Genomic Analysis of a Strain Collection Containing Multidrug-, Extensively Drug-, Pandrug-, and Carbapenem-Resistant Modern Clinical Isolates of Acinetobacter baumannii.

In this study, we characterize a new collection that comprises multidrug-resistant (MDR), extensively drug-resistant (XDR), pandrug-resistant (PDR), and carbapenem-resistant modern clinical isolates of Acinetobacter baumannii collected from hospitals through national microbiological surveillance in Belgium. Bacterial isolates (n = 43) were subjected to whole-genome sequencing (WGS), combining Illumina (MiSeq) and Nanopore (MinION) technologies, from which high-quality genomes (chromosome and plasmids) were de novo assembled. Antimicrobial susceptibility testing was performed along with genome analyses, which identified intrinsic and acquired resistance determinants along with their genetic environments and vehicles. Furthermore, the bacterial isolates were compared to the most prevalent A. baumannii sequence type 2 (ST2) (Pasteur scheme) genomes available from the BIGSdb database. Of the 43 strains, 40 carried determinants of resistance to carbapenems; blaOXA-23 (n = 29) was the most abundant acquired antimicrobial resistance gene, with 39 isolates encoding at least two different types of OXA enzymes. According to the Pasteur scheme, the majority of the isolates were globally disseminated clones of ST2 (n = 25), while less frequent sequence types included ST636 (n = 6), ST1 (n = 4), ST85 and ST78 (n = 2 each), and ST604, ST215, ST158, and ST10 (n = 1 each). Using the Oxford typing scheme, we identified 22 STs, including two novel types (ST2454 and ST2455). While the majority (26/29) of blaOXA-23 genes were chromosomally carried, all blaOXA-72 genes were plasmid borne. Our results show the presence of high-risk clones of A. baumannii within Belgian health care facilities with frequent occurrences of genes encoding carbapenemases, highlighting the crucial need for constant surveillance.

Plasmidome analysis of a hospital effluent biofilm: Status of antibiotic resistance.

Plasmids are widely involved in the dissemination of characteristics within bacterial communities. Their genomic content can be assessed by high-throughput sequencing of the whole plasmid fraction of an environment, the plasmidome. In this study, we analyzed the plasmidome of a biofilm formed in the effluents of the teaching hospital of Clermont-Ferrand (France). Our analysis discovered >350 new complete plasmids, with a length ranging from 1219 to 40,193 bp. Forty-two plasmid incompatibility (Inc) groups were found among all the plasmid contigs. Ten large plasmids, described here in detail, were reconstructed from plasmid contigs, seven of which carried antibiotic resistance genes. Four plasmids potentially confer resistance to numerous families of antibiotics, including carbapenems, aminoglycosides, colistin, and chloramphenicol. Most of these plasmids were affiliated to Proteobacteria, a phylum of Gram-negative bacteria. This study therefore illustrates the composition of an environmental mixed biofilm in terms of plasmids and antibiotic resistance genes.

Spectrum and resistance in bacterial infections of the ocular surface in a German tertiary referral center 2009-2019.

PURPOSE: Aim of this study was to evaluate the frequencies, trends, and antibiotic resistance of bacteria collected from ocular surface or contact lens material in a German tertiary referral center from 2009 to 2019. METHODS: Microbiological data from 2009 to 2019 was analyzed. Culture-dependent microbial identification and analysis of antibiotic sensitivity was completed by the Institute of Microbiology. Statistical analysis of age- and sex-specific differences as well as changes in the microbial spectrum and resistance over the study period was performed with GraphPad Prism 9.0 applying nonparametric tests (level of significance: p ≦ 0.05). RESULTS: A total of 6361 specimens were analyzed. Positivity rate was 18.6%. Sixty-three percent (n = 680) of the bacterial isolates were derived from ocular surface and 37% (n = 399) from contact lens material. The ratio of gram-negative bacteria was significantly higher in contact lens material. Multiresistant bacteria showed a significant increase with patient age (p < 0.0001). An overall increase in resistance to levofloxacin (p = 0.0239) was detected. Only 2.4% and 3.1% isolates were resistant to a combination of moxifloxacin and gentamicin, respectively, levofloxacin and gentamicin. CONCLUSIONS: The reported bacterial spectrum is similar to comparable centers. Our data show that it should not be assumed that the newest classes of antibiotics have the best efficacy or lowest resistance levels. In suspected bacterial conjunctivitis, we propose using gentamicin as first-line therapy. In therapy refractive cases and in involvement of the cornea, we recommend a combination of gentamicin and ofloxacin or moxifloxacin. Overall, the evaluated organisms showed good sensitivity to the regularly used antibiotics.

Plasmid stability of potential probiotic Lactobacillus plantarum strains in artificial gastric juice, at elevated temperature, and in the presence of novobiocin and acriflavine.

In this study, the presence of plasmids responsible for carbohydrate fermentation and antibiotic resistance and the stability of these plasmids in artificial gastric juice were investigated in 20 Lactobacillus plantarum strains with probiotic properties. Plasmid curing was performed with novobiocin, acriflavine and elevated incubation temperature to identify plasmids encoded with carbohydrate fermentation and antibiotic resistance genes and to compare them with artificial gastric juice. Plasmid profiling of the strains revealed that 100% of the strains were harbouring plasmids in varying sizes and numbers. The plasmid number of the potential probiotic strains ranged between 1 and 4, and the plasmid size ranged between 5.779 and 16.138 kb. The potential probiotic strains could not survive in the artificial gastric juice at pH 2.0. Although the strains maintained their viability in an artificial gastric juice at pH 2.5 and 3.0, and their derivatives lost their plasmids at a high rate (100%). Similarly, high levels of cured derivatives were obtained with 8 µg/mL novobiocin and 100 µg/mL acriflavine applications, and 24 h incubation at 43 °C. All the experiments were also performed to compare with two L. plantarum-type strains containing plasmids responsible for tetracycline and tetracycline + erythromycin resistances. Artificial gastric juice and other plasmid curing treatments caused a high-frequency loss in the antibiotic resistances of type strains. Determining plasmid stability in artificial gastric juice is a novel approach. Plasmid stability in the gastrointestinal tract is important for maintaining the plasmid-encoded probiotic properties.

Characterization and genome sequencing of a Citrobacter freundii phage CfP1 harboring a lysin active against multidrug-resistant isolates.

Citrobacter spp., although frequently ignored, is emerging as an important nosocomial bacterium able to cause various superficial and systemic life-threatening infections. Considered to be hard-to-treat bacterium due to its pattern of high antibiotic resistance, it is important to develop effective measures for early and efficient therapy. In this study, the first myovirus (vB_CfrM_CfP1) lytic for Citrobacter freundii was microbiologically and genomically characterized. Its morphology, activity spectrum, burst size, and biophysical stability spectrum were determined. CfP1 specifically infects C. freundii, has broad host range (>85 %; 21 strains tested), a burst size of 45 PFU/cell, and is very stable under different temperatures (-20 to 50 °C) and pH (3 to 11) values. CfP1 demonstrated to be highly virulent against multidrug-resistant clinical isolates up to 12 antibiotics, including penicillins, cephalosporins, carbapenems, and fluroquinoles. Genomically, CfP1 has a dsDNA molecule with 180,219 bp with average GC content of 43.1 % and codes for 273 CDSs. The genome architecture is organized into function-specific gene clusters typical for tailed phages, sharing 46 to 94 % nucleotide identity to other Citrobacter phages. The lysin gene encoding a predicted D-Ala-D-Ala carboxypeptidase was also cloned and expressed in Escherichia coli and its activity evaluated in terms of pH, ionic strength, and temperature. The lysine optimum activity was reached at 20 mM HEPES, pH 7 at 37 °C, and was able to significantly reduce all C. freundii (>2 logs) as well as Citrobacter koseri (>4 logs) strains tested. Interestingly, the antimicrobial activity of this enzyme was performed without the need of pretreatment with outer membrane-destabilizing agents. These results indicate that CfP1 lysin is a good candidate to control problematic Citrobacter infections, for which current antibiotics are no longer effective.

Large Scale Screening of Ethnomedicinal Plants for Identification of Potential Antibacterial Compounds.

The global burden of bacterial infections is very high and has been exacerbated by increasing resistance to multiple antibiotics. Antibiotic resistance leads to failed treatment of infections, which can ultimately lead to death. To overcome antibiotic resistance, it is necessary to identify new antibacterial agents. In this study, a total of 662 plant extracts (diverse parts) from 222 plant species (82 families, 177 genera) were screened for antibacterial activity using the agar cup plate method. The aqueous and methanolic extracts were prepared from diverse plant parts and screened against eight bacterial (two Gram-positive and six Gram-negative) species, most of which are involved in common infections with multiple antibiotic resistance. The methanolic extracts of several plants were shown to have zones of inhibition ≥ 12 mm against both Gram-positive and Gram-negative bacteria. The minimum inhibitory concentration was calculated only with methanolic extracts of selected plants, those showed zone of inhibition ≥ 12 mm against both Gram-positive and Gram-negative bacteria. Several extracts had minimum inhibitory concentration ≤ 1 mg/mL. Specifically Adhatoda vasica, Ageratum conyzoides, Alangium salvifolium, Alpinia galanga, Andrographis paniculata, Anogeissus latifolia, Annona squamosa, A. reticulate, Azadirachta indica, Buchanania lanzan, Cassia fistula, Celastrus paniculatus, Centella asiatica, Clausena excavate, Cleome viscosa, Cleistanthus collinus, Clerodendrum indicum, Croton roxburghii, Diospyros melanoxylon, Eleutherine bulbosa, Erycibe paniculata, Eryngium foetidum, Garcinia cowa, Helicteres isora, Hemidesmus indicus, Holarrhena antidysenterica, Lannea coromandelica, Millettia extensa, Mimusops elengi, Nyctanthes arbor-tristis, Oroxylum indicum, Paederia foetida, Pterospermum acerifolium, Punica granatum, Semecarpus anacardium, Spondias pinnata, Terminalia alata and Vitex negundo were shown to have significant antimicrobial activity. The species listed here were shown to have anti-infective activity against both Gram-positive and Gram-negative bacteria. These results may serve as a guide for selecting plant species that could yield the highest probability of finding promising compounds responsible for the antibacterial activities against a broad spectrum of bacterial species. Further investigation of the phytochemicals from these plants will help to identify the lead compounds for drug discovery.

Antimicrobial resistance and virulence traits in Enterococcus strains isolated from dogs and cats.

We investigated presence and prevalence of antibiotic-resistances and other biological characters in enterococci isolated from faeces of healthy dogs and cats because these microorganisms represent important human and veterinary pathogens/opportunists, and a significant burden for healthcare systems. In all samples (n=115) we detected enterococci, with a predominance of Enterococcus faecium (42; 36.5%) and Enterococcus faecalis (36; 31.3%) species, endowed with virulence traits and multidrug-resistance. The two predominant resistance patterns (erythromycin, tetracycline) were examined by polymerase chain reaction for tet and erm genes. Only tetM for tetracycline, and ermA and ermB for erythromycin were detected. PCR for gelatinase gene (gelE) was positive in 62.6% of isolates, but only 26.1% produce gelatinase suggesting the existence of silent genes. efaAfs and efaAfm genes were found in E. faecalis and E. faecium respectively. 89.6% of isolates produced bacteriocin-like substances with a prevailing action against Listeria genus and, among these, 33.9% were positive for the bacteriocin structural genes entA, entL50 or entP. According to our study, pet animals can be considered a reservoir of potentially pathogenic enterococci and we cannot exclude that those microorganisms may be responsible for opportunistic infections in high-risk pet owners.

Removal of selected sulfonamides and sulfonamide resistance genes from wastewater in full-scale constructed wetlands.

Sulfonamides are high-consumption antibiotics that reach the aquatic environment. The threat related to their presence in wastewater and the environment is not only associated with their antibacterial properties, but also with risk of the spread of drug resistance in bacteria. Therefore, the aim of this work was to evaluate the occurrence of eight commonly used sulfonamides, sulfonamide resistance genes (sul1-3) and integrase genes intI1-3 in five full-scale constructed wetlands (CWs) differing in design (including hybrid systems) and in the source of wastewater (agricultural drainage, domestic sewage/surface runoff, and animal runs runoff in a zoo). The CWs were located in low-urbanized areas in Poland and in Czechia. No sulfonamides were detected in the CW treating agricultural tile drainage water. In the other four systems, four sulfonamide compounds were detected. Sulfamethoxazole exhibited the highest concentration in those four CWs and its highest was 12,603.23 ± 1000.66 ng/L in a CW treating a mixture of domestic sewage and surface runoff. Despite the high removal efficiencies of sulfamethoxazole in the tested CWs (86 %-99 %), it was still detected in the treated wastewater. The sul1 genes occurred in all samples of raw and treated wastewater and their abundance did not change significantly after the treatment process and it was, predominantly, at the level 105 gene copies numbers/mL. Noteworthy, sul2 genes were only found in the influents, and sul3 were not detected. The sulfonamides can be removed in CWs, but their elimination is not complete. However, hybrid CWs treating sewage were superior in decreasing the relative abundance of genes and the concentration of SMX. CWs may play a role in the dissemination of sulfonamide resistance genes of the sul1 type and other determinants of drug resistance, such as the intI1 gene, in the environment, however, the magnitude of this phenomenon is a matter of further research.

Occurrence and fate of CECs (OMPs, ARGs and pathogens) during decentralised treatment of black water and grey water.

Decentralised wastewater treatment is becoming a suitable strategy to reduce cost and environmental impact. In this research, the performance of two technologies treating black water (BW) and grey water (GW) fractions of urban sewage is carried out in a decentralised treatment of the wastewater produced in three office buildings. An Anaerobic Membrane Bioreactor (AnMBR) treating BW and a Hybrid preanoxic Membrane Bioreactor (H-MBR) containing small plastic carrier elements, treating GW were operated at pilot scale. Their potential on reducing the release of contaminants of emerging concern (CECs) such as Organic Micropollutants (OMPs), Antibiotic Resistance Genes (ARGs) and pathogens was studied. After 226 d of operation, a stable operation was achieved in both systems: the AnMBR removed 92.4 ± 2.5 % of influent COD, and H-MBR removed 89.7 ± 3.5 %. Regarding OMPs, the profile of compounds differed between BW and GW, being BW the matrix with more compounds detected at higher concentrations (up to µg L-1). For example, in the case of ibuprofen the concentrations in BW were 23.63 ± 3.97 µg L-1, 3 orders of magnitude higher than those detected in GW. The most abundant ARGs were sulfonamide resistant genes (sul1) and integron class 1 (intl1) in both BW and GW. Pathogenic bacteria counts were reduced between 1 and 3 log units in the AnMBR. Bacterial loads in GW were much lower than in BW, being no bacterial re-growth observed for the GW effluents after treatment in the H-MBR. None of the selected enteric viruses was detected in GW treatment line.

Interrogating Salmonella Typhi biofilm formation and dynamics to understand antimicrobial resistance.

AIMS: Salmonella Typhi biofilm-mediated infections are globally rising. Due to the emergence of drug resistance antibiotics did not show effective results against S. Typhi biofilm. Therefore, there is an urgent need for an in-depth interrogation of S. Typhi biofilm to understand its formation kinetics, compositions, and surface charge value. METHODS: This study utilized the S. Typhi MTCC-733 strain from a microbial-type culture collection in India. The S. Typhi biofilm was formed on a glass slide in a biofilm development apparatus. Typhoidal biofilm analysis was done with the help of various assays such as a crystal violet assay, SEM analysis, FTIR analysis, Raman analysis, and zeta potential analysis. KEY FINDING: This article contained a comprehensive assessment of the typhoid biofilm formation kinetics, biofilm compositions, and surface charge which revealed that cellulose was a major molecule in the typhoidal biofilm which can be used as a major biofilm drug target against typhoidal biofilm. SIGNIFICANCE: This study provided interrogations about typhoidal biofilm kinetics which provided ideas about the biofilm composition. The cellulose molecule showed a major component of S. Typhi biofilm and it could potentially involved in drug resistance, and offer a promising avenue for developing a new antibiofilm therapeutic target to conquer the big obstacle of drug resistance. The obtained information can be instrumental in designing novel therapeutic molecules in the future to combat typhoidal biofilm conditions effectively for overcoming antibiotic resistance against bacterial infection Salmonella.

Rapid Phenotypic Antibiotics Susceptibility Analysis by a 3D Printed Prototype.

One of the most important public health concerns is the increase in antibiotic-resistant pathogens and corresponding treatment of associated infections. Addressing this challenge requires more efficient use of antibiotics, achievable by the use of evidence-based, effective antibiotics identified by antibiotic susceptibility testing (AST). However, the current standard method of phenotypic AST used for this purpose requires 48 h or more from sample collection to result. Until results are available, broad-spectrum antibiotics are used to avoid delaying treatment. The turnaround time must therefore be shortened in order for the results to be available before the second administration of antibiotics. The phenotypic electrochemical AST method presented here identifies effective antibiotics within 5-10 h after sampling. Spiked serum samples, including polymicrobial samples, with clinically relevant pathogens and respective concentrations commonly found in bloodstream infections (Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa) are used. Direct loading of the test with diluted serum eliminates the need for a pre-culture, as required by existing methods. Furthermore, by combining several electrochemical measurement procedures with computational analysis, allowing the method to be used both online and offline, the AST achieves a sensitivity of 94.44% and a specificity of 95.83% considering each replicate individually.

Antibiotic resistances from slaughterhouse effluents and enhanced antimicrobial blue light technology for wastewater decontamionation.

The frequencies of 6 different facultative pathogenic bacteria of the ESKAPE group (priority list WHO) and a total of 14 antibiotic resistance genes (ARGs) with different priorities for human medicine were quantified in wastewaters of poultry and pig slaughterhouses using molecular biological approaches. Raw sewage from poultry and pig slaughterhouses was found to be contaminated not only with facultative pathogenic bacteria but also with various categories of clinically relevant ARGs, including ARGs against the reserve antibiotics group. The concentration of the different gene targets decreased after on-site conventional biological or advanced oxidative wastewater treatments, but was not eliminated. Hence, the antimicrobial BlueLight (aBL) in combination with a porphyrin photo-sensitizer was studied with ESKAPE bacteria and real slaughterhouse wastewaters. The applied broad LED-based blue light (420-480 nm) resulted in groups of sensitive, intermediate, and non-sensitive ESKAPE bacteria. The killing effect of aBL was increased in the non-sensitive bacteria Klebsiella pneumoniae and Enterococcus faecium due to the addition of porphyrins in concentrations of 10-6 M. Diluted slaughterhouse raw wastewater was treated with broad spectrum aBL and in combination with porphyrin. Here, the presence of the photo-sensitizer enhanced the aBL biocidal impact.

Antibiotic Resistances of Enterobacteriaceae with Chromosomal Ampc in Urine Cultures: Review and Experience of a Spanish Hospital.

The Enterobacteriaceae Citrobacter freundii, Enterobacter cloacae, Klebsiella aerogenes, Morganella morganii, Providencia stuartii, and Serratia marcescens (CESPM group) produce numerous urinary tract infections (UTIs) which are difficult to treat due to their high multiresistance rate. The objectives of this study were to carry out a systematic review of antibiotic resistances by UTIs and to determine changes over time in urine cultures from a reference hospital in southern Spain. The literature was searched for European data on the resistance rates of each microorganism, and a retrospective cross-sectional descriptive study was performed in samples with suspicion of UTI from patients in Virgen de las Nieves University Hospital (Granada, Spain) between 2016 and the first half of 2021. Among 21,838 positive urine cultures, 1.85% were caused by E. cloacae, 0.77% by M. Morganii, 0.65% by K. aerogenes, 0.46% by C. freundii, 0.29% by P stuartii, and 0.25% by S. marcescens. The lowest resistance rates by microorganism were: E. cloacae to amikacin (3.47%) and imipenem (5.28%); M. morganii to piperacillin-tazobactam (1.79%), cefepime (4.76%), and tobramycin (7.74%); K. aerogenes to tobramycin (3.55%), gentamicin (4.25%), trimethoprim-sulfamethoxazole (4.96%), imipenem (5.75%), and cefepime (6.43%); C. freundii to imipenem (no resistance), nitrofurantoin (1.96%), fosfomycin (2.80%), and ertapenem (6.12%); P. stuartii to cefepime (3.28%) and ceftazidime (3.28%); and S. marcescens to gentamicin (1.8%), ciprofloxacin (3.64%), cefepime (3.70%), piperacillin-tazobactam (3.70%), and trimethoprim-sulfamethoxazole (5.45%). In our setting, CESMP Enterobacteriaceae showed the lowest resistance to piperacillin-tazobactam, cefepime, imipenem, gentamicin, and colistin, which can therefore be recommended for the empirical treatment of UTIs. The COVID-19 pandemic may have had a clinical impact in relation to the increased resistance of E. cloacae and M. morgani to some antibiotics.

Antibiotic Resistance Changes in Gram-Positive Bacteria from Urine Cultures: Development Analysis in a Health Area of South-East Spain.

This study analyzed the epidemiology and antibiotic susceptibility profile of significant bacteriuria and assessed the impact of adopting EUCAST criteria on antibiotic resistances. A systematic review was performed on publications in English or Spanish between 1 January 2010 and 30 June 2021 on the susceptibility of Gram-positive bacteria isolated in urinary samples in Europe. A retrospective descriptive study was also conducted on the results of 21,838 urine cultures with presumptive urinary tract infection (UTI) obtained during the past five years by the Department of Microbiology of the Virgen de las Nieves University Hospital (Granada, Spain). The activity of various antibiotics was determined, differentiated among various populations, and interpretations compared according to the application of EUCAST or CLSI criteria. Among 21,838 cases of significant bacteriuria, 27.69% were by Gram-positive bacteria, which were Enterococcus faecalis in 19.04% and Enterococcus faecium in 3.92%. The susceptibility profile remained stable for most antibiotics except for levofloxacin for E. faecalis and Staphylococcus aureus and nitrofurantoin for E. faecium. The resistance of Enterococcus spp. and Staphylococcus spp. to glycopeptides was exceptionally low in our setting. No significant difference in the prevalence of methicillin-resistant Staphylococcus aureus was observed between hospital (26.67%) and community (28.85%) samples. Resistances in our local setting remain stable and appear to be lower than reported in other studies. The adoption of EUCAST vs. CLSI criteria did not produce a general change in resistance rates. Findings suggest the need to revise certain empirical criteria, such as aminoglycoside synergy for Enterococcus and for community-origin S. aureus.

The efficacies of non-bismuth containing quadruple therapies in the treatment of first-line anti-Helicobacter pylori across 4-year time interval with changing antibiotics resistance.

BACKGROUND: Non-bismuth containing quadruple therapy (concomitant therapy) is an alternative treatment for Helicobacter pylori (H. pylori) eradication with increasing clarithromycin-resistant strains over times. This study compared the efficacies of non-bismuth containing quadruple therapy (concomitant therapy) in the treatment of first-line anti-Helicobacter Pylori between two time intervals (January 2013 to June 2014 and June 2016 to December 2017). METHODS: H. pylori-infected patients were recruited in the intention-to-treat (ITT analysis) and divided into EACM-A group (enrolled from January 2013 to June 2014, N = 98) and EACM-B group (enrolled from June 2016 to December 2017, N = 99). Patients were prescribed with 7-day esomeprazole 40 mg bid., clarithromycin 500 mg bid., amoxicillin 1 g bid. and metronidazole 500 mg bid. Ninety patients and 93 patients were analyzed in the per protocol (PP) analysis (8 and 6 patients lost follow-up in each group). Urea breath tests were performed 4-8 weeks thereafter. RESULTS: The eradication rates for EACM-A and EACM-B groups were 87.8% (95% confidence interval [CI] = 79.7%-93.5%) and 84.8% (95% CI = 76.2%-91.2%) (p = 0.55) in intention-to-treat (ITT) analysis; 95.6% (95% CI = 89.1%-98.8%) and 90.3% (95% CI = 82.4%-95.5%) (p = 0.17) in per protocol (PP) analysis. The adverse event rates were 16.7% vs. 10.8% in the 2 groups (p = 0. 0.24). The antibiotic resistance rates between the 2 groups were amoxicillin (0%), tetracycline (0%); clarithromycin (11.8% vs. 17.8%, p = 0.46); metronidazole (32.4% vs. 33.3%, p = 0.93) and levofloxacin (14.7% vs. 37.8%, p = 0.02). CONCLUSION: The success rate of 7-days concomitant therapy encountered an approximately 5% decrease across 4-year time interval (2013-2017) with the changes of clarithromycin resistance from 11.8% to 17.8% in Taiwan.

Antibiotic-Resistant Genes and Bacteria as Evolving Contaminants of Emerging Concerns (e-CEC): Is It Time to Include Evolution in Risk Assessment?

The pressing issue of the abundance of antibiotic resistance genes and resistant bacteria in the environment (ARGs and ARB, respectively) requires procedures for assessing the risk to health. The chemo-centric environmental risk assessment models identify hazard(s) in a dose-response manner, obtaining exposure, toxicity, risk, impact and policy. However, this risk assessment approach based on ARGs/ARB evaluation from a quantitative viewpoint shows high unpredictability because ARGs/ARB cannot be considered as standard hazardous molecules: ARB duplicate and ARGs evolve within a biological host. ARGs/ARB are currently listed as Contaminants of Emerging Concern (CEC). In light of such characteristics, we propose to define ARGs/ARB within a new category of evolving CEC (or e-CEC). ARGs/ARB, like any other evolving determinants (e.g., viruses, bacteria, genes), escape environmental controls. When they do so, just one molecule left remaining at a control point can form the origin of a new dangerous and selection-responsive population. As a consequence, perhaps it is time to acknowledge this trait and to include evolutionary concepts within modern risk assessment of e-CEC. In this perspective we analyze the evolutionary responses most likely to influence risk assessment, and we speculate on the means by which current methods could measure evolution. Further work is required to implement and exploit such experimental procedures in future risk assessment protocols.

Antibiotic-Resistant Bacteria in Clams-A Study on Mussels in the River Rhine.

Bacterial infections have been treated effectively by antibiotics since the discovery of penicillin in 1928. A worldwide increase in the use of antibiotics led to the emergence of antibiotic resistant strains in almost all bacterial pathogens, which complicates the treatment of infectious diseases. Antibiotic-resistant bacteria play an important role in increasing the risk associated with the usage of surface waters (e.g., irrigation, recreation) and the spread of the resistance genes. Many studies show that important pathogenic antibiotic-resistant bacteria can enter the environment by the discharge of sewage treatment plants and combined sewage overflow events. Mussels have successfully been used as bio-indicators of heavy metals, chemicals and parasites; they may also be efficient bio-indicators for viruses and bacteria. In this study an influence of the discharge of a sewage treatment plant could be shown in regard to the presence of E. coli in higher concentrations in the mussels downstream the treatment plant. Antibiotic-resistant bacteria, resistant against one or two classes of antibiotics and relevance for human health could be detected in the mussels at different sampling sites of the river Rhine. No multidrug-resistant bacteria could be isolated from the mussels, although they were found in samples of the surrounding water body.

Rapid and Ultrasensitive Detection of Mutations and Genes Relevant to Antimicrobial Resistance in Bacteria.

The worldwide emergence of multidrug-resistant (MDR) bacteria is associated with significant morbidity, mortality, and healthcare costs. Rapid and accurate diagnostic methods to detect antibiotic resistance are critical for antibiotic stewardship and infection control measurements. Here a cantilever nanosensor-based diagnostic assay is shown to detect single nucleotide polymorphisms (SNPs) and genes associated with antibiotic resistance in Gram negative (Pseudomonas aeruginosa) and positive (Enterococcus faecium) bacteria, representing frequent causes for MDR infections. Highly specific RNA capture probes for SNPs (ampRD135G or ampRG154R ) or resistance genes (vanA, vanB, and vanD) allow to detect the binding of bacterial RNA within less than 5 min. Serial dilutions of bacterial RNA indicate an unprecedented sensitivity of 10 fg µL-1 total RNA corresponding to less than ten bacterial cells for SNPs and 1 fg µL-1 total RNA for vanD detection equivalent to single bacterial cell sensitivity.

Genetic and Phenotypic Diversity of Morganella morganii Isolated From Cheese.

The bacterium Morganella morganii can produce the biogenic amines (BA) cadaverine, putrescine, and histamine in vitro and is responsible for high histamine concentrations in fish products. These BA can have toxic effects upon ingestion and are undesired in food. The purpose of this study was to characterize the phenotype and genotype of 11 M. morganii isolated from cheese in regard to the BA formation. In addition, we investigated the phylogeny, trehalose fermentation ability, and antibiotic resistance of the cheese isolates. To do so, we sequenced their genomes using both long and short read technologies. Due to the presence of the trehalose operon and the ability to ferment trehalose, the cheese isolates can be assigned to the subsp. sibonii. Comparative genomics with public available M. morganii genomes shows that the genomes of the cheese isolates cluster together with other subsp. sibonii genomes. All genomes between subsp. morganii and subsp. sibonii are separated by an average nucleotide identity (ANI) of less than 95.0%. Therefore, the subspecies could represent two distinct species. Nine of the strains decarboxylated lysine yielding cadaverine in vitro. This metabolic activity is linked to a previously unknown gene cluster comprising genes encoding a lysine-tRNA ligase (lysS), an HTH-transcriptional regulator (argP), a cadaverine-lysine antiporter (cadB), and a lysine decarboxylase (cadA). The formation of putrescine is linked to the speF gene encoding an ornithine decarboxylase. The gene is disrupted in five strains by an insertion sequence, and these strains only exhibit a weak putrescine production. Antimicrobial susceptibility profiling revealed that all cheese strains are resistant to tetracycline, chloramphenicol, tigecycline, colistin, and ampicillin. These phenotypes, except for colistin which is intrinsic, could be linked to antimicrobial resistance genes located on the chromosome.

3rd Generation of Cephalosporins and Monobactam Resistant Among Pathogenic Bacteria Collected from Ilam Hospitals During 2008 to 2015: A Systematic Review and Meta-Analysis.

BACKGROUND: Recently, increasing antibiotic resistance is causing serious publichealth concerns worldwide. This phenomenon increased patient's morbidity and mortality rate, particularly in immunocompromised individuals. METHODS: Considering that antimicrobial resistance can vary according to geographical location, we investigated the status of antibiotic resistance in Ilam province from 2008 to 2013. RESULTS: The results of our study showed that antibiotic resistance rates to aztreonam, cefotaxime, ceftazidime and ceftriaxone were 38%, 39%, 43% and 48% respectively. CONCLUSION: Antibiotic resistance had increased (except for aztreonam) during the period from 2008 to 2013.