Widespread occurrence of fecal indicator bacteria in oligotrophic tropical streams. Are common culture-based coliform tests appropriate?

Monitoring of stream water quality is a key element of water resource management worldwide, but methods that are commonly used in temperate habitats may not be appropriate in humid tropical systems. We assessed the influence of four land uses on microbial water quality in 21 streams in the Panama Canal Watershed over a one-year period, using a common culture-based fecal indicator test and 16S rDNA metabarcoding. Each stream was located within one of four land uses: mature forest, secondary forest, silvopasture, and traditional cattle pasture. Culturing detected total coliforms and Escherichia coli across all sites but found no significant differences in concentrations between land uses. However, 16S rDNA metabarcoding revealed variability in the abundance of coliforms across land uses and several genera that can cause false positives in culture-based tests. Our results indicate that culture-based fecal indicator bacteria tests targeting coliforms may be poor indicators of fecal contamination in Neotropical oligotrophic streams and suggest that tests targeting members of the Bacteroidales would provide a more reliable indication of fecal contamination.

Effectiveness of selective antibiotics use in ESBL-related UTIs.

BACKGROUND: Urinary tract infections (UTIs) are the second most common infection, affecting 150 million people each year worldwide. Enterobacteriaceae species expressing extended-spectrum ß-lactamases (ESBLs) are on the rise across the globe and are becoming a severe problem in the therapeutic management of clinical cases of urinary tract infection. Knowledge of the prevalence and antibiogram profile of such isolates is essential to develop an appropriate treatment methodology. This study aimed to investigate the prevalence of Enterobacteriaceae isolates exhibiting ESBL and their selective oral antibiogram profile at the district general hospital, Polonnaruwa. RESULTS: A total of 4386 urine specimens received to the Microbiology Laboratory during the study period. Among them, 1081 (24.6%) showed positive results for urine culture while 200/1081 specimens showed ESBL isolates. Out of the selected 200 specimen's majority (67.5%) of samples received from the In-Patient Department. There were 200 patients and reported that 115 (57.5%) were females and 85 (42.5%) were males. The majority (51%) of the patients belong to the age group of 55-74 years. Among the ESBLs positive specimens, the majority 74.5% (n = 149) identified organisms were E. coli followed by Klebsiella spp.17.5% (n = 35), Enterobacteriaceae 7% (n = 14) and only1% (n = 2) isolate of Proteus spp. Mecillinam (87.92%) and Nitrofurantoin (83.2%) showed higher effectiveness against E. coli. Nitrofurantoin showed the highest effectiveness against Klebsiella spp. (40%), other Enterobacteriaceae spp. (100%). Proteus spp. showed 100% effectiveness and resistance respectively against Ciprofloxacin, Cotrimoxazole and Nitrofurantoin. CONCLUSION: The most predominant ESBLs producing uro-pathogen was the E. coli in the study setting and E. coli had higher sensitivity rate against Mecillinam. Among currently used oral antibiotics Nitrofurantoin was the best choice for UTIs caused by ESBL producers.

Efficacy assessment of different cryoprotectants for preserving the viability of Enterobacterales strains at - 20 °C.

The preservation of microorganisms is pivotal in microbiological practice. Currently, cryopreservation is assumed to be an effective and inexpensive approach for the storage of microorganisms, including bacteria. The key point of cryopreservation is optimal cryoprotectant selection. In the present study, different cryoprotectant compositions were tested for long-term storage of 15 Enterobacterales bacterial strains at - 20 °C. The survival rates of the bacterial strains were evaluated in four different cryoprotectant solutions containing 70% glycerin only (cryoprotectants 1 and 4), 10% dimethyl sulfoxide (DMSO) with 70% glycerin (cryoprotectant 2), and 10% DMSO (cryoprotectant 3). In addition, cryoprotectants 1 and 2 contained peptone and yeast extract as nutritional supplements. The general survival rates of the bacterial strains were evaluated after 12 months of storage. After 12 months, the survival rates of the different cryoprotectants were as follows: cryoprotectant 1-88.87%; cryoprotectant 2-84.85%; cryoprotectant 3-83.50%; and cryoprotectant 4-44.81%. Thus, the composition of cryoprotectant 1 (70% glycerin with nutrient supplements) was optimal for preserving 15 tested strains of the order Enterobacterales. Despite these findings, the biochemical properties of the tested strains changed after cryopreservation for 12 months in the presence of 1 or 3 cryoprotectants. Alterations in the biochemical profile could be related to changes in environmental conditions and cold adaptation. We assume that the composition of cryoprotectant 1 can be optimal for storing the order Enterobacterales at - 20 °C. However, further investigations are needed to elucidate the problem of cryopreservation and to support our assumption.

Biocontrol of rusted root rot in Panax ginseng by a combination of extracts from Bacillus amyloliquefaciens YY8 crude protein and Enterobacteriaceae YY115 ethyl acetate.

BACKGROUND: Rusted root rot is one of the most common root diseases in Panax ginseng, and Cylindrocarpon destructans is one of the main pathogenic fungus. The objective of this study was to screen and explore the extracts of biocontrol bacteria isolated from ginseng rhizosphere soil against Cylindrocarpon destructans. RESULTS: Bacterial strains Bacillus amyloliquefaciens YY8 and Enterobacteriacea YY115 were isolated and found to exhibit in vitro antifungal activity against C. destructans. A combination of crude protein extract from B. amyloliquefaciens YY8 and ethyl acetate extract from Enterobacteriacea YY115 in a 6:4 ratio exhibited the strongest antifungal activity against C. destructans. Measurements of electrical conductivity, protein content, and nucleic acid content in suspension cultures of C. destructans treated with a mixture extracts indicated that the extracts disrupted the cell membranes of rusted root rot mycelia, resulting in the leakage of electrolytes, proteins, and nucleic acids from the cells, and ultimately inhibiting the growth of C. destructans. The combined extracts suppressed the infection of ginseng roots discs by C. destructans effectively. CONCLUSION: The extracts obtained from the two bacterial strains effectively inhibited C. destructans in P. ginseng. It can provide scientific basis for the development of new biological control pesticides, reduce the use of chemical pesticides, and promote the sustainable development of agriculture.

One decade of point-prevalence surveys for carriage of extended-spectrum beta-lactamase-producing enterobacterales: whole genome sequencing based prevalence and genetic characterization in a large Dutch teaching hospital from 2013 to 2022.

OBJECTIVES: To determine the prevalence, trends, and potential nosocomial transmission events of the hidden reservoir of rectal carriage of extended-spectrum beta-lactamase-producing Enterobacterales (ESBL-E). METHODS: From 2013 to 2022, yearly point prevalence surveys were conducted in a large Dutch teaching hospital. On the day of the survey, all admitted patients were screened for ESBL-E rectal carriage using peri-anal swabs and a consistent and sensitive selective culturing method. All Enterobacterales phenotypically suspected of ESBL production were analysed using whole genome sequencing for ESBL gene detection and clonal relatedness analysis. RESULTS: On average, the ESBL-E prevalence was 4.6% (188/4,119 patients), ranging from 2.1 to 6.6% per year. The ESBL-prevalence decreased on average 5.5% per year. After time trend correction, the prevalence in 2016 and 2020 was lower compared to the other year. Among the ESBL-E, Escherichia coli (80%) and CTX-M genes (85%) predominated. Potential nosocomial transmission events could be found in 5.9% (11/188) of the ESBL-E carriers. CONCLUSIONS: The ESBL-E rectal carriage prevalence among hospitalized patients was 4.6% with a downward trend from 2013 to 2022. The decrease in ESBL-E prevalence in 2020 could have been due to the COVID-19 pandemic and subsequent countrywide measures as no nosocomial transmission events were detected in 2020. However, the persistently low ESBL-E prevalences in 2021 and 2022 suggest that the decline in ESBL-E prevalence goes beyond the COVID-19 pandemic, indicating that overall ESBL-E carriage rates are declining over time. Continuous monitoring of ESBL-E prevalence and transmission rates can aid infection control policy to keep antibiotic resistance rates in hospitals low.

Biological studies of the activity of Manuka honey against Carbapenem-resistant Enterobacterales (CRE) bacteria.

OBJECTIVES: To evaluate the potency of Manuka honey UMF +15 against Carbapenem-resistant Enterobacterales (CRE). Bacterial resistance is a worldwide problem that is increasing year by year, especially Carbapenem resistance. Alternatives to antibiotics are needed to both reduce costs, and to reduce the spread of antibiotic resistance, with the ultimate goal of saving lives. METHODS: The efficacy of Manuka honey UMF +15 was tested by 2 methods; Well diffusion assay and minimum bactericidal concentration (MBC) against twenty Carbapenem-resistant isolates which collected from Makkah city hospitals during three months of study from 1st of September 2023 up to 1st of December 2023. RESULTS: The growth of all isolates of Carbapenem-resistant Enterobacterales (CRE) was severely inhibited by low concentrations of Manuka honey, affecting 25% of isolates at 15% and 75% of isolates at 18% of Manuka honey. In addition, using the honey at different concentrations in a well diffusion assay resulted, as expected, in a variable zone diameter, ranging from large zones(14mm) to small zones (2 mm) according to the concentration of the honey. CONCLUSION: This study shows the remarkable antibacterial activity of Manuka honey and suggests that this natural remedy might be used in the future as an alternative treatment option against Carbapenem-resistant Enterobacterales (CRE); however, further clinical trials should be performed to corroborate our initial findings.

Prevalence and epidemiological distribution of indicators of pathogenic bacteria in households drinking water in Ethiopia: a systematic review and meta-analysis.

BACKGROUND: Ensuring the availability of safe drinking water remains a critical challenge in developing countries, including Ethiopia. Therefore, this paper aimed to investigate the prevalence of fecal coliform and E. coli bacteria and, geographical, children availability, and seasonal exposure assessment through a meta-analysis. METHODS: Two independent review groups extensively searched internet databases for English-language research articles published between 2013 and 2023. This systematic review and meta-analysis followed PRISMA guidelines. The methodological quality of each included study was evaluated using the STROBE guidelines. Publication bias was assessed by visual inspection of a funnel plot and then tested by the Egger regression test, and meta-analysis was performed using DerSimonian and Laird random-effects models with inverse variance weighting. Subgroup analyses were also conducted to explore heterogeneity. RESULTS: Out of 48 potentially relevant studies, only 21 fulfilled the inclusion criteria and were considered for meta-analysis. The pooled prevalence of fecal coliform and E. coli was 64% (95% CI: 56.0-71.0%, I2 = 95.8%) and 54% (95% CI: 45.7-62.3%, I2 = 94.2%), respectively. Subgroup analysis revealed that the prevalence of fecal coliform bacteria increased during the wet season (70%) compared to the dry season (60%), particularly in households with under-five children (74%) compared to all households (61%), in rural (68%) versus urban (66%) areas, and in regions with high prevalence such as Amhara (71%), Gambela (71%), and Oromia (70%). Similarly, the prevalence of E. coli was higher in households with under-five children (66%) than in all households (46%). CONCLUSIONS: The analysis highlights the higher prevalence of fecal coliform and E. coli within households drinking water, indicating that these bacteria are a significant public health concern. Moreover, these findings emphasize the critical need for targeted interventions aimed at improving drinking water quality to reduce the risk of fecal contamination and enhance public health outcomes for susceptible groups, including households with under-five children, in particular geographical areas such as the Amhara, Gambela, and Oromia regions, as well as rural areas, at point-of-use, and during the rainy season. REGISTRATION: This review was registered on PROSPERO (registration ID - CRD42023448812).

Prevalence, risk factors, and characterisation of extended-spectrum ß-lactamase -producing Enterobacterales (ESBL-E) in horses entering an equine hospital and description of longitudinal excretion.

BACKGROUND: Extended-spectrum ß-lactamase -producing Enterobacterales (ESBL-E) are important zoonotic pathogens that can cause serious clinical infections, also in horses. Preventing the spread of ESBL-E, especially in the equine hospital environment, is key to reducing the number of difficult-to-treat infections. Estimating the local prevalence of ESBL-E in horses is crucial to establish targeted infection control programs at equine hospitals. We conducted a prevalence and risk factor study in equine patients on admission to an equine teaching hospital in Finland through a rectal ESBL-E screening specimen of the horse and a questionnaire. RESULTS: The prevalence of ESBL-E in admitted horses was 3% (5/161, 95% CI 1-7%); none of the tested factors remained statistically significant in multivariate analysis, although antimicrobial treatment within three months was borderline significant (p = 0.052). Extended-spectrum ß-lactamase -producing Klebsiella pneumoniae ST6179:CTX-M-15 was detected in three horses using whole-genome sequencing, which in combination with patient records suggested nosocomial transmission. Escherichia coli isolates were ST1250:CTX-M-1 (n = 1), ST1079:CTX-M-1 (n = 1), and ST1245:CTX-M-14 (n = 1). Multiple virulence genes were detected in the ESBL-E isolates. In the ESBL-E positive horses enrolled in a one-year follow-up study, ESBL-E were unlikely to be isolated in rectal screening specimens after the initial positive specimen. CONCLUSIONS: The prevalence of ESBL-E in horses visiting a veterinary teaching hospital in Finland is low, indicating an overall low prevalence estimate in the country's equine population. No statistically significant risk factors were identified, likely due to the low number of cases. The duration of ESBL-E carriage is likely to be very short in horses.

Antibiotic-resistant bacteria contaminating leafy vegetables in Saudi Arabia's eastern region.

BACKGROUND: Food-associated antibiotic-resistant bacteria can cause infections that may critically impact human health. The objectives of this study were to determine the microbial contamination level of green leafy vegetables and their antibiotic resistance pattern. METHODS: Sixty-three samples of leafy vegetables were collected from Dammam Central Fruit and Vegetables Market from January to June 2023. The vegetables included lettuce (Lactuca sativa), parsley (Petroselinum crispum), and watercress (Nasturtium officinale). Samples were tested by standard microbiological techniques for identification and antibiotic susceptibility testing. RESULT: Eight types of bacteria belonging to six different genera were detected. Enterobacteriaceae family was represented by four genera: Klebsiella, Proteus, Morganella, and Enterobacter. The other two genera were Pseudomonas and Aeromonas. Enterobacter cloacae was the most abundant organism, followed by Pseudomonas putida and Aeromonas sobria. On the other hand, Morganella morganii, Aeromonas hydrophila, and Proteus mirabilis were the least abundant. The three vegetable types had different levels of bacterial contamination. All isolated organisms were sensitive to penicillin, cephalosporin, aminoglycoside, and fluoroquinolone. However, Klebsiella oxytoca, M. morganii, and K. pneumonia showed resistance to ampicillin. A. hydrophila, Morganella morganii, and E. cloacae showed resistance to amoxicillin. M. morganii and E. cloacae were found to be resistant to cefalotin. Moreover, A. hydrophila, M. morganii, and E. cloacae were resistant to cefoxitin. Again, A. hydrophila was found to be resistant to imipenem. Only M. morganii was resistant to Ciprofloxacin. Two isolates, P. mirabilis and M. morganii were resistant to tigecycline. Another two, M. morganii and P. mirabilis were resistant to Nitrofurantoin. Only M. morganii was found to be resistant to trimethoprim. CONCLUSION: This study aligns with the broad consensus in the literature about the significance of bacterial contamination in vegetables and the public health implications. The unique focus on antibiotic resistance patterns adds an essential dimension to the existing body of knowledge.

Modification of carbapenemase inhibition test and comparison of its performance with NG-Test CARBA 5 for detection of carbapenemase-producing Enterobacterales.

AIMS: Adequately and accurately identifying carbapenemase-producing Enterobacterales (CPE) is vital for selecting appropriate antimicrobial therapy and implementing effective infection control measures. This study aims to optimize the phenotypic detection method of carbapenemase for routine diagnostics in clinical microbiology laboratories. METHODS AND RESULTS: Carbapenemase genes in 2665 non-duplicate CRE clinical strains collected from various regions of China were confirmed through whole-genome sequencing (WGS). The carbapenemase inhibition test (CIT) was conducted and interpreted using different methods and breakpoints, then compared with the NG-Test CARBA 5 for carbapenemase detection. The diagnostic performance of the CIT method was optimal when the carbapenemase types were determined by comparing the inhibition zone diameters of the imipenem disc with 3-aminophenylboronic acid (APB) plus ethylenediaminetetraacetic acid (EDTA) to those of the imipenem disc with either APB or EDTA alone, with a breakpoint of 4 mm. The overall sensitivities of the current CIT, the modified CIT, and NG-Test CARBA 5 were 91.4%, 94.9%, and 99.9%, respectively. For detecting isolates co-producing Klebsiella pneumoniae carbapenemase (KPC) and metallo-ß-lactamases (MBLs), the modified CIT method had higher sensitivity than the current method (70.0% vs. 53.3%), though this difference was not statistically significant (P = 0.063). The NG-Test CARBA 5 showed excellent performance for multi-carbapenemases diagnosis, with sensitivity and specificity of 97.1% and 100%, respectively. CONCLUSIONS: Optimizing and standardizing the CIT method for clinical use is necessary. It has certain advantages in diagnosing multi-carbapenemase and rare carbapenemase production. However, for identifying common carbapenemase types, the NG-Test CARBA 5 demonstrated superior performance.

Scandinavium lactucae sp. nov. Isolated from Healthy Lettuce in South Korea.

Four Gram-negative, facultative anaerobic, oxidase-negative and catalase-positive strains were isolated from lettuce sample collected from test beds at the National Institute of Agricultural Sciences in Wanju, South Korea. The whole genome sequences of the strains ranged from 4,624,629 to 4,849,846 bp in size, with DNA G + C contents of 54.32 to 54.56 mol%. Phylogenetic analyses based on 16S rRNA gene and four housekeeping (atpD, gyrB, infB, and rpoB) gene sequences showed that the four strains clustered closely together with Scandinavium type strains within the Enterobacteriaceae family. Moreover, the average nucleotide identity and digital DNA-DNA hybridization value of the proposed type strain (V105_6T) with the closely related Scandinavium type strains were in the range of 85.71-86.16% and 30.2-31.2%, respectively, which were all below the species delineation threshold values. The major cellular fatty acid of V105_6T was C16:0. Growth was observed at 7, 10 and 35 °C, and in the presence of 7% NaCl concentration. Based on phenotypic and genotypic results, strain V105_6T represents a novel species of the genus Scandinavium, for which the name Scandinavium lactucae sp. nov. is proposed. The type strain is V105_6T (= LMG 33389T = DSM 117134T).

Cellular Effects of Enterobacteriaceae Polysaccharide Colanic Acid.

Colanic acid (CA) is an exopolysaccharide found in Enterobacteriaceae. Recently, its ability to stimulate physical activity in mice and to prolong the lifespan of invertebrates has been described. In the current work, we use standard MTT assay, fluorescence microscopy, and flow cytometry to describe CA action on several cell lines of different origins. We observed slight antiproliferative activity against colorectal cancer (HCT-116), neuroblastoma (IMR-32), and myoblast (C2C12) cell lines at a concentration of 256 µg/mL, while other cell lines of non-cancerous origin (Vero, HPF) did not show any decrease in the MTT assay. In all cell lines, we observed a rearrangement of mitochondria localization using fluorescence microscopy. CA induces cell differentiation in the myoblast cell line (C2C12) at concentrations of 50-200 µg/mL. Briefly, we observed that the number of apoptotic cells increased and the metabolic activity in the MTT assay decreased, which was accompanied by changes in cell morphology, the quantity of ROS, and the potential of the mitochondrial membrane. Taken together, these results indicate that CA is specific in cytotoxicity to cell lines of different origins and can impact mitochondria and differentiation, consistent with its potential geroprotective function.

Antimicrobial Susceptibility and Genetic Epidemiology of Extended-Spectrum ß-Lactamase-Positive Enterobacterales Clinical Isolates in Central Poland.

The extended-spectrum ß-lactamases (ESßLs) are bacterial enzymes capable of hydrolyzing penicillins, cephalosporins, and aztreonam. The prevalence of ESßL is increasing among clinically significant microorganisms worldwide, drastically reducing the therapeutic management of infectious diseases. The study aimed to determine the drug susceptibility of ESßL-positive clinical isolates acquired from patients hospitalized in Lodz, central Poland, and analyze the prevalence of specific genes, determining acquired resistance in these bacteria. The samples of ESßL-positive clinical isolates were gathered in 2022 from medical microbiological laboratories in the city of Lodz, central Poland. The strains were subjected to biochemical identification and antimicrobial susceptibility testing following EUCAST guidelines. The presence of studied genes (blaCTX-M, blaSHV, blaTEM, blaPER, blaVEB) was confirmed by PCR. Over 50% of studied isolates were resistant to gentamicin, cefepime, ceftazidime and ciprofloxacin. The most common ESßL gene was blaCTX-M. In most isolates, the resistance genes occurred simultaneously. The blaPER was not detected in any of the tested strains. ESßL-producing strains are largely susceptible to the currently available antibiotics. The observation of the coexistence of different genes in most clinical isolates is alarming.

Adapting nature's own solution: The effect of rhamnolipid and lytic bacteriophage cocktail on enteric pathogens that proliferate in mucilage.

The mucilage event witnessed in the Sea of Marmara in 2021 has emerged as a prominent environmental concern, capturing public attention due to its detrimental effects on ecological, economic, and aesthetic dimensions. Addressing the multifaceted impacts of mucilage demands a nature-centric scientific approach, given its global ramifications spanning economy, public health, international relations, and tourism. Consequently, this study sought to explore alternative approaches for the removal of pathogenic enteric bacteria associated with mucilage occurrences, diverging from conventional methodologies. Specifically, the primary objective was to assess the efficacy of rhamnolipid and a bacteriophage cocktail in mitigating the proliferation of enteric pathogens within mucilaginous environments. During the study, 91 phage isolations were obtained from 45 water samples taken and 10 phages were selected for the broad host range and because of the efficacy tests, a phage cocktail was created with 5 phages. It was found that the mixture of rhamnolipid, phage cocktail and rhamnolipid-phage cocktail reduced bacterial load by 7-9 log10, 9-12 log10 and 9-11 log10 respectively under laboratory conditions. When the study was carried out in seawater, reductions of 4-5 log10, 3 log10 and 4 log10 were achieved. This study has shown that the combined use of rhamnolipid, phage cocktail and rhamnolipid-phage cocktail can be considered as the most effective natural solution proposal for reducing bacterial load, both in laboratory conditions and in sea surface water.

Natural products from food sources can alter the spread of antimicrobial resistance plasmids in Enterobacterales.

Antimicrobial resistance (AMR) poses a significant threat to global public health. Notably, resistance to carbapenem and extended-spectrum ß-lactam antibiotics in Gram-negative bacteria is a major impediment to treating infections. Genes responsible for antibiotic resistance are frequently carried on plasmids, which can transfer between bacteria. Therefore, exploring strategies to prevent this transfer and the prevalence of AMR plasmids is timely and pertinent. Here, we show that certain natural product extracts and associated pure compounds can reduce the conjugation of AMR plasmids into new bacterial hosts. Using our established high-throughput fluorescence-based flow cytometry assay, we found that the natural products were more active in reducing transmission of the IncK extended-spectrum ß-lactamase-encoding plasmid pCT in Escherichia coli EC958c, compared to Klebsiella pneumoniae Ecl8 carrying the IncFII carbapenemase-encoding plasmid pKpQIL. The exception was the natural product rottlerin, also active in K. pneumoniae. In classical conjugation assays, rottlerin also reduced the conjugation frequency of the IncFII bla NDM-1 carrying plasmid pCPE16_3 from a clinical K. pneumoniae isolate. Our data indicate that the natural products tested here, in their current molecular structure, reduced conjugation by a small amount, which is unlikely to achieve a large-scale reduction in AMR in bacterial populations. However, certain natural products like rottlerin could provide a foundation for further research into compounds with effective anti-plasmid activity.

Transmission chains and molecular characterizations of extended-spectrum ß-lactamase producing Enterobacteriaceae at a veterinary hospital in Chengdu, China.

The rapid emergence of Extended-spectrum ß-lactamase producing Enterobacteriaceae (ESBL-E) is a major global public health concern. Previous studies have identified that intensive medical care of dogs and cats in veterinary hospitals have accelerated the infections and spread of ESBL-E. To investigate the spread of ESBL-E in a veterinary hospital, a total of 202 samples including hospitalized animals, veterinary healthcare workers and environment were collected from a veterinary hospital in Chengdu, China. ESBL-E were identified by antimicrobial susceptibility testing and 16 s rRNA sequencing and were further conducted on ESBL gene detection and multilocus sequence typing (MLST). At last, strains with transmission potential were analyzed by whole genome sequencing (WGS). Our results showed that the overall prevalence of ESBL-positive isolates was 34.7% (70/202), with 55.3% (26/47) in animals, 29.3% (12/41) in healthcare workers and 28.1% (32/114) in environment swabs. Twenty diverse MLST types were detected, with ST744, ST231 as the most prevalent ones. Transmission chains of two ESBL-E.coli (ST744 blaCTX-M-18, blaTEM-1) from cat_21 to cat_14, and two ESBL-Kp (ST231 blaCTX-M-27, blaTEM-1, blaSHV-1) from cat_20 to cat_37 were further confirmed by WGS. Furthermore, interdisciplinary investigation and cooperation of AMR are needed to better limit the transmissions of high-risk strains and to implement effective public health interventions.

D-galactonate metabolism in enteric bacteria: a molecular and physiological perspective.

D-galactonate, a widely prevalent sugar acid, was first reported as a nutrient source for enteric bacteria in the 1970s. Since then, decades of research enabled a description of the modified Entner-Doudoroff pathway involved in its degradation and reported the structural and biochemical features of its metabolic enzymes, primarily in Escherichia coli K-12. However, only in the last few years, the D-galactonate transporter has been characterized, and the regulation of the dgo operon, encoding the structural genes for the transporter and enzymes of D-galactonate metabolism, has been detailed. Notably, in recent years, multiple evolutionary studies have identified the dgo operon as a dominant target for adaptation of E. coli in the mammalian gut. Despite considerable research on dgo operon, numerous fundamental questions remain to be addressed. The emerging relevance of the dgo operon in host-bacterial interactions further necessitates the study of D-galactonate metabolism in other enterobacterial strains.

Occurrence of blaNDM-1, blaNDM-5, blaNDM-7, and blaKPC-2 genes in clinical isolates of enterobacterales with high genetic variability, from colonization and infection in patients with or without COVID-19, from a hospital in Brazil.

AIMS: This study aimed to investigate the presence of beta-lactams resistance genes and the clonal relationship of clinical isolates of Enterobacterales obtained from patients with and without COVID-19, in a hospital in northeastern Brazil. METHODS AND RESULTS: The study analyzed 45 carbapenem-resistant clinical isolates using enterobacterial repetitive intergenic consensus (ERIC-PCR), PCR, and amplicon sequencing to detect resistance genes (blaKPC, blaGES, blaNDM, blaVIM, and blaIMP). The main species were Klebsiella pneumoniae, Serratia marcescens, and Proteus mirabilis. Detected genes included blaNDM (46.66%), blaKPC (35.55%), and both (17.79%). ERIC-PCR showed multiclonal dissemination and high genetic variability. The main resistance gene was blaNDM, including blaNDM-5 and blaNDM-7. CONCLUSIONS: The presence of Enterobacterales carrying blaKPC and blaNDM in this study, particularly K. pneumoniae, in infections and colonizations of patients with COVID-19 and non-COVID-19, highlights genetic variability and resistance to carbapenems observed in multiple species of this order.

Fitness factor genes conserved within the multi-species core genome of Gram-negative Enterobacterales species contribute to bacteremia pathogenesis.

There is a critical gap in knowledge about how Gram-negative bacterial pathogens, using survival strategies developed for other niches, cause lethal bacteremia. Facultative anaerobic species of the Enterobacterales order are the most common cause of Gram-negative bacteremia, including Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Citrobacter freundii, and Enterobacter hormaechei. Bacteremia often leads to sepsis, a life-threatening organ dysfunction resulting from unregulated immune responses to infection. Despite a lack of specialization for this host environment, Gram-negative pathogens cause nearly half of bacteremia cases annually. Based on our existing Tn-Seq fitness factor data from a murine model of bacteremia combined with comparative genomics of the five Enterobacterales species above, we prioritized 18 conserved fitness genes or operons for further characterization. Mutants were constructed for all genes in all five species. Each mutant was used to cochallenge C57BL/6 mice via tail vein injection along with each respective wild-type strain to determine competitive indices for each fitness gene. Five fitness factor genes, when mutated, attenuated mutants in four or five species in the spleen and liver (tatC, ruvA, gmhB, wzxE, arcA). Five additional fitness factor genes or operons were validated as outcompeted by wild-type in three, four, or five bacterial species in the spleen (xerC, prc, apaGH, atpG, aroC). Overall, 17 of 18 fitness factor mutants were attenuated in at least one species in the spleen or liver. Together, these findings allow for the development of a model of bacteremia pathogenesis that may include future targets of therapy against bloodstream infections.

Metabolite-based inter-kingdom communication controls intestinal tissue recovery following chemotherapeutic injury.

Cytotoxic chemotherapies have devastating side effects, particularly within the gastrointestinal tract. Gastrointestinal toxicity includes the death and damage of the epithelium and an imbalance in the intestinal microbiota, otherwise known as dysbiosis. Whether dysbiosis is a direct contributor to tissue toxicity is a key area of focus. Here, from both mammalian and bacterial perspectives, we uncover an intestinal epithelial cell death-Enterobacteriaceae signaling axis that fuels dysbiosis. Specifically, our data demonstrate that chemotherapy-induced epithelial cell apoptosis and the purine-containing metabolites released from dying cells drive the inter-kingdom transcriptional re-wiring of the Enterobacteriaceae, including fundamental shifts in bacterial respiration and promotion of purine utilization-dependent expansion, which in turn delays the recovery of the intestinal tract. Inhibition of epithelial cell death or restriction of the Enterobacteriaceae to homeostatic levels reverses dysbiosis and improves intestinal recovery. These findings suggest that supportive therapies that maintain homeostatic levels of Enterobacteriaceae may be useful in resolving intestinal disease.