Franz Mascher

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.

Waterborne diseases are a serious threat because of their ability to infect a high number of individuals in a short time span, such as during outbreaks of Legionellosis. This significantly highlights the need for the rapid detection and quantification of bacteria in environmental water samples. The aim of this study was to investigate the feasibility of quantitative Polymerase Chain Reaction (qPCR) for the detection of Legionellapneumophila (L. pneumophila) in environmental water samples and comparison of standard culture methods for Legionella detection with qPCR. Our study reached a negative predictive value (NPV) for L. pneumophila of 80.7% and for L. pneumophila serogroup 1 (sg1) the calculated NPV was 87.0%. The positive predictive value (PPV) for L. pneumophila was 53.9% and for L. pneumophila sg1 PPV was 21.4%. Results showed a correlation between qPCR and culture with an R² value of 0.8973 for L. pneumophila, whereas no correlation was observed for the detection of L. pneumophila sg1. In our study, qPCR proved useful for the identification of L. pneumophila negative samples. However, despite the obvious benefits (sample handling, rapid generation of results), qPCR needs to be improved regarding the PPV before it can replace culture in water quality assessment.

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.

Within the framework of a one-year study the treatment capacity of a municipal wastewater treatment plant (WWTP) was evaluated, with regard to fecal indicator bacteria (FIB) and to their influence on the recipient. The logarithmic reduction rates for fecal coliforms (FC), Escherichia coli (EC) and intestinal enterococci (IE) were 2.84, 2.90 and 2.93. In the investigated period of time, the tested treatment plant released 4.3% of the total annual load flow volume as combined sewer overflow (CSO), that is, when the influent into the combined sewer exceeds the capacity of the treatment plant and coarsely cleaned wastewater arrives at the recipient. This CSO discharge increased the number of FIB significantly by 1.2 × 10² MPN/100 mL for EC, and by 1.8 × 10¹ MPN/100 mL for IE. For the Styrian part of the Mur River (1.6 million inhabitants), a calculation of FIB of all sewage treatment plants estimating the same ratio of CSO (4.3%) and a given mean flow rate (QM) results in a significant increase of the FIB load in the recipient: 3.8 × 10³ MPN/100 mL for EC and 5.8 × 10² MPN/100 mL for IE. On the basis of these values the standards of water quality for recreational purposes cannot be met.