Members Of Enterobacteriaceae Research Articles

Membrane-targeting, ultrashort lipopeptide acts as an antibiotic adjuvant and sensitizes MDR gram-negative pathogens toward narrow-spectrum antibiotics

Globally, infections due to multi-drug resistant (MDR) Gram-negative bacterial (GNB) pathogens are on the rise, negatively impacting morbidity and mortality, necessitating urgent treatment alternatives. Herein, we report a detailed bio-evaluation of an ultrashort, cationic lipopeptide ‘SVAP9I’ that demonstrated potent antibiotic activity and acted as an adjuvant to potentiate existing antibiotic classes towards GNBs. Newly synthesized lipopeptides were screened against ESKAPE pathogens and cytotoxicity assays were performed to evaluate the selectivity index (SI). SVAP9I exhibited broad-spectrum antibacterial activity against critical MDR-GNB pathogens including members of Enterobacteriaceae (MIC 4–8 mg/L), with a favorable CC50 value of ≥100 mg/L and no detectable resistance even after 50th serial passage. It demonstrated fast concentration-dependent bactericidal action as determined via time-kill analysis and also retained full potency against polymyxin B-resistant E. coli, indicating distinct mode of action. SVAP9I targeted E. coli's outer and inner membranes by binding to LPS and phospholipids such as cardiolipin and phosphatidylglycerol. Membrane damage resulted in ROS generation, depleted intracellular ATP concentration and a concomitant increase in extracellular ATP. Checkerboard assays showed SVAP9I's synergism with narrow-spectrum antibiotics like vancomycin, fusidic acid and rifampicin, potentiating their efficacy against MDR-GNB pathogens, including carbapenem-resistant Acinetobacter baumannii (CRAB), a WHO critical priority pathogen. In a murine neutropenic thigh infection model, SVAP9I and rifampicin synergized to express excellent antibacterial efficacy against MDR-CRAB outcompeting polymyxin B. Taken together, SVAP9I's distinct membrane-targeting broad-spectrum action, lack of resistance and strong in vitro andin vivopotency in synergism with narrow spectrum antibiotics like rifampicin suggests its potential as a novel antibiotic adjuvant for the treatment of serious MDR-GNB infections.

Rapidly engineering an osmotic-pressure-tolerant gut bacterium for efficient non-sterile production of bulk chemicals

The concept of next-generation industrial biotechnology (NGIB) has been presented as a green biomanufacturing approach based on the use of low-cost substrates in an open and continuous non-sterilization fermentation process. However, the realization of NGIB, partially through the utilization of extremophiles, has been impeded by the challenges in engineering these extremophiles. A previously unexplored solution for advancing NGIB is to obtain readily engineerable microorganisms with osmotic-pressure tolerance. Here, Jejubacter sp. L23 strain, a Gram-negative, rod-shaped, osmotic-pressure-tolerant bacterium, was isolated from the gut of superworm Zophobas atratus and identified as a new member of the family Enterobacteriaceae, with high 16S rDNA similarity to Escherichia coli (95.2 %). The growth of L23 strain was observed at 10 to 45 °C and pH 4.0 to 10.0 in the presence of 0 to 12 % (w/v) NaCl. We systematically investigated the genetic parts, molecular tools, and manipulation approaches applicable to the L23 strain, and successfully conducted chromosomal genetic engineering, e.g. base-editing and deletion, by using high-efficiency CRISPR systems under low-salt conditions. Furthermore, the metabolic network of L23 strain was rewired to obtain the engineered L23R7 strain, which could display stable expression of exogenous genes and produce bulk chemicals at high yields using inulin, abundant non-food biomass, and salt water without sterilization. For example, 41 g/L isobutanol was produced by utilizing inulin in a 3-L bioreactor through a non-sterile fermentation process. This potent industrial strain holds the promise of promoting the development of NGIB as a sustainable biomanufacturing solution.

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).

Structural insight into Escherichia coli CsgA amyloid fibril assembly.

The discovery of functional amyloids in bacteria dates back several decades, and our understanding of the Escherichia coli curli biogenesis system has gradually expanded over time. However, due to its high aggregation propensity and intrinsically disordered nature, CsgA, the main structural component of curli fibrils, has eluded comprehensive structural characterization. Recent advancements in cryo-electron microscopy (cryo-EM) offer a promising tool to achieve high-resolution structural insights into E. coli CsgA fibrils. In this study, we outline an approach to addressing the colloidal instability challenges associated with CsgA, achieved through engineering and electrostatic repulsion. Then, we present the cryo-EM structure of CsgA fibrils at 3.62 Å resolution. This structure provides new insights into the cross-β structure of E. coli CsgA. Additionally, our study identifies two distinct spatial arrangements within several CsgA fibrils, a 2-CsgA-fibril pair and a 3-CsgA-fibril bundle, shedding light on the intricate hierarchy of the biofilm extracellular matrix and laying the foundation for precise manipulation of CsgA-derived biomaterials.IMPORTANCEThe visualization of the architecture of Escherichia coli CsgA amyloid fibril has been a longstanding research question, for which a high-resolution structure is still unavailable. CsgA serves as a major subunit of curli, the primary component of the extracellular matrix generated by bacteria. The support provided by this extracellular matrix enables bacterial biofilms to resist antibiotic treatment, significantly impacting human health. CsgA has been identified in members of Enterobacteriaceae, with pathogenic E. coli being the most well-known model system. Our novel insights into the structure of E. coli CsgA protofilaments form the basis for drug design targeting diseases associated with biofilms. Additionally, CsgA is widely researched in biomaterials due to its self-assembly characteristics. The resolved spatial arrangements of CsgA amyloids revealed in our study will further enhance the precision design of functional biomaterials. Therefore, our study uniquely contributes to the understanding of CsgA amyloids for both microbiology and material science.

Prevalence and antimicrobial susceptibility of Enterobacteriaceae isolated from ready-to-eat foods retailed in Damietta, Egypt

ABSTRACT The presence of Enterobacteria in food in large numbers indicates fecal contamination. This study was carried out to determine the susceptibility of Enterobacteriaceae strains isolated from ready-to-eat foods from the Damietta market. A total of 40 random ready-to-eat food samples were collected from different localities at Damietta Governorate, Egypt. These samples were examined for Enterobacteriaceae count, and identification of members by conventional biochemical methods and antimicrobial susceptibility was determined by disc diffusion method. Enterobacteriaceae isolates were detected among 34/40 (85%) examined Ready-to-eat food samples. A total of 139 isolates were identified. The most frequent genus was Klebsiella followed by Enterobacter. Other members such as Citrobacter spp., Proteus spp., Providencia spp., and E. coli spp. were also identified. The isolates showed different susceptibility to 12 tested antimicrobials. Most of the tested isolates showed high resistance percentages to cefepime and ampicillin. Imipenem, ciprofloxacin, amikacin, gentamicin, and cefotaxime were the most effective antimicrobials. In addition, 29% of the isolates were multiple-drug resistant (MDR). The high prevalence of members of Enterobacteriaceae in different food sources is a reliable index of food contamination. So, it is recommended to reconsider restrictions on hygiene and sanitary control in the current food acts and regulations.

CTX-M-127 with I176F mutations found in bacteria isolates from Bangladeshi circulating banknotes.

Extended-spectrum beta-lactamase (ESBL)-producing organisms are widely recognized as clinically relevant causes of difficult-to-treat infections. CTX-M has formed a rapidly growing family distributed worldwide among a wide range of clinical bacteria, particularly members of Enterobacteriaceae. Circulating banknotes, exchanged daily among people, pose a potential vehicle for transmitting multidrug resistance. We screened for ESBL-carrying bacteria in the present study and reported CTX-M mutations in Bangladesh's banknotes. We sequenced the genes and performed homology modeling using the Swiss model with CTX-M-15 (4HBT) as a template. Then, we performed molecular docking of mecillinam with the template and the generated model using Autodock 4.2 (Release 4.2.6). After docking, we visually inspected the complexes built using Autodock tools for polar contacts and pi-pi interactions in PyMOL 2.5.4. Our partially sequenced blaCTX-M was related to blaCTX-M-10 and blaCTX-M-15. We observed multiple single-nucleotide substitution mutations, i.e., G613T (silent mutation), A626T (I176F), and A503G (N135D). Homology modeling showed high similarity when the model was superimposed over the template. The orientation of Asn (135) in the template and Asp (135) in the model does not show a significant difference. Likewise, Ile (176) in the template and Phe (176) in the model offer the same orientation. Our generated model could bind to Lys237, Ser240, and Asp135 residues with the lowest binding energy on docking. Our predicted binding of the mecillinam to the mutated D-135 residue in the model indicates contributions and supports previous reports proposing CTX-M-15 to CTX-M-127 mutational conversion on the mecillinum resistance phenotype.

Global phylogeography and genomic characterization of blaKPC and blaNDM-positive clinical Klebsiella aerogenes isolates from China, 2016-2022

Carbapenem-resistant Klebsiella aerogenes (CRKA), being one of the members of carbapenem-resistant Enterobacteriaceae (CRE), has caused great public health concern, but with fewer studies compared to other CRE members. Furthermore, studies on phylogenetic analysis based on whole genome Single-Nucleotide Polymorphism (SNP) of CRKA were limited. Here, 20 CRKA isolates (11 blaKPC-2-bearing and 9 blaNDM-1/5-harboring) were characterized by antimicrobial susceptibility testing, conjugation assay, whole genome sequencing (WGS) and bioinformatics analysis. Additionally, the phylogeographic relationships of K. aerogenes were further investigated from public databases. All isolates were multidrug-resistant (MDR) bacteria, and they demonstrated susceptibility to colistin. Most blaKPC-2 or blaNDM-1/5-carrying plasmids were found to be conjugative. Phylogenetic analysis revealed the clonal dissemination of K. aerogenes primarily occurred within clinical settings. Notably, some strains in this study showed the potential for clonal transmission, sharing few SNPs between K. aerogenes and KPC- and/or NDM-positive K. aerogenes isolated from various countries. The STs of K. aerogenes strains had significant diversity. WGS analysis showed that the IncFIIK plasmid was the most prevalent carrier of blaKPC-2, and, blaNDM-1/5 were detected on the IncX3 plasmids. The Tn6296 and Tn3000 transposons were most common vehicles for facilitating the transmission of blaKPC-2 and blaNDM-1/5, respectively. This study highlights the importance of continuous screening and surveillance by WGS for analysis of drug-resistant strains in hospital settings, and provide clinical information that supports epidemiological and public health research on human pathogens.

Colicins produced by Escherichia coli, a review

Bacteria produce proteins called bacteriocins and can inhibit or even kill closely related species. Among the bacteriocins, Colicins are by far the most well-characterized group. They are generated by Escherichia coli and other Enterobacteriaceae family members, and they are effective against them. At least 34 Colicins have been identified, with 21 of them being more thoroughly studied, and they share not common set of properties. A group of carcinogenic bacteria generates colicin proteins under stressful conditions. The kind of stress that is causing the inductive activity must be specified. The release of colicins does not always result in the death of the cell that generates them.

Carbapenem Resistant Pseudomonas aeruginosa, Enterobacteriaceae, and Acinetobacter baumannii , Prevalence, Biochemical Identification and Clinical Characteristics in Karachi, Pakistan

Antibiotic resistant pathogens are affecting the community and healthcare institutions all over the world. Pakistan is a developing country and resistance to drugs is the main issue and is of great importance. Current study is focused on isolation and identification of bacterial pathogens, i.e. member of Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii for the evaluation of prevalence, distribution of sensitivity and antibiogram of different antibiotics and carbapenem resistance isolates with phenotypic detection of resistant gene. Total 200 samples of different sources were collected and tested for bacterial pathogens. Out of 200 samples, 83 (41.5%) were found positive for different bacterial pathogens while 117 (58.5%) were negative. Among these 83 positive samples, Urine 43 (51.8%), Pus 22 (26.5%), Blood 8 (9.8%), Tissue 3 (3.6%), wound swab 2 (2.4%), Sputum 3 (3.6%) and HVS 2 (2.4%). E. coli 37 (44.6%), Klebsiella species 23 (27.7%), Proteus vulgaris 8 (9.6%), Ps. aeruginosa 4 (4.8%), Acinetobacter baumannii 4 (4.8%), C. freundii 2 (2.4%), S. typhi 2 (2.4%), P. mirabilis 1 (1.2%), M. morganii 1 (1.2%) and member of Enterobacteriaceae 1 (1.2%) were identified. Out of 83 (41.5%) positive samples there are 17 samples which showed resistance against Imipenem (IPM) and were further processed by phenotypic method Modified Hodge Test MHT. After Modified Hoge Test (MHT) among all these 17 samples there were only 07 (39%) positive and the remaining 11 (61%) were negative, its mean there was no gene involve in 11 samples.

Antibacterial Resistance and Phenotypic Detection of Extended Spectrum Beta-Lactamase (ESBL) Producing Enterobacteriaceae Isolated from Environmental Sources in Nasarawa State, Nigeria

The multidrug resistant member of Enterobacteriaceae is common causes of community related infections. A study on antibacterial resistance and phenotypic detection of Extended Spectrum Beta-Lactamase (ESBL) producing Enterobacteriaceae isolated from environmental sources in Nasarawa state, Nigeria was carried out. A total of 400 samples comprised of air, water, soil, vegetable, and sewage were collected and 89 samples were multidrug resistant. Enterobacteriaceae were isolated and identified using commercial biochemical kit Eneterosystem 18R. The antibacterial susceptibility testing of Enterobacteriaceae isolates was carried out using the clinical and laboratory standards institute (CLSI). The phenotypic confirmation test of ESBL was carried out by double disc synergy test (DDST) method. The occurrence of multidrug resistant isolates shows that Escherichia coli (24.71%) and Klebsiella pneumoniae (19.10%) find to be high while the occurrence of Proteus mirabilis and Citrobacter freundii (6.74%) were low. The Enterobacteriaceae isolates were more resistant to Cefuroxime, Cefexime, Amoxicillin Clavulanate and Imipenem/Cilastatin with percentage resistance ranges from 62.5%–100%. The occurrence of ESBL producers shows that Klebsiella pneumoniae (66.7%) and Shigella flexneri (57.14%) were found to be high while the occurrence of ESBL was low in Proteus mirabilis (20.0%). The Beta Lactam and gentamycin antibiotics were not effective against the MDR isolates and most of the isolates were ESBL producers.

Staining-Enhanced Peroxidase-Mimicking Gold Nanoparticles in Nano-ELISA for Highly Sensitive Detection of Klebsiella pneumoniae.

Klebsiella pneumoniae, a member of the family Enterobacteriaceae, is a rod-shaped, Gram-negative bacterium, mainly found in the hospital environment and medical tools. It is the leading cause of nosocomial infection, characterized by bloodstream infection, wound site infection, urinary tract infection, and sepsis, mostly in older adults, newborn infants, and immunocompromised patients. This present study demonstrated a novel diagnostic method for K. pneumoniae detection based on the gold nanozyme activity for the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2. The nanozyme activity of AuNPs with staining enhancement was statistically three times higher than that of the bare AuNPs in solid absorption at 650 nm. Nano-ELISA with staining enhancement could detect as low as 102 CFUs/mL of K. pneumoniae concentration, as the cutoff value was determined to be 0.158, which boosted the sensitivity of the immunoreactions by up to 100-fold. The detection limit of our assays was 26.023 CFUs/mL, and the limit of quantification was 78.857 CFUs/mL. There was no cross-reaction against other bacteria, which proved the immunoassays' remarkable specificity for recognizing K. pneumoniae. Taken together, we successfully developed and optimized the highly sensitive and decently specific nano-ELISA strategy that might be applicable for detecting various other bacterial pathogens.

Molecular characterization and in-depth genomic analysis to unravel the pathogenic features of an environmental isolate Enterobacter sp. S-33.

Enterobacter species represent widely distributed opportunistic pathogens, commonly associated with plants and humans. In the present study, we performed a detailed molecular characterization as well as genomic study of a type VI secretion system (T6SS) bacterium belonging to member of the family Enterobacteriaceae and named Enterobacter sp. S-33. The comparative sequence analysis of the 16S rRNA gene showed that the strain was closely related to other Enterobacter species. The complete genome of the strain with a genome size of 4.6 Mbp and GC-content of 55.63% was obtained through high-quality sequencing. The genomic analysis with online tools unravelled the various genes belonging to the bacterial secretion system, antibiotic resistance, virulence, efflux pumps, etc. The isolate showed the motility behavior that contributes to Enterobacter persistence in a stressed environment and further supports infections. PCR amplification and further sequencing confirmed the presence of drug-efflux genes acrA, acrB, and outer membrane genes, viz. OmpA, OmpC, and OmpF. The cell surface hydrophobicity and co-aggregation assay against different bacterial strains illustrated its putative pathogenic nature. Genome mining identified various biosynthetic gene clusters (BGCs) corresponding to non-ribosomal proteins (NRPS), siderophore, and arylpolyene production. Briefly, genome sequencing and detailed characterization of environmental Enterobacter isolate will assist in understanding the epidemiology of Enterobacter species, and the further prevention and treatment of infectious diseases caused by these broad-host range species.

Obesity is the main driver of altered gut microbiome functions in the metabolically unhealthy

ABSTRACT Obesity (OB) and cardiometabolic disease are major public health issues linked to changes in the gut microbiome. OB and poor cardiometabolic health status (CHS) are often comorbid, which hinders efforts to identify components of the microbiome uniquely linked to either one. Here, we used a deeply phenotyped cohort of 408 adults from Colombia, including subjects with OB, unhealthy CHS, or both, to validate previously reported features of gut microbiome function and diversity independently correlated with OB or CHS using fecal metagenomes. OB was defined by body mass index, waist circumference, and body fat; CHS as healthy or unhealthy according to blood biochemistry and anthropometric data. We found that OB, more so than metabolic status, drove associations with gut microbiome structure and functions. The microbiome of obese individuals with and without co-existing unhealthy CHS was characterized by reduced metagenomic diversity, reduced fermentative potential and elevated capacity to respond to oxidative stress and produce bacterial antigens. Disease-linked features were correlated with increased host blood pressure and inflammatory markers, and were mainly contributed by members of the family Enterobacteriaceae. Our results link OB with a microbiome able to tolerate an inflammatory and oxygenated gut state, and suggest that OB is the main driver of microbiome functional differences when poor CHS is a comorbidity.

Probiotic Escherichia coli Nissle 1917 protect chicks from damage caused by Salmonella enterica serovar Enteritidis colonization.

As a foodborne pathogen of global importance, Salmonella enterica serovar Enteritidis (S. Enteritidis) is a threat to public health that is mainly spread by poultry products. Intestinal Enterobacteriaceae can inhibit the colonization of S. Enteritidis and are regarded as a potential antibiotic substitute. We investigated, in chicks, the anti-S. Enteritidis effects of Escherichia coli (E.coli) Nissle 1917, the most well-known probiotic member of Enterobacteriaceae. Eighty 1-d-old healthy female AA broilers were randomly divided into 4 groups, with 20 in each group, namely the negative control (group P), the E.coli Nissle 1917-treated group (group N), the S. Enteritidis-infected group (group S) and the E.coli Nissle 1917-treated and S. Enteritidis-infected group (group NS). From d 5 to 7, chicks in groups N and NS were orally gavaged once a day with E.coli Nissle 1917 and in groups P and S were administered the same volume of sterile PBS. At d 8, the chicks in groups S and NS were orally gavaged with S. Enteritidis and in groups P and N were administered the same volume of sterile PBS. Sampling was conducted 24h after challenge. Results showed that gavage of E.coli Nissle 1917 reduced the spleen index, Salmonella loads, and inflammation (P<0.05). It improved intestinal morphology and intestinal barrier function (P<0.05). S. Enteritidis infection significantly reduced mRNA expression of angiotensin-converting enzyme 2 (ACE2) and solute carrier family 6-member 19 (SLC6A19) in the cecum and the content of Gly, Ser, Gln, and Trp in the serum (P<0.05). Pretreatment with E.coli Nissle 1917 yielded mRNA expression of ACE2 and SLC6A19 in the cecum and levels of Gly, Ser, Gln, and Trp in the serum similar to that of uninfected chicks (P<0.05). Additionally, E.coli Nissle 1917 altered cecum microbiota composition and enriched the abundance of E.coli, Lactobacillales, and Lachnospiraceae. These findings reveal that the probiotic E.coli Nissle 1917 reduced S. Enteritidis infection and shows enormous potential as an alternative to antibiotics.

The Prevalence of Extended Spectrum Beta Lactamase Producing Citrrobacter Freundii and Pprovidencia Stuartii from Medical Laboratories in Parts of Lagos State, Nigeria

Citrobacter freundii and Providencia stuartii are members of the family Enterobacteriaceae which are emerging as important Extended Spectrum Beta Lactamase (ESBL) producing pathogens. This study was aimed to determine the prevalence of ESBL and carbapenemase production in C. freundii and P. stuartii. from laboratories in the northwest of Lagos. A total of 134 isolates were obtained from laboratories. Isolate identification, antibiotics susceptibility testing, and phenotypic detection of ESBL production were done using standardized procedures. Multiplex polymerase chain reaction (PCR) was performed on ESBL producing isolates to detect blaTEM, blaSHV, blaCTX-M, blaKPC, blaVIM, blaIMP and blaOXA. Out of 134 isolates 15 (11.2%) were Citrobacter freundii and only 2 (1.5%) were of the Providencia genus both of which were Providencia rettgeri and Providencia stuartii. A total of 9 (60%) tested positive for the production of Extended Spectrum Beta Lactamase (ESBL) enzyme while only Providencia stuartii of the Providencia genus also produced ESBL. Also, 8 (47%) of the initial Citrobacter and Providencia isolates produced carbapenemase Molecular characterization showed that 4 (44.4%) and 1 (100%) of the Citrobacter freundii and Providencia stuartii isolates respectively, displayed the presence of CTX-M gene while 2 (20%) of all ESBL producing isolates also possessed SHV gene including 1 (11.1%) of ESBL producing Citrobacter freundii and 1 (100%) of ESBL producing Providencia stuartii. Antibiotic susceptibility testing showed that Citrobacter freundii displayed the highest resistance to Ceftazidime and Cefotaxime at 71.4% and the least resistance to Amoxicillin-Clavulanate at 28.6%. Providencia stuartii also displayed absolute resistance (100%), to cefotaxime and ciprofloxacin and absolute susceptibility (100%) to Ceftazidime. Also, Citrobacter freundii showed the same resistance patterns with 57.1% resistance to Gentamicin, Cefuroxime and Ceftriaxone antibiotics while Providencia stuartii showed varied resistance patterns with absolute resistance (100%), absolute susceptibility (100%) and absolute resistance (100%) to Gentamicin, Cefuroxime and Ceftriaxone antibiotics respectively. In conclusion, the Multiple Antibiotic Resistance Index values of both bacterial genera showed that 70.6% were from High-Risk sources indicating that the majority of bacteria analysed were from environments where they have been subjected to a high burden of antibiotic exposure.

Broad-Host Dissemination of Plasmids Coharboring the fos Operon for Fructooligosaccharide Metabolism with Antibiotic Resistance Genes.

The fos operon encoding short-chain fructooligosaccharide (scFOS) utilization enables bacteria of the family Enterobacteriaceae to grow and be sustained in environments where they would struggle to survive. Despite several cases of the detection of the fos operon in isolates of avian and equine origins, its global distribution in bacterial genomes remains unknown. The presence of the plasmid-harbored fos operon among resistant bacteria may promote the spread of antibiotic resistance. A collection of 11,538 antimicrobial-resistant Enterobacteriaceae isolates from various sources was screened for the fosT gene encoding the scFOS transporter. Out of 307 fosT-positive isolates, 80% of them originated from sources not previously linked to fosT (humans, wastewater, and animals). The chromosomally harbored fos operon was detected in 163/237 isolates subjected to whole-genome sequencing. In the remaining 74 isolates, the operon was carried by plasmids. Further analyses focusing on the isolates with a plasmid-harbored fos operon showed that the operon was linked to various incompatibility (Inc) groups, including the IncHI1, IncF-type, IncK2, IncI1, and IncY families. Long-read sequencing of representative plasmids showed the colocalization of fos genes with antibiotic resistance genes (ARGs) in IncHI1 (containing a multidrug resistance region), IncK2 (blaTEM-1A), IncI1 [sul2 and tet(A)], and IncY [aadA5, dfrA17, sul2, and tet(A)] plasmids, while IncF-type plasmids had no ARGs but coharbored virulence-associated genes. Despite the differences in the locations and structures of the fos operons, all isolates except one were proven to utilize scFOSs. In this study, we show that the fos operon and its spread are not strictly bound to one group of plasmids, and therefore, it should not be overlooked. IMPORTANCE It was believed that members of the family Enterobacteriaceae are unable to grow under conditions with short-chain fructooligosaccharides as the only source of carbon. Nevertheless, the first Escherichia coli isolate from chicken intestine was able to utilize these sugars owing to the chromosomally harbored fos operon. Studies on E. coli isolates from horses discovered the horizontal transfer of the fos operon on IncHI1 plasmids along with genes for antibiotic resistance. The first plasmid detected was pEQ1, originating from the feces of a hospitalized horse in the Czech Republic. Follow-up studies also revealed the dissemination of the IncHI1 plasmid-harbored fos operon in the Netherlands, Germany, Denmark, and France among healthy horses. Despite several cases of detection of the fos operon, its global distribution in bacterial genomes remains unknown. The fos operon possibly plays a role in the adaptation of plasmids among resistant bacteria and therefore may promote the spread of antibiotic resistance.

Primary Carrier Status and Alteration in the Colonization of the Extended-Spectrum β-Lactamase Producing Enterobacteriaceae Among Children in Pediatric Intensive Care Unit During the Hospital Stay

Background: Widespread and inappropriate use of antibiotics has led to an increase in antibiotic-resistant bacteria worldwide. Extended-spectrum β-lactamases (ESBLs) are among the most important resistance mechanisms in members of Enterobacteriaceae, which can pose a threat to patients. Objectives: This study aimed to investigate the carrier status and alteration in the fecal transmission of ESBL-producing Enterobacteriaceae (ESBL-E) on admission and during the hospital stay, as well as its correlation with the usage of antibiotics among children in a pediatric intensive care unit (PICU). Molecular typing between the primary and secondary isolates was done to detect the homotypic clonal strains. Methods: Demographic and medical data of PICU children were collected, and the carrier status of ESBL-E was investigated in pairs of their rectal swab samples at the admission and discharge time. Detection of ESBL phenotype and antimicrobial susceptibility to 12 antibiotics were performed by double-disk synergy and disc diffusion methods, respectively. Polymerase chain reaction for detection of blaTEM, blaSHVblaPER, blaCTX-M, and blaVEB genes was performed using specific primers. The phylogenetic relations were analyzed by the enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) method. Results: Extended-spectrum β-lactamase-producing Enterobacteriaceae was detected in 48% of the samples at admission and 42% at discharge time. The highest frequency of resistance was observed in cephazolin, amoxicillin-clavulanic acid, and ampicillin. Children with a history of meropenem administration showed a significantly higher frequency of meropenem resistance Enterobacteriaceae in their samples. Moreover, the administration of metronidazole increased the isolation of ESBL-Escherichia species in hospitalized children. The most common gene associated with the ESBL phenotype was blaCTX-M, while blaPER and blaVEB were not detected in the ESBL-E isolates. The phylogenetic analysis did not confirm the occurrence of the cross-transmission of ESBL-E in the patients during hospitalization. Conclusions: Our results showed a high frequency of ESBL-E in the feces of children upon admission to the PICU, which did not change significantly during the hospital stay. Although the prescription of metronidazole showed an association with the enrichment of ESBL-E due to observed diversity in the molecular types and resistance phenotypes of the isolates, the community seems to be the primary source of ESBL-E transmission in children. Further investigations are needed to understand the role of hospital stay in the colonization and enrichment of ESBL-E in the intestinal tract of children and their association with infections during the medical interventions in the PICU.

A Study of Antibacterial Effect of Nigella sativa Seed Extracts on Bacterial Isolates from Cases of Wound Infection.

With an increasing trend of pathogenic bacteria developing resistance to the existing drugs, there is a need for newer therapeutic measures. Nigella sativa seeds and oil have been used for decades as Ayurveda, Unani Tibb and other forms of traditional medicine for various disorders. Thymoquinone is one of the active components of the N. sativa seeds. The present study determines the antibacterial effect of crude methanolic extract N. sativa seeds and thymoquinone against bacteria causing wound infection. Samples obtained from cases of wound infection received at a Microbiology laboratory attached to a tertiary care hospital over a period of six months were included in the study. The antibacterial effect of crude methanolic extract of N. sativa seeds was determined by the Punch Well method. The minimum inhibitory concentration (MIC) of thymoquinone against bacteria isolated from cases of wound infection was determined by the Micro Broth Dilution technique. A total of 60 isolates were collected from 60 samples of wound infection. By the Punch Well method, Staphylococcus aureus showed varying zones of inhibition whereas all gram-negative bacilli and Enterococcus faecalis did not show any zone of inhibition. Thymoquinone showed good antibacterial activity against S. aureus with MIC values ranging from 2-8μg/ml for most of the isolates. Uniformly, MIC of thymoquinone against all gram-negative bacilli and E. faecalis was >128μg/ml, p<0.001. It was found that methicillin-resistant S. aureus (MRSA) isolates showed higher MIC than methicillin sensitive S. aureus (MSSA) isolates p<0.05. Antibacterial activity of thymoquinone was very good against S. aureus but showed limited activity against Enterobacteriaceae members and E. faecalis isolated from patients with wound infection. Thymoquinone may be considered a potential antibacterial agent against wound infection caused by S. aureus.

Resistance profiles and genotyping of extended-spectrum beta-lactamase (ESBL) -producing and non-ESBL-producing E. coli and Klebsiella from retail market fishes.

Studies on antimicrobial resistance (AMR) profiles and epidemiological affirmation for AMR transmission are limited in fisheries and aquaculture settings. Since 2015, based on Global Action Plan on AMR by World Health Organization (WHO) and World Organization for Animal Health (OIE), several initiatives have been under taken to enhance the knowledge, skills and capacity to establish AMR trends through surveillance and strengthening of epidemiological evidence. The focus of this study was to determine the prevalence of antimicrobial resistance (AMR), its resistance profiles and molecular characterization with respect to phylogroups, antimicrobial resistance genes (ARGs), virulence genes (VGs), quaternary ammonium compounds resistance (QAC) genes and plasmid typing in retail market fishes. Pulse field gel electrophoresis (PFGE) to understand the genetic lineage of the two most important Enterobacteriaceae members, E. coli and Klebsiella sp. was performed. 94 fish samples were collected from three different sites viz., Silagrant (S1), Garchuk (S2) and North Guwahati Town Committee (NGTC) Region (S3) in Guwahati, Assam. Out of the 113 microbial isolates from the fish samples, 45 (39.82%) were E. coli; 23 (20.35%) belonged to Klebsiella genus. Among E. coli, 48.88% (n=22) of the isolates were alerted by the BD Phoenix M50 instrument as ESBL, 15.55% (n=7) as PCP and 35.55% (n=16) as non-ESBL. E. coli (39.82%) was the most prevalent pathogen among the Enterobacteriaceae members screened and showed resistance to ampicillin (69%) followed by cefazoline (64%), cefotaxime (49%) and piperacillin (49%). In the present study, 66.66% of E. coli and 30.43% of Klebsiella sp. were categorized as multi drug resistance (MDR) bacteria. CTX-M-gp-1, with CTX-M-15 variant (47%), was the most widely circulating beta-lactamase gene, while other ESBL genes blaTEM (7%), blaSHV (2%) and blaOXA-1-like (2%) were also identified in E. coli. Out of the 23 isolates of Klebsiella, 14(60.86%) were ampicillin (AM)-resistant (11(47.82%) K. oxytoca, 3(13.04%) K. aerogenes), whereas 8(34.78%) isolates of K. oxytoca showed intermediate resistance to AM. All Klebsiella isolates were susceptible to AN, SCP, MEM and TZP, although two K. aerogenes were resistant to imipenem. DHA and LAT genes were detected, respectively, in 7(16%) and 1(2%) of the E. coli strains while a single K. oxytoca (4.34%) isolate carried MOX, DHA and blaCMY-2 genes. The fluoroquinolone resistance genes identified in E. coli included qnrB (71%), qnrS (84%), oqxB (73%) and aac(6)-Ib-cr (27%); however, in Klebsiella, these genes, respectively, had a prevalence of 87%, 26%, 74% and 9%. The E. coli isolates belonged to phylogroup A(47%), B1(33%) and D(14%). All of the 22(100%) ESBL E. coli had chromosome-mediated disinfectant resistance genes viz., ydgE, ydgF, sugE(c), mdfA while 82% of ESBL E. coli had emrE. Among the non-ESBL E. coli isolates, 87% of them showed the presence of ydgE, ydgF and sugE(c) genes, while 78% of the isolates had mdfA and 39% had emrE genes respectively. 59% of the ESBL and 26% of the non-ESBL E. coli had showed the presence of qacEΔ1. The sugE(p) was present in 27% of the ESBL-producing E. coli and in 9% of non-ESBL isolates. Out of the 3 ESBL-producing Klebsiella isolates, 2(66.66%) K. oxytoca isolates were found harboring plasmid-mediated qacEΔ1 gene while one (33.33%) K. oxytoca isolate had sugE(p) gene. IncFI was the most prevalent plasmid type detected in the isolates studied, with A/C (18%), P (14%), X, Y (9% each) and I1-Iγ (14%, 4%). 50% (n=11) of the ESBL and 17% (n=4) of the non-ESBL E. coli isolates harboured IncFIB and 45% (n=10) ESBL and one (4.34%) non-ESBL E. coli isolates harboured IncFIA. Dominance of E. coli over other Enterobacterales and diverse phylogenetic profiles of E. coli and Klebsiella sp. suggests the possibility of contamination and this may be due to compromised hygienic practices along the supply chain and contamination of aquatic ecosystem. Continuous surveillance in domestic markets must be a priority in addressing antimicrobial resistance in fishery settings and to identify any unwarranted epidemic clones of E. coli and Klebsiella that can challenge public health sector.

Dried Baby Formula as a Source of Opportunistic Members of Enterobacteriaceae for Infants

For evaluating the prevalence of Enterobacteriaceae in baby formulas, we examined 60 different samples of powdered infant milk formula and milk-cereal based infant formula that is recommended for first-day birth and from 6-month age, respectively, and collected from various supermarkets, shops, and pharmacies. Six (20.0%) and thirteen (43.3%) powdered infant milk formulas and milk-cereal based infant formulas, respectively, were contaminated by different Enterobacteriaceae involved Enterobacter cloacae ssp. cloacae, Pantoea spp., Enterobacter hormaechei, and Raoultella ornithinolytica, which were identified using traditional biochemical tests and the vitek2 compact system. Only five samples of investigating milk-cereal based infant formula type have coliforms count of 4 MPN/g. All species were susceptible to gentamycin, while all were resistant to cephalothin antibiotics. Eventually, 20.0 % of powdered infant milk formula samples were unacceptable referred to Egyptian standards (2072/2006) and most of the comparable international standards. In the study, we proved the contamination of powdered milk baby formula by Enterobacteriaceae, which mainly causes health problems in immature and immunocompromised infants. The dried infant formula factories are more needful for good hygiene and safety systems applications. This study is one of the fewest studies conducted in Egypt to isolate Enterobacteriaceae members from dried foods; especially milk-cereal based infant formula.