Real time detection of pathogenic bacteria in veterinary microbiology using isothermal microcalorimetry - A different approach.

With today's challenges regarding antibiotic resistance and the importance of the implementation of prudent use of antibiotics, fast and reliable diagnostic tools for bacterial infections and subsequent antimicrobial susceptibility testing are of utmost relevance. Isothermal microcalorimetry (IMC) is a broadly applicable method, with which metabolic heat flow in reproducing bacteria can be measured in real time. To the best of the authors' knowledge, this is the first report on examination of 124 urine samples from feline and canine urinary tract infection with an IMC-based prototype instrument. A concentration-dependent time of peak heat flow by dilution series with Escherichia coli and Enterococcus faecalis reference strains demonstrated the general good performance of the prototype for detection of these bacteria. With diagnostic culture being set as a gold standard, the diagnostic sensitivity of IMC compared to bacteriological culture was 80 %, the diagnostic specificity was 97 %. With a Cohens' kappa value (κ) of 0.80, the two methods show good concordance. The results from our study demonstrate that the IMC technology is suitable to allow reliable, but much faster detection of bacteria than conventional culture, especially for Escherichia coli. Thus, implementing IMC technology could markedly speed up the bacteriological diagnostic process in veterinary medicine

The increase of methicillin-resistant Staphylococcus aureus (MRSA) and the presence of an unusual sequence type ST49 in slaughter pigs in Switzerland

<p>Abstract</p> <p>Background</p> <p>In years past, methicillin-resistant <it>S. aureus </it>(MRSA) has been frequently detected in pigs in Europe, North America and Asia. Recent, yet sporadic studies have revealed a low occurrence of MRSA in Switzerland. In 2009, a monitoring survey of the prevalence and genetic diversity of methicillin-resistant <it>S. aureus </it>(MRSA) in slaughter pigs in Switzerland was conducted using methods recommended by the EU guidelines, and using a sampling strategy evenly distributed throughout the year and representative of the Swiss slaughter pig population. Monitoring should determine if the overall prevalence of MRSA in the entire country is increasing over the years and if specific multi-resistant MRSA clones are spreading over the country.</p> <p>Results</p> <p>In 2009, the nasal cavities of eight out of 405 randomly selected pigs were positive for MRSA, representing a prevalence of 2.0% (95% CI 0.9-3.9). The following year, 23 out of 392 pigs were positive for MRSA [5.9% prevalence (95% CI 3.8-8.7)]. Three multilocus sequence types (ST), four <it>spa </it>types and two types of staphylococcal cassette chromosome <it>mec </it>(SCC<it>mec</it>) elements were detected. The most frequent genotypes were ST398 (MLST)-(<it>spa</it>)t034-V(SCC<it>mec</it>) (n = 18) and ST49-t208-V (n = 7), followed by ST398-t011-V (n = 4), ST398-t1451-V (n = 1), and ST1-t2279-IVc (n = 1). The isolates displayed resistance to ß-lactams [<it>mecA</it>, (31/31); <it>blaZ</it>, (19/31)]; tetracycline [<it>tet</it>(M), (31/31); <it>tet</it>(K), (30/31)] (n = 31); macrolides and lincosamides [<it>erm</it>(C) (4/31) or <it>erm</it>(A) (18/31)] (n = 22); tiamulin [<it>vga</it>(A)<it>v </it>(9/31) or unknown mechanism (18/31)] (n = 27); trimethoprim [<it>dfr</it>(G) (18/31); spectinomycin [<it>ant(9)-Ia </it>(19/31) or unknown mechanism (3/31)] (n = 22); streptomycin [<it>str </it>(19/31)]; sulphamethoxazole (7/31) and ciprofloxacin (n = 1) (mechanisms not determined).</p> <p>Conclusions</p> <p>This study is the first to describe the presence of MRSA ST49 in slaughter pigs, and to demonstrate a significant and nearly three-fold increase of MRSA prevalence in pigs within two years. The presence of a specific clonal lineage of MRSA from Switzerland suggests that it has been selected in Swiss pig husbandry. Effective hygiene measures should be enhanced within the entire pig production chain to suppress the spread of these pathogens into the community.</p

Campylobacter jejuni from Canine and Bovine Cases of Campylobacteriosis Express High antimicrobial Resistance Rates against (Fluoro)quinolones and Tetracyclines

Campylobacter (C.) spp. from poultry is the main source of foodborne human campylobacteriosis, but diseased pets and cattle shedding Campylobacter spp. may contribute sporadically as a source of human infection. As fluoroquinolones are one of the drugs of choice for the treatment of severe human campylobacteriosis, the resistance rates of C. jejuni and C. coli from poultry against antibiotics, including fluoroquinolones, are monitored within the European program on antimicrobial resistance (AMR) in livestock. However, much less is published on the AMR rates of C. jejuni and C. coli from pets and cattle. Therefore, C. jejuni and C. coli isolated from diseased animals were tested phenotypically for AMR, and associated AMR genes or mutations were identified by whole genome sequencing. High rates of resistance to (fluoro)quinolones (41%) and tetracyclines (61.1%) were found in C. jejuni (n = 29/66). (Fluoro)quinolone resistance was associated with the known point mutation in the quinolone resistance-determining region (QRDR) of gyrA, and tetracycline resistance was mostly caused by the tet(O) gene. These high rates of resistance, especially to critically important antibiotics in C. jejuni and C. coli, are worrisome not only in veterinary medicine. Efforts to preserve the effcacy of important antimicrobial treatment options in human and veterinary medicine have to be strengthened in the future

Prevalence and antimicrobial resistance of Salmonella enterica subsp. diarizonae serovar 61:k:1,5,(7) in Swiss sheep flocks.

Salmonella (S.) enterica subspecies diarizonae (IIIb) serovar 61:k:1,5,(7) (S. IIIb 61:k:1,5,(7)) is considered to be sheep-associated, as it can be found in the intestine, tonsils and nose of clinically healthy sheep, but it has also been described in separate clinical disorders in sheep. In particular, S. IIIb 61:k:1,5,(7) is described as the causative agent of chronic proliferative rhinitis (CPR) in sheep. In Switzerland, CPR in sheep due to S. IIIb 61:k:1,5,(7) was first described in 2017 in a flock of Texel sheep. Therefore, we assessed the prevalence of S. IIIb 61:k:1,5,(7) within the Swiss sheep population using a representative sampling strategy. From May 2017 to June 2018 a total of 681 nasal swabs from individual clinically healthy sheep of 141 different flocks throughout Switzerland were taken. Swabs were analysed by selective enrichment for the presence of S. IIIb 61:k:1,5,(7). Additionally, antimicrobial resistance of the isolates was determined by broth microdilution. A total of 146 out of 681 nasal swabs tested positive for S. IIIb 61:k:1,5,(7), which corresponds to a prevalence on animal level of 21% (95%CI 18%-25%). In 73 out of 141 flocks tested, at least one sheep tested positive for S. IIIb 61:k:1.5,(7), resulting in a minimal prevalence on flock level of 52% (95%CI 43%-60%). Positive flocks were found in all cantons except the canton of Jura. Adults were significantly more affected than sheep under one year/lambs and positive sheep were found in several breeds. No microbiologically resistant isolates were detected, except for one isolate showing resistance against ampicillin. Because of its widespread occurrence in the Swiss sheep population, further research should focus on the pathogenic impact of S. IIIb 61:k:1,5,(7) on the health status of sheep

Surgical site infection after 769 Tibial Plateau Leveling Osteotomies

ObjectiveTo report surgical site infections (SSI) after Tibial Plateau Leveling Osteotomy (TPLO), treatment course, associated risk factors, bacterial isolates and antimicrobial resistance.Study designRetrospective clinical cohort study.Study populationSix hundred and twenty seven dogs and 769 TPLO procedures.MethodsData from electronic medical records of dogs undergoing TPLO between 2005 and 2015 at a single institution have been retrospectively reviewed. A generalized mixed logistic regression was used to determine possible risk factors. The Chi-Square test of independence was used to examine the relationship between the isolation of multidrug-resistant (MDR) bacteria and the development of major infections undergoing additional surgical treatment. To assess the correlation between number of SSI and number MDR isolate per year, Pearson's correlation coefficient was calculated.ResultsThe overall complication rate was 19.3% (n = 149). SSI was most frequent with 8.5% (n = 65). Major SSI occurred in 6.8% (n = 52) TPLO (80.0% SSI). Staphylococcus (S.) pseudintermedius (n = 37) and S. aureus (n = 10) were most frequently isolated. Multidrug-resistant bacteria were identified in 2.7% (n = 21) TPLO (32.3% SSI) but were not associated with major SSI (p = 0.426). There was a strong positive correlation between number of MDR isolates per year and number of SSI per year [r(9) = 0.79, p = 0.004]. Factors associated with SSI were previous TPLO in the contralateral stifle (p = 0.02, OR = 2.01, 95% CI = 1.11–3.64) and German Shepherd dogs (p = 0.035, OR = 4.41, 95% CI = 1.11–17.54). The use of non-locking implants was found to be protective (p = 0.02, OR = 0.179, 95% CI = 0.18–0.77).Clinical significanceInfection with multidrug-resistant bacteria is an emerging problem in veterinary practice and treatment is challenging. The incidence of major SSI was found to be high but was not associated with the isolation of MDR bacteria

Dynamics of extended-spectrum cephalosporin-resistant Escherichia coli in pig farms: A longitudinal study

Point prevalence estimates of extended-spectrum cephalosporin-resistant Escherichia coli (ESC-R-Ec) are important surveillance measures but may not uncover the ESC-R-Ec dynamics within pig farms. A longitudinal study was therefore performed by sampling individual pigs, pig farmers and the environment. On average, 30 (range 10-46) piglets of 31 Swiss farms were sampled during the suckling, weaning and fattening stages (n= 2437 samples). In addition, stool from pig farmers and environmental samples were obtained and metadata collected by questionnaires. ESC-R-Ec was identified by routine culture, and clonal relationships and resistance genes were derived from whole genome sequencing data. Working on pig farms was not associated with an increased prevalence of ESC-R-Ec in humans. ESC-R-Ec prevalence significantly decreased from 6.2% to 3.9% and 1.8% for the suckling, weaned and fattening pigs, respectively (P &lt; 0.001). Within the 57 ESC-R-positive suckling piglets, persisting carriage was detected in 25 animals at two consecutive time points and one animal at three consecutive time points. Clonal spread (n=7 farms, 22.6%) and horizontal gene transfer (n=1 farm, 3%) within pigs but not between humans and animals was detected. Liquid manure (n=10 samples, 16.7%) was identified as the major environmental reservoir of ESC-R-Ec in the pig farm environment. Pig farming practices like all-in-all-out systems, but not antimicrobial usage, were associated with reduced risk of ESC-R-Ec at the farm level. As carriage duration is normally short within the individual pigs, the risk of recolonisation and clonal spread of ESC-R-Ec might be reduced by applying appropriate decontamination strategies

Characterization of third-generation cephalosporin-resistant Escherichia coli from slaughter calves and fattening pigs: A pilot study for monitoring antimicrobial resistance by whole genome sequencing in Switzerland.

Whole genome sequencing (WGS) was introduced into Swiss antimicrobial resistance monitoring in 2022 as an additional method to phenotypic antimicrobial susceptibility testing by broth microdilution to characterize presumptive third-generation cephalosporin-resistant (3GC-R) Escherichia coli. Caecal samples from Swiss slaughter calves and fattening pigs, as well as beef and pork meat from Swiss retail taken in 2021, were analyzed for the presence of 3GC-R E. coli according to European harmonized protocols. In 2021, 3GC-R E. coli was detected in 23,8 % of slaughter calves, 5,9 % of fattening pigs, and 0 % of meat. Comparative analysis of the antimicrobial resistance results obtained by phenotypic measurement and those obtained by the detection of corresponding underlying molecular mechanisms by WGS showed very high agreement (99 %). Resistance to third-generation cephalosporins (3GCs) was mainly associated with the presence of blaCTX-M-15 in E. coli isolates from calves and blaCTX-M-1 in E. coli isolates from pigs and mutations in the ampC-promoter (g.-42 C>T) in E. coli isolates from both animal species. Moreover, WGS data were used for phylogenetic analysis based on multi locus sequence types (MLST) and core genome MLST(cgMLST) revealing that 3GC-R E. coli isolated from Swiss slaughter calves and fattening pigs were genetically diverse. In this study, it was shown that using WGS alone to monitor antimicrobial resistance could detect trends in known molecular antimicrobial resistance mechanisms while also providing other valuable information about the isolates, such as genetic relatedness. However, only by combining phenotypic susceptibility testing and WGS early detection of previously unknown resistance mechanisms will be possible

Feedborne Salmonella enterica Serovar Jerusalem Outbreak in Different Organic Poultry Flocks in Switzerland and Italy Linked to Soya Expeller

Poultry feed is a leading source of Salmonella infection in poultry. In Switzerland, heat-treated feed is used to reduce Salmonella incursions into flocks in conventional poultry production. By contrast, organic feed is only treated with organic acids. In 2019, the Swiss National Reference Center for Enteropathogenic Bacteria identified the rare serovar S. Jerusalem from samples of organic soya feed. Further, in July 2020, the European Union’s Rapid Alert System for Food and Feed published a notification of the detection of S. Jerusalem in soya expeller from Italy. During 2020, seven S. Jerusalem isolates from seven different poultry productions distributed over six cantons in Switzerland were reported, providing further evidence of a possible outbreak. Using whole-genome sequencing (WGS), S. Jerusalem isolates from feed and from animals in Switzerland were further characterized and compared to S. Jerusalem from organic poultry farm environments in Italy. WGS results showed that feed isolates and isolates from Swiss and Italian poultry flocks belonged to the sequence type (ST)1028, grouped in a very tight cluster, and were closely related. This outbreak highlights the risk of spreading Salmonella by feed and emphasizes the need for a heat-treatment process for feed, also in organic poultry production

Genomic Characterization and Antimicrobial Susceptibility of Dromedary-Associated Staphylococcaceae from the Horn of Africa.

Members of the Staphylococcaceae family, particularly those of the genus Staphylococcus, encompass important human and animal pathogens. We collected and characterized Staphylococcaceae strains from apparently healthy and diseased camels (n = 84) and cattle (n = 7) in Somalia and Kenya. We phenotypically characterized the strains, including their antimicrobial inhibitory concentrations. Then, we sequenced their genomes using long-read sequencing, closed their genomes, and subsequently compared and mapped their virulence- and resistance-associated gene pools. Genome-based phylogenetics revealed 13 known Staphylococcaceae and at least two novel species. East African strains of different species encompassed novel sequence types and phylogenetically distant clades. About one-third of the strains had non-wild-type MICs. They were resistant to at least one of the following antimicrobials: tetracycline, benzylpenicillin, oxacillin, erythromycin, clindamycin, trimethoprim, gentamicin, or streptomycin, encoded by tet(K), blaZ/blaARL, mecA/mecA1, msrA/mphC, salA, dfrG, aacA-aphD, and str, respectively. We identified the first methicillin- and multidrug-resistant camel S. epidermidis strain of sequence type (ST) 1136 in East Africa. The pool of virulence-encoding genes was largest in the S. aureus strains, as expected, although other rather commensal strains contained distinct virulence-encoding genes. We identified toxin-antitoxin (TA) systems such as the hicA/hicB and abiEii/abiEi families, reported here for the first time for certain species of Staphylococcaceae. All strains contained at least one intact prophage sequence, mainly belonging to the Siphoviridae family. We pinpointed potential horizontal gene transfers between camel and cattle strains and also across distinct Staphylococcaceae clades and species. IMPORTANCE Camels are a high value and crucial livestock species in arid and semiarid regions of Africa and gain importance giving the impact of climate change on traditional livestock species. Our current knowledge with respect to Staphylococcaceae infecting camels is very limited compared to that for other livestock species. Better knowledge will foster the development of specific diagnostic assays, guide promising antimicrobial treatment options, and inform about potential zoonotic risks. We characterized 84 Staphylococcaceae strains isolated from camels with respect to their antimicrobial resistance and virulence traits. We detected potentially novel Staphylococcus species, resistances to different classes of antimicrobials, and the first camel multidrug-resistant S. epidermidis strain of sequence type 1136

Driving laboratory standardisation of bacterial culture and antimicrobial susceptibility testing in veterinary clinical microbiology in Europe and beyond.

Globally, antimicrobial resistance is one of the most important public health challenges in which the clinical microbiology laboratory plays a critical role by providing guidance for antimicrobial treatment. Despite the recognition of its importance, there is still a real need for standardized training of clinical microbiologists and harmonisation of diagnostic procedures. This is particularly true for veterinary clinical microbiology where additional challenges exist when microbiologists are trying to fulfil a professional role very similar to their colleagues working in human microbiology laboratories. The specific points that need addressing to improve the outputs of veterinary microbiology laboratories discussed here include 1) harmonisation of methodologies used by veterinary laboratories for antimicrobial susceptibility testing (AST); 2) specific guidelines for interpretation and reporting of AST results for animal pathogens; 3) guidelines for detection of antimicrobial resistance mechanisms in animal isolates; 4) standardisation of diagnostic procedures for animal clinical specimens and 5) the need to train more veterinary clinical microbiology specialists. However, there is now a plan to address these issues led by the European Network for Optimisation of Veterinary Antimicrobial Treatment (ENOVAT) which is bringing together experts in veterinary microbiology, pharmacology, epidemiology and antimicrobial stewardship from Europe and wider afield. ENOVAT is aiming to work with project partners towards standardisation and harmonisation of laboratory methodologies and optimisation of veterinary antimicrobial treatment. Ultimately, the project may provide a mechanism for standardisation and harmonisation of veterinary clinical microbiology methodologies, which could then be used as a template for implementation at a wider international level