The Role of nmcR, ampR, and ampD in the Regulation of the Class A Carbapenemase NmcA in Enterobacter ludwigii

Various carbapenemases have been identified in the Enterobacteriaceae. However, the induction and corresponding regulator genes of carbapenemase NmcA has rarely been detected in the Enterobacter cloacae complex (ECC). The NmcA-positive isolate ECC NR1491 was first detected in Japan in 2013. It was characterized and its induction system elucidated by evaluating its associated regulator genes nmcR, ampD, and ampR. The isolate was highly resistant to all β-lactams except for third generation cephalosporins (3GC). Whole-genome analysis revealed that blaNmcA was located on a novel 29-kb putatively mobile element called EludIMEX-1 inserted into the chromosome. The inducibility of β-lactamase activity by various agents was evaluated. Cefoxitin was confirmed as a strong concentration-independent β-lactamase inducer. In contrast, carbapenems induced β-lactamase in a concentration-dependent manner. All selected 3GC-mutants harboring substitutions on ampD (as ampR and nmcR were unchanged) were highly resistant to 3GC. The ampD mutant strain NR3901 presented with a 700 × increase in β-lactamase activity with or without induction. Similar upregulation was also observed for ampC and nmcA. NR1491 (pKU412) was obtained by transforming the ampR mutant (135Asn) clone plasmid whose expression increased by ∼100×. Like NR3901, it was highly resistant to 3GC. Overexpression of ampC, rather than nmcA, may have accounted for the higher MIC in NR1491. The ampR mutant repressed nmcA despite induction and it remains unclear how it stimulates nmcA transcription via induction. Future experiments should analyze the roles of nmcR mutant strains.

Isolation and Identification of Bacteria with Surface and Antibacterial Activity from the Gut of Mediterranean Grey Mullets

Fish gut represents a peculiar ecological niche where bacteria can transit and reside to play vital roles by producing bio-compounds with nutritional, immunomodulatory and other functions. This complex microbial ecosystem reflects several factors (environment, feeding regimen, fish species, etc.). The objective of the present study was the identification of intestinal microbial strains able to produce molecules called biosurfactants (BSs), which were tested for surface and antibacterial activity in order to select a group of probiotic bacteria for aquaculture use. Forty-two bacterial isolates from the digestive tracts of twenty Mediterranean grey mullets were screened for testing emulsifying (E-24), surface and antibiotic activities. Fifty percent of bacteria, ascribed to Pseudomonas aeruginosa, Pseudomonas sp., P. putida and P. anguilliseptica, P. stutzeri, P. protegens and Enterobacter ludwigii were found to be surfactant producers. Of the tested strains, 26.6% exhibited an antibacterial activity against Staphylococcus aureus (10.0 ± 0.0–14.5 ± 0.7 inhibition zone), and among them, 23.3% of isolates also showed inhibitory activity vs. Proteus mirabilis (10.0 ± 0.0–18.5 ± 0.7 mm inhibition zone) and 6.6% vs. Klebsiella pneumoniae (11.5 ± 0.7–17.5 ± 0.7 mm inhibition zone). According to preliminary chemical analysis, the bioactive compounds are suggested to be ascribed to the class of glycolipids. This works indicated that fish gut is a source of bioactive compounds which deserves to be explored.

Isolating and Characterizing Phosphorus Solubilizing Bacteria From Rhizospheres of Native Plants Grown in Calcareous Soils

Many soils including urban soils have high legacy soil phosphorus (P) due to repeated applications of P fertilizers, but a large portion legacy soil P is fixed by calcium in the calcareous soils. Phosphorus solubilizing bacteria (PSB) have the ability to transfer fixed (non-labile) soil P into bioavailable P. The aim of this study was to isolate P solubilizing bacteria from the rhizospheres of four local native plants [broomsedge bluestem (Andropogon virginicus), giant sword ferns (Nephrolepis biserrata), sawgrass (Cladium jamaicense), and sea ox-eye daisy (Borrichia frutescens)] grown in low bioavailable P calcareous soils and to determine their ability to solubilize P. A total of 44 strains of PSB were isolated with 15 of them being identified by sequencing the 16S rRNA genes as Bacillus flexus, Beijerinckia fluminensis, Enterobacter ludwigii, Enterobacter sp., and Pantoea cypripedii. After a 7-day incubation, these strains reduced pH to <4.27 and increased water-soluble P up to 588 mg L−1. Enterobacter ludwigii showed superior P solubilizing ability amount PSB isolated. Therefore, the isolated strains from the local native environment have the potential to thrive in local calcareous soils and possess strong ability to transform non-labile P into bioavailable forms for plants to uptake.

A hospital-wide outbreak of IMI-17-producing Enterobacter ludwigii in an Israeli hospital

Absract Background To describe the course and intervention of an hospital-wide IMI-Producing Enterobacter ludwigii outbreak. Methods This was an outbreak interventional study, done at a tertiary care center in Tel-Aviv, Israel. Data was collected on the course of the outbreak and the demographic and clinical characteristics of all patients involved in the outbreak. The intervention measures included patients’ cohorting, contact isolation precautions, environmental cleaning and screening of contacts. The molecular features and phylogeny of outbreak-related isolates were studied by whole-genome based analysis. Results The outbreak included 34 patients that were colonized by IMI-Producing E. ludwigii and were identified in 24 wards throughout the hospital. Colonization was identified in the first 72 h of admission in 13/34 patients (38.2%). Most patients (91.2%) were admitted from home and had relatively low level of comorbidities. The majority of them (88%) had no recent use of invasive catheters and none had previous carriage of other multi-drug resistant bacteria. All available isolates harbored the blaIMI-17 allele and belonged to Sequence-Type 385. With the exception of two isolates, all isolates were closely related with less than a 20-SNP difference between them. Conclusions This outbreak had most likely originated in the community and subsequently disseminated inside our institution. More studies are required in order to elucidate the epidemiology of IMI-Producing E. ludwigii and the possible role of environmental sources in its dissemination.

Re-examining the association of AmpC variants with Enterobacter species in the context of updated taxonomy

We performed whole genome sequencing for 17 Enterobacter clinical strains and analyzed all available Enterobacter genomes and its closely-related genera (n=3,389) from NCBI. The exact origin of plasmid-borne bla CMH and bla MIR genes is Enterobacter cloacae and Enterobacter roggenkampii , respectively, while plasmid-borne bla ACT genes originated from multiple other Enterobacter species including Enterobacter xiangfangensis , Enterobacter hoffmannii , and Enterobacter asburiae , Enterobacter ludwigii , and Enterobacter kobei . The genus of Enterobacter represents a large reservoir of plasmid-borne AmpC β-lactamase.

The Profile Analysis of Lactic Acid Bacteria (LAB) from Sumbawa White Honey and Its Potential Producing Antibacterial Compounds

Honey acts as an antibacterial without side effects, and also contains antiseptic substances which function to inhibit bacterial growth. This study aimed to isolate the Lactic Acid Bacteria (LAB) in Sumbawa white honey and the bioactive compounds produced as pathogenic antibacteria. The 1st stage in this study was the isolation of lactic acid bacteria (LAB) in Sumbawa white honey, then continued with a grading test, morphological test, catalase test, methyl red test, and the last test, namely the antimicrobial test against 5 pathogenic bacteria (Salmonellatyhposa, Staphylococcus aureus, Escherichia coli, Enterobacter ludwigii, and Leclerciaadecarboxylata). Data analysis was performed using the Analysis of variance (ANOVA) test at a confidence level of 0.05 with SPSS 24. Based on the results of sequencing analysis, it was found that the 5 selected isolates were Enterococcus faecium species. The Enterococcus faecium species obtained from the sequencing results had different strains. The accession numbers of the 5 Enterococcus faecium were: Isolate-03 with a percentage of 97.29 % (accession number: KU324920.1), Isolate-07 has a percent identity of 97.36 % (accession number: MF108201.1), Isolate-09 of 97.73 % (accession number: CP041261.3), Isolate-20 with a percentage of 96.40 % (accession number: MN511819.1), and Isolate-24 with a percentage of 98.61 % (accession number: KM495938.1). These isolates can inhibit the growth of all tested pathogenic bacteria treated with 100 % LAB metabolites and were not significantly different (p > 0.05) compared to a positive control (Ampicillin). HIGHLIGHTS Antibacterial compound of Lactic Acid Bacteria (LAB) from Sumbawa white honey Lactic acid bacteria isolation, characterization, and biosprosprection against pathogens Identified LAB by 16s rRNA sequencing gives five strains of Enterococcus faecium All identified LAB metabolites can inhibit all pathogens by similar inhibition percentage with Ampicillin GRAPHICAL ABSTRACT

Selective Adsorption of Amino Acids in Crystals of Monohydrocalcite Induced by the Facultative Anaerobic Enterobacter ludwigii SYB1

The morphology, crystal structure, and elemental composition of biominerals are commonly different from chemically synthesized minerals, but the reasons for these are not fully understood. A facultative anaerobic bacterium, Enterobacter ludwigii SYB1, is used in experiments to document the hydrochemistry, mineral crystallization, and cell surface characteristics of biomineralization. It was found that carbonate anhydrase and ammonia production were major factors influencing the alkalinity and saturation of the closed biosystem. X-ray diffraction (XRD) spectra showed that calcite, monohydrocalcite (MHC), and dypingite formed in samples with bacterial cells. It was also found that the (222) plane of MHC was the preferred orientation compared to standard data. Scanning transmission electron microscopy (STEM) analysis of cell slices provides direct evidence of concentrated calcium and magnesium ions on the surface of extracellular polymeric substances (EPS). In addition, high-resolution transmission electron microscopy (HRTEM) showed that crystallized nanoparticles were formed within the EPS. Thus, the mechanism of the biomineralization induced by E. ludwigii SYB1 can be divided into three stages: (i) the production of carbonate anhydrase and ammonia increases the alkalinity and saturation state of the milieu, (ii) free calcium and magnesium ions are adsorbed and chelated onto EPS, and (iii) nanominerals crystallize and grow within the EPS. Seventeen kinds of amino acids were identified within both biotic MHC and the EPS of SYB1, while the percentages of glutamic and aspartic acid in MHC increased significantly (p < 0.05). Furthermore, the adsorption energy was calculated for various amino acids on seven diffracted crystal faces, with preferential adsorption demonstrated on (111) and (222) faces. At the same time, the lowest adsorption energy was always that of glutamic and aspartic acid for the same crystal plane. These results suggest that aspartic and glutamic acid always mix preferentially in the crystal lattice of MHC and that differential adsorption of amino acids on crystal planes can lead to their preferred orientation. Moreover, the mixing of amino acids in the mineral structure may also have a certain influence on the mineral lattice dislocations, thus enhancing the thermodynamic characteristics.

Isolation and Molecular Characterization of Klebsiella And Enterobacter Species Recovered in Sunflower Seed Agar from Cases Resembling Respiratory Cryptococcosis

Respiratory cryptococcosis caused by Cryptococcus species can present with symptoms indistinguishable from bacterial or viral etiology. Cryptococcus species produce typical colonial features on Sunflower Seed Agar (SSA), which aids in rapid diagnoses of cryptococcosis. In studying respiratory cryptococcosis, we observed bacterial growths on SSA that resembled Cryptococcus species in colonial characteristics. This study aimed at identifying and characterizing those bacterial isolates for documentation. Sputum samples were collected from 201 patients with symptoms suggestive of respiratory cryptococcosis. The samples were inoculated onto SSA, incubated at 37oC for two weeks. Suspected colonies were further evaluated. Of the samples, none yielded Cryptococcus species, although a total of twenty Cryptococcus-resembling bacterial colonies were encountered and isolated. Eight of the isolates could not amplify by PCR techniques. The other twelve were identified as follows: Klebsiella pneumonia (8 or 67%), Klebsiella ozaneae (3 or 25%), and Enterobacter ludwigii (1 or 8%). All isolates were susceptible to Ertapenem, Meropenem, and Fosfomycin but resistant to ampicillin. Results show that Klebsiella and Enterobacter pneumonia-like illnesses can be misidentified as cryptococcosis using SSA. Reliance on microscopic rather than macroscopic, colonial features on SSA will prevent misdiagnosis.

Biofilm Formation, Production of Matrix Compounds and Biosorption of Copper, Nickel and Lead by Different Bacterial Strains

Bacterial biofilms play a key role in metal biosorption from wastewater. Recently, Enterobacter asburiae ENSD102, Enterobacter ludwigii ENSH201, Vitreoscilla sp. ENSG301, Acinetobacter lwoffii ENSG302, and Bacillus thuringiensis ENSW401 were shown to form air–liquid (AL) and solid–air–liquid (SAL) biofilms in a static condition at 28 and 37°C, respectively. However, how environmental and nutritional conditions affect biofilm formation; production of curli and cellulose; and biosorption of copper (Cu), nickel (Ni), and lead (Pb) by these bacteria have not been studied yet. In this study, E. asburiae ENSD102, E. ludwigii ENSH201, and B. thuringiensis ENSW401 developed the SAL biofilms at pH 8, while E. asburiae ENSD102 and Vitreoscilla sp. ENSG301 constructed the SAL biofilms at pH 4. However, all these strains produced AL biofilms at pH 7. In high osmolarity and ½-strength media, all these bacteria built fragile AL biofilms, while none of these strains generated the biofilms in anaerobic conditions. Congo red binding results showed that both environmental cues and bacterial strains played a vital role in curli and cellulose production. Calcofluor binding and spectrophotometric results revealed that all these bacterial strains produced significantly lesser amounts of cellulose at 37°C, pH 8, and in high osmotic conditions as compared to the regular media, at 28°C, and pH 7. Metal biosorption was drastically reduced in these bacteria at 37°C than at 28°C. Only Vitreoscilla sp. ENSG301 and B. thuringiensis ENSW401 completely removed (100%) Cu and Ni at an initial concentration of 12.5 mg l–1, while all these bacteria totally removed (100%) Pb at concentrations of 12.5 and 25 mg l–1 at pH 7 and 28°C. At an initial concentration of 100 mg l–1, the removal of Cu (92.5 to 97.8%) and Pb (89.3 to 98.3%) was the highest at pH 6, while it was higher (84.7 to 93.9%) for Ni at pH 7. Fourier transform infrared spectroscopy results showed metal-unloaded biomass biofilms contained amino, hydroxyl, carboxyl, carbonyl, and phosphate groups. The peak positions of these groups were shifted responding to Cu, Ni, and Pb, suggesting biosorption of metals. Thus, these bacterial strains could be utilized to remove Cu, Ni, and Pb from aquatic environment.