Jacek Osek

Antimicrobial resistance mechanisms among Campylobacter.

Campylobacter jejuni and Campylobacter coli are recognized as the most common causative agents of bacterial gastroenteritis in the world. Humans most often become infected by ingesting contaminated food, especially undercooked chicken, but also other sources of bacteria have been described. Campylobacteriosis is normally a self-limiting disease. Antimicrobial treatment is needed only in patients with more severe disease and in those who are immunologically compromised. The most common antimicrobial agents used in the treatment of Campylobacter infections are macrolides, such as erythromycin, and fluoroquinolones, such as ciprofloxacin. Tetracyclines have been suggested as an alternative choice in the treatment of clinical campylobacteriosis but in practice are not often used. However, during the past few decades an increasing number of resistant Campylobacter isolates have developed resistance to fluoroquinolones and other antimicrobials such as macrolides, aminoglycosides, and beta-lactams. Trends in antimicrobial resistance have shown a clear correlation between use of antibiotics in the veterinary medicine and animal production and resistant isolates of Campylobacter in humans. In this review, the patterns of emerging resistance to the antimicrobial agents useful in treatment of the disease are presented and the mechanisms of resistance to these drugs in Campylobacter are discussed.

Prevalence, Characterization, and Antimicrobial Resistance of Listeria monocytogenes Isolates from Bovine Hides and Carcasses

ABSTRACT Listeria monocytogenes isolates from bovine hides and carcasses (n = 812) were mainly of serogroup 1/2a. All strains were positive for internalin genes. Several isolates were resistant to oxacillin (72.2%) or clindamycin (37.0%). These findings indicate that L. monocytogenes of beef origin can be considered a public health concern.

Determination of K88 antigens and enterotoxins of Escherichia coli strains isolated from Polish piglets with diarrhea by the use of enzyme-linked immunosorbent assays.

We studied 103 Escherichia coli strains isolated from suckling and weaned piglets with diarrhea using different ELISA tests. K88 fimbrial antigen was determined by the slide agglutination test and the ELISA inhibition method. LT and STa enterotoxins were tested directly in the microtiter plates using monoclonal antibodies. It was found that 56.3% strains possessed K88 antigen, all of which were of the K88ac type. There was 100% correlation between the slide agglutination and ELISA tests. Of the 103 strains tested 68.9% produced LT or STa or both toxins. LT-positive strains were the most common ones in both groups of piglets. All K88-positive strains were enterotoxigenic and elaborated LT (56 strains) or LT and STa (2 strains); STb production was not determined in this study. Our ELISA tests were easy to perform, specific and can be used for determination of K88 and enterotoxins in E. coli strains isolated from piglets.

Prevalence and antimicrobial resistance of Campylobacter spp. isolated from poultry carcasses in Poland.

The purpose of the present study was to determine the prevalence of Campylobacter in poultry carcasses at slaughter in Poland. For the isolated strains, resistance to selected antibiotics and the associated genetic determinants were identified. A total of 498 Campylobacter isolates were obtained from 802 poultry samples during the 2-year study period. Strains were identified to species with the PCR method; 53.6% of the strains were Campylobacter jejuni and 46.4% were Campylobacter coli. A high percentage of the tested Campylobacter strains were resistant to ciprofloxacin and nalidixic acid (74.1 and 73.5%, respectively) followed by tetracycline (47.4%) and streptomycin (20.5%). Only one C. jejuni and two C. coli isolates were resistant to gentamicin. Seventy-nine (15.9%) of the 498 strains were resistant to three or more classes of antibiotics examined. Higher levels of resistance, irrespective of the antimicrobial agent tested, were found within the C. coli group. Almost all strains resistant to quinolones (99.5%) and to tetracycline (99.6%) carried the Thr-86-to-Ile mutation in the gyrA gene and possessed the tet(O) marker, respectively. All isolates resistant to erythromycin had the A2075G mutation in the 23S rRNA gene. These results reveal that poultry carcasses in Poland are a reservoir of potentially pathogenic and antimicrobial-resistant Campylobacter strains for humans, which may pose a public health risk.

Prevalence and Antimicrobial Resistance of Vibrio parahaemolyticus Isolated from Raw Shellfish in Poland.

Vibrio parahaemolyticus is a marine bacterium recognized as an important cause of gastroenteritis in humans consuming contaminated shellfish. In recent years, increasing resistance to ampicillin and aminoglycosides has been observed among V. parahaemolyticus isolates. However, the first-line antimicrobials such as tetracyclines and fluoroquinolones remained highly effective against these bacteria. The aim of this study was to evaluate the occurrence of V. parahaemolyticus in live bivalve molluscs available on the Polish market and to determine the antimicrobial resistance of the recovered isolates. A total of 400 shellfish samples (mussels, oysters, clams, and scallops) from 2009 to 2012 were tested using the International Organization for Standardization standard 21872-1 method and PCR for the species-specific toxR gene. Antimicrobial susceptibility of the isolates was determined using a microbroth dilution method. V. parahaemolyticus was identified in 70 (17.5%) of the 400 samples, and the toxR gene was confirmed in 64 (91.4%) of these isolates. Most of the isolates were recovered from clams (31 isolates; 48.4% prevalence) followed by mussels (17 isolates; 26.6% prevalence). More V. parahaemolyticus-positive samples were found between May and September (22.7% prevalence) than between October and April (11.4% prevalence). Antibiotic profiling revealed that most isolates were resistant to ampicillin (56 isolates; 87.5%) and to streptomycin (45 isolates; 70.3%), but all of them were susceptible to tetracycline and chloramphenicol. Forty-one isolates (64.1%) were resistant to two or more antimicrobials; however, only one isolate (1.6%) was resistant to three antimicrobial classes. The antimicrobials used in treatment of human V. parahaemolyticus infection had high efficacy against the bacterial isolates tested. This study is the first concerning antibiotic resistance of V. parahaemolyticus isolates in Poland, and the results obtained indicate that these bacteria may pose a health risk to consumers.

Occurrence of Staphylococcus aureus on Farms with Small Scale Production of Raw Milk Cheeses in Poland.

This paper describes the results of a 3-year study on the prevalence, enterotoxinogenicity and resistance to antimicrobials of S. aureus isolated on dairy farms with small scale production of raw cow milk cheeses. The samples of raw milk, semi-finished products and the final products as well as swabs were collected between 2011 and 2013 from nine dairy farms in Poland. A total of 244 samples were examined, of which 122 (50.0%) were contaminated with S. aureus including 18 of 26 (69.2%) mature cheese samples with log10 CFU g−1 between <1- and 7.41. In swabs collected from the staff and production environment the highest contamination rate with coagulase positive staphylococci (CPS) was detected on hands of cheese makers (4.34 log10 CFU/swab). None of the cheese samples contaminated with CPS contained staphylococcal enterotoxins (SEs). However, 55 of 122 (45.1%) S. aureus isolates possessed SEs genes, mainly (26 of 55; 47.3%) a combination of the sed, sej and ser genes. Furthermore, the sep (15 of 55; 27.3%) as well as seg and sei (9 of 55; 16.4%) genes were also identified. The remaining S. aureus isolates possessed the sea gene (one isolate), the combination of sec, seg and sei (three isolates) as well as the sed, sej, sep and ser markers together (one CPS). Resistance to penicillin (62 of 122 isolates; 50.8%) was the most common among the tested isolates. Some CPS were also resistant to chloramphenicol (7; 5.7%) and tetracycline (5; 4.1%). The obtained results indicated that the analyzed cheeses were safe for consumers. To improve the microbiological quality of traditional cheese products more attention should be paid to animal welfare and hygiene practices during the process of cheese manufacturing in some dairy farms.

Characterization and Antimicrobial Resistance of Listeria monocytogenes Isolated from Retail Beef Meat in Poland

Four hundred seventeen retail beef meat samples purchased in the eastern part of Poland during October 2009 to January 2011 were tested for the presence of Listeria monocytogenes. It was found that 81 (19.4%) of them were positive for this microorganism as identified by the culture and polymerase chain reaction (PCR) methods. Molecular serotyping performed by PCR revealed that the majority of the isolates (50 strains; 61.7%) were of 1/2a serotype. Furthermore, 26 (32.1%) L. monocytogenes strains were classified as 1/2c serotype, and only five strains belonged to serotypes 1/2b or 4b (four and one isolates, respectively). All the isolates were positive for the inlA, inlC, inlJ, and lmo2672 sequences, whereas two L. monocytogenes (both of 4b serotype) had another virulence marker gene--llsX. The results of the antimicrobial resistance revealed that the strains were sensitive to most of the antimicrobials used in the study except oxacillin (62.7% resistant strains). Several isolates (17.3%) were also resistant to ceftriaxone. Our results indicate that L. monocytogenes identified in raw beef meat possessed virulence markers that make them potentially pathogenic for humans. Therefore, this kind of food may create a public health concern.

Comparative analysis of antimicrobial resistance and genetic diversity of Campylobacter from broilers slaughtered in Poland.

In the current study, the relationship of Campylobacter jejuni and Campylobacter coli strains isolated at slaughter was investigated using comparative analysis of antimicrobial resistance (AMR), virulence gene (VG) and PFGE profiling. A total of 254 Campylobacter isolates from poultry caeca and corresponding carcasses, including 139 C. jejuni and 115 C. coli strains were tested. The most prevalent resistance profiles observed in C. jejuni were ciprofloxacin, nalidixic acid and tetracycline (46 out of 139, 33.1% isolates) as well as ciprofloxacin, nalidixic acid, tetracycline and streptomycin among C. coli strains (34 out of 115, 29.6%). Multi-resistance was found more frequently among C. coli than C. jejuni (P<0.05). The presence of 11 virulence genes exhibited 19 different VG profiles in Campylobacter isolates tested. All Campylobacter strains were classified into 154 different PFGE types. Among them, 56 profiles (28 C. jejuni and 28 C. coli) were common for at least two isolates including 9 clusters covering from 4 to 9 strains. Campylobacter composite types generated by a combination of 154 PFGE types, 10 AMR profiles and 19 VG patterns divided 178 distinct types with 95% similarity. The majority of the composite profiles (76 for C. jejuni and 58 for C. coli; 75.3% in total) included only one bacterial isolate. Furthermore, 11 pairs of C. jejuni and 12 pairs of C. coli from caeca and the corresponding carcasses isolated from the same places possessed the identical PFGE, AMR and VG patterns. This study demonstrated that C. jejuni and C. coli isolated from poultry in Poland showed to have a high genetic diversity and a weak clonal population structure. However, the composite analysis revealed a strong evidence for cross-contamination of chicken carcasses during the slaughter process. Additionally, our results confirm that Campylobacter may easily contaminate poultry carcasses at slaughter process and spread around country. More than half of Campylobacter strains tested (50.4%) were resistant to at least two classes of antimicrobials, i.e. quinolones and tetracyclines, which may cause a public health risk.

A five-year study on prevalence and antimicrobial resistance of Campylobacter from poultry carcasses in Poland.

During 2009-2013 a total of 2114 swab samples collected from broiler carcasses in all 16 voivodeships (administrative districts) of Poland were examined for the presence of Campylobacter jejuni and Campylobacter coli. The antimicrobial resistance of the isolates to ciprofloxacin, tetracycline and erythromycin using the MIC method was also tested. It was found that 1151 (54.4%) carcasses were contaminated with Campylobacter, with 50% of C. jejuni and C. coli species isolated from positive samples. The temporal trend in the prevalence of Campylobacter-positive samples demonstrated that the highest percentage of carcasses was contaminated during the first year of the survey (70.5%) whereas in the last year (2013) only 36.3% of broilers contained these bacteria. Antimicrobial resistance analysis showed that overall 939 (81.6%) of isolates were resistant to ciprofloxacin, 646 (56.1%) to tetracycline but only 28 (2.4%) to erythromycin. Significant differences in resistance profiles between C. jejuni and C. coli were observed with greater resistance level observed in the latter species. Furthermore, a significant increase in the percentage of C. jejuni resistant to ciprofloxacin (from 59.6% in 2009 to 85.9% in 2014) and to tetracycline (from 23.2% to 70.4%, respectively) was identified. Only 20 (1.7%) Campylobacter isolates displayed a multiresistance pattern.

Improved medium for large-scale production of recombinant cholera toxin B subunit for vaccine purposes

Abstract Cholera toxin B subunit (CTB) is an important component of the new oral cholera vaccine. CTB can also be used as a potential carrier protein for foreign antigens or epitopes covalently linked to this molecule, such as carbohydrates or peptides. In order to obtain large amounts of CTB, high-level expressions, which facilitate its production, have been introduced. We were able to develop a plasmid expression system that, when used in conjunction with an optimized culture medium, provided yield of CTB more than 800 mg per liter. Based on syncase medium, the higher levels of CTB production were obtained by raising the concentration of casamino acids twice and reduction of the sucrose level down to 50% of the original formula. This simple change resulted in 3-fold increasement of CTB expression into the medium. Using our method, production and purification of cholera toxin B subunit become simple, inexpensive and very effective.