Whole-Genome Sequencing-Based Profiling of Antimicrobial Resistance Genes and Core-Genome Multilocus Sequence Typing of Campylobacter jejuni from Different Sources in Lithuania.

Campylobacter jejuni is known as one of the main causative agents of gastroenteritis in humans worldwide, and the rise of antimicrobial resistance (AMR) in Campylobacter is a growing public health challenge of special concern. Whole-genome sequencing (WGS) was used to characterize genetic determinants of AMR in 53 C. jejuni isolates from dairy cattle, broiler products, wild birds, and humans in Lithuania. The WGS-based study revealed 26 C. jejuni AMR markers that conferred resistance to various antimicrobials. Genetic markers associated with resistance to beta-lactamases, tetracycline, and aminoglycosides were found in 79.3%, 28.3%, and 9.4% of C. jejuni isolates, respectively. Additionally, genetic markers associated with multidrug resistance (MDR) were found in 90.6% of C. jejuni isolates. The WGS data analysis revealed that a common mutation in the quinolone resistance-determining region (QRDR) was R285K (854G > A) at 86.8%, followed by A312T (934G > A) at 83% and T86I (257C > T) at 71.7%. The phenotypic resistance analysis performed with the agar dilution method revealed that ciprofloxacin (CIP) (90.6%), ceftriaxone (CRO) (67.9%), and tetracycline (TET) (45.3%) were the predominant AMR patterns. MDR was detected in 41.5% (22/53) of the isolates tested. Fifty-seven virulence genes were identified in all C. jejuni isolates; most of these genes were associated with motility (n = 28) and chemotaxis (n = 10). Additionally, all C. jejuni isolates harbored virulence genes related to adhesion, invasion, LOS, LPS, CPS, transportation, and CDT. In total, 16 sequence types (STs) and 11 clonal complexes (CC) were identified based on core-genome MLST (cgMLST) analysis. The data analysis revealed distinct diversity depending on phenotypic and genotypic antimicrobial resistance of C. jejuni.

Whole Genome Sequence-Based Prediction of Resistance Determinants in High-Level Multidrug-Resistant Campylobacter jejuni Isolates in Lithuania.

Spread of antibiotic resistance via mobile genetic elements associates with transfer of genes providing resistance against multiple antibiotics. Use of various comparative genomics analysis techniques enables to find intrinsic and acquired genes associated with phenotypic antimicrobial resistance (AMR) in Campylobacter jejuni genome sequences with exceptionally high-level multidrug resistance. In this study, we used whole genome sequences of seven C. jejuni to identify isolate-specific genomic features associated with resistance and virulence determinants and their role in multidrug resistance (MDR). All isolates were phenotypically highly resistant to tetracycline, ciprofloxacin, and ceftriaxone (MIC range from 64 to ≥256 µg/mL). Besides, two C. jejuni isolates were resistant to gentamicin, and one was resistant to erythromycin. The extensive drug-resistance profiles were confirmed for the two C. jejuni isolates assigned to ST-4447 (CC179). The most occurring genetic antimicrobial-resistance determinants were tetO, beta-lactamase, and multidrug efflux pumps. In this study, mobile genetic elements (MGEs) were detected in genomic islands carrying genes that confer resistance to MDR, underline their importance for disseminating antibiotic resistance in C. jejuni. The genomic approach showed a diverse distribution of virulence markers, including both plasmids and phage sequences that serve as horizontal gene transfer tools. The study findings describe in silico prediction of AMR and virulence genetics determinants combined with phenotypic AMR detection in multidrug-resistant C. jejuni isolates from Lithuania.

Draft Genome Sequence of Ciprofloxacin and Ceftriaxone Resistant Campylobacter jejuni MM26-781 Assigned to Novel ST Isolated From Common Pigeon in Lithuania.

Campylobacter jejuni is an important zoonotic pathogen known to be resistant to a wide range of antibiotics worldwide. Campylobacter jejuni may be intrinsically resistant to antibiotics or can acquire antibiotic resistance determinants through gene transfer. However, the knowledge of molecular mechanisms of antimicrobial resistance among Campylobacter isolates from wild birds, especially in Lithuania, is limited. Whole genome sequencing (WGS) is a tool for better understanding the evolutionary and epidemiologic dynamics of C jejuni. This study describes a draft whole genome sequence of C jejuni MM26-781 isolated from a common pigeon (Columba livia) in Lithuania in 2011 and assigned to ST-6424 (CC179) sequence type. The draft genome sequence contained 1.68 Mb, comprising 1651 coding genes, 40 transfer RNAs, 1 ribosomal RNA, and 69 pseudogenes with an average G + C content of 30.4%. The RAST (Rapid Annotation using Subsystem Technology) pipeline annotated (NCTC11168) a total of 305 subsystems in the genome of C jejuni MM26-781 strain, with most of the genes associated with amino acids and derivatives related to metabolism (18.93%) and protein metabolism (14.43%). The genes and mutations related to antibiotic resistance, including gyrA and gyrB genes associated with quinolone resistance, blaOXA-448 gene (locus tag C9371_07715) associated with resistance to ß-lactams, rpoB gene associated with resistance to rifamycin, vgaE gene associated with resistance to streptogramin and efflux system CmeABC (cmeA, cmeB, cmeC), efflux pump PmrA, and transcriptional regulator CmeR responsible for multidrug resistance in C jejuni MM26-781 chromosome, were identified. Also, the virulence factors, including ciaB, cadF, ceuE, pldA, motB, and bd1A genes, were identified by WGS data analysis.

Overlap of Antibiotic Resistant Campylobacter jejuni MLST Genotypes Isolated From Humans, Broiler Products, Dairy Cattle and Wild Birds in Lithuania.

Antimicrobial resistance was determined for 341 thermophilic Campylobacter jejuni isolates obtained from human clinical cases (n = 101), broiler products (n = 98), dairy cattle (n = 41) and wild birds (n = 101) with known multilocus sequence types (MLST) in Lithuania. The minimum inhibitory concentration (MIC) values for ciprofloxacin, tetracycline, gentamicin, ceftriaxone and erythromycin were determined with the agar dilution method. MIC values were compared with MLST types to find possible associations among isolation source, sequence type and resistance to antibiotics. The proportions of resistant strains were 94.2% (human), 95% (wild birds), 100% (broiler products) and 100% (dairy cattle) for one of the tested antibiotics. Most frequently, resistance to ciprofloxacin was observed (91.5%), followed by ceftriaxone with 60.4%, and tetracycline (37.8%). However only three C. jejuni strains were resistant to erythromycin (0.9%) and all tested thermophilic Campylobacter strains were sensitive to gentamicin. Most of the examined C. jejuni isolates (80.6%) showed resistance to at least one of three profiles: CIP+AXO (28.1%), TET+CIP+AXO (26.7%) and CIP (25.8%). Statistically significant differences in resistance to tetracycline were found between C. jejuni strains obtained from cattle (85.4%) and broiler products (64.3%) (P < 0.05). The majority (87.1%) of the tested strains from wild birds were resistant to ciprofloxacin (P < 0.05). The results showed that strains of novel ST's showed significantly lower resistance to ceftriaxone (P < 0.05). The ST-21 (CC21) (78.8%) was identified with significantly higher multidrug resistance relatively to other tested ST's in this study. Our results emphasize the high antimicrobial resistance of phylogenetically diverse C. jejuni strains isolated from different sources including specific genotypes of wild bird's strains in Lithuania. The results support the opinion that not only broiler products but cattle and wild birds may be a reservoir of resistant C. jejuni and stipulate a risk of spread or resistant bacteria. There is increasing need for broad surveillance and control measures to track changes and pathways of antimicrobial resistance of C. jejuni in epidemiologically distinct populations.

Prevalence of Foodborne Pathogens in Freshwater Fish in Latvia.

The aim of this study was to detect the prevalence of Salmonella spp., Listeria monocytogenes, and Yersinia enterocolitica in freshwater fish in Latvia. In total, 235 samples, including freshly caught fish from fives lakes (n = 129) and fish from retail markets (n = 106), were collected from April 2014 to December 2014 in Latvia. Samples were tested according to International Organization for Standardization methods. No Salmonella spp. were found in fresh fish from lakes or in commercially available fish. In contrast, the overall prevalence of L. monocytogenes and Y. enterocolitica in freshwater fish was 13% (30 of 235) and 14% (34 of 235), respectively, and no significant difference between the prevalence of L. monocytogenes and Y. enterocolitica was observed (P > 0.05). All Y. enterocolitica isolates belonged to the nonpathogenic 1A biotype. Molecular serotyping of L. monocytogenes revealed that the most distributed serogroup was 1/2a-3a (65%), followed by 1/2c-3c (25%), 1/2b-3b (5%), and 4b, 4d, 4e (5%). The prevalence of L. monocytogenes and Y. enterocolitica in freshwater lake fish was 2% (2 of 129) and 3% (4 of 129), respectively. In contrast, the prevalence of L. monocytogenes and Y. enterocolitica in fish at retail markets was 26% (28 of 106) and 28% (30 of 106), respectively. In retail samples, 9 of 58 positive fish contained both L. monocytogenes and Y. enterocolitica. In general, differences in the prevalences of L. monocytogenes and Y. enterocolitica in retail samples were significantly higher than those in freshly caught fish (P < 0.05). The results of this study indicate that freshwater fish could be an important source of Y. enterocolitica and L. monocytogenes for consumers in Latvia.

High Prevalence and Genetic Diversity of Campylobacter jejuni in Wild Crows and Pigeons.

The occurrence, seasonal variation and genetic diversity of Campylobacter spp. in pigeons and crows over a 1-year period were evaluated. Campylobacter spp. were isolated from 166 (34.6 %) out of 480 wild bird faecal samples. The occurrence of Campylobacter spp. in faecal samples was higher among crows (39.2 %) than pigeons (30.0 %), (P < 0.05). Campylobacter jejuni was the most common species detected among wild bird faecal samples (98.2 %). Meanwhile, Campylobacter coli prevalence in wild bird faecal samples was low-6 %. The Simpson's diversity index of C. jejuni flaA RFLP types was lower in pigeons (D = 0.88) compared with C. jejuni isolates detected in crows (D = 0.97). Obtained results revealed that C. jejuni are widely prevalent among crows and pigeons, indicating these wild birds as potential infection sources to humans. Further studies are required to determine crows and pigeons role in zoonotic transmission of Campylobacter.

The antimicrobial effect of spice-based marinades against Campylobacter jejuni on contaminated fresh broiler wings.

The antimicrobial effect of spice-based marinades against Campylobacter jejuni on inoculated fresh broiler wings was investigated. Experiments were carried out with 1 strain of C. jejuni and 6 marinades. Four experimental marinades were composed for the study and contained spices (thyme, rosemary, basil, marjoram, and so on) and different combination of bioactive compounds. Two marinades were commercial and contained spices (black pepper, sweet red pepper, and so on) and chemical additives (monosodium glutamate, sodium diacetate, calcium lactate), 1 commercial marinade was also enriched with bioactive compounds (linalool, cinnamaldehyde, lactic acid). Total aerobic bacterial count was examined to estimate the possible effect of tested marinades on the shelf-life of marinated broiler wings. Study revealed that thyme-based marinade was the most effective against C. jejuni on broiler wings and reduced the numbers of campylobacters by 1.04 log colony forming unit (CFU)/g (P ≤ 0.05) during storage for 168 h at 4 °C temperature. Moreover, it was more effective against C. jejuni than commercial marinade with 0.47 log CFU/g (P ≤ 0.05) reduction effect. Both experimental and commercial marinades had very similar effect on the total aerobic bacterial count. Although experimental and commercial marinades had different effect on pH of broiler wings, this parameter did not show a major impact on the antimicrobial effect of tested marinades (P ≥ 0.05). Our study shows that experimental natural thyme-based marinade can reduce numbers of C. jejuni more effectively than tested commercial marinades.

Prevalence and genetic diversity of enteropathogenic Yersinia spp. in pigs at farms and slaughter in Lithuania.

The prevalence of enteropathogenic Yersinia spp. in pigs at farms and slaughter in relation to potential farming risk factors in Lithuania was examined. Pig faeces and carcase swab samples from 11 farms were studied at slaughterhouses. Nine of the 11 farms were visited again 3-5 months later, and pooled feacal samples and environmental samples were collected. Pathogenic Yersinia enterocolitica was found in 64% and Yersinia pseudotuberculosis in 45% of the sampled pig farms. All obtained isolates belonged to bioserotypes 4/O:3 and 2/O:3, respectively. Low biosecurity level was associated with a high prevalence of Y. enterocolitica on farms. Characterization with PFGE of 64 Y. enterocolitica and 27 Y. pseudotuberculosis isolates revealed seven and two different genotypes, respectively. Dominant enteropathogenic Yersinia spp. genotypes were obtained in both pig feacal and carcase samples. The high contamination of pig carcases (25%) with enteropathogenic Yersinia spp. may be an important factor contributing to the high incidence of human yersiniosis in Lithuania.

Genetic diversity and antimicrobial resistance of Yersinia enterocolitica isolated from pigs and humans in Lithuania.

BACKGROUND: Yersiniosis is one of the three leading foodborne zoonoses in Lithuania, and the incidence of 12.86 per 100,000 population was the highest among EU member states in 2010. Contaminated pig carcasses and subsequently undercooked pig meat are considered to be the primary transmission vehicle of enteropathogenic Y. enterocolitica to consumers. With the aim of evaluating pigs as a possible source of human yersiniosis in Lithuania, this study investigated the genetic diversity of Y. enterocolitica isolated from pigs and human cases of yersiniosis. In addition, the antimicrobial resistance of selected isolates from both sources was compared. RESULTS: In total, 83 Y. enterocolitica strains were characterised using pulsed field gel electrophoresis. Overall, 68% of Y. enterocolitica 4/O:3 pulsotypes found in human clinical samples were identical to 81% of pulsotypes found in the pig production chain. Yersinia enterocolitica pulsotype II was confirmed as the dominant pulsotype in the pig production chain and was identical to nine of 19 Y. enterocolitica strains found in humans. All tested Y. enterocolitica 4/O:3 strains were resistant to ampicillin and erythromycin and sensitive to ciprofloxacin. Of the strains studied, 5% were resistant to tetracycline and streptomycin. CONCLUSION: This study showed that pigs may be the main source of human yersiniosis in Lithuania. In addition, Y. enterocolitica 4/O:3 strains isolated from the pig production chain and from yersiniosis patients shared similar resistance to different antimicrobials.