Hemolysin-Producing Strains among Diarrheagenic Escherichia coli Isolated from Children under 2 Years Old with Diarrheal Disease

Even if serotyping based on O antigens is still routinely used by most laboratories for the detection of diarrheagenic Escherichia coli, this method can provide false-positive reactions, due to the high diversity of O antigens. Molecular methods represent a valuable tool that clarifies these situations. In the Bacteriology Laboratory of Mureș County Hospital, between May 2016 and July 2019, 160 diarrheagenic E. coli strains were isolated from children under 2 years old with diarrheic disease. The strains were identified as Shiga toxin-producing E. coli (STEC)/enteropathogenic Escherichia coli (EPEC) via agglutination with polyvalent sera. STEC strains were serotyped using monovalent sera for serogroup O157. Simplex PCR was performed on the strains to determine the presence of the hlyA gene, and, for the positive ones, the hemolytic activity was tested. Antibiotic susceptibility of the identified diarrheagenic E. coli strains was also investigated. STEC strains were the most frequently identified (49.1%), followed by EPEC (40.2%). The hlyA gene was identified in 12 cases, representing 18.2% of the STEC strains. Even if the extended-spectrum β-lactamase (ESBL)-producing strains represented only 10%, a relevant percentage of multidrug-resistant (MDR) strains (24%) was identified.

Antibiotic susceptibility and phylogenetic analyses for the origins and serotypes of Listeriamonocytogenes strains isolated from ice cream and cream cakes

Listeria monocytogenes is a zoonotic bacterium which also infects humans. The aim of this study was to isolate this organism from cream cakes and ice cream and obtain 16S rRNA and hlyA gene sequences from isolates in order to perform phylogenetic analyses. Serotypes and antibiotic susceptibility were also determined. The cream cake and ice cream samples were examined for L. monocytogenes according to ISO 11290-1 and using the mini Vidas LMO 2 kit procedure. Antibiotic susceptibilities were investigated using the disc diffusion method according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Clinical and Laboratory Standards Institute (CLSI) standards. The Sanger DNA sequencing method for phylogenetic analysis was used for L. monocytogenes isolates. A total of 16 (n =128, 12.50%) L. monocytogenes strains, 9 (12.16%) from cream cake samples and 7 (12.96%) from ice cream samples, were isolated. This was the first investigation involving sequencing of L. monocytogenes isolated from cream cakes and ice creams in Turkey. All isolates were susceptible to sulfamethoxazole/trimethoprim, tetracycline, streptomycin, gentamicin, meropenem, and erythromycin. The numbers of isolates resistant to sulbactam/ampicillin, penicillin G, chloramphenicol, and ampicillin were 16, 2, 1, and 1, respectively. Moreover, 3 isolates showed intermediate resistance to amikacin and 2 to ciprofloxacin. The hlyA gene sequences of 11 of the L. monocytogenesisolates isolated from milk were closely related to the hlyA gene sequences of the GenBank reference strains. The comparison of the 16s rRNA gene sequences of the L. monocytogenes strains with the GenBank reference serotypes identified 1 isolate as serotype 1/2c, 1 as serotype 1/2a, and 1 as serotype 4. Nucleic acid sequencing is useful for identification of L. monocytogenes. The 16S rRNAand hlyA sequences can be used to determine the origin and relationship between L. monocytogenesisolates, as well as the serotype.

Prevalence, virulence and antibiotic susceptibility of Listeria monocytogenes isolated from sheep

Objective: This study was undertaken to determine the prevalence, virulence, and antibiotics susceptibility of Listeria monocytogenes isolated from hindbrain, spinal cord, milk, and intestinal content collected from sheep in the Dakahlia Governorate, Egypt. Design: Observational study. Samples: We analyzed 472 samples, including 72 hindbrain/spinal cord samples from emergency-slaughtered sheep, 300 raw-milk samples from apparently healthy sheep, and 100 intestinal content samples from slaughtered sheep at three abattoirs. Procedures: Isolation and identification of L. monocytogenes were performed using conventional techniques. The biochemically identified isolates were confirmed by 16SrRNA gene sequencing and examined for virulence-associated genes (hlyA and iap) as well as for antimicrobial susceptibility. Results: In total, 16 (3.39%) out of 472 sheep samples [5.56% (4/72) in hindbrain/spinal cord, 4% (12/300) in milk, and 0% (0/100) in intestinal content samples] were found to be positive for L. monocytogenes. All the confirmed isolates were positive for the hlyA gene (100%); meanwhile, none of them exhibited the iap gene. Antibiotic susceptibility testing showed high resistance rates to amoxicillin, cefotaxime, erythromycin (50% each), and vancomycin (37.5%). Sulfamethoxazole–trimethoprim and tetracycline resistance rates were 25% and 12.5%, respectively. On the contrary, all isolates were susceptible to amikacin, ciprofloxacin, and norfloxacin. Interestingly, 37.5% (6/16) of L. monocytogenes isolates exhibited multidrug resistance (MDR). The multiple antibiotic resistances (MAR) index of isolates ranged from 0.1 to 0.6. Conclusion and clinical relevance: Our data highlights the importance of awareness of virulent strains of MDR L. monocytogenes of sheep samples and potentially samples from other domestic animals in Egypt.

A Novel Strategy for Enhance Potentiation of Meglumine antimo-niate against Leishmania major In Vitro

Background: We aimed to design a different method of drug delivery for increased transfer of the choice drug (meglumine antimoniate) within the host cells. Therefore, listeriolysin O (LLO), a bacterial product which is a member of pore-forming peptides was used as an enhancer factor with meglumine antimoniate in order to facilitate the transition of the drug across macrophage membrane. Methods: LLO was produced in Isfahan University of Medical Sciences in 2016, by expressing the hlyA gene in Escherichia coli and purified using affinity chromatography. Cytotoxicity of the purified protein was investigated in an in vitro model of macrophage Leishmania infection. Results: LLO was cytotoxic against murine macrophage cells (J774-A1) and amastigote forms of L. major (MRHO/IR/75/ER). It was less toxic to macrophages (CC50=2.56 μg ml-1 ±0.09) than to the parasites (IC50=1.72 μg ml-1 ±0.07). Moreover, non-cytotoxic concentration of LLO (0.006 ug ml-1) potentiated the cytotoxicity induced by low dose concentration of meglumine antimoniate. Very little dose of meglumine antimoniate was needed when combined with the LLO (IC50=12.63 μg ml-1 ±0.13) in comparison with the cytotoxicity induced when the drug is used alone (IC50=46.17 μg ml-1 ±0.28). Conclusion: The combination of pore-forming proteins with anti-leishmanial agents could increase the advantage of anti-leishmanial drugs. Since lower concentrations of anti-leishmanial drugs can reduce undesirable side effects of chemotherapy trials carried out in animal models and then in humans with this system.

A COMPARATIVE STUDY OF SOME VIRULENCE FACTORS AND PHYLOGENETIC CHARACTRIZATION OF Escherichia.coli ISOLATES CAUSING URINARY TRACT INFECTION AND THE COMMENSAL GUT MICROBIOTA

Variety of virulence factors are involved in the pathogenicity of Escherichia coli isolates, the common cause of the urinary tract infections.(UTIs). This study was aimed.to determine some virulence factors.involved in the pathogenicity.and the phylogenetic.grouping of uropathogenic E. coli isolates.from UTIs in compare.with isolates.from gut.microbiota (fecal flora) In.this study ,E coli isolates were collected from samples .urine (n = 25), fecal samples (n = 25) samples of the same patients with UTI, and from fecal samples (n= 5 as control )of patients without UTI. The detection of phylogenetic grouping and some virulence genes among the all isolates were confirmed by PCR technique. The results.showed that.phylogenetic groups B2,B1(36%) and D (28%) were predominated.among uropathogenic E.coli in comparison with group A (8%) ,whereas in .commensal isolates.groups B1(36%), B2(32%) ,D (28%) were.more prevalent in compare.with group A (4%).The prevalence.of cnf1 and fimH .genes were higher in.UPEC in comparsion with.commensal isolates. However, the.prevalence of kpsMT II gene.was similar among.both groups, while hlyA gene was.higher in fecal.isolates. According to this results., microbiota may considered the main source of UPEC bacteria.

Primer reporte de Escherichia coli Productora de Toxina Shiga no O157 que codifica el gen de la enterohemolisina en carne cruda en Colombia

Dentro de los patotipos de Escherichia coli, el grupo STEC puede producir en el ser humano desde diarrea hemorrágica hasta insuficiencia renal aguda e incluso la muerte; el ganado bovino es el principal reservorio de este agente patógeno y por ende la ingestión de alimentos derivados de estos animales de abasto son una fuente muy importante de infección para el hombre. El objetivo principal de este estudio fue determinar la prevalencia de STEC en muestras de carne cruda comercializada en Pamplona-Colombia y en cepas obtenidas a partir de las muestras. Se analizaron cien muestras de carne cruda aplicando la técnica de Reacción en Cadena de la Polimerasa para la detección de los siguientes genes en muestras y en cepas STEC: stx1, stx2, eae y hlyA. Adicionalmente, se estableció el patrón de resistencia-susceptibilidad antibiótica de cepas STEC aisladas empleando métodos regulados. En el 39% de las muestras fue posible detectar el gen stx2; en el 38%, de ellas, se detectaron los genes stx1 y stx2. Además, se aislaron cepas STEC en el 13% de las muestras analizadas, 85% de ellas portaban el gen hlyA. No se detectó la presencia del gen eae o del serogrupo O157. Las cepas aisladas demostraron resistencia frente a algunos antibióticos de primera y segunda generación. En conclusión, se detectó la presencia de genes que codifican factores de virulencia en las muestras de carne analizadas que representan un riesgo potencial para la salud de los consumidores. Este es el primer reporte de STEC no O157 que codifica el gen de la enterohemolisina en alimentos en Colombia. Within the Escherichia coli patotypes, the STEC group can produce in humans from hemorrhagic diarrhea to acute renal failure and even death; cattle are the main reservoir of this pathogen and therefore the ingestion of food derived from these stock animals are a very important source of infection for man. The main objective of this study was to determine the prevalence of STEC in raw meat samples marketed in Pamplona-Colombia and in strains obtained from those samples. One hundred raw meat samples were analyzed using the Polymerase Chain Reaction technique for the detection of the following genes in samples and in STEC strains: stx1, stx2, eae and hlyA. In addition, STEC strains were isolated in 13% of the analyzed samples, 85% of them carried the hlyA gene. The presence of the eae gene or serogroup O157 was not detected. The isolated strains demonstrated resistance against some first and second generation antibiotics. In conclusion, the presence of genes encoding virulence factors in the meat samples analyzed, that represent a potential health risk factor to consumers, was confirmed. This is the first report of STEC non-O157 that encodes the enterohemolysin gene in foods in Colombia.

Analysis of gene encoding haemolysin A of Vibrio cholerae isolated in Vietnam

Vibrio cholerae is the cholera causing agent, divided into two biotypes, including the classical biotype and ElTor biotype. Both of these biotypes caused cholera epidemics in the world. The classical biotype caused 6th cholera pandemic (from 1921 to 1961), and ElTor biotype caused 7th cholera pandemic (from 1961 to the 70s). Haemolysin A, a hemolytic protein of V. cholerae ElT or biotype, is encoded by the hlyA gene. This gene is often used for analyzing genetic relationship between strains in the same species or between species in the same Vibrio genus. Results of analyzing nucleotide and amino acid sequences of hlyA gene of V. cholerae strain causing cholera in Vietnam (named hlyA.VN) showed that: the hlyA.VN gene sequence was similar to the hlyA gene sequences of V. cholerae strains of the 6th and 7th cholera epidemics. The hlyA gene of the 6th cholera epidemic strain was deficient in 11 nuleotides (this deficiency leading to the loss of 4 amino acids in the haemolysin A protein) comparing to hlyA.VN gene and hlyA gene of the 7th cholera epidemic strain. The results of genetic distance analysis as well as phylogenetic tree construction also confirmed V. cholerae causing cholera in Vietnam was closely relationship to the strains causing cholera pandemics in the world. It is great significance for the surveillance of molecular epidemiology to prevent cholera effectively. Vibrio cholerae là tác nhân gây bệnh tả, được chia thành hai typ sinh học, đó là typ sinh học cổ điển và typ sinh học ElTor. Cả hai typ này đã từng gây ra các đại dịch tả trên thế giới. Typ sinh học cổ điển đã từng gây ra đại dịch tả lần thứ 6 (từ năm 1921 đến 1961), còn typ sinh học ElTor đã từng gây ra đại dịch tả lần thứ 7 (từ 1961 đến những năm 70). Haemolysin A, một protein có chức năng làm tan máu của V. cholerae typ sinh học ElTor, được mã hóa bởi gen hlyA. Gene này thường được sử dụng cho các phân tích quan hệ di truyền giữa các chủng trong cùng một loài V. cholerae hay giữa các loài trong cùng một chi Vibrio. Kết quả phân tích trình tự nucleotide và axit amin gen hlyA của chủng V. cholerae gâybệnh ở Việt Nam (hlyA.VN) cho thấy: trình tự gen hlyA.VN có sự tương đồng lớn với trình tự gen hlyA của chủng gây đại dịch tả 6 và 7. Gen hlyA của chủng gây đại dịch tả 6 bị thiếu hụt 11 nuleotide (sự thiếu hụt này dẫn tới sự mất đi 4 axit amin trong phân tử haemolysin A) so với gen hlyA.VN và gene hlyA của chủng gây đại dịch tả 7. Kết quả phân tích khoảng cách di truyền cũng như xây dựng cây phát sinh chủng loại cũng đã khẳng định: chủng gây bệnh ở Việt Nam có quan hệ rất gần với các chủng gây đại dịch tả trên thế giới. Nhận định này có ý nghĩa rất lớn đối với công tác giám sát dịch tễ học phân tử để ngăn chặn bệnh tả hiệu quả.