Azithromycin resistance in Escherichia coli and Salmonella from food-producing animals and meat in Europe.

OBJECTIVES: To characterize the genetic basis of azithromycin resistance in Escherichia coli and Salmonella collected within the EU harmonized antimicrobial resistance (AMR) surveillance programme in 2014-18 and the Danish AMR surveillance programme in 2016-19. METHODS: WGS data of 1007 E. coli [165 azithromycin resistant (MIC > 16 mg/L)] and 269 Salmonella [29 azithromycin resistant (MIC > 16 mg/L)] were screened for acquired macrolide resistance genes and mutations in rplDV, 23S rRNA and acrB genes using ResFinder v4.0, AMRFinder Plus and custom scripts. Genotype-phenotype concordance was determined for all isolates. Transferability of mef(C)-mph(G)-carrying plasmids was assessed by conjugation experiments. RESULTS: mph(A), mph(B), mef(B), erm(B) and mef(C)-mph(G) were detected in E. coli and Salmonella, whereas erm(C), erm(42), ere(A) and mph(E)-msr(E) were detected in E. coli only. The presence of macrolide resistance genes, alone or in combination, was concordant with the azithromycin-resistant phenotype in 69% of isolates. Distinct mph(A) operon structures were observed in azithromycin-susceptible (n = 50) and -resistant (n = 136) isolates. mef(C)-mph(G) were detected in porcine and bovine E. coli and in porcine Salmonella enterica serovar Derby and Salmonella enterica 1,4, [5],12:i:-, flanked downstream by ISCR2 or TnAs1 and associated with IncIγ and IncFII plasmids. CONCLUSIONS: Diverse azithromycin resistance genes were detected in E. coli and Salmonella from food-producing animals and meat in Europe. Azithromycin resistance genes mef(C)-mph(G) and erm(42) appear to be emerging primarily in porcine E. coli isolates. The identification of distinct mph(A) operon structures in susceptible and resistant isolates increases the predictive power of WGS-based methods for in silico detection of azithromycin resistance in Enterobacterales.

Rare Plasmid-Mediated AmpC Beta-Lactamase DHA-1 Located on Easy Mobilized IS26-Related Genetic Element Detected in Escherichia coli from Livestock and Food in Germany.

AmpC beta-lactamases cause resistance to third-generation cephalosporins, including beta-lactamase inhibitors. In Escherichia coli from the German food production chain, the majority of AmpC beta-lactamase activity can be attributed to plasmid-mediated CMY-2 or overproduction of chromosomal AmpC beta-lactamase, but occasionally other enzymes like DHA-1 are involved. This study investigated the prevalence of the AmpC beta-lactamase DHA-1 in ESBL/AmpC-producing E. coli (n = 4706) collected between 2016 and 2021 as part of a German antimicrobial resistance monitoring program along the food chain. Eight isolates (prevalence < 0.2%) were detected and further characterized by PFGE, transformation and conjugation experiments as well as short-read and long-read sequencing. All eight strains harbored blaDHA-1 together with qnrB4, sul1 and mph(A) resistance genes on an IS26 composite transposon on self-transferable IncFII or IncFIA/FIB/II plasmids. During laboratory experiments, activation of the translocatable unit of IS26-bound structures was observed. This was shown by the variability of plasmid sizes in original isolates, transconjugants or transferred plasmids, and correspondingly, duplications of resistance fragments were found in long-read sequencing. This activation could be artificial due to laboratory handling or naturally occurring. Nevertheless, DHA-1 is a rare AmpC beta-lactamase in livestock and food in Germany, and its dissemination will be monitored in the future.

Biocide Susceptibility and Antimicrobial Resistance of Escherichia coli Isolated from Swine Feces, Pork Meat and Humans in Germany.

Phenotypic susceptibility testing of Escherichia (E.) coli is an essential tool to gain a better understanding of the potential impact of biocide selection pressure on antimicrobial resistance. We, therefore, determined the biocide and antimicrobial susceptibility of 216 extended-spectrum ß-lactamase-producing (ESBL) and 177 non-ESBL E. coli isolated from swine feces, pork meat, voluntary donors and inpatients and evaluated associations between their susceptibilities. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of benzalkonium chloride, chlorhexidine digluconate (CHG), chlorocresol (PCMC), glutaraldehyde (GDA), isopropanol (IPA), octenidine dihydrochloride and sodium hypochlorite (NaOCl) showed unimodal distributions, indicating the absence of bacterial adaptation to biocides due to the acquisition of resistance mechanisms. Although MIC95 and MBC95 did not vary more than one doubling dilution step between isolates of porcine and human origin, significant differences in MIC and/or MBC distributions were identified for GDA, CHG, IPA, PCMC and NaOCl. Comparing non-ESBL and ESBL E. coli, significantly different MIC and/or MBC distributions were found for PCMC, CHG and GDA. Antimicrobial susceptibility testing revealed the highest frequency of resistant E. coli in the subpopulation isolated from inpatients. We observed significant but weakly positive correlations between biocide MICs and/or MBCs and antimicrobial MICs. In summary, our data indicate a rather moderate effect of biocide use on the susceptibility of E. coli to biocides and antimicrobials.

MRSA in bulk tank milk of dairy herds in Germany - changes over time.

OBJECTIVE: Methicillin-resistant Staphylococcus aureus (MRSA) have repeatedly been isolated from dairy herds. It was the purpose of this study to compare the results of 3 subsequent national scale cross-sectional investigations in dairy herds in Germany on the prevalence of MRSA in bulk tank milk and the characteristics of the isolates. MATERIAL AND METHODS: The investigations were carried out in 2010, 2014 and 2019, respectively. MRSA were isolated from 25 ml of bulk tank milk using a double selective enrichment protocol. Samples were distributed across the country according to the regional dairy cattle population. RESULTS: The prevalence of MRSA in bulk tank milk samples was lower in 2010 than in 2014 and tended to decrease until 2019. Prevalence was higher in samples from conventional than from organic herds and increased with herd size. Most isolates (75/78) were assigned to the clonal complex 398 and the spa-types t011 and t034. Resistance of the isolates to other antimicrobials than beta-lactams decreased over time. CONCLUSIONS: MRSA remain present in the German dairy population and are found more frequently in larger vs. smaller herds and in conventional vs. organic herds. CLINICAL RELEVANCE: MRSA should be considered in biosecurity protocols and with respect to occupational health of farm staff. Presence of MRSA in raw milk supports the recommendation not to drink unpasteurized raw milk.

Plasmid-Coded Linezolid Resistance in Methicillin-Resistant Staphylococcus aureus from Food and Livestock in Germany

Resistance of methicillin-resistant Staphylococcus aureus (MRSA) from food and livestock to last resort antibiotics such as linezolid is highly concerning, since treatment options for infections in humans might be diminished. Known mechanisms of linezolid resistance include point mutations in the 23S rRNA gene and in the ribosomal proteins L3, L4 and L22 as well as an acquisition of the cfr, optrA or poxtA gene. The objective of our study was to characterize antimicrobial resistance (AMR) determinants and phylogenetic relationships among linezolid-resistant (LR-) MRSA from food and livestock. In total, from more than 4000 incoming isolates in the years 2012 to 2021, only two strains from 2015 originating from pig samples exhibited linezolid resistance in the antimicrobial susceptibility testing with MICs of ≥8 mg/L. These LR-MRSA were characterized in detail by whole-genome sequencing and phylogenetic analyses using cgMLST. The LR-MRSA strains showed resistances to ten and eight different antibiotics, respectively. Both strains harbored plasmid-coded cfr genes mediating the linezolid resistance. The cfr genes showed identical sequences in both strains. In addition to the cfr gene, genes for phenicol and clindamycin resistance were detected on the respective plasmids, opening the possibility for a co-selection. The LR-MRSA differed distantly in the phylogenetic analyses and also to other MRSA from pig samples in the year 2015. In conclusion, the occurrence of LR-MRSA in food and livestock seems to be very rare in Germany. However, carriage of plasmids with linezolid resistance determinants could lead to further linezolid-resistant strains by horizontal gene transfer.

Comparison of Antimicrobial Resistances in Escherichia coli from Conventionally and Organic Farmed Poultry from Germany.

In this study, resistance rates in Escherichia coli from organic and conventional poultry in Germany were compared. Isolates were randomly collected from organic and conventional broiler and turkey flocks at the farm and from turkey meat at retail. Resistance testing was performed as prescribed by Commission implementing decision 2013/652/EU. Logistic regression analyses were performed for the resistance to the different antimicrobials. Overall, resistance rates for the antimicrobials tested were lower in E. coli from organic than from conventionally raised animals. In turkeys, the percentage of isolates susceptible to all antimicrobials tested from animals and meat was twice as high from organic than from conventional origin (~50% vs. <25%). In broilers, the percentage of susceptible isolates from organic farms was five times higher than from conventional farms (70.1% vs. 13.3%) and resistance to three or more classes of antimicrobials was 1.7- to 5.0-fold more common in isolates from conventional farms. The differences between organic and conventional farming were more pronounced in broilers than in turkeys. More studies on turkeys are needed to determine whether this difference is confirmed.

Different fosA genes were found on mobile genetic elements in Escherichia coli from wastewaters of hospitals and municipals in Turkey.

AIMS: The increasing number of globally established fosfomycin-resistant (FosR) Gram-negative bacteria inspired us to investigate the occurrence of FosREnterobacterales populations (esp. E. coli) in samples of city wastewater treatment plants (WWTPs) and hospital sewage in Hatay, Turkey. FosR target bacteria were further characterized for their clonal relatedness, resistomes and mobile genetic elements (MGEs) to evaluate their impact on fosfomycin resistance dissemination. METHODS: A total of 44 samples from raw and treated waters of WWTPs as well as of two hospitals in the Hatay province were subjected to selective cultivation for recovering FosREnterobacterales. The presence of fosA was verified by PCR and Sanger amplicon sequencing. Detected E. coli were further evaluated against antimicrobial susceptibility-testing, macrorestriction profiling (PFGE) and whole-genome sequencing (WGS). Bioinformatics analysis was performed for genome subtyping (i.e., MLST, serotype), resistome/virulome determination and dissection of the genetic determinants of plasmidic fosA3/4 resistances. RESULTS: Besides ten non-E. coli Enterobacterales, 29 E. coli were collected within this study. In silico-based subtyping revealed that E. coli isolates were assigned to six different serovars and 14 sequence types (ST), while O8:H21 and ST410 represented the major prevalent types, respectively. Fosfomycin resistance in the isolates was found to be mediated by the fosA4 (n = 18), fosA3 (n = 10) and fosA (n = 1), which are frequently associated with transmissible MGEs. Reconstruction of plasmid-associated fosA gene context revealed a linkage between the resistance cassette and IS6 (IS26 family) transposases, which might represent a major driver for the distribution of the genes and the generation of novel fosA-carrying plasmids. CONCLUSIONS: The occurrence of plasmid-mediated, transmissible FosR in E. coli from wastewater pose a foreseeable threat to "One-Health". To minimize further spread of the resistances in bacterial populations associated with environmental, animal and human health further resistance monitoring and management strategies must be developed.

What WGS Reveals about Salmonella enterica subsp. enterica in Wildlife in Germany.

The aim of this study was to gain an overview of the genetic diversity of Salmonella found in wildlife in Germany. We were particularly interested in exploring whether wildlife acts as a reservoir of certain serovars/subtypes or antimicrobial resistance (AMR) genes. Moreover, we wanted to explore the potential of Salmonella in spreading from wildlife to livestock and humans. To answer these questions, we sequenced 260 Salmonella enterica subsp. enterica isolates sampled between 2002 and 2020 from wildlife across Germany, using short-read whole genome sequencing. We found, consistent with previous findings, that some Salmonella sequence types are associated with certain animal species, such as S. Choleraesuis ST145 with wild boar and S. Enteritidis ST183 with hedgehogs. Antibiotic resistance was detected in 14.2% of all isolates, with resistance against important WATCH group antibiotics present in a small number of isolates. We further found that wildlife isolates do not form separate phylogenetic clusters distant to isolates from domestic animals and foodstuff, thus indicating frequent transmission events between these reservoirs. Overall, our study shows that Salmonella in German wildlife are diverse, with a low AMR burden and close links to Salmonella populations of farm and food-production environments.

Phenotypic and Genotypic Properties of Fluoroquinolone-Resistant, qnr-Carrying Escherichia coli Isolated from the German Food Chain in 2017.

Fluoroquinolones are the highest priority, critically important antimicrobial agents. Resistance development can occur via different mechanisms, with plasmid-mediated quinolone resistance (PMQR) being prevalent in the livestock and food area. Especially, qnr genes, commonly located on mobile genetic elements, are major drivers for the spread of resistance determinants against fluoroquinolones. We investigated the prevalence and characteristics of qnr-positive Escherichia (E.) coli obtained from different monitoring programs in Germany in 2017. Furthermore, we aimed to evaluate commonalities of qnr-carrying plasmids in E. coli. We found qnr to be broadly spread over different livestock and food matrices, and to be present in various sequence types. The qnr-positive isolates were predominantly detected within selectively isolated ESBL (extended spectrum beta-lactamase)-producing E. coli, leading to a frequent association with other resistance genes, especially cephalosporin determinants. Furthermore, we found that qnr correlates with the presence of genes involved in resistance development against quaternary ammonium compounds (qac). The detection of additional point mutations in many isolates within the chromosomal QRDR region led to even higher MIC values against fluoroquinolones for the investigated E. coli. All of these attributes should be carefully taken into account in the risk assessment of qnr-carrying E. coli from livestock and food.

Comparative Analysis of Consumer Exposure to Resistant Bacteria through Chicken Meat Consumption in Germany.

Human exposure to bacteria carrying antimicrobial resistance (AMR) genes through the consumption of food of animal origin is a topic which has gained increasing attention in recent years. Bacterial transmission can be enhanced, particularly in situations in which the consumer pays less attention to hygiene practices, and consumer exposure to foodborne resistant bacteria through ready-to-eat foods could be increased. It has been demonstrated that even methicillin-resistant Staphylococcus aureus (MRSA) bacteria, which have low prevalence and concentration in raw chicken meat in Germany, may reach the consumer during barbecue events after failures in hygiene practices. This study aimed to quantify the consumer exposure to extended-spectrum beta-lactamase- (ESBL) or ampicillinase class C (AmpC) beta-lactamase-producing E. coli in Germany through the consumption of chicken meat and bread during household barbecues. The study considered cross-contamination and recontamination processes from raw chicken meat by using a previously-developed probabilistic consumer exposure model. In addition, a comparative analysis of consumer exposure was carried out between ESBL-/AmpC-producing E. coli and MRSA. Our results demonstrated that the probability of ESBL-/AmpC-producing E. coli reaching the consumer was 1.85 × 10-5 with the number of bacteria in the final serving averaging 332. Given the higher prevalence and concentration of ESBL-/AmpC-producing E. coli in raw chicken meat at retail compared to MRSA, comparative exposure assessment showed that the likelihood and extent of exposure were significantly higher for ESBL-/AmpC-producing E. coli than for MRSA. ESBL-/AmpC-producing E. coli was determined to be 7.6 times likelier (p-value < 0.01) than MRSA to reach the consumer, with five times the concentration of bacteria in the final serving (p-value < 0.01).

Isolation Procedure for CP E. coli from Caeca Samples under Review towards an Increased Sensitivity.

Due to the increasing reports of carbapenemase-producing Enterobacteriaceae (CPE) from livestock in recent years, the European Reference Laboratory for Antimicrobial Resistances (EURL-AR) provided a protocol for their recovery from caecum and meat samples. This procedure exhibited limitations for the detection of CPE with low carbapenem MIC values. Therefore, it was modified by a second, selective enrichment in lysogeny broth with cefotaxime (CTX 1 mg/L) and with meropenem (MEM 0.125 mg/L) at 37 °C under microaerophilic conditions. By Real-time PCR, these enrichments are pre-screened for the most common carbapenemase genes. Another adaptation was the use of in-house prepared MacConkey agar with MEM and MEM+CTX instead of commercial selective agar. According to the EURL-method, we achieved 100% sensitivity and specificity using the in-house media instead of commercial agar, which decreased the sensitivity to ~75%. Comparing the method with and without the second enrichment, no substantial influence on sensitivity and specificity was detected. Nevertheless, this enrichment has simplified the CPE-isolation regarding the accompanying microbiota and the separation of putative colonies. In conclusion, the sensitivity of the method can be increased with slight modifications.

Comparison of Phenotypical Antimicrobial Resistance between Clinical and Non-Clinical E. coli Isolates from Broilers, Turkeys and Calves in Four European Countries.

Livestock data on antimicrobial resistance (AMR) are commonly collected from bacterial populations of clinical and non-clinical isolates. In contrast to data on non-clinical isolates from livestock, data on clinical isolates are not harmonized in Europe. The Normalized Resistance Interpretation (NRI) method was applied to overcome the lack of harmonization of laboratory methods and interpretation rules between monitoring systems. Statistical analyses were performed to identify associations between the isolate type (clinical vs. non-clinical) and resistance to four antimicrobials (ampicillin, tetracycline, gentamicin, and nalidixic acid) per animal category in Germany and France. Additional statistical analyses comparing clinical and non-clinical isolates were performed with the available data on the same antimicrobial panel and animal categories from the UK and Norway. Higher resistance prevalence was found in clinical isolates compared to non-clinical isolates from calves to all antimicrobials included in Germany and France. It was also found for gentamicin in broilers from France. In contrast, in broilers and turkeys from Germany and France and in broilers from the UK, a higher resistance level to ampicillin and tetracycline in non-clinical isolates was encountered. This was also found in resistance to gentamicin in isolates from turkeys in Germany. Resistance differed within countries and across years, which was partially in line with differences in antimicrobial use patterns. Differences in AMR between clinical and non-clinical isolates of Escherichia coli are associated with animal category (broiler, calf, and turkey) and specific antimicrobials. The NRI method allowed comparing results of non-harmonized AMR systems and might be useful until international harmonization is achieved.

Comparison of Consumption Data and Phenotypical Antimicrobial Resistance in E. coli Isolates of Human Urinary Samples and of Weaning and Fattening Pigs from Surveillance and Monitoring Systems in Germany.

Antimicrobial resistance (AMR) data from humans are mostly collected from clinical isolates, whereas from livestock data also exist from colonizing pathogens. In Germany, livestock data are collected from clinical and nonclinical isolates. We compared resistance levels of clinical and nonclinical isolates of Escherichia coli from weaning and fattening pigs with clinical outpatient isolates of humans from urban and rural areas. We also studied the association of AMR with available antimicrobial use (AMU) data from humans and pigs. Differences between rural and urban isolates were minor and did not affect the comparison between human and pig isolates. We found higher resistance levels to most antimicrobials in human isolates compared to nonclinical isolates of fattening pigs. Resistance to ampicillin, however, was significantly more frequent in clinical isolates of fattening pigs and in clinical and nonclinical isolates of weaning pigs compared to isolates from humans. The opposite was observed for ciprofloxacin. Co-trimoxazole resistance proportions were higher in clinical isolates of weaning and fattening pigs as compared to isolates from humans. Resistance proportions were higher in clinical isolates than in nonclinical isolates from pigs of the same age group and were also higher in weaner than in fattening pigs. Significant associations of AMU and AMR were found for gentamicin resistance and aminoglycoside use in humans (borderline) and for ampicillin resistance in clinical isolates and penicillin use in fattening pigs. In summary, we found significant differences between isolates from all populations, requiring more detailed analyses supported by molecular data and better harmonized data on AMU and AMR.

Phenotypical antimicrobial resistance data of clinical and non-clinical Escherichia coli from poultry in Germany between 2014 and 2017.

Antimicrobial resistance (AMR) is a global threat in humans and animals, and antimicrobial usage (AMU) has been identified as a main trigger of AMR. The purpose of this work was to compare data on AMR in clinical and non-clinical isolates of Escherichia coli in German broilers and turkeys between 2014 and 2017. Furthermore, we investigated AMR changes over time and the association of changes in AMU with changes in AMR. Data on clinical and non-clinical isolates together with data on therapy frequency of broilers and turkeys were collected from German monitoring systems. Logistic regression analyses were performed to assess the association between the explanatory factors (AMU, year and isolate type) and the dependent variable (AMR). In broilers, the analysis showed lower resistance proportions of clinical isolates of E. coli to ampicillin and colistin (ampicillin: Odds ratio (OR) and 95% confidence interval (CI) = 0.44 (0.3-0.64), p<0.001; colistin: OR and 95% CI = 0.75 (0.73-0.76), p<0.001) but higher proportions for cefotaxime (OR and 95% CI = 4.58 (1.56-15.1), p = 0.007). Resistance to ampicillin, gentamicin and tetracycline was less frequent in clinical isolates in turkeys (ampicillin: OR and 95% CI = 0.4 (0.29-0.53), p<0.001; gentamicin: OR and 95% CI = 0.5 (0.26-0.94), p = 0.035; tetracycline: OR and 95% CI = 0.4 (0.29-0.55), p<0.001). The analysis found decreasing associations of AMU with resistance to tetracycline in turkeys and to colistin in broilers. Year was associated with a decrease in resistance to colistin in broilers and to tetracycline in turkeys. Differences in resistance found in this study between clinical and non-clinical isolates might play an important role in resistance prevalence. This study indicated that further data analyses over longer time intervals are required to clarify the differences found between clinical and non-clinical isolates and to assess the long-term effects of changes in AMU on the prevalence of AMR.

Identification of a blaVIM-1-Carrying IncA/C2 Multiresistance Plasmid in an Escherichia coli Isolate Recovered from the German Food Chain.

Within the German national monitoring of zoonotic agents, antimicrobial resistance determination also targets carbapenemase-producing (CP) Escherichia coli by selective isolation from food and livestock. In this monitoring in 2019, the CP E. coli 19-AB01133 was recovered from pork shoulder. The isolate was assigned to the phylogenetic group B1 and exhibited the multi-locus sequence-type ST5869. Molecular investigations, including whole genome sequencing, of 19-AB01133 revealed that the isolate carried the resistance genes blaVIM-1, blaSHV-5 and blaCMY-13 on a self-transmissible IncA/C2 plasmid. The plasmid was closely related to the previously described VIM-1-encoding plasmid S15FP06257_p from E. coli of pork origin in Belgium. Our results indicate an occasional spread of the blaVIM-1 gene in Enterobacteriaceae of the European pig population. Moreover, the blaVIM-1 located on an IncA/C2 plasmid supports the presumption of a new, probably human source of carbapenemase-producing Enterobacteriaceae (CPE) entering the livestock and food chain sector.

First Detection of GES-5-Producing Escherichia coli from Livestock-An Increasing Diversity of Carbapenemases Recognized from German Pig Production.

Resistance to carbapenems due to carbapenemase-producing Enterobacteriaceae (CPE) is an increasing threat to human health worldwide. In recent years, CPE could be found only sporadically from livestock, but concern rose that livestock might become a reservoir for CPE. In 2019, the first GES carbapenemase-producing Escherichia coli from livestock was detected within the German national monitoring on antimicrobial resistance. The isolate was obtained from pig feces and was phenotypically resistant to meropenem and ertapenem. The isolate harbored three successive blaGES genes encoding for GES-1, GES-5 and GES-5B in an incomplete class-I integron on a 12 kb plasmid (pEC19-AB02908; Acc. No. MT955355). The strain further encoded for virulence-associated genes typical for uropathogenic E. coli, which might hint at an increased pathogenic potential. The isolate produced the third carbapenemase detected from German livestock. The finding underlines the importance CPE monitoring and detailed characterization of new isolates.

Antibiotic Resistance in Escherichia coli from Broiler Chickens After Amoxicillin Treatment in an Experimental Environment.

Groupwise antibiotic treatments are common in broiler chicken production. They induce selection for antibiotic resistance in commensal Escherichia coli. This study aimed to investigate antibiotic resistance after individual (I, drenching) or groupwise treatment (G, by water) with amoxicillin, and after contact with I or G (KI or KG), compared with untreated broilers without contact with treated broilers (C), and pretreatment values. Finally, we compared antibiotic resistance from broilers (G) after a second treatment, with a treatment in the contact animals (KG), and a first treatment in the control animals (C). Resistance to ampicillin and other antibiotics was significantly increased in groups G and I within 2 days, suggesting (co-)selection of resistance. The increase was lower in groups KI, KG, and C during the first treatment (days 1-5). The increased resistance in group C was interpreted as a change in the microbiota after initial moving and first feeding. After treatment, resistance rates decreased to initial or lower values in all groups. During the second treatment period (days 34-38), all three groups' (G, KG, and C) resistance levels increased to equally high levels. Cephalosporin resistance was low, and did not change over the experimental period. On days 3 and 38, resistance rates of E. coli from duodenum, jejunum, and cecum did not differ between segments and treatment routes. Overall, the baseline levels of antibiotic resistance in E. coli were high. Amoxicillin triggered an increase in resistance levels, irrespective of the mode of treatment. Substantial resistance dynamics in untreated controls warrant further investigation.

ChromID® CARBA Agar Fails to Detect Carbapenem-Resistant Enterobacteriaceae With Slightly Reduced Susceptibility to Carbapenems.

After first detections of carbapenemase-producing Enterobacteriaceae (CPE) in animals, the European Union Reference Laboratory for Antimicrobial Resistance has provided a protocol for the isolation of carbapenemase-producing Escherichia (E.) coli from cecum content and meat. Up to now, only few isolates were recovered using this procedure. In our experience, the choice of the selective agar is important for the efficacy of the method. Currently, the use of the prevailing method fails to detect CPE that exhibit a low resistance against carbapenems. Thus, this study aims to evaluate the suitability of selective media with antibiotic supplements and commercial ChromID® CARBA agar for a reliable CPE detection. For comparative investigations, detection of freeze-dried carbapenemase-resistant bacteria was studied on different batches of the ChromID® CARBA agar as well as on MacConkey agar supplemented with 1 mg/L cefotaxime and 0.125 mg/L meropenem (McC+CTX+MEM). The suitability of the different media was assessed within a time of 25 weeks, starting at least six weeks before expiration of the media. Carbapenem-resistant isolates exhibiting a serine-based hydrolytic resistance mechanism (e.g., bla KPC genes) were consistently detected over 25 weeks on the different media. In contrast, carbapenemase producers with only slightly reduced susceptibility and exhibiting a zinc-catalyzed activity (e.g., bla VIM, bla NDM, and bla IMP) could only be cultivated on long-time expired ChromID® CARBA, but within the whole test period on McC+CTX+MEM. Thus, ChromID® CARBA agar appears to be not suitable for the detection of CPE with slightly increased minimum inhibitory concentrations (MIC) against carbapenems, which have been detected in German livestock and thus, are of main interest in the national monitoring programs. Our data are in concordance with the results of eleven state laboratories that had participated in this study with their ChromID® CARBA batches routinely used for the German CPE monitoring. Based on the determined CPE detection rate, we recommend the use of McC+CTX+MEM for monitoring purposes. This study indicates that the use of ChromID® CARBA agar might lead to an underestimation of the current CPE occurrence in food and livestock samples.

[Antimicrobial resistance in E. coli from different cattle populations in Germany]. / Antibiotikaresistenz von E. coli aus Rinderpopulationen in Deutschland.

OBJECTIVE: It was the objective of this study to describe and compare antimicrobial resistance in clinical and non-clinical E. coli from different cattle populations. MATERIAL AND METHODS: For this, the minimum inhibitory concentrations (MIC) of antimicrobials against clinical and non-clinical E. coli isolates from dairy cows, beef cattle, veal calves and other calves were analyzed. The MIC examinations were performed in the framework of a monitoring program for clinical isolates from animals in Germany (GERM-Vet) and as part of the national zoonosis monitoring between 2009 and 2019 using broth microdilution. The MIC were evaluated based on the epidemiological cut off values provided by the European Centre for Antimicrobial Susceptibility Testing (EUCAST). RESULTS: A total of 5127 isolates was included in the analysis. The highest resistance rates were observed in isolates from calves with diarrhea, followed by non-clinical isolates from cattle slaughtered at an age under 1 year. The lowest resistance rates were encountered in E. coli from bulk tank milk of dairy herds and from beef cattle. Resistance rates of isolates from cases of mastitis were higher than of non-clinical isolates from bulk tank milk, but lower than the rates observed in calves. CONCLUSION AND CLINICAL RELEVANCE: Resistance rates to the critically important antimicrobials fluoroquinolones and 3rd generation cephalosporins were high in isolates from mastitis samples and in isolates from calves with diarrhea. This may be linked to the high use of these substances in dairy cows and the feeding of wastemilk from treated cows to calves, exposing the latter to drug residues and resistant bacteria from the milk. The use of these substances has to be reduced to a minimum in order to avoid further spread of this kind of resistance in the cattle population.

Spill-Over from Public Health? First Detection of an OXA-48-Producing Escherichia coli in a German Pig Farm.

Resistance to carbapenems is a severe threat to human health. These last resort antimicrobials are indispensable for the treatment of severe human infections with multidrug-resistant Gram-negative bacteria. In accordance with their increasing medical impact, carbapenemase-producing Enterobacteriaceae (CPE) might be disseminated from colonized humans to non-human reservoirs (i.e., environment, animals, food). In Germany, the occurrence of CPE in livestock and food has been systematically monitored since 2016. In the 2019 monitoring, an OXA-48-producing E. coli (19-AB01443) was recovered from a fecal sample of a fattening pig. Phenotypic resistance was confirmed by broth microdilution and further characterized by PFGE, conjugation, and combined short-/long-read whole genome sequencing. This is the first detection of this resistance determinant in samples from German meat production. Molecular characterization and whole-genome sequencing revealed that the blaOXA-48 gene was located on a common pOXA-48 plasmid-prototype. This plasmid-type seems to be globally distributed among various bacterial species, but it was frequently associated with clinical Klebsiella spp. isolates. Currently, the route of introduction of this plasmid/isolate combination into the German pig production is unknown. We speculate that due to its strong correlation with human isolates a transmission from humans to livestock has occurred.