Comparison of the Antibiotic Resistance of Escherichia coli 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.

Cefsulodin and Vancomycin: A Supplement for Chromogenic Coliform Agar for Detection of Escherichia coli and Coliform Bacteria from Different Water Sources.

Background microorganism growth on Chromogenic Coliform Agar (CCA) can be challenging. For this reason, a new alternative method with a Cefsulodin/Vancomycin (CV)-supplemented CCA should be developed in this study. CCA supplemented with CV was validated according to ÖNORM EN ISO 16140-4:2021 using water from natural sources in Styria, Austria. Results show that the alternative method using the supplemented CCA has similar values in relation to sensitivity (82.2%), specificity (98.6%) and higher selectivity (59%) compared to the reference method. Repeatability and reproducibility were acceptable for the alternative method and showed similar results with the reference method. The alternative method shows a very low false positive rate and a low false negative rate paired with good performance regarding the inclusion study. The exclusion study shows the advantage of our method by suppressing background microorganisms and facilitating the process of enumeration of Escherichia coli and other coliform bacteria on CCA plates. Aeromonas hydrophila and Pseudomonas aeruginosa growth was inhibited using the supplement. To conclude, the coliform CV selective supplement combined with CCA is an appropriate tool for coliform bacteria detection in water samples.

Analysis of Extended Spectrum Beta Lactamase (ESBL) Genes of Non-Invasive ESBL Enterobacterales in Southeast Austria in 2017.

Extended spectrum beta lactamases producing Enterobacteriaceae are a major player in the antibiotic resistance challenge. In general, the situation regarding antibiotic resistance in Austria is very good compared to many other countries. Perhaps this is why there is a lack of data on the distribution of ESBL genes in the clinical setting. The aim of this study was to collect data on ESBL genes from a larger sample of human non-invasive clinical isolates from one region in Austria. In total, 468 isolates from different sample materials isolated at the Medical University of Graz from 2017 were examined. The most frequent organisms were Escherichia coli and Klebsiella pneumoniae. Among the enzymes produced, CTX-M-15 was clearly dominant, exotic ESBLs were only represented by three Proteus mirabilis isolates harboring genes for VEB-6 and one P. mirabilis for CTX-M-2, respectively. Compared to other countries, the results are in line with the expectations. The data help to better classify the many studies from the non-clinical field in Austria and to shift the focus slightly away from the exotic results and sample sites.

Troubled water under the bridge: Screening of River Mur water reveals dominance of CTX-M harboring Escherichia coli and for the first time an environmental VIM-1 producer in Austria.

Antibiotic resistant bacteria (ARB) in the aquatic environment are reported from all over the world and their presence in the environment has become quite common. The current most prominent example is the presence of beta-lactamases harboring Enterobacteriaceae. The aim of this study was to investigate the presence and diversity (on the genetic and phenotypic levels) of extended spectrum beta-lactamases (ESBL) and carbapenemases harboring Enterobacteriaceae from the River Mur in the center of Graz, Austria's second largest city. Thus over a period of four months water samples were taken, filtrated and screened for these bacteria. All samples revealed ESBL harboring Enterobacteriaceae, of which all with only one exception were Escherichia coli. Dominant ESBL gene family was CTX-M, represented by subgroups CTX-M-1 group, CTX-M-2 group and CTX-M-9 group. Surprisingly co-resistances to non-beta-lactam antibiotics were low, only resistance to trimethoprim was detected in 50% of all (70) isolates. One Klebsiella oxytoca with GES-1 was isolated. To date, GES ESBL has never been reported from Austria before and only rarely from other European countries. Screening for carbapenemase harboring Enterobacteriaceae revealed one Enterobacter cloacae with the gene for VIM-1. Members sharing the same multi-locus-sequence-type (MLST) as well as members of the same rep PCR clusters occurred at different sampling time points. ESBL-harboring Enterobacteriaceae are common in Austrian river water, is dominated by Escherichia coli and CTX-M enzymes. Furthermore, some of the isolates could be linked to different origins.

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.

Enterobacteriaceae Isolated from the River Danube: Antibiotic Resistances, with a Focus on the Presence of ESBL and Carbapenemases.

In a clinical setting it seems to be normal these days that a relevant proportion or even the majority of different bacterial species has already one or more acquired antibiotic resistances. Unfortunately, the overuse of antibiotics for livestock breeding and medicine has also altered the wild-type resistance profiles of many bacterial species in different environmental settings. As a matter of fact, getting in contact with resistant bacteria is no longer restricted to hospitals. Beside food and food production, the aquatic environment might also play an important role as reservoir and carrier. The aim of this study was the assessment of the resistance patterns of Escherichia coli and Klebsiella spp. out of surface water without prior enrichment and under non-selective culture conditions (for antibiotic resistance). In addition, the presence of clinically important extended spectrum beta lactamase (ESBL) and carbapenmase harboring Enterobacteriaceae should be investigated. During Joint Danube Survey 3 (2013), water samples were taken over the total course of the River Danube. Resistance testing was performed for 21 different antibiotics. Samples were additionally screened for ESBL or carbapenmase harboring Enterobacteriaceae. 39% of all isolated Escherichia coli and 15% of all Klebsiella spp. from the river Danube had at least one acquired resistance. Resistance was found against all tested antibiotics except tigecycline. Taking a look on the whole stretch of the River Danube the proportion of multiresistances did not differ significantly. In total, 35 ESBL harboring Enterobacteriaceae, 17 Escherichia coli, 13 Klebsiella pneumoniae and five Enterobacter spp. were isolated. One Klebsiella pneumoniae harboring NMD-1 carbapenmases and two Enterobacteriaceae with KPC-2 could be identified. Human generated antibiotic resistance is very common in E. coli and Klebsiella spp. in the River Danube. Even isolates with resistance patterns normally associated with intensive care units are present.

Antibiotic Resistance Patterns of Pseudomonas spp. Isolated from the River Danube.

Spread and persistence of antibiotic resistance pose a severe threat to human health, yet there is still lack of knowledge about reservoirs of antibiotic resistant bacteria in the environment. We took the opportunity of the Joint Danube Survey 3 (JDS3), the world's biggest river research expedition of its kind in 2013, to analyse samples originating from different sampling points along the whole length of the river. Due to its high clinical relevance, we concentrated on the characterization of Pseudomonas spp. and evaluated the resistance profiles of Pseudomonas spp. which were isolated from eight sampling points. In total, 520 Pseudomonas isolates were found, 344 (66.0%) isolates were identified as Pseudomonas putida, and 141 (27.1%) as Pseudomonas fluorescens, all other Pseudomonas species were represented by less than five isolates, among those two P. aeruginosa isolates. Thirty seven percent (37%) of all isolated Pseudomonas species showed resistance to at least one out of 10 tested antibiotics. The most common resistance was against meropenem (30.4%/158 isolates) piperacillin/tazobactam (10.6%/55 isolates) and ceftazidime (4.2%/22 isolates). 16 isolates (3.1%/16 isolates) were multi-resistant. For each tested antibiotic at least one resistant isolate could be detected. Sampling points from the upper stretch of the River Danube showed more resistant isolates than downriver. Our results suggest that antibiotic resistance can be acquired by and persists even in Pseudomonas species that are normally not in direct contact with humans. A possible scenario is that these bacteria provide a reservoir of antibiotic resistance genes that can spread to related human pathogens by horizontal gene transfer.

Antimicrobial activity of gentamicin palmitate against high concentrations of Staphylococcus aureus.

The reduction of implant related infections plays a pivotal role in orthopaedic surgery as an increasing number of people require implants (up to 200,000 per year in the United States (source: Joint Implant Surgery & Research Foundation 2010)). The aim of the current study is to prevent and thus decrease the number of bacterial infections. Both pre and post operative systemic antibiotic treatment and gentamicin containing bone cements (polymethylmethacrylate, PMMA) are commonly used strategies to overcome infections. In this study, the antimicrobial efficacy of gentamicin sulfate loaded bone cement was compared with titan discs coated with a new form of gentamicin, gentamicin palmitate. Adherence prevention, killing rates and killing kinetics were compared in an in vitro model, using Staphylococcus aureus (S. aureus), which together with Staphylococcus epidermidis (S. epidermidis) represents 60% of bacteria found responsible for hip implant infections (An and Friedman, 1996, J Hosp Infect 33(2):93-108). In our experiments gentamicin, which was applied as gentamicin palmitate on the surface of the implants, showed a high efficacy in eliminating bacteria. In contrast to gentamicin sulfate containing bone cements, gentamicin palmitate is released over a shorter period of time thus not inducing antibiotic resistance. Another benefit for clinical application is that it achieves high local levels of active ingredient which fight early infections and minimize toxic side effects. Furthermore, the short term hydrophobic effect of gentamicin palmitate can successfully impede biofilm formation. Thus, the use of self-adhesive antibiotic fatty acid complexes like gentamicin palmitate represents a new option for the anti-infective coating of cementless titan implants.