Effects of Ribosomal Protein S10 Flexible Loop Mutations on Tetracycline and Tigecycline Susceptibility of Escherichia coli

Tigecycline is a tetracycline derivative that is being used as an antibiotic of last resort. Both tigecycline and tetracycline bind to the small (30S) ribosomal subunit and inhibit translation. Target mutations leading to resistance to these antibiotics have been identified both in the 16S ribosomal RNA and in ribosomal proteins S3 and S10 (encoded by the rpsJ gene). Several different mutations in the S10 flexible loop tip residue valine 57 (V57) have been observed in tigecycline-resistant Escherichia coli isolates. However, the role of these mutations in E. coli has not yet been characterized in a defined genetic background. In this study, we chromosomally integrated 10 different rpsJ mutations into E. coli, resulting in different exchanges or a deletion of S10 V57, and investigated the effects of the mutations on growth and tigecycline/tetracycline resistance. While one exchange, V57K, decreased the minimal inhibitory concentration (MIC) (Etest) to tetracycline to 0.75 μg/ml (compared to 2 μg/ml in the parent strain) and hence resulted in hypersensitivity to tetracycline, most exchanges, including the ones reported previously in resistant isolates (V57L, V57D, and V57I) resulted in slightly increased MICs to tigecycline and tetracycline. The strongest increase was observed for the V57L mutant, with a MIC (Etest) to tigecycline of 0.5 μg/ml (compared to 0.125 μg/ml in the parent strain) and a MIC to tetracycline of 4.0 μg/ml. Nevertheless, none of these exchanges increased the MIC to the extent observed in previously described clinical tigecycline-resistant isolates. We conclude that, next to S10 mutations, additional mutations are necessary in order to reach high-level tigecycline resistance in E. coli. In addition, our data reveal that mutants carrying S10 V57 exchanges or deletion display growth defects and, in most cases, also thermosensitivity. The defects are particularly strong in the V57 deletion mutant, which is additionally cold-sensitive. We hypothesize that the S10 loop tip residue is critical for the correct functioning of S10. Both the S10 flexible loop and tigecycline are in contact with helix h31 of the 16S rRNA. We speculate that exchanges or deletion of V57 alter the positioning of h31, thereby influencing both tigecycline binding and S10 function

Antibiotic Resistance of Acinetobacter spp. Isolates from the River Danube: Susceptibility Stays High

Acinetobacter spp. occur naturally in many different habitats, including food, soil, and surface waters. In clinical settings, Acinetobacter poses an increasing health problem, causing infections with limited to no antibiotic therapeutic options left. The presence of human generated multidrug resistant strains is well documented but the extent to how widely they are distributed within the Acinetobacter population is unknown. In this study, Acinetobacter spp. were isolated from water samples at 14 sites of the whole course of the river Danube. Susceptibility testing was carried out for 14 clinically relevant antibiotics from six different antibiotic classes. Isolates showing a carbapenem resistance phenotype were screened with PCR and sequencing for the underlying resistance mechanism of carbapenem resistance. From the Danube river water, 262 Acinetobacter were isolated, the most common species was Acinetobacter baumannii with 135 isolates. Carbapenem and multiresistant isolates were rare but one isolate could be found which was only susceptible to colistin. The genetic background of carbapenem resistance was mostly based on typical Acinetobacter OXA enzymes but also on VIM-2. The population of Acinetobacter (baumannii and non-baumannii) revealed a significant proportion of human-generated antibiotic resistance and multiresistance, but the majority of the isolates stayed susceptible to most of the tested antibiotics

Preliminary Toxicological Evaluation of the River Danube Using in Vitro Bioassays

The Joint Danube Survey 3, carried out in 2013 was the world’s biggest river research expedition of its kind. The course of the second largest river of Europe passes large cities like Vienna, Budapest and Belgrade and is fed from many tributaries like Inn, Thisza, Drava, Prut, Siret and Argeș. During the 6 weeks of shipping the 2375 km downstream the River Danube from Germany to the Black Sea an enormous number of water samples were analyzed and collected. A wide spectrum of scientific disciplines cooperated in analyzing the River Danube waters. For toxicological analysis, water samples were collected on the left, in the middle, and on the right side of the river at 68 JDS3 sampling points and frozen until the end of the Danube survey. All samples were analyzed with two in vitro bioassays tests (umuC and MTS). Testing umuC without S9 activation and MTS test did not show positive signals. But umuC investigations of the water samples came up with toxic signals on two stretches, when activated with S9 enzymes. The override of the limiting value of the umuC investigation with prior S9 activation started downstream Vienna (Austria) and was prolonged until Dunaföldvar (Hungary). This stretch of the River Danube passes a region that is highly industrialized, intensively used for agricultural purposes and also highly populated (Vienna, Bratislava and Budapest). The elevated values may indicate these influences

Tigecycline Resistant Klebsiella pneumoniae Isolated from Austrian River Water

Abstract: Antibiotic-resistant bacteria are spreading worldwide in medical settings but also in the environment. These resistant bacteria illustrate a major health problem in our times, and last-line antibiotics such as tigecycline represent an ultimate therapy option. Reports on tigecycline non-susceptible Enterobacteriaceae are presented with regard to medical settings but are rare with that for the environment. The aim of this study was to characterize two tigecycline non-susceptible Klebsiella pneumoniae isolates from the river Mur, and to question the resistance mechanism. The screening for chromosomal mutations revealed a deletion and a silent point mutation in one isolate and a point mutation in the other isolate all within the ramR allele. RamR acts as repressor and prevents overexpression of ramA. These mutations are likely to cause a resistant phenotype due to the overexpression of AcrAB-TolC. MLST revealed that the isolates belonged to two unrelated MLST types (ST2392 and ST2394). Both isolates only revealed resistance to tigecycline and tetracycline. This is one of the rare reports of tigecycline-resistant Klebsiella pneumoniae from surface water. The presence of two genetically different isolates suggests that the river water may bear substances that favor mutations that can lead to this efflux pump-driven resistance

Pseudomonas Species Diversity Along the Danube River Assessed by rpoD Gene Sequence and MALDI-TOF MS Analyses of Cultivated Strains

A collection of 611 Pseudomonas isolated from 14 sampling sites along the Danube River were identified previously by MALDI-TOF MS with the VITEK MS system and were grouped in 53 clusters by their main protein profiles. The strains were identified in the present study at the phylospecies level by rpoD gene sequencing. Partial sequences of the rpoD gene of 190 isolates representatives of all clusters were analyzed. Strains in the same MALDI-TOF cluster were grouped in the same phylospecies when they shared a minimum 95% similarity in their rpoD sequences. The sequenced strains were assigned to 34 known species (108 strains) and to 32 possible new species (82 strains). The 611 strains were identified at the phylospecies level combining both methods. Most strains were assigned to phylospecies in the Pseudomonas putida phylogenetic group of species. Special attention was given to 14 multidrug resistant strains that could not be assigned to any known Pseudomonas species and were considered environmental reservoir of antibiotic resistance genes. Coverage indices and rarefaction curves demonstrated that at least 50% of the Pseudomonas species in the Danube River able to grow in the isolation conditions have been identified at the species level. Main objectives were the confirmation of the correlation between the protein profile clusters detected by MALDI-TOF MS and the phylogeny of Pseudomonas strains based on the rpoD gene sequence, the assessment of the higher species discriminative power of the rpoD gene sequence, as well as the estimation of the high diversity of Pseudomonas ssp. along the Danube river. This study highlights the Pseudomonas species diversity in freshwater ecosystems and the usefulness of the combination of MALDI-TOF mass spectrometry for the dereplication of large sets of strains and the rpoD gene sequences for rapid and accurate identifications at the species level.Financial support to the Spanish Research Group was obtained from the Spanish MINECO through Project CGL2015-70925-P (AEI/FEDER, UE), with Fondo Europeo de Desarrollo Regional (FEDER) co-funding

Phenotypic Stability of Staphylococcus aureus Small Colony Variants (SCV) Isolates from Cystic Fibrosis (CF) Patients

One of the most interesting features of Staphylococcus aureus is its ability to switch to a small colony variant (SCV). This switch allows the pathogen to survive periods of antibiotic treatment or pressure from the immune system of the host and further enables it to start the infection once again after the environmental stress declines. However, so far only little is known about this reversion back to the more virulent wild type phenotype. Therefore, this study aimed to analyze the frequency of reversion to the wild type phenotype of thymidine auxotroph S. aureus SCV isolates (TD-SCVs) obtained from patients with cystic fibrosis (CF). With the use of single cell starting cultures, the occurrence of the thymidine prototroph revertants was monitored. The underlying mutational cause of the SCVs and subsequent revertants were analyzed by sequencing the gene coding for thymidylate synthase (ThyA), whose mutations are known to produce thymidine auxotroph S. aureus SCV. In our study, the underlying mutational cause for the switch to the TD-SCV phenotype was primarily point mutations. Out of twelve isolates, seven isolates showed an occurrence of revertants with a frequency ranging from 90.06% to 0.16%. This high variability in the frequency of reversion to the wild type was not expected. However, this variability in the frequency of reversion may also be the key to successful re-infection of the host. Sometimes quick reversion to the wild type proves necessary for survival, whereas other times, staying hidden for a bit longer leads to success in re-colonization of the host

Comparison of Updated Methods for Legionella Detection in Environmental Water Samples

The difficulty of cultivation of Legionella spp. from water samples remains a strenuous task even for experienced laboratories. The long incubation periods for Legionellae make isolation difficult. In addition, the water samples themselves are often contaminated with accompanying microbial flora, and therefore require complex cultivation methods from diagnostic laboratories. In addition to the recent update of the standard culture method ISO 11731:2017, new strategies such as quantitative PCR (qPCR) are often discussed as alternatives or additions to conventional Legionella culture approaches. In this study, we compared ISO 11731:2017 with qPCR assays targeting Legionella spp., Legionella pneumophila, and Legionella pneumophila serogroup 1. In samples with a high burden of accompanying microbial flora, qPCR shows an excellent negative predictive value for Legionella pneumophila, thus making qPCR an excellent tool for pre-selection of negative samples prior to work-intensive culture methods. This and its low limit of detection make qPCR a diagnostic asset in Legionellosis outbreak investigations, where quick-risk assessments are essential, and are a useful method for monitoring risk sites

Inactivation of Bacteriophage ɸ6 and SARS-CoV-2 in Antimicrobial Surface Tests

Due to the COVID-19 pandemic, researchers have focused on new preventive measures to limit the spread of SARS-CoV-2. One promising application is the usage of antimicrobial materials on often-touched surfaces to reduce the load of infectious virus particles. Since tests with in vitro-propagated SARS-CoV-2 require biosafety level 3 (BSL-3) laboratories with limited capacities and high costs, experiments with an appropriate surrogate like the bacteriophage ɸ6 are preferred in most studies. The aim of this study was to compare ɸ6 and SARS-CoV-2 within antiviral surface tests. Different concentrations of copper coatings on polyethylene terephthalate (PET) were used to determine their neutralizing activity against ɸ6 and SARS-CoV-2. The incubation on the different specimens led to similar inactivation of both SARS-CoV-2 and ɸ6. After 24 h, no infectious virus particles were evident on any of the tested samples. Shorter incubation periods on specimens with high copper concentrations also showed a complete inactivation. In contrast, the uncoated PET foils resulted only in a negligible reduced inactivation during the one-hour incubation. The similar reduction rate for ɸ6 and SARS-CoV-2 in our experiments provide further evidence that the bacteriophage ɸ6 is an adequate model organism for SARS-CoV-2 for this type of testing

Comparison of the Antibiotic Resistance of <i>Escherichia coli</i> Populations from Water and Biofilm in River Environments

Antibiotic-resistant, facultative pathogenic bacteria are commonly found in surface water; however, the factors influencing the spread and stabilization of antibiotic resistance in this habitat, particularly the role of biofilms, are not fully understood. The extent to which bacterial populations in biofilms or sediments exacerbate the problem for specific antibiotic classes or more broadly remains unanswered. In this study, we investigated the differences between the bacterial populations found in the surface water and sediment/biofilm of the Mur River and the Drava River in Austria. Samples of Escherichia coli were collected from both the water and sediment at two locations per river: upstream and downstream of urban areas that included a sewage treatment plant. The isolates were subjected to antimicrobial susceptibility testing against 21 antibiotics belonging to seven distinct classes. Additionally, isolates exhibiting either extended-spectrum beta-lactamase (ESBL) or carbapenemase phenotypes were further analyzed for specific antimicrobial resistance genes. E. coli isolates collected from all locations exhibited resistance to at least one of the tested antibiotics; on average, isolates from the Mur and Drava rivers showed 25.85% and 23.66% resistance, respectively. The most prevalent resistance observed was to ampicillin, amoxicillin–clavulanic acid, tetracycline, and nalidixic acid. Surprisingly, there was a similar proportion of resistant bacteria observed in both open water and sediment samples. The difference in resistance levels between the samples collected upstream and downstream of the cities was minimal. Out of all 831 isolates examined, 13 were identified as carrying ESBL genes, with 1 of these isolates also containing the gene for the KPC-2 carbapenemase. There were no significant differences between the biofilm (sediment) and open water samples in the occurrence of antibiotic resistance. For the E. coli populations in the examined rivers, the different factors in water and the sediment do not appear to influence the stability of resistance. No significant differences in antimicrobial resistance were observed between the bacterial populations collected from the biofilm (sediment) and open-water samples in either river. The different factors in water and the sediment do not appear to influence the stability of resistance. The minimal differences observed upstream and downstream of the cities could indicate that the river population already exhibits generalized resistance