Uwe Roesler

Salmonella enterica subsp. enterica producing VIM-1 carbapenemase isolated from livestock farms

Sir, Thirdand fourth-generation cephalosporins and carbapenems are ‘critically important’ antimicrobials as classified by the WHO (www.who.int). In fact, carbapenems are last-line clinical antibiotics against infections caused by multidrug-resistant Gram-negative bacteria. In contrast to cephalosporins, carbapenems are not hydrolysed by most b-lactamases, including AmpC b-lactamases and extended-spectrum b-lactamases (ESBLs). However, during the last decade the prevalence of carbapenem resistance in Enterobacteriaceae has increased worldwide. Whereas the increase in the prevalence of ESBL-producing Enterobacteriaceae isolated from livestock is becoming an important public health problem, the increasing prevalence of carbapenemases has only affected hospitals and the community. Recently, however, the occurrence of carbapenemase-carrying commensal Escherichia coli isolated from livestock and their environment has been reported, and this could be the beginning of a new era in the antibiotic resistance field. Within the national RESET project (www.reset-verbund.de) several longitudinal and cross-sectional studies, collecting potential ESBL-carrier organisms from German farms, have been performed (using MacConkey agar with 1 mg/L cefotaxime as the selective medium). From the 221 isolates collected during 2011, 3 of them were ascribed to Salmonella enterica subsp. enterica (Table 1). The three Salmonella isolates (R3, R25 and R27) were obtained from two pig-fattening farms (R25 was collected outside the farm) and one broiler farm (Table 1). The three farms were distributed in different locations in the same German federal region, and although there was no apparent link between them, a common source cannot be excluded. The three isolates were tested for their susceptibility to 35 antimicrobials, including b-lactams/b-lactamase inhibitors (Table 1), phenicols, aminoglycosides, quinolones/fluoroquinolones, tetracycline, folate pathway antagonists, lipopeptides and fosfomycin, as previously described. For the present study, tigecycline (15 mg) and nitrofurantoin (300 mg) were included as well. The presence of ESBLs, AmpC b-lactamases and/or carbapenemase-encoding genes, class 1 and 2 integrons and other resistance genes was screened by PCR/sequencing, as previously described (Table S1, available as Supplementary data at JAC Online). The MIC values for some carbapenemase producers can be lower than the currently recommended breakpoints, and the results of the carbapenem susceptibility tests can be influenced by the genetic background. The Salmonella isolates R3, R25 and R27 showed decreased susceptibility to these antimicrobials [non-wild-type by the EUCAST epidemiological cut-off (ECOFF), but susceptible or intermediate according to the CLSI clinical breakpoint; Table 1]. This ‘decreased susceptibility’ could be transformed to a competent E. coli recipient, but conjugation or mobilization under the conditions used was unsuccessful. The three isolates carried both the AmpC-encoding gene blaACC-1 and the carbapenemase gene blaVIM-1, like in the previously reported E. coli isolates R178 and R29. When Salmonella R3 and the control strain E. coli R178 were grown in liquid medium with carbapenems (Luria-Bertani broth with 16 mg/L imipenem or 8 mg/L ertapenem inoculated with 1:1000 overnight culture), both isolates grew well, showing full carbapenem resistance (clinical breakpoints, CLSI versus EUCAST: imipenem ≥4 versus .8 mg/L; and ertapenem ≥1 versus .1 mg/L). Several class 1 integrons (In31, In70, In71, In110 and In450), transposons (Tn3, Tn402 and Tn21) and plasmids (incompatibility groups IncHI2, IncN, IncI1 and IncW) carrying blaVIM genes have been described. Like in E. coli R178 and R29, in the three Salmonella isolates the blaVIM-1 gene was located on a class 1 integron (variable region with blaVIM-1-aacA4-aadA1 gene cassettes) harboured by an 300 kb IncHI2 plasmid (determined by S1-nuclease PFGE analysis, PCR-based replicon typing and Southern blot hybridization, as previously described; HI2 double plasmid sequence typing failed). The plasmid also carried blaACC-1, strA/B, catA1 and a trimethoprim resistance gene (not identified with the primers used; see Table S1, available as Supplementary data at JAC Online). The sequence of the complete integron of Salmonella R3 (5436 bp, including complete sul1 and orf5, obtained using as template the pRHR3 plasmid of this isolate; see Table S1, available as Supplementary data at JAC Online) was identical to the one from E. coli R178 (accession number HE663536) and was related to Tn402 (like in GQ422826). Salmonella R27 was isolated from the same farm as both E. coli R178 and R29 (Table 1). However, the IncHI2 plasmids harboured by these Salmonella and E. coli isolates were different in size and gene content ( 300 versus 220 kb and presence versus absence of chloramphenicoland trimethoprimresistance genes), suggesting different plasmid evolutions. The three isolates were classified as S. enterica group C, antigenic formula ‘6,7:–:– ’ (www.pasteur.fr) at the National Salmonella Reference Laboratory (NRL-Salm, BfR). The sequence type ST32 (http://mlst.ucc.ie/mlst/dbs/Senterica) and the PFGE patterns found (Figure S1, available as Supplementary data at JAC Online) are typical of Salmonella Infantis (6,7:r:1,5) and have also been detected in German isolates from humans, poultry/ poultry meat and pig/pork meat. Salmonella Infantis is among the top 10 Salmonella serovars implicated in human salmonellosis worldwide (ranking in third place in 2011 in Europe; www.ecdc. europa.eu). Isolates from this serovar also caused disease with Research letters

Escherichia coli producing VIM-1 carbapenemase isolated on a pig farm

and the source from which the isolate was obtained. Further target gene mutations were detected in single isolates: (i) a mutation that resulted in the Glu471Asp exchange in GrlB was present in an avian ST5 MRSA; (ii) a mutation at codon 517 in the gyrB gene (resulting in an Arg to Lys exchange) was found in the ST1791 MRSA of turkey meat origin; and (iii) the ST2269 isolate had an additional grlA mutation at codon 84 that caused a Glu to Asp exchange, and a gyrA mutation that resulted in a Glu88Asp exchange. The GrlA alterations Ser80Leu and Glu84Asp and the GyrA exchange Glu88Asp have not been identified so far in S. aureus. The role in fluoroquinolone resistance of the Glu422Asp exchange in GrlB needs further investigation as the corresponding mutation was present along with other mutations in staphylococci that varied in their enrofloxacin MICs between 1 and 8 mg/L, but it was also the only mutation detected in two porcine MRSA isolates with an enrofloxacin MIC of 1 mg/L. All but one of the MRSA and MSSA isolates investigated in this study showed one of two types of point mutations (A and B in Figure S1, available as Supplementary data at JAC Online) in the norA promoter region; these, however, affected neither the 235 and 210 positions, nor the norA-associated ribosome binding site. For one avian MSSA isolate, no norA-specific PCR product could be obtained in repeated attempts. The results of this study show that increased MICs of enrofloxacin among MRSA and MSSA isolates from diseased foodproducing animals or food of animal origin is mainly mediated by grlA and/or gyrA mutations, which—aside from the four novel mutations detected during this study—correspond to those reported previously in S. aureus isolates of different origin. 2

Longitudinal Monitoring of Extended-Spectrum-Beta-Lactamase/AmpC-Producing Escherichia coli at German Broiler Chicken Fattening Farms

ABSTRACT Antimicrobial resistance of Escherichia coli to modern beta-lactam antibiotics due to the production of extended-spectrum beta-lactamases (ESBL) and/or plasmid-mediated AmpC beta-lactamases (AmpC) represents an emerging and increasing resistance problem that dramatically limits therapeutic options in both human and veterinary medicine. The presence of ESBL/AmpC genes in commensal E. coli from food-producing animals like broilers may pose a human health hazard. However, there are no data available concerning the prevalence of ESBL/AmpC-producing E. coli in German broiler flocks using selective methods. In this longitudinal study, samples were taken from seven conventional broiler fattening farms at three different times within one fattening period. Various samples originating from the animals as well as from their direct environment in the barn were investigated for the occurrence of ESBL/AmpC-producing E. coli. Average detection levels of 51, 75, and 76% in animal samples collected during the three samplings in the course of the fattening period demonstrate a colonization of even 1-day-old chicks, as well as a continuous significant (P < 0.001) increase in prevalence thereafter. The detection frequencies in housing environmental samples were relatively high, with an increase over time, and ranged between 54.2 and 100%. A total of 359 E. coli isolates were characterized by PCR and partly via the disc diffusion method. This study shows that prevalence of ESBL/AmpC-producing E. coli increases during the fattening period of the broiler flocks examined. Both colonized day-old chicks and contaminated farm environments could represent significant sources of ESBL/AmpC-producing E. coli in German broiler fattening farms.

Subgrouping of ESBL-producing Escherichia coli from animal and human sources: An approach to quantify the distribution of ESBL types between different reservoirs

Escherichia (E.) coli producing extended-spectrum beta-lactamases (ESBLs) are an increasing problem for public health. The success of ESBLs may be due to spread of ESBL-producing bacterial clones, transfer of ESBL gene-carrying plasmids or exchange of ESBL encoding genes on mobile elements. This makes it difficult to identify transmission routes and sources for ESBL-producing bacteria. The objectives of this study were to compare the distribution of genotypic and phenotypic properties of E. coli isolates from different animal and human sources collected in studies in the scope of the national research project RESET. ESBL-producing E. coli from two longitudinal and four cross-sectional studies in broiler, swine and cattle farms, a cross-sectional and a case-control study in humans and diagnostic isolates from humans and animals were used. In the RESET consortium, all laboratories followed harmonized methodologies for antimicrobial susceptibility testing, confirmation of the ESBL phenotype, specific PCR assays for the detection of bla(TEM), bla(CTX), and bla(SHV) genes and sequence analysis of the complete ESBL gene as well as a multiplex PCR for the detection of the four major phylogenetic groups of E. coli. Most ESBL genes were found in both, human and non-human populations but quantitative differences for distinct ESBL-types were detectable. The enzymes CTX-M-1 (63.3% of all animal isolates, 29.3% of all human isolates), CTX-M-15 (17.7% vs. 48.0%) and CTX-M-14 (5.3% vs. 8.7%) were the most common ones. More than 70% of the animal isolates and more than 50% of the human isolates contained the broadly distributed ESBL genes bla(CTX-M-1), bla(CTX-M-15), or the combinations bla(SHV-12)+bla(TEM) or bla(CTX-M-1)+bla(TEM). While the majority of animal isolates carried bla(CTX-M-1) (37.5%) or the combination bla(CTX-M-1)+bla(TEM) (25.8%), this was the case for only 16.7% and 12.6%, respectively, of the human isolates. In contrast, 28.2% of the human isolates carried bla(CTX-M-15) compared to 10.8% of the animal isolates. When grouping data by ESBL types and phylogroups bla(CTX-M-1) genes, mostly combined with phylogroup A or B1, were detected frequently in all settings. In contrast, bla(CTX-M-15) genes common in human and animal populations were mainly combined with phylogroup A, but not with the more virulent phylogroup B2 with the exception of companion animals, where a few isolates were detectable. When E. coli subtype definition included ESBL types, phylogenetic grouping and antimicrobial susceptibility data, the proportion of isolates allocated to common clusters was markedly reduced. Nevertheless, relevant proportions of same subtypes were detected in isolates from the human and livestock and companion animal populations included in this study, suggesting exchange of bacteria or bacterial genes between these populations or a common reservoir. In addition, these results clearly showed that there is some similarity between ESBL genes, and bacterial properties in isolates from the different populations. Finally, our current approach provides good insight into common and population-specific clusters, which can be used as a basis for the selection of ESBL-producing isolates from interesting clusters for further detailed characterizations, e.g. by whole genome sequencing.

Occurrence of MRSA in air and housing environment of pig barns

A high prevalence of MRSA among farm animals, especially pigs, has been observed for some time. However, knowledge on transmission routes of MRSA in livestock production is still scarce. Therefore, the aim of this study was to determine the occurrence of MRSA in pig house air as well as in samples from pigs and their housing environment in 27 MRSA positive pig barns of different sizes and production types. In 85.2% of all barns MRSA was detected in the animal house air. Impingement turned out to be a more sensitive sampling technique than filtration. Other environmental samples such as boot swabs or faeces showed prevalences of MRSA from 55.6% to 85.2% at sample level. The level of MRSA was 88.3% for pooled and 82.1% for single nasal swabs, in skin swabs the one was 87.7%, the others was 78.7%. Spa typing of isolates from air and nasal swabs showed predominantly spa types t011 and t034. MRSA prevalences in pigs as well as in various environmental samples were significantly higher in fattening farms than in breeding farms. This study provides good reference that there could be an airborne transmission of MRSA within pig herds indicating a potential contamination of the environment of barns.

Prevalence of mcr-1 in E. coli from Livestock and Food in Germany, 2010–2015

Since the first description of a plasmid-mediated colistin resistance gene (mcr-1) in November 2015 multiple reports of mcr-1 positive isolates indicate a worldwide spread of this newly discovered resistance gene in Enterobacteriaceae. Although the occurrence of mcr-1 positive isolates of livestock, food, environment and human origin is well documented only few systematic studies on the prevalence of mcr-1 are available yet. Here, comprehensive data on the prevalence of mcr-1 in German livestock and food isolates are presented. Over 10.600 E. coli isolates from the national monitoring on zoonotic agents from the years 2010–2015 were screened for phenotypic colistin resistance (MIC value >2 mg/l). Of those, 505 resistant isolates were screened with a newly developed TaqMan-based real-time PCR for the presence of the mcr-1 gene. In total 402 isolates (79.8% of colistin resistant isolates) harboured the mcr-1 gene. The prevalence was depending on the food production chain. The highest prevalence was detected in the turkey food chain (10.7%), followed by broilers (5.6%). A low prevalence was determined in pigs, veal calves and laying hens. The mcr-1 was not detected in beef cattle, beef and dairy products in all years investigated. In conclusion, TaqMan based real-time PCR provides a fast and accurate tool for detection of mcr-1 gene. The overall detection rate of 3.8% for mcr-1 among all E. coli isolates tested is due to high prevalence of mcr-1 in poultry production chains. More epidemiological studies of other European countries are urgently needed to assess German prevalence data.

Emission of ESBL/AmpC-producing Escherichia coli from pig fattening farms to surrounding areas.

The presence of ESBL/AmpC-producing Escherichia coli in livestock such as pigs has been known for some time. However, to date there is little information about the transmission of these resistant bacteria between pig farms and their surroundings. Thus, the aim of this study was to explore this topic by investigating seven German pig fattening farms. Samples from outside (including ground surfaces, ambient air, slurry and digestate from biogas plants) and, in parallel, from inside the pig barns (including pig feces, dust, barn air, flies and mice feces) were examined for ESBL/AmpC-producing E. coli and selected isolates were compared by pulsed-field gel electrophoresis (PFGE) analysis. 14/17 (82.4%) slurry samples and three of four samples of digestate from biogas plants tested positive for ESBL/AmpC-producing E. coli. In the vicinity of the pig barns these resistant bacteria were detected in 14/87 (16.1%) boot swabs taken from various ground surfaces and in 2/36 (6%) ambient air samples. Inside the pig barns, 6/63 (9.5%) barn air samples and a small proportion of flies and mice feces samples were ESBL/AmpC-positive. PFGE analysis proved fecal emission as well as a possible spread via flies, as identical ESBL-E. coli isolates were detected in slurry and on fertilized fields, as well as in flies and pooled feces from inside the barn and slurry. Contaminated slurry presented the major emission source for ESBL/AmpC-producing E. coli in the pig fattening farms, but a spread via the airborne route or via different vectors also seems possible.

Development of a Multiplex Real-Time PCR for the Rapid Detection of the Predominant Beta-Lactamase Genes CTX-M, SHV, TEM and CIT-Type AmpCs in Enterobacteriaceae

Beta-lactamase resistant bacteria and especially ESBL producing Enterobacteriaceae are an increasing problem worldwide. For this reason a major interest in efficient and reliable methods for rapid screening of high sample numbers is recognizable. Therefore, a multiplex real-time PCR was developed to detect the predominant class A beta-lactamase genes bla CTX-M, bla SHV, bla TEM and CIT-type AmpCs in a one-step reaction. A set of 114 Enterobacteriaceae containing previously identified resistance gene subtypes and in addition 20 undefined animal and environmental isolates were used for the validation of this assay. To confirm the accessibility in variable settings, the real-time runs were performed analogous in two different laboratories using different real-time cyclers. The obtained results showed complete accordance between the real-time data and the predetermined genotypes. Even if sequence analyses are further necessary for a comprehensive characterization, this method was proofed to be reliable for rapid screening of high sample numbers and therefore could be an important tool for e. g. epidemiological purposes or support infection control measures.

Longitudinal Analysis of Prototheca zopfii-Specific Immune Responses: Correlation with Disease Progression and Carriage in Dairy Cows

ABSTRACT In order to characterize the humoral and cellular immune responses to bovine mammary protothecosis, serum and whey samples obtained from 72 dairy cows assigned to four different clinical stages of infection were examined for specific antibodies by indirect enzyme-linked immunosorbent assay techniques. Milk samples were analyzed for the total numbers of excreted algal cells and somatic cells. After characterization of the course of immune induction in bovine protothecal mastitis, a long-term sentinel study was performed in an affected herd in order to investigate disease progression. A total of 61 dairy cows with protothecal mastitis were examined for shedding of algae cells and for local immune responses three times in 6-month intervals. During acute and chronic stages of protothecosis, significantly elevated specific antibody activities in sera were detected. A strong correlation of whey immunoglobulin A (IgA) and whey IgG1 antibody activity with the total counts of somatic cells in milk was observed, whereas only a weak correlation of whey IgA and whey IgG1 concentrations to the number of algal cells excreted with the milk was seen. Our results from the sentinel long-term study of infected cows revealed that 70.5% of the persistently infected animals were continuously shedding the pathogen. About 4.9% of the animals showed an intermittent shedding, whereas 18% of the cows were tested culturally negative throughout the study. It can be assumed that Prototheca zopfii mastitis in dairy cows is maintained on the herd level by subclinically infected alga-shedding cows.

Identification of harmless and pathogenic algae of the genus Prototheca by MALDI-MS

The only plants infectious for mammals, green algae from the genus Prototheca, are often overseen or mistaken for yeast in clinical diagnosis. To improve this diagnostical gap, a method was developed for fast and reliable identification of Prototheca. A collection of all currently recognized Prototheca species, most represented by several strains, were submitted to a simple extraction by 70% formic acid and ACN; the extracts were analyzed by means of MALDI‐MS. Most of the peaks were found in the range from 4 to 20 kDa and showed a high reproducibility, not in absolute intensities, but in their peak pattern. The selection of measured peaks is mostly due to the technique of ionization in MALDI‐MS, because proteins in the range up to 200 kDa were detected using gel electrophoresis. Some of the proteins were identified by peptide mass fingerprinting and MS2 analysis and turned out to be ribosomal proteins or other highly abundant proteins such as ubiquitin. For the preparation of a heatmap, the intensities of the peaks were plotted and a cluster analysis was performed. From the peak‐lists, a principal component analysis was conducted and a dendrogram was built. This dendrogram, based on MALDI spectra, was in fairly good agreement with a dendrogram based on sequence information from 18S DNA. As a result, pathogenic and nonpathogenic species from the genus Prototheca can be identified, with possible consequences for clinical diagnostics by MALDI‐typing.