In vitro antimicrobial resistance of Escherichia coli, Serratia marcescens, Klebsiella oxytoca and Klebsiella pneumoniae on Bavarian dairy farms between 2014-2022.

The objective of this study was to describe the prevalence of antimicrobial resistance of E. coli, K. oxytoca, K. pneumoniae, and S. marcescens from quarter milk samples submitted to the udder health laboratory of the Bavarian Animal Health Services (TGD) in Southern Germany between 2014 and 2022. All samples were tested with the California Mastitis Test and analyzed with a standard microbroth dilution to determine the minimum inhibitory concentrations (MIC). The antimicrobials tested were amoxicillin/clavulanate, cefazoline, kanamycin/cefalexin, cefoperazone, cefquinome, and marbofloxacin. Breakpoints were chosen in accordance with CLSI. Over the study period, E. coli, K. oxytoca, and K. pneumoniae showed only few resistances to all antimicrobials tested. For those pathogens MIC 50 and MIC 90 were below breakpoint for all antimicrobials except cefoperazone over the 9 years. A decrease in MIC could be seen for E. coli and K. oxytoca for all of the antimicrobials. While the MIC for K. pneumoniae stayed more stagnant, the prevalence of resistance still decreased overall. S. marcescens isolates were proven intrinsically resistant to amoxicillin/clavulanate and cefazolin and while in vitro resistances were low for all other antimicrobials tested, S. marcescens tended toward higher MIC for most of the antimicrobials over the years. Over time, there was also an overall increase in the number of isolates for all 4 pathogens per year. Starting 2018 there was steep increase in the number of isolates particularly from clinical cases. This jump in numbers coincided with a change of the regulation for veterinary drug prescriptions in Germany in 2018 that required, among other things, antimicrobial resistance testing before a change of antibiotics in the course of treatment and the use of critically important antimicrobials. Overall, while the pathogens increased in numbers, the prevalence of their antimicrobial resistance remained low.

Phenotypic and Genotypic Properties of Vibrio cholerae non-O1, non-O139 Isolates Recovered from Domestic Ducks in Germany.

Vibrio cholerae non-O1, non-O139 bacteria are natural inhabitants of aquatic ecosystems and have been sporadically associated with human infections. They mostly lack the two major virulence factors of toxigenic V. cholerae serogroups O1 and O139 strains, which are the causative agent of cholera. Non-O1, non-O139 strains are found in water bodies, sediments, and in association with other aquatic organisms. Occurrence of these bacteria in fecal specimens of waterfowl were reported, and migratory birds likely contribute to the long-distance transfer of strains. We investigated four V. cholerae non-O1, non-O139 isolates for phenotypic traits and by whole genome sequencing (WGS). The isolates were recovered from organs of domestic ducks with serious disease symptoms. WGS data revealed only a distant genetic relationship between all isolates. The isolates harbored a number of virulence factors found in most V. cholerae strains. Specific virulence factors of non-O1, non-O139 strains, such as the type III secretion system (TTSS) or cholix toxin, were observed. An interesting observation is that all isolates possess multifunctional autoprocessing repeats-in-toxin toxins (MARTX) closely related to the MARTX of toxigenic El Tor O1 strains. Different primary sequences of the abundant OmpU proteins could indicate a significant role of this virulence factor. Phenotypic characteristics such as hemolysis and antimicrobial resistance (AMR) were studied. Three isolates showed susceptibility to a number of tested antimicrobials, and one strain possessed AMR genes located in an integron. Knowledge of the environmental occurrence of V. cholerae non-O1, non-O139 in Germany is limited. The source of the infection of the ducks is currently unknown. In the context of the 'One Health' concept, it is desirable to study the ecology of V. cholerae non-O1, non-O139, as it cannot be excluded that the isolates possess zoonotic potential and could cause infections in humans.

Antibiotic resistance in Escherichia coli from pigs from birth to slaughter and its association with antibiotic treatment.

The purpose of this longitudinal study was to describe the occurrence of antibiotic resistance in faecal Escherichia coli isolated from pigs between birth and slaughter and its association with antibiotic treatment. Four objectives were addressed: comparison of antibiotic resistance in isolates from a) treated vs. non-treated pigs, b) follow-up vs. initial samples of treated and non-treated pigs, c) pigs receiving treatments via different administration routes and d) sows and their piglets. Each comparison addressed the following antibiotic groups used for treatment: beta-lactams, tetracyclines, polymyxins and macrolides, and the susceptibility of E. coli isolates to the respective agents: ampicillin, tetracycline, colistin and azithromycin. Between 2014 and 2016, 406 focal animals from 29 commercial breeding herds were followed from birth to the end of the relevant fattening periods. All antibiotic treatments in these pigs were documented. Faecal samples were collected from the focal pigs once while suckling, once after weaning and three times during fattening, and from their dams once around farrowing. Escherichia coli isolated from these samples was tested for antibiotic susceptibility. In total, 264 animals from 19 breeding herds were treated with an antibiotic at least once during their lifetime. Beta-lactams, tetracyclines and colistin were used most frequently. Piglets were treated individually by injection (n = 108 treatments) or via drench (9); weaners via feed (192) or water (56) and fatteners via feed (30) or injection (15). Resistance to ampicillin and tetracycline in E. coli was already common prior to antibiotic treatment. Resistance proportions were higher for beta-lactam-, tetracycline-, colistin- and macrolide-treated pigs compared to untreated pigs at different sampling periods (p < 0.05; Fisher's exact test). In the logistic analysis, the difference was confirmed for beta-lactam-treated vs. untreated pigs. In E. coli from macrolide-untreated pigs, resistance to azithromycin was more frequent compared to pre-treatment values. Route of application did not affect rates of antibiotic resistance in the logistic analysis even though Fisher's exact test indicated associations for beta-lactams (feed/water vs. injection), tetracyclines (feed/water vs. non-treatment) and macrolides (tulathromycin-injection vs. tylosin in feed). Piglets were more likely to carry an E. coli resistant to ampicillin or azithromycin if their dams did so as well. Our results suggest further research on resistance effects by administration routes is required. Reducing antibiotic resistance in sows might lead to a lower level of beta-lactam or macrolide-resistant E. coli among their progeny. To preserve treatment options for bacterial infections, antibiotic use should be restricted to necessary cases.

The impact of Q fever-phase-specific milk serology for the diagnosis of puerperal and chronic milk shedding of C. burnetii in dairy cows.

C. burnetii infection might be associated with puerperal shedding; additionally, the chronic shedding of this pathogen in milk has been observed in individual animals. A longitudinal survey was performed in an endemically infected dairy cow herd with 100 cows in order to compare phase-specific milk-serology with pathogen shedding. From March 2010 through December 2011, 870 individual milk samples from 212 cows were analysed using both quantitative (q) PCR and phase-specific antibody-ELISA. The mean milk-shedding/cow was calculated for 137 cows with > or = 3 milk samples per cow. In addition, 110 puerperal swabs were collected after August 2010. The cows yielding three successive qPCR-positive milk samples or > 3 qPCR-positive milk samples, irrespective of the sequence of positive/negative results, were classified as chronic shedders (CS). Milk shedding was observed during the entire study, but a major period of puerperal shedding occurred from February through October 2011; 35/52 swabs tested positive, whereas only 3/58 swabs collected outside this period were positive. The PhI/PhII(+)-pattern in primiparous cows (< 36 months old) was consistent with puerperal shedding in the herd, but not at the individual level. This pattern was observed in older cows, irrespective of the period of puerperal shedding. Four primiparous CS-cows showed low-level mean shedding < 100 C.b./ml milk, and the PhI-titre increased from negative or weakly positive to more than 500 at the end of the first lactation. Puerperal shedding during the second parturition was observed in three of these cows. Six multiparous CS-cows with mean shedding exceeding 100 C.b./ml milk were characterised with stable PhI-titres of > or = 500. The three available puerperal swabs tested negative. Only one multiparous CS-cow showed low-level shedding and a PhI-titre below 500 for the entire study. In conclusion, the PhI-/PhII(+)-pattern in primiparous cows indicated puerperal shedding at the herd level, and a PhI-titre > or = 500 is a suitable screening method for the detection of chronic shedding in milk.

Insights into the dynamics of endemic Coxiella burnetii infection in cattle by application of phase-specific ELISAs in an infected dairy herd.

Serological diagnosis of acute and chronic Q fever in humans relies on detection of antibodies to phase I (PhI) and II (PhII) antigens of Coxiella (C.) burnetii. Although phase-specific antigens are available, they are not yet used in ruminants as they are in humans. This study focuses on phase-specific serology as a tool for analysis of the dynamics of infection in cattle. As a prerequisite, sero-prevalence in Bavarian cattle (1) and sero-prevalences for age-groups (2) were determined by ELISA (CHEKIT Q-Fever; mix of PhI/PhII-antigen). Subsequently, phase-specific antigens were coated onto ELISA plates individually and tests were simultaneously applied in an endemically infected herd with about 90 dairy cows and 250 calves/heifers in April 2005, March 2006 and retrospectively in May and October 2004. From April 2005 onward, placentas were analysed for C. burnetii by PCR (3). (1) Sero- and herd prevalences based on 21,051 sera from 603 Bavarian dairy farms collected in 2003 were 14.8% ± 0.48% and 72.3% ± 3.6%, respectively. (2) Analysis of 3965 animals from 105 farms for which age was reported revealed a base level of sero-prevalence of less than 5% in 1-2 years old animals, it increased to 15% in 2-3 years old and reached a plateau (25-30%) in cows four years and older. (3) In May 2004 and April 2005 a peak of PhI(-)/PhII(+)-prevalence in primiparous cows (2.0-3.5 years) was observed; but not in October 2004 and March 2006. The PhI(-)/PhII(+)-pattern in primiparous cows changed to negative (one-third), PhI(+)/PhII(+) (1/3) or persisted (1/3). In contrast, sero-conversion was rare in multiparous cows (>3.5 years). If the PhI(-)/PhII(+) pattern was detected, it was due to an infection in preceding years. This pattern persisted (2/3) or changed to negative (1/3); a change to PhI(+)/PhII(+) did not occur. PhI(-)/PhII(+) in heifers (1-2 years) always changed to negative. Detection of PhII-antibodies was significantly associated with PCR-positive placentas. Remarkably, 45% of sera with the PhI(-)/PhII(+) pattern were negative for the CHEKIT Q-Fever ELISA, thus this test missed an important group of infected animals.

Bone marrow depletion with haemorrhagic diathesis in calves in Germany: characterization of the disease and preliminary investigations on its aetiology.

Since 2007 a new fatal haemorrhagic diathesis in calves has been observed in all areas of Germany. Analysis of 56 cases submitted for necropsy allowed its characterization. Calves fell ill within the first month of life independent of breed and sex. Only single or a few animals per herd were affected. Petechial and ecchymotic haemorrhages in many organs and tissues, particularly in skin, subcutis and gastrointestinal tract, were major findings in all animals. Microscopically a severe depletion of bone marrow cells was always observed. Lymphocytic depletion (43%) and inflammatory lesions (46%) were less frequently observed. Blood analysis of five animals indicated an aplastic pancytopenia. The resulting thrombocytopenia is regarded as major pathomechanism of this Haemorrhagic Disease Syndrome (HDS). Pedigree analysis gave no indication of hereditary disease. Tests for specific toxins such as S-(1,2-Dichlorovinyl)-L-cysteine (DCVC), furazolidone, or mycotoxins resulting in bone marrow depletion were negative. Bacterial infections, Bovine Viral Diarrhoea Virus, and Bluetongue Virus were ruled out as cause of the disease. HDS shares similarities with a circoviral infection in chickens (chicken infectious anaemia). A broad-spectrum PCR allowed detection of circoviral DNA in 5 of 25 HDS cases and in 1 of 8 non-HDS cases submitted for necropsy. Sequencing of the whole viral genome revealed a high similarity (up to 99%) with Porcine Circovirus type 2b. Single bone marrow cells stained weakly positive for PCV2 antigen by immunohistochemistry in 1 of 8 tested HDS animals. This is the first report of circovirus detection in cattle in Germany. The exact cause of HDS still remains unknown. A multifactorial aetiology involving infection, poisoning, immunopathy, or a genetic predisposition is conceivable. Additional research is necessary to clarify the pathogenesis and the potential role of PCV2 in HDS.

[Layout proposal for a microtitre plate to be used in routine antimicrobial susceptibility testing of bacteria from infections of dogs and cats]. / Empfindlichkeitsprüfung von Bakterien gegenüber antimikrobiellen Wirkstoffen in der Routinediagnostik: Vorschlag für ein Layout für Mikrotiterplatten zur Testung von Bakterien von Hunden und Katzen.

The determination of minimum inhibitory concentrations by broth microdilution is recommended as method of choice for susceptibility testing of veterinary bacterial pathogens. Accordingly, broth microdilution is used in veterinary routine diagnostic laboratories at a progressive rate. To reduce the costs of susceptibility testing, it is reasonable to develop widely accepted uniform microtitre plate layouts that are produced in large quantities. Such microtitre plate layouts have already been developed and published for the susceptibility testing of pathogens from food-producing animals. However, a microtitre plate layout, especially designed for the testing of bacteria from dogs and cats, should be available, too. The choice of the antimicrobial agents or combinations of antimicrobial agents to be included in a suitable layout should be based on the following criteria: (1) the approval and availability of an antimicrobial agent or combination of agents, (2) known cross-resistances, and (3) availability of approved clinical breakpoints. The latter point is of particular importance for the choice of the numbers of concentrations per antimicrobial agent tested and the range of test concentrations. Taking into account these aspects, a science-based layout proposal for microtitre plates, which are suitable for routine testing of bacteria from dogs and cats, is presented and discussed.