Determination of salmonella serotypes by conventional and molecular methods

Salmonella enterica serotiplerinin belirlenmesi epidemiyolojik çalışmalar açısından önem taşımaktadır. Salmonella serotipleri, hücre duvarında yer alan somatik (O) ve flajel (H) antijenleri temel alınarak belirlenir. Bu çalışmanın amacı multipleks polimeraz zincir reaksiyonu (mPCR) yöntemi ile belirlenen moleküler serotiplendirme sonuçlarının konvansiyonel serotiplendirme sonuçları ile karşılaştırılmasıdır. Konvansiyonel serotiplendirme Ulusal Enterik Patojenler Laboratuvar Sürveyans Ağı (UEPLA) kapsamında Sağlık Bakanlığı Refik Saydam Hıfzıssıhha Merkezi Başkanlığı'nda yapılmıştır. Referans laboratuvarı tarafından 14 farklı serotipte tanımlanmış toplam 100 Salmonella suşu, Salmonella serogrupları (A, B, C1, D, E) ve Vi antijen gen bölgelerine özgü primerler kullanılarak mPCR yöntemi ile moleküler olarak serogruplandırılmıştır. H antijenleri (H: a, -b, -d, -g,m, -i, -r, -z10) ve iki antijen kompleksinde yer alan (H: 1,2, -1,5, -1,6, - 1,7 ve H: enx, enz15) antijenleri kodlayan fliC ve fliB genlerine yönelik ardışık dört mPCR yöntemi uygulanarak serotipler belirlenmiştir. Multipleks PCR sonuçları, konvansiyonel yöntem ile belirlenen serogruplar ile %100 uyum göstermiş; serogruplara yönelik mPCR’nin duyarlılık ve özgüllüğü %100 olarak bulunmuştur. Moleküler yöntemle elde edilen antijenik formüle göre belirlenen serotiplendirmede; 2 (%2) izolatta kesin sonuç, 91 (%91) izolatta muhtemel sonuç, 7 (%7) izolatta ise tamamlanamayan formül elde edilmiştir. Klinik mikrobiyoloji laboratuvarlarında en sık izole edilen Salmonella serotipleri olan S.Enteritidis, S.Typhimurium ve S.Paratyphi için moleküler serotiplendirme sonuçları muhtemel sonuç olarak de ğerlendiriliştir. Bu serotipler için mPCR ile elde edilen antijenik formüle göre belirlenen muhtemel sonuçlar epidemiyolojik veriler göz önünde bulundurularak yorumlandığında elde edilen sonuçların konvansiyonel serotiplendirme sonuçları ile uyumlu olduğu gözlenmiştir. Moleküler yöntem ile kesin sonuç elde edilen S.Typhi’nin serogruplandırma ve serotiplendirilmesi açısından mPCR’nin duyarlılığı %100 olarak bulunmuştur. Multipleks PCR klinik laboratuvarlarda izole edilen suşların tanımlanmasında konvansiyonel serotiplendirmeye göre daha ucuz ve daha hızlı bir yöntem olarak değerlendirilmektedir. Moleküler tiplendirme ile bütün serotiplerin kesin olarak belirlenmesi mümkün olmadığından, bugün için moleküler tiplendirme konvansiyonel serotiplendirmenin alternatifi değildir. Ancak konvansiyonel serotiplendirme sonuçları alınıncaya kadar veri sunması bakımından yararlı görünmektedir.Determination of Salmonella enterica serotypes is crutial for epidemiological studies. Salmonella serotypes are defined on the basis of somatic (O) and flagellar (H) antigens, both of which are present in the cell wall of Salmonella. The aim of this study was to compare the results of molecular serotyping obtained by multiplex polymerase chain reaction (mPCR) with conventional serotyping results. Conventional serotyping has been performed in Ministry of Health Refik Saydam Hygiene Center as part of the National Laboratory of Enteric Pathogenes Surveillance Network (UEPLA). A total of 100 Salmonella strains, thay comprise 14 different serotypes by the reference laboratory have been investigated by using specific primers for Salmonella serogroups (A, B, Cl, D and E) and Vi antigen gene clusters via mPCR method. Serotypes have been determined by applying four sequential mPCR targeting the fliC and fliB genes encoding the H1 antigens (H1: a, -b, -d, -g,m, -r, -z(10)) and H2 antigen complexes (H2: 1,2, 1,5, -1,6, -1,7 and H: enx, enz(15)). The results of mPCR showed 100% consistency with the serogroups determined by the conventional method. Both sensitivity and specificity of mPCR according to each serogroups were found to be 100%. Results of serotyping that have been determined with the molecular antigenic formula showed accurate results for 2 (2%), probable results for 91(91%) and incomplete formula for 7 (7%) isolates. Molecular serotyping results of the most common isolated Salmonella serotypes of which S.Enteritidis, S.Typhimurium and S.Paratyphi from clinical microbiology laboratories have been determined as probable results. Antigenic formula of these serotypes that detected using mPCR were considered to be consistent with the results of conventional serotyping when interpreted with epidemiologic data. The sensitivity of mPCR to identify S.Typhi which have been determined as accurate result with molecular serotyping was 100% for serogrouping and serotyping. Multiplex PCR is cheaper and faster for the serotyping of strains isolated in clinical laboratories, compared to the conventional methods. However since it is not possible to detect all serotypes by using molecular typing, this technique can not be currently considered as an alternative for conventional serotyping. Nevertheless molecular typing could be beneficial in providing the preliminary results earlier

Global monitoring of antimicrobial resistance based on metagenomics analyses of urban sewage

Antimicrobial resistance (AMR) is a serious threat to global public health, but obtaining representative data on AMR for healthy human populations is difficult. Here, we use meta-genomic analysis of untreated sewage to characterize the bacterial resistome from 79 sites in 60 countries. We find systematic differences in abundance and diversity of AMR genes between Europe/North-America/Oceania and Africa/Asia/South-America. Antimicrobial use data and bacterial taxonomy only explains a minor part of the AMR variation that we observe. We find no evidence for cross-selection between antimicrobial classes, or for effect of air travel between sites. However, AMR gene abundance strongly correlates with socio-economic, health and environmental factors, which we use to predict AMR gene abundances in all countries in the world. Our findings suggest that global AMR gene diversity and abundance vary by region, and that improving sanitation and health could potentially limit the global burden of AMR. We propose metagenomic analysis of sewage as an ethically acceptable and economically feasible approach for continuous global surveillance and prediction of AMR.Peer reviewe

Setting a baseline for global urban virome surveillance in sewage

The rapid development of megacities, and their growing connectedness across the world is becoming a distinct driver for emerging disease outbreaks. Early detection of unusual disease emergence and spread should therefore include such cities as part of risk-based surveillance. A catch-all metagenomic sequencing approach of urban sewage could potentially provide an unbiased insight into the dynamics of viral pathogens circulating in a community irrespective of access to care, a potential which already has been proven for the surveillance of poliovirus. Here, we present a detailed characterization of sewage viromes from a snapshot of 81 high density urban areas across the globe, including in-depth assessment of potential biases, as a proof of concept for catch-all viral pathogen surveillance. We show the ability to detect a wide range of viruses and geographical and seasonal differences for specific viral groups. Our findings offer a cross-sectional baseline for further research in viral surveillance from urban sewage samples and place previous studies in a global perspective

Shigella and salmonella contamination in various foodstuffs in Turkey

The prevalence of Shigella and Salmonella in a range of foodstuffs purchased from supermarkets and smaller units in Bursa province (Turkey) over a 7-month period between December 2004 and June 2005 was evaluated. In total 416 food samples composed from chicken parts, minced meats, ready-to-eat salads, raw vegetables and raw milks were analysed. Among the samples only one chicken thigh sample (0.24%) was found to be contaminated with Salmonella whereas Shigella was not isolated from any samples. Isolated Salmonella strain was serotyped as Salmonella enterica subsp. enterica serovar Infantis (S. Infantis) and displayed multidrug resistance to several antibiotics including streptomycin, tetracycline, sulphonamides, trimethoprim, trimethoprim-sulphamethoxazole and nalidixic acid. Decreased susceptibility to ciprofloxacin (MIC 0.38 mg/L by E-test) was also determined. The present study revealed that despite low contamination rate, foodstuffs particularly chicken parts could be a potential vehicle for foodborne infections and implementation of preventive measures and consumer food safety education efforts are needed

Antimicrobial Susceptibilities and Molecular Characterization of Toxin-Positive Clostridium difficile Isolates: The First Report on the Presence of Hypervirulent Strains from Turkey

WOS: 000408311400004PubMed ID: 28929960Clostridium difficile infection is one of the most important hospital-acquired infections. Infections caused by hypervirulent C. difficile strains which produce toxins at high levels, have higher morbidity and mortality rates, more complications and relapses. They are characterized by higher sporulation ratios and resistance rates for fluoroquinolones. In order to prevent serious morbidities, mortalities and remarkable increase in health costs, highly pathogenic C. difficile strains must be identified before causing severe outbreaks. The aim of this study was to determine the antimicrobial susceptibilities and molecular characteristics of 61 C. difficile strains isolated by culture from toxin-positive fecal samples of patients who were admitted to three different laboratories in Ankara, between September 2012 and November 2014. Antimicrobial susceptibilities were determined by using gradient test strips and results were interpreted according to the current CLSI and EUCAST criteria. The presence of toxin genes was investigated by polymerase chain reaction (PCR), and mutations in the tcdC gene were determined by sequence analysis following PCR amplification. Genetic characterization of one hypervirulent strain was performed by Public Health Institution of Turkey using the GenoType CDiff (Hain Lifescience, Germany) test. All strains were susceptible to vancomycin and metronidazole. Three (4.9%) isolates were resistant to moxifloxacin with a minimum inhibitory concentration (MIC) of > 8 mu g/ml. The MIC 50 and MIC 90 values for erythromycin and clindamycin were 1.5-3 mu g/ml, and 2-4 mu g/ml, respectively. All strains carried the tcdA and tcdB genes, and 1 (1.6%) was positive for the binary-toxin (cdtA and cdtB) genes. The binary-toxin positive strain carried a 54 bp deletion as well as a single nucleotide change in the tcdC gene. Various single nucleotide changes were found in the tcdC gene of 12 strains (19.6%). Our results have shown that, hypervirulent strains exist in our country, but we have no evidence for the presence of ribotype 027 yet. On the other hand, when the increasing incidence of these strains through out the world is taken into consideration, it would be of great importance to perform surveillance studies and characterize the isolated strains

A case of urinary tract infection due to Salmonella enterica serovar Virchow and review of the related literature

Nontyphoid salmonella (NTS) serotypes can cause gastroenteritis, bacteriemia, and focal infections. However, these focal infections, including urinary tract infections (UTI), are occasionally observed; in particular, the presence of several predisposing factors, such as immunodeficiency and structural abnormality in the urinary tract, increase the possibility of the occurrence of infection. We present a case of UTI caused by Salmonella enterica serovar Virchow in an elderly and debilitated patient with benign prostatic hyperplasia (BPH). Administration of appropriate antibiotic treatment resulted in recovery of the patient’s clinical course.</jats:p

A case of urinary tract infection due to Salmonella enterica serovar Virchow and review of the related literature

Nontyphoid salmonella (NTS) serotypes can cause gastroenteritis, bacteriemia, and focal infections. However, these focal infections, including urinary tract infections (UTI), are occasionally observed; in particular, the presence of several predisposing factors, such as immunodeficiency and structural abnormality in the urinary tract, increase the possibility of the occurrence of infection. We present a case of UTI caused by Salmonella enterica serovar Virchow in an elderly and debilitated patient with benign prostatic hyperplasia (BPH). Administration of appropriate antibiotic treatment resulted in recovery of the patient’s clinical course

Characterization of Shiga Toxin-Producing Escherichia coli Isolated from Humans between 2011 and 2014

WOS: 000384869100007PubMed ID: 26902213Although E. coli O157:H7 is the major serotype among Shiga toxin-producing Escherichia coli (STEC) strains, non-O157 serotypes have caused numerous outbreaks worldwide. We aimed to evaluate the distribution of serogroups, serotypes, virulence genes, and antimicrobial resistance of STEC strains recovered from stool samples. A total of 395 stool samples characterized by watery/bloody diarrhea and/or symptoms of hemolytic-uremic syndrome were included in this study. Strains compatible with E. coli, based on biochemical tests, were tested for the presence of Shiga toxin by ELISA. Toxigenic strains were tested by serotyping and serogrouping. Virulence genes, stxl, stx2, aggR, hlyA, and eae were detected by polymerase chain reaction. Overall, 26 (6.6%) stool culture samples tested positive for STEC. Shiga toxin was positive in 28 (7.1%) patient isolates based on ELISA and PCR. Two isolates could not be serotyped. STEC strains were distributed into 10 serogroups and 14 serotypes. Of the serotyped strains, 92.3% were non-O157, with the major distribution in O104:H4 and O26:HNM. All were negative for extended-spectrum beta-lactamase enzyme and 62.5% were resistant to at least 1 drug. This study demonstrated the wide distribution of non-O157 STEC strains from our patient group. Further studies should be performed to better understand STEC characteristics on a larger scale

Nanoparticle Enhanced Antibody and DNA Biosensors for Sensitive Detection of Salmonella

Bacteria-related pathogenic diseases are one of the major health problems throughout the world. Salmonella is a genus of rod-shaped Gram-negative enterobacteria of which more than 2600 serotypes have been identified. Infection with Salmonella can cause salmonellosis, a serious bacterial toxi-infection syndrome associated with gastroenteritis, and paralyphoid and typhoid fevers. Its rapid and sensitive detection is a key to the prevention of problems related to health. This paper describes the development of antibody and DNA sensors for Salmonella detection using a microfluidic-based electrochemical system. Commercial Salmonella typhimurium and Salmonella typhimurium from human stool samples were investigated using standard and nanomaterial-amplified antibody sensors. S. typhimurium could be detected down to 1 cfu mL&minus;1. The specificity of immunoassay was tested by studying with non-specific bacteria including E. coli and S. aureus that revealed only 2.01% and 2.66% binding when compared to the target bacterium. On the other hand, the quantification of Salmonella DNA was investigated in a concentration range of 0.002&ndash;200 &micro;M using the developed DNA biosensor that demonstrated very high specificity and sensitivity with a detection limit of 0.94 nM. Our custom-designed microfluidic sensor offers rapid, highly sensitive, and specific diagnostic assay approaches for pathogen detection