Joachim Mankertz

Diversity of antimicrobial resistance pheno- and genotypes of methicillin-resistant Staphylococcus aureus ST398 from diseased swine

OBJECTIVESnFifty-four methicillin-resistant Staphylococcus aureus (MRSA) ST398 isolates from unrelated diseased swine collected all over Germany were comparatively investigated for their antimicrobial resistance and virulence properties, and for their genomic relatedness.nnnMETHODSnMICs of 30 antimicrobial agents were determined by broth microdilution. Resistance and virulence genes were detected via a diagnostic DNA microarray and specific PCRs. The genomic relationships were determined by ApaI-PFGE, spa typing and SCCmec typing.nnnRESULTSnTwenty-two distinct resistance patterns were observed. All 54 isolates were tetracycline resistant, mediated by tet(M), tet(K) and/or tet(L), with 14 isolates being only resistant to beta-lactam antibiotics and tetracyclines. Trimethoprim resistance, seen in 28 isolates, was mostly due to the gene dfrK or dfrG. Among the 24 macrolide/lincosamide-resistant isolates, the genes erm(A), erm(B) and/or erm(C) were detected. The two chloramphenicol/florfenicol-resistant isolates harboured the gene fexA. The eight gentamicin-resistant isolates carried the gene aacA/aphD. Fifty-three isolates harboured SCCmec type V elements while the remaining one carried mecA and ugpQ, but no recombinase genes. All isolates were PVL negative, but one and three isolates, respectively, were positive for the enterotoxin B and enterotoxin K and Q genes. Eight different spa types were identified with t011 being the most predominant. Six ApaI-PFGE clusters with up to nine individual patterns were detected.nnnCONCLUSIONSnMRSA ST398 isolates varied slightly in their virulence properties and spa types but differed distinctly in their antimicrobial resistance pheno- and genotypes as well as their ApaI-PFGE patterns. These data underline the ability of ST398 to acquire genetic material that might increase antimicrobial resistance and virulence.

A practical approach to screen for authorised and unauthorised genetically modified plants

In routine analysis, screening methods based on real-time PCR are most commonly used for the detection of genetically modified (GM) plant material in food and feed. In this paper, it is shown that the combination of five DNA target sequences can be used as a universal screening approach for at least 81 GM plant events authorised or unauthorised for placing on the market and described in publicly available databases. Except for maize event LY038, soybean events DP-305423 and BPS-CV127-9 and cotton event 281-24-236u2009×u20093006-210-23, at least one of the five genetic elements has been inserted in these GM plants and is targeted by this screening approach. For the detection of these sequences, fully validated real-time PCR methods have been selected. A screening table is presented that describes the presence or absence of the target sequences for most of the listed GM plants. These data have been verified either theoretically according to available databases or experimentally using available reference materials. The screening table will be updated regularly by a network of German enforcement laboratories.

Analysis of extended-spectrum-β-lactamase-producing Escherichia coli isolates collected in the GERM-Vet monitoring programme

OBJECTIVESnThe aims of this study were (i) to detect extended-spectrum β-lactamase (ESBL) genes among 1378 Escherichia coli isolates from defined disease conditions of companion and farm animals and (ii) to determine the localization and organization of ESBL genes.nnnMETHODSnE. coli isolates from the German resistance monitoring programme GERM-Vet were included in the study. Plasmids were transferred by conjugation or transformation and typed by PCR-based replicon typing. ESBL genes were detected by PCR; the complete ESBL genes and their flanking regions were sequenced by primer walking. Phylogenetic grouping and multilocus sequence typing (MLST) were performed for all ESBL-producing E. coli isolates.nnnRESULTSnOf the 27 ESBL-producing E. coli isolates detected, 22 carried blaCTX-M-1 genes on IncN (nu200a=u200a16), IncF (nu200a=u200a3), IncI1 (nu200a=u200a2) or multireplicon (nu200a=u200a1) plasmids. A blaCTX-M-3 gene was located on an IncN plasmid and a blaCTX-M-15 gene was located on an IncF plasmid. A multireplicon plasmid and an IncHI1 plasmid harboured blaCTX-M-2. A blaTEM-52c gene was identified within Tn2 on an IncI1 plasmid. The blaCTX-M genes located within the same or related genetic contexts showed differences due to the integration of insertion sequences. Various MLST types were detected, with ST10 (nu200a=u200a7), ST167 (nu200a=u200a4) and ST100 (nu200a=u200a3) being the most common.nnnCONCLUSIONSnThis study showed that the blaCTX-M-1 gene is the predominant ESBL gene among E. coli isolates from diseased animals in Germany and a considerable structural heterogeneity was found in the regions flanking the blaCTX-M-1 gene. Insertion sequences, transposons and recombination events are likely to be involved in alterations of the ESBL gene regions.

A proposal of interpretive criteria for cefoperazone applicable to bovine mastitis pathogens

The correct assessment of mastitis pathogens for their susceptibility/resistance to cefoperazone is currently hampered by the lack of harmonized test conditions and interpretive criteria. The aim of this study was to provide a proposal for clinical breakpoints of cefoperazone which are applicable to Staphylococcus aureus, coagulase-negative staphylococci, Escherichia coli, Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis from cases of bovine mastitis and better reflect the situation in the bovine udder than breakpoints adopted from human medicine. For this, pharmacological data and clinical efficacy data of the documents submitted for approval of cefoperazone have been revisited. In addition, 1086 bacterial pathogens of the aforementioned six species/groups collected in Germany and in the USA during recent years were tested in parallel for their cefoperazone MICs and the zone diameters using a 75 μg disk. Subsequently, MICs were plotted against zone diameters. Based on the pharmacological data, the clinical efficacy and the microbiological data, a proposal was made for veterinary-specific breakpoints which classify members of the aforementioned species/groups as (a) susceptible to cefoperazone when their MIC is ≤ 2 μg/ml and their zone diameters are ≥ 27 mm (staphylococci or E. coli) or ≥ 21 mm (streptococci), (b) intermediate when their MIC is 4 μg/ml and their zone diameters are 22-26 mm (staphylococci or E. coli) or 16-20mm (streptococci), and (c) resistant when their MIC is ≥ 8 μg/ml and their zone diameters are ≤ 21 mm (staphylococci or E. coli) or ≤ 15 mm (streptococci).

Target gene mutations among methicillin-resistant Staphylococcus aureus and methicillin-susceptible S. aureus with elevated MICs of enrofloxacin obtained from diseased food-producing animals or food of animal origin

Sir, Fluoroquinolones are broad-spectrum antimicrobial agents that are effective against Gram-positive and Gram-negative bacteria. Enrofloxacin, the first fluoroquinolone approved for veterinary use, is still commonly used in veterinary practice to treat infections in food-producing, pet and companion animals, caused by a wide variety of bacterial pathogens including methicillinresistant Staphylococcus aureus (MRSA) and methicillinsusceptible S. aureus (MSSA). However, CLSI-approved clinical breakpoints for enrofloxacin applicable to Staphylococcus spp. are restricted to staphylococci from cats and dogs (susceptible, ≤0.5 mg/L; intermediate, 1–2 mg/L; resistant, ≥4 mg/L). No CLSI-approved clinical breakpoints applicable to staphylococci from pigs, cattle or poultry are currently available. DNA gyrase and DNA topoisomerase IV are the target enzymes of fluoroquinolones in S. aureus, and resistance in staphylococci is mainly due to mutations in the quinolone resistance-determining regions (QRDRs) of the genes gyrA, gyrB, grlA and grlB, which code for the corresponding subunits of these two enzymes. Another mechanism involved in fluoroquinolone resistance of S. aureus is overexpression of the norA gene, which encodes a multidrug efflux pump, by mutations in the norA promoter region. The aim of the present study was to gain insight into target gene mutations and norA regulator mutations present among MRSA and MSSA from diseased pigs, cattle and poultry, as well as food of poultry origin. For this, all MRSA and MSSA isolates that exhibited enrofloxacin MICs of ≥1 mg/L, from five different strain collections, were comparatively analysed. This cut-off was chosen based on the breakpoint for enrofloxacin-non-susceptible canine and feline Staphylococcus spp. isolates. The test population included 8/54 MRSA from diseased pigs and 9/32 MRSA from poultry meat and poultry meat products characterized in two previous studies, 2/11 MRSA from diseased cattle and 1/2 MRSA from diseased poultry collected in the GERM-Vet programme 2008–09, 1/2 MRSA and 12/24 MSSA from diseased poultry collected in the GERM-Vet programme 2006–07, and 3/27 MSSA from diseased pigs collected in the BfT-GermVet study 2006–08. In total, 36 isolates with enrofloxacin MICs of 1 to ≥32 mg/L were identified (Table 1). If not already done in previous studies, isolates were subjected to spa typing (http ://spaserver.ridom.de) as well as to two clonal complex (CC) 398-specific PCRs as described previously. Isolates that were negative in these PCRs were subjected to multilocus sequence typing (MLST; http://saureus.mlst.net). Of the 36 isolates, 24 were assigned to sequence type (ST) 398 or CC398, depending on whether MLST or the CC398-specific PCRs had been conducted. Five isolates had ST9, another five isolates had ST5 and one isolate had ST1791 (both ST5 and ST1791 belong to CC5). One avian isolate had ST2269, which is a novel ST (allelic profile 1-4-1-4-243-250-10) that has been identified for the first time during the course of this study. Nine different spa types (t002, t011, t034, t214, t899, t1197, t1419, t1430 and t2346) were detected (Table 1). The QRDRs of the gyrA, gyrB, grlA and grlB genes and the promoter region of the norA gene of all 36 S. aureus isolates were amplified by PCR using previously described primers. Mutations were identified by sequencing the PCR products, and the results are summarized in Table 1 in relation to the enrofloxacin MICs. Regardless of their origin and their methicillinresistance status, all isolates with enrofloxacin MICs of ≥4 mg/L exhibited mutations in both grlA at codon 80 (resulting in a Ser to Phe exchange) and gyrA at codon 84 (resulting in exchange of Ser to Leu or Ala) (Table 1). The Ser84Ala exchange in GyrA observed in two MRSA isolates has not been reported before in staphylococci. In contrast, isolates with enrofloxacin MICs of 1 or 2 mg/L also exhibited a mutation in grlA at codon 80 (resulting in the exchange of Ser to Phe, Tyr or Leu), but lacked the amino acid substitutions at position 84 in GyrA. Another interesting observation was the presence of the Glu422Asp exchange in GrlB in all isolates assigned to CC398/ ST398, independent of enrofloxacin MIC, the year of isolation,

In-house and interlaboratory validation of a method for the extraction of DNA from pollen in honey

In this work, the in-house and interlaboratory validation of a DNA extraction method from pollen in an unifloral rape honey as well as several multifloral honeys is described. The amplifiability of plant and rape DNA amplifiable by real-time PCR was used as a parameter for the evaluation of the method. The practical (i.e., relative) limit of detection was used as a tool for assessing the suitability of the extraction method for further GMO analysis. In a collaborative study with 14 participating labs the results of the in-house validation could be confirmed. The amount of amplifiable plant and rape DNA varied depending on the type of honey. For rape honey, a mean practical LOD of 0.12xa0% was obtained.ZusammenfassungDie Einzellabor- sowie Ringversuchsvalidierung einer DNA-Extraktionsmethode für Pollen in Honigen wird vorgestellt. Als wesentlicher Parameter für die Auswertung wird die Amplifizierbarkeit von pflanzlicher DNA sowie von Raps-DNA herangezogen, die mittels real-time PCR vervielfältigt werden kann. Die praktische (d.xa0h. relative) Nachweisgrenze wird als Kriterium zur Bewertung der Eignung für eine nachfolgende GVO-Analytik verwendet. In einem Ringversuch mit 14 teilnehmenden Labors wurden die Ergebnisse der In-house-Validierung bestätigt. Je nach Art des Honigs kann die Menge an extrahierbarer DNA stark variieren. Im Ringversuch wurde bei Rapshonig im Mittel eine praktische Nachweisgrenze von 0,12xa0% erhalten.

Pheno- and genotypic analysis of antimicrobial resistance properties of Yersinia ruckeri from fish.

Enteric red-mouth disease, caused by Yersinia ruckeri, is an important disease in rainbow trout aquaculture. Antimicrobial agents are frequently used in aquaculture, thereby causing a selective pressure on bacteria from aquatic organisms under which they may develop resistance to antimicrobial agents. In this study, the distribution of minimal inhibitory concentrations (MICs) of antimicrobial agents for 83 clinical and non-clinical epidemiologically unrelated Y. ruckeri isolates from north west Germany was determined. Antimicrobial susceptibility was conducted by broth microdilution at 22 ± 2°C for 24, 28 and 48 h. Incubation for 24h at 22 ± 2°C appeared to be suitable for susceptibility testing of Y. ruckeri. In contrast to other antimicrobial agents tested, enrofloxacin and nalidixic acid showed a bimodal distribution of MICs, with one subpopulation showing lower MICs for enrofloxacin (0.008-0.015 μg/mL) and nalidixic acid (0.25-0.5 μg/mL) and another subpopulation exhibiting elevated MICs of 0.06-0.25 and 8-64 μg/mL, respectively. Isolates showing elevated MICs revealed single amino acid substitutions in the quinolone resistance-determining region (QRDR) of the GyrA protein at positions 83 (Ser83-Arg or -Ile) or 87 (Asn87-Tyr), which raised the MIC values 8- to 32-fold for enrofloxacin or 32- to 128-fold for nalidixic acid. An isolate showing elevated MICs for sulfonamides and trimethoprim harbored a ∼ 8.9 kb plasmid, which carried the genes sul2, strB and a dfrA14 gene cassette integrated into the strA gene. These observations showed that Y. ruckeri isolates were able to develop mutations that reduce their susceptibility to (fluoro)quinolones and to acquire plasmid-borne resistance genes.

Tylosin susceptibility of staphylococci from bovine mastitis

Although the 16-membered macrolide tylosin is commonly used for the treatment of bovine mastitis, little information is currently available about the susceptibility of mastitis pathogens to tylosin. In the present study, 112 Staphylococcus aureus and 110 coagulase-negative Staphylococcus (CoNS) spp. isolates from cases of bovine mastitis were tested by broth microdilution and agar disk diffusion with 30 μg tylosin disks. Susceptibility to erythromycin was tested by broth microdilution and disk diffusion using 15 μg disks. Both test populations showed bimodal distributions of minimal inhibitory concentrations (MICs) and zone diameters with eleven S. aureus and eight CoNS isolates showing tylosin MICs of ≥ 256 μg/ml and no zones of growth inhibition around the tylosin 30 μg disks. All 19 isolates with tylosin MICs of ≥ 256 μg/ml were also resistant to erythromycin. For six additional erythromycin-resistant isolates, tylosin MICs of 1-8 μg/ml were observed. One S. aureus and two CoNS isolates showed inducible macrolide resistance. PCR analysis of the 25 erythromycin-resistant staphylococcal isolates identified the resistance genes erm(A), erm(B), erm(C), erm(T), mph(C) and msr(A) alone or in different combinations. An excellent correlation between the results of the different tylosin susceptibility tests (broth microdilution versus disk diffusion) was seen for S. aureus and CoNS isolates. Since tylosin does not induce the expression of the aforementioned erm genes, isolates with an inducible resistance phenotype may - if only tylosin is tested - be falsely classified as tylosin-susceptible. Thus, erythromycin should be tested in parallel and tylosin should only be used for the treatment of infections caused by erythromycin-susceptible staphylococci.

In-house validation of a liquid chromatography - tandem mass spectrometry method for the determination of selective androgen receptor modulators (SARMS) in bovine urine

ABSTRACT A sensitive and robust LC–MS/MS method allowing the rapid screening and confirmation of selective androgen receptor modulators in bovine urine was developed and successfully validated according to Commission Decision 2002/657/EC, chapter 3.1.3 ‘alternative validation’, by applying a matrix-comprehensive in-house validation concept. The confirmation of the analytes in the validation samples was achieved both on the basis of the MRM ion ratios as laid down in Commission Decision 2002/657/EC and by comparison of their enhanced product ion (EPI) spectra with a reference mass spectral library by making use of the QTRAP technology. Here, in addition to the MRM survey scan, EPI spectra were generated in a data-dependent way according to an information-dependent acquisition criterion. Moreover, stability studies of the analytes in solution and in matrix according to an isochronous approach proved the stability of the analytes in solution and in matrix for at least the duration of the validation study. To identify factors that have a significant influence on the test method in routine analysis, a factorial effect analysis was performed. To this end, factors considered to be relevant for the method in routine analysis (e.g. operator, storage duration of the extracts before measurement, different cartridge lots and different hydrolysis conditions) were systematically varied on two levels. The examination of the extent to which these factors influence the measurement results of the individual analytes showed that none of the validation factors exerts a significant influence on the measurement results.

Detection of Transgenic Atlantic and Coho Salmon by Real-time PCR

Genetic modifications (GM) have been applied to salmon to generate fast-growing strains for potential use in aquaculture. In November 2015, the first transgenic salmon (AquAdvantage® Atlantic salmon) was accepted for commercialization in the USA under defined conditions. The presence of GM food products in the marketplace stimulates the need for detection methods to allow screening for the presence of genetic modifications in seafood products. This paper first shows that it is possible to obtain amplifiable DNA from raw and processed products containing salmon. Detection methods by real-time PCR are proposed in this work. An endogenous gene target was designed to detect salmonid species DNA in samples. In addition, detection methods using real-time PCR were developed for two GM salmon possessing growth hormone transgenes: the AquAdvantage® Atlantic salmon (Salmo salar) developed by AquaBounty for commercial purposes, and the coho salmon (Oncorhynchus kisutch) developed for research purposes by Fisheries and Oceans Canada. The methods are able to detect at least 20 copies of the target. It was found however that one of the construct-specific methods for the AquAdvantage® salmon detection did not work on AquAdvantage® genomic DNA even though it works on the sequence published in GenBank. The other assay however was found to reliably detect AquAdvantage® transgenic sequences in genomic DNA.