Minimum inhibitory concentrations of chlorhexidine- and lactic acid-based teat disinfectants: An intervention trial assessing bacterial selection and susceptibility.

Teat disinfection is a recommended preventive tool to improve udder health and to prevent new intramammary infections. However, side effects are discussed, such as bacterial selection of less-susceptible bacteria with the application of certain teat disinfectants. The objective of this study was to assess the species composition and bacterial in vitro susceptibility by means of an interventive trial. For this purpose, 3 different postmilking teat treatments (disinfection with 0.215% chlorhexidine or 3.5% lactic acid, or control group with no dipping) were applied to 28 cows in a 6-d intervention approach using a split-udder design. Milk samples were taken before and after intervention. Bacteria were cultured and differentiated to species or genus level by MALDI-TOF mass spectrometry. Minimum inhibitory concentrations (MIC) were determined, and MIC changes over time were recorded. Susceptibilities to chlorhexidine and lactic acid were compared between species of the genera Staphylococcus, Streptococcus, Corynebacterium, and others. Species composition changed during the intervention. Under the treatment of chlorhexidine and lactic acid, the proportion of coagulase-negative staphylococci (CNS) decreased. An increased proportion of species belonging to the genus Corynebacterium was observed especially under the application of lactic acid. Although both teat disinfectants were basically effective, isolates differed in their susceptibility to both teat disinfectants. Populations of CNS, Staphylococcus aureus, and Corynebacterium spp. showed significantly lower absolute MIC values for chlorhexidine. Compared with other species, Corynebacterium spp. showed the lowest susceptibility for chlorhexidine as well as for lactic acid. A significant increase in MIC values after 6 d of intervention was observed with the lactic acid treatment in all isolates, as well as in CNS. This increase can be interpreted as either adaptation of isolates or displacement of more-susceptible species by less-susceptible species. Further studies using long-term intervention might reveal more pronounced effects on MIC values and species composition.

Susceptibility to disinfectants in antimicrobial-resistant and -susceptible isolates of Escherichia coli, Enterococcus faecalis and Enterococcus faecium from poultry-ESBL/AmpC-phenotype of E. coli is not associated with resistance to a quaternary ammonium compound, DDAC.

AIMS: The spread of bacteria that are simultaneously resistant to disinfectants and antimicrobials would constitute an unsettling scenario. In order to explore an association between antimicrobial resistance and reduced susceptibility to biocides/microbicides (disinfectants) in agriculture, we investigated Escherichia coli (n = 438) and enterococci (n = 120) isolated from six different flocks of the same poultry farm with known history of antimicrobial treatment. METHODS AND RESULTS: Susceptibility to disinfectants (formic acid and a quaternary ammonium compound (QAC), didecyldimethylammoniumchloride-DDAC) was assessed by macrodilution according to guidelines of the German Veterinary Society. Escherichia coli, Enterococcus faecalis and Enterococcus faecium were screened (i) for reduced biocide susceptibility and (ii) for an association of biocide susceptibility and antimicrobial resistance including the production of extended-spectrum beta-lactamases (ESBL) and the hyperproduction of AmpC-type beta-lactamases. DDAC inhibited ESBL/AmpC(hyper)-producing E. coli (n = 53) from poultry at similar or slightly lower inhibitory concentrations, compared with non-ESBL/AmpC strains (median MIC = 0·36 vs 1·44 mg l-1 ). In contrast, DDAC-MICs were positively correlated with several other antibiotic MICs (e.g. piperacillin and sulphamethoxazole + trimethoprim in E. coli, chloramphenicol in E. faecalis) and increased DDAC-MICs were statistically linked to high-level aminoglycoside resistance in enterococci (streptomycin high level). DDAC-MICs did not correlate with the presence of the integron marker qacEDelta1. CONCLUSIONS: This study provides indication that residual disinfectant might be able to select antimicrobial-resistant enterococci, but not ESBL-/AmpC (hyper)producing E. coli from poultry. SIGNIFICANCE AND IMPACT OF THE STUDY: While ESBL-/AmpC-E. coli were inhibited at disinfectant concentrations comparable to or lower than wildtype values, low concentrations of QACs might be able to select other antimicrobial-resistant E. coli or enterococci-a finding with special significance for the food processing industry, where QACs are regularly used.

Hot topic: Bovine milk samples yielding negative or nonspecific results in bacterial culturing--the possible role of PCR-single strand conformation polymorphism in mastitis diagnosis.

A large proportion of mastitis milk samples yield negative or nonspecific results (i.e., no mastitis pathogen can be identified) in bacterial culturing. Therefore, the culture-independent PCR-single strand conformation polymorphism method was applied to the investigation of bovine mastitis milk samples. In addition to the known mastitis pathogens, the method was suitable for the detection of fastidious bacteria such as Mycoplasma spp., which are often missed by conventional culturing methods. The detection of Helcococcus ovis in 4 samples might indicate an involvement of this species in pathogenesis of bovine mastitis. In conclusion, PCR-single-strand conformation polymorphism is a promising tool for gaining new insights into the bacteriological etiology of mastitis.

Phenotypic and genotypic bacterial antimicrobial resistance in liquid pig manure is variously associated with contents of tetracyclines and sulfonamides.

AIMS: Antibiotic residues as well as antibiotic-resistant bacteria in environmental samples might pose a risk to human health. This study aimed to investigate the association between antibiotic residues and bacterial antimicrobial resistance in liquid pig manure used as fertilizer. METHODS AND RESULTS: Concentrations of tetracyclines (TETs) and sulfonamides (SULs) were determined by liquid chromatography-mass spectrometry in 305 pig manure samples; antibiotic contents were correlated to the phenotypic resistance of Escherichia coli (n = 613) and enterococci (n = 564) towards up to 24 antibiotics. In 121 samples, the concentration of the TET resistance genes tet(M), tet(O) and tet(B) was quantified by real-time-PCR. TETs were found in 54% of the samples. The median sum concentration of all investigated TETs in the positive samples was 0.73 mg kg(-1). SULs were found with a similar frequency (51%) and a median sum concentration of 0.15 mg kg(-1) in the positive samples. Associated with the detection of TETs and/or SULs, resistance rates were significantly elevated for several substances - some of them not used in farm animals, e.g. chloramphenicol and synercid. In addition, multiresistant isolates were found more often in samples containing antibiotics. Analysis of the resistance genes tet(M) and tet(O) already showed a significant increase in their concentrations - but not in tet(B) - in the lowest range of total TET concentration. Mean tet(M) concentrations increased by the factor of 4.5 in the TET concentration range of 0.1-1 mg kg(-1), compared to negative manure samples. CONCLUSIONS: Antibiotic contamination of manure seems to be associated with a variety of changes in bacterial resistance, calling for a prudent use of antibiotics in farm animals. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides an interdisciplinary approach to assess antimicrobial resistance by combining the microbiological analysis of bacterial resistance with high quality chemical analysis of antibiotic residues in a representative number of environmental samples.