Harmonisation of in-silico next-generation sequencing based methods for diagnostics and surveillance.

Improvements in cost and speed of next generation sequencing (NGS) have provided a new pathway for delivering disease diagnosis, molecular typing, and detection of antimicrobial resistance (AMR). Numerous published methods and protocols exist, but a lack of harmonisation has hampered meaningful comparisons between results produced by different methods/protocols vital for global genomic diagnostics and surveillance. As an exemplar, this study evaluated the sensitivity and specificity of five well-established in-silico AMR detection software where the genotype results produced from running a panel of 436 Escherichia coli were compared to their AMR phenotypes, with the latter used as gold-standard. The pipelines exploited previously known genotype-phenotype associations. No significant differences in software performance were observed. As a consequence, efforts to harmonise AMR predictions from sequence data should focus on: (1) establishing universal minimum to assess performance thresholds (e.g. a control isolate panel, minimum sensitivity/specificity thresholds); (2) standardising AMR gene identifiers in reference databases and gene nomenclature; (3) producing consistent genotype/phenotype correlations. The study also revealed limitations of in-silico technology on detecting resistance to certain antimicrobials due to lack of specific fine-tuning options in bioinformatics tool or a lack of representation of resistance mechanisms in reference databases. Lastly, we noted user friendliness of tools was also an important consideration. Therefore, our recommendations are timely for widespread standardisation of bioinformatics for genomic diagnostics and surveillance globally.

Validation of an antimicrobial susceptibility testing protocol for Brachyspira hyodysenteriae and Brachyspira pilosicoli in an international ring trial.

Brachyspira hyodysenteriae and Brachyspira pilosicoli cause economically important enteric disease in pigs. Treatment of these infections often includes antimicrobial administration, which can be most effective when therapeutic options are informed by antimicrobial susceptibility testing data. Here we describe a method for broth dilution antimicrobial susceptibility testing of these bacteria, both of which are difficult to culture in vitro. The protocol was evaluated for its fitness for use in an inter-laboratory ring trial involving eight laboratories from seven countries, and employing eleven test strains (5 Brachyspira hyodysenteriae including the type strain B78T and 6 Brachyspira pilosicoli) and six antibiotics. Overall intra- and inter-laboratory reproducibility of this method was very good (>90 % MICs at mode +/- 1 log2). Whole genome sequencing revealed good correspondence between reduced susceptibility and the presence of previously defined antimicrobial resistance determinants. Interestingly, lnu(C) was identified in B. pilosicoli isolates with elevated MICs of lincomycin, whilst tva(B) was associated with elevated MICs of pleuromutilins in this species. We designated two new control strains with MICs lying within currently tested ranges, including for the pleuromutilins, in contrast to the control strain B. hyodysenteriae B78T. These were deposited at the DSMZ-German Collection of Microorganisms and Cell Cultures GmbH. The validation of a standard protocol and identification of new control strains facilitates comparisons between studies, establishment of robust interpretative criteria, and ultimately contributes to rational antimicrobial use when treating infected livestock.

Changing epidemiology of invasive non-typhoid Salmonella infection: a nationwide population-based registry study.

OBJECTIVES: Non-typhoid Salmonella (NTS) may invade beyond the intestine, causing bacteraemia, sepsis, and infection of normally sterile sites. The epidemiology of invasive NTS (iNTS) infection is under-researched. We determined trends, risk factors, serotype distribution, antimicrobial resistance (AMR), and attributable sources of iNTS infection in a high-income setting. METHODS: 22,837 records of culture-confirmed human salmonellosis cases and 10,008 serotyped Salmonella isolates from five putative animal reservoirs (pigs, cattle, broilers, layers, reptiles) in the Netherlands during 2005-2018 were retrieved from national surveillance registries. Risk factors for iNTS infection were identified using logistic regression analysis. Source attribution modelling was based on serotyping, prevalence, and exposure data. RESULTS: The average annual percentage of iNTS infections was 4.6% (range 3.5-5.7%). An increase in iNTS infections was observed since 2012 (odds ratio (OR) 1.09, 95% confidence interval (95% CI) 1.04-1.14). Increased iNTS infection risk was associated with wintertime (OR 1.37, 95% CI 1.12-1.66), male sex (OR 1.73, 95% CI 1.51-1.99), older age (ORs: 3.27 to 16.33, depending on age groups), and living in rural areas (OR 1.54, 95% CI 1.23-1.93). While 52% of iNTS infections (n = 950) were caused by serotypes Enteritidis and Typhimurium, those displaying the highest invasiveness relative to their occurrence were Dublin (32.9%, n = 163), Panama (21.6%, n = 106), and Poona (14.1%, n = 71). Cattle were a larger source of iNTS than non-iNTS infections (12.2% vs. 7.6%). Lower AMR and multi-resistance rates were observed among iNTS (37.9%) than non-iNTS isolates (48.6%). DISCUSSION: The increase in iNTS infections, which is reported also in other countries, is of public health and clinical concern. The underlying reasons seem to be multi-factorial in nature. iNTS infection risk depends more on the infecting serotypes and patient demographics, and less on the attributable reservoirs and AMR profiles.

Occurrence and characteristics of extended-spectrum-ß-lactamase- and AmpC-producing clinical isolates derived from companion animals and horses.

OBJECTIVES: To investigate the occurrence and characteristics of extended-spectrum ß-lactamase (ESBL)- and AmpC-producing Enterobacteriaceae isolates in clinical samples of companion animals and horses and compare the results with ESBL/AmpC-producing isolates described in humans. METHODS: Between October 2007 and August 2009, 2700 Enterobacteriaceae derived from clinical infections in companion animals and horses were collected. Isolates displaying inhibition zones of ≤ 25 mm for ceftiofur and/or cefquinome by disc diffusion were included. ESBL/AmpC production was confirmed by combination disc tests. The presence of resistance genes was identified by microarray, PCR and sequencing, Escherichia coli genotypes by multilocus sequence typing and antimicrobial susceptibility by broth microdilution. RESULTS: Sixty-five isolates from dogs (n = 38), cats (n = 14), horses (n = 12) and a turtle were included. Six Enterobacteriaceae species were observed, mostly derived from urinary tract infections (n = 32). All except 10 isolates tested resistant to cefotaxime and ceftazidime by broth microdilution using clinical breakpoints. ESBL/AmpC genes observed were bla(CTX-M-1, -2, -9, -14, -15,) bla(TEM-52), bla(CMY-2) and bla(CMY-)(39). bla(CTX-M-1) was predominant (n = 17). bla(CTX-M-9) occurred in combination with qnrA1 in 3 of the 11 Enterobacter cloacae isolates. Twenty-eight different E. coli sequence types (STs) were found. E. coli carrying bla(CTX-M-1) belonged to 13 STs of which 3 were previously described in Dutch poultry and patients. CONCLUSIONS: This is the first study among a large collection of Dutch companion animals and horses characterizing ESBL/AmpC-producing isolates. A similarity in resistance genes and E. coli STs among these isolates and isolates from Dutch poultry and humans may suggest exchange of resistance between different reservoirs.

[Multiresistant Brachyspira hyodysenteriae in a Dutch sow herd]. / Multiresistentie bij Brachyspira hyodysenteriae-isolaten op een varkensvermeerderingsbedrijf in Nederland.

This case study describes the isolation ofa multiresistant strain ofBrachyspira hyodysenteriae in April 2007 in a Dutch sow herd with recurrent diarrhoea. Examination of faecal samples taken from 7-month-old breeding gilts with diarrhoea revealed the presence of resistance against tiamulin, lincomycin, tylosin, doxycycline, and tylvalosin (the active substance in Aivlosin) in four of five samples. Tiamulin resistance has not been reported in The Netherlands before. The repeated use of tiamulin on the affected farm was assumed to be the main cause of the development of resistance to the drug. The farmer was advised to adopt a medication strategy and to implement management practices that would prevent an ongoing cycle of infection on the farm. It is important that the Dutch swine industry appreciates that tiamulin-resistant strains of B. hyodysenteriae may be found on other farms as well. The appropriate and prudent use of antibiotics is essential in order to prevent the development of resistance against the last option left to cure B. hyodysenteriae infections: valnemulin.

In vitro antibiotic susceptibility of Dutch Mycoplasma synoviae field isolates originating from joint lesions and the respiratory tract of commercial poultry.

The in vitro susceptibility of 17 Dutch Mycoplasma synoviae isolates from commercial poultry to enrofloxacin, difloxacin, doxycycline, tylosin and tilmicosin was examined. Three isolates originated from joint lesions and 14 were from the respiratory tract. The type strain M. synoviae WVU 1853 was included as a control strain. Antibiotic susceptibility was tested quantitatively using the broth microdilution test. Based on initial and final minimum inhibitory concentration values, all tested isolates were susceptible to doxycycline, tylosin and tilmicosin. Two isolates from the respiratory tract were resistant to enrofloxacin and showed intermediate resistance to difloxacin.

[Antibiotic resistance in Escherichia coli O157 isolated between 1998 and 2003 in The Netherlands]. / Antibioticumresistentie bij Escherichia coli O157 geïsoleerd tussen 1998 en 2003 in Nederland.

Little is known about the antibiotic resistance of E. coli O157 in The Netherlands. In this study, 218 human and 247 nonhuman samples, isolated between 1998 and 2003, were tested for antimicrobial resistance. About 5.5% of E. coli O157 isolates from human samples were resistant, as were about 4.00% of E. coli O157 isolates from non-human samples. These figures are lower than those reported in the literature. Class I integrons were found in six multiresistant isolates. This type of integron is also found in commensal E. coli in food animals and Salmonella spp. One of the integron-positive isolates contained the beta-lactamase bla(TEM-1b) and an extended spectrum beta-lactamase (ESBL), which belongs to the group 2 CTX-M enzymes. This is the first report of these beta-lactamases in E. coli O157 isolated from chicken.

Quality-control ranges for antimicrobial susceptibility testing by broth dilution of the Brachyspira hyodysenteriae type strain (ATCC 27164T).

There are no approved standards for antimicrobial susceptibility testing of the fastidious spirochete Brachyspira hyodysenteriae. An interlaboratory study was performed to establish MIC quality control ranges for six antimicrobial agents for the type strain of B. hyodysenteriae using broth dilution. The results showed that B. hyodysenteriae B78T ATCC 27164T is a suitable quality control strain. This is a first step toward standardization of methods regarding this anaerobe.

Investigations of Enterococcus faecalis-induced bacteraemia in brown layer pullets through different inoculation routes in relation to the production of arthritis.

In the present aerosol experiment, assessment of the respiratory tract of 1-day-old birds as a natural route of infection for induction of Enterococcus faecalis bacteraemia and arthritis was performed. Second, the severity and type of arthritis produced through intramuscular infection in two different inoculation sites (musculus pectoralis versus musculus gastrocnemius) was studied. Third, the resulting bacteraemia was assessed qualitatively and quantitatively in relation to the occurrence of arthritis. Exposure of 1-day-old brown layer pullets to aerosolized E. faecalis with an estimated uptake of 10(4) to 10(5) colony forming units per chick resulted in bacteraemia; however, joint lesions were not induced. In contrast, 3/10 birds inoculated intratracheally with 10(8) colony forming units developed both bacteraemia and arthritis. This suggests the occurrence of a dose effect and a role for the respiratory tract as a natural infection route in young chickens. In both intramuscularly inoculated groups the incidence of arthritis was 10/10 birds and 9/10 birds, respectively. Birds inoculated in the m. pectoralis developed symmetric polyarthritis, which harmonizes with haematogenous colonization of joints. In contrast, m. gastrocnemius-inoculated chicks mostly had asymmetric (poly)arthritis of the injected leg and varus deformation of the contralateral leg, suggesting predominantly local spread. The qualitative and quantitative results of bacteriology of blood samples show that arthritis develops in those groups with the highest number of bacteraemic birds and the highest median bacterial colony forming units per millilitre of blood during the first 24 to 36 h after treatment.

Molecular epidemiology of unilateral amyloid arthropathy in broiler breeders associated with Enterococcus faecalis.

Although symmetrical polyarticular amyloidosis has been described extensively in brown layers, spontaneous unilateral amyloid arthropathy has not been described previously in chickens. Birds from nine flocks of broiler parent stock (PS) had unilateral lameness associated with severe swelling of the left hock joint and the caudal aspect of the metatarsus. Gross pathology was restricted to the left hock joint and the left digital flexor tendons in almost all cases, suggesting an association with administration of Marek's disease vaccine. Amyloid deposits were found in 83% (25/30) of affected joints by histological examination of Congo red stained sections. Systemic amyloidosis, involving mainly the liver and spleen, was found in 59% (10/17) of birds. Enterococcus faecalis was isolated from joints in 77% (23/30) of cases and Staphylococcus aureus was isolated from the joint in one case (1/30). Thirty-five E. faecalis isolates from joints, tendons and blood samples from birds in five affected PS flocks were compared using pulsed-field gel electrophoresis (PFGE) to separate genomic fragments after digestion with SmaI. All but one isolate had identical or closely related restriction endonuclease digestion (RED) patterns that were very similar to a known arthropathic and amyloidogenic E. faecalis isolate. A further 30 E. faecalis isolates from seven grandparent stock (GPS) flocks and two isolates from two unaffected PS flocks of the same genetic background were analysed by PFGE. Among these isolates, 11 originating from four GPS flocks had RED patterns identical to or closely related to the reference amyloid-inducing strain. Moreover, one E. faecalis isolate from amyloidotic joints of brown layers housed in California, USA was included in the analysis and appeared to be identical to the reference strain. This study showed that the E. faecalis isolates involved in these outbreaks of unilateral amyloid arthropathy in broiler breeders belonged to the same clone as that responsible for outbreaks in brown layers.

Study on the vertical transmission of arthropathic and amyloidogenic Enterococcus faecalis in a flock of brown layer chickens.

Enterococcus faecalis-related amyloid arthropathy was diagnosed in a sample of birds from a flock of brown layer parent chickens aged 57 weeks. E. faecalis was isolated from amyloidotic knee joints and from blood samples from lame birds. From this flock a smaller one was kept for production purposes to study the vertical transmission of arthropathic and amyloidogenic E. faecalis. Three batches of non-soiled and thoroughly disinfected eggs, to prevent egg-shell contamination, were collected for 6 weeks and submitted for incubation every 2 weeks. No joint pathology was found in the offspring chicks (n = 458) monitored for joint disease until 16 or 21 weeks of age. Fresh and candled eggs (infertiles and dead embryos) obtained at day 18 of the incubation period had negative results at bacteriological examination of the yolk sac, except one non-hatched egg (late embryonal death in shell) from which E. faecalis was isolated. Genotyping by pulsed-field gel electrophoresis (PFGE) of all E. faecalis isolates obtained from blood and joints (except one) of the parent birds and of the non-hatched egg after Sma I digestion showed that all isolates were genetically closely related or identical to a known arthropathic and amyloidogenic E. faecalis strain. In this study, vertical transmission of E. faecalis, although it may occur on a small scale as shown by PFGE, did not seem to play a significant role. Conversely, the chronicity of the condition and the development of an immune response may have affected the efficiency of its transmission.

Aerosol transmission of arthropathic and amyloidogenic Enterococcus faecalis.

One-day-old brown layer chicks were exposed to an aerosol of an arthropathic and amyloidogenic Enterococcus faecalis strain alone or after being subjected to treatment with formaldehyde gas (100-200 ppm). Four-day-old chicks were also treated with the same aerosol but after treatment with a Newcastle disease vaccine virus (NDVV) aerosol or intramuscular injection with methylprednisolon at day 1. The same E. faecalis strain was inoculated intramuscularly in day-old chicks as positive control. Bacteremia with time showed that 24 hr after the aerosol the day-old exposed chicks had the highest rate of positive blood cultures (70%-80%). Lower numbers of bacteremic birds at this point in time were found in the chicks treated with E. faecalis aerosol at day 4 (3/10 in the methylprednisolon-treated group and 0/10 in the NDVV-treated group) and the E. faecalis intramuscular-injected group at day 1 (2/10). Formaldehyde gas treatment did not favor the occurrence of bacteremia. NDVV aerosol exposure or injection with corticosteroids did not favor the occurrence of bacteremia 24 hr after E. faecalis aerosol exposure at day 4 either, although 66 days after aerosol, one bird (1/14) treated with NDVV showed bacteremia. A few bacteremic birds were found 10 days after aerosol in the NDVV- and methylprednisolon-treated groups, whereas at 14 days after aerosol, one bacteremic bird was seen in the group subjected to E. faecalis aerosol at day 1, indicating the occurrence of chronic bacteremia. In contrast to the E. faecalis intramuscular-inoculated birds, no joint pathology was seen in the aerosol-exposed groups in spite of the occurrence of chronic bacteremia.

[Preliminary results of antibiotic resistance monitoring in the Netherlands]. / Eerste resultaten van de monitoring van antibioticumresistentie in Nederland.

Qualitative tests are used to monitor antimicrobial resistance in bacteria of animal origin in the Netherlands. Quantitative information on trends in resistance is thus not obtained. Moreover, in general a limited panel of antibiotics is tested. The present study describes resistance in zoonotic food-borne pathogens Salmonella, Campylobacter, and Escherichia coli O157 isolated from human clinical cases and from faeces of healthy food animals in 1998 and 1999, as determined with quantitative susceptibility tests. The resistance of the indicator organisms E. coli and Enterococcus faecium isolated from faecal samples of broilers and pigs randomly sampled at slaughterhouses was also determined. For this end, faecal samples from veal calves were sampled in 1996 and 1997 at the three main Dutch veal calf slaughterhouses. In 1998 only a limited number of faecal samples of veal calves were taken at farms. For E. coli and Salmonella the following antibiotics were tested: amoxicillin, amoxicillin-clavulanic acid, piperacillin, cefotaxime, ceftazidime, imipenem, gentamicin, doxycycline, trimethoprim, trimethoprim/sulphamethoxazole, ciprofloxacin, chloramphenicol, florfenicol, carbadox, and flumequine. For E. faecium the following antibiotics were tested: amoxicillin, amoxicillin-clavulanic acid, chloramphenicol, doxycycline, erythromycin, vancomycin, teicoplanin, streptomycin ('high level' > 2000 mg/ml), gentamicin ('high level' > 500 mg/ml), ciprofloxacin, bacitracin, flavofosfolipol, salinomycin, quinupristin-dalfopristin, virginiamycin, tilmicosin, avilamycin, and everninomycin. For Campylobacter the following antibiotics were tested: erythromycin, doxycycline, gentamicin, carbadox, flavofosfolipol, ciprofloxacin, trimethoprim/sulphamethoxazole, amoxicillin, and metronidazole.

Contamination of Marek's disease vaccine suspensions with Enterococcus faecalis and its possible role in amyloid arthropathy.

Samples of hatchery air (hatcher and processing room), Marek's disease vaccine suspensions and injection needles collected during chick processing, revealed variable levels (< 500 to 10(6) colony forming units (cfu)/m(3) air, < 10 to 10(6) cfu/ml vaccine suspension, and 9500 to 61000 cfu/needle) of Enterococcus faecalis contamination. This observation suggests a possible infection route in 1-day-old chickens through intramuscular vaccination of Marek's disease vaccine contaminated with arthropathic and amyloidogenic E. faecalis, which could lead to amyloid arthropathy. Pulsed-field gel electrophoresis (PFGE) DNA restriction endonuclease fragment analysis of E. faecalis strains obtained from two hatcheries revealed a predominant PFGE pattern in one hatchery, while one isolate with an almost identical PFGE pattern to an amyloid arthropathy inducing isolate was found.

Sensitivity testing of veterinary pathogens with a semi-automatic image analysis system compared with tablet diffusion and agar dilution tests.

Recently a commercial computer-controlled image analysis system (IAS) was introduced to measure automatically the diameters of inhibition zones in the agar diffusion test. However, there is little information on the precision of this method. In the present study clinical isolates of Salmonella spp. (N = 104), Escherichia coli (N = 100), Pasteurella spp. (N = 99), Actinobacillus pleuropneumoniae (N = 85), porcine streptococci (N = 100), and Staphylococcus aureus (N = 95) were tested in the agar diffusion test, using nineteen different antibiotics in tablets. All inhibition zone diameters were first measured by a laboratory technician and then by the IAS. Although the zone diameters of all bacteria-antibiotic combinations measured by the IAS and those measured by the laboratory technician showed a significant positive correlation, the size of the inhibition zone diameters measured by the technician and the IAS differed significantly in 59% of the combinations. However, these differences were very small and may have no clinical relevance. The IAS was also used to calculate minimum inhibitory concentrations (MIC values) from the zone diameters. In 82% of the bacteria-antibiotic combinations MIC values calculated by the IAS showed a significant positive correlation with MIC values obtained with the reference agar dilution test. However, in 92% of the bacteria-antibiotic combinations, the calculated MIC values differed significantly from the reference values. In some cases these differences were so large that they could be of clinical relevance. The IAS was unable to measure the diameter of inhibition zones of porcine streptococci properly, due to poor contrast. We concluded that when tablets are used as antibiotic carriers the IAS accurately measures the diameter of inhibition zones for bacteria species that give good contrast between the agar and bacterial growth. MIC values determined with the IAS were only indicative of those determined with the reference agar dilution test.

The effect of flavophospholipol (Flavomycin) and salinomycin sodium (Sacox) on the excretion of Clostridium perfringens, Salmonella enteritidis, and Campylobacter jejuni in broilers after experimental infection.

Intestinal colonization and shedding of pathogenic bacteria in animal feces is an important factor in both human food safety and animal health. The effect of broiler feed additives flavophospholipol (FPL; Flavomycin, bambermycins) and salinomycin sodium (SAL; Sacox) given singly on the excretion of Salmonella enteritidis, Campylobacter jejuni, and Clostridium perfringens was studied following controlled infection. The incidence of shedding (number of birds with positive fecal cultures) and the degree of shedding (cfu per gram of feces in positive birds) were measured to determine the influence of these two common feed additive antibiotics on shedding rates of potential pathogens. A total of 216 Ross broiler chickens, housed in battery cages, were fed either an unmedicated feed (controls), feed containing FPL, or feed containing SAL. Feed treatment groups were subdivided into three bacterial challenge groups of 24 chicks, each receiving only one of the pathogens. Bacterial challenge was administered orally on Days 11 and 12 for Salmonella and Campylobacter and on Days 2 and 3 for Clostridium. Fecal samples were collected weekly up to 6 wk of age and cultured for presence of the target organism. The shedding rate was determined by decimal dilutions of the fecal samples. Feeding FPL resulted in a reduced (P < or = 0.05) degree and incidence of Salmonella and Clostridium shedding at 6 wk. Feeding SAL reduced (P < or = 0.05) the incidence of Salmonella shedding at 6 wk. Neither feed additive affected the incidence nor the degree of Campylobacter shedding. The results of this study indicate that these feed additives may reduce the incidence of these potential human and animal pathogens in preslaughter broilers.

A study on the vertical transmission of arthropathic and amyloidogenic Enterococcus faecalis.

Ten brown layer parent hens were injected intravenously with arthropathic and amyloidogenic Enterococcus faecalis at 27 weeks of age to assess its vertical transmission during the subsequent 6-week production period. All inoculated hens developed chronic bacteraemia and arthritis, four died due to septicaemia and two of the remaining six showed amyloid arthropathy. The egg production was maintained at a lower level than the controls. Of eggs collected during the first 2 weeks after inoculation, E. faecalis was re-isolated from the yolk sac of 76% (13/17) of infertile eggs and dead embryos detected at the 18-day candling, and 100% (6/6) of non-hatching eggs, and from arthritic joints of 3% (2/66) offspring chicks of the same batch, although the latter did not develop joint amyloidosis by 8 weeks of age. E. faecalis was also re-isolated from ovary and oviduct of parent birds that died due to septicaemia. The E. faecalis organisms re-isolated from blood, ovaries and joints of diseased parent stock, yolk sac of infertile eggs and dead embryos detected at the 18-day candling, and non-hatching eggs, as well as organs and joints of offspring, had the same pulsed-field gel electrophoresis patterns as the E. faecalis isolate used to infect the parent birds. These findings indicate that vertical transmission of arthropathic and amyloidogenic E. faecalis may occur on a small scale.