and coliforms in tap water, well water, lake water, and bottled water, and for enumeration in tap water, well water, and lake water. Three production lots of AquaCHROM ECC were tested for product consistency and stability. Variations in incubation time and temperature were evaluated in robustness testing. Inclusivity/exclusivity results demonstrated expected performance with the exception of three strains of Salmonella enterica, two species of Shigella, and one strain of Aeromonas, which turned the media blue or yellow. Results from the matrix studies demonstrated that AquaCHROM ECC and the reference methods are not statistically different for detection of E. coli and coliforms and statistically equivalent for enumeration of E. coli and coliforms. The AquaCHROM ECC powder production was proven to be consistent with a 24-month shelf life. Variation in temperature did not affect the method performance. Shortening the incubation time is not recommended. AquaCHROM ECC is an effective method for the detection and enumeration of E. coli and coliforms for the water matrixes evaluated. The AquaCHROM ECC method is a quick, one-step method for the recovery and enumeration of E. coli and coliforms in 100 mL water samples. It is a non-agar-based chromogenic medium which provides a clear result without the use of a UV lamp.

Candida auris and some Candida parapsilosis strains exhibit similar characteristics on CHROMagarTMCandida Plus.

Candida auris is considered a public health problem due to its resistance and its tendency to cause nosocomial outbreaks. CHROMagarTMCandida Plus has recently been marketed as capable of presumptively identifying C. auris. The objective of this work was to analyze the ability of this new chromogenic medium to differentiate C. auris from other members of the C. haemulonii complex and from other yeasts commonly isolated in clinical practice. A collection of 220 strains including species of the C. haemulonii (n=83) and C. parapsilosis (n=80) complexes was studied. The strains were identified by molecular methods and cultured as individual or as mixed aqueous inoculum on CHROMagarTMCandida Plus plates. Colony morphotypes were evaluated at 5 time points. CHROMagarTMCandida Plus was a helpful tool for presumptive identification for C. auris. Better reading results were obtained after 48 hours of incubation at 35°C. It is able to easily differentiate C. auris from other closely related species of the C. haemulonii complex and other yeasts. This chromogenic medium would be also useful as screening and surveillance tool for C. auris colonization. However, we demonstrated that it would be a possible misidentification of C. parapsilosis as C. auris (44.3% showed similar morphotypes). To reduce false positives when it is used in a context of a C. auris outbreak, we propose to supplement the chromogenic medium with 8 μg/ml fluconazole. This modified medium was tested and it clearly differentiate C. parapsilosis from C. auris.

Occurrence and Quantitative Microbial Risk Assessment of Methicillin-Resistant Staphylococcus aureus (MRSA) in a Sub-Catchment of the Yodo River Basin, Japan.

The occurrence of Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA) in a sub-catchment of the Yodo River Basin, a representative water system of a drinking water source in Japan, was investigated. The chromogenic enzyme-substrate medium method was used for the detection of S. aureus and MRSA by the presence or absence of antimicrobials in the medium for viable bacteria in a culture-based setting. The contributions of S. aureus and MRSA from wastewater to the rivers were estimated based on mass flux-based analysis, and quantitative microbial risk assessment (QMRA) was further conducted for S. aureus and MRSA in river environments. The mean abundance of S. aureus and MRSA was 31 and 29 CFU/mL in hospital effluent, 124 and 117 CFU/mL in sewage treatment plant (STP) influent, 16 and 13 CFU/mL in STP effluent, and 8 and 9 CFU/mL in river water, respectively. Contribution of the pollution load derived from the target STP effluent to river water ranged from 2% to 25%. The QMRA showed that to achieve the established health benchmarks, the drinking water treatment process would need to yield 1.7 log10 and 2.9 log10 inactivation in terms of infection risk and disability-adjusted life year (DALY) indexes, respectively. These findings highlight the link between medical environment and the importance of environmental risk management for antimicrobial-resistant bacteria in aquatic environments.

P200 Lodderomyces elongisporus fungemia in a late post-operative patient with ventricular septal defect: case report

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PMObjectivesTo report a case of Lodderomyces elongisporus fungemia in a late post-operative patient with the ventricular septal defect.MethodsInformed consent was obtained from the parents of the child. Clinical history was collected. Routine biochemical tests were conducted. Blood samples were sent to the bacteriology and mycology laboratory for culture. Radiological examination of the head was done to ascertain the cause of neurological manifestations.ResultsAn 11-year-old boy, previously diagnosed as a case of ventricular septal defect (VSD) and severe aortic regurgitation, had undergone VSD closure in December 2010 and aortic valve replacement in January 2011. He was discharged on oral anticoagulants. However, he did not follow up.In August 2018, he presented with fever and right-sided hemiparesis. Hemogram was within normal limits except for decreased hemoglobin. Bacteriological blood culture was sterile, but I.V. antibiotics were administered empirically. However, the patient started to have epileptic attacks and therefore was intubated and antiepileptics were administered. An NCCT of the head revealed a large intraparenchymal bleed. Echocardiography revealed intra-cardiac vegetation measuring 8 × 7 mm and moderate aortic regurgitation.Fungal blood culture inoculated in BACTEC Mycosis IC/F bottle flagged positive after 5 days of incubation. A smear and Gram stain from the same revealed budding yeast cells. Sub-culture was done on Sabouraud dextrose agar and HiCromeTM Candida Differential Agar, and blue-tinged colonies were observed on the latter. The isolate was identified as L. elongisporus when subjected to MALDI-TOF analysis. Identification was confirmed by sequencing the internal transcribed spacer (ITS) region of the ribosomal DNA.Antifungal susceptibility test was performed by broth microdilution as per CLSI guidelines. Antifungal therapy was initiated with liposomal amphotericin B, but he continued to have fever even after 1 week and consequently developed status epilepticus. CT scan of the brain revealed massive intracranial hemorrhage. Parents were advised neurosurgery, but they requested discharge against medical advice. The patient was lost to follow-up.Conclusions Lodderomyces elongisporus is a rare cause of invasive bloodstream infections and should not be ignored as a contaminant when isolated from sterile sites. It is often misidentified as Candida parapsilosis by conventional methods and commercially available systems but can be distinguished from it using chromogenic culture media and MALDI-TOF-MS. The current case report highlights the significance of L. elongisporus as a rare cause of invasive fungal infections, the difficulties faced in the identification of this pathogen, and the importance of newer diagnostic methods in identifying it.

P385 Candida auris: a growing threat to global health

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM Background and ObjectivesThe emerging pathogen, C. auris, has been associated with nosocomial outbreaks in recent times. The true scale of the problem is difficult to comprehend due to several issues with the identification of C. auris using both phenotypic and molecular techniques. Most commonly, these isolates have been misidentified as C. haemulonii. Biofilm formation is strongly suggested given its association with intensive care settings, especially in patients with CVCs and long-term urinary catheters. Many isolates of C. auris have also shown raised MICs to multiple classes of antifungal agents, raising the possibility of pan-drug resistance.ObjectiveTo study the demographic characteristics, risk factors, and outcomes in patients with C. auris infection.MethodologyThis is a retrospective study from a tertiary care hospital (JPNATC, AIIMS) including all patients from the time period of 2018-2022 that showed growth of C. auris in any site. C. auris was identified using conventional methods (pale-pink growth on chromogenic medium, no pseudohyphae on germ-tube test, growth in presence of 10% NaCl) and VITEK-2. To reduce the misidentification and the intertest variability, the results were confirmed with MALDI-TOF. The risk factors and other patient information were taken from the HIS. Statistical analysis was performed.ResultsDuring the study period, a total of 31 patients had a C. auris infection. The most common age group was 20-40 years (n = 11,44%) with a preponderance in males (n = 23,74%). A total of 74% of the infections were found in blood, which was the most common site of infection followed by urine (10%). The other sites were pus-from-wound (n = 2), groin, nailbeds, and CVP tip (n = 1). Most of the cases were ICU patients (86%). All the patients with candidemia due to C. auris (n = 17 100%) had CVC, had surgery within the past 30 days, and were on broad-spectrum antibiotics and TPN. 71% (n = 12) had a history of immunosuppression and 18% (n = 14) had a history of prior antifungal therapy. Although 100% (n = 17) had the presence of an indwelling urinary catheter, none of them had candiduria due to C. auris. No patient with C. auris infection had neutropenia. The median LOS was 34.5 days. Most of the isolates were resistant to fluconazole (n = 13,93%), amphotericin B (n = 13,93%), voriconazole (n = 6,55%), flucytosine (n = 10,71%). A total of 87% (n = 12,87%) of isolates were sensitive to caspofungin and micafungin by VITEK-2 (limitation of this study). In all, 28% (n = 7) of the patients died whereas 40% (n = 10) were discharged. A total of 75% patients had clearing of the persistent candidemia when treated with caspofungin whereas only 25% patients had clearing of the candidemia when treated with voriconazole.ConclusionMost cases of C. auris infection were found in critical patients with the most common presentation being candidemia. The risk factors are similar to any other Candida infection. C.auris is the leading antimicrobial-resistant fungi and poses an additional burden to the healthcare system. The fungus has a high crude-mortality rate and we are running out of treatment options. A comprehensive intervention program with ongoing surveillance and good AMR practices is the need of the hour to reduce the burden of this dangerous pathogen.

P367 Candida sepsis in neonates a neglected pathogen: study from neonatal intensive care unit tertiary care hospital

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM IntroductionSignificance of Candida species in neonatal intensive care units (NICU) is increasingly being recognized. It is the third most common cause of late-onset sepsis in NICU patients and accounts for 9 to 13% of bloodstream infections (BSI) in neonates. Although Candida albicans has historically been the most frequently isolated species, recently non-albicans Candida (NAC) have emerged as an important opportunistic pathogen, notably Candida tropicalis, C. parapsilosis, C. krusei, C. glabrata, and C auris. This changing trend and the increasing use of azoles agents is now a matter of concern due to the emerging resistance of the non-albicans species to azoles.AimTo evaluate the Candida sepsis in neonates from neonatal intensive care units from a tertiary care hospital.Material and MethodsA cross-sectional study was conducted in the Department of Microbiology, University College of Medical Sciences and Guru Teg Bahadur Hospital, East Delhi, India, during the period of July 2021 to December 2021. Overall, 30 suspected Candida sepsis patients from neonatal intensive care units were enrolled in the present study. The Candida spp. Isolated was identified as per standard mycological techniques. Preliminary identification was done by colony morphology on SDA, chromogenic media (Hichrome, Himedia Pvt. Ltd.), grown at 45°C, germ tube test, chlamydospore formation, and was confirmed by carbohydrate fermentation and assimilation tests. Antifungal susceptibility testing (AFST) was performed for Fluconazole (25 μg), and amphotericin B (AMB, 100 units) using the disc diffusion method on Muller-Hinton agar supplemented with 2% glucose and methylene blue (5 mg/ml). Zone diameters were interpreted as per the approved Clinical Laboratory Standards (CLSI-M44-A) guidelines. Quality control for AFST was performed using C. albicans-ATCC 90028 and C. parapsilosis-ATCC 22019.Statistical analysisThe statistical analysis was done using Statistical Package for Social Sciences (SPSS) version. The P-value ˂0.05 were considered as statistically significant.ResultOut of 30 Candida species from suspected septicemia neonates, non-albicans Candida (NAC) species accounted for (87%) compared to C. albicans (13.4%). Candida tropicalis was predominant at 9 (30%) followed by C. glabrata 8 (27%), C.parapsilosis 4 (13.3%), and C. krusei 3(10%), Pichia species (4.2%), and C.auris (3.4%). Antifungal sensitivity of the NAC isolates demonstrated >85% resistance to fluconazole amongst C. glabrata, C. parapsilosis, though amphotericin B had 100% sensitivity in all Candida species. The risk factor for candidemia as observed in our study were low birth weight 26(87%), use of broad-spectrum antibiotics 25 (83.4%), presence of an orogastric tube 18 (60%), and total parental nutrition 9 (30%) were significantly associated statistically (P <.05, paired t-test). The overall mortality amongst these neonates was 16.6%.ConclusionFungal BSI has emerged as an important cause of morbidity and mortality in neonates. Reporting of fungal bloodstream infections and the spectrum of species involved are essential measures in neonatal intensive care units in order to implement appropriate preventive and therapeutic strategies.

P159 The impact of COVID-19 pandemic on invasive Candida auris infections in Kuwait

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PM ObjectivesTo evaluate the impact of the coronavirus disease (COVID-19) pandemic on the incidence of Candida auris invasive infections at a national level.MethodsThis is a laboratory-based, three years-period retrospective study. The demographic and mycological data were obtained from the mycology reference laboratory (MRL) active surveillance system. The number of C. auris blood isolates was retrieved for the following years: 2019, 2020, and 2021. C. auris blood isolates from all hospitals were sent to the MRL as part of patient care. Species-level identification was confirmed by chromogenic media, VITEK 2 yeast identification system, and/or MALDI-TOF MS (VITEK MS). Antifungal susceptibility was performed using Etest according to manufacturer instructions. The following antifungal agents were tested: amphotericin B, fluconazole, voriconazole, and caspofungin. If the strain was resistant or intermediate to caspofungin, other echinocandins (micafungin or anidulafungin) were tested for confirmation. Since there were no established clinical breakpoints for C. auris, tentative minimum inhibitory concentrations were used based on experts’ opinions. Incidence rates of COVID-19 during the years 2020 and 2021 (COVID-19) were obtained from the World Health Organization website. All data were numerically coded and labelled for each variable using Microsoft Excel (Version 16.57, 2021), then analyzed by SAS 9.4 (SAS Institute., USA). P-value <.05 was considered statistically significant.ResultsA total of 307 samples isolated from invasive infections were reported with C. auris in this study (50 in 2019, 102 in 2020, and 155 in 2021). The number of patients reported with C. auris invasive infection increased significantly (P <.05) from 50 in 2019 (pre-COVID-19) to 155 in 2021 (post-COVID-19). This was associated with the waves of the COVID-19 pandemic in Kuwait. Of 307 C. auris isolates tested, all were resistant to fluconazole. Resistance to amphotericin B and caspofungin was 29/307 (9%) and 3/285 (1%) respectively.ConclusionPatients infected with COVID-19 are at considerable risk of invasive candidiasis especially critically ill patients staying in ICU. The significant increase in C. auris invasive infections as seen during this pandemic clearly demonstrates the great ability of this opportunistic pathogen to cause secondary lethal infections among COVID-19 patients. In addition, the spread of C. auris among patients in care facilities results in outbreaks, which further compromises the health care services and threatens patient safety. Therefore, a greater understanding of its epidemiology and awareness among healthcare staff about its identification methods, antifungal resistance profile and infection control measures are essential to improve outcomes and prevent further hospital transmission.

P494 Comparison of Candida colonization in intensive care unit patients with and without COVID-19: First prospective cohort study from Turkey

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjectives Candida species, as the main component of human mycobiome, are the most common cause of fungal infections in intensive care units (ICU). ICU patients with COVID-19 are more prone to fungal infections, due to various causes like mechanical ventilation, use of steroids, or long-term hospitalization. There is yet no extended prospective study examining Candida colonization rates, epidemiology of species, and predisposing factors in this population. This is the first prospective cohort study comparing the time-varying colonization features of Candida species in ICU patients with and without COVID-19.MethodsThis study was performed between March 2021-December 2021 in ICUs of Istanbul University, Istanbul Faculty of Medicine, Department of Anesthesiology and Reanimation. COVID-19 and non-COVID-19 ICU patients who were ≥ 18 years and expected to stay in the ICU for at least 7 days were included in the study.Samples were taken at certain time intervals from different body parts of the patients [mouth, skin (axilla), rectal, and urine] (Table 1) and evaluated at Istanbul University, Istanbul Faculty of Medicine, Department of Medical Microbiology, Mycology Laboratory. All specimens were inoculated on CHROMagar Candida media (CHROMagar Candida, France) to detect mixed growth and CHROMagar Candida Plus media (CHROMagar Candida Plus, France) to avoid missing Candida auris.Cultures were incubated at 35-37⁰C for 48 h and phenotypically different colonies on primary media were subcultured on corn meal-tween-80 agar for determining their morphology. All strains were identified to the species level using MALDI-TOF MS (Version 4.1.80; Biotyper Bruker) in Yeditepe University, Faculty of Engineering, Genetics and Bioengineering Department. Patient groups were compared statistically in terms of isolated Candida species and distribution according to regions.ResultsThe study consisted of 122 ICU patients including 62 COVID-19 (25 female; 37 male; mean age:63.29 years) and 60 non-COVID-19 (24 female; 36 male; mean age:63.9 years).A total of 1464 samples (756 COVID-19 and 708 non-COVID-19 patients) were taken (Table 1) and fungi grew in 340 (23.2%) samples. Mixed growth was observed in 108 cultures; was more frequently in COVID-19 patients (P < .05), and significantly higher in oral specimens (P < .05).Out of a total of 471 strains that were obtained from fungal cultures, C. albicans (42.25%) and C. glabrata (24.2%) were most frequently isolated. Candida auris was not observed in this period (Table 2).Patients with COVID-19 were found more frequently colonized in oral (P < .001), rectal (P < .05) regions and urine (P < .001) compared with non-COVID-19 patients. There was no growth in the axillary region in any of the patients.Non-albicans Candida strains were found significantly more frequent in patients with COVID-19 in oral (P < .001) and rectal regions (P < .05).ConclusionIn this study, we found significantly higher oral, rectal, and urine Candida colonization rates in COVID-19 ICU patients compared with non-COVID-19 individuals. Increased oral Candida colonization can be the result of insufficient oral care application to these patients in the ICUs due to infection control anxiety, and also mechanical ventilation.Because non-albicans Candida strains were found significantly more frequent in COVID-19 patients, intrinsically resistant isolates should be kept in mind before administering antifungals.The high mixed growth rate detected in all individuals and especially in COVID-19 patients will affect the antifungal therapy and therefore emphasized the importance of using chromogenic media for routine evaluation.

Temporo-spatial variations in resistance determinants and clonality of Acinetobacter baumannii and Pseudomonas aeruginosa strains from Romanian hospitals and wastewaters

BackgroundRomania is one of the European countries reporting very high antimicrobial resistance (AMR) rates and consumption of antimicrobials. We aimed to characterize the AMR profiles and clonality of 304 multi-drug resistant (MDR) Acinetobacter baumannii (Ab) and Pseudomonas aeruginosa (Pa) strains isolated during two consecutive years (2018 and 2019) from hospital settings, hospital collecting sewage tanks and the receiving wastewater treatment plants (WWTPs) located in the main geographical regions of Romania.MethodsThe strains were isolated on chromogenic media and identified by MALDI-TOF-MS. Antibiotic susceptibility testing and confirmation of ESBL- and CP- producing phenotypes and genotypes were performed. The genetic characterization also included horizontal gene transfer experiments, whole-genome sequencing (WGS), assembling, annotation and characterization.ResultsBoth clinical and aquatic isolates exhibited high MDR rates, especially the Ab strains isolated from nosocomial infections and hospital effluents. The phenotypic resistance profiles and MDR rates have largely varied by sampling point and geographic location. The highest MDR rates in the aquatic isolates were recorded in Galați WWTP, followed by Bucharest. The Ab strains harbored mostly blaOXA-23, blaOXA-24, blaSHV, blaTEM and blaGES, while Pa strains blaIMP, blaVIM, blaNDM, blaVEB, blaGES and blaTEM, with high variations depending on the geographical zone and the sampling point. The WGS analysis revealed the presence of antibiotic resistance genes (ARGs) to other antibiotic classes, such as aminoglycosides, tetracyclines, sulphonamides, fosfomycin, phenicols, trimethoprim-sulfamethoxazole as well as class 1 integrons. The molecular analyses highlighted: (i) The presence of epidemic clones such as ST2 for Ab and ST233 and ST357 for Pa; (ii) The relatedness between clinical and hospital wastewater strains and (iii) The possible dissemination of clinical Ab belonging to ST2 (also proved in the conjugation assays for blaOXA-23 or blaOXA-72 genes), ST79 and ST492 and of Pa strains belonging to ST357, ST640 and ST621 in the wastewaters.ConclusionOur study reveals the presence of CP-producing Ab and Pa in all sampling points and the clonal dissemination of clinical Ab ST2 strains in the wastewaters. The prevalent clones were correlated with the presence of class 1 integrons, suggesting that these isolates could be a significant reservoir of ARGs, being able to persist in the environment.

Faecal carriage of multidrug-resistant bacteria and associated risk factors: results from a point prevalence study.

Since 2003, incidences of carbapenemase-producing Gram-negative bacilli (CP-GNB) and vancomycin-resistant Enterococcus faecium (VRE) have steadily increased in France. We therefore conducted a point prevalence study to estimate carriage rates of CP-GNB, VRE and ESBL-producing Enterobacterales (ESBL-PE) and associated risk factors. Between September 2019 and January 2020, all inpatients hospitalized on a given day in 11 teaching hospitals in the Paris urban area were eligible. Patient interviews and rectal swab screening results were recorded by dedicated nurses. The swabs were plated onto selective chromogenic media and processed using the GeneXpert® system. Of 2396 patients, 364 (15.2%) yielded at least one multiresistant bacterial isolate, including 29 CP-GNB carriers (1.2%), 13 VRE carriers (0.5%) and 338 ESBL-PE carriers (14%). In 15 patients (4.4% of ESBL-PE carriers and 36.6% of CP-GNB/VRE carriers), concomitant CP-GNB/VRE and ESBL-PE carriage was observed. In 7/29 CP-GNB and 7/13 VRE carriers, carbapenemase production and vanA in the screening samples was only detected with Xpert® tests. The OXA-48 gene was predominant in 13/34 CP-GNB isolates from 29 carriers. From the 338 ESBL-PE carriers, 372 isolates were recovered, mainly Escherichia coli (61.2%). Among 379 children, 1.1% carried a CP-GNB/VRE strain, and 12.4% carried an ESBL strain. Previous hospitalization outside mainland France, previous antimicrobial treatment and previous ESBL-PE carriage were the main risk factors associated with CP-GNB and/or VRE carriage. The low CP-GNB and VRE prevalence likely reflects the French policy to limit intrahospital spread of CP-GNB and VRE strains.

High frequency of colonization by extended-spectrum beta-lactamase-producing Gram-negative bacilli in hemodialysis patients and their household contacts in Colombia: dissemination between the community and the hospital.

Increasing colonization by beta-lactam-resistant Gram-negative bacilli (BR-GNB) represents a risk for infections and bacterial resistance spread, both in hospitals and the community. Hemodialysis patients and their household contacts regularly transit between these environments. This study investigated the clinical and epidemiological characteristics of BR-GNB colonization in hemodialysis patients and their household contacts, as well as the genetic relationship between their isolates. A cross-sectional study was conducted on hemodialysis patients at a hospital-associated dialysis center in Medellín, Colombia and their household contacts. Clinical and epidemiological information was collected. Colonization was assessed from stool or rectal swab samples. Bacterial identification and susceptibility were determined using chromogenic media and Vitek-2. Molecular characterization included beta-lactamase detection by polymerase chain reaction, multiple-locus sequence typing (MLST), pulsed-field gel electrophoresis, and identification of Escherichia coli phylogroups by the Clermont protocol. This study included 36 hemodialysis patients and 90 household contacts. Colonization by BR-GNB occurred in 58.3% of patients and 22.2% of household contacts. The main beta-lactamase detected was CTX-M group-1 (40.5%). In 3 of the 9 homes that had more than 1 colonized individual, a genetic relationship was found. MLST showed a high diversity in E. coli isolates, and the most frequent phylogroups were B1 and B2. These results show a high frequency of colonization and the presence of potentially pathogenic BR-GBN both in hospitals and the community. This highlights the importance of populations who move between those 2 environments, and the need to prevent the spread of bacterial resistance outside hospitals.

Comparison of Classical methods and Chromogen Media for Detection of Stool Colonisation by Carbapenem-resistant Enterobacteriaceae

Abstract Objectives: We aimed to compare classical methods and chromogenic media to detect carbapenem-resistant Enterobacteriaceae (CRE) colonization among hospitalized patients and determine the risk factors causing infection in colonized patients. Methods: Between January and August 2017, 100 patients over the age of 18 who were hospitalized in the Reanimation Intensive Care Unit and the Trauma Emergency Intensive Care Unit of a university hospital were examined. From the first day of intensive care unit (ICU) admission, rectal swabs were collected once every week and were tested for the presence of CRE by using the classical method defined by the Centers for Disease Control and Prevention (CDC), ChromID CARBA chromogenic medium, and direct inoculation into MacConkey agar plates. In addition, MIC values for imipenem, ertapenem, meropenem and colistin were determined by using the Etest. Results: Rectal BDE carriage was detected by at least one method in 46 (46%) of 100 patients included in the study. Sensitivity and specificity values of the CDC classical method, direct MacConkey inoculation, and ChromID CARBA medium in the first 24 hours were found as 78%-42%, 87%-80%, and 91%-98%, respectively. Sensitivity and specificity values of these methods after 72 hours were determined as 78%-100%, 87%-100%, and 91%-100%, respectively. Conclusion: We observed that, although the ChromID CARBA method performed better than classical CDC and direct MacConkey inoculation methods, direct MacConkey inoculation can still be employed, especially in areas with limited resources. Keywords: carbapenem, carbapenemase, rectal colonization

False-positive detection of Group B Streptococcus (GBS) in chromogenic media (Strep B Carrot Broth) due to presence of Enterococcus faecalis in High Vaginal swabs.

Introduction. Vaginal colonization of Group B Streptococcus (GBS) is associated with preterm births and neonatal sepsis. Thus routine screening of GBS in prenatal care is recommended.Hypothesis. Chromogenic media (carrot broth) aids in specific and rapid detection of GBS.Aim. To investigate the efficiency of Strep B Carrot Broth for detection of GBS in high vaginal swabs from pregnant women.Methods. In this study 201 vaginal swab samples were collected from pregnant women. Swabs were inoculated in chromogenic media (Strep B Carrot Broth). The positive and negative cultures were inoculated on blood agar and crome agar plates. The colonies were subjected to 16S rRNA sequencing and gene-specific PCR for confirmation. The Christie Atkins Munch Peterson (CAMP) and bile esculin agar (BEA) tests were used for biochemical confirmation. PCR was performed on genomic DNA isolated from uncultured vaginal swabs.Results. It was found that 20/201 (9.9 %) vaginal swab samples were positive in the Strep B Carrot Broth and 17/20 (85 %) and 19/20 (95 %) of these samples yielded colonies on blood agar and crome agar, respectively. Of the 181 carrot broth-negative samples, 1 (0.5 %) and 38 (20.9 %) yielded colonies on blood agar and crome agar plates, respectively. However, 16 s rRNA sequencing revealed that none of the 20 carrot broth-positive cultures were GBS and had sequence similarities to Enterococcus faecalis. This was also confirmed by using gene-specific PCR and BEA positivity. Furthermore, E. faecalis was detected by PCR in DNA isolated from 57 uncultured vaginal swabs samples, GBS could only be detected by PCR in four samples.Conclusion. Carrot broth-based culture can lead to false-positive detection due to the presence of E. faecalis. Thus GBS-positive results in carrot broth must be confirmed by the other molecular and biochemical tests before making a final diagnosis.

Identification, characterization and antibiotic susceptibility testing for Bacillus species.

With the increase in immunosuppressed patients and antimicrobial misuse, Bacillus species have risen as opportunistic pathogens in hospitalized patients. The present study aimed at comparing chromogenic media, automated identification cards versus MALDI-TOF as the gold standard method for identification of different Bacillus species and determining minimal inhibitory concentration (MIC) of different antibiotics by broth microdilution method (BMD) to suggest recommendations for Bacillus treatment. The study included 30 Bacillus species isolates recovered from normally sterile sites of the human body and were subjected to identification by MALDI-TOF, Vitek-2c, and HiCrome Bacillus Agar. BMD test was performed to determine the MIC of vancomycin, gentamicin, and ciprofloxacin. Our study showed B. cereus was the most commonly isolated species (76.66%) followed by B. subtilis (23.33%). Regarding the different methods of identification, the highest agreement with MALDI-TOF was exhibited by HiCrome agar without polymyxin B (93.3%) followed by Vitek-2C and HiCrome agar with polymyxin with an agreement of 83.3%. Concerning the antibiogram, the tested isolates showed a susceptibility of 93.3%, 86.6%, and 83.3% towards vancomycin, gentamicin, and ciprofloxacin respectively. In conclusion, we spotlight that Bacillus species should no longer be considered contaminant bacteria in cultures, particularly in immunosuppressed patients. HiCrome Bacillus Agar without polymyxin B displayed the highest agreement with MALDI-TOF. Hence, it represents a good option for identification for routine laboratories where expensive instruments are unavailable. The high susceptibility towards the tested antibiotics can suggest the possibility of empirical use of vancomycin, gentamicin, and ciprofloxacin.

Diagnostic performance of two chromogenic media for Streptococcus agalactiae screening in pregnant women.

Diagnostic performance of two chromogenic media for Streptococcus agalactiae screening in pregnant women.

Extended Spectrum Beta-Lactamase (ESBL) Resistant genes in Salmonella species Isolated from Ready-to-eat Foods and Food Handlers in Port Harcourt Metropolis, Nigeria

Aim: To assess the Extended Spectrum Beta-Lactamase (ESBL) resistant genes in Salmonella spp isolated from ready-to-eat foods and food handlers in Port Harcourt Metropolis, Nigeria.&#x0D; Study Design: A cross-sectional study.&#x0D; Place and Duration of Study: This study was conducted in selected places in Port Harcourt, between November 2019 and June 2021.&#x0D; Methodology: A total of 350 food specimens and 230 food handlers were collected and the social demographic information were collected using a questionnaire survey. The samples were analysed for contamination with Salmonella species using conventional and molecular methods. Salmonella species were isolated from samples using Salmonella-Shigella agar (SSA), Xylose-lysine desoxycholate agar (XLD), MacConkey agar (MA), Blood agar (BA) after pre-enrichment and enrichment method has been done using peptone water broth and Selenite F. Salmonella Chromogenic medium (SCM) was also used to confirm the isolate. Antibiotic susceptibility patterns of the Salmonella isolates were determined using Kirby Bauer disk diffusion method. The presence of Extended Spectrum Beta Lactamase genes (CTX-M, SHV, and TEM) in the Salmonella isolates were screened using double disc synergy method and also investigated using Polymerase chain reaction (PCR) technique.&#x0D; Results: Results showed that of the 11 Salmonella species isolated from the food handlers, 7(63.6%) harboured extended spectrum beta-lactamase (ESBL) genes while 18 (64.3%) out of 28 Salmonella isolated from street vended food sample were positive for ESBL genes. However, there was no significant difference in the proportion of ESBL producers observed in the street vended food and those in food handlers’ sample (P= 0.964) PCR result revealed the presence of genes encoding for TEM, SHV and CTX-M.&#x0D; Conclusion: Street vended foods such as white rice/stew, jollof rice, rice/beans stew, porridge beans, beans/stew, moi moi, abacha, and roasted plantain in Port Harcourt harbour Salmonella species that possess the following Extended Spectrum Beta Lactamase (ESBL) genes as CTX-M, SHV and TEM. This should receive particular attention, as the presence of extended spectrum beta-lactamase genes isolated, indicate public health hazard and gives a warning signal for the possible occurrence of food borne diseases and multi-drug resistance.

CAMPYAIR, a New Selective, Differential Medium for Campylobacter spp. Isolation without the Need for Microaerobic Atmosphere.

Campylobacter spp. are considered the most frequent bacterial cause of acute gastroenteritis worldwide. Although the diarrhea produced by these bacteria is self-limiting, the pathogen has been associated with severe long-term sequelae following acute signs and symptoms of the illness. However, research on Campylobacter is hampered by costs and technical requirements for isolating and culturing the bacterium, especially in low and middle-income countries. Therefore, attempts have been made to simplify these culture methods and to reduce costs associated with conducting research on Campylobacter. Recently, a liquid medium which allows selective enrichment of Campylobacter using aerobic incubation has been described. However, a solid medium is also needed for the isolation of pure colonies, enumeration of bacterial populations, and other studies on the pathogen. Therefore, a new medium (CAMPYAIR) was developed, based on the formulation of the liquid medium. CAMPYAIR is a solid chromogenic medium that supports the growth of Campylobacter isolates within 48 h of incubation in aerobic atmospheres. Moreover, CAMPYAIR contains antibiotic supplements with an enhanced ability to recover Campylobacter from environmental samples that may also contain non-campylobacter bacteria. The addition of the indicator 2,3,5-triphenyltetrazolium (TTC) to the medium differentiates Campylobacter from other bacteria growing on the media. The findings from studies on CAMPYAIR suggest that the utilization of the new selective, differential medium could help to reduce the costs, equipment, and technical training required for Campylobacter isolation from clinical and environmental samples.

Prevalence and Antimicrobial Susceptibility of Salmonella spp Isolated from Ready-to-eat foods and Food Handlers in Port Harcourt Metropolis, Nigeria

Aim: To assess the prevalence and antimicrobial susceptibility of Salmonella species isolated from ready-to-eat foods and food handlers in Port Harcourt Metropolis, Nigeria.&#x0D; Study Design: Cross-sectional study&#x0D; Place and Duration of Study: This study was conducted in selected places in Port Harcourt, between November 2019 and June 2021.&#x0D; Methodology: A total of 350 food specimens and 230 food handlers’ specimens were collected. The following street vended food were analysed: White rice/stew, Jollof rice, Rice/beans stew, Porridge beans, Beans/stew, Moi Moi, Africa salad and Roasted plantain. The social demographic information was collected using a questionnaire survey. The samples were analysed for contamination with Salmonella species using conventional protocol. Salmonella species were isolated from samples using Salmonella-Shigella agar (SSA), Xylose-lysine desoxycholate agar (XLD), MacConkey agar (MA), Blood agar (BA) after pre-enrichment and enrichment method has been done using peptone water broth and selenite F broth. Salmonella Chromogenic medium (SCM) was also used to confirm the isolate. Antibiotic susceptibility patterns of the Salmonella isolates were determined using Kirby Bauer disk diffusion method. Data collected was analyzed with the Statistical Package for Social Sciences (SPSS, V25, IBM, USA). The prevalence and distribution of Salmonella sp and antibiotic resistance patterns were presented in frequencies and percentages. All analysis was done at a 95% confidence interval and p-values less than 0.05 were considered significant.&#x0D; Results: The prevalence of Salmonella species in the street vended foods was 8.2% and 4.8% among the food handlers. However, there was no statistically significant difference in the proportion of Salmonella growth observed in street vended food and food handlers (P= 0.2900). The isolates from street vended foods and handlers were susceptible to Sulfamethioxazole/Trimetoprin and Meropeneme and resistant to Amoxiclave, Ceftiaxone, Ampicillin, Cefotaxime, Ceftazidine, Levofloxacin and Tetracycline. &#x0D; Conclusion: Salmonella isolates identified from the samples and their handlers showed susceptibility to ciprofloxacin, erythromycin, gentamicin, meropenem, sulfamethroxazole/ Trimethoprim with more isolates being sensitive to meropenem. However, they were resistant to amoxiclav, ceftazidine, cefotaxime, levofloxacin and ceftriaxone.

Prevalence and Molecular Characterisation of Extended-Spectrum Beta-Lactamase-Producing Shiga Toxin-Producing Escherichia coli, from Cattle Farm to Aquatic Environments.

Extended-spectrum beta-lactamase (ESBL)-producing bacteria are a major problem for public health worldwide because of limited treatment options. Currently, only limited information is available on ESBL-producing Shiga toxin-producing Escherichia coli (STEC) in cattle farms and the surrounding aquatic environment. This study sought to track and characterise ESBL-producing STEC disseminating from a cattle farm into the water environment. Animal husbandry soil (HS), animal manure (AM), animal drinking water (ADW), and nearby river water (NRW) samples were collected from the cattle farm. Presumptive ESBL-producing STEC were isolated and identified using chromogenic media and mass spectrophotometry methods (MALDI-TOF-MS), respectively. The isolates were subjected to molecular analysis, and all confirmed ESBL-producing STEC isolates were serotyped for their O serogroups and assessed for antibiotic resistance genes (ARGs) and for the presence of selected virulence factors (VFs). A phylogenetic tree based on the multilocus sequences was constructed to determine the relatedness among isolates of ESBL-producing STEC. The highest prevalence of ESBL-producing STEC of 83.33% was observed in HS, followed by ADW with 75%, NRW with 68.75%, and the lowest was observed in AM with 64.58%. Out of 40 randomly selected isolates, 88% (n = 35) belonged to the serogroup O45 and 13% (n = 5) to the serogroup O145. The multilocus sequence typing (MLST) analysis revealed four different sequence types (STs), namely ST10, ST23, ST165, and ST117, and the predominant ST was found to be ST10. All 40 isolates carried sul1 (100%), while blaOXA, blaCTX-M, sul2, blaTEM, and qnrS genes were found in 98%, 93%, 90%, 83%, and 23% of the 40 isolates, respectively. For VFs, only stx2 was detected in ESBL-producing STEC isolates. The results of the present study indicated that a cattle environment is a potential reservoir of ESBL-producing STEC, which may disseminate into the aquatic environment through agricultural runoff, thus polluting water sources. Therefore, continual surveillance of ESBL-producing STEC non-O157 would be beneficial for controlling and preventing STEC-related illnesses originating from livestock environments.