Two microbes assisting Miscanthus floridulus in remediating multi-metal(loid)s-contaminated soil.

Miscanthus has good tolerance to multi-metal(loid)s and has received increasing attention in remediated studies of metal(loid)s-contaminated soil. In this study, we conducted phytoextraction techniques to investigate the synergic effects of remediation of multi-metal(loid)s-contaminated soil by Miscanthus floridulus (Lab.) and two plant growth-promoting bacteria (PGPB), TS8 and MR2, affiliated to Enterobacteriaceae. The results exhibited a decrease of arsenic (15.27-21.50%), cadmium (8.64-15.52%), plumbum (5.92-12.76%), and zinc (12.84-24.20%) except for copper contents in the soil in bacterial inoculation groups, indicating that MR2 and TS8 could enhance the remediation of metal(loid)s. Moreover, increased fresh/dry weight and height indicated that inoculated bacteria could promote Miscanthus growth. Although the activities of antioxidant enzymes and the content of chlorophyll in the overground tissues showed no significant increase or even decrease, the activities of antioxidant enzymes in the underground tissues and soil were elevated by 48.95-354.17%, available P by 19.07-23.02%, and available K by 15.34-17.79% (p < 0.05). Bacterial inoculants could also decrease the soil pH. High-throughput sequencing analysis showed that the bacterial inoculant affected the rhizosphere bacterial community and reduced community diversity, but the relative abundance of some PGPB was found to increase. Phylogenetic molecular ecological networks indicated that bacterial inoculants reduced interactions between rhizosphere bacteria and thereby led to a simpler network structure but increased the proportion of positive-correlation links and enhanced the metabiosis and symbiosis of those bacteria. Spearman's test showed that OTUs affiliated with Enterobacteriaceae and soil nutrients were critical for metal(loid) remediation and Miscanthus growth. The results of this study provide a basis for the synergic remediation of multi-metal(loid)s-contaminated soils by Miscanthus and PGPB and provide a reference for the subsequent regulation of Miscanthus remediation efficiency by the other PGPB or critical bacteria.

Clinical distribution of carbapenem genotypes and resistance to ceftazidime-avibactam in Enterobacteriaceae bacteria.

Introduction: Bacterial resistance is a major threat to public health worldwide. To gain an understanding of the clinical infection distribution, drug resistance information, and genotype of CRE in Dongguan, China, as well as the resistance of relevant genotypes to CAZ-AVI, this research aims to improve drug resistance monitoring information in Dongguan and provide a reliable basis for the clinical control and treatment of CRE infection. Methods: VITEK-2 Compact automatic analyzer was utilized to identify 516 strains of CRE collected from January 2017 to June 2023. To determine drug sensitivity, the K-B method, E-test, and MIC methods were used. From June 2022 to June 2023, 80 CRE strains were selected, and GeneXpert Carba-R was used to detect and identify the genotype of the carbapenemase present in the collected CRE strains. An in-depth analysis was conducted on the CAZ-AVI in vitro drug sensitivity activity of various genotypes of CRE, and the results were statistically evaluated using SPSS 23.0 and WHONET 5.6 software. Results: This study identified 516 CRE strains, with the majority (70.16%) being K.pneumoniae, followed by E.coli (18.99%). Respiratory specimens had highest detection rate with 53.77% identified, whereas urine specimens had the second highest detection rate with 17.99%. From June 2022 to June 2023, 95% of the strains tested using the CRE GeneXpert Carba-R assay possessed carbapenemase genes, of which 32.5% were blaNDM strains and 61.25% blaKPC strains. The results showed that CRE strains containing blaKPC had a significantly higher rate of resistance to amikacin, cefepime, and aztreonam than those harboring blaNDM. Conclusions: The CRE strains isolated from Dongguan region demonstrated a high resistance rate to various antibiotics used in clinical practice but a low resistance rate to tigecycline. These strains produce Class A serine carbapenemases and Class B metals ß-lactamases, with the majority of them carrying blaNDM and blaKPC. Notably, CRE strains with blaKPC and blaNDM had significantly lower resistance rates to tigecycline. CAZ-AVI showed a good sensitivity rate with no resistance to CRE strains carrying blaKPC. Therefore, CAZ-AVI and tigecycline should be used as a guide for rational use of antibiotics in clinical practice to effectively treat CRE.

A systematic review and meta-analysis of carbapenem resistance and its possible treatment options with focus on clinical Enterobacteriaceae: Thirty years of development in Pakistan.

Background: Carbapenem resistance is epidemic worldwide, these last resort antimicrobials are listed in the WHO 'watch group' with higher resistance potential. During the years 2017-18 Pakistan Antimicrobial Resistance Surveillance System reported an increase in carbapenem resistance. However, a comprehensive information on prevalence and molecular epidemiology of carbapenem resistance in Pakistan is not available. This systematic review and meta-analysis is aimed to report the current carbapenem resistance situation in Pakistan and its treatment options. Methods: In this systematic review and meta-analysis, we investigated the pooled prevalence (PPr) of carbapenem resistance in Enterobacteriaceae and non-Enterobacteriaceae by organizing available data, from Web of Science and PubMed by April 2, 2020, in various groups and subgroups including species, years, provinces, extended spectrum ß-lactamase production, clinical presentation, carbapenemase and metallo-ß-lactamase production, and New Delhi metallo-ß-lactamase (NDM) prevalence. Literature review was updated for the studies publisehd by December 07, 2023. Moreover, we descriptively reviewed the molecular epidemiology of carbapenem resistance in Enterobacteriaceae and non-Enterobacteriaceae in Pakistan. Lastly, we statistically explored different treatment options available for carbapenem resistant infections. We used R package 'metafor' for performing meta-analysis and influence diagnostics and determining treatment options. Results: From two academic databases Web of Science and PubMed we identified 343 studies. Eighty-eight studies were selected for the systematic review and meta-analysis. Seventy-four studies were selected for phenotypic analysis, 36 for genotypic analysis, and 31 for available treatment options. PPr-ID of 12% [0.12 (0.07, 0.16)] was observed for phenotypic carbapenem resistance in Enterobacteriaceae with more prevalence recorded in Klebsiella pneumoniae 24% [0.24 (0.05, 0.44)] followed by 9% [0.09 (-0.03, 0.20)] in Escherichia coli. During the last two decades we observed a striking increase in carbapenem resistance PPr i.e., from 0% [0.00 (-0.02, 0.03)] to 36% [0.36 (0.17, 0.56)]. blaNDM with PPr 15% [0.15 (0.06, 0.23)] in naive isolates was found to be the fundamental genetic determinant for carbapenem resistance in Enterobacteriaceae in Pakistan. Polymyxin B, colistin, tigecycline, and fosfomycin were identified as the suggested treatment options available for multidrug resistant infections not responding to carbapenems. Various studies reported carbapenem resistance from human, animal, and environment sources. Conclusion: In conclusion, we found that NDM-1 producing carbapenem resistant Enterobacteriaceae are increasing in Pakistan. Meta-analysis showed that metallo-ß-lactamases producing E. coli ST405 and K. pneumoniae sequence type11 are the major resistant clones. Number of reported studies in various subgroups and inconsistency in following CLSI guidelines are the potential limitations of this meta-analysis. A National antimicrobial resistance (AMR) surveillance strategy based on One Health is urgently needed to check any future AMR crisis in Pakistan.

[Key microbial monitoring and clinical analysis of bloodstream infections and CRO colonization after hematopoietic stem cell transplantation in hematological patients].

Objective: To investigate the distribution and clinical characteristics of pathogenic bacteria following hematopoietic stem cell transplantation (HSCT), as well as to provide a preliminary research foundation for key microbial monitoring, and clinical diagnosis and treatment of infections after HSCT in hematological patients. Methods: We retrospectively analyzed the clinical data of 190 patients who tested positive for microbial testing [G-bacteria blood culture and/or carbapenem-resistant organism (CRO) screening of perianal swabs] at our center from January 2018 to December 2022. Patients were divided into blood culture positive, perianal swab positive, and double positive groups based on the testing results. The three patient groups underwent statistical analysis and comparison. Results: The top four pathogenic bacteria isolated from sixty-three patients with G-bacteria bloodstream infection (BSI) were Escherichia coli (28 strains, 43.75% ), Klebsiella pneumonia (26 strains, 40.63% ), Pseudomonas aeruginosa (3 strains, 4.69% ), and Enterobacter cloacae (3 strains, 4.69% ). The top three pathogenic bacteria isolated from 147 patients with CRO perianal colonization were carbapenem-resistant Klebsiella pneumoniae (58 strains, 32.58% ), carbapenem-resistant Escherichia coli (49 strains, 27.53% ), and carbapenem-resistant Enterobacter cloacae (20 strains, 11.24% ). The 3-year disease-free survival (DFS ) and overall survival (OS) of double positive group patients were significantly lower compared to those in the blood culture and perianal swab positive groups (DFS: 35.6% vs 53.7% vs 68.6%, P=0.001; OS: 44.4% vs 62.4% vs 76.9%, P<0.001), while non-relapse mortality (NRM) was significantly higher (50.0% vs 34.9% vs 10.6%, P<0.001). Failed engraftment of platelets and BSI are independent risk factors for NRM (P<0.001). Using polymyxin and/or ceftazidime-avibactam for more than 7 days is an independent protective factor for NRM (P=0.035) . Conclusion: This study suggests that the occurrence of BSI significantly increases the NRM after HSCT in patients with hematological diseases; CRO colonization into the bloodstream has a significant impact on the DFS and OS of HSCT patients.

3-O-Substituted Quercetin: an Antibiotic-Potentiating Agent against Multidrug-Resistant Gram-Negative Enterobacteriaceae through Simultaneous Inhibition of Efflux Pump and Broad-Spectrum Carbapenemases.

The discovery of safe and efficient inhibitors against efflux pumps as well as metallo-ß-lactamases (MBL) is one of the main challenges in the development of multidrug-resistant (MDR) reversal agents which can be utilized in the treatment of carbapenem-resistant Gram-negative bacteria. In this study, we have identified that introduction of an ethylene-linked sterically demanding group at the 3-OH position of the previously reported MDR reversal agent di-F-Q endows the resulting compounds with hereto unknown multitarget inhibitory activity against both efflux pumps and broad-spectrum ß-lactamases including difficult-to-inhibit MBLs. A molecular docking study of the multitarget inhibitors against efflux pump, as well as various classes of ß-lactamases, revealed that the 3-O-alkyl substituents occupy the novel binding sites in efflux pumps as well as carbapenemases. Not surprisingly, the multitarget inhibitors rescued the antibiotic activity of a carbapenem antibiotic, meropenem (MEM), in NDM-1 (New Delhi Metallo-ß-lactamase-1)-producing carbapenem-resistant Enterobacteriaceae (CRE), and they reduced MICs of MEM more than four-fold (synergistic effect) in 8-9 out of 14 clinical strains. The antibiotic-potentiating activity of the multitarget inhibitors was also demonstrated in CRE-infected mouse model. Taken together, these results suggest that combining inhibitory activity against two critical targets in MDR Gram-negative bacteria, efflux pumps, and ß-lactamases, in one molecule is possible, and the multitarget inhibitors may provide new avenues for the discovery of safe and efficient MDR reversal agents.

IncX3 plasmid-mediated spread of blaNDM gene in Enterobacteriaceae among children in China.

BACKGROUND: blaNDM gene was prevalent among children, and became the predominant cause of severe infection in infants and children. This study aimed to investigate the epidemiology and molecular characteristics of blaNDM in Enterobacteriaceae among children in China. METHODS: Carbapenem-resistant Enterobacteriaceae (CRE) were collected in the Children's Hospital of Fudan University from January 2016 to December 2022. Five carbapenemase genes (blaKPC, blaNDM, blaVIM, blaIMP, blaOXA-48) were screened by PCR method. Multilocus sequence typing (MLST) was conducted for phylogenetic analyses. blaNDM-carrying plasmids were typed by PCR-based Incompatibility (Inc) typing method. Moreover, plasmid comparison was performed with 213 publicly available IncX3 plasmids. RESULTS: A total of 330 CRE strains were enrolled, 96.4% of which carried carbapenemase genes. blaNDM gene accounted for 64.8% (214 strains) and included four variants, including blaNDM-1 (59.8%), blaNDM-5 (39.3%), blaNDM-7 (0.5%) and blaNDM-9 (0.5%). There were no predominant MLST lineages of blaNDM carrying strains. IncX3 was the major plasmid carrying blaNDM-1 (68.0%) and blaNDM-5 (72.6%), and was dominant in blaNDM-K. penumoniae (79.8%), blaNDM-E. coli (58.2%) and blaNDM-E. cloacae (61.0%), respectively. Majority (79.0%) of clinical IncX3 plasmids in the world carried blaNDM, and the prevalence of blaNDM in IncX3 plasmids was more common in China (95.8%) than other countries (58.1%, p<0.01). CONCLUSION: blaNDM is highly prevalent in CREs among children in China. The spread of blaNDM was mainly mediated by IncX3 plasmids. Surveillance and infection control on the spread of blaNDM among children are important.

Proton pump inhibitors increase the risk of carbapenem-resistant Enterobacteriaceae colonization by facilitating the transfer of antibiotic resistance genes among bacteria in the gut microbiome.

Carbapenem-resistant Enterobacteriaceae (CRE) pose a global health threat; however, there is still limited understanding of the risk factors and underlying mechanisms of CRE colonization in the gut microbiome. We conducted a matched case-control study involving 282 intensive care unit patients to analyze influencing covariates on CRE colonization. Subsequently, their effects on the gut microbiome were analyzed in a subset of 98 patients (47 CRE carriers and 51 non-CRE carriers) using whole metagenome sequences. The concomitant use of proton pump inhibitors (PPIs) and antibiotics was a significant risk factor for CRE colonization. The gut microbiome differed according to PPI administration, even within the CRE and non-CRE groups. Moreover, the transfer of mobile genetic elements (MGEs) harboring carbapenem resistance genes (CRGs) between bacteria was higher in the PPI-treated group than in the PPI-not-treated group among CRE carriers. The concomitant use of PPIs and antibiotics significantly alters the gut microbiome and increases the risk of CRE colonization by facilitating the transfer of CRGs among bacteria of the gut microbiome. Based on these findings, improved stewardship of PPIs as well as antibiotics can provide strategies to reduce the risk of CRE colonization, thereby potentially improving patient prognosis.

Landscape of plasmids encoding ß-lactamases in disinfection residual Enterobacteriaceae from wastewater treatment plants.

Conventional disinfection processes, such as chlorination and UV radiation, are ineffective in controling antibiotic-resistant bacteria, especially disinfection residual Enterobacteriaceae (DRE) encoding ß-lactamases, some of which have been classified as "critical priority pathogens" by the World Health Organization. However, few studies have focused on the transferability, phenotype, and genetic characteristics of DRE-derived plasmids encoding ß-lactamases, especially extended-spectrum ß-lactamases and carbapenemases. In this study, we isolated 10 typical DRE harboring plasmid-mediated blaNDM, blaCTX-M, or blaTEM in post-disinfection effluent from two wastewater treatment plants (WWTPs), with transfer frequency ranging from 1.69 × 10-6 to 3.02 × 10-5. According to genomic maps of plasmids, all blaNDM and blaTEM were cascaded with IS26, and blaCTX-M was adjacent to ISEcp1 or IS26, indicating the important role of these elements in the movement of ß-lactamase-encoding genes. The presence of intact class 1 integrons on pWTPN-01 and pWTPC-03 suggested the ability of these DRE-derived plasmids to integrate other exogenous antibiotic resistance genes (ARGs). The coexistence of antibiotic, disinfectant, and heavy metal resistance genes on the same plasmid (e.g., pWTPT-03) implied the facilitating role of disinfectants and heavy metals in the transmission of DRE-derived ARGs. Notably, two plasmid transconjugants exhibited no discernible competitive fitness cost, suggesting a heightened environmental persistence. Furthermore, enhanced virulence induced by ß-lactamase-encoding plasmids in their hosts was confirmed using Galleria mellonella infection models, which might be attributed to plasmid-mediated virulence genes. Overall, this study describes the landscape of ß-lactamase-encoding plasmids in DRE, and highlights the urgent need for advanced control of DRE to keep environmental and ecological security.

Antibiotic Resistance Trends Among Enterobacteriaceae in Saudi Arabia: A Systematic Review.

Antibiotic resistance is a global public health concern that poses a significant threat to the effective treatment of bacterial infections. Enterobacteriaceae, a family of gram-negative bacteria, are associated with a wide range of infections, including urinary tract infections, bloodstream infections, and respiratory tract infections. This systematic review aimed to examine the antibiotic resistance trend among Enterobacteriaceae in Saudi Arabia in the period between 2003 and 2023. Five databases (PubMed, Medline, Ovid, Scopus, and Cochrane) were searched using the keywords "Resistance AND Enterobacteriaceae AND Saudi Arabia" in the title and abstract. All papers assessing the prevalence of resistance among Enterobacteriaceae in Saudi Arabia were included in the systematic review. Out of 97 papers that were extracted through the database search, 22 articles were considered suitable for the systematic review. The articles included 17027 Enterobacteriaceae isolates, out of which 7592 isolates were identified as resistant bacteria. The studies included various resistant strains, such as Escherichia coli and Klebsiella pneumoniae, that were responsible for various clinical conditions, including urinary tract infections, blood infections, surgical site infections, and pneumonia. In addition, the review highlighted the dynamic nature of antibiotic resistance, with the identification of new resistant bacterial species and the emergence of resistance to newer antibiotic classes over the last decade. Continued surveillance, rational antibiotic use, and the development of alternative treatment options are crucial to address the evolving landscape of antibiotic resistance among Enterobacteriaceae bacteria in the country.

Targeted remodeling of the human gut microbiome using Juemingzi (Senna seed extracts).

The genus Senna contains globally distributed plant species of which the leaves, roots, and seeds have multiple traditional medicinal and nutritional uses. Notable chemical compounds derived from Senna spp. include sennosides and emodin which have been tested for antimicrobial effects in addition to their known laxative functions. However, studies of the effects of the combined chemical components on intact human gut microbiome communities are lacking. This study evaluated the effects of Juemingzi (Senna sp.) extract on the human gut microbiome using SIFR® (Systemic Intestinal Fermentation Research) technology. After a 48-hour human fecal incubation, we measured total bacterial cell density and fermentation products including pH, gas production and concentrations of short chain fatty acids (SCFAs). The initial and post-incubation microbial community structure and functional potential were characterized using shotgun metagenomic sequencing. Juemingzi (Senna seed) extracts displayed strong, taxon-specific anti-microbial effects as indicated by significant reductions in cell density (40%) and intra-sample community diversity. Members of the Bacteroidota were nearly eliminated over the 48-hour incubation. While generally part of a healthy gut microbiome, specific species of Bacteroides can be pathogenic. The active persistence of the members of the Enterobacteriaceae and selected Actinomycetota despite the reduction in overall cell numbers was demonstrated by increased fermentative outputs including high concentrations of gas and acetate with correspondingly reduced pH. These large-scale shifts in microbial community structure indicate the need for further evaluation of dosages and potential administration with prebiotic or synbiotic supplements. Overall, the very specific effects of these extracts may offer the potential for targeted antimicrobial uses or as a tool in the targeted remodeling of the gut microbiome.

Impact of suppression of the SOS response on protein expression in clinical isolates of Escherichia coli under antimicrobial pressure of ciprofloxacin.

Introduction/objective: Suppression of the SOS response in combination with drugs damaging DNA has been proposed as a potential target to tackle antimicrobial resistance. The SOS response is the pathway used to repair bacterial DNA damage induced by antimicrobials such as quinolones. The extent of lexA-regulated protein expression and other associated systems under pressure of agents that damage bacterial DNA in clinical isolates remains unclear. The aim of this study was to assess the impact of this strategy consisting on suppression of the SOS response in combination with quinolones on the proteome profile of Escherichia coli clinical strains. Materials and methods: Five clinical isolates of E. coli carrying different chromosomally- and/or plasmid-mediated quinolone resistance mechanisms with different phenotypes were selected, with E. coli ATCC 25922 as control strain. In addition, from each clinical isolate and control, a second strain was created, in which the SOS response was suppressed by deletion of the recA gene. Bacterial inocula from all 12 strains were then exposed to 1xMIC ciprofloxacin treatment (relative to the wild-type phenotype for each isogenic pair) for 1 h. Cell pellets were collected, and proteins were digested into peptides using trypsin. Protein identification and label-free quantification were done by liquid chromatography-mass spectrometry (LC-MS) in order to identify proteins that were differentially expressed upon deletion of recA in each strain. Data analysis and statistical analysis were performed using the MaxQuant and Perseus software. Results: The proteins with the lowest expression levels were: RecA (as control), AphA, CysP, DinG, DinI, GarL, PriS, PsuG, PsuK, RpsQ, UgpB and YebG; those with the highest expression levels were: Hpf, IbpB, TufB and RpmH. Most of these expression alterations were strain-dependent and involved DNA repair processes and nucleotide, protein and carbohydrate metabolism, and transport. In isolates with suppressed SOS response, the number of underexpressed proteins was higher than overexpressed proteins. Conclusion: High genomic and proteomic variability was observed among clinical isolates and was not associated with a specific resistant phenotype. This study provides an interesting approach to identify new potential targets to combat antimicrobial resistance.

Apirhabdus apintestini gen. nov., sp. nov., a member of a novel genus of the family Enterobacteriaceae, isolated from the gut of the western honey bee Apis mellifera.

A Gram-negative, motile, rod-shaped bacterial strain, CA-0114T, was isolated from the midgut of a western honey bee, Apis mellifera. The isolate exhibited ≤96.43 % 16S rRNA gene sequence identity (1540 bp) to members of the families Enterobacteriaceae and Erwiniaceae. Phylogenetic trees based on genome blast distance phylogeny and concatenated protein sequences encoded by conserved genes atpD, fusA, gyrB, infB, leuS, pyrG and rpoB separated the isolate from other genera forming a distinct lineage in the Enterobacteriaceae. In both trees, the closest relatives were Tenebrionicola larvae YMB-R21T and Tenebrionibacter intestinalis BIT-L3T, which were isolated previously from Tenebrio molitor L., a plastic-eating mealworm. Digital DNA-DNA hybridization, orthologous average nucleotide identity and average amino acid identity values between strain CA-0114T and the closest related members within the Enterobacteriaceae were ≤23.1, 75.45 and 76.04 %, respectively. The complete genome of strain CA-0114T was 4 451669 bp with a G+C content of 52.12 mol%. Notably, the apparent inability of strain CA-0114T to ferment d-glucose, inositol and l-rhamnose in the API 20E system is unique among closely related members of the Enterobacteriaceae. Based on the results obtained through genotypic and phenotypic analysis, we propose that strain CA-0114T represents a novel species and genus within the family Enterobacteriaceae, for which we propose the name Apirhabdus apintestini gen. nov., sp. nov. (type strain CA-0114T=ATCC TSD-396T=DSM 116385T).

Analysis of Microbial Composition of Edible Insect Products Available for Human Consumption within the United States Using Traditional Microbiological Methods and Whole Genome Sequencing.

Edible insects offer a promising protein source for humans, but their food safety risks have not been previously investigated within the United States. Therefore, the aim of this study was to investigate the microbial content of processed edible insect products. A total of eight different types of edible insect products, including diving beetles, silkworms, grasshoppers, Jamaican crickets, mealworms, mole crickets, whole roasted crickets, and 100% pure cricket powder, were purchased from a large online retailer for the analysis. All the products were purchased in August 2022 and examined between August 2022 and November 2022. Traditional microbiological methods were employed to determine microbial counts for each product type using three replicates (total number of samples = 24). This included assessing aerobic bacterial spore, lactic acid bacteria, Enterobacteriaceae, total viable counts, and the presence of Salmonella. Additionally, whole genome sequencing was employed to further characterize selected colonies (n = 96). Microbial counts data were statistically analyzed using one-way ANOVA, while sequence data were taxonomically classified using Sepia.Bacilluscereusgroup isolates underwent additional characterization with Btyper3. Product type significantly influenced total viable counts, bacterial spore counts, and lactic acid bacteria counts (P = 0.00391, P = 0.0065, and P < 0.001, respectively), with counts ranging from < 1.70 to 6.01 Log10 CFU/g, <1.70 to 5.25 Log10 CFU/g, and < 1.70 to 4.86 Log10 CFU/g, respectively. Enterobacteriaceae were only detected in mole crickets (<2.30 Log10 CFU/g) and house cricket powder (<2.15 Log10 CFU/g). All samples were negative for Salmonella. Whole genome sequencing revealed the presence of 12 different bacterial genera among the analyzed isolates, with a majority belonging to the Bacillus genus. Some of the isolates of Bacillus cereus group were identified as biovar Emeticus. Overall, although edible insects offer a promising food alternative, the presence of Bacillus cereus group in some products could raise concerns regarding food safety.

Preclinical characterization and in silico safety assessment of three virulent bacteriophages targeting carbapenem-resistant uropathogenic Escherichia coli.

Phage therapy has recently been revitalized in the West with many successful applications against multi-drug-resistant bacterial infections. However, the lack of geographically diverse bacteriophage (phage) genomes has constrained our understanding of phage diversity and its genetics underpinning host specificity, lytic capability, and phage-bacteria co-evolution. This study aims to locally isolate virulent phages against uropathogenic Escherichia coli (E. coli) and study its phenotypic and genomic features. Three obligately virulent Escherichia phages (øEc_Makalu_001, øEc_Makalu_002, and øEc_Makalu_003) that could infect uropathogenic E. coli were isolated and characterized. All three phages belonged to Krischvirus genus. One-step growth curve showed that the latent period of the phages ranged from 15 to 20 min, the outbreak period ~ 50 min, and the burst size ranged between 74 and 127 PFU/bacterium. Moreover, the phages could tolerate a pH range of 6 to 9 and a temperature range of 25-37 °C for up to 180 min without significant loss of phage viability. All phages showed a broad host spectrum and could lyse up to 30% of the 35 tested E. coli isolates. Genomes of all phages were approximately ~ 163 kb with a gene density of 1.73 gene/kbp and an average gene length of ~ 951 bp. The coding density in all phages was approximately 95%. Putative lysin, holin, endolysin, and spanin genes were found in the genomes of all three phages. All phages were strictly virulent with functional lysis modules and lacked any known virulence or toxin genes and antimicrobial resistance genes. Pre-clinical experimental and genomic analysis suggest these phages may be suitable candidates for therapeutic applications.

Enterobacteriaceae in the Human Gut: Dynamics and Ecological Roles in Health and Disease.

The human gut microbiota plays a crucial role in maintaining host health. Our review explores the prevalence and dynamics of Enterobacteriaceae, a bacterial family within the Proteobacteria phylum, in the human gut which represents a small fraction of the gut microbiota in healthy conditions. Even though their roles are not yet fully understood, Enterobacteriaceae and especially Escherichia coli (E. coli) play a part in creating an anaerobic environment, producing vitamins and protecting against pathogenic infections. The composition and residency of E. coli strains in the gut fluctuate among individuals and is influenced by many factors such as geography, diet and health. Dysbiosis, characterized by alterations in the microbial composition of the gut microbiota, is associated with various diseases, including obesity, inflammatory bowel diseases and metabolic disorders. A consistent pattern in dysbiosis is the expansion of Proteobacteria, particularly Enterobacteriaceae, which has been proposed as a potential marker for intestinal and extra-intestinal inflammatory diseases. Here we develop the potential mechanisms contributing to Enterobacteriaceae proliferation during dysbiosis, including changes in oxygen levels, alterations in mucosal substrates and dietary factors. Better knowledge of these mechanisms is important for developing strategies to restore a balanced gut microbiota and reduce the negative consequences of the Enterobacteriaceae bloom.

Predictive Modeling of Phenotypic Antimicrobial Susceptibility of Selected Beta-Lactam Antimicrobials from Beta-Lactamase Resistance Genes.

The outcome of bacterial infection management relies on prompt diagnosis and effective treatment, but conventional antimicrobial susceptibility testing can be slow and labor-intensive. Therefore, this study aims to predict phenotypic antimicrobial susceptibility of selected beta-lactam antimicrobials in the bacteria of the family Enterobacteriaceae from different beta-lactamase resistance genotypes. Using human datasets extracted from the Antimicrobial Testing Leadership and Surveillance (ATLAS) program conducted by Pfizer and retail meat datasets from the National Antimicrobial Resistance Monitoring System for Enteric Bacteria (NARMS), we used a robust or weighted least square multivariable linear regression modeling framework to explore the relationship between antimicrobial susceptibility data of beta-lactam antimicrobials and different types of beta-lactamase resistance genes. In humans, in the presence of the blaCTX-M-1, blaCTX-M-2, blaCTX-M-8/25, and blaCTX-M-9 groups, MICs of cephalosporins significantly increased by values between 0.34-3.07 µg/mL, however, the MICs of carbapenem significantly decreased by values between 0.81-0.87 µg/mL. In the presence of carbapenemase genes (blaKPC, blaNDM, blaIMP, and blaVIM), the MICs of cephalosporin antimicrobials significantly increased by values between 1.06-5.77 µg/mL, while the MICs of carbapenem antimicrobials significantly increased by values between 5.39-67.38 µg/mL. In retail meat, MIC of ceftriaxone increased significantly in the presence of blaCMY-2, blaCTX-M-1, blaCTX-M-55, blaCTX-M-65, and blaSHV-2 by 55.16 µg/mL, 222.70 µg/mL, 250.81 µg/mL, 204.89 µg/mL, and 31.51 µg/mL respectively. MIC of cefoxitin increased significantly in the presence of blaCTX-M-65 and blaTEM-1 by 1.57 µg/mL and 1.04 µg/mL respectively. In the presence of blaCMY-2, MIC of cefoxitin increased by an average of 8.66 µg/mL over 17 years. Compared to E. coli isolates, MIC of cefoxitin in Salmonella enterica isolates decreased significantly by 0.67 µg/mL. On the other hand, MIC of ceftiofur increased in the presence of blaCTX-M-1, blaCTX-M-65, blaSHV-2, and blaTEM-1 by 8.82 µg/mL, 9.11 µg/mL, 8.18 µg/mL, and 1.04 µg/mL respectively. In the presence of blaCMY-2, MIC of ceftiofur increased by an average of 10.20 µg/mL over 14 years. The ability to predict antimicrobial susceptibility of beta-lactam antimicrobials directly from beta-lactamase resistance genes may help reduce the reliance on routine phenotypic testing with higher turnaround times in diagnostic, therapeutic, and surveillance of antimicrobial-resistant bacteria of the family Enterobacteriaceae.

Detection of Extended-Spectrum Beta-Lactamase (ESBL)-Producing Enterobacteriaceae from Diseased Broiler Chickens in Lusaka District, Zambia.

Poultry products in Zambia form an integral part of the human diet in many households, as they are cheap and easy to produce. The burden of poultry diseases has, however, remained a major challenge. Growing consumer demand for poultry products in Zambia has resulted in non-prudent antimicrobial use on farms, intending to prevent and treat poultry diseases for growth optimisation and maximising profits. This cross-sectional study aimed to identify the different types of bacteria causing diseases in chickens in Lusaka and to detect the extended-spectrum lactamase (ESBL)-encoding genes. We collected 215 samples from 91 diseased chickens at three post-mortem facilities and screened them for Gram-negative bacteria. Of these samples, 103 tested positive for various clinically relevant Enterobacteriaceae, including Enterobacter (43/103, 41.7%), Escherichia coli (20/103, 19.4%), Salmonella (10/103, 9.7%), and Shigella (8/103, 7.8%). Other isolated bacteria included Yersinia, Morganella, Proteus, and Klebsiella, which accounted for 21.4%. E. coli, Enterobacter, Salmonella, and Shigella were subjected to antimicrobial susceptibility testing. The results revealed that E. coli, Enterobacter, and Shigella were highly resistant to tetracycline, ampicillin, amoxicillin, and trimethoprim-sulfamethoxazole, while Salmonella showed complete susceptibility to all tested antibiotics. The observed resistance patterns correlated with antimicrobial usage estimated from sales data from a large-scale wholesale and retail company. Six (6/14, 42.9%) E. coli isolates tested positive for blaCTX-M, whilst eight (8/14, 57.1%) Enterobacter samples tested positive for blaTEM. Interestingly, four (4/6, 66.7%) of the E. coli isolates carrying blaCTX-M-positive strains were also positive for blaTEM. Sanger sequencing of the PCR products revealed that five (5/6, 83.3%) of the abovementioned isolates possessed the blaCTX-M-15 allele. The results suggest the presence of potentially pathogenic ESBL-producing Enterobacteriaceae in poultry, threatening public health.

Genomic Insights into the expansion of Carbapenem-Resistant Klebsiella pneumoniae within Portuguese Hospitals.

Carbapenem-resistant Klebsiella pneumoniae complex (CR-KP) are a public health concern, causing infections with a high mortality rate, limited therapeutic options and challenging infection control strategies. In Portugal CR-KP rate has increased steeply, but the factors associated to this expansion are poorly explored. To address this question we compared, by phylogenetic and resistome analysis, the draft genomes of 200 CR-KP isolates collected in 2017-2019 from five hospitals in the Lisbon region, Portugal. We found that CR-KP belonged mainly to ST13 (29%), ST17 (15%), ST348 (13%), ST231 (12%) and ST147 (7%). Carbapenem resistance was conferred mostly by KPC-3 (74%) or OXA-181 (18%) presence, which were associated with IncF/IncN and IncX plasmids, respectively. Almost all isolates were multidrug resistant harbouring resistance determinants to aminoglycosides, beta-lactams, trimethoprim, fosfomycin, quinolones and sulphonamides. In addition, 11% of isolates were resistant to colistin. Colonizing and infection isolates were highly related and most colonized patients (89%) reported a previous hospitalization. Moreover, among the 171 events of cross-dissemination identified, by cgMLST data analysis (<5 alleles), 41 occurred between different hospitals and 130 within the same hospital. Our results suggest that CR-KP dissemination in the Lisbon region result from acquisition of carbapenemases in mobile genetic elements, influx of CR-KP into the hospitals by colonized ambulatory patients and transmission of CR-KP within and between hospitals. Our data reinforces that the prudent use of carbapenems, patients screening at hospital entrance, and improvement of infection control will be needed to decrease the burden of CR-KP infection in Portugal.

Clinical characteristics and risk factors for septic shock in patients with pyometra: A retrospective multicenter cohort study.

BACKGROUND: Pyometra is a disease characterized by the collection of pus in the uterus. The clinical characteristics and etiology of pyometra have not been sufficiently described. In this study, we investigated the clinical characteristics, epidemiology, outcomes, and risk factors of septic shock in patients with pyometra. METHODS: Patients with pyometra admitted to one of four university-affiliated hospitals between January 2010 to August 2022 were enrolled. Pyometra cases associated with peripartum infection and surgical site infection were excluded. Clinical characteristics and outcomes of pyometra were described, and pyometra patients with or without septic shock were compared. RESULTS: A total of 192 patients was included. Twenty-eight-day all-cause mortality was 5.0%, and the 1-year recurrence rate was 6.3%. Median patient age was 77.5 years. The two most common symptoms were abdominal pain (49.0%) and vaginal discharge (47.9%). Escherichia coli (40.1%), Klebsiella pneumoniae (16.7%), and Streptococcus spp.(16.0%) were the pathogens most frequently isolated by conventional culture; those isolated from polymerase chain reaction were Mycoplasma hominis (48.0%), and Ureaplasma spp. (32.0%). In multivariable analysis, fever, uterine perforation, and dementia were associated with increased incidence of septic shock, while vaginal discharge was associated with a lower incidence of septic shock. CONCLUSIONS: Our findings suggest that pyometra is a unique gynecological infectious syndrome in post-menopausal individuals. The most common associated pathogens are similar to those involved in urinary tract infections rather than those of sexually transmitted diseases. Decreased cognitive function could delay early diagnosis of pyometra and lead to septic shock and higher mortality.

Comparative effectiveness and mortality of colistin monotherapy versus colistin-fosfomycin combination therapy for the treatment of carbapenem-resistant Enterobacteriaceae (CRE) infections: A propensity score analysis.

BACKGROUND: Carbapenem-resistant Enterobacteriaceae (CRE) infections pose a significant threat to global health due to limited treatment options and high mortality rates. Colistin-based regimens have emerged as a primary treatment approach, but the effectiveness and mortality outcomes of colistin monotherapy versus colistin-fosfomycin combination therapy remain uncertain. This study aims to compare the effectiveness and mortality of colistin monotherapy and colistin-fosfomycin combination therapy for CRE infections. Notably, our study is the first to undertake a comprehensive examination of the effectiveness and mortality outcomes between colistin monotherapy and colistin-fosfomycin combination therapy in the context of CRE infections. METHODS: A retrospective cohort study was conducted using data from patients diagnosed with carbapenem-resistant Enterobacteriaceae (CRE) infections at Nakornping Hospital during 2015 to 2022. Inverse probability weighting (IPW) was employed to create balanced cohorts of patients receiving either colistin monotherapy or colistin-fosfomycin combination therapy. The primary outcome measure was treatment effectiveness, assessed by 30-day mortality. Secondary outcome measures included clinical response, mortality at the end of treatment, and microbiologic response. Univariate and multivariate logistic regression analysis were employed after applying propensity score weighting using inverse probability of weighting (IPW). RESULTS: A total of 220 patients were included in the analysis, with 67 receiving colistin monotherapy and 153 receiving colistin-fosfomycin combination therapy. Propensity score weighting using IPW balanced the baseline characteristics between the two groups. The effectiveness of treatment, as measured by 30-day mortality, was not significantly different between the colistin monotherapy group and the colistin-fosfomycin combination therapy group (adjusted odds ratio [aOR] = 1.51, 95% confidence interval [CI]: 0.60-3.78, p = 0.383). Similarly, no significant difference was observed in the mortality at the end of treatment between the two groups (aOR = 1.26, 95% CI: 0.55-2.90, p = 0.576). The clinical response (aOR = 1.48, 95% CI: 0.61-3.59, p = 0.383) and microbiologic response (aOR = 0.66, 95% CI: 0.18-2.38, p = 0.527) were similar between the colistin monotherapy and colistin-fosfomycin combination therapy groups. CONCLUSION: The propensity score analysis among 220 matched patients showed comparable treatment effectiveness and mortality between colistin monotherapy and colistin-fosfomycin combination therapy for CRE infections. These results suggest that colistin monotherapy may be as effective as combination therapy. More prospective randomized controlled trials are needed to confirm these findings and establish optimal CRE treatment strategies.