Assessing the mechanisms of multi-drug resistant non-typhoidal Salmonella (NTS) serovars isolated from layer chicken farms in Nigeria.

BACKGROUND: In Nigeria, there have been reports of widespread multiple antimicrobial resistance (AMR) amongst Salmonella isolated from poultry. To mitigate the impact of mortality associated with Salmonella on their farms, farmers resort to the use of antimicrobials without sound diagnostic advice. We conducted this study to describe the AMR patterns, mechanisms and genetic similarities within some Salmonella serovars isolated from different layer farms. METHOD: We determine the AMR profiles of two hundred Salmonella isolates, selected based on frequency, serovar, and geographical and sample type distribution. We also assessed the mechanisms of multi-drug resistance for specific genetic determinants by using PCR protocols and gene sequence analysis. Pulsed-field gel electrophoresis (PFGE) was conducted on seven selected serovars to determine their genetic relatedness. RESULTS: Of 200 isolates, 97 (48.5%) revealed various AMR profiles, with the multiple antibiotic resistance (MAR) index ranging from 0.07-0.5. Resistance to ciprofloxacin was common in all the multi-drug resistant isolates, while all the isolates were susceptible to cefotaxime, ceftazidime, and meropenem. Genotypic characterization showed the presence of resistance genes as well as mutations in the nucleotide genes with subsequent amino acid substitutions. Fifteen isolates (43%) of S. Kentucky were indistinguishable, but were isolated from four different states in Nigeria (Ogun, n = 9; Kaduna, n = 6; Plateau, n = 3, and: Bauchi, n = 2). PFGE revealed 40 pulsotype patterns (Kentucky, n = 12; Larochelle, n = 9; Virchow, n = 5; Saintpaul, n = 4; Poona, n = 3; Isangi, n = 2, and; Nigeria, n = 2). CONCLUSION: This study recorded strictly related but diversely distributed Salmonella serovars with high AMR rates in poultry. We recommend strict regulation on antimicrobial use and regular monitoring of AMR trends among bacteria isolated from animals and humans to inform public policy.

Improving the Reliability of Literature Reviews: Detection of Retracted Articles through Academic Search Engines.

Nowadays, a multitude of scientific publications on health science are being developed that require correct bibliographic search in order to avoid the use and inclusion of retracted literature in them. The use of these articles could directly affect the consistency of the scientific studies and could affect clinical practice. The aim of the present study was to evaluate the capacity of the main scientific literature search engines, both general (Gooogle Scholar) and scientific (PubMed, EMBASE, SCOPUS, and Web of Science), used in health sciences in order to check their ability to detect and warn users of retracted articles in the searches carried out. The sample of retracted articles was obtained from RetractionWatch. The results showed that although Google Scholar was the search engine with the highest capacity to retrieve selected articles, it was the least effective, compared with scientific search engines, at providing information on the retraction of articles. The use of different scientific search engines to retrieve as many scientific articles as possible, as well as never using only a generic search engine, is highly recommended. This will reduce the possibility of including retracted articles and will avoid affecting the reliability of the scientific studies carried out.

Health Plans for Suicide Prevention in Spain: A Descriptive Analysis of the Published Documents.

The number of deaths by suicide worldwide each year is more than 800,000 people, which is equivalent to one death every 40 seconds. Suicide prevention has been listed by the World Health Organisation as a global imperative and has become a priority for global public health. This descriptive study describes and compares the intervention components included in the suicide prevention plans in the different provinces of Spain. We analysed the published documents through an extensive literature search and summarised the findings using descriptive content analysis. The search was carried out through the official websites of the government and health departments of each province in addition to consulting other official digital platforms such as the National Suicide Observatory, the World Health Organisation and the National Institute of Statistics. The results show the most relevant differences between the prevention plans, revealing that although all the activities included were related to the health sector, not all of them include prevention aimed at the general population level. We conclude that there is a lack of interventions related to the application of universal prevention, while selective and indicated prevention are the most developed tools in Spain.

Polycyclic Aromatic Hydrocarbon-DNA Adducts in Gulf of Mexico Sperm Whale Skin Biopsies Collected in 2012.

The northern Gulf of Mexico has a long history of polycyclic aromatic hydrocarbon (PAH) contamination from anthropogenic activities, natural oil seepages, and the 2010 Deepwater Horizon explosion and oil spill. The continental shelf of the same area is a known breeding ground for sperm whales (Physeter macrocephalus). To evaluate PAH-DNA damage, a biomarker for potential cancer risk, we compared skin biopsies collected from Gulf of Mexico sperm whales in 2012 with skin biopsies collected from sperm whales in areas of the Pacific Ocean in 1999-2001. All samples were obtained by crossbow and comprised both epidermis and subcutaneous blubber. To evaluate exposure, 7 carcinogenic PAHs were analyzed in lipids extracted from Pacific Ocean sperm whale blubber, pooled by sex, and location. To evaluate PAH-DNA damage, portions of all tissue samples were formalin-fixed, paraffin-embedded, sectioned, and examined for PAH-DNA adducts by immunohistochemistry (IHC) using an antiserum elicited against benzo[a]pyrene-modified DNA, which crossreacts with several high molecular weight carcinogenic PAHs bound to DNA. The IHC showed widespread epidermal nuclear localization of PAH-DNA adducts in the Gulf of Mexico whales (n = 15) but not in the Pacific Ocean whales (n = 4). A standard semiquantitative scoring system revealed significantly higher PAH-DNA adducts in the Gulf of Mexico whales compared to the whales from the Pacific Ocean study (p = .0002).

Insight into an outbreak of Salmonella Choleraesuis var. Kunzendorf in wild boars.

An unusual mortality of wild boars occurred in Italy from 2012 to 2015 due to Salmonella Choleraesuis infection. In order to confirm the occurrence of an outbreak of S. Choleraesuis in wild boars and to epidemically characterise the unique S. Choleraesuis biovar, a collection of isolates belonging to wild boars was investigated from the phenotypic, molecular and genomic points of view (PFGE and WGS). Moreover, the possibility of transmission to domestic pigs and humans, temporally and geographically close to the wild boar epidemic, was tested by also including in the panel isolates from infected domestic pigs and from one human case of infection. Wild boar isolates displayed a high genetic correlation, thus suggesting they are part of the same outbreak, with a common invasiveness potential. Conversely, no correlation between pig isolates and those from the other sources (wild boars and human) was found. However, the phylogenetic and PFGE analyses suggest a high degree of similarity between the human and the investigated wild boar outbreak isolates, implying the potential for the spread of Salmonella Choleraesuis among these species.

Different Resolution Power of Multilocus Variable-Number Tandem Repeat Analysis and Whole-Genome Sequencing in the Characterization of S. 1,4,[5],12:i:- Isolates.

Salmonella enterica serovar 1,4,[5],12:i:- has emerged over the last two decades as one of the most common serovars causing human salmonellosis in Europe. It is supposed to originate from Salmonella enterica serovar Typhimurium due to antigenic and genotypic similarities between the two serovars. Due to the high level of similarity, the multilocus variable-number tandem repeat analysis (MLVA) protocol designed for Salmonella Typhimurium routine typing is commonly used also for the characterization of S. 1,4,[5],12:i. Nevertheless, the Salmonella Typhimurium-based MLVA protocol often shows poor discriminatory power for S. 1,4,[5],12:i. Indeed, only a limited number of MLVA profiles have been described for S. 1,4,[5],12:i:-. Moreover, based on the MLVA clustering, S. 1,4,[5],12:i:- is supposed to display high clonality. The aim of the present work was to assess whether the five loci of Salmonella Typhimurium investigated by MLVA are sufficiently accurate to correctly assign S. 1,4,[5],12:i:- isolates. For this purpose, 38 epidemiologically unrelated S. 1,4,[5],12:i:- were subjected to whole-genome sequencing. Isolates were selected among a collection of monophasic strains isolated in Italy from different sources over the period 2014-2016 and belonging to the five most commonly detected MLVA profiles. Results confirmed the possible clonality for S. 1,4,[5],12:i:- serovar in the light of the scarce difference observed in terms of single-nucleotide polymorphisms (SNPs) among investigated isolates. Nevertheless, unrelated isolates on the basis of the difference of SNP number were characterized as indistinguishable by MLVA profile, thus suggesting an insufficient resolution of MLVA. Hence, we can conclude that MLVA-based approach does not seem a valuable proxy to deepen into the epidemiological relationship among S. 1,4,[5],12:i:- isolates. These evidences can be useful to avoid incorrect assignment especially when surveillance data are used for outbreak investigations.

Intestinal polycyclic aromatic hydrocarbon-DNA adducts in a population of beluga whales with high levels of gastrointestinal cancers.

Carcinogenic polycyclic aromatic hydrocarbons (PAHs) were disposed directly into the Saguenay River of the St. Lawrence Estuary (SLE) by local aluminum smelters (Quebec, Canada) for 50 years (1926-1976). PAHs in the river sediments are likely etiologically related to gastrointestinal epithelial cancers observed in 7% of 156 mature (>19-year old) adult beluga found dead along the shorelines. Because DNA adduct formation provides a critical link between exposure and cancer induction, and because PAH-DNA adducts are chemically stable, we hypothesized that SLE beluga intestine would contain PAH-DNA adducts. Using an antiserum specific for DNA modified with several carcinogenic PAHs, we stained sections of paraffin-embedded intestine from 51 SLE beluga (0-63 years), 4 Cook Inlet (CI) Alaska beluga (0-26 years), and 20 beluga (0-46 years) living in Arctic areas (Eastern Beaufort Sea, Eastern Chukchi Sea, Point Lay Alaska) and aquaria, all with low PAH contamination. Stained sections showed nuclear light-to-dark pink color indicating the presence of PAH-DNA adducts concentrated in intestinal crypt epithelial lining cells. Scoring of whole tissue sections revealed higher values for the 51 SLE beluga, compared with the 20 Arctic and aquarium beluga (P = 0.003). The H-scoring system, applied to coded individual photomicrographs, confirmed that SLE beluga and CI beluga had levels of intestinal PAH-DNA adducts significantly higher than Arctic and aquarium beluga (P = 0.003 and 0.02, respectively). Furthermore, high levels of intestinal PAH-DNA adducts in four SLE beluga with gastrointestinal cancers, considered as a group, support a link of causality between PAH exposure and intestinal cancer in SLE beluga. Environ. Mol. Mutagen. 60:29-41, 2019. Published 2018. This article is a U.S. Government work and is in the public domain in the USA.

Ascertaining the relationship between Salmonella Typhimurium and Salmonella 4,[5],12:i:- by MLVA and inferring the sources of human salmonellosis due to the two serovars in Italy.

The current picture of human salmonellosis shows Salmonella Typhimurium and S. 4,[5],12:i:- as the most common serovars in Italy. The aims of this study were to investigate the genetic relationship between these serovars, as well as to test the possibility of inferring sources of human salmonellosis due to S. Typhimurium and S. 4,[5],12:i:- by using multilocus variable-number tandem repeat analysis (MLVA) subtyping data. Single isolates from 268 human sporadic cases and 325 veterinary isolates (from pig, cattle, chicken, and turkey) collected over the period 2009-2011 were typed by MLVA, and the similarities of MLVA profiles were investigated using different analytical approaches. Results showed that isolates of S. 4,[5],12:i:- were more clonal compared to S. Typhimurium and that clones of both serovars from different non-human sources were very close to those which were responsible for human infections, suggesting that source attribution by MLVA typing should be possible. However, using the Asymmetric Island Model it was not possible to obtain a confident ranking of sources responsible for human infections based on MLVA profiles. The source assignments provided by the model could have been jeopardized by the high heterogeneity found within each source and the negligible divergence between sources as well as by the limited source data available, especially for some species.

Tamoxifen-DNA adduct formation in monkey and human reproductive organs.

The estrogen analog tamoxifen (TAM), used for adjuvant therapy of breast cancer, induces endometrial and uterine tumors in breast cancer patients. Proliferation stimulus of the uterine endometrium is likely involved in tumor induction, but genotoxicity may also play a role. Formation of TAM-DNA adducts in human tissues has been reported but remains controversial. To address this issue, we examined TAM-DNA adducts in uteri from two species of monkeys, Erythrocebus patas (patas) and Macaca fascicularis (macaque), and in human endometrium and myometrium. Monkeys were given 3-4 months of chronic TAM dosing scaled to be equivalent to the daily human dose. In the uteri, livers and brains from the patas (n = 3), and endometrium from the macaques (n = 4), TAM-DNA adducts were measurable by TAM-DNA chemiluminescence immunoassay. Average TAM-DNA adduct values for the patas uteri (23 adducts/10(8) nucleotides) were similar to those found in endometrium of the macaques (19 adducts/10(8) nucleotides). Endometrium of macaques exposed to both TAM and low-dose estradiol (n = 5) averaged 34 adducts/10(8) nucleotides. To examine TAM-DNA persistence in the patas, females (n = 3) were exposed to TAM for 3 months and to no drug for an additional month, resulting in low or non-detectable TAM-DNA in livers and uteri. Human endometrial and myometrial samples from women receiving (n = 8) and not receiving (n = 8) TAM therapy were also evaluated. Women receiving TAM therapy averaged 10.3 TAM-DNA adducts/10(8) nucleotides, whereas unexposed women showed no detectable TAM-DNA. The data indicate that genotoxicity, in addition to estrogen agonist effects, may contribute to TAM-induced human endometrial cancer.

Molecular characterization of Salmonella enterica serovar 4,[5],12:i:- DT193 ASSuT strains from two outbreaks in Italy.

Salmonella enterica subsp. enterica serovar 4,[5],12:i:- DT193 is recognized as an emerging monophasic variant of Salmonella Typhimurium in many European countries. Resistance to ampicillin, streptomycin, sulphonamides, and tetracycline (R-type ASSuT) is described as one of the most common profiles of resistance within this clone. Recently, strains presenting such features were isolated from two unrelated outbreaks in Italy. Strains were characterized by pulsed-field gel electrophoresis (PFGE), performed with XbaI, BlnI, and SpeI, and multiple-locus variable-number tandem repeat analysis (MLVA). XbaI-PFGE showed strains related to the two outbreaks as indistinguishable. Conversely, both BlnI-PFGE and MLVA characterized the strains related the two outbreaks as different. XbaI-PFGE identified two profiles, differing by one band, within strains isolated from one of the two outbreaks. Also BlnI-PFGE and MLVA generated different profiles among the strains related to that outbreak. Combining the PFGE profiles obtained by XbaI and BlnI and comparing them with the MLVA profiles, the two methods grouped the same isolates based on identity. Moreover, genomic deletions of the genes included in the operon fljAB, the flanking iroB gene, and the closely located STM2757 gene were investigated. For all strains, the same profile of deletion characterized by the absence of fljA, fljB, and hin genes and the presence of STM2757 and iroB genes was identified. This profile of deletion represents a mixture between two profiles of Salmonella 4,[5],12:i:- described as the "Spanish" and the "U.S." clones. This study demonstrated that although strains of Salmonella 4,[5],12:i:- DT193 ASSuT are highly clonal, minor differences between strains may be seen during the same outbreak by using in parallel PFGE with different restriction enzymes, MLVA, and the analysis of molecular markers related to the operon fljAB. The combination of these different molecular approaches was essential to clarify the epidemiological relationship among the strains.

A rapid and sensitive method to identify and differentiate Salmonella enterica serotype Typhimurium and Salmonella enterica serotype 4,[5],12:i:- by combining traditional serotyping and multiplex polymerase chain reaction.

Salmonella enterica subspecies enterica serotype 4,[5],12:i:- is an emerging serovar considered as a monophasic variant of Salmonella enterica serotype Typhimurium. The antigenic and genetic similarity between Salmonella 4,[5],12:i:- and Salmonella Typhimurium suggests that they may behave in a similar way and represent a comparable threat to public health. As serotyping alone does not necessarily provide for identification of Salmonella 4,[5],12:i:- and its differentiation from Salmonella Typhimurium, a method that combines traditional serotyping and a multiplex polymerase chain reaction has been tested on 208 strains serotyped as Salmonella 4,[5],12:i:-, Salmonella Typhimurium, and similar serovars of serogroup B sharing the same phase-1 antigen "i." For 191 strains, the combined method fully confirmed the results provided by traditional serotyping, whereas for 17 strains of Salmonella 4,[5],12:i:- and Salmonella Typhimurium some inconsistencies emerged between the two methods. The combined method resulted in a more accurate and faster identification of these two relevant serovars.

Fluoroquinolone resistance detection in Campylobacter coli and Campylobacter jejuni by Luminex xMAP technology.

The proportion of Campylobacter spp. isolates that are resistant to fluoroquinolones, the drugs of choice for campylobacteriosis, has been increasing worldwide. We developed an innovative method based on a Luminex xMAP DNA suspension array that allows the identification of Campylobacter species and, simultaneously, the detection of the most common point mutation in the gyrA gene (substitution from threonine 86 to isoleucine 86) that is responsible for fluoroquinolone resistance. Ninety-six Campylobacter coli and Campylobacter jejuni isolates collected from turkeys were first investigated by microdilution test to characterize the antimicrobial resistance patterns. The isolates, amplified for the quinolone resistance determining region of the gyrA gene, were then tested using Luminex suspension array. The reliability of the method was demonstrated by the total concordance between the results obtained using Luminex and those of the sequencing of gyrA polymerase chain reaction products. The genotypic characterization of fluoroquinolone resistance using Luminex was also consistent with the data on phenotypical resistance obtained by microdilution test. The results of this study strongly support the potential of Luminex xMAP technology as an efficient molecular method for the rapid and accurate identification of C. coli and C. jejuni isolates and the characterization of the major determinant of fluoroquinolone resistance.

Dimerization of the human papillomavirus type 16 E2 N terminus results in DNA looping within the upstream regulatory region.

Papillomavirus E2 proteins play a central role in regulating viral gene expression and replication. DNA-binding activity is associated with the C-terminal domain of E2, which forms a stable dimer, while the N-terminal domain is responsible for E2's replication and transactivation functions. The crystal structure of the latter domain revealed a second dimerization interface on E2 which may be responsible for DNA loop formation in the regulatory region of the human papillomavirus (HPV) genome. We investigated the biological significance of the N-terminal dimerization by introducing single amino acid substitutions into the dimerization interface. As expected, these substitutions did not influence the C-terminal dimerization and DNA-binding functions of E2. However, the mutations led to reduced transactivation of a synthetic E2-responsive reporter gene, while HPV DNA replication was unaffected. The effect of the mutations on DNA looping was visualized by atomic force microscopy. While wild-type E2 was able to generate DNA loops, all three mutant E2 proteins were defective in this ability. Our results suggest that N-terminal dimerization plays a role in E2-mediated transactivation, probably via DNA looping, a common mechanism for remote regulation of gene transcription.

The rate of peptidyl-tRNA dissociation from the ribosome during minigene expression depends on the nature of the last decoding interaction.

The expression of some very short open reading frames (ORFs) in Escherichia coli results in peptidyl-tRNA accumulation that is lethal to cells defective in peptidyl-tRNA hydrolase activity. In an attempt to understand the factors that affect this phenotype, we have surveyed the toxicity of a complete set of two-codon ORFs cloned as minigenes in inducible expression vectors. The minigenes were tested in hydrolase-defective hosts and classified according to their degree of toxicity. In general, minigenes harboring codons belonging to the same box in the standard table of the genetic code mediated similar degrees of toxicity. Moreover, the levels of peptidyl-tRNA accumulation for synonymous minigenes decoded by the same tRNA were comparable. However, two exceptions were observed: (i) expression of minigenes harboring the Arg codons CGA, CGU, and CGC, resulted in the accumulation of different levels of the unique peptidyl-tRNAArg-2 and (ii) the toxicity of minigenes containing CUG and UCU codons, each recognized by two different tRNAs, depended on peptidyl-tRNA accumulation of only one of them. Non-toxic, or partly toxic, minigenes prompted higher accumulation levels of peptidyl-tRNA upon deprivation of active RF1, implying that translation termination occurred efficiently. Our data indicate that the nature of the last decoding tRNA is crucial in the rate of peptidyl-tRNA release from the ribosome.