Acinetobacter stercoris sp. nov. isolated from output source of a mesophilic german biogas plant with anaerobic operating conditions.

The Gram-stain-negative, oxidase negative, catalase positive strain KPC-SM-21T, isolated from a digestate of a storage tank of a mesophilic German biogas plant, was investigated by a polyphasic taxonomic approach. Phylogenetic identification based on the nearly full-length 16S rRNA gene revealed highest gene sequence similarity to Acinetobacter baumannii ATCC 19606T (97.0%). Phylogenetic trees calculated based on partial rpoB and gyrB gene sequences showed a distinct clustering of strain KPC-SM-21T with Acinetobacter gerneri DSM 14967T = CIP 107464T and not with A. baumannii, which was also supported in the five housekeeping genes multilocus sequence analysis based phylogeny. Average nucleotide identity values between whole genome sequences of strain KPC-SM-21T and next related type strains supported the novel species status. The DNA G + C content of strain KPC-SM-21T was 37.7 mol%. Whole-cell MALDI-TOF MS analysis supported the distinctness of the strain to type strains of next related Acinetobacter species. Predominant fatty acids were C18:1 ω9c (44.2%), C16:0 (21.7%) and a summed feature comprising C16:1 ω7c and/or iso-C15:0 2-OH (15.3%). Based on the obtained genotypic, phenotypic and chemotaxonomic data we concluded that strain KPC-SM-21T represents a novel species of the genus Acinetobacter, for which the name Acinetobacter stercoris sp. nov. is proposed. The type strain is KPC-SM-21T (= DSM 102168T = LMG 29413T).

Acinetobacter baumannii in manure and anaerobic digestates of German biogas plants.

Studies considering environmental multidrug-resistant Acinetobacter spp. are scarce. The application of manure on agricultural fields is one source of multidrug-resistant bacteria from livestock into the environment. Here, Acinetobacter spp. were quantified by quantitative polymerase chain reaction in manure applied to biogas plants and in the output of the anaerobic digestion, and Acinetobacter spp. isolated from those samples were comprehensively characterized. The concentration of Acinetobacter 16S ribosomal ribonucleic acid (rRNA) gene copies per g fresh weight was in range of 106-108 in manure and decreased (partially significantly) to a still high concentration (105-106) in digestates. 16S rRNA, gyrB-rpoB and blaOXA51-like gene sequencing identified 17 different Acinetobacter spp., including six A. baumannii strains. Multilocus sequence typing showed no close relation of the six strains with globally relevant clonal complexes; however, they represented five novel sequence types. Comparative genomics and physiological tests gave an explanation how Acinetobacter could survive the anaerobic biogas process and indicated copper resistance and the presence of intrinsic beta-lactamases, efflux-pump and virulence genes. However, the A. baumannii strains lacked acquired resistance against carbapenems, colistin and quinolones. This study provided a detailed characterization of Acinetobacter spp. including A. baumannii released via manure through mesophilic or thermophilic biogas plants into the environment.

Moheibacter stercoris sp. nov., isolated from an input sample of a biogas plant.

A Gram-stain-negative, rod-shaped bacterium, strain 784B1_12E-CasoT, was isolated from a mixed manure sample used as input material of a German biogas plant. Phylogenetic identification based on the nearly full-length 16S rRNA gene sequence placed the strain within the family Flavobacteriaceae(Bacteroidetes) with highest sequence similarity to the type strain of Moheibacter sediminis (93.7 %) followed by Empedobacter brevis(90.7 %). Major cellular fatty acids of strain 784B1_12E-CasoT were iso-C15 : 0 and iso-C17 : 0 3-OH. The polyamine pattern contained predominantly sym-homospermidine and the quinone system consisted exclusively of menaquinone MK-6. Major polar lipids were phosphatidylethanolamine and an unidentified polar lipid only detectable after staining for total lipids. The DNA G+C content was 34.5 mol%. Based on phylogenetic, chemotaxonomic and physiological analyses, a novel species of the genus Moheibacter is proposed, Moheibacterstercoris sp. nov. (type strain 784B1_12E-CasoT=CIP 110830T=LMG 28502T).

Empedobacter stercoris sp. nov., isolated from an input sample of a biogas plant.

Two Gram-stain-negative, rod-shaped bacteria, strains 990B6_12ER2AT and 994B6_12ER2A, were isolated during microbiological analysis of a mixed manure sample which was used as input material for a German biogas plant. Phylogenetic identification based on nearly full-length 16S rRNA gene sequences placed the isolates into the family Flavobacteriaceae within the phylum Bacteroidetes. Strains 990B6_12ER2AT and 994B6_12ER2A shared identical 16S rRNA gene sequences and showed highest 16S rRNA gene sequence similarity to the type strains of Empedobacter falsenii (97.3 %) and Empedobacter brevis (96.8 %).The major cellular fatty acids of strains 990B6_12ER2AT and 994B6_12ER2A were iso-C15 : 0, summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and iso-C17 : 0 3-OH.The polyamine pattern contained predominantly sym-homospermidine and the quinone system was menaquinone MK-6. Major polar lipids were phosphatidylethanolamine, one unidentified aminolipid and one unidentified polar lipid not containing an amino residue, a phosphate residue or a sugar moiety. In addition, moderate to minor amounts of several unidentified lipids were detected. The DNA G+C content was 31.7 and 29.0 mol%, for strains 990B6_12ER2AT and 994B6_12ER2A, respectively. On the basis of phylogenetic, chemotaxonomic and physiological analysis we propose a novel species of the genus Empedobacter, Empedobacter stercoris sp. nov. (type strain 990B6_12ER2AT = CIP 110833T = LMG 28501T).

Improved detection of extended spectrum beta-lactamase (ESBL)-producing Escherichia coli in input and output samples of German biogas plants by a selective pre-enrichment procedure.

The presence of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli was investigated in input (manure from livestock husbandry) and output samples of six German biogas plants in 2012 (one sampling per biogas plant) and two German biogas plants investigated in an annual cycle four times in 2013/2014. ESBL-producing Escherichia coli were cultured by direct plating on CHROMagar ESBL from input samples in the range of 100 to 104 colony forming units (CFU) per g dry weight but not from output sample. This initially indicated a complete elimination of ESBL-producing E. coli by the biogas plant process. Detected non target bacteria were assigned to the genera Acinetobacter, Pseudomonas, Bordetella, Achromobacter, Castellaniella, and Ochrobactrum. A selective pre-enrichment procedure increased the detection efficiency of ESBL-producing E. coli in input samples and enabled the detection in five of eight analyzed output samples. In total 119 ESBL-producing E. coli were isolated from input and 46 from output samples. Most of the E. coli isolates carried CTX-M-type and/or TEM-type beta lactamases (94%), few SHV-type beta lactamase (6%). Sixty-four blaCTX-M genes were characterized more detailed and assigned mainly to CTX-M-groups 1 (85%) and 9 (13%), and one to group 2. Phylogenetic grouping of 80 E. coli isolates showed that most were assigned to group A (71%) and B1 (27%), only one to group D (2%). Genomic fingerprinting and multilocus sequence typing (MLST) showed a high clonal diversity with 41 BOX-types and 19 ST-types. The two most common ST-types were ST410 and ST1210. Antimicrobial susceptibility testing of 46 selected ESBL-producing E. coli revealed that several isolates were additionally resistant to other veterinary relevant antibiotics and some grew on CHROMagar STEC but shiga-like toxine (SLT) genes were not detected. Resistance to carbapenems was not detected. In summary the study showed for the first time the presence of ESBL-producing E. coli in output samples of German biogas plants.