CHROMagar ESBL Research Articles

High prevalence and genetic diversity of multidrug-resistant and extended-spectrum ß-lactamase-producing Escherichia coli and Klebsiella pneumoniae in mothers and neonates in a Cameroonian labor ward

Escherichia coli and Klebsiella pneumoniae rank among the primary bacterial culprits in neonatal infections and fatalities in sub-Saharan Africa. This study characterized the phenotypic and genotypic features of E coli and K pneumoniae in a labor ward in Yaoundé, Cameroon. A prospective and cross-sectional study spanning 5months, from February 21, 2022 to June 30, 2022. Rectovaginal swabs were obtained from expectant mothers, and nasopharyngeal swabs were collected from their babies. Hand swabs of health care workers and environmental samples were also collected. The samples were cultured on eosin methylene blue agar. Extended-spectrum ß-lactamase (ESBL) production was assessed using CHROMAgar ESBL and the double-disk synergy test. A polymerase chain reaction was employed to detect ß-lactamase genes. A total of 93 mothers and 90 neonates were collected. Almost all pregnant women (90%) were colonized by one or more multidrug-resistant (MDR) isolates with 58% being concomitantly ESBL producers. Altogether, 14 of 22 (64%) neonates were colonized by MDR isolates, while out of the 5 workers positive to Enterobacterales, all were colonized by MDR isolates. E coli predominated in pregnant women (55%) and neonates (73%), while K pneumoniae (83%) predominated in health care workers. The blaCTX-M (75%) was the leading ß-lactamase gene detected. Our study suggests that drug-resistant E coli and K pneumoniae are circulating at high prevalence in thelabor ward in Yaoundé and emphasizes the necessity for effective infection prevention and control along with antimicrobial stewardship measures.

Detection of antibiotic resistant Enterobacterales in commercial raw pet food: a preliminary study.

Raw diets have become popular in companion animal nutrition, but these diets may be contaminated with harmful bacteria because heat processing is not utilized to mitigate pathogens during the production process. We analyzed 24 commercially available frozen raw canine and feline diets for extended-spectrum beta-lactamase-producing Enterobacterales (ESBL-E). Samples were incubated in tryptic soy broth augmented with 50 μg/mL ampicillin to enrich for ESBL-E. ESBL-E were isolated using CHROMagar ESBL plates and isolate identification and antibiotic susceptibility testing were confirmed using the VITEK®2 instrument. ESBL-E were isolated from 42% (10/24) of raw diets, with E. coli, Enterobacter cloacae complex and Klebsiella pneumoniae predominating. Most ESBL-E isolates (71%, 32/45) were multidrug-resistant. Direct plating of samples onto tryptic soy agar yielded bacterial counts >6 log10 for 2 samples from two different manufacturers. This preliminary study justifies further investigation into the potential contribution of raw diets to the dissemination of antibiotic resistant bacteria in companion animals and domestic living spaces.

High carriage of plasmid-mediated quinolone resistance (PMQR) genes by ESBL-producing and fluoroquinolone-resistant Escherichia coli recovered from animal waste dumps.

There has been a rise in the consumption of fluoroquinolones in human and veterinary medicine recently. This has contributed to the rising incidence of quinolone resistance in bacteria. This study aimed at the determination of the antibiotic resistance profile of ESBL-producing and fluoroquinolone-resistant E. coli (FQEC) isolated from animal waste obtained from the waste dumps of an agricultural farm and their carriage of genes encoding PMQR. Isolation of ESBL-producing E. coli from animal waste samples was done on CHROMagar ESBL, while presumptive isolates were purified, and identified via the detection of uidA gene. Susceptibility to a panel of ten antibiotics was done using the disc diffusion method, and detection of PMQR genes (qnrA, qnrB, qnrS, aac(6')-lb-cr, qepA and oqxAB) was done using monoplex and duplex PCR. Twenty-five ESBL-producing and FQEC were obtained from the cattle (6), piggery (7) and poultry (12) waste dumps of the farm. There was 100% resistance to cefpodoxime, cefotaxime, enrofloxacin, trimethoprim-sulfamethoxazole and penicillin by the isolates. The resistance to the other antibiotics was streptomycin (48%), ceftazidime (24%), while no isolate resisted amoxicillin-clavulanate and imipenem. The frequencies of PMQR genes detected were; qnrA (96%), oqxAB (96%), qnrB (92%), while qnrS was detected in 88% (22) of the isolates. Aminoglycoside acetyltransferase (aac(6')-lb-cr) and quinolone efflux pump (qepA) were each detected in 20 (80%) of the isolates. This study showed that animal wastes disposed indiscriminately into dumps could be a budding 'hotspot' for multidrug resistant, ESBL-producing and fluoroquinolone-resistant E. coli carrying multiple genes encoding resistance to fluoroquinolone antibiotics.

Metagenome and Resistome Analysis of Beta-Lactam-Resistant Bacteria Isolated from River Waters in Surabaya, Indonesia.

Antimicrobial agents are administered to humans and livestock, and bacterial antimicrobial resistance (AMR) and antimicrobial agents are released into the environment. In this study, to investigate the trend of AMR in humans, livestock, and the environment, we performed a metagenomic analysis of multidrug-resistant bacteria with CHROMagar ESBL in environmental river water samples, which were collected using syringe filter units from waters near hospitals, downtown areas, residential areas, and water treatment plants in Surabaya, Indonesia. Our results showed that Acinetobacter, Pseudomonas, Aeromonas, Enterobacter, Escherichia, and Klebsiella grew in CHROMagar ESBL; they were most frequently detected in water samples from rivers surrounding hospitals contaminated with various AMR genes (ARGs) in high levels. These results identified bacteria as ARG reservoirs and revealed that hospitals could be sources for various ARGs disseminated into the environment. In conclusion, this study details a novel metagenomic analysis of collected bacteria in environmental water samples using a syringe filter unit for an AMR epidemiological study based on the One Health approach.

Rapid and automated screening of carbapenemase- and ESBL-producing Gram-negative bacteria from rectal swabs using chromogenic agar media and the ScanStation device.

The ScanSation 100 device (Interscience, France) is an incubator allowing real-time detection of bacterial colony growth by frequently imaging agar plates over time, counting CFU, and detecting colony color. This study evaluated its performance for the early detection of carbapenemase-producing bacteria (CPB) and extended-spectrum β-Lactamase-producing bacteria (ESBL-PB) from rectal swabs inoculated on CHROMagar mSuperCARBA and ESBL media, respectively. Rectal screening ESwabs collected from patients admitted to Grenoble University Hospital between January and June 2021 were analyzed. After inoculation, chromogenic media were incubated for 24 h in the automaton, with image acquisition every 30 min. ScanStation results were compared to visual observations of the plates after 24 h of incubation. In total, 501 rectal swabs were tested. ScanStation showed 100% positive percent agreement (PPA) for the detection of CPB and ESBL-PB, whereas the PPA of color categorization ranged between 45% and 100%. Negative percent agreement (NPA) ranged between 70% and 98%. Negative predictive values (NPVs) were 100% for both bacterial groups, whereas positive predictive values (PPVs) were 70.3% for CPB and 81.0% for ESBL-PB. Importantly, real-time screening allowed detection of the first suspected colony within 10-14 h of growth, on average, whereas visual observation is usually only performed once a day after 18-24 h of incubation. Our study demonstrates the potential use of early images to accelerate the detection of CPB and ESBL-PB and implement effective and timely infection control measures. IMPORTANCE The ScanStation 100 device is an incubator able to follow the real-time growth of bacterial colonies on agar plates through digital imaging, allowing users to sort plates according to the presence or absence of colonies, and to distinguish their color using four numeric color filters. Real-time screening shows that first colony detection is possible much earlier (after 10-14 h of growth, on average), whereas visual observation is usually performed only once a day after 18-24 h of incubation. The ScanStation device, combined with chromogenic agar media, is an efficient automated screening method to accelerate the detection of Gram-negative multidrug-resistant bacteria in laboratories that do not have access to larger laboratory automation systems. Our study shows that setting the image acquisition to one or two early images may allow for the detection of positive samples that were inoculated in the morning, by the end of the working day.

Detection of carbapenem-resistant Enterobacterales from nursing home wastewater effluent from September 2021 to November 2021

Background: Surveillance and early detection of antibiotic resistance genes and multidrug-resistant organisms (MDROs), such as carbapenem-resistant Enterobacterales (CRE), are important to quell outbreaks early, as antibiotic resistance continues to be an increasing threat. Wastewater surveillance in general has gained attention in the United States, but scientific evidence demonstrating the feasibility to assess antibiotic resistance genes and MDROs is limited. In this study, wastewater effluent was used to screen a nursing home facility, which housed a population at increased risk for colonization with MDROs, for the presence of β-lactam–resistant genes.Methods: Wastewater effluent samples (24-hour composite, n = 19; grab samples, n = 6) collected from a skilled nursing home facility from September 2021 to November 2021 in DeKalb County, Georgia, were screened for carbapenem-resistant and extended-spectrum β-lactam (ESBL)–resistant Enterobacterales using 2 selective chromogenic media: mSuperCARBA (mSC) for selection of CRE and CHROMagar ESBL for selection of gram-negative bacteria producing extended-spectrum β-lactamases. Colilert-18 (IDEXX) was applied to detect and quantify total coliforms and Escherichia coli, serving as an enrichment approach for potential gram-negative organisms (Enterobacterales) containing antibiotic resistance genes. E. coli–positive Colilert-18 (IDEXX) samples (n = 24) had a composite of 1.0 mL total from 5 positive wells or 20% per plate collected and stored at −80°C in 25% glycerol. The E. coli–positive Colilert-18 samples were later thawed and plated on mSC and CHROMagar ESBL, where a random subset of all the colonies (ie, mixture of typical and atypical colonies; n = 28) were selected for matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) to confirm identification. Additionally, a non-enrichment approach, filtered wastewater samples (10 mL, n = 23) were frozen (−80°C) until DNA extraction, followed by multiplex real-time PCR for the blaKPC, blaNDM, blaVIM, and blaOXA-48–like carbapenemase genes. Results: Among 24 E. coli–positive Colilert-18 samples, 16 (67%) of 24 contained carbapenem-resistant Klebsiella, Enterobacter, or Citrobacter (KEC), 88% contained ESBL-resistant KEC (21 of 24), 4% (1 of 24) contained carbapenem-resistant E. coli, and 67% contained ESBL-resistant E.coli (16 of 24). In the 28 colonies picked from mSC or ESBL, 10 different genera were confirmed using MALDI-TOF: Aeromonas, Citrobacter, Enterobacter, Escherichia, Klebsiella, Providencia, and Raoultella. Of 23 filtered samples, 18 (78%) were positive for the blaKPC gene, whereas all samples were negative for blaNDM, blaVIM, and blaOXA-48–like genes. In this nursing home, these findings suggest a concerning frequency of bacteria resistant to last-line antibiotics. Wastewater surveillance can potentially serve as an approach to identify antibiotic resistance and track its presence over time.Disclosure: None

Whole genome sequencing of multidrug resistant Enterobacterales identified in children and their household members within Siem Reap, Cambodia.

To explore the association of recent hospitalization and asymptomatic carriage of multidrug-resistant Enterobacterales (MDRE) and determine the prevailing strains and antibiotic resistance genes in Siem Reap, Cambodia using WGS. In this cross-sectional study, faecal samples were collected from two arms: a hospital-associated arm consisted of recently hospitalized children (2-14 years), with their family members; and a community-associated arm comprising children in the matching age group and their family members with no recent hospitalization. Forty-two families in each study arm were recruited, with 376 enrolled participants (169 adults and 207 children) and 290 stool specimens collected from participants. The DNA of ESBL- and carbapenemase-producing Enterobacterales cultured from the faecal samples was subject to WGS on the Illumina NovaSeq platform. Of the 290 stool specimens, 277 Escherichia coli isolates and 130 Klebsiella spp. were identified on CHROMagar ESBL and KPC plates. The DNA of 276 E. coli (one isolate failed quality control test), 89 Klebsiella pneumoniae, 40 Klebsiella quasipneumoniae and 1 Klebsiella variicola was sequenced. CTX-M-15 was the most common ESBL gene found in E. coli (n = 104, 38%), K. pneumoniae (n = 50, 56%) and K. quasipneumoniae (n = 16, 40%). The prevalence of bacterial lineages and ESBL genes was not associated with any specific arm. Our results demonstrate that MDRE is likely to be endemic within the Siem Reap community. ESBL genes, specifically blaCTX-M, can be found in almost all E. coli commensals, indicating that these genes are continuously propagated in the community through various unknown channels at present.

Occurrence of extended spectrum β-lactamases, AmpC β-lactamase and sulfonamide resistance genes in ESBL-producing Escherichia coli recovered from faecal matter of lizards

ABSTRACT The occurrence of β-lactamases and sulfonamide resistance genes in ESBL-producing E. coli from faecal matter of lizards was investigated. Isolation of ESBL-producing E. coli from lizard faecal matter was done on CHROMagar ESBL, and identification was done using molecular method. Disc diffusion method was used for the antibiotic susceptibility testing and detection of β-lactamases and sulfonamide resistance genes was done using PCR. Six ESBL-producing E. coli isolates were obtained. All the isolates obtained were multidrug resistant, showing a high level of resistance to the tested antibiotics. Five isolates carried at least one of the variants of bla CTX-M and sul2, one isolate carried sul1, while bla CMY was detected in three isolates, with two isolates carrying bla SHV and bla TEM concurrently. One isolate did not carry any of the genes. This study showed that faecal matter from lizards could be a reservoir of antibiotic resistant bacteria and resistance genes.

Comprehensive Genome and Plasmidome Analysis of Antimicrobial Resistant Bacteria in Wastewater Treatment Plant Effluent of Tokyo.

To characterize environmental antimicrobial resistance (AMR) in urban areas, extended-spectrum β-lactamase- (ESBL)/carbapenemase-producing bacteria (EPB/CPB, respectively) from urban wastewater treatment plant effluents in Tokyo were isolated on CHROMagar ESBL plate. Complete genome sequence analysis, including plasmids, indicated that 126 CTX-M-positive isolates (31%) were identified among the 404 obtained isolates. The CTX-M-9 group was predominant (n = 65, 52%), followed by the CTX-M-1 group (n = 44, 35%). Comparative genome analysis revealed that CTX-M-27-positive E. coli O16:H5-ST131-fimH41 exhibited a stable genome structure and clonal-global dissemination. Plasmidome network analysis revealed that 304 complete plasmid sequences among 85 isolates were grouped into 14 incompatibility (Inc) network communities (Co1 to Co14). Co10 consisted of primarily IncFIA/IncFIB plasmids harboring blaCTX-M in E. coli, whereas Co12 consisted primarily of IncFIA(HI1)/Inc FIB(K) plasmids harboring blaCTX-M, blaKPC, and blaGES in Klebsiella spp. Co11 was markedly located around Co10 and Co12. Co11 exhibited blaCTX-M, blaKPC, and blaNDM, and was mainly detected in E. coli and Klebsiella spp. from human and animal sources, suggesting a mutual role of Co11 in horizontal gene transfer between E. coli and Klebsiella spp. This comprehensive resistome analysis uncovers the mode of relational transfer among bacterial species, highlighting the potential source of AMR burden on public health in urban communities.

Wastewater Surveillance Detected Carbapenemase Enzymes in Clinically Relevant Gram-Negative Bacteria in Helsinki, Finland; 2011-2012.

Antimicrobial resistance profiling of pathogens helps to identify the emergence of rare or new resistance threats and prioritize possible actions to be taken against them. The analysis of wastewater (WW) can reveal the circulation of antimicrobial-resistant bacteria (ARB) and antimicrobial resistance genes (ARG) among the catchment communities. Here, we analyzed WW influent samples to determine the prevalence of carbapenemase genes-carrying Gram-negative bacteria (Carba-GNB) in Helsinki, Finland. This study set important historical reference points from the very early stage of the carbapenemase era, during the period 2011–2012. A total of 405 bacterial isolates grown on CHROMagarKPC (n = 195) and CHROMagarESBL (n = 210) from WW influent samples were collected between October 2011 and August 2012 and were analyzed. The bacterial DNA from the isolates was extracted, and the prevalence of carbapenemases genes blaKPC, blaNDM, blaGES, blaOXA-48, blaIMP, blaIMI, and blaVIM were screened with multiplexed PCR. All carbapenemase-positive isolates were identified taxonomically to species or genus level with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The nucleic acid extraction was successful for 399 isolates, of which 59 (14.8%) were found to carry carbapenemase genes. A total of 89.8% of the carbapenemase positive isolates (53 out of 59) were obtained from CHROMagarKPC plates and only 10.2% (six out of 59) were obtained from CHROMagar ESBL plates. Among the Carba-GNB isolates, 86.4% were blaGES (51 out of 59), 10.2% were blaKPC (six out of 59), and 3.4% were blaVIM (two out of 59). The most common carba-gene, blaGES, was carried by 10 different bacterial species, including Aeromonas spp., Enterobacter spp., and Kluyvera spp.; the blaKPC gene was carried by Escherichia coli, Klebsiella pneumoniae, and Kluyvera cryocescens; and the blaVIM gene was carried by Aeromonas hydrophila/caviae and Citrobacter amalonaticus. This study emphasizes that wastewater surveillance (WWS) can be an additional tool for monitoring antimicrobial resistance (AMR) at the population level.

Comprehensive Genomic Survey of Antimicrobial-Resistance Bacteria in the Sewage Tank Replacement with Hospital Relocation.

BackgroundExcrement containing antimicrobial-resistant bacteria (ARB) is discharged from the hospital sewage through wastewater treatment plants (WWTP) into rivers, increasing the antimicrobial resistance (AMR) burden on the environment.PurposeWe illustrate the contamination of hospital sewage tanks with ARB harboring antimicrobial resistance genes (ARGs) using comprehensive metagenomic sequencing. During the study period, we moved to a new hospital building constructed for renovation. Therefore, we investigated the difference in bacterial flora in the sewage tanks for each building with different departments, and the change in bacterial flora over time in new sewage tanks. Furthermore, we performed a comparative genome analysis of extended spectrum β-lactamase (ESBL)-producing organisms (EPOs) from hospital sewage and clinical samples. Residual antibiotics in the sewage tank were also measured.MethodsMetagenomic analysis was performed on the hospital sewage samples, followed by whole genome sequencing of EPOs.ResultsThe bacterial composition of new sewage tanks was comparable with that of old tanks within 1 month after relocation and was instantly affected by excrement. The bacterial composition of sewage tanks in the old and new buildings, containing rooms where seriously ill patients were treated, was similar. Selection on CHROMagar ESBL allowed detection of EPOs harboring blaCTX-M and carbapenemase genes in all sewage tanks. One of the sewage Escherichia coli strain comprising ST393 harboring blaCTX-M-27 corresponded to the clinical isolates based on core genome analysis. Moreover, the levels of levofloxacin and clarithromycin in the hospital sewage were 0.0325 and 0.0135 µg/mL, respectively.ConclusionHospital sewage was contaminated with many ARB species, ARGs and residual antibiotics, which can cause a burden on WWTP sewage treatment. The bacterial flora in the sewage tank was rapidly affected, especially by the ward with seriously ill patients. AMR monitoring of hospital sewage may help detect carriers prior to nosocomial ARB-associated outbreaks and control the outbreaks.

Isolation of Human Lineage, Fluoroquinolone-Resistant and Extended-β-Lactamase-Producing Escherichia coli Isolates from Companion Animals in Japan.

An increase in human and veterinary fluoroquinolone-resistant Escherichia coli is a global concern. In this study, we isolated fluoroquinolone-resistant E. coli isolates from companion animals and characterized them using molecular epidemiological analysis, multiplex polymerase chain reaction to detect E. coli ST131 and CTX-M type extended-spectrum β-lactamases (ESBL), and multi-locus sequence typing analysis. Using plain-CHROMagar ECC, 101 E. coli isolates were isolated from 34 rectal swabs of dogs and cats. The prevalence of resistance to fluoroquinolone and cefotaxime was 27.7% and 24.8%, respectively. The prevalence of fluoroquinolone-resistant isolates (89.3%) was higher when CHROMagar ECC with CHROMagar ESBL supplement was used for E. coli isolation. The prevalence of cefotaxime resistance was also higher (76.1%) when 1 mg/L of ciprofloxacin-containing CHROMagar ECC was used for isolation. The cefotaxime-resistant isolates possessed CTX-M type β-lactamase genes (CTX-M-14, CTX-M-15, or CTX-M-27). Seventy-five percent of fluoroquinolone-resistant isolates were sequence types ST131, ST10, ST1193, ST38, or ST648, which are associated with extensive spread in human clinical settings. In addition, we isolated three common fluoroquinolone-resistant E. coli lineages (ST131 clade C1-M-27, C1-nM27 and ST2380) from dogs and their respective owners. These observations suggest that companion animals can harbor fluoroquinolone-resistant and/or ESBL-producing E. coli, in their rectums, and that transmission of these isolates to their owners can occur.

Antimicrobial susceptibility testing profiles of ESBL-producing Enterobacterales isolated from hospital and community adult patients in Blantyre, Malawi

ObjectiveThere is a paucity of data on antimicrobial resistance (AMR) in Malawi. Here we present a study of AMR of extended-spectrum β-lactamases-producing Enterobacterales (ESBL-E) isolated from hospital and community settings in Blantyre, Malawi. Design and MethodsA cross-sectional study was conducted between March and November 2020, involving 403 adult participants aged ≥18 years. Screening for ESBL-E was performed using CHROMagar ESBL medium. Production of ESBLs was confirmed by a combination disk test method. Antimicrobial susceptibility was tested using the agar disk diffusion method in accordance with the Clinical Laboratory Standards Institute's 2019 guidelines. ResultsThe mean resistance rate of ESBL-E to antimicrobial agents tested was 49.2% (range from 1.4%–92%). The highest resistance rates were observed for trimethoprim-sulfamethoxazole (92%), amoxicillin and ceftriaxone (79%), doxycycline (75%) and gentamicin (72%). Carbapenems (meropenem and imipenem) were highly active against isolates. The overall rate of multi-drug resistant (MDR) ESBL-E was 47%. The highest MDR was found in Yersinia enterocolitica (51%) and the least in Serratia spp. (40%). ConclusionsWe found a high resistance rate of ESBL-E isolates to antimicrobial agents; the majority were MDR. Surveillance systems are recommended to monitor AMR in Malawi.

Escherichia coli and Serratia fonticola ESBLs as a potential source of antibiotics resistance dissemination in the Tricity water reservoirs.

Despite the fact that cephalosporins are rarely used in medical or veterinary treatment, the presence of Enterobacterales strains resistant to this group of anti-bacterial drugs (ESBL) is an important issue that requires attention. Between 2019 and 2021, 14 retention reservoirs, 12 streams, 3 rivers and 1 lake situated in the Tricity area (in northern Poland) were sampled for the presence of ESBL strains. Out of 40 water samples, characteristic growth (E. coli and the KESC group) on Chromagar ESBL plates was observed for 33 samples. The average number of ESBL E. coli was 42±132 CFU/100 ml, while the KESC group was 73±147 CFU/100 ml. Out of 33 positive samples, 57 ESBL Enterobacterales strains were isolated, of which the most abundant species were E. coli (13 isolates) and S. fonticola (23 isolates). The E. coli ESBL isolates not only showed resistance to third generation cephalosporins but also to antibiotics from other groups, such as fluoroquinolones, aminoglycosides and sulfonamides. The S. fonticola ESBL isolates were also found to be mainly resistant to the third generation cephalosporins, with the exception of 5 imipenem and 2 ertapenem-resistant strains. These strains presented highly diverse fingerprinting profiles, as well as significant differences in phenotypic traits helpful for survival in the environment, such as biofilm formation and motility. Moreover, biofilm formation and the swimming ability were species and temperature dependent. We confirmed the presence of highly diverse ESBL strains with multiple drug resistance patterns in the Tricity water reservoirs. This could possibly pose a threat to human health and create a suitable ground for acquiring antibiotics resistance in the natural environment.

Comparison of the novel Uroquattro HB&L™ system and classical phenotypic method for rapid screening of multidrug-resistant organism colonization at the University Hospital Centre Split, Croatia

Background. Infections caused by multidrug-resistant organisms (MDRO) are difficult to treat and associated with poor outcomes for patients. Therefore, early identification and management of colonization are essential as first steps in infection prevention. Culture-based methods have been widely used for MDRO screening. The turnaround time (TAT) for the identification of carriers varies between 48-72 h with this method. The aim of our study was to compare the performance of the new rapid semiautomatic method for detection of MDRO (HB&L Uroquattro, Alifax) with standard cultivation on selective media. Methods. Twenty-one axillary, 20 nose and 19 rectal swabs were taken in duplicate on two selected days at the University Hospital Centre Split, Croatia. Swabs were cultivated and MDRO isolates were identified on selective media (Chromagar MRSA and Chromagar ESBL) according to the standard operating procedure. Novel Alifax method was performed according to manufacturer’s instructions. Results. TAT for the new method was significantly lower (6.5 h) in comparison to the classical method. With classical method, 10 extended spectrum ß-lactamases (ESBL) producing strains from 10 different rectal specimens were isolated on Chromagar ESBL media. Exactly the same specimens were positive for the presence of ESBL-producing bacteria by rapid Alifax method. On selective Chromagar MRSA media, 11 MDRO were isolated, while rapid method detected 11 MDRO from the same specimens. Conclusions. High concordance of positive and negative results obtained with classical and rapid method is encouraging. However, our study was performed on a small sample size and further research with larger sample size is needed.

Extended-Spectrum β-Lactamase-Producing Enterobacterales Shedding by Dogs and Cats Hospitalized in an Emergency and Critical Care Department of a Veterinary Teaching Hospital.

Extended-spectrum β-lactamase-producing Enterobacterales (ESBL-PE) gut shedding in human medicine is considered as a major reservoir for ESBL-associated infections in high risk patients. In veterinary medicine, data regarding ESBL-PE gut shedding on admission to emergency and critical care department is scarce. We aimed to determine ESBL-PE shedding rates by dogs and cats in this setting and to determine the risk factors for shedding, at two separate periods, three-years apart. Rectal swabs were collected from animals, on admission and 72 h post admission, enriched and plated on Chromagar ESBL plates, followed by bacterial identification. ESBL phenotype was confirmed and antibiotic susceptibility profiles were determined (Vitek 2). Medical records were reviewed for risk factor analysis (SPSS). Overall, 248 animals were sampled, including 108 animals on period I (2015–2016) and 140 animals on period II (2019). In both periods combined, 21.4% of animals shed ESBL-PE on admission, and shedding rates increased significantly during hospitalization (53.7%, p-value < 0.001). The main ESBL-PE species were Escherichia coli and Klebsiella pneumoniae, accounting for more than 85% of the isolates. In a multivariable analysis, previous hospitalization was a risk factor for ESBL-PE gut shedding (p-value = 0.01, Odds ratio = 3.05, 95% Confidence interval 1.28–7.27). Our findings demonstrate significant ESBL-PE gut shedding among small animals in the emergency and critical care department, posing the necessity to design and implement control measures to prevent transmission and optimize antibiotic therapy in this setting.

AMR Escherichia coli and its temporal and spatial variability within the aquatic environment

Phenotypic and genotypic identification methods have been used to determine the temporal and spatial dynamics of AMR-Escherichia coli in a mainly rural watercourse that receives WWTP-effluent compared to a parallel river which does not. We aimed to investigate the incidence of plasmid-mediated mcr-1and β-lactamase-genes in E. coli recovered from both water and Asellus aquaticus samples throughout two-calendar-years. Samples of the water and A. aquaticus were recovered from the relevant locations each month. CHROMagar ESBL agar was used throughout to isolate and identify ESBL-E. coli. The presence of AMR-genes was confirmed using the ‘BSAC’ antibiotic-disk-synergy method and PCR analysis to confirm the presence of mcr-1and ESBL-genes. The CHROMagar ESBL agar was found to be 99.7% (n=578) accurate when confirmed with a PCR analysis of the ESBL-genes. Seventy-six-point-six percent (n=449) of the isolated ESBL-E. coli were correctly identified as ESBL-producing organisms using the ‘BSAC’ method. Interestingly 61.9% (n=358) of the ESBL-E. coli were also found to carry the mcr-1 gene. Our data shows that AMR levels were highest at the WWTP-effluent throughout the two-years for both water and A. aquaticus samples. The incidence of AMR-E. coli 1km downstream of the effluent discharge was equivalent to the parallel river sites, suggesting that the dispersal of AMR from the WWTP-effluent is limited, although AMR-E. coli were found in relatively high numbers at the WWTP-effluent. We argue that the presence of AMR in the freshwater invertebrate A. aquaticus could represent an important route by which AMR can spread from the aquatic environment to the terrestrial environment.

Molecular Characterization of Extended Spectrum Beta-lactamase from &lt;i&gt;Enterobacter cloacae, E. coli&lt;/i&gt; and &lt;i&gt;Klebsiella pneumoniae&lt;/i&gt; from Pregnant Women in South-south Nigeria

Extended spectrum beta-lactamase prevalence is still on the increase across the world and has been implicated in urinary tract infections in Nigeria. TEM, SHV and CTX-M are becoming more common with CTX-M-15 becoming more significant as it is associated with complicated urinary tract infections. This study was conducted to evaluate distribution of blaTEM, blaCTX-M-15 and blaSHV genes among Enterobacter cloacae, E. coli and Klebsiella pneumoniae. A total of 660 urine samples were collected from pregnant women in 3 general hospitals from Akwa Ibom state. Enterobacter cloacae, E. coli and Klebsiella pneumoniae were identified using Microbact 24E. The disc diffusion and combined discs methods were used for testing antimicrobial susceptibility. The presence of ESBL was detected using Double Disk Synergy Test (DDST) and CHROMagar ESBL, respectively. Plasmid extraction was carried out following the protocol of ZR Plasmid Miniprep-Classic extraction kit. The blaCTX-M-15, blaTEM and blaSHV was identified by PCR with specific primers in selected 50 ESBL producing isolates. A total of 252 clinical isolates were collected from three General Hospitals in South-South, Nigeria. ESBLs were found in 231 (92%) isolates. blaCTX-M-15 was the commonest genotype (84%), followed by blaSHV (60%) and blaTEM (60%). ESBL positive strains of E. cloacae, E. coli and K pneumoniae are increasingly found in isolates from pregnant women. The current study demonstrated the predominance of gene encoding blaCTX-M-15 with a percentage frequency of (84%) commonly with plasmid in the setting.

Evaluation of CHROMagar ESBL and Double Disk Synergy Test (DDST) for Screening of Extended Spectrum Beta-lactamase Producing Uropathogens in South-South Nigeria

Background/Purpose: The aim of this study was to evaluate the effectiveness of CHROMagar ESBL in comparison with Double Disc Synergy Test (DDST) for the detection of ESBL producing uropathogens.&#x0D; Methods: Six hundred and sixty urine samples were collected from pregnant women attending antenatal at General hospital Ikot Ekpene, Eket and Oron. Two hundred and fifty eight isolates were obtained while two hundred and thirty one isolates were ESBL producers. Microbact 24E(Oxoid, UK) was used in the identification of bacterial isolates, antibiotic susceptibility test was done using Kirby-Bauer disk diffusion method following CLSI guidelines using commercially available disc (Oxoid Ltd). Double disk synergy test was carried out on the isolates and inoculation was done using CHROMagar ESBL (France).&#x0D; Results: The prevalence of ESBL 35% was recorded. The sensitivity and specificity of DDST was 88% and 89%, respectively. CHROMagar showed an increase in sensitivity and specificity at 48 h with 98% and 99.0%, respectively. 80% of the ESBL producing isolates were multi drugs resistant. The predominant bacterial pathogens were Enterobacter cloacae (23%), Proteus mirabilis (14%) and Acinetobacter baumanii (13.4%).The ESBL producing isolates showed maximum resistance against Ceftazidime (90%), Cefotaxime (91%), Azetronam (95%), Amikacin (68.2%) followed by ofloxacin (70%) while maximum sensitivity was seen for imipenem (90%) and Augumentin (80%). The study demonstrated that CHROMagar was superior and more sensitive than DDST.&#x0D; Conclusion: CHROMagar ESBL seems to be the most reliable method among phenotypic methods for detection of ESBL in the absence of PCR.

Clonal Spread of Extended-Spectrum Cephalosporin-Resistant Enterobacteriaceae Between Companion Animals and Humans in South Korea

Extended-spectrum cephalosporin (ESC)-resistant Enterobacteriaceae is an increasingly important problem in both human and veterinary medicine. The aims of this study were to describe a comparative molecular characterization of Enterobacteriaceae carrying ESC resistance genes, encoding extended-spectrum β-lactamase (ESBL) and AmpC, isolated from human stool samples, rectal swabs from companion animals, and swabs from the environment of veterinarian hospitals in South Korea, and to examine their possible dissemination and transmission. The ESC resistance genes were identified by PCR and sequencing. Isolates with the predominant ESC resistance genes were assessed for their genetic relatedness by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing. A total of 195 Escherichia coli and 41 Klebsiella pneumoniae isolates that exhibited ESC resistance were recovered on CHROMagar ESBL from human, companion animal, and the veterinary hospital environmental samples. In companion animals, most of the ESC resistance genes were blaCMY–2–like (26.4%), followed by blaCTX –M–55 (17.2%) and blaCTX–M–14 (16.1%), whereas blaCTX–M–15 (28.6%) was predominant in human samples. The epidemiological relatedness of isolates carrying ESC resistance genes, including 124 E. coli and 23 K. pneumoniae isolates carrying CMY-2-like, DHA-1-like, or/and CTX-M-type, were analyzed by PFGE. The pulsotypes of five E. coli isolates (three from dogs and two from humans) carrying blaCMY–2–like, which were attributed to sequence type 405, from different veterinary clinics showed >85% similarity. Our results indicate direct transmission and dissemination of ESC-resistant Enterobacteriaceae between humans and companion animals.