Anika Friese

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 Study of the Contamination of Air and of Soil Surfaces in the Vicinity of Pig Barns by Livestock-Associated Methicillin-Resistant Staphylococcus aureus

ABSTRACT During 1 year, samples were taken on 4 days, one sample in each season, from pigs, the floor, and the air inside pig barns and from the ambient air and soil at different distances outside six commercial livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA)-positive pig barns in the north and east of Germany. LA-MRSA was isolated from animals, floor, and air samples in the barn, showing a range of airborne LA-MRSA between 6 and 3,619 CFU/m3 (median, 151 CFU/m3). Downwind of the barns, LA-MRSA was detected in low concentrations (11 to 14 CFU/m3) at distances of 50 and 150 m; all upwind air samples were negative. In contrast, LA-MRSA was found on soil surfaces at distances of 50, 150, and 300 m downwind from all barns, but no statistical differences could be observed between the proportions of positive soil surface samples at the three different distances. Upwind of the barns, positive soil surface samples were found only sporadically. Significantly more positive LA-MRSA samples were found in summer than in the other seasons both in air and soil samples upwind and downwind of the pig barns. spa typing was used to confirm the identity of LA-MRSA types found inside and outside the barns. The results show that there is regular airborne LA-MRSA transmission and deposition, which are strongly influenced by wind direction and season, of up to at least 300 m around positive pig barns. The described boot sampling method seems suitable to characterize the contamination of the vicinity of LA-MRSA-positive pig barns by the airborne route.

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.

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.

Transmission of ESBL/AmpC-producing Escherichia coli from broiler chicken farms to surrounding areas

Although previous studies have demonstrated high carriage of ESBL/AmpC-producing Escherichia coli in livestock, especially in broiler chickens, data on emission sources of these bacteria into the environment are still rare. Therefore, this study was designed to systematically investigate the occurrence of ESBL/AmpC-producing E. coli in slurry, air (inside animal houses), ambient air (outside animal houses) and on soil surfaces in the areas surrounding of seven ESBL/AmpC-positive broiler chicken fattening farms, including investigation of the possible spread of these bacteria via the faecal route and/or exhaust air into the environment. Seven German broiler fattening farms were each investigated at three points in time (3-36 h after restocking, 14-18 and 26-35 days after housing) during one fattening period. The occurrence of ESBL/AmpC genes in the investigated samples was confirmed by PCR, detecting blaCTX-M, blaSHV, blaTEM and blaCMY-genes, and, if necessary, by sequencing and/or the disc diffusion method. The results showed a wide spread of ESBL/AmpC-producing E. coli in broiler farms, as well as emissions into the surroundings. 12 out of 14 (86%) slurry samples were positive for ESBL/AmpC-producing E. coli. Additionally, 28.8% (n=23/80) of boot swabs taken from various surfaces in the areas surrounding of the farms as well as 7.5% (n=3/40) of the exhaust air samples turned out to be positive for these microorganisms. Moreover, a small proportion of air samples from inside the barns were ESBL/AmpC-positive. By comparing selected isolates using pulsed field gel electrophoresis, we proved that faecal and airborne transfer of ESBL/AmpC-producing microorganisms from broiler fattening farms to the surrounding areas is possible. Two isolates from farm G2 (slurry and boot swab 50 m downwind), two isolates from farm G3 (slurry and individual animal swab) as well as two isolates from farm G6 (air sample in the barn and air sample 50 m downwind) showed 100% similarity in PFGE analysis.

Prevalence and potential risk factors for the occurrence of cefotaxime resistant Escherichia coli in German fattening pig farms—A cross-sectional study

A cross-sectional study concerning farm prevalence and risk factors for the count of cefotaxime resistant Escherichia coli (E. coli) (CREC) positive samples per sampling group on German fattening pig farms was performed in 2011 and 2012. Altogether 48 farms in four agricultural regions in the whole of Germany were investigated. Faecal samples, boot swabs and dust samples from two sampling groups per farm were taken and supplemental data were collected using a questionnaire. On 85% of the farms, at least one sample contained cefotaxime resistant E. coli colonies. Positive samples were more frequent in faeces (61%) and boot swabs (54%) than in dust samples (11%). Relevant variables from the questionnaire were analysed in a univariable mixed effect Poisson regression model. Variables that were related to the number (risk) of positive samples per sampling group with a p-value <0.2 were entered in a multivariable model. This model was reduced to statistically significant variables via backward selection. Factors that increased the risk for positive samples involved farm management and hygienic aspects. Farms that had a separate pen for diseased pigs had a 2.8 higher mean count of positive samples (95%-CI [1.71; 4.58], p=0.001) than farms without an extra pen. The mean count was increased on farms with under-floor exhaust ventilation compared to farms with over floor ventilation (2.22 [1.43; 3.46], p=0.001) and more positive samples were observed on farms that controlled flies with toxin compared to farms that did not (1.86 [1.24; 2.78], p=0.003). It can be concluded, that CREC are wide spread on German fattening pig farms. In addition the explorative approach of the present study suggests an influence of management strategies on the occurrence of cefotaxime resistant E. coli.

Colonization Kinetics of Different Methicillin-Resistant Staphylococcus aureus Sequence Types in Pigs and Host Susceptibilities

ABSTRACT In this study we investigated the kinetics of colonization, the host susceptibility and transmissibility of methicillin-resistant Staphylococcus aureus (MRSA) after nasal treatment of pigs with three different MRSA strains of distinctive clonal lineages (sequence type 398 [ST398], ST8, and ST9), and origin in weaning piglets. The colonization dose of 5.0 × 108 CFU/animal was determined in preliminary animal studies. A total of 57 piglets were randomly divided into four test groups and one control group. Each of three test groups was inoculated intranasally with either MRSA ST8, MRSA ST9, or MRSA ST398. The fourth group was a mixture of animals inoculated with MRSA ST398 and noninoculated “sentinel” animals. Clinical signs, the nasal, conjunctival, and skin colonization of MRSA, fecal excretion, and organ distribution of MRSA, as well as different environmental samples were examined. After nasal inoculation with MRSA piglets of all four test groups showed no clinical signs of an MRSA infection. MRSA was present on the nasal mucosa, skin, and conjunctiva in all four test groups, including sentinel animals. Likewise, fecal excretion and internal colonization of MRSA ST8, ST9, and ST398 could be shown in each group. However, fecal excretion and the colonization rate of the nasal mucosa with MRSA ST9 were significantly lower in the first days after infection than in test groups infected with ST8 and ST398. The results of this study suggest differences in colonization potential of the different MRSA types in pigs. Furthermore, colonization of lymph nodes (e.g., the ileocecal lymph node) with MRSA of the clonal lineage ST398 was demonstrated.