[Investigation of Azithromycin Minimum Inhibitory Concentration Values and Carbapenem Resistance in Salmonella and Shigella Clinical Isolates]. / Salmonella ve Shigella Klinik Izolatlarinda Azitromisin Minimum Inhibitör Konsantrasyon Degerlerinin ve Karbapenem Direncinin Arastirilmasi.

Increasing resistance to first-line antibiotics used in the treatment of infections caused by Salmonella and Shigella species is emerging. Azithromycin presents a good alternative treatment option for Salmonella and Shigella infections. However, there are limited data regarding the susceptibility of azithromycin in Turkey. In this study, we aimed to evaluate the susceptibility of Salmonella and Shigella species to azithromycin, to determine and compare the minimum inhibitory concentration (MIC) values and disk diffusion zone diameters. In addition, susceptibility to meropenem and first-line antibiotic options in isolates was also investigated. A total of 170 Salmonella, 76 Shigella clinical isolates collected between 2014 and 2018 in our hospital were tested for their susceptibility to azithromycin, meropenem, ampicillin, pefloxacin, trimetoprim-sulfamethoxazole, ceftazidime, and cefotaxime. Isolates were identified by matrix-assisted laser desorption ionization time of flight mass spectrometry. The isolates were confirmed and serotyped by the reference laboratory using the conventional slide agglutination method. Susceptibility of the isolates to azithromycin and other antibiotics was evaluated by Kirby-Bauer disk diffusion method. MIC values of azithromycin were determined by the reference broth microdilution method. Combined disk diffusion test was used for the detection of extended spectrum beta-lactamase (ESBL) production. Polymerase chain reaction was performed for macrolide and carbapenem resistance genes and the detected resistance genes were confirmed by sequencing. Of the 76 Shigella isolates tested, 64 (84.2%) were identified as Shigella sonnei, 10 (13.2%) as Shigella flexneri, one (1.3%) as Shigella boydii, and one (1.3%) as Shigella dysenteriae. Among the 170 Salmonella isolates, 131 (77%) were identified as Salmonella enteritidis, 11(6.5%) as Salmonella Typhimurium, 8 (4.7%) as Salmonella Kentucky, 5 (2.9%) as Salmonella Paratyphi B, 4 (2.4%) as Salmonella Infantis, 3 (1.8%) as Salmonella Cholerasuis, and 8 (4.7%) as other serovars (Salmonella Agona, Salmonella Dabou, Salmonella Gallinarum, Salmonella Hadar, Salmonella Muenchen, Salmonella Newport, Salmonella Paratyphi C, Salmonella Senftenberg), respectively. ESBL production was determined as 7.9% (6/76) in Shigella isolates and 2.9% (5/170) in Salmonella isolates. A carbapenem resistant S.Senftenberg isolate positive for the blaOXA-48 resistance gene was detected in our study. Meropenem MIC value of the isolate was detected as > 32 µg/ml with gradient diffusion test. Among all isolates, only one S.boydii isolate was detected as resistant to azithromycin with a MIC value of 128 µg/ml. The isolate was positive for the existence of mphA gene by PCR. In the disk diffusion test, azithromycin inhibition zone diameters were ≥ 12 mm in all of the tested isolates, except for the azithromycin-resistant isolate, and the azithromycin MICs were determined as ≤ 16 µg/ ml by broth microdilution. Increasing resistance to commonly used antibiotics in Salmonella and Shigella species is emerging. The detection of a carbapenem-resistant Salmonella isolate in our study indicates that the spread of carbapenem resistance to other Enterobacterales species may cause global problems. Antimicrobial susceptibility testing of azithromycin for Salmonella and Shigella species has been difficult to establish due to the lack of approval in vitro breakpoints for all species. Consequently, our data shows that azithromycin exhibits as a good alternative therapeutic choice for the treatment of gastrointestinal diseases caused by Salmonella and Shigella species. Further studies are needed to provide appropriate in vitro breakpoints supported by clinical data.

Pyelonephritis Caused by Salmonella Anatum: An Unusual Case.

Salmonella species are a very rare cause of urinary tract infection in healthy children. This species is associated with a high incidence of genitourinary abnormalities and immune deficiencies. We report the first pediatric case of a healthy 9-year-old girl with pyelonephritis due to Salmonella Anatum.

[Antimicrobial susceptibilities and molecular characterization of toxin-positive Clostridium difficile isolates: the first report on the presence of hypervirulent strains from Turkey]. / Toksin Pozitif Clostridium difficile Izolatlarinin Antimikrobiyal Duyarliliklari ve Moleküler Karakterizasyonu: Türkiye'den Hipervirülan Suslarin Varligi ile Iliskili Ilk Bildirim.

Clostridium 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 µg/ml. The MIC50 and MIC90 values for erythromycin and clindamycin were 1.5-3 µg/ml, and 2-4 µg/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.

[Serotype distribution and antimicrobial susceptibilities of Salmonella strains recovered from environmental samples between 2008-2014]. / 2008-2014 yillari arasinda çevresel örneklerden izole edilen Salmonella suslarinin serotip dagilimi ve antimikrobiyal duyarliliklari.

Despite the measures taken and control applications worldwide, Salmonella infections continue to threat the public health. Since these infections also cause significant economical loss, the salmonellas continue to be forefront globally. The determination of Salmonella serotypes and their sources is important for epidemiological point of view. In this study, serotype distribution and antimicrobial resistance of environmental isolates of Salmonella spp. recovered from the poultry farms, that were send for confirmation and serotyping between seven years period, 2008-2014, were evaluated. Strains isolated from environmental samples that were sent to Public Health Institute, Department of Microbiology Reference Laboratory, National Reference Laboratory for Enteric Pathogens, were inoculated onto Salmonella-Shigella and Xylose Lysine Desoxycholate agar and evaluated after 18-24 hours of incubation at 37°C. The identification of the strains was performed by using standard biochemical tests from the suspected colonies. Strains compatible with Salmonella spp. were serotyped using polyvalent and monovalent Salmonella O and H antisera by slide agglutination method. Antibiotic susceptibility tests were performed and evaluated according to CLSI recommendation using Kirby-Bauer disk diffusion method. In our study, a total of 2011 Salmonella strains were evaluated and 15 different serogroups and 75 different serotypes were identified. The most common Salmonella serotypes were S.Infantis (30.6%), followed by S.Enteritidis (21.8%), S.Typhimurium (6.5%), S.Kottbus (5.2%), S.Tennessee (4.3%), S.Mbandaka (4.1%), S.Indiana (3.9%), S.Kentucky (3%), S.Corvallis (2.5%), S.Paratyphi B (1.9%) and S.Hadar (1.7%). Among the isolates, 50.1% (1008/2011) were found susceptible to all of the tested antimicrobials. The rate of isolates that were resistant to only one drug was found to be 15.6%, whereas 30.9% of the strains showed multi-drug resistance (resistant to ≥ 3 antimicrobial drugs). Antimicrobial resistance rates of the Salmonella strains were as follows; nalidixic acid 35.9%, tetracycline 30%, sulfonamides 27.5%, trimethoprim 25.6%, trimethoprim/sulfamethoxazole 25.4%, streptomycin 23.4% and ampicillin 13.5%, respectively. The highest resistance rates for streptomycin (91.4%) ampicillin (88.6%) and tetracycline (88.6%) were observed in S.Hadar strains; for sulfonamide (82.2%) and trimethoprim/sulfamethoxazole (78.2%) in S.Infantis strains, and for nalidixic acid in S.Indiana (97.4%), S.Hadar (91.4%) and S.Infantis (88.8%) strains. In conclusion, the origins, serotypes and antibiotic susceptibility patterns of the strains should be defined for the management of Salmonella infections which are still today a global problem.

[Determination of Salmonella serotypes by conventional and molecular methods]. / Salmonella Serotiplerinin Konvansiyonel ve Moleküler Yöntemler ile Belirlenmesi.

Determination of Salmonella enterica serotypes is crucial 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, C1, 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, -i, -r, -z10) and H2 antigen complexes (H2: 1,2, -1,5, -1,6, -1,7 and H: enx, enz15). 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.

[Investigation of verotoxigenic Escherichia coli O157:H7 incidence in gastroenteritis patients]. / Gastroenteritli olgularda verotoksijenik escherichia coli O157:H7 insidansinin arastirilmasi.

Escherichia coli O157:H7 is the most common serotype among verotoxigenic E.coli (VTEC) strains that cause haemolytic uremic syndrome. Although sporadic VTEC cases originating from Turkey and small outbreaks have been reported from our country, VTEC has not been routinely investigated in most of the diagnostic microbiology laboratories in Turkey and studies related to this topic are limited. In this study, the incidence of E.coli O157:H7 in patients who were admitted to Alanya Research and Application Hospital of Baskent University with the complaints of acute gastroenteritis between September 2005 and September 2008, was investigated. Stool samples collected from 1815 diarrheal patients (of them 50.5% were male; 49.3% were ? 5 years old; 10.2% were tourists) were evaluated initially by direct microscopy and then inoculated to hectoen enteric agar, EMB agar, Skirrow agar and cefixime tellurite sorbitol MacConkey (CT-SMC) agar media for cultivation. The sorbitol-negative colonies which were compatible with E.coli according to the conventional methods were tested with E.coli polyvalent and 0157 and H7 monovalent antisera and agglutination positive strains were also investigated for verotoxin production in Vero cell cultures. VTEC RPLA toxin detection kit (Oxoid, UK) was used for further identification of toxin type of verotoxin positive strains. Fecal leukocytes were detected in 41.3% of the samples in direct microscopy, while 27% (173/639) of the samples were also found positive for amoeba antigen, 6% (24/396) for rotavirus antigen, 1.2% (22/1815) for Salmonella spp., 0.6% (11/1815) for Shigella spp., 0.2% (4/1815) for Giardia trophozoites and 0.06% (1/1815) for Campylobacter jejuni. The isolation rate of sorbitol-negative E.coli strains was %0.8 (14/1815), and two of them were identified as E.coli O157:H7 by monovalent antisera, and both of them were determined as verotoxin-producers in cell culture. Verotoxin types of those isolates were found as verotoxin 1 in one strain and verotoxin 1 + verotoxin 2 in the other. The two patients infected with verotoxigenic E.coli O157:H7 were both tourists (one was 7 and the other was 35 years old) and admitted to the emergency room of hospital with complaints of bloody diarrhea. No further investigation directed towards the origin of the pathogen could be performed in the hotels of these patients. These data indicated that VTEC O157:H7 incidence was low (2/1815; 0.1%) in our area during the study period. Thus, routine testing of stool samples for E.coli O157:H7 does not seem to be cost-effective. However, E.coli O157:H7 should necessarily be investigated at least in bloody diarrhea cases since this pathogen has serious morbidity, mortality and complications like haemolytic uremic syndrome, hemorrhagic colitis and also due to its epidemiological significance.

Enterohemorrhagic Escherichia coli O157:H7: case report.

Enterohemorrhagic Escherichia coli (EHEC) is a significant bacterial pathogen of bloody diarrhea. Not only does it cause systemic complications, such as hemolytic uremic syndrome (HUS) (the most common cause of potentially preventable pediatric renal failure), but it also leads to large outbreaks of bloody diarrhea. Among EHEC serotypes that cause HUS, E. coli O157:H7 is the most common. Herein, we present the case of a young girl with E. coli O157:H7 infection and review the related literature.

[Typing of Shigella sonnei strains isolated in some provinces of Turkey using antimicrobial resistance and pulsed field gel electrophoresis methods]. / Türkiye'nin bazi illerinde izole edilen Shigella sonnei suslarinin antimikrobiyal direnç ve "pulsed field" jel elektroforezi yöntemleri ile tiplendirilmesi.

Shigella species may lead to large epidemics owing to their low infective doses and frequent transmission from person to person with high secondary attack rates. Shigella sonnei is one of the most prevalent causative agent of infectious gastroenteritis in developing and developed countries and it is the most frequently reported Shigella serotype from Turkey in recent years. The aim of this study was to determine the types of S. sonnei strains isolated in different provinces of Turkey [in Marmara earthquake regions (Izmit, n=5; Adapazari, n=6; Yalova, n=2) in 1999 and in Ankara (n=17) in 1997, 2000 and 2001] according to their antimicrobial resistance and pulsed field gel electrophoresis (PFGE) patterns. All isolates were found sensitive to gentamicin, ceftriaxone, imipenem, nalidixic acid and ciprofloxacin. Twenty three (76.6%) of isolates were found resistant to streptomycin, 21 (70%) to trimethoprim-sulfamethoxazole, 20 (66.6%) to tetracycline, 6 (20%) to ampicillin, 3 (10%) to ampicillin/sulbactam and 1 (3.3%) to chloramphenicol. Three (%10) isolates were detected as intermediate susceptible to tetracycline and cefoperazone, while four isolates (13.3%) were susceptible to all antimicrobial agents tested. A total of nine different patterns were obtained according to antimicrobial resistance patterns. PFGE was performed by Xbal restriction enzyme. Isolates were grouped into A (n=24) and B (n=6) main PFGE types and into 13 closely or possibly related types. A total of 15 different PFGE patterns were identified among the isolates. It was determined that isolates from the same clone disseminated in Ankara during the years 2000-2001. Overall, different clones of S. sonnei strains were in dissemination in the provinces included. This study indicated that different S. sonnei clones were in circulation in Turkey and these results constitute the basic molecular preliminary data for the National Enteric Pathogens Laboratory Network in Turkey.

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-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 0.002⁻200 µM 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.

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

Although 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, stx1, 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 ß-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.

[A case with fever of unknown origin during treatment for malaria: multi-drug resistant Salmonella typhi infection]. / Sitma tedavisi alan bir olguda nedeni bilinmeyen ates: cok ilaca direncli Salmonella typhi enfeksiyonu.

Typhoid fever is an acute infectious disease caused by Salmonella serotype Typhi, leading to endemic or epidemic outbreaks in tropical/ subtropical countries (especially in India, Southeast Asia, Central and South Africa). In this report, a 27 years old male patient with malaria has been presented. The patient was diagnosed to have malaria while working in Afghanistan, and received malaria treatment since one month. He admitted to our hospital because of still continuing high fever, and other complaints (weight loss, night sweats, weakness, anorexia). His fever was 39.5 degrees C at admission, and blood smears were negative for Plasmodium sp. On the third day of admission, rose spots were detected on the skin of the abdomen and chest, and group agglutination tests gave positive results for S. Typhi O (titer: 1/800), and S. Typhi H (titer: 1/3200). Blood cultures revealed growth of Salmonella enterica serotip Typhi. The isolate was found to be resistant to ampicillin, chloramphenicol, tetracyclin and trimethoprimsulfamethoxazole, and sensitive to ciprofloxacin. The patient was treated successfully with ciprofloxacin for 14 days.

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.

Analysis of an outbreak of Salmonella enteritidis by repetitive-sequence-based PCR and pulsed-field gel electrophoresis.

OBJECTIVE: The aim of this study was to investigate a large food-borne outbreak associated with eggs contaminated by Salmonella Enteritidis in a military unit using pulse field gel electrophoresis (PFGE) and the Repetitive-sequence-based PCR (rep-PCR) employing the DiversiLab system. MATERIALS AND METHODS: In mid-January 2008, a food-borne outbreak associated with S. Enteritidis occurred in a military unit located in the city centre of Isparta. A total of 2,469 patients were registered to six hospitals with gastrointestinal disease symptoms such as diarrhea and abdominal pain. Of those registered, 445 were hospitalized. S. Enteritidis was isolated from 276 stool samples and a blood sample of the hospitalized patients and from a food item. The PFGE patterns after XbaI digestion and rep-PCR profiles produced by the DiversiLab system were determined for eight randomly selected stool isolates, one blood isolate and one food isolate. RESULTS: The PFGE patterns of all isolates were identical. The Rep-PCR profiles produced by using the DiversiLab system showed that all isolates were indistinguishable. The PFGE and rep-PCR interpretations were concordant for the S. Enteritidis isolates. All stool isolates, one blood isolate and one food isolate were susceptible to all antibiotics tested. CONCLUSION: This data suggest that the DiversiLab system may be a reasonable alternative to PFGE for investigation and control of S. Enteritidis outbreaks, since it is easy to use, rapid and does not require highly skilled operators.

External quality assessment for the determination of diphtheria antitoxin in human serum.

Accurate determination of diphtheria toxin antibodies is of value in determining the rates of immunity within broad populations or the immune status of individuals who may be at risk of infection, by assessing responses to vaccination and immunization schedule efficacy. Here we report the results of an external quality assessment (EQA) study for diphtheria serology, performed within the dedicated surveillance network DIPNET. Twelve national laboratories from 11 European countries participated by testing a standard panel of 150 sera using their current routine method: Vero cell neutralization test (NT), double-antigen enzyme-linked immunosorbent assay (ELISA; DAE), dual double-antigen time-resolved fluorescence immunoassay (dDA-DELFIA), passive hemagglutination assay (PHA), toxin binding inhibition assay (ToBI), and in-house or commercial ELISAs. The objective of the study was not to identify the best assay, as the advantages and drawbacks of methods used were known, but to verify if laboratories using their routine method would have categorized (as negative, equivocal, or positive) a serum sample in the same way. The performance of each laboratory was determined by comparing its results on a quantitative and qualitative basis to NT results from a single reference laboratory, as this test is considered the in vitro "gold standard." The performance of laboratories using NT was generally very good, while the laboratories' performance using other in vitro methods was variable. Laboratories using ELISA and PHA performed less well than those using DAE, dDA-DELFIA, or ToBI. EQA is important for both laboratories that use in vitro nonstandardized methods and those that use commercial ELISA kits.