Phytochemical Evaluation of Terminalia canescens DC. Radlk. Extracts with Antibacterial and Antibiotic Potentiation Activities against Selected ß-Lactam Drug-Resistant Bacteria.

Terminalia canescens DC. Radlk. (family: Combretaceae) is native to northern Australia. Species of the genus Terminalia are widely used as traditional medicines to treat diverse ailments, including bacterial infections. However, we were unable to find any studies that had examined the antimicrobial activity of T. canescens. In this study, T. canescens was screened against a panel of bacterial pathogens, including multi-antibiotic-resistant strains. Solvents with different polarities were used to extract different complements of phytochemicals from T. canescens leaves. Methanolic and aqueous extracts exhibited substantial antimicrobial activity against various pathogens, including those that are multidrug-resistant strains. When combined with some selected clinical antibiotics, some extracts potentiated the antibacterial inhibitory activity. This study identified two synergistic, eleven additive, eleven non-interactive and eight antagonistic interactions. The toxicities of the plant extracts were examined in the Artemia franciscana nauplii assay and were found to be non-toxic, except the aqueous extract, which showed toxicity. Metabolomic liquid chromatography-mass spectrometry (LC-MS) analyses highlighted and identified several flavonoids, including vitexin, quercetin, orientin and kaempferol, as well as the tannins ellagic acid and pyrogallol, which may contribute to the antibacterial activities observed herein. The possible mechanism of action of these extracts was further explored in this study.

Occurrence and molecular characteristics of antimicrobial resistance, virulence factors, and extended-spectrum ß-lactamase (ESBL) producing Salmonella enterica and Escherichia coli isolated from the retail produce commodities in Bangkok, Thailand.

The incidence of antimicrobial resistance (AMR) in the environment is often overlooked and leads to serious health threats under the One Health paradigm. Infection with extended-spectrum ß-lactamase (ESBL) producing bacteria in humans and animals has been widely examined, with the mode of transmission routes such as food, water, and contact with a contaminated environment. The purpose of this study was to determine the occurrence and molecular characteristics of resistant Salmonella enterica (S. enterica) (n = 59) and Escherichia coli (E. coli) (n = 392) isolated from produce commodities collected from fresh markets and supermarkets in Bangkok, Thailand. In this study, the S. enterica isolates exhibited the highest prevalence of resistance to tetracycline (11.9%) and streptomycin (8.5%), while the E. coli isolates were predominantly resistant to tetracycline (22.5%), ampicillin (21.4%), and sulfamethoxazole (11.5%). Among isolates of S. enterica (6.8%) and E. coli (15.3%) were determined as multidrug resistant (MDR). The prevalence of ESBL-producing isolates was 5.1% and 1.0% in S. enterica and E. coli, respectively. A minority of S. enterica isolates, where a single isolate exclusively carried blaCTX-M-55 (n = 1), and another isolate harbored both blaCTX-M-55 and blaTEM-1 (n = 1); similarly, a minority of E. coli isolates contained blaCTX-M-55 (n = 2) and blaCTX-M-15 (n = 1). QnrS (11.9%) and blaTEM (20.2%) were the most common resistant genes found in S. enterica and E. coli, respectively. Nine isolates resistant to ciprofloxacin contained point mutations in gyrA and parC. In addition, the odds of resistance to tetracycline among isolates of S. enterica were positively associated with the co-occurrence of ampicillin resistance and the presence of tetB (P = 0.001), while the E. coli isolates were positively associated with ampicillin resistance, streptomycin resistance, and the presence of tetA (P < 0.0001) in this study. In summary, these findings demonstrate that fresh vegetables and fruits, such as cucumbers and tomatoes, can serve as an important source of foodborne AMR S. enterica and E. coli in the greater Bangkok area, especially given the popularity of these fresh commodities in Thai cuisine.

High frequency of antimicrobial resistance in Salmonella and Escherichia coli causing diarrheal diseases at the Yirimadio community health facility, Mali.

BACKGROUND: Diarrhoea is a public health problem, especially in developing countries where it is the second leading cause of child mortality. In Low Income Countries like in Mali, self-medication and inappropriate use of antibiotics due to the scarcity of complementary diagnostic systems can lead to the development of multidrug-resistant bacteria causing diarrhoea. The objective of this work was to determine the microorganisms responsible for diarrhoea in children under 15 years of age and to characterize their sensitivity to a panel of antibiotics used in a peri-urban community in Mali. The study involved outpatient children visiting the Yirimadio Community Health Centre and diagnosed with diarrhoea. Stool samples from those patients were collected and analysed by conventional stools culture and the susceptibility to antibiotics of detected bacteria was determined by the disc diffusion method in an agar medium. RESULT: Overall, 554 patients were included. Children under the age of 3 years accounted for 88.8% (492 of 554) of our study population. Two bacterial species were isolated in this study, Escherichia coli 31.8% (176 of 554) and Salmonella 2.9% (16 of 554). In the 176, E. coli strains resistance to amoxicillin and to cotrimoxazole was seen in 93.8% (165 of 176) and 92.6% ( 163 of 176), respectively. The ESBL resistance phenotype accounted for 39,8% (70 of 176) of E. coli. Sixteen (16) strains of Salmonella were found, of which one strain (6.3%) was resistant to amoxicillin and to amoxicillin + clavulanic acid. Another one was resistant to chloramphenicol (6.3%). Two strains of Salmonella were resistant to cotrimoxazole (12.5%) and two others were resistant to cefoxitin (12.5%). CONCLUSIONS: The data suggest that E. coli is frequently involved in diarrhoea in children under 3 years of age in this peri-urban setting of Bamako, Mali, with a high rate of resistance to amoxicillin and cotrimoxazole, the most widely used antibiotics in the management of diarrhoea in this setting.

Comparison of three doses of amikacin on alternate days with a daily dose of meropenem during the same period for the treatment of urinary tract infection with E. coli: a double-blind clinical trial.

OBJECTIVES: Urinary tract infections (UTIs) are very common infections in humans, and Escherichia coli (E. coli) is the commonest pathogen leading to UTIs. The generation of beta-lactamase enzymes in this bacterium results in its resistance against many antibiotics. This study compares three doses of amikacin on alternate days with a daily dose of meropenem in the same period for the treatment of UTIs with E. coli in a double-blind clinical trial. METHODS: The current double-blind clinical trial compares three doses of amikacin on alternate days with a daily dose of meropenem in the same period for the treatment of UTIs with E. coli. The patients were assigned to two groups: Intervention (receiving a single dose of amikacin once a day at 48-h intervals for a week, three doses) and control (receiving meropenem for 1/TDS for a week). RESULTS: The E. coli infection frequency was 61 (21 cases of non-ESBL and 40 cases of ESBL-positive infections) and the frequency of the other infections was 52 (46%). In the patients with ESBL E. coli infection, ciprofloxacin (21; 70%) showed the highest antibiotic resistance, and nitrofurantoin (33; 91.7%) showed the highest sensitivity. The baseline variables between the control and intervention groups indicated no significant difference (p > 0.05). The frequency of signs and symptoms showed no significant difference between the amikacin and meropenem groups in the first 24 h and the first week. In the second week of follow-up, no clinical signs or symptoms were observed in the two groups. CONCLUSION: The results of this study showed that treatment with amikacin, 1 g q48h, for one week (three doses) has the same result as meropenem, 1 g q8h, for one week (21 doses). The results are the same for the treatment of UTIs with ESBL positive and ESBL negative. Amikacin can be used once every 48 h to treat UTIs, is less expensive and can be administered on an outpatient basis. TRIAL REGISTRATION: This study was registered in the Iranian Registry of Clinical Trials (IRCT) with ID number: IRCT20170417033483N2 on the date 2018-02-13.

Comparative analysis of a rapid diagnostic test and scoring tools for ESBL detection in Enterobacterales bloodstream infections for optimizing antimicrobial therapy.

IMPORTANCE: Our study addresses a significant issue in the medical and scientific community-the delayed administration of appropriate antimicrobial treatments due to the time-consuming process of phenotypic susceptibility data collection in gram-negative bloodstream infections. Our research indicates that a multiplex PCR rapid diagnostic test (RDT) significantly outperformed two clinical scoring tools in predicting ceftriaxone susceptibility. Multiplex PCR also led to reduced instances of undertreatment with ceftriaxone and minimized overtreatment with carbapenems. Furthermore, multiplex PCR demonstrated high sensitivity and specificity in predicting ceftriaxone susceptibility. The results of our study underscore the potential RDTs to reduce the time to appropriate antimicrobial therapy, leading to improved patient outcomes and reduced healthcare costs.

Terminalia petiolaris A.Cunn ex Benth. Extracts Have Antibacterial Activity and Potentiate Conventional Antibiotics against ß-Lactam-Drug-Resistant Bacteria.

Terminalia petiolaris A. Cunn. Ex Benth. (genus: Terminalia, family: Combretaceae) is native to Australia. Terminalia spp. have traditionally been used to treat various ailments, including bacterial infections. Solvents of varying polarity were used to extract compounds from leaves of this species, and the extracts were tested against a panel of bacteria, including antibiotic-resistant strains. The methanolic and water extracts showed substantial inhibitory activity against several bacteria, including antibiotic-resistant strains in both disc diffusion and liquid dilution assays. Combining these extracts with selected conventional antibiotics enhanced the inhibition of bacterial growth for some combinations, while others showed no significant interaction. In total, two synergistic, twenty-five additive, twenty-three non-interactive and one antagonistic interaction were observed. The methanolic and ethyl acetate plant extracts were found to be non-toxic in Artemia franciscana nauplii toxicity assays. A liquid chromatography-mass spectrometry metabolomics analysis identified several flavonoid compounds, including miquelianin, trifolin and orientin, which might contribute to the observed activities. The potential modes of these active extracts are further discussed in this study.

Resistance and Virulence Surveillance in Escherichia coli Isolated from Commercial Meat Samples: A One Health Approach.

Escherichia coli is a key indicator of food hygiene, and its monitoring in meat samples points to the potential presence of antimicrobial-resistant strains capable of causing infections in humans, encompassing resistance profiles categorized as serious threats by the Centers for Disease Control and Prevention (CDC), such as Extended-Spectrum Beta-Lactamase (ESBL)-a problem with consequences for animal, human, and environmental health. The objective of the present work was to isolate and characterize ESBL-producing E. coli strains from poultry, pork, and beef meat samples, with a characterization of their virulence and antimicrobial resistance profiles. A total of 450 meat samples (150 chicken, 150 beef, and 150 pork) were obtained from supermarkets and subsequently cultured in medium supplemented with cefotaxime. The isolated colonies were characterized biochemically, followed by antibiogram testing using the disk diffusion technique. Further classification involved biofilm formation and the presence of antimicrobial resistance genes (blaCTX-M, AmpC-type, mcr-1, and fosA3), and virulence genes (eaeA, st, bfpA, lt, stx1, stx2, aggR, iss, ompT, hlyF, iutA, iroN, fyuA, cvaC, and hylA). Statistical analysis was performed via the likelihood-ratio test. In total, 168 strains were obtained, with 73% originating from chicken, 22% from pork, and 17% from beef samples. Notably, strains exhibited greater resistance to tetracycline (51%), ciprofloxacin (46%), and fosfomycin (38%), apart from ß-lactams. The detection of antimicrobial resistance in food-isolated strains is noteworthy, underscoring the significance of antimicrobial resistance as a global concern. More than 90% of the strains were biofilm producers, and strains carrying many ExPEC genes were more likely to be biofilm formers (OR 2.42), which increases the problem since the microorganisms have a greater chance of environment persistence and genetic exchange. Regarding molecular characterization, bovine samples showed a higher prevalence of blaCTX-M-1 (OR 6.52), while chicken strains were more likely to carry the fosA3 gene (OR 2.43, CI 1.17-5.05) and presented between 6 to 8 ExPEC genes (OR 2.5, CI 1.33-5.01) compared to other meat samples. Concerning diarrheagenic E. coli genes, two strains harbored eae. It is important to highlight these strains, as they exhibited both biofilm-forming capacities and multidrug resistance (MDR), potentially enabling colonization in diverse environments and causing infections. In conclusion, this study underscores the presence of ß-lactamase-producing E. coli strains, mainly in poultry samples, compared to beef and pork samples. Furthermore, all meat sample strains exhibited many virulence-associated extraintestinal genes, with some strains harboring diarrheagenic E. coli (DEC) genes.

Prevalence and characterization of ESBL-producing Escherichia coli in healthy pregnant women and hospital environments in Benin: an approach based on Tricycle.

Introduction: Extended-Spectrum Beta-Lactamase (ESBL)-producing Enterobacterales are recognized as significant pathogens due to their resistance to multiple antibiotics. This study aimed to determine the prevalence of ESBL-producing Escherichia coli (E. coli) in different settings, including healthy pregnant women, the food chain, and the environment of tertiary hospitals in Benin. Methods: Samples were collected from various sources, including fecal samples from healthy pregnant women, food samples from hospital canteens, and hospital effluents from four tertiary hospitals in southern Benin. Fecal samples were plated on MacConkey agar supplemented with cefotaxime (4 µg/mL), while food and water samples were plated on Tryptone Bile X agar supplemented with cefotaxime (4 µg/mL). Urea indole tests were used for preliminary identification of E. coli colonies, followed by confirmation of ESBL production using the double disk synergy technique. Antibiotic susceptibility testing of ESBL-producing E. coli strains was conducted using the disk diffusion method on MH agar. Polymerase Chain Reaction (PCR) was used to investigate the presence of ESBL-encoding genes. Results: Among the 296 fecal samples collected from four tertiary hospitals, ESBL-producing E. coli was isolated from 22.30% (66) of the samples. All E. coli isolates from hospital effluents exhibited ESBL production, while ESBL-producing E. coli was not detected in food and drinking water samples. The analysis of variable associations showed no significant associations (p > 0.05) for the studied factors. Antibiotic susceptibility testing revealed high resistance rates among the ESBL-Ec isolates against several tested antibiotics, including amoxicillin, aztreonam, ceftriaxone, ciprofloxacin, and trimethoprim-sulfamethoxazole. However, most isolates remained susceptible to ertapenem, amoxicillin-clavulanate, and imipenem. The most prevalent ESBL-encoding genes were blaTEM (37.50%), blaOXA-1 (19.44%), and blaSHV (11.11%), while a smaller proportion of isolates carried blaCTXM-1/blaCTXM-15 (5.55%) and blaCTXM-9. Discussion: This study provides insights into the prevalence of ESBL-producing E. coli carriage in the feces of healthy pregnant women in southern Benin. Additionally, it highlights hospital wastewater as a potential reservoir of ESBL-producing bacteria in the environment. The detection of ESBL-producing E. coli in hospital effluents raises concerns about the dissemination of antibiotic resistance genes into the environment. The high resistance rates observed among ESBL-Ec isolates against commonly used antibiotics emphasize the urgent need for antimicrobial stewardship and infection control measures. The identification of prevalent ESBL-encoding genes contributes to understanding the genetic basis of ESBL resistance in the studied population. Further research is warranted to explore the mechanisms of transmission and potential interventions to mitigate the spread of ESBL-producing Enterobacterales.

Detection of blaCTX-M and blaDHA genes in stool samples of healthy people: comparison of culture- and shotgun metagenomic-based approaches.

We implemented culture- and shotgun metagenomic sequencing (SMS)-based methods to assess the gut colonization with extended-spectrum cephalosporin-resistant Enterobacterales (ESC-R-Ent) in 42 volunteers. Both methods were performed using native and pre-enriched (broth supplemented with cefuroxime) stools. Native culture screening on CHROMID® ESBL plates resulted in 17 positive samples, whereas the pre-enriched culture (gold-standard) identified 23 carriers. Overall, 26 ESC-R-Ent strains (24 Escherichia coli) were identified: 25 CTX-M and 3 DHA-1 producers (2 co-producing CTX-Ms). Using the SMS on native stool ("native SMS") with thresholds ≥60% for both identity and coverage, only 7 of the 23 pre-enriched culture-positive samples resulted positive for blaCTX-M/blaDHA genes (native SMS reads mapping to blaCTX-M/blaDHAs identified in gold-standard: sensitivity, 59.0%; specificity 100%). Moreover, an average of 31.5 and 24.6 antimicrobial resistance genes (ARGs) were detected in the 23 pre-enriched culture-positive and the 19 negative samples, respectively. When the pre-enriched SMS was implemented, more blaCTX-M/blaDHA genes were detected than in the native assay, including in stools that were pre-enriched culture-negative (pre-enriched SMS reads mapping to blaCTX-M/blaDHAs identified in gold-standard: sensitivity, 78.3%; specificity 75.0%). In addition, the pre-enriched SMS identified on average 38.6 ARGs/sample, whereas for the corresponding native SMS it was 29.4 ARGs/sample. Notably, stools resulting false-negative by using the native SMS had lower concentrations of ESC-R-Ent (average: ~105 vs. ~107 CFU/g) and E. coli classified reads (average: 193,959 vs. 1.45 million) than those of native SMS positive samples. Finally, the detection of blaCTX-M/blaDHA genes was compared with two well-established bioinformatic tools. In conclusion, only the pre-enriched SMS assured detection of most carriers of ESC-R-Ent. However, its performance was not comparable to the pre-enriched culture-based approach.

Hamamelis virginiana L. Leaf Extracts Inhibit the Growth of Antibiotic-Resistant Gram-Positive and Gram-Negative Bacteria.

Virginian witch hazel (WH; Hamamelis virginiana L.; family: Hamamelidaceae) is a North American plant that is used traditionally to treat a variety of ailments, including bacterial infections. Solvents of varying polarity (water, methanol, ethyl acetate, hexane and chloroform) were used to prepare extracts from this plant. Resuspensions of each extract in an aqueous solution were tested for growth-inhibitory activity against a panel of bacteria (including three antibiotic-resistant strains) using agar disc diffusion and broth microdilution assays. The ethyl acetate, hexane and chloroform extracts were completely ineffective. However, the water and methanolic extracts were good inhibitors of E. coli, ESBL E. coli, S. aureus, MRSA, K. pneumoniae and ESBL K. pneumoniae growth, with the methanolic extract generally displaying substantially greater potency than the other extracts. Combining the active extracts with selected conventional antibiotics potentiated the bacterial growth inhibition of some combinations, whilst other combinations remained non-interactive. No synergistic or antagonistic interactions were observed for any WH extracts/antibiotic combinations. Gas chromatography-mass spectrometry analysis of the extracts identified three molecules of interest that may contribute to the activities observed, including phthalane and two 1,3-dioxolane compounds. Putative modes of action of the active WH extracts and these molecules of interest are discussed herein.

Effects of Melaleuca alternifolia Chell (Tea Tree) and Eucalyptus globulus Labill. Essential Oils on Antibiotic-Resistant Bacterial Biofilms.

In the present investigation, the anti-biofilm potential of two essential oils (EOs), Melaleuca alternifolia Chell (Tea-Tree) (TTO) and Eucalyptus globulus Labill. (EEO) was characterized and tested "in vitro" against both mature biofilms and biofilms in the process of formation, produced by strains belonging to three main categories of antibiotic resistant bacteria (ARB): Vancomycin-resistant enterococci (VRE), methicillin-resistant Staphylococcus aureus (MRSA) and broad-spectrum ß-lactamase-producing Escherichia coli (ESBL). The study was carried out in 96-well microtiter-plates using EOs alone, in association with each other and in combination with antibiotics against both single and multi-species biofilm. The study demonstrated the ability of TTO and EEO to counteract the ARB strains in sessile form, with promising results in particular against the biofilm in formation. Mature biofilm by ESBL E. coli was the most sensitive in the results from the quantification study of viable cells performed in multi-species biofilms. Lastly, in all tests, carried out using TTO/EEO associations and EOs/antibiotic combinations, the synergistic effect which emerged from the FIC-index has been confirmed, and both the reduction of biofilm in formation, and the removal of mature structure was obtained at very low concentrations, with values from 4 to >512-fold lower than the minimum inhibitory concentration (MIC) of the single compounds.

Clinical and cost-effectiveness analysis of carbapenems versus ciprofloxacin in the treatment of urinary tract infections due to extended spectrum ß-lactamase-producing Enterobacterales.

BACKGROUND: Urinary tract infections (UTIs) are the most common infectious diseases in both hospital and community settings. The management of UTIs caused by extended spectrum ß-lactamase-producing Enterobacterales (ESBL-PE) has become more complicated given the limited options of effective antibiotic agents besides the amplification of total healthcare costs. METHODS: This was a retrospective cohort study conducted among hospitalized patients between January 2018 and March 2020. Adults diagnosed with UTI due to ESBL-PE with at least 2 days of admission were included. Excluded were patients with concomitant infection, polymicrobial UTI, and pregnant women. The primary endpoints were clinical cure and incremental cost-effectiveness ratio (ICER). Clinical cure, hospitalization, and antibiotics costs were considered to evaluate ICER. The secondary endpoints included microbiological eradication, length of stay (LOS), and 30-day readmission. RESULTS: Of 102 patients, 89 received a carbapenem and 13 received ciprofloxacin. The patients had similar baseline characteristics, including history of hospitalization and UTI within 3 months. No difference was observed in clinical cure rates (86.5% vs. 100%, P = 0.159), microbiological eradication (93.1% vs. 100%, P = 0.639), median LOS (6 days in both groups, P = 0.773), and 30-day readmission rates (41.6% vs. 46.2%, P = 0.755). The ICER of carbapenem to ciprofloxacin was - 7,626.05, indicating that ciprofloxacin was more cost-effective compared with carbapenems. CONCLUSION: Ciprofloxacin had comparable cure rates with carbapenems, lower risk of 30-day readmission, and was more cost-effective for the treatment of UTI due to ESBL-PE. Therefore, it should be considered as a valuable option if ESBL-PE showed susceptibility to it.

Metagenomic Analysis of Urban Wastewater Treatment Plant Effluents in Tokyo.

Purpose: Urban wastewater treatment plant (WWTP) effluents, even with proper treatment, may cause antimicrobial resistance (AMR) burden, with a high frequency of acquired antimicrobial resistance genes (ARGs). The dissemination of ARGs into the environment increases the risk of infectious diseases; however, there is little direct evidence regarding their epidemiological effects. This study aimed to assess effluents from urban WWTPs around the Tama River and Tokyo Bay using metagenomic analysis of (AMR) genes (ARGs) and heavy-metal resistance genes. Methods: Metagenomic DNA-seq analysis of water samples and resistome analysis were performed. Results: The most prevalent ARG was the sulfonamide resistance gene, sul1, followed by the quaternary ammonium compound resistance gene, qacE, suggesting that basic gene sets (sul1 and ∆qacE) in the class 1 integrons are the predominant ARGs. The aminoglycoside resistance genes, aadA and aph, and macrolide resistance genes, msr(E) and mph(E), were the predominant ARGs against each antimicrobial. bla OXA and bla GES were frequently detected, whereas the bla CTX-M cluster was faintly detected. Non-metric multidimensional scaling plot analysis and canonical correspondence analysis results suggested that marked differences in ARGs could be involved in the seasonal differences; qnrS2, aac(6')-Ib, and mef(C) increased markedly in summer, whereas msr(E) was more frequently detected in winter. Heavy-metal (Hg and Cu) resistance genes (HMRGs) were significantly detected in effluents from all WWTPs. Conclusion: We characterized a baseline level of the environmental ARG/HMRG profile in the overall community, suggesting that environmental AMR surveillance, particularly in urban WWTPs, is a valuable first step in monitoring the AMR dissemination of bacteria from predominantly healthy individuals carrying notable ARG/Bs.

Prevalence and distribution of extended-spectrum ß-lactamase and AmpC-producing Escherichia coli in two New Zealand dairy farm environments.

Antimicrobial resistance (AMR) is a global threat to human and animal health, with the misuse and overuse of antimicrobials being suggested as the main driver of resistance. In a global context, New Zealand (NZ) is a relatively low user of antimicrobials in animal production. However, the role antimicrobial usage on pasture-based dairy farms, such as those in NZ, plays in driving the spread of AMR within the dairy farm environment remains equivocal. Culture-based methods were used to determine the prevalence and distribution of extended-spectrum ß-lactamase (ESBL)- and AmpC-producing Escherichia coli from farm environmental samples collected over a 15-month period from two NZ dairy farms with contrasting management practices. Whole genome sequencing was utilised to understand the genomic epidemiology and antimicrobial resistance gene repertoire of a subset of third-generation cephalosporin resistant E. coli isolated in this study. There was a low sample level prevalence of ESBL-producing E. coli (faeces 1.7%; farm dairy effluent, 6.7% from Dairy 4 and none from Dairy 1) but AmpC-producing E. coli were more frequently isolated across both farms (faeces 3.3% and 8.3%; farm dairy effluent 38.4%, 6.7% from Dairy 1 and Dairy 4, respectively). ESBL- and AmpC-producing E. coli were isolated from faeces and farm dairy effluent in spring and summer, during months with varying levels of antimicrobial use, but no ESBL- or AmpC-producing E. coli were isolated from bulk tank milk or soil from recently grazed paddocks. Hybrid assemblies using short- and long-read sequence data from a subset of ESBL- and AmpC-producing E. coli enabled the assembly and annotation of nine plasmids from six E. coli, including one plasmid co-harbouring 12 antimicrobial resistance genes. ESBL-producing E. coli were infrequently identified from faeces and farm dairy effluent on the two NZ dairy farms, suggesting they are present at a low prevalence on these farms. Plasmids harbouring several antimicrobial resistance genes were identified, and bacteria carrying such plasmids are a concern for both animal and public health. AMR is a burden for human, animal and environmental health and requires a holistic "One Health" approach to address.

The bactericidal effect of far-UVC on ESBL-producing Escherichia coli.

Because extended-spectrum beta-lactamase (ESBL) infections can cause life-threatening disease and effective treatments need to be developed, we examined the bactericidal effect of far-ultraviolet C (far-UVC) light therapy on ESBL-producing Escherichia coli (E. coli). The bactericidal effect on 2 types of ESBL-producing E. coli was the same as that on the wild strain although the results of drug resistance tests varied among these strains. We believe that irradiation with far-UVC is effective in preventing infection by ESBL-producing E. coli in health care settings.

Ceftriaxone treatment failure in two cases of Enteric Fever caused by CTX-M15 positive Salmonella Typhi and its gastrointestinal carriage.

We report two cases of culture positive typhoid fever caused by ceftriaxone resistant Salmonella Typhi. Bacterial isolates from both the cases were positive for ESBL by phenotypic methods. Both patients didn't respond to ceftriaxone and were finally treated with meropenem. Screening of family members of one patient isolated a similar strain from a healthy carrier with the same antibiogram pattern. All isolates were subjected to PCR, which confirmed the presence of blaCTX-M15 ESBL gene. These two cases confirm emergence of ESBL-producing Salmonella Typhi causing Enteric Fever in India and also their presence in the gut flora of healthy carriers.

Prevalence of enteric opportunistic pathogens and extended-spectrum cephalosporin- and carbapenem-resistant coliforms and genes in wastewater from municipal wastewater treatment plants in Croatia.

Extended-spectrum ß-lactamase (ESBL)- and carbapenemase-producing Enterobacterales are a critical global health problem and wastewater treatment plants (WWTPs) can promote their spread into the environment; yet their efficacy is not well characterized. Here, we have used conventional culturing to monitor coliform bacteria and quantitative PCR to monitor 2 ESBL and 5 carbapenemase (CP) genes and 4 enteric opportunistic pathogens (EOPs) in the influent and effluent of 7 Croatian WWTPs in two seasons. In general, levels of total, cefotaxime- and carbapenem-resistant coliforms were significantly reduced but not eliminated by conventional treatment in most WWTPs. Most WWTPs efficiently removed EOPs such as K. pneumoniae and A. baumannii, while E. coli and Enterococcus spp. were reduced but still present in relatively high concentrations in the effluent. ESBL genes (blaTEM and blaCTX-M-32) were only slightly reduced or enriched after treatment. CP genes, blaKPC-3, blaNDM and blaOXA-48-like, were sporadically detected, while blaIMP and blaVIM were frequently enriched during treatment and correlated with plant size, number or size of hospitals in the catchment area, and COD effluent concentration. Our results suggest that improvements in wastewater treatment technologies are needed to minimize the risk of environmental contamination with top priority EOPs and ARGs and the resulting public health.

Using Molecular Diagnostics to Develop Therapeutic Strategies for Carbapenem-Resistant Gram-Negative Infections.

Infections caused by multidrug-resistant Gram-negative organisms have become a global threat. Such infections can be very difficult to treat, especially when they are caused by carbapenemase-producing organisms (CPO). Since infections caused by CPO tend to have worse outcomes than non-CPO infections, it is important to identify the type of carbapenemase present in the isolate or at least the Ambler Class (i.e., A, B, or D), to optimize therapy. Many of the newer beta-lactam/beta-lactamase inhibitor combinations are not active against organisms carrying Class B metallo-enzymes, so differentiating organisms with Class A or D carbapenemases from those with Class B enzymes rapidly is critical. Using molecular tests to detect and differentiate carbapenem-resistance genes (CRG) in bacterial isolates provides fast and actionable results, but utilization of these tests globally appears to be low. Detecting CRG directly in positive blood culture bottles or in syndromic panels coupled with bacterial identification are helpful when results are positive, however, even negative results can provide guidance for anti-infective therapy for key organism-drug combinations when linked to local epidemiology. This perspective will focus on the reluctance of laboratories to use molecular tests as aids to developing therapeutic strategies for infections caused by carbapenem-resistant organisms and how to overcome that reluctance.

Colonization with selected antibiotic resistant bacteria among a cohort of Sri Lankan university students.

BACKGROUND: Antibiotic Resistance is an imminent global public health threat. Antibiotic resistance emerged in healthcare settings and has now moved on to the community settings. This study was conducted to identify the rates of asymptomatic colonization with selected antibiotic resistant organisms, (Methicillin Resistant Staphylococcus aureus (MRSA), Extended Spectrum Beta Lactamase (ESBL) producing Escherichia coli and Klebsiella spp and carbapenem resistant E.coli and Klebsiella spp) - among a group of university students in Sri Lanka. Identification of genetic determinants of MRSA and ESBL was an additional objective of the study. METHODS: A self - collected nasal swab and a peri-rectal swab collected after passing stools were obtained. Routine microbiological methods were used for the isolation S.aureus from the nasal swab and E.coli and Klebsiella species from the peri-rectal swab. Antibiotic sensitivity testing was performed as recommended by clinical and laboratory standard institute (CLSI). Three (3) genes that are responsible for ESBL production; blaCTX-M, blaSHV, and blaTEM were tested using previously described primers and PCR procedures. Identification of MecA and PVL genes attributed to MRSA was also done with PCR. RESULTS: A total of 322 participants between 21 and 28 years were recruited representing 5 different faculties of study. Seventy one (22.0%) were colonized with S.aureus and 14 among them with MRSA, making the MRSA colonization rate of 4.3%. Forty five (15%) of the participants were colonized with an ESBL producing E.coli or Klebsiella spp. No one was colonized with carbapenem resistant E.coli or Klebsiella species. Of the 45 ESBL producers the commonest genetic determinant identified was blaCTX-M (n = 36), while 16 isolates had blaTEM and 7 had blaSHV. Similarly, of the 14 isolates identified as MRSA, 3 (21.4%) were found to be PVL positive while 11 (78.6%) were MecA positive. CONCLUSIONS: A high rate of colonization with ESBL producing E.coli and Klebsiella species was noted in our study group.

Unusual antibiotic resistance pattern among blood culture isolates of Salmonella Paratyphi A.

INTRODUCTION: With increasing fluoroquinolone resistance, extended spectrum cephalosporins are recommended for the treatment of invasive Salmonella infections. However, Extended spectrum beta-lactamases (ESBL) producing Salmonella Paratyphi A causing enteric fever is on the rise and constitutes a major therapeutic challenge. Hence, we aimed to assess the incidence of ESBL production, fluoroquinolone resistance in S. Paratyphi A and to compare the fluoroquinolone resistance detection methods. METHODOLOGY: Seventeen blood-culture isolates of S. Paratyphi A were tested for susceptibility to ampicillin, chloramphenicol, co-trimoxazole, streptomycin and tetracycline (ACCuST), fluoroquinolones, azithromycin and ceftriaxone by disk diffusion method. We compared and correlated between disk diffusion of ciprofloxacin and pefloxacin with ciprofloxacin MIC. Combined disk test was employed to determine ESBL production. RESULTS: In this study, 13(76.5%) isolates were nalidixic acid resistant (NAR), 16 (94.1%) were pefloxacin resistant, while 7 (41.2%), 9 (52.9%) exhibited resistance and intermediate susceptibility to ciprofloxacin respectively. The MIC50, MIC90 of ciprofloxacin was 1 µg/mL, 2 µg/mL respectively. Among the NAR, 76.92% were DSC (MIC 0.5-1 µg/mL) and 23.08% had an MIC of 2-4 µg/mL. Of note, 4 isolates with DSC were NAS. Of the 17 S. Paratyphi A isolates, 14 (82.4%) were ESBL producers and 11 (64.7%) isolates were ceftriaxone susceptible. CONCLUSIONS: Multidrug resistant (AmpRChlRSxtR) S. Paratyphi A with combined resistance to fluoroquinolones and ESBL production is a cause of concern. We found S. Paratyphi A isolates with a relatively unusual phenotype: nalidixic acid susceptible but exhibited DSC; pefloxacin susceptible but ciprofloxacin resistant. Of note one multidrug resistant (AmpRChlRSxtR) isolate, an ESBL producer exhibited resistance to azithromycin, cephalosporins and fluoroquinolones but was susceptible to carbapenems and streptomycin.