PCR Protocol Research Articles

Post-mortem detection of hemoplasmas (hemotropic Mycoplasma spp.) in South American fur seal (Arctocephalus australis) sampled in Rio Grande do Sul State, southern Brazil

Hemotropic mycoplasmas are bacteria that attaches to erythrocytes surface, which some species presents zoonotic concerns. In the suborder Pinnipedia, genera Otaria and Arctocephalus are prominent in Brazil. This study investigated the occurrence of hemoplasmas in Arctocephalus sp. and Otaria flavescens found dead along the coast of a Southern Brazilian State. DNA from 135 spleen samples were extracted and subjected to conventional PCR protocols, targeting the 16 S rRNA and 23 S rRNA gene. Three (2.22 %) Arctocephalus australis were positive in the 16 S rRNA gene, and no samples amplified in the 23 S rRNA gene. Samples from this study clustered with Zalophus californianus and Arctocephalus tropicalis mycoplasmas on a Bayesian phylogenetic analysis. Genetic diversity analysis suggested distinct genotypes, indicating A. australis as a new host for hemoplasma, and also a potential putative novel hemoplasma genotype. These findings raises future awareness for pinnipeds conservation, and adds Mycoplasma spp. to be taken into consideration when clinically evaluating rescued animals.

Optimization and Testing of a Commercial Viability PCR Protocol to Detect Escherichia coli in Whole Blood.

Bacteremia, specifically if progressed to sepsis, poses a time-sensitive threat to human and animal health. Escherichia coli is a main causative agent of sepsis in humans. The objective was to evaluate a propidium monoazide (PMA)-based viability PCR (vPCR) protocol to detect and quantify live E. coli from whole blood. We optimized the protocol by adding a eukaryotic-specific lysis step prior to PMA exposure, then used spiking experiments to determine the lower limit of detection (LOD) and linear range of quantification. We also compared the vPCR quantification method to standard colony count of spiked inoculum. Lastly, we calculated percent viability in spiked samples containing 50% live cells or 0% live cells. The LOD was 102 CFU/mL for samples containing live cells only and samples with mixed live and heat-killed cells. The linear range of quantification was 102 CFU/mL to 108 CFU/mL (R2 of 0.997) in samples containing only live cells and 103 CFU/mL to 108 CFU/mL (R2 of 0.998) in samples containing live plus heat-killed cells. A Bland-Altman analysis showed that vPCR quantification overestimates compared to standard plate count of the spiked inoculum, with an average bias of 1.85 Log10 CFU/mL across the linear range when only live cells were present in the sample and 1.98 Log10 CFU/mL when live plus heat-killed cells were present. Lastly, percent viability calculations showed an average 89.5% viable cells for samples containing 50% live cells and an average 19.3% for samples containing 0% live cells. In summary, this optimized protocol can detect and quantify viable E. coli in blood in the presence of heat-killed cells. Additionally, the data presented here provide the groundwork for further development of vPCR to detect and quantify live bacteria in blood in clinical settings.

Indian and Chinese Origins of West African Village Chickens as Revealed by Mitochondrial DNA Partial D-Loop Regions

The study assessed the maternal genetic origins of West African village chickens using a 920-bp mtDNA D-loop region fragment of 127 samples from four populations (Gambia, Togo, Liberia and Nigeria). The mtDNA D-loop region was amplified following standard PCR protocols, using AV1F2: 5′-AGGACTACGGCTTGAAAAGC-3′ and 5′-TGCTTAAGGTTAATTACTGCTG-3′. as the forward and reverse primers respectively. Sequencing was done using Sanger’s dideoxy chain termination method. In addition, 268 sequence samples harvested from Genbank representing Gallus gallus from Africa, Mediterranean and different regions of Asia, were included in the analysis, bringing the total sample size to 395. 24 polymorphic sites and 13 West African chicken haplotypes (WAC1-13) were detected. The evolutionary relationships amongst studied populations and other African and global chicken populations were investigated using MEGA 7 and NETWORK 4.6 softwares. The results grouped 11 of the haplotypes including the dominant one (WAC1) with India, Pakistan and Egypt into haplogroup E, indicating an Indian sub-continental origin and Egypt as an entry point, while the other haplotypes (WAC8 and 10) clustered with haplogroup B widely believed to be of Chinese matrilineage and reported for the first time in West Africa by this study. Haplogroup B is thought to be a heritage of recent introgression of exotic alleles from commercial lines. The study shows that India and China were most likely geographic origins of the matriarchs of the West African village chickens.

A foundation for comparative genomics and evolutionary studies in Nucella lapillus based on complete mitogenome assembly

The Atlantic dog whelk, Nucella lapillus, is a marine snail that exhibits divergent evolution in response to habitat adaptation, resulting in distinct populations at the phenotypic, genotypic, and karyotypic levels. In this study, we utilized short- and long-read NGS data to perform a de novo assembly of the entire mitochondrial genome of N. lapillus and developed a multiplex PCR protocol to sequence most of its length using ONT sequencing. Our analysis revealed a typical circular configuration of 16,474 bp in length with 13 protein-coding genes, 22 different tRNA genes, 2 of them showing two copies, 2 rRNA genes, and a control region. Long-read sequencing enabled us to identify a 1826 bp perfect inverted repeat within the control region. Comparative analysis of the mitogenomes of related species in the Muricidae family revealed a conserved gene configuration for N. lapillus. We found a low genetic diversity, as well as a moderate genetic differentiation among the studied populations. Interestingly, there was no observed differentiation between the two chromosomal races, suggesting either introgression and permanent incorporation of the mitochondrial DNA haplotype from one of the chromosomal races into the other or a slower evolutionary rate of the mtDNAs with respect to that of the karyotypes. Our study serves as a foundation for comparative genomics and evolutionary investigations in this species.

Ultrasensitive sequencing of STR markers utilizing unique molecular identifiers and the SiMSen-Seq method

Massively parallel sequencing (MPS) is increasingly applied in forensic short tandem repeat (STR) analysis. The presence of stutter artefacts and other PCR or sequencing errors in the MPS-STR data partly limits the detection of low DNA amounts, e.g., in complex mixtures. Unique molecular identifiers (UMIs) have been applied in several scientific fields to reduce noise in sequencing. UMIs consist of a stretch of random nucleotides, a unique barcode for each starting DNA molecule, that is incorporated in the DNA template using either ligation or PCR. The barcode is used to generate consensus reads, thus removing errors. The SiMSen-Seq (Simple, multiplexed, PCR-based barcoding of DNA for sensitive mutation detection using sequencing) method relies on PCR-based introduction of UMIs and includes a sophisticated hairpin design to reduce unspecific primer binding as well as PCR protocol adjustments to further optimize the reaction. In this study, SiMSen-Seq is applied to develop a proof-of-concept seven STR multiplex for MPS library preparation and an associated bioinformatics pipeline. Additionally, machine learning (ML) models were evaluated to further improve UMI allele calling. Overall, the seven STR multiplex resulted in complete detection and concordant alleles for 47 single-source samples at 1 ng input DNA as well as for low-template samples at 62.5 pg input DNA. For twelve challenging mixtures with minor contributions of 10 pg to 150 pg and ratios of 1–15% relative to the major donor, 99.2% of the expected alleles were detected by applying the UMIs in combination with an ML filter. The main impact of UMIs was a substantially lowered number of artefacts as well as reduced stutter ratios, which were generally below 5% of the parental allele. In conclusion, UMI-based STR sequencing opens new means for improved analysis of challenging crime scene samples including complex mixtures.

Optimised PCR assays for detecting elusive waterfowl from environmental DNA.

For many aquatic and semiaquatic mammal, amphibian and fish species, environmental DNA (eDNA) methods are employed to detect species distribution and to monitor their presence, but eDNA is much less employed for avian species. Here, we developed primers for the detection of true geese and swan species using eDNA and optimised a PCR protocol for eDNA. We selected taiga bean goose (Anser fabalis fabalis) as our focal (sub)species and sampled water from lakes, from which the presence of taiga bean goose was visually confirmed. To test, if taiga bean goose DNA could be detected among DNA of other goose species, we similarly sampled eDNA from a zoo pond housing several Anatidae species. We were able to detect taiga bean goose DNA in all but one of the tested lakes, including the zoo pond. The primers developed are not species-specific, but rather specific to the genus Anser, due to the close relatedness of Anser species, which prevented the development of species-specific primers and the use of, for example, quantitative PCR. We also developed eDNA primers for Branta species and Cygnus species and tested these primers using the same samples. Canada goose (B. canadensis) and barnacle goose (B. leucopsis) DNA were only detected in the zoo pond (in which they were present), as the sampled natural lakes fall outside the range of these species. We detected whooper swan (C. cygnus) DNA in three lakes and the zoo pond (in which the species was present). The eDNA method presented here provides a potential means to monitor elusive goose species and to study the co-occurrence of large waterfowl.

New Genetic Determinants for qPCR Identification and the Enumeration of Selected Lactic Acid Bacteria in Raw-Milk Cheese.

Lactic acid bacteria (LAB) play an important role in the ripening of cheeses and contribute to the development of the desired profile of aroma and flavor compounds. Therefore, it is very important to monitor the dynamics of bacterial proliferation in order to obtain an accurate and reliable number of their cells at each stage of cheese ripening. This work aimed to identify and conduct a quantitative assessment of the selected species of autochthonous lactic acid bacteria from raw cow's milk cheese by the development of primers and probe pairs based on the uniqueness of the genetic determinants with which the target microorganisms can be identified. For that purpose, we applied real-time quantitative PCR (qPCR) protocols to quantify Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, and Lactococcus lactis subsp. cremoris cells in cheese directly after production and over three-month and six-month ripening periods. While L. lactis subsp. cremoris shows good acidification ability and the ability to produce antimicrobial compounds, L. delbrueckii subsp. bulgaricus has good proteolytic ability and produces exo-polysaccharides, and S. thermophilus takes part in the formation of the diacetyl flavor compound by metabolizing citrate to develop aroma, they all play an important role in the cheese ripening. The proposed qPCR protocols are very sensitive and reliable methods for a precise enumeration of L. delbrueckii subsp. bulgaricus, S. thermophilus, and L. lactis subsp. cremoris in cheese samples.

Detection of Hbv Dna By Direct Pcr Protocol And Nested Pcr Protocol And Comparing Them. In Chronic Patients and Healthy Carriers of Hepatitis B Virus.

Hepatitis B virus is a major public health problem, and the outcome of infection depends on the effectiveness of the virus's interaction with the host, and in particular on the strength of the natural and conditioned response of humoral and cellular immunity. This study was conducted to assess the clinical status, genetic immunity and viral (viral) traits of Iraqi patients with chronic hepatitis B virus and healthy carriers of hepatitis B surface antigen, as well as to show the relationship between the immune status and the chronic condition of the disease. The study included (60) patients with chronic hepatitis B virus type (B) and (60) healthy hepatitis B surface antigen carriers who attended the Teaching Hospital for Gastrointestinal and Liver Diseases in Nasiriya City Al-Hussein Teaching Hospital, Thalassemia Center and the Central Blood Bank in Nasiriya Citysouthern of Iraq. for the period from the first period. From February 2022 until the end of February 2022, the average age of patients infected with chronic hepatitis B virus was 64.5 years, and the average age of healthy carriers of hepatitis B surface antigen was 35. 1 year, and the ratio of males to females was (12.2:) for chronic patients and (13.6:) for carriers of the disease. As for the deoxyribonucleic acid, it was (100%) in chronic patients and healthy carriers using the Nested type polymerase chain reaction, and at a rate of (30.0%) and (46.7%) in chronic patients and healthy carriers, respectively using the polymerase chain reaction technique. direct with a significant difference between the two interactions. The current results confirm that a nested PCR protocol to detect HBV DNA is a more sensitive way of detecting HBV in Iraq CHB patients and carriers. Recent advanced techniques such as nucleic acid assays, quantitative assays for viral load and RT-PCR for Iraqi viral hepatitis laboratories are recommended.

Ticks (Acari: Ixodidae) and rickettsiae associated with wild boars in a rural area of Minas Gerais, Brazil

Wild boars or feral pigs are classified by the Brazilian Institute for the Environment and Renewable Resources (IBAMA) in “Category I of invasive exotic species”. They cause economic losses, harm the environment, serve as hosts and reservoirs for several zoonotic disease agents, and provide a blood meal for tick species that act as vectors for zoonotic diseases. The objective of this study was to identify tick species on wild boars, assess host-seeking ticks in the related environment, and identify other potential tick hosts coexisting with wild boars on a farm located in the state of Minas Gerais, southeastern Brazil. Additionally, the study aimed to determine the presence of rickettsiae in these arthropods and assess the exposure of wild boars to rickettsiae species from the Spotted Fever Group and Rickettsia bellii through serology. A total of 3585 host-seeking ticks from three species (Amblyomma sculptum – 41.58%; Amblyomma dubitatum – 0.39% and Rhipicephalus microplus – 0.05%) were collected in the environment and A. sculptum was the most abundant species. Thirty-one wild boars were evaluated, resulting in the collection of 415 ticks, all of which were A. sculptum. Rickettsia DNA was not detected in samples of A. sculptum and R. microplus from the environment or in A. sculptum ticks from wild boars. However, all A. dubitatum ticks (n = 14) had Rickettsia bellii DNA confirmed by the species-specific PCR protocol. Out of the 31 serum samples from wild boars, 24 reacted with at least one Rickettsia antigen. Among these, seven individuals exhibited a reaction to a probable homologous antigen (PHA) of three rickettsiae species: R. rickettsii (n = 3), R. amblyommatis (n = 3) and R. rhipicephali (n = 1). Despite the high prevalence of seroreactivity, titers were low, indicating limited exposure to Rickettsia spp. Camera traps generated 874 animal records, capturing a total of 1688 individuals. At least 11 species of birds and 14 species of mammals (12 wild and two domestic) shared the environment with wild boars and potentially shared ticks with them. These findings provide baseline information for understanding the sharing of ticks and tick-borne pathogens between wild boars and other animals within the Cerrado biome. Further studies are necessary to monitor the potential and actual risk of wild boars to harbor infected ticks and their role in the transmission and maintenance cycle of Rickettsia spp.

Evaluation of metal ions and DNA recovery from the surface of fired and unfired brass ammunition to improve STR profiling.

Interest in recovering DNA from the surface of ammunition evidence for genotyping has increased over the past few years. Numerous studies have examined a variety of methods to maximize DNA recovery from these types of challenging samples, but successful DNA profiling has been inconsistent. Low amounts of DNA and PCR inhibition due to metal ions have been suggested as the leading causes of poor results; however, no study quantitatively examined the presence of metal ions at various stages of the DNA analysis workflow from DNA collection through to amplification. In this study, the effectiveness of six different DNA collection and purification methods commonly used by forensic laboratories to process brass ammunition for DNA evidence was investigated. The amount of copper, zinc, and other metals co-recovered from fired and unfired brass casings during DNA collection (using numerous soaking, swabbing, and direct PCR protocols) was quantified via Inductively Coupled Plasma - Optical Emission Spectrometry (ICP-OES). This same panel of metals was subsequently quantified after DNA lysis and purification steps. Results demonstrated that low amounts of DNA, DNA damage, and degradation are more detrimental to STR typing results than PCR inhibition, as metal ions were successfully removed by all DNA purification methods tested. Furthermore, the use of metal ion chelators increased the amount of DNA recovered and number of reportable STR alleles. This research informs the forensic community on the most effective way to collect and process trace amounts of biological material from brass ammunition and similar evidence.

Three novel multiplex PCR assays for rapid detection of virulence, antimicrobial resistance, and toxin genes in Acinetobacter calcoaceticus-baumannii complex species.

The Acinetobacter calcoaceticus-baumannii (ACB) complex is an often-overlooked group of nosocomial pathogens with a significant environmental presence. Rapid molecular screening methods for virulence, antimicrobial resistance, and toxin (VAT) genes are required to investigate the potential pathogenicity of environmental isolates. This study aimed to develop and apply novel ACB complex-specific multiplex PCR (mPCR) primers and protocols for the rapid detection of eight VAT genes. We optimized three single-tube mPCR assays using reference DNA from ACB complex and other Acinetobacter species. These assays were then applied to detect VAT genes in cultured ACB complex isolates recovered from clinical and environmental sources. Widespread detection of VAT genes in environmental isolates confirmed the validity, functionality, and applicability of these novel assays. Overall, the three newly developed ACB complex species-specific mPCR assays are rapid and simple tools that can be adopted in diagnostic and clinical lab settings. The detection of VAT genes in environmental isolates suggests that environmental niches could serve as a reservoir for potentially pathogenic ACB complex and warrants further investigation. The newly developed mPCR assays are specific, sensitive, and efficient, making them well-suited for high-throughput screening in epidemiological studies and evaluating the potential pathogenicity of ACB complex recovered from various sources.

Validation of a real-time polymerase chain reaction for the detection and quantification of the nucleic acid of Histoplasma from equine clinical samples.

Histoplasmosis is a neglected yet major cause of morbidity and mortality in both equids and people in resource-scarce settings. One of the major hindrances to the control of histoplasmosis is a lack of readily available diagnostic tests. Tests are needed to support clinical decision-making and to be applied in population-based research to further understand this disease in situ. This paper reports, for the first time, the validation and application of a qPCR to detect Histoplasma directly from equine clinical samples, bypassing the need to culture this notoriously difficult organism. We report and comment on the performance of the qPCR in comparison with our previously developed nested PCR.

Development and validation of a universal primer pair for the taxonomic and phylogenetic studies of vertebrates.

Recent studies in the field of molecular identification have described 16S rRNA gene as a highly informative fragment of mitochondrial DNA for species discrimination. This study presents a newly developed universal primer pair yielding an approximately 350bp fragment of mitochondrial 16S rRNA, variable enough to encompass and identify all vertebrate classes. The primers were designed by aligning and analyzing over 1500 16S rRNA sequences downloaded from the NCBI nucleotide database. A total of 93 vertebrate species, spanning 27 orders and 55 families, were PCR-amplified to validate the primers. All the target species were successfully amplified and identified when aligned with reference sequences from the NCBI nucleotide database. Using the Kimura 2-parameter model, low intra-species genetic divergence of the target region was observed - from 0 to 4.63%, whereas relatively higher inter-species genetic divergence was observed, ranging from 4.88% to 69.81%. Moreover, the newly developed primers were successfully applied to a direct PCR protocol, making the workflow very cost-effective, time-saving and less laborious in comparison to conventional PCR. The short length, high variability and conserved priming sites of the target fragment across all vertebrate species make it a highly desirable marker for species identification and discrimination.

Identifying Molecular Markers for Ficus erecta Thunb. Based on Complete Plastome Sequences of Korean Figs (Ficus L., Moraceae)

Plastome sequences are crucial in plant studies due to their role in examining genomic evolution, understanding phylogenetic relationships, and developing molecular markers. Despite the collection of information about Korean figs, their genomic data remain underexplored. We utilize next-generation sequencing and PCR techniques to investigate genomic data and to develop and validate molecular markers. In this study, we characterize the complete plastomes of Korean figs: F. erecta, F. erecta var. sieboldii, F. sarmentosa var. nipponica, and F. sarmentosa var. thunbergii, which range in length from 160,276 to 160,603 bp. These genomes comprise 78 plastid protein-coding genes, 30 tRNA, and four rRNA, with the exception of one pseudogene, infA. We discovered that F. erecta and F. erecta var. sieboldii share identical plastome sequences. Phylogenomic analysis indicates the monophyly of Ficus, although the relationships among its subgenera remain unclear. We discovered that Ficus possesses 467 molecular diagnostic characters in its plastid protein-coding genes compared to other Moraceae groups, and F. erecta exhibits 33 molecular diagnostic characters. Single nucleotide polymorphisms in ndhD, petA, and rbcL were effectively used to develop molecular markers for distinguishing F. erecta from other figs. Additionally, we provided a straightforward PCR protocol for utilizing these newly developed molecular markers.

High diversity, novel genotypes, and vertical transmission of hemotropic Mycoplasma in micromammals

Hemotropic mycoplasmas (hemoplasmas) are emerging zoonotic pathogens. Micromammals have received little attention as hosts for hemoplasmas despite their ubiquitous presence, high population abundances, and close association with humans. A PCR protocol targeting a fragment of the 16 S rRNA gene and direct sequencing in blood samples of 189 adult specimens and 35 fetuses belonging to three species of Eulipotyphla (shrews) and seven species of Rodentia, captured in three ecologically diverse habitats in North-Eastern Spain (Steppe, High Mountain, Mediterranean) yielded and occurrence of 26%, including 36% of 39 shrews and 23% of 150 rodents. Sequencing revealed the presence of 14 nucleotide sequence types (ntST) among the 56 readable sequences. In general, each ntST was associated with a given host species, although in some cases, the same ntST was sequenced in different species (chiefly rodents). Most ntST were closely related to rodent and/or bat hemoplasmas, but one was identical with Mycoplasma haemocanis/haemofelis, and others can be considered novel genotypes. High sequence diversity was detected in rodents, whereas in the white-toothed shrew (Crocidura russula), 9/11 sequences from two distant areas were identical. Phylogenetic and network analyses classified our sequences in different clades including hemoplasmas of rodents, carnivores, bats, and humans. Twelve of the fetuses (34.2%) of 9/12 litters (75.0%) of shrews and rodents were hemoplasma-positive, indicating frequent vertical transmission. Our study contributes to expanding our knowledge about the distribution, diversity, and transmission of hemoplasmas.

Pluck-pools as diagnostic samples for detecting porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 in porcine abortion material and stillbirths

Investigating infectious agents in porcine abortion material and stillborn piglets poses challenges for practitioners and diagnostic laboratories. In this study, pooled samples of individual reference organs (thymus and heart) from a total of 1000 aborted fetuses and stillborn piglets were investigated using quantitative PCR protocols for porcine reproductive and respiratory syndrome virus 1 (PRRSV-1) and porcine circovirus type 2 (PCV2). Simultaneously, a pluck-pool containing equivalent portions of fetal thymus, heart, and lung tissue was collected, frozen at − 20 °C, and re-analyzed when a certain amount of either PRRSV-1 RNA or PCV2 DNA was detected in individual reference organs. Thirteen pluck-pools were assessed for PRRSV-1, all being PCR-positive. For PCV2, 11 of 15 pluck-pools investigated were PCR-positive. In all pluck-pools testing negative, viral loads in individual pools were low. This study indicates that pluck-pools can be valuable diagnostic material and the consolidation of multiple organs through a single RNA/DNA extraction optimizes the utilization of available laboratory resources. Additional research is required to assess the feasibility of follow-up investigations and to accurately define criteria for interpretation of viral loads in a clinical context.

Saliva direct PCR protocol for HLA-DQB1*02 genotyping.

Celiac disease (CD) is an immune disorder, that is triggered by gluten ingestion in genetically predisposed individuals. The HLA-DQB1*02 allele is the main predisposing genetic factor and a candidate for first-line genotyping screening. We designed and validated a simple, DNA purification-free PCR protocol directly from crude saliva, enabling the detection of the DQB1*02 allele. This assay also distinguishes homozygous from heterozygous carriers. We propose this method for use in mass screening and/or epidemiological studies.

Standardisation of an In-house Multiplex PCR Assay for Identifying Diffusely Adherent E. coli and ESBL Producing Resistant Isolates

We aimed to develop a multiplex PCR (mPCR) assay utilizing the afaC gene for the detection of DAEC pathotypes and the blaTEM gene for the identification of ESBL production in clinical E. coli isolates along with an Internal Amplification Control (IAC) to rule out false negative test results. Monoplex PCR assays were first established for the afaC gene and blaTEM gene using 60 characterized E. coli isolates from various clinical samples. Subsequently, an mPCR assay was designed to detect both the genes simultaneously along with an IAC to rule out false negative reactions. The effectiveness of this assay was validated using 80 additional clinical isolates. The overall occurrence of DAEC in the study was found to be 0.7% (1/140). ESBL production was detected in 40.7% of the tested isolates, indicating a concerning prevalence of drug-resistant strains. This study emphasizes the value of an in-house mPCR assay as a crucial tool for simultaneously identifying DAEC and ESBL E. coli strains. The inclusion of an IAC in the PCR protocol bolstered the assay’s reliability. This innovation offers a vital resource for effective infection management and contributes to the comprehension of pathogenicity and resistance mechanisms in clinical E. coli isolates.

Amplifying PCR productivity and environmental sustainability through shortened cycling protocols

Since its inception in the 1980s, advancements in PCR technology using improved thermal cyclers, engineered DNA polymerases and commercial master mixes, have led to increased PCR productivity. Despite these advancements, PCR cycling protocols have largely remained unchanged over the same period. This study aimed to systemically evaluate the effect of reduced PCR cycling parameters on amplicon production. The 1466bp fragment from the 16S rRNA gene present in low-, medium- and high-CG bacteria was amplified using three commercially available PCR master mixes. The shortest cycling parameters required to successfully amplify the 16S fragment from all bacteria and master mixes comprised 30-cycles of 5 s denaturation, 25 s annealing, and 25 s extension. While all produced an amplicon with sufficient yield to enable downstream sequence analysis, the PCRBIO Ultra Mix in conjunction with the shortened parameters was found to achieve the highest amplicon yield across low-, medium- and high CG bacteria. Comparing the run times to that of a typical 16S PCR protocol, the shortened cycling parameters reduced the program duration by 46 % and consumed 50 % less electricity, translating into increased productivity and helping to improve laboratory environmental sustainability.

Wild Birds as Drivers of Salmonella Braenderup and Multidrug Resistant Bacteria in Wetlands of Northern Italy

In this study, the antimicrobial resistance profiles of bacterial strains obtained from wild avian species recovered in wetlands of Northern Italy were described. Cloacal swabs collected from 67 aquatic birds, hunted or found dead in two private hunting grounds, were submitted to microbiological investigations and antimicrobial susceptibility testing using the Vitek 2 system, while specific PCR protocols were applied to screen for genes associated with the resistance. One hundred fifty-seven bacterial strains were characterized. The most frequent isolates were Enterococcus faecalis (36/157; 22.9%) and Escherichia coli (23/157; 14.6%). Seventy-seven isolates (77/157; 49%) were resulted resistant to at least one antibiotic, and eight isolates (8/157; 5%) were classified as multidrug resistant bacteria. Resistance for critically important antibiotics (linezolid, vancomycin, carbapenems, third-generation cephalosporins, and fluoroquinolones) was also described. Salmonella spp. was obtained from a Eurasian teal (Anas crecca), and it was subsequently analyzed by whole genome sequencing, revealing the serovar Salmonella Braenderup ST22. The phylogenetic analysis, performed with all ST22 described in 2021 and 2022, placed the strain under study in a large clade associated with human salmonellosis cases. These results suggest that migratory aquatic birds may be considered as relevant carriers of critically important antibiotic resistant bacteria and zoonotic food-borne pathogens potentially able to impact public health.