Magdalena Zając

The global establishment of a highly-fluoroquinolone resistant Salmonella enterica serotype Kentucky ST198 strain

While the spread of Salmonella enterica serotype Kentucky resistant to ciprofloxacin across Africa and the Middle-East has been described recently, the presence of this strain in humans, food, various animal species (livestock, pets, and wildlife) and in environment is suspected in other countries of different continents. Here, we report results of an in-depth molecular epidemiological study on a global human and non-human collection of S. Kentucky (n = 70). We performed XbaI-pulsed field gel electrophoresis and multilocus sequence typing, assessed mutations in the quinolone resistance-determining regions, detected β-lactam resistance mechanisms, and screened the presence of the Salmonella genomic island 1 (SGI1). In this study, we highlight the rapid and extensive worldwide dissemination of the ciprofloxacin-resistant S. Kentucky ST198-X1-SGI1 strain since the mid-2000s in an increasingly large number of contaminated sources, including the environment. This strain has accumulated an increasing number of chromosomal and plasmid resistance determinants and has been identified in the Indian subcontinent, Southeast Asia and Europe since 2010. The second substitution at position 87 in GyrA (replacing the amino acid Asp) appeared helpful for epidemiological studies to track the origin of contamination. This global study provides evidence leading to the conclusion that high-level resistance to ciprofloxacin in S. Kentucky is a simple microbiological trait that facilitates the identification of the epidemic clone of interest, ST198-X1-SGI1. Taking this into account is essential in order to detect and monitor it easily and to take rapid measures in livestock to ensure control of this infection.

Antimicrobial resistance in commensal Escherichia coli isolated from animals at slaughter

Monitoring of antimicrobial resistance in commensal Escherichia coli (N = 3430) isolated from slaughtered broilers, laying hens, turkeys, swine, and cattle in Poland has been run between 2009 and 2012. Based on minimal inhibitory concentration (MIC) microbiological resistance to each of 14 tested antimicrobials was found reaching the highest values for tetracycline (43.3%), ampicillin (42.3%), and ciprofloxacin (39.0%) whereas the lowest for colistin (0.9%), cephalosporins (3.6 ÷ 3.8%), and florfenicol (3.8%). The highest prevalence of resistance was noted in broiler and turkey isolates, whereas it was rare in cattle. That finding along with resistance patterns specific to isolation source might reflect antimicrobial consumption, usage preferences or management practices in specific animals. Regression analysis has identified changes in prevalence of microbiological resistance and shifts of MIC values. Critically important fluoroquinolone resistance was worrisome in poultry isolates, but did not change over the study period. The difference (4.7%) between resistance to ciprofloxacin and nalidixic acid indicated the scale of plasmid-mediated quinolone resistance. Cephalosporin resistance were found in less than 3.8% of the isolates but an increasing trends were observed in poultry and MIC shift in the ones from cattle. Gentamycin resistance was also increasing in E. coli of turkey and cattle origin although prevalence of streptomycin resistance in laying hens decreased considerably. Simultaneously, decreasing MIC for phenicols observed in cattle and layers isolates as well as tetracycline values in E. coli from laying hens prove that antimicrobial resistance is multivariable phenomenon not only directly related to antimicrobial usage. Further studies should elucidate the scope of commensal E. coli as reservoirs of resistance genes, their spread and possible threats for human and animal health.

High-level fluoroquinolone resistant Salmonella enterica serovar Kentucky ST198 epidemic clone with IncA/C conjugative plasmid carrying blaCTX-M-25 gene

Multidrug resistant Salmonella Kentucky strains have been isolated from turkeys in Poland since 2009. Multiple mutations within chromosomal genes gyrA and parC were responsible for high-level ciprofloxacin resistance. One of the isolates was extended spectrum β-lactamase- (ESBL) positive: the strain 1643/2010 carried a conjugative 167,779 bps plasmid of IncA/C family. The sequence analysis revealed that it carried a blaCTX-M-25 gene and an integron with another β-lactamase encoding gene-blaOXA-21. This is the first known report of a CTX-M-25 encoding gene both in Poland and in Salmonella Kentucky world-wide, as well as in the IncA/C plasmid. Analysis of the integron showed a novel arrangement of gene cassettes-aacA4, aacC-A1 and blaOXA-21 where the latter might result from an intergeneric gene transfer. The study confirmed Salmonella Kentucky population isolated in Poland belongs to global epidemics of high level fluoroquinolone resistant clone ST198 that can carry rare β-lactamase genes.

Genetic lineages of Salmonella enterica serovar Kentucky spreading in pet reptiles

The purpose of the study was to define genetic diversity of reptilian Salmonella enterica serovar (S.) Kentucky isolates and their epidemiological relations to the ones from poultry, food, and environmental origin in Poland. Between 2010 and 2012 twenty-four S. Kentucky isolates derived from snakes (N=8), geckos (N=7), chameleons (N=4), agamas (N=1), lizard (N=1), and environmental swabs taken from reptile exhibition (N=3) were identified. They were characterized with antimicrobial minimal inhibitory concentration testing, XbaI-PFGE and MLST typing. The profiles compared to S. Kentucky available in BioNumerics local laboratory database (N=40) showed 67.3% of relatedness among reptile isolates. Three genetic lineages were defined. The first lineage gathered 20 reptile isolates with 83.4% of similarity and wild-type MICs for all antimicrobials tested but streptomycin in single case. The remaining three reptilian and one post-exhibition environment S. Kentucky isolates were clustered (87.2%) with isolates originating from poultry, mainly turkey, food, and environment and presented variable non-wild type MICs to numerous antimicrobials. The third S. Kentucky lineage was composed of two isolates from feed (96.3%). The results suggest diverse sources and independent routes of infection. Most of the isolates belonged to reptile-associated clones spread both horizontally and vertically. Simultaneously, PFGE profiles and MLST type indistinguishable from the ones observed in poultry point out carnivore reptiles as possible vector of infection with multidrug and high-level ciprofloxacin resistant (MIC≥8 mg/L) S. Kentucky. Public awareness and education are required to prevent potential reptile-associated S. Kentucky infections in humans.

Fifteen years of successful spread of Salmonella enterica serovar Mbandaka clone ST413 in Poland and its public health consequences

In the 1990s, Salmonella enterica serovar (S.) Mbandaka occurred in feed and poultry in Poland. In the following years, the serovar also gained epidemiological importance in other EU countries. The objectives of current study were to evaluate the genetic relationship of contemporary S. Mbandaka with isolates originating from the beginning of the epidemics, and to assess the contribution of poultry as the source of infections in humans. Seventy S. Mbandaka isolated mainly in 2009 - 2010 from humans, poultry, food, and feed were typed with API ID32 (®), MIC, plasmid profiling, PFGE, and MLST. PCR and sequencing were used to identify plasmid mediated quinolone and cephalosporin resistance mechanisms. Six biochemical profiles were identified and 59 of S. Mbandaka proved to be susceptible to the applied antimicrobials. Eight strains carried plasmids and a few of them were positive for blaCMY-2 and qnrS1 genes. Two clusters of 15 XbaI-PFGE profiles with similarity of 77.5% were found. The first cluster, gathered 7 profiles involving historical isolates and several contemporary non-human S. Mbandaka. The predominant profile in the second cluster consisted of 28 human and 1 broiler isolate. MLST analysis showed sequence type ST413 occurring among all tested isolates. The identification of close genetic relationships between S. Mbandaka of human and poultry origin indicates animals as a primal human infection route. Despite Salmonella control programmes, the S. Mbandaka ST413 clone has been circulating for several years in Poland. Salmonella control polices in food production chain should be continuously updated to target serovars of major epidemiological importance. Resistance noted in S. Mbandaka to such antimicrobials as fluoroquinolones and cephalosporins may hinder public health.

Mechanisms of cephalosporin resistance in indicator Escherichia coli isolated from food animals

Resistance to β-lactams is considered one of the major global problems and recently it became the most frequently studied topic in the area of antimicrobial resistance. The study was focused on phenotypic and genetic characterisation of commensal Escherichia coli (E. coli), including those producing cephalosporinases, isolated from gut flora of healthy slaughter animals. E. coli were cultured simultaneously on MacConkey agar (MCA) and cefotaxime supplemented MCA. The isolates were confirmed with ONPG and indol tube tests as well as PCR targeting uspA gene. Microbroth dilution method was applied for determination of Minimal Inhibitory Concentrations and interpreted according to EUCAST epidemiological cut-off values. Cephalosporin resistance phenotypes were defined by E-tests (BioMerieux) and relevant gene amplicons from selected strains were sequenced. A total of 298 E. coli isolates with cephalosporin resistance (ESC) found in 99 ones, were obtained from 318 cloacal or rectal swabs deriving from broilers, layers, turkeys, pigs and cattle. Both extended spectrum β-lactamase (ESBL) and ampC-cephalosporinase resistance phenotypes were noted in all tested animal species but cattle. At least one of the analysed genes was identified in 90 out of 99 cephalosporin-resistant isolates: blaTEM (n=44), blaCMY (n=38), blaCTX-M (n=33) and blaSHV (n=12). None of the phenotypes was identified in nine isolates. Sequencing of PCR products showed occurrence of ESBL-genes: blaCTX-M-1/-61, blaSHV-12, blaTEM-1,-52/-92,-135 and ampC-gene blaCMY-2. They were located on numerous and diverse plasmids and resistance transferability was proved by electroporation of blaSHV-12 and blaCTX-M-1/-61 located on X1 plasmids. Detection of cephalosporin resistant E. coli confirms the existence of resistance genes reservoir in farm animals and their possible spread (i.e. via IncX1 plasmids) to other bacteria including human and animal pathogens. The identified genetic background indicates on ecological aspects of selection and dissemination of cephalosporin resistance in E. coli isolated from food-producing animals rather than its potential role for public health threats.

Molecular Epidemiology of Salmonella Enterica Serovar Saintpaul Isolated from Animals, Food, and Humans in 12 European Countries

Abstract The molecular epidemiological relationship among isolates of Salmonella enterica serovar (S.) Saintpaul, which was identified in animals, food, and humans in several EU countries, was investigated. Pulsed-field gel electrophoresis (PFGE) revealed a high degree of genetic diversity (82 XbaI PFGE profiles with 42.6% similarity) among 159 S. Saintpaul isolates from animals (n=91), food of animal origin (n=29), and humans (n=36) in 12 European countries during 2005 to 2009. Most frequent profiles (n=12) comprised almost 50% of the tested isolates. Profiles obtained in isolates from a single source within a particular geographical region or particular period of time were indistinguishable or closely related. Turkeys were confirmed as the major reservoir for S. Saintpaul. Indistinguishable PFGE profiles were identified in up to 19 isolates from turkey breeding and fattening flocks and food over the study period. Other animals, including food and pets, may also contribute to S. Saintpaul spread. International trade of animals and food, as well as travelling contributes to the spread of a specific clone to different geographical areas. Although control programmes in breeding turkey flocks, together with improved biosecurity, may interrupt the major transmission routes, it was concluded that S. Saintpaul will continue to represent a potential threat to human health.

Distribution of Salmonella serovars along the food chain in Poland, 2010–2015

Abstract Introduction: Data collection on the Salmonella occurrence is crucial in effective implementation of different actions or control programmes aiming to protect consumers’ health and to reduce the level of Salmonella prevalence in farm animals. The goal was to describe Salmonella serovar distribution along the food chain in Poland during 2010–2015 and to identify their epidemiological importance. Material and Methods: Slide agglutination according to White-Kauffmann-Le Minor scheme was used to identify Salmonella serovars of 6,928 isolates originating from animals, food, feeds, and fertilisers. Results: In total, 160 Salmonella serovars were identified. Differences in serovar distribution were observed depending on animal species. Among isolates from hens, S. Enteritidis and S. Infantis were the most prevalent. Serovar pattern in turkeys differed from those in hens, with S. Kentucky, S. Newport, S. Saintpaul being the most prevalent. Monophasic S. Typhimurium was predominant in pigs. Serovars found in food reflected those observed among livestock animals. Nine out of the ten most prevalent serovars in animals and humans were also found in organic fertilisers. Conclusion: Serotyping of large number of isolates from different sources is essential for insight on emerging serovars and trends of Salmonella occurrence. This may increase the value of epidemiological data and result in updating of Salmonella control programmes to target further epidemiologically important serovars in animals and better protection of consumers’ health.