Emergence of methicillin resistance predates the clinical use of antibiotics.

The discovery of antibiotics more than 80 years ago has led to considerable improvements in human and animal health. Although antibiotic resistance in environmental bacteria is ancient, resistance in human pathogens is thought to be a modern phenomenon that is driven by the clinical use of antibiotics1. Here we show that particular lineages of methicillin-resistant Staphylococcus aureus-a notorious human pathogen-appeared in European hedgehogs in the pre-antibiotic era. Subsequently, these lineages spread within the local hedgehog populations and between hedgehogs and secondary hosts, including livestock and humans. We also demonstrate that the hedgehog dermatophyte Trichophyton erinacei produces two ß-lactam antibiotics that provide a natural selective environment in which methicillin-resistant S. aureus isolates have an advantage over susceptible isolates. Together, these results suggest that methicillin resistance emerged in the pre-antibiotic era as a co-evolutionary adaptation of S. aureus to the colonization of dermatophyte-infected hedgehogs. The evolution of clinically relevant antibiotic-resistance genes in wild animals and the connectivity of natural, agricultural and human ecosystems demonstrate that the use of a One Health approach is critical for our understanding and management of antibiotic resistance, which is one of the biggest threats to global health, food security and development.

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.

High molecular/low acetylated chitosans reduce adhesion of Campylobacter jejuni to host cells by blocking JlpA.

Infections caused by Campylobacter spp. are a major cause of severe enteritis worldwide. Multifactorial prevention strategies are necessary to reduce the prevalence of Campylobacter. In particular, antiadhesive strategies with specific inhibitors of early host-pathogen interaction are promising approaches to reduce the bacterial load. An in vitro flow cytometric adhesion assay was established to study the influence of carbohydrates on the adhesion of C. jejuni to Caco-2 cells. Chitosans with a high degree of polymerization and low degree of acetylation were identified as potent antiadhesive compounds, exerting significant reduction of C. jejuni adhesion to Caco-2 cells at non-toxic concentrations. Antiadhesive and also anti-invasive effects were verified by confocal laser scanning microscopy. For target identification, C. jejuni adhesins FlpA and JlpA were expressed in Escherichia coli ArcticExpress, and the influence of chitosan on binding to fibronectin and HSP90α, respectively, was investigated. While no effects on FlpA binding were found, a strong inhibition of JlpA-HSP90α binding was observed. To simulate real-life conditions, chicken meat was inoculated with C. jejuni, treated with antiadhesive chitosan, and the bacterial load was quantified. A strong reduction of C. jejuni load was observed. Atomic force microscopy revealed morphological changes of C. jejuni after 2 h of chitosan treatment, indicating disturbance of the cell wall and sacculi formation by electrostatic interaction of positively charged chitosan with the negatively charged cell surface. In conclusion, our data indicate promising antiadhesive and anti-invasive potential of high molecular weight, strongly de-acetylated chitosans for reducing C. jejuni load in livestock and food production. KEY POINTS: • Antiadhesive effects of chitosan with high DP/low DA against C. jejuni to host cells • Specific targeting of JlpA/Hsp90α interaction by chitosan • Meat treatment with chitosan reduces C. jejuni load.

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.

Proposal of a Method for Harmonized Broth Microdilution Antimicrobial Susceptibility Testing of Avibacterium gallinarum.

Avibacterium (Av.) gallinarum is an opportunistic pathogen in poultry, which, however, has also been associated with human disease. There is currently no approved method for antimicrobial susceptibility testing of this pathogen, so this study aimed at developing a harmonized broth microdilution method for Av. gallinarum that is suitable for diagnostic laboratories. For this, the Av. gallinarum CCUG 12391T type strain and 42 field isolates were collected and their species was confirmed by using a species-specific PCR assay and biochemical reactions. To select epidemiologically unrelated isolates, ApaI macrorestriction analysis was performed. Preliminary growth experiments were conducted with six culture media, and based on the results, four media were selected to compile growth curves with four isolates. Independent repetitions of MIC determinations were then performed to evaluate the reproducibility of the values. Cation-adjusted Mueller-Hinton broth (CAMHB) was initially selected as broth medium, but did not show sufficient homogeneity of MICs. Therefore, CAMHB plus 1% chicken serum and 0.0025% NADH was selected and showed a good homogeneity of MICs after 20 h and 24 h of incubation at 35 ± 2°C. This was reflected in essential MIC agreements ranging between 96% and 100%. Testing of a larger Av. gallinarum collection (n = 43) revealed that easily readable MICs could be obtained for the type strain and all isolates. Some Av. gallinarum showed elevated MICs of enrofloxacin (n = 35), nalidixic acid (n = 35), penicillin (n = 2), tetracycline (n = 19), and/or trimethoprim-sulfamethoxazole (n = 1). By using PCR analyses, the following antimicrobial resistance genes were detected: blaTEM, dfrA14, sul2, tet(B), tet(H). The study demonstrated that the proposed medium is suitable for a harmonized broth microdilution susceptibility testing of Av. gallinarum with a recommended incubation time of 20 to 24 h.

Development of a harmonized method for antimicrobial susceptibility testing of Bordetella avium using broth microdilution and detection of resistance genes.

AIMS: In response to a request from the Clinical and Laboratory Standards Institute (CLSI), the objective of this study was to develop a harmonized method for broth microdilution susceptibility testing of Bordetella (B.) avium, the major causative agent of infectious coryza in poultry. METHODS AND RESULTS: To find a suitable test medium, growth curves with four epidemiologically unrelated B. avium isolates were created in cation-adjusted Mueller-Hinton broth (CAMHB), CAMHB + 2.5% lysed horse blood and veterinary fastidious medium. All isolates showed good growth in CAMHB, therefore MIC values were determined using this medium and the homogeneity of the values was determined. An essential MIC agreement of 99.7% was calculated. Testing of a larger strain collection (n = 49) for their susceptibility to 24 antimicrobials confirmed the suitability of the tested method and revealed some isolates with elevated MICs of florfenicol (n = 1), streptomycin (n = 2), tetracyclines (n = 5), and trimethoprim/sulfamethoxazole (n = 6). PCR assays detected the resistance genes aadA1, dfrB1, floR, sul1, sul2 and tet(A). CONCLUSIONS: The method used enables easy reading and a good reproducibility of MIC values for B. avium. SIGNIFICANCE AND IMPACT OF STUDY: Application of the tested method allows harmonized resistance testing of B. avium and identification of isolates with elevated MIC values.

The Lytic Siphophage vB_StyS-LmqsSP1 Reduces the Number of Salmonella enterica Serovar Typhimurium Isolates on Chicken Skin.

Phage-based biocontrol of bacteria is considered a natural approach to combat foodborne pathogens. Salmonella spp. are notifiable and highly prevalent pathogens that cause foodborne diseases worldwide. In this study, six bacteriophages were isolated and further characterized that infect food-derived Salmonella isolates from different meat sources. The siphovirus VB_StyS-LmqsSP1, which was isolated from a cow's nasal swab, was further subjected to in-depth characterization. Phage-host interaction investigations in liquid medium showed that vB_StyS-LmqsSP1 can suppress the growth of Salmonella species isolates at 37°C for 10 h and significantly reduce the bacterial titer at 4°C. A reduction of 1.4 to 3 log units was observed in investigations with two food-derived Salmonella isolates and one reference strain under cooling conditions using multiplicities of infection (MOIs) of 104 and 105. Phage application on chicken skin resulted in a reduction of about 2 log units in the tested Salmonella isolates from the first 3 h throughout a 1-week experiment at cooling temperature and with an MOI of 105. The one-step growth curve analysis using vB_StyS-LmqsSP1 demonstrated a 60-min latent period and a burst size of 50 to 61 PFU/infected cell for all tested hosts. Furthermore, the genome of the phage was determined to be free from genes causing undesired effects. Based on the phenotypic and genotypic properties, LmqsSP1 was assigned as a promising candidate for biocontrol of Salmonella enterica serovar Typhimurium in food. IMPORTANCE Salmonella enterica is one of the major global causes of foodborne enteritis in humans. The use of chemical sanitizers for reducing bacterial pathogens in the food chain can result in the spread of bacterial resistance. Targeted and clean-label intervention strategies can reduce Salmonella contamination in food. The significance of our research demonstrates the suitability of a bacteriophage (vB_StyS-LmqsSP1) for biocontrol of Salmonella enterica serovar Typhimurium on poultry due to its lytic efficacy under conditions prevalent in food production environments.

Campylobacter sp.: Pathogenicity factors and prevention methods-new molecular targets for innovative antivirulence drugs?

Infections caused by bacterial species from the genus Campylobacter are one of the four main causes of strong diarrheal enteritis worldwide. Campylobacteriosis, a typical food-borne disease, can range from mild symptoms to fatal illness. About 550 million people worldwide suffer from campylobacteriosis and lethality is about 33 million p.a. This review summarizes the state of the current knowledge on Campylobacter with focus on its specific virulence factors. Using this knowledge, multifactorial prevention strategies can be implemented to reduce the prevalence of Campylobacter in the food chain. In particular, antiadhesive strategies with specific adhesion inhibitors seem to be a promising concept for reducing Campylobacter bacterial load in poultry production. Antivirulence compounds against bacterial adhesion to and/or invasion into the host cells can open new fields for innovative antibacterial agents. Influencing chemotaxis, biofilm formation, quorum sensing, secretion systems, or toxins by specific inhibitors can help to reduce virulence of the bacterium. In addition, the unusual glycosylation of the bacterium, being a prerequisite for effective phase variation and adaption to different hosts, is yet an unexplored target for combating Campylobacter sp. Plant extracts are widely used remedies in developing countries to combat infections with Campylobacter. Therefore, the present review summarizes the use of natural products against the bacterium in an attempt to stimulate innovative research concepts on the manifold still open questions behind Campylobacter towards improved treatment and sanitation of animal vectors, treatment of infected patients, and new strategies for prevention. KEY POINTS: • Campylobacter sp. is a main cause of strong enteritis worldwide. • Main virulence factors: cytolethal distending toxin, adhesion proteins, invasion machinery. • Strong need for development of antivirulence compounds.

Occurrence of extended-spectrum beta-lactamase-producing Enterobacteriaceae, microbial loads, and endotoxin levels in dust from laying hen houses in Egypt.

BACKGROUND: Poultry houses are often highly contaminated with dust, which might contain considerable amounts of microorganisms and endotoxins. The concentrations of microorganisms and endotoxins in dust from laying hen houses in Egypt are unknown. However, to estimate the risks for birds, the environment, and people working in laying hen houses, it is important to gather information about the composition of these dusts. Here we report the microbial loads, the occurrence of antimicrobial-resistant bacteria, and endotoxin concentrations in dust samples from 28 laying hen farms in Dakahliya Governorate, Egypt, and discuss the results relevant to the literature. RESULTS: Pooled settled dust samples (n = 28) were analyzed for total viable counts of bacteria and fungi (CFU/g), the occurrence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae, Salmonella spp., and methicillin-resistant Staphylococcus aureus (MRSA), and endotoxin concentrations (ng/g). The means and standard deviations of total viable counts were 7.10 × 108 ± 2.55 × 109 CFU/g for bacteria and 5.37 × 106 ± 7.26 × 106 CFU/g for fungi. Endotoxin levels varied from 2.9 × 104 to 6.27 × 105 ng/g. None of the tested samples contained Salmonella spp. or MRSA. In contrast, by direct plating, Enterobacteriaceae were found frequently (57%; n = 16), and suspected ESBL-producing Enterobacteriaceae occurred in 21% (n = 6) of the sampled barns. Using an enrichment method, the detection of Enterobacteriaceae and suspected ESBL-producing Enterobacteriaceae increased to 20 and 16 positive barns, respectively. Taking results from both methods into account, Enterobacteriaceae and suspected ESBL-producing Enterobacteriaceae were detected in 23 barns Overall, 100 ESBL suspected isolates (Escherichia coli, n = 64; Enterobacter cloacae, n = 20; and Klebsiella pneumoniae n = 16) were identified to species level by MALDI-TOF MS. Isolates from 20 barns (71% positive barns) were confirmed as ESBL producing Enterobacteriaceae by the broth microdilution test. CONCLUSIONS: Dust in Egyptian laying hen houses contains high concentrations of microorganisms and endotoxins, which might impair the health of birds and farmers when inhaled. Furthermore, laying hens in Egypt seem to be a reservoir for ESBL-producing Enterobacteriaceae. Thus, farmers are at risk of exposure to ESBL-producing bacteria, and colonized hens might transmit these bacteria into the food chain.

Curcumin Mitigates Immune-Induced Epithelial Barrier Dysfunction by Campylobacter jejuni.

Campylobacter jejuni (C. jejuni) is the most common cause of foodborne gastroenteritis worldwide. The bacteria induce diarrhea and inflammation by invading the intestinal epithelium. Curcumin is a natural polyphenol from turmeric rhizome of Curcuma longa, a medical plant, and is commonly used in curry powder. The aim of this study was the investigation of the protective effects of curcumin against immune-induced epithelial barrier dysfunction in C. jejuni infection. The indirect C. jejuni-induced barrier defects and its protection by curcumin were analyzed in co-cultures with HT-29/B6-GR/MR epithelial cells together with differentiated THP-1 immune cells. Electrophysiological measurements revealed a reduction in transepithelial electrical resistance (TER) in infected co-cultures. An increase in fluorescein (332 Da) permeability in co-cultures as well as in the germ-free IL-10-/- mouse model after C. jejuni infection was shown. Curcumin treatment attenuated the C. jejuni-induced increase in fluorescein permeability in both models. Moreover, apoptosis induction, tight junction redistribution, and an increased inflammatory response-represented by TNF-α, IL-1ß, and IL-6 secretion-was observed in co-cultures after infection and reversed by curcumin. In conclusion, curcumin protects against indirect C. jejuni-triggered immune-induced barrier defects and might be a therapeutic and protective agent in patients.

Susceptibility of Methicillin-Resistant and -Susceptible Staphylococcus aureus Isolates of Various Clonal Lineages from Germany to Eight Biocides.

Few studies have been conducted on the susceptibility of bacteria to biocides. A total of 182 methicillin-resistant and -susceptible Staphylococcus aureus isolates collected from healthy or diseased humans and animals in Germany were included in the present study. Sixty-three isolates of animal origin and 119 human isolates were tested for their MICs to eight biocides or heavy metals by the broth microdilution method. The MIC50 and MIC90 values of human and animal isolates were equal or differed by not more than 1 dilution step, and statistical analysis revealed that differences between MICs of human and animal isolates were not significant. However, when taking into account the multilocus sequence type (MLST), a strong tendency (P = 0.054) to higher MICs of silver nitrate was detected for clonal complex 398 (CC398) isolates from humans compared to those from animals. Furthermore, a comparison of MIC values from isolates belonging to different clonal lineages revealed that important human lineages such as CC22 and CC5 exhibited significantly (P < 0.05) higher MICs for the biocides chlorhexidine, benzethonium chloride, and acriflavine than the main animal lineage sequence type 398 (ST398). Isolates with elevated MIC values were tested for the presence of biocide and heavy metal tolerance-mediating genes by PCR assays, and the following genes were detected: mepA (n [no. of isolates containing the gene] = 44), lmrS (n = 36), norA (n = 35), sepA (n = 22), mco (n = 5), czrC (n = 3), smr (n = 2), copA (n = 1), qacA and/or -B (n = 1), qacG (n = 2), and qacJ (n = 1). However, only for some compounds was a correlation between the presence of a biocide tolerance gene and the level of MIC values detected.IMPORTANCE Biocides play an essential role in controlling the growth of microorganisms and the dissemination of nosocomial pathogens. In this study, we determined the susceptibility of methicillin-resistant and -susceptible S. aureus isolates from humans and animals to various biocides and heavy metal ions and analyzed differences in susceptibilities between important clonal lineages. In addition, the presence of biocide or heavy metal tolerance-mediating genes was investigated. We demonstrated that important human lineages such as CC22 and CC5 had significantly higher MIC values for chlorhexidine, benzethonium chloride, and acriflavine than the main farm animal lineage, ST398. In addition, it was shown that for some combinations of biocides and tolerance genes, significantly higher MICs were detected for carriers. These findings provide new insights into S. aureus biocide and heavy metal tolerance.

Towards a Standardized Method for Broth Microdilution Susceptibility Testing of Haemophilus parasuis.

Currently, there is no agreed method available for broth microdilution susceptibility testing of Haemophilus parasuis, one of the most important bacterial pathogens in pig production. Therefore, the aim of this study was to develop a method that could be easily performed by diagnostic laboratories and that appears suitable for a harmonized susceptibility testing. Growth determinations using one type strain and three field isolates revealed no visible growth of H. parasuis in media which have proven to be suitable for susceptibility testing of fastidious organisms. Therefore, a new medium, cation-adjusted Mueller-Hinton broth (CAMHB) plus NADH and sterile filtered heat-inactivated chicken serum, was developed. The reproducibility of MICs obtained in this medium was evaluated and statistically analyzed, considering a model with two different variables (precondition of five identical MICs and MIC mode accepting a deviation of ±1 dilution step, respectively). No significant differences for both variables were seen between two time points investigated and between results obtained with the recently proposed test medium broth (TMB). Nearly all MICs of quality control strains were in the acceptable range. Subsequently, 47 H. parasuis isolates representing 13 serovars were tested with the newly developed medium and TMB. Statistical analysis of all isolates and 15 antimicrobial agents and antimicrobial combinations showed no significant difference between MICs obtained in supplemented CAMHB and TMB. Because of a simplified implementation in routine diagnostic and a lower chance of interference between medium components and antimicrobial agents, supplemented CAMHB is recommended with an incubation time of 24 h.

Influence of modified atmosphere and vacuum packaging with and without nanosilver-coated films on different quality parameters of pork.

Pork is often marketed in packages with high oxygen atmosphere (MAP) or vacuum to improve shelf life and appearance. As silver ions have antibacterial effects, food contact films coated with silver might improve the shelf life of meat. In the present study, pork was wrapped in commercially available films, coated with nanosilver particles, and stored in the two packaging variants MAP and vacuum for 12 days. During storage, samples were analyzed on days 1 (before packaging), 4, 8 and 12 for microbiological contamination, meat quality (e.g., pH, color), and for the percentages of the myoglobin (Mb) redox forms. In addition, the effects of the film were examined after inoculation of the meat with high quantities of methicillin-resistant Staphylococcus aureus (MRSA) cells before vacuum storage for 8 days. MAP storage resulted in higher lightness (L*) values, lower liquid loss and higher Mb oxidation compared to vacuum. Microbiological spoilage was partly affected by the packaging variants with reducing effects of the MAP. The nanosilver-coating only affects the Mb redox form percentages of the pork cutlets and on day 4 the L* values, whereas microbiological parameters were not influenced. As the nanosilver coating had no influence on the total viable bacteria counts as well as Pseudomonas spp., Enterobacteriaceae and MRSA counts, an advantage of the nanosilver coating on the shelf life could be excluded.

Yersinia enterocolitica Isolates from Wild Boars Hunted in Lower Saxony, Germany.

Yersiniosis is strongly associated with the consumption of pork contaminated with enteropathogenic Yersinia enterocolitica, which is harbored by domestic pigs without showing clinical signs of disease. In contrast to data on Y. enterocolitica isolated from conventionally reared swine, investigations into the occurrence of Y. enterocolitica in wild boars in Germany are rare. The objectives of the study were to get knowledge about these bacteria and their occurrence in wild boars hunted in northern Germany by isolation of the bacteria from the tonsils, identification of the bioserotypes, determination of selected virulence factors, macrorestriction analysis, multilocus sequence typing (MLST), and testing of antimicrobial susceptibility. Altogether, tonsils from 17.1% of 111 tested wild boars were positive for Y. enterocolitica by culture methods. All but two isolates belonged to biotype (BT) 1A, with the majority of isolates bearing a ystB nucleotide sequence which was revealed to have 85% identity to internal regions of Y. enterocolitica heat-stable enterotoxin type B genes. The remaining Y. enterocolitica isolates were identified to be BT 1B and did not carry the virulence plasmid. However, two BT 1A isolates carried the ail gene. Macrorestriction analysis and results from MLST showed a high degree of genetic diversity of the isolates, although the region where the samples were taken was restricted to Lower Saxony, Germany, and wild boars were shot during one hunting season. In conclusion, most Y. enterocolitica isolates from wild boars investigated in this study belonged to biotype 1A. Enteropathogenic Y. enterocolitica bioserotypes 4/O:3 and 2/O:9, usually harbored by commercially raised pigs in Europe, could not be identified.

Comparative analysis and limitations of ethidium monoazide and propidium monoazide treatments for the differentiation of viable and nonviable campylobacter cells.

The lack of differentiation between viable and nonviable bacterial cells limits the implementation of PCR-based methods for routine diagnostic approaches. Recently, the combination of a quantitative real-time PCR (qPCR) and ethidium monoazide (EMA) or propidium monoazide (PMA) pretreatment has been described to circumvent this disadvantage. In regard to the suitability of this approach for Campylobacter spp., conflicting results have been reported. Thus, we compared the suitabilities of EMA and PMA in various concentrations for a Campylobacter viability qPCR method. The presence of either intercalating dye, EMA or PMA, leads to concentration-dependent shifts toward higher threshold cycle (CT) values, especially after EMA treatment. However, regression analysis resulted in high correlation coefficient (R(2)) values of 0.99 (EMA) and 0.98 (PMA) between Campylobacter counts determined by qPCR and culture-based enumeration. EMA (10 µg/ml) and PMA (51.10 µg/ml) removed DNA selectively from nonviable cells in mixed samples at viable/nonviable ratios of up to 1:1,000. The optimized EMA protocol was successfully applied to 16 Campylobacter jejuni and Campylobacter coli field isolates from poultry and indicated the applicability for field isolates as well. EMA-qPCR and culture-based enumeration of Campylobacter spiked chicken leg quarters resulted in comparable bacterial cell counts. The correlation coefficient between the two analytical methods was 0.95. Nevertheless, larger amounts of nonviable cells (>10(4)) resulted in an incomplete qPCR signal reduction, representing a serious methodological limitation, but double staining with EMA considerably improved the signal inhibition. Hence, the proposed Campylobacter viability EMA-qPCR provides a promising rapid method for diagnostic applications, but further research is needed to circumvent the limitation.

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.

Genotypic and phenotypic characterization of Staphylococcus aureus isolates from wild boars.

Eight Staphylococcus aureus isolates collected from 117 wild boars were characterized and compared to livestock isolates. They belonged to sequence types ST133, ST425, and the new type ST1643. The spa types were t1181, t6782, and the new types t6384, t6385, and t6386. Antimicrobial susceptibility testing and microarray-based genotyping confirmed the absence of important virulence/resistance genes.

Towards a Standardized Antimicrobial Susceptibility Testing Method for Mycoplasma hyorhinis.

Conducting antimicrobial susceptibility testing (AST) in a comparable manner requires the availability of a standardized method. Organizations, such as the Clinical and Laboratory Standards Institute (CLSI) or the European Committee on Antimicrobial Susceptibility Testing (EUCAST), provide standardized protocols for a range of fastidious bacteria but not for Mycoplasma hyorhinis. We developed a broth microdilution method for testing M. hyorhinis in a standardized and harmonized way using a modified Friis broth devoid of antimicrobial or otherwise bacterial growth-inhibiting agents. The type strain M. hyorhinis DSM 25591 was chosen to establish the methodology. The antimicrobial agents of interest were doxycycline, enrofloxacin, erythromycin, florfenicol, gentamicin, marbofloxacin, tetracycline, tiamulin, tilmicosin, tulathromycin, and tylosin, tested by using commercial SensititreTM microtiter plates. In addition, the suitability of the methodology was evaluated via variation of the individual ingredients of the modified Friis broth by either using different batches or choosing other distributors. Despite these alterations, the method provided reliable results. We obtained repeatable minimal inhibitory concentrations for all six tested field isolates and the M. hyorhinis type strain. With this newly proposed method, we aim to provide an improved AST method for diagnostic laboratories and monitoring purposes with better comparability between times and countries. In addition, this new method will allow for an improvement of targeted treatments using antimicrobial agents and thereby reduce the options for resistance development.

Characterization of Temperate LPS-Binding Bordetella avium Phages That Lack Superinfection Immunity.

Bordetella avium causes a highly infectious upper respiratory tract disease in turkeys and other poultry with high economic losses. Considering the antimicrobial resistance crisis, bacteriophages (phages) may be an alternative approach for treating bacterial infections such as bordetellosis. Here, we describe seven B. avium phages, isolated from drinking water and feces from chicken and turkey farms. They showed strong bacteriolytic activity with a broad host range and used lipopolysaccharides (LPS) as a host receptor for their adsorption. All phages are myoviruses based on their structure observed by transmission electron microscopy. Genome sequence analyses revealed genome assembly sizes ranging from 39,087 to 43,144 bp. Their permutated genomes were organized colinearly, with a conserved module order, and were packed according to a predicted headful packing strategy. Notably, they contained genes encoding putative markers of lysogeny, indicative of temperate phages, despite their lytic phenotype. Further investigation revealed that the phages could indeed undergo a lysogenic life cycle with varying frequency. However, the lysogenic bacteria were still susceptible to superinfection with the same phages. This lack of stable superinfection immunity after lysogenization appears to be a characteristic feature of B. avium phages, which is favorable in terms of a potential therapeutic use of phages for the treatment of avian bordetellosis. IMPORTANCE To maintain the effectiveness of antibiotics over the long term, alternatives to treat infectious diseases are urgently needed. Therefore, phages have recently come back into focus as they can specifically infect and lyse bacteria and are naturally occurring. However, there is little information on phages that can infect pathogenic bacteria from animals, such as the causative agent of bordetellosis of poultry, B. avium. Therefore, in this study, B. avium phages were isolated and comprehensively characterized, including whole-genome analysis. Although phenotypically the phages were thought to undergo a lytic cycle, we demonstrated that they undergo a lysogenic phase, but that infection does not confer stable host superinfection immunity. These findings provide important information that could be relevant for potential biocontrol of avian bordetellosis by using phage therapy.

Evaluation of a Method for Standardized Antimicrobial Susceptibility Testing with Mycoplasma hyorhinis Field Isolates.

Organizations like the Clinical and Laboratory Standards Institute (CLSI) or the European Committee of Antimicrobial Susceptibility Testing (EUCAST) provide standardized methodologies for antimicrobial susceptibility testing of a wide range of nonfastidious and fastidious bacteria, but so far not for Mycoplasma spp. of animal origin. Recently, a proposed method for the standardized broth microdilution testing of Mycoplasma hyorhinis using commercial Sensititre microtiter plates was presented. In this study, we evaluated this broth microdilution method with 37 field isolates and tested their susceptibility toward the following antimicrobial agents: doxycycline, enrofloxacin, erythromycin, florfenicol, gentamicin, marbofloxacin, tetracycline, tiamulin, tilmicosin, tulathromycin, and tylosin. The isolates originated from different countries, isolation sites, and years. The broth microdilution method was carried out using a modified Friis broth as the culture and test medium. For macrolides and lincosamides, a bimodal distribution with elevated MIC values could be observed for almost half of the tested field isolates, deducing reduced susceptibility toward these substances. With a recently published protocol, we were able to test a variety of field isolates, and consistent data could be obtained. Using this method, monitoring studies of Mycoplasma hyorhinis isolates can be carried out in a comparable manner, and the observed susceptibility profiles can be screened for possible changes in MIC values in the future.