Search Results (64)

Microbial forensics involves analyzing biological evidence to evaluate weaponized microorganisms or their toxins. This study aimed to detect and type Yersinia pestis from four simulated forensic samples—human plasma diluted in phosphate-buffered saline (#24-2), tomato juice (#24-5), grape juice (#24-8), and a surgical mask (#24-10). Notably, samples #24-10 may have contained live bacteria other than Y. pestis. A real-time polymerase chain reaction confirmed the presence of Y. pestis in all samples; however, whole-genome sequencing (WGS) coverage of the Y. pestis chromosome ranged from 0.46% to 97.1%, largely due to host DNA interference and low abundance. To address these limitations and enable strain-level identification, we designed a hybridization-based target enrichment approach focused on multilocus variable number tandem repeat analysis (MLVA). Next-generation sequencing (NGS) using whole-genome amplification revealed that the accuracy of the 25 MLVA profiles of Y. pestis for samples #24-2, #24-5, #24-8, and #24-10 was 4%, 100%, 52%, and 0%, respectively. However, all samples showed 100% accuracy with target-enriched NGS, confirming they all belong to the same strain. These findings demonstrate that a targeted enrichment strategy for MLVA loci can overcome common obstacles in microbial forensics, particularly when working with trace or degraded samples where conventional WGS proves challenging. Full article

A post-COVID surge of Bordetella pertussis was observed globally. China has reported a high level of macrolide-resistant Bordetella pertussis (MRBP) in recent years; however, the epidemiology of MRBP in Hong Kong remains unknown. We retrieved archived B. pertussis isolates from respiratory samples collected at five regional public hospitals in Hong Kong between 2015 and 2024 and tested their minimum inhibitory concentration (MIC) for macrolides and other non-macrolide antibiotics using the Etest method. All isolates were also subjected to whole genome sequencing for genotypic resistance, Multi-locus Antigen Sequence Typing (MLST) and Multi-locus Variable Number of Tandem Repeat Analysis (MLVA) typing. Twenty-nine isolates of B. pertussis were included in the study. All isolates demonstrating phenotypic macrolide resistance harbored the A2047G mutation while showing low MIC to trimethoprim-sulfamethoxazole, doxycycline, levofloxacin, piperacillin-tazobactam and meropenem. In 2023 and 2024, 100% were MRBP and all belonged to the MT28 clone with the ptxP3 antigenic type. The MRBP isolates in Hong Kong were phylogenetically related to those from mainland China during the same period. There was no obvious correlation between macrolide resistance and clinical presentation, laboratory findings, management and outcome. Phylogenetic analysis suggests that MRBP isolates in Hong Kong and mainland China are closely related. Full article

A Streptococcus suis strain isolated from the blood of a patient in Zhejiang Province, China, was analysed using whole-genome sequencing and tested for antimicrobial resistance. The isolated strain was identified as S. suis serotype 2, and classified to ST25 on multilocus sequence typing (MLST). The minimum core genome group of the strain was identified as Group 4, and multilocus variable-number tandem-repeat analysis (MLVA) assigned it as type 2, 4.4, 0, 9, 3, 2, 0, 0. An antimicrobial resistance analysis showed that the strain was resistant to clindamycin, tetracycline, azithromycin, and erythromycin but sensitive to 11 other antibiotics. In a genomic evolution analysis, this isolate clustered on the same branch as North American pig isolate, Chinese pig isolates from Tianjin, and Hubei pig isolates. Full article

Brucellosis is caused by Brucella spp.; it can result in fetal loss and abortion, resulting in economic losses and negative effects on human health. Herein, a cross-sectional study on the epidemiology of Brucella spp. in aborted livestock in Ningxia from 2022 to 2023 was conducted. A total of 749 aborted tissue samples from 215 cattle and 534 sheep were collected from farmers who reported abortions that were supported by veterinarians trained in biosecurity. The samples were analyzed using qPCR and were cultured for Brucella spp. when a positive result was obtained; the samples were speciated using AMOS-PCR. MLST and MLVA were employed for genotype identification. The results demonstrated that 8.68% of the samples were identified as being positive for Brucella spp. based on qPCR results. In total, 14 field strains of Brucella spp. were subsequently isolated, resulting in 11 B. melitensis, 2 B. abortus, and 1 B. suis. being identified via AMOS-PCR. Four sequence types were identified via MLST—ST7 and ST8 (B. melitensis), ST2 (B. abortus), and ST14 (B. suis)—with ST8 predominating. Five MLVA-8 genotypes and seven MLVA-11 genotypes were identified, with MLVA-11 GT116 predominating in livestock. Thus, at least three Brucella species are circulating in aborted livestock in Ningxia. This suggests a significant risk of transmission to other animals and humans. Therefore, disinfection and safe treatment procedures for aborted livestock and their products should be carried out to interrupt the transmission pathway; aborted livestock should be examined to determine zoonotic causes and targeted surveillance should be strengthened to improve the early detection of infectious causes, which will be of benefit to the breeding industry and public health security. Full article

Cryptosporidium is a significant cause of foodborne outbreaks. The 60 kDa glycoprotein gene (gp60) is most often used for subtyping Cryptosporidium species but is not always sufficient for defining clusters and infections sources. The Multilocus Variable-Number Tandem-Repeat Analysis (MLVA) scheme has been developed to better differentiate between subtypes. A cryptosporidiosis outbreak, with 35 cases, was detected in Finland in September 2022. At the same time, in Sweden, three cryptosporidiosis outbreaks, with 107 cases, were detected, leading to international collaboration. In both countries, salad mixes were suspected as being the outbreak source. In the Finnish outbreak, the suspected salad mixes were produced in Sweden. In the Swedish outbreaks, salad mixes from two different producers were suspected. Twenty-nine patient samples which were positive for Cryptosporidium parvum (11 from Finland and 18 from Sweden) were sent for MLVA. The Finnish outbreak samples had different gp60 subtypes and MLVA profiles compared to the Swedish samples. In our investigation, MLVA differentiated C. parvum subtypes in more detail than gp60 typing. MLVA suggested no connection between the Finnish and Swedish outbreaks. A traceback investigation supported this conclusion. To detect outbreaks and identify infection sources, the timely subtyping of patient samples is crucial and should be implemented in routine surveillance and outbreak investigations. Full article

Brucellosis is a zoonotic disease caused by bacteria of the Brucella species. This infectious disease represents a significant public health and economic challenge in many regions of the world, including Ecuador. Brucella abortus is the most common species in cattle. Transmission mainly occurs through direct contact with secretions, aborted fetuses, or contaminated reproductive fluids. In this study, to evaluate the circulating strains of Brucella in continental Ecuador, Brucella strains were cultured and isolated from retromammary lymph nodes and milk samples collected over the past three years from six Ecuadorian provinces within the National Brucellosis Program of Ecuador. Brucella cultures were performed on two specific media, CITA and Farrell, followed by molecular identification using PCR and multiple-locus variable-number tandem repeat analysis 16 (MLVA-16) diagnostic techniques. Out of a total of 25 retromammary lymph nodes collected at slaughterhouses and 50 milk samples obtained from serologically positive animals on farms, Brucella was isolated from 35 milk samples and 19 retromammary lymph node samples and identified as Brucella abortus by PCR. Subsequent MLVA-16 genotyping enabled accurate discrimination among the Brucella strains present in Ecuador. This study confirmed the presence of Brucella abortus strains of biovars 1 and 4 and, for the first time, detected the presence of biovar 2 in Ecuador. The isolation and accurate detection of Brucella, along with the implementation of advanced genotyping techniques, such as MLVA, are crucial for future epidemiological studies, outbreak tracing, and the development of control strategies to mitigate animal and human infection in Ecuador. Full article

This study investigates Brucella ceti infection in marine mammals stranded along the Lisbon and Tagus Valley coast between 2022 and mid-2024, marking the first report of Brucella presence in Portuguese waters. Out of 59 examined marine mammals, B. ceti was isolated in three common dolphins (5.1%), a prevalence rate consistent with previous studies from other coastlines. PCR-based detection indicated a higher infection rate (23.7%), suggesting an underestimation of the prevalence of B. ceti infection in this population. Multi-locus Sequence Typing (MLST) and Multiple-Locus Variable-Number Tandem-Repeat Analysis (MLVA) revealed distinct genetic profiles and close relationships to B. ceti strains from the Atlantic, supporting the hypothesis of specific host-adapted lineages in dolphins. Virulence genes, including those for host interaction (bspE, btpB) and intracellular survival (virB7, vceA), were consistent across isolates, highlighting the pathogenic potential. Additionally, antimicrobial resistance (AMR) genes, such as mprF and efflux proteins (bepC-G), were also identified. These findings underscore the need for further research and surveillance to understand B. ceti transmission, host range, and impacts on Atlantic cetaceans, as well as to develop effective diagnostic and management strategies to mitigate infection risks in marine environments. Full article

Brucellosis is a neglected zoonotic disease that has a significant economic and public health impact, especially in endemic countries. This review delves deeply into brucellosis’s current epidemiological situation and potential sources of livestock infection in Egypt during the last two decades. MLVA-16 and Whole Genome Sequencing based on core-genome SNP analyses confirm the presence of different B. abortus and B. melitensis outbreak strains, both older widely disseminated Brucella strains and newly introduced ones. Despite implementing the test-and-slaughter control strategy over forty years, the disease is still endemic, and different Brucella species circulate among several animal species. The raising of mixed animal species in the same households or farms, exposure to aborted animals, and lack of public awareness about brucellosis transmission are among the main risk factors for increasing livestock brucellosis prevalence in Egypt. Young animals’ voluntary vaccination, lack of a nationwide animal identification system, and uncontrolled animal movement stand beyond the ineffectively applied control strategy and may be subdued by applying mass vaccination to decrease disease prevalence dramatically and target imported camels, domestic pigs, and dogs (housed and stray) in the national control surveillance. Increasing awareness through educational campaigns is compulsory to reduce brucellosis transmission risk to livestock/humans. Full article

Leptospirosis is a widespread disease throughout the world, presenting in severe clinical forms in dogs. The pathogenicity of the different serovars in field infections is not fully documented, and clinical diagnosis is often limited to a combination of serological tests and molecular analyses. The latter, although a fundamental tool, cannot identify the infecting strain without further analysis. This study reports the use of various indirect (microscopic agglutination test, MAT) and direct (microbiological culture, real-time PCR) laboratory techniques, followed by typing protocols (Multi-locus Sequence Typing (MLST), Multiple Loci Variable number tandem repeat Analysis (MLVA), serotyping) that allowed for the identification of the Leptospira serovar Australis in a symptomatic and previously vaccinated dog (vaccine containing heterologous strains). This study reports long-term clinical follow-up (0–640 days) and describes the possible role of the infection in the development of chronic renal failure. This study aims to highlight how a combination of different techniques can be useful to better characterise the environmental circulation of zoonotic agents. Therefore, the identification and isolation of circulating L. strains would facilitate the updating of epidemiological data, enhance the knowledge of pathogenicity and long-term clinical effects, and provide a valuable resource for improving the efficacy of a specific serovar vaccination. Full article

Antimicrobial resistance (AMR) in Mycoplasma hyopneumoniae, the causative agent of Enzootic Pneumonia in swine, poses a significant challenge to the swine industry. This review focuses on the genetic foundations of AMR in M. hyopneumoniae, highlighting the complexity of resistance mechanisms, including mutations, horizontal gene transfer, and adaptive evolutionary processes. Techniques such as Whole Genome Sequencing (WGS) and multiple-locus variable number tandem repeats analysis (MLVA) have provided insights into the genetic diversity and resistance mechanisms of M. hyopneumoniae. The study underscores the role of selective pressures from antimicrobial use in driving genomic variations that enhance resistance. Additionally, bioinformatic tools utilizing machine learning algorithms, such as CARD and PATRIC, can predict resistance traits, with PATRIC predicting 7 to 12 AMR genes and CARD predicting 0 to 3 AMR genes in 24 whole genome sequences available on NCBI. The review advocates for a multidisciplinary approach integrating genomic, phenotypic, and bioinformatics data to combat AMR effectively. It also elaborates on the need for refining genotyping methods, enhancing resistance prediction accuracy, and developing standardized antimicrobial susceptibility testing procedures specific to M. hyopneumoniae as a fastidious microorganism. By leveraging contemporary genomic technologies and bioinformatics resources, the scientific community can better manage AMR in M. hyopneumoniae, ultimately safeguarding animal health and agricultural productivity. This comprehensive understanding of AMR mechanisms will be beneficial in the adaptation of more effective treatment and management strategies for Enzootic Pneumonia in swine. Full article

Background/objectives: Pseudomonas aeruginosa can cause community-acquired infections affecting various body sites. The present retrospective study investigated the genetic diversity of 173 isolates (166 clinical, 7 environmental) of P. aeruginosa collected from clinical pathology laboratories in Abidjan, Côte d’Ivoire (2001–2011). Methods: Multiple-Locus Variable Number of Tandem Repeats (VNTR) Analysis (MLVA) using 13 loci was applied to all isolates and compared to published MLVA data. The antibiotics status of the isolates was compiled when available and compared to published profiles. Results: Among 95 isolates analyzed for their antibiotics status, 14 displayed concerning resistance profiles: five multidrug-resistant (MDR) and nine extensively drug-resistant (XDR). MLVA typing revealed a high genetic diversity (>130 genotypes), with many genotypes represented by a single strain. Notably, thirteen clusters (≥4 related isolates) were observed. Some clusters displayed close genetic relatedness to isolates from France, Korea, and well-studied strains (ST560, LES and PA14). Comparative analysis suggested the presence of international high-risk MDR clones (CC233, CC111) in Côte d’Ivoire. Importantly, MLVA clustering revealed a close relationship of CC235-MDR strains with a locally identified cluster (group 9). Conclusions: These findings support MLVA as a reliable and cost-effective tool for low-resource settings, allowing the selection of relevant strains for future whole genome sequence analyses. This approach can improve outbreak investigations and public health interventions aimed at curbing MDR P. aeruginosa transmission within hospitals and at the national level. Full article

Q fever is a zoonosis caused by Coxiella burnetii, primarily affecting those in close contact with domestic ruminants, the main source of human infection. Coxiella burnetii has also been detected in various wildlife species globally. In Australia, serological and molecular studies have shown exposure to and infection by C. burnetii in macropods, bandicoots, and koalas. However, the extent to which these species contribute to human infection remains unclear. An unpublished public health investigation into a Q fever case in a person involved in koala care could not conclusively link the infection to koalas due to the patient’s broad animal exposure. This study aimed to explore the potential role of koalas in transmitting C. burnetii to humans by investigating the presence of C. burnetii DNA in urogenital tract (UGT) swabs from koalas. DNA was extracted from UGT swabs from koalas in three regions in New South Wales, Australia. An optimised multiplex qPCR assay detected C. burnetii DNA in 2 out of 225 samples (0.89%) at approximately 10 genome equivalents per reaction. Both positive samples amplified all three gene targets. MLVA genotyping identified two distinct C. burnetii genotypes previously isolated from Australian Q fever cases. These findings highlight the need for vaccination against Q fever for those in close contact with koalas. Full article

Brucellosis is one of the most important zoonotic diseases in Greece, causing a significant burden on both human and animal vitality as well as economic loss. The present study was conducted from 2015 to 2022 on 711,415 serum samples by determining the seroepidemiology of Brucellosis among livestock in 24 geographical areas in Greece using the Rose Bengal Test (RBT) and the complement fixation test (CFT) and further performing genetic analysis of Brucella spp. by species-specific real-time PCR and MLVA Brucella analysis. A total of 3086 serum samples from goats, sheep, and cattle showed positive results using the RBT and CFT, and only strongly positive samples (n = 800) were preserved in the Βlood Bank of the Veterinary Laboratory of Brucellosis. From these, 212 sera samples were randomly selected for molecular and genetic analysis. The results indicated that the incidence rate of Brucellosis is higher in cattle herds in comparison with other animal species. Overall, 48 samples tested positive by real-time PCR, of which forty-seven of them were B. abortus and one was B. melitensis. Genetic analysis of two B. abortus samples revealed a common pattern, indicating two Bruce04, two Bruce18, four Bruce07, two Bruce09, three Bruce16, and four Bruce30 for both samples, which, interestingly, were not identical with the known genotypes in the public MLVA Brucella database. Our findings substantiate that animal Brucellosis remains a health issue in Greece, with a stable but apparent incidence rate, and further investigation is needed to fully characterize the newly identified Brucella strains in Greece. Full article

Brucellosis is a disease caused by the Brucella (B.) species. It is a zoonotic disease that affects farm animals and causes economic losses in many countries worldwide. Brucella has the ability to persist in the environment and infect the host at low doses. Thus, it is more important to trace brucellosis outbreaks, identify their sources of infection, and interrupt their transmission. Some countries already have initial data, but most of these data are based on a Multiple-Locus Variable-Number Tandem-Repeat Analysis (MLVA), which is completely unsuitable for studying the Brucella genome. Since brucellosis is an endemic disease in Turkey, this study aimed to examine the genome of Turkish Brucella isolates collected between 2018 and 2020, except for one isolate, which was from 2012. A total of 28 strains of B. melitensis (n = 15) and B. abortus (n = 13) were analyzed using a core-genome single-nucleotide polymorphism (cgSNP) analysis. A potential connection between the Turkish isolates and entries from Sweden, Israel, Syria, Austria, and India for B. melitensis was detected. For B. abortus, there may be potential associations with entries from China. This explains the tight ties found between Brucella strains from neighboring countries and isolates from Turkey. Therefore, it is recommended that strict measures be taken and the possible effects of uncontrolled animal introduction are emphasized. Full article

Tularemia is an acute febrile disease caused by the Gram-negative bacillus Francisella tularensis. Based on genetic and phenotypic characteristics, three subspecies are distinguished: tularensis, holarctica, and mediasiatica. F. tularensis subsp. mediasiatica remains the least studied subspecies. Over the past decade, new foci of distribution of F. tularensis subsp. mediasiatica have been discovered in Russia (Siberia), expanding the possible distribution area by thousands of kilometers. This article provides whole genome single nucleotide polymorphism (wgSNP) and polymorphic tandem repeats (MLVA) analyses of 28 mediasiatica strains isolated between 1965 and 2004 in Kazakhstan. Despite high genetic homogeneity, MLVA with eleven loci (MLVA11) demonstrates a high discriminatory ability (diversity index, 0.9497). The topological structure of the trees based on wgSNP and MLVA is not comparable; however, clustering remains congruent for most outbreaks, with the exception of two strains from one outbreak that are identical in terms of wgSNP but differ at three tandem repeat loci. Based on wgSNP, the strains are assigned to one of the three currently known mediasiatica sublineages, lineage M.I, together with other historical strains maintained in collections in Russia and Sweden. wgSNP shows limited previously unknown genetic diversity, with the M.I lineage size being only 118 SNPs. The wgSNP genotype is not strongly correlated with year and place of isolation. Full article