Antibiotic concentrations in raw hospital wastewater surpass minimal selective and minimum inhibitory concentrations of resistant Acinetobacter baylyi strains.

Antibiotics are essential for modern medicine, they are employed frequently in hospitals and, therefore, present in hospital wastewater. Even in concentrations, that are lower than the minimum inhibitory concentrations (MICs) of susceptible bacteria, antibiotics may exert an influence and select resistant bacteria, if they exceed the MSCs (minimal selective concentrations) of resistant strains. Here, we compare the MSCs of fluorescently labelled Acinetobacter baylyi strains harboring spontaneous resistance mutations or a resistance plasmid with antibiotic concentrations determined in hospital wastewater. Low MSCs in the µg/L range were measured for the quinolone ciprofloxacin (17 µg/L) and for the carbapenem meropenem (30 µg/L). A 24 h continuous analysis of hospital wastewater showed daily fluctuations of the concentrations of these antibiotics with distinctive peaks at 7-8 p.m. and 5-6 a.m. The meropenem concentrations were always above the MSC and MIC values of A. baylyi. In addition, the ciprofloxacin concentrations were in the range of the lowest MSC for about half the time. These results explain the abundance of strains with meropenem and ciprofloxacin resistance in hospital wastewater and drains.

Detection of methicillin-resistant coagulase-negative staphylococci harboring the class A mec complex by MALDI-TOF mass spectrometry.

The aim of this study was to test the identification of methicillin resistance in coagulase-negative staphylococci by routine matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). SCCmec cassettes of type II, III and VIII encode a small peptide called PSM-mec in the vicinity of mecA. It is visible at m/z 2415 during MALDI-TOF MS of whole cells of Staphylococcus aureus. In view of the fact that psm-mec has been identified in methicillin-resistant coagulase-negative staphylococci, we evaluated a collection of clinical coagulase-negative staphylococci, that contained 77.03% of methicillin-resistant isolates, for the presence of the structural gene encoding PSM-mec and the appearance of the corresponding signal during mass spectroscopy. In MALDI-TOF MS spectra, 89.65% of the strains that harbored the gene yielded the correct signal, corresponding to a sensitivity of 0.897 and a specificity of 1.0. However, regarding detection of methicillin resistance, i. e. considering all resistant strains as positive regardless of the presence of the gene, the overall sensitivity of the test decreased to 0.285, due to the fact that only 29.43% of all resistant isolates contained psm-mec. In conclusion, the presence of the signal in MALDI-TOF MS quickly indicates methicillin-resistance in coagulase-negative staphylococci but its absence does not indicate susceptibility to methicillin.

Bacteria isolated from hospital, municipal and slaughterhouse wastewaters show characteristic, different resistance profiles.

Multidrug-resistant bacteria cause difficult-to-treat infections and pose a risk for modern medicine. Sources of multidrug-resistant bacteria include hospital, municipal and slaughterhouse wastewaters. In this study, bacteria with resistance to 3rd generation cephalosporins were isolated from all three wastewater biotopes, including a maximum care hospital, municipal wastewaters collected separately from a city and small rural towns and the wastewaters of two pig and two poultry slaughterhouses. The resistance profiles of all isolates against clinically relevant antibiotics (including ß-lactams like carbapenems, the quinolone ciprofloxacin, colistin, and trimethoprim/sulfamethoxazole) were determined at the same laboratory. The bacteria were classified according to their risk to human health using clinical criteria, with an emphasis on producers of carbapenemases, since carbapenems are prescribed for hospitalized patients with infections with multi-drug resistant bacteria. The results showed that bacteria that pose the highest risk, i. e., bacteria resistant to all ß-lactams including carbapenems and ciprofloxacin, were mainly disseminated by hospitals and were present only in low amounts in municipal wastewater. The isolates from hospital wastewater also showed the highest rates of resistance against antibiotics used for treatment of carbapenemase producers and some isolates were susceptible to only one antibiotic substance. In accordance with these results, qPCR of resistance genes showed that 90% of the daily load of carbapenemase genes entering the municipal wastewater treatment plant was supplied by the clinically influenced wastewater, which constituted approximately 6% of the wastewater at this sampling point. Likewise, the signature of the clinical wastewater was still visible in the resistance profiles of the bacteria isolated at the entry into the wastewater treatment plant. Carbapenemase producers were not detected in slaughterhouse wastewater, but strains harboring the colistin resistance gene mcr-1 could be isolated. Resistances against orally available antibiotics like ciprofloxacin and trimethoprim/sulfamethoxazole were widespread in strains from all three wastewaters.

Dissemination of multi-resistant Gram-negative bacteria into German wastewater and surface waters.

Carbapenem antibiotics constitute the mainstay therapy of nosocomial infections with extended spectrum beta-lactamase producing Gram-negative bacteria; however, resistance against these compounds is increasing. This study was designed to demonstrate that carbapenemase-producing bacteria are disseminated from hospitals into the environment. To this end, resistant bacteria were isolated from a clinical/urban and from a rural catchment system in Germany in 2016/17. The study followed the dissemination of resistant bacteria from the wastewater through the wastewater treatment plant (WWTP) into the receiving surface waters. The bacteria were cultivated on selective agar and characterized by antibiotic testing, real-time PCR targeting carbapenemase genes and typing. Bacteria with resistance to third generation cephalosporins were isolated from all sample sites. 134 isolates harboring carbapenemase genes encoding VIM, NDM and OXA-48 and 26 XDR (extensively drug-resistant) strains with susceptibility to only one or two antibiotics were isolated from the clinical/urban system. The rural system yielded eight carbapenemase producers and no XDR strains. In conclusion, clinical wastewaters were charged with a high proportion of multidrug resistant bacteria. Although most of these bacteria were eliminated during wastewater treatment, dissemination into surface waters is possible as single carbapenemase producers were still present in the effluent of the WWTP.