Molecular characterization of Escherichia coli isolates recovered from broilers with cellulitis.

Avian cellulitis in broilers, caused by avian pathogenic Escherichia coli, is a major cause for carcass rejections during meat inspection, resulting in significant economic losses. In this study, we analysed E. coli isolates obtained from broiler chickens affected by cellulitis for their genetic relatedness and antimicrobial resistance phenotype and genotype. The objective was to determine whether there is a clonal spread or whether these clinical isolates differ. For this purpose, E. coli was isolated from swab samples collected from diseased broilers across 77 poultry farms in Germany, resulting in 107 isolates. These isolates were subjected to serotyping, PCR-based phylotyping and macrorestriction analysis with subsequent pulsed-field gel-electrophoresis for typing purposes. In addition, the presence of virulence genes associated with avian pathogenic E. coli (APEC) was investigated by PCR. Antimicrobial susceptibility of the isolates was examined by the disk diffusion method according to CLSI guidelines and subsequently, the presence of corresponding resistance genes was investigated by PCR. Typing results revealed that a significant proportion of the isolates belonged to serotype O78:K80, which is one of the major APEC serotypes. Phylogenetic grouping showed that phylogenetic group D was most commonly represented (n = 49). Macrorestriction analysis showed overall heterogenous results, however, some clustering of closely related isolates was observed. The level of antimicrobial resistance was high, with 83.8% of isolates non-susceptible to at least one class of antimicrobial agents and 40% of isolates showing resistance to at least three classes. The most frequently observed resistance was to ampicillin, mediated by blaTEM (n = 56). However, few isolates were non-susceptible to ciprofloxacin (n = 8) and none of the isolates was resistant to 3rd generation cephalosporins or carbapenems. Overall, the results show that genetically diverse APEC associated with avian cellulitis can be found among and within German poultry farms. While most isolates were antimicrobial resistant, resistance levels to high(est) priority critically important antimicrobials were low.

Recommendation of a standardized broth microdilution method for antimicrobial susceptibility testing of Avibacterium paragallinarum and resistance monitoring.

This study aimed to develop a method for standardized broth microdilution antimicrobial susceptibility testing (AST) of Avibacterium (Av.) paragallinarum, the causative agent of infectious coryza in chickens. For this, a total of 83 Av. paragallinarum isolates and strains were collected from 15 countries. To select unrelated isolates for method validation steps, macrorestriction analyses were performed with 15 Av. paragallinarum. The visible growth of Av. paragallinarum was examined in six broth media and growth curves were compiled. In Veterinary Fastidious Medium and cation-adjusted Mueller-Hinton broth (CAMHB) + 1% chicken serum + 0.0025% NADH (CAMHB + CS + NADH), visible growth of all isolates was detected and both media allowed adequate bacterial growth. Due to the better readability of Av. paragallinarum growth in microtiter plates, CAMHB + CS + NADH was chosen for AST. Repetitions of MIC testing with five epidemiologically unrelated isolates using a panel of 24 antimicrobial agents resulted in high essential MIC agreements of 96%-100% after 48-h incubation at 35 ± 2°C. Hence, the remaining 78 Av. paragallinarum were tested and demonstrated easily readable MICs with the proposed method. Differences in MICs were detected between isolates from different continents, with isolates from Africa showing lower MICs compared to isolates from America and Europe, which more often showed elevated MICs of aminoglycosides, quinolones, tetracyclines, and/or trimethoprim/sulfamethoxazole. PCR analyses of isolates used for method development revealed that isolates with elevated MICs of tetracyclines harbored the tetracycline resistance gene tet(B) but none of the other tested resistance genes were detected. Therefore, whole-genome sequencing data from 62 Av. paragallinarum were analyzed and revealed the presence of sequences showing nucleotide sequence identity to the genes aph(6)-Id, aph(3″)-Ib, blaTEM-1B, catA2, sul2, tet(B), tet(H), and mcr-like. Overall, the proposed method using CAMHB + CS + NADH for susceptibility testing with 48-h incubation time at 35 ± 2°C in ambient air was shown to be suitable for Av. paragallinarum. Due to a variety of resistance genes detected, the development of clinical breakpoints is highly recommended. IMPORTANCE: Avibacterium paragallinarum is an important pathogen in veterinary medicine that causes infectious coryza in chickens. Since antibiotics are often used for treatment and resistance of the pathogen is known, targeted therapy should be given after resistance testing of the pathogen. Unfortunately, there is currently no accepted method in standards that allows susceptibility testing of this fastidious pathogen. Therefore, we have worked out a method that allows harmonized susceptibility testing of the pathogen. The method meets the requirements of the CLSI and could be used by diagnostic laboratories.

Toward a Method for Harmonized Susceptibility Testing of Mycoplasma bovis by Broth Microdilution.

Mycoplasma bovis is a fastidious pathogen of cattle causing massive economic losses in the calf and dairy industries worldwide. Since there is no approved standard method for antimicrobial susceptibility testing (AST) of M. bovis, the Clinical and Laboratory Standards Institute has requested the development of a suitable method. Therefore, this study aimed at developing a method for harmonized broth microdilution AST of M. bovis. For this, 131 M. bovis field isolates and M. bovis strain DSM 22781T were collected and macrorestriction analysis was performed to select 15 epidemiologically unrelated M. bovis strains for method validation steps. To select a suitable broth for AST of M. bovis, growth determinations were performed using five media and growth curves were compiled. Then, susceptibility testing was performed considering the exact (precondition of five identical MICs) and essential (MIC mode, accepting a deviation of ±1 dilution step) MIC agreements to evaluate the reproducibility of MIC values using a panel of 16 antimicrobial agents. Subsequently, the remaining field isolates were tested and the suitability of quality control (QC) strains was assessed. Growth experiments showed that SP4 broth was the only one of the five media that yielded sufficient growth of M. bovis. Therefore, it was selected as the test medium for AST and homogeneous MIC values were obtained (exact and essential agreements of 36 to 100% and 92 to 100%, respectively). For all other isolates tested, easy-to-read MIC endpoints were determined with this medium. High overall MIC50 and/or MIC90 values were observed for aminoglycosides and macrolides, and some isolates showed elevated MICs of fluoroquinolones, gentamicin, and/or tiamulin. Since the MICs of four commonly used QC strains were partially not within their ranges, a 20-fold MIC testing of M. bovis DSM 22781T was performed and met the criteria for a new QC strain. For harmonized AST of M. bovis, SP4 broth seems to be suitable with an incubation time of 72 ± 2 h and further validation of M. bovis DSM 22781T as a future QC strain is recommended.