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.

Isolation and Characterization of a Novel Aeromonas salmonicida-Infecting Studiervirinae Bacteriophage, JELG-KS1.

Representatives of the bacterial genus Aeromonas are some of the most notorious aquaculture pathogens associated with a range of diseases in different fish species. As the world forges toward the post-antibiotic era, alternative options for combating bacterial pathogens are needed. One such alternative option is phage biocontrol. In this study, a novel podophage-JELG-KS1-infecting Aeromonas salmonicida was retrieved from wastewater along with its host strain. The genome of the JELG-KS1 phage is a 40,505 bp dsDNA molecule with a GC% of 53.42% and 185 bp direct terminal repeats and encodes 53 predicted proteins. Genomic analysis indicates that JELG-KS1 might represent a novel genus within the subfamily Studiervirinae. Podophage JELG-KS1 is a strictly lytic phage without any identifiable virulence or AMR genes that quickly adsorbs onto the surface of host cells to initiate a 48 min long infectious cycle, resulting in the release of 71 ± 12 JELG-KS1 progeny virions per infected cell. JELG-KS1 effectively lyses its host population in vitro, even at very low multiplicities of infection. However, when challenged against a panel of Aeromonas spp. strains associated with diseases in aquaculture, JELG-KS1 shows host-specificity that is confined only to its isolation strain, immediately compromising its potential for Aeromonas spp. biocontrol in aquaculture.

Antimicrobial Resistance in Enterococcus spp. Isolates from Red Foxes (Vulpes vulpes) in Latvia.

Antimicrobial resistance (AMR) is an emerging public health threat and is one of the One Health priorities for humans, animals, and environmental health. Red foxes (Vulpes vulpes) are a widespread predator species with great ecological significance, and they may serve as a sentinel of antimicrobial resistance in the general environment. The present study was carried out to detect antimicrobial resistance, antimicrobial resistance genes, and genetic diversity in faecal isolates of red foxes (Vulpes vulpes). In total, 34 Enterococcus isolates, including E. faecium (n = 17), E. faecalis (n = 12), E. durans (n = 3), and E. hirae (n = 2), were isolated. Antimicrobial resistance to 12 antimicrobial agents was detected with EUVENC panels using the minimum inhibitory concentration (MIC). The presence of antimicrobial resistance genes (ARGs) was determined using whole-genome sequencing (WGS). Resistance to tetracycline (6/34), erythromycin (3/34), ciprofloxacin (2/34), tigecycline (2/34), and daptomycin (2/34) was identified in 44% (15/34) of Enterococcus isolates, while all the isolates were found to be susceptible to ampicillin, chloramphenicol, gentamicin, linezolid, teicoplanin, and vancomycin. No multi-resistant Enterococcus spp. were detected. A total of 12 ARGs were identified in Enterococcus spp., with the presence of at least 1 ARG in every isolate. The identified ARGs encoded resistance to aminoglycosides (aac(6')-I, ant(6)-Ia, aac(6')-Iih and spw), tetracyclines (tet(M), tet(L) and tet(S)), and macrolide-lincosamide-streptogramin AB (lnu(B,G), lsa(A,E), and msr(C)), and their presence was associated with phenotypical resistance. Core genome multilocus sequence typing (cgMLST) revealed the high diversity of E. faecalis and E. faecium isolates, even within the same geographical area. The distribution of resistant Enterococcus spp. in wild foxes in Latvia highlights the importance of a One Health approach in tackling AMR.

Genetic diversity and known virulence genes in Listeria innocua strains isolated from cattle abortions and farm environment.

Listeria innocua is considered as non-pathogenic bacteria living in an environment although several cases of immunocompromised humans and ruminant listeriosis infections have been reported. Previously, L. innocua was identified as a potential pathogen and virulence in association with L. monocytogenes PrfA dependent virulence (LIPI-1) gene cluster was demonstrated in hemolytic L. innocua. L. innocua usually considered non-pathogenic versus pathogenic L. monocytogenes and L. ivanovii because of the main virulence gene loss. There are limited studies and reports available about L. innocua-caused illness in cattle. A total of 18 STs were identified in cattle abortions while 17 STs in the farm environment with majority of STs were present in both abortions and environmental samples. Genome sequencing showed that in one farm identical L. innocua clones were represented in water, feed, soil, and faeces sample groups, suggesting that animals most likely through the faecal shedding may remain as the main source of L. innocua in a farm environment. Out of all L. innocua isolates PrfA-dependent virulence genes were not found in aborted foetuses isolates and environmental L. innocua isolate groups; however, in 20% of isolates a complete LIPI-3 pathogenicity island encoding listeriolysin S was identified. In this study, we demonstrated that genetically diverse L. innocua clones were widely distributed in cattle farm environment and certain isolates had a significant pathogenicity potential for cattle, thus causing adverse health effects, including abortions.

Characterization and Genetic Diversity of Listeria monocytogenes Isolated from Cattle Abortions in Latvia, 2013-2018.

Listeria monocytogenes can cause disease in humans and in a wide range of animal species, especially in farm ruminants. The aim of the study was to determine the prevalence and genetic diversity of L. monocytogenes related to 1185 cattle abortion cases in Latvia during 2013-2018. The prevalence of L. monocytogenes among cattle abortions was 16.1% (191/1185). The seasonality of L. monocytogenes abortions was observed with significantly higher occurrence (p < 0.01) in spring (March-May). In 61.0% of the cases, the affected cattle were under four years of age. L. monocytogenes abortions were observed during the third (64.6%) and second (33.3%) trimesters of gestation. Overall, 27 different sequence types (ST) were detected, and four of them, ST29 (clonal complex, CC29), ST37 (CC37), ST451 (CC11) and ST7 (CC7), covered more than half of the L. monocytogenes isolates. Key virulence factors like the prfA-dependent virulence cluster and inlA, inlB were observed in all the analyzed isolates, but lntA, inlF, inlJ, vip were associated with individual sequence types. Our results confirmed that L. monocytogenes is the most important causative agent of cattle abortions in Latvia and more than 20 different STs were observed in L. monocytogenes abortions in cattle.

Prevalence, Genetic Diversity and Factors Associated with Distribution of Listeria monocytogenes and Other Listeria spp. in Cattle Farms in Latvia.

Listeria spp. is a diverse genus of Gram-positive bacteria commonly present in the environment while L. monocytogenes and L. ivanovii are well known human and ruminant pathogens. The aim of the present study was to reveal the prevalence and genetic diversity of L. monocytogenes and other Listeria spp. and to identify the factors related to the abundance of pathogen at cattle farms. A total of 521 animal and environmental samples from 27 meat and dairy cattle farms were investigated and the genetic diversity of L. monocytogenes isolates was studied with WGS. The prevalence of Listeria was 58.9%, while of L. monocytogenes it was -11%. The highest prevalence of L. monocytogenes was found in the environment-soil samples near to manure storage (93%), mixed feed from the feeding trough and hay (29%), water samples from farms drinking trough (28%) and cattle feces (28%). Clonal complexes (CC) of CC37 (30%), CC11 (20%) and CC18 (17%) (all IIa serogroup) were predominant L. monocytogenes clones. CC18, CC37 and CC8 were isolated from case farms and CC37, CC11 and CC18 from farms without listeriosis history. Only one hypervirulent CC4 (1%) was isolated from the case farm. Sequence types (STs) were not associated with the isolation source, except for ST7, which was significantly associated with soil (p < 0.05). The contamination of soil, feeding tables and troughs with L. monocytogenes was associated with an increased prevalence of L. monocytogenes at farms. Our study indicates the importance of hygienic practice in the prevention of the dissemination of L. monocytogenes in the cattle farm environment.

Determination of methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis by MALDI-TOF MS in clinical isolates from Latvia.

Rapid identification of methicillin-resistant Staphylococcus could ensure appropriate medical care. A total of 409 Staphylococcus spp. strains were used to develop a reliable MALDI-TOF method for species identification. We tested twelve S. aureus strains to compare three different sample preparation methods and the reproducibility of the methicillin-resistant m/z 2414 ± 2 indicator peak with direct method in triplicate. A total of 65 Staphylococcus spp. strains (including 37 methicillin-resistant strains) from clinical and hospital environment isolates were used to confirm the presence of phenol-soluble modulin (PSM-mec) peptide. All 272 S. aureus strains from 409 samples were correctly identified at species level by MALDI-TOF. The samples prepared by three methods gave spectra with differences in the intensities and presence of certain peaks. The PSM-mec peak was not visible after the extraction method. The peak m/z 2414 ± 2 was only detected in 61% of the methicillin-resistant strains and in none of the methicillin-sensitive strains. The peak reproducibility for the five analyzed S. aureus strains showing the peak at m/z 2414 ± 2 was 87%. The delta-toxin was observed in 49 out of 65 samples regardless of methicillin susceptibility, as well as in all the samples exhibiting the PSM-mec peak. The peak m/z 2414 ± 2 is specific to methicillin-resistant strains carrying the mecA gene, but the absence of peak m/z 2414 ± 2 does not exclude the possibility of resistance to methicillin. Thus, implementing MALDI-TOF analysis in routine laboratory work, especially with clinical samples, would in many cases provide rapid warning about the presence of methicillin-resistant strains.

Campylobacter species prevalence, characterisation of antimicrobial resistance and analysis of whole-genome sequence of isolates from livestock and humans, Latvia, 2008 to 2016.

BackgroundCampylobacter is the main cause of bacterial gastroenteritis worldwide. The main transmission route is through consumption of food contaminated with Campylobacter species or contact with infected animals. In Latvia, the prevalence of campylobacteriosis is reported to be low (4.6 cases per 100,000 population in 2016).AimTo determine prevalence, species spectrum and antimicrobial resistance (AMR) of Campylobacter spp. in Latvia, using data from various livestock and human clinical samples.MethodsWe analysed data of Campylobacter microbiological monitoring and AMR (2008 and 2014-16) in Latvia. Data from broilers, poultry, pigs, calves and humans were used to determine prevalence of Campylobacter. Additionally, 45 different origin isolates (22 human) were sequenced on the Illumina MiSeq platform; for each isolate core genome multilocus sequence typing was used and relevant antimicrobial resistance mechanisms were identified.ResultsOverall, Campylobacter prevalence in was 83.3% in pigs, 50.2% in broilers, 16.1% in calves and 5.3% in humans; C. jejuni was the predominant species in all sources except pigs where C. coli was main species. High level of resistance in Campylobacter were observed against fluoroquinolones, tetracycline and streptomycin, in most of sequenced isolates genetic determinants of relevant AMR profiles were identified.ConclusionsIn Latvia, prevalence of Campylobacter in livestock is high, especially in pigs and broilers; prevalence in poultry and humans were lower than in other European countries. AMR analysis reveals increase of streptomycin and tetracycline resistant broiler origin C. jejuni strains. WGS demonstrates a high compliance between resistance phenotype and genotype for quinolones and tetracyclines.

Prevalence and Antimicrobial Resistance of Escherichia coli, Enterococcus spp. and the Major Foodborne Pathogens in Calves in Latvia.

The aim of the present study was to detect the prevalence and antimicrobial resistance of fecal indicators and major foodborne pathogens in feces of calves and to identify the factors associated with increased prevalence of resistant bacteria on farms. Altogether, 180 rectal swabs were collected from 18 farms in Latvia. Samples were investigated to detect the prevalence and antimicrobial resistance of Escherichia coli, Enterococcus spp., Listeria monocytogenes, Yersinia enterocolitica, Salmonella spp., Staphylococcus aureus, and Campylobacter spp. Among all, 64% (74/110) of commensal E. coli, 100% (78/78) Enterococcus faecalis and 96% (22/23) Enterococcus faecium isolates were resistant at least to one antibiotic. The prevalence of extended-spectrum ß-lactamase (ESBL)/AmpC-producing E. coli were 11.1% (20/180) with blaCTX-M, blaTEM, and blaCMY genes identified. Campylobacter jejuni (12.8%, 23/180) and Campylobacter coli (2.8%, 5/180) were the most resistant to tetracycline (61%, 14/23; 100%, 5/5) and fluoroquinolones (61%, 14/23; 100%, 5/5). Prevalence of L. monocytogenes was 0.6% (1/180) and S. aureus 1.7% (3/180). All samples were Salmonella and Y. enterocolitica negative. Farm size, bought calves, contact with other calves, and antimicrobial treatment of cows were associated with increased prevalence of resistant E. coli and Enterococcus spp. Despite the low overall usage of antimicrobials in Latvia, the high rates of antimicrobial resistance in fecal indicators and Campylobacter, in addition to the high prevalence of ESBL-producing E. coli, highlights the necessity for the prudent use of antimicrobials in dairy farms in Latvia.

Seroprevalence of Brucella suis in eastern Latvian wild boars (Sus scrofa).

Brucellosis due to Brucella suis biovar 2 is one of the most important endemic diseases in wild boar (Sus scrofa) populations in Europe. The aim of the present study was to determine the seroprevalence of brucellosis in wild boars in the eastern part of Latvia. Wild boars killed by hunters in the period from January to April 2015 (n = 877) and from March to April in 2016 (n = 167) were examined for antibodies against B. suis by the Rose Bengal test (RBT), a complement fixation test (CFT), and by enzyme-linked immunosorbent assays. In 2015, 199 samples (22.7%) were positive by RBT and/or CFT while 36 samples (21.6%) were seropositive in 2016. Of the Brucella seropositive samples from 2015 and 2016 (n = 235), 162 (68.9%) were also seropositive to Yersinia enterocolitica. Considering cross-reactivity of serological tests, the seroprevalence of B. suis biovar 2 exposure in wild boars in the eastern part of Latvia was calculated to 14.0% in 2015 and 9.6% in 2016. From selected seropositive samples (42 in 2015 and 36 in 2016) total DNA was extracted and analyzed with an IS711-based nested polymerase chain reaction (PCR) assay. Species and biovar identification was conducted for bacteria isolated in monoculture from PCR positive samples by species specific primers and Bruce-ladder multiplex PCR. Brucella suis biovar 2 was isolated from 12/20 samples in 2015 and 9/9 samples in 2016. The average seroprevalence was relatively low compared to that found in certain other European countries. Males and females had an equal level of seropositivity, but a positive age-trend was observed for both males and females.

High occurrence rates of enrofloxacin and ciprofloxacin residues in retail poultry meat revealed by an ultra-sensitive mass-spectrometric method, and antimicrobial resistance to fluoroquinolones in Campylobacter spp.

An ultra-sensitive mass spectrometric confirmation and quantification method for the determination of selected fluoroquinolones-enrofloxacin and its main metabolite ciprofloxacin-was developed and validated in poultry meat samples. The achieved limits of quantification were 1 ng kg-1 for enrofloxacin and 10 ng kg-1 for ciprofloxacin. The analysis of 40 retail poultry samples originating from Estonia, Latvia, Lithuania, Poland and France revealed that 93% of samples contained residues of enrofloxacin in the range from 3.3 to 1126 ng kg-1. Previous studies have shown high levels of antimicrobial resistance to fluoroquinolones, particularly in Campylobacter spp. and various faecal indicators isolated from broiler meat. Consequently, the revealed widespread usage of fluoroquinolones in the poultry industry may result in the further emergence of antimicrobial resistance of Campylobacter in the food chain.