Search Results (40)

Legionella pneumophila (L. pneumophila), the primary causative agent of Legionnaires’ disease, is a waterborne bacterial pathogen that poses significant public health concern. This opportunistic pathogen commonly inhabits both natural and man-made water systems, particularly drinking water distribution systems (DWDSs), where it can proliferate and pose a risk to human health. In this study, we evaluated the potential of Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) for rapid and accurate subtyping of L. pneumophila. Our analysis included 70 L. pneumophila strains collected from the Middle East, representing one of the largest and most comprehensive MALDI-TOF MS-based subtyping of strains from this geographically underrepresented region. These strains, representing three Multi-Locus Variable Number Tandem Repeat Analysis (MLVA-8) genotypic groups (GT4, GT6, and GT15), have been extensively characterized in previous studies for their virulence traits, cytotoxicity patterns, and antimicrobial susceptibility profiles. Our findings revealed distinct genotype-associated spectral signatures with 30 discriminatory m/z peaks (p ≤ 0.005). These markers enabled accurate genotype-level classification, achieving over 85% classification accuracy with a Random Forest model and over 71% accuracy using a Decision Tree algorithm. Importantly, the m/z peak at 5358 was uniquely present in the GT15 strains, whereas m/z 5353 was consistently detected in both GT4 and GT6 isolates, demonstrating the potential of specific mass peaks to serve as reliable genotype markers. Furthermore, GT15 strains consistently formed a separate cluster in both Principal Component Analysis (PCA) and hierarchical analyses, whereas GT4 and GT6 exhibited partial overlap, reflecting their exceptionally high genomic similarity. Full article

Several different pathogens, including Escherichia coli, are strongly associated with calf diarrhoea. The population diversity of intestinal E. coli within each diarrhetic calf and between diarrhetic calves is not well understood. In the present study, 391 faecal samples were obtained during 2023–2024 from Danish dairy calves with diarrhoea. Semi-quantified growth estimates of E. coli after culturing did not reflect the diarrhetic grade nor whether E. coli was the only pathogen observed in the sample. From each sample, five isolates were subjected to multiple-locus variable-number tandem repeat analysis (MLVA) and revealed that 70% of faecal samples contained more than one type of E. coli. Genotyping, sequence typing and in silico serotyping showed a large diversity of E. coli between faecal samples. Surprisingly, isolates with a genotype representing mixed features of Diffusely adhering E. coli/Extraintestinal pathogenic E. coli were found in 25% of the isolates, while the classic Enterotoxigenic E. coli genotype was only observed in 5% of the isolates, and only 4% of the faecal samples were positive for E. coli F5 (K99) fimbriae, as determined by PCR. In conclusion, a diverse population of (non-F5) E. coli is associated with diarrhoea in calves. High genomic diversity of E. coli within samples needs to be considered when selecting only one isolate for antimicrobial resistance profiling and vaccination measurements. 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

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

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

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

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

Ochrobactrum anthropi (O. anthropi) is found in water, soil, plants and animals. Even though it has low virulence, it has increasingly been found to cause a number of infectious diseases in people with low immunity. The identification of O. anthropi mainly uses biochemical methods, such as the API 20NE or Vitek-2. The typing studies of O. anthropi have mainly utilized PFGE, rep-PCR, AFLP, 16s rDNA sequencing, RecA-PCR RFLP, and MALDI-TOF MS. This study aims to evaluate the polymorphisms of variable-number tandem-repeats (VNTRs) within genomic DNA of O. anthropi strains. The tandem repeats (TRs) in genomic DNA are discovered using Tandem Repeat Finder software (version 4.09). Twelve different VNTRs are designated and assigned to the nomenclature. The primers for PCR of 12 loci are designed. The PCR product size is converted to the number of tandem repeats in every locus. The relatedness of 65 O. anthropi strains from geographically different countries are analyzed by means of 12-variable-number tandem-repeat analysis(MLVA-12). A total of 51 different genotypes are found in 65 O. anthropi strains. These strains, which were collected from the same environmental samples, hospitals, and countries, are clustered within the same or closely genotypes. The MLVA-12 assay has a good discriminatory power for species determination, typing of O. anthropi, and inferring the origin of bacteria. Full article

The control and eradication of brucellosis represents a critical objective for Veterinary and Health Authorities across several countries globally. Efficient surveillance programs play a pivotal role in detecting and managing outbreaks. Epidemiological investigations significantly benefit from standardized and efficient molecular typing techniques and analytical tools, enabling public health laboratories to identify the origin of outbreaks. This study aimed to sequence Brucella spp. strains isolated in Iraq from different ruminant species to verify their molecular epidemiological correlations and, above all, to shed a light on how these Iraqi isolates are positioned in the phylogenetic context of Brucella spp. The 35 isolates under study were from abortion, milk, placenta, and the fetal membranes of sheep, cattle, and buffalo. Genotyping involved various techniques: MLVA-16, Whole Genome Sequencing, MLST, and cgMLST. All the Iraqi isolates from our study clustered in MLVA-16 within the East Mediterranean clade, and all but one grouped together in the same branch of the MST tree. MST analysis showed the minimum distance of one allele between the studied isolates, except for one strain from buffalo, which was positioned farther away from the rest of the isolates. In cgMLST, the majority of strains grouped within a large cluster predominantly comprising genotypes from the Middle East. The application of different control measures in different territories based on molecular epidemiological studies would increase the chances of maximizing public health benefits and minimizing the spread of infection to disease-free or lower prevalence areas. Full article

The obligate intracellular bacterial pathogen Coxiella burnetii has been identified in a few species of marine mammals, some of which are showing population declines. It has been hypothesized that C. burnetii in marine mammals is a distinct genotype that varies significantly from the typical terrestrial genotypes. It appears to lack an IS1111. Isolates originating from Australian marine animals have a distinctly non-Australian profile of multiple-locus variable-number tandem-repeat analysis (MLVA). Extracted Coxiella DNA of Australian fur seal placental origin was sequenced using the Novaseq platform. Illumina 150 bp paired-end reads were filtered and trimmed with Trimgalore. The microbial community present in the sequenced genome was evaluated with Kraken and Bracken software using the NCBI database. A phylogenetic analysis was performed using 1131 core genes. Core genes were identified using Panaroo and inputted into Iqtree to determine the maximum-likelihood tree. A second phylogenetic tree was created using Rickettsiella grylii and using seven housekeeping genes. Results were compared with the C. burnetii Nine Mile RSA439 virulent genome. This new Australian marine mammal isolate of Coxiella (PG457) appears to be a novel genotype that lacks IS1111 and has a distinct MLVA signature (ms26, ms27, ms28, ms30, and ms31). The presence of genes for multiple virulence factors appears to give this genotype sufficient pathogenicity for it to be considered a possible causative agent of abortion in Australian fur seals as well as a potential zoonotic risk. Full article

Legionella pneumophila is an environmental bacterium and clinical pathogen that causes many life-threating outbreaks of an atypical pneumonia called Legionnaires’ disease (LD). Studies of this pathogen have focused mainly on Europe and the United States. A shortage in L. pneumophila data is clearly observed for developing countries. To reduce this knowledge gap, L. pneumophila isolates were studied in two widely different geographical areas, i.e., the West Bank and Germany. For this study, we sequenced and compared the whole genome of 38 clinical and environmental isolates of L. pneumophila covering different MLVA-8(12) genotypes in the two areas. Sequencing was conducted using the Illumina HiSeq 2500 platform. In addition, two isolates (A194 and H3) were sequenced using a Pacific Biosciences (PacBio) RSII platform to generate complete reference genomes from each of the geographical areas. Genome sequences from 55 L. pneumophila strains, including 17 reference strains, were aligned with the genome sequence of the closest strain (L. pneumophila strain Alcoy). A whole genome phylogeny based on single nucleotide polymorphisms (SNPs) was created using the ParSNP software v 1.0. The reference genomes obtained for isolates A194 and H3 consisted of circular chromosomes of 3,467,904 bp and 3,691,263 bp, respectively. An average of 36,418 SNPs (min. 8569, max. 70,708 SNPs) against our reference strain L. pneumophila str. Alcoy, and 2367 core-genes were identified among the fifty-five strains. An analysis of the genomic population structure by SNP comparison divided the fifty-five L. pneumophila strains into six branches. Individual isolates in sub-lineages in these branches differed by less than 120 SNPs if they had the same MLVA genotype and were isolated from the same location. A bioinformatics analysis identified the genomic islands (GIs) for horizontal gene transfer and mobile genetic elements, demonstrating that L. pneumophila showed high genome plasticity. Four L. pneumophila isolates (H3, A29, A129 and L10-091) contained well-defined plasmids. On average, only about half of the plasmid genes could be matched to proteins in databases. In silico phage findings suggested that 43 strains contained at least one phage. However, none of them were found to be complete. BLASTp analysis of proteins from the type IV secretion Dot/Icm system showed those proteins highly conserved, with less than 25% structural differences in the new L. pneumophila isolates. Overall, we demonstrated that whole genome sequencing provides a molecular surveillance tool for L. pneumophila at the highest conceivable discriminatory level, i.e., two to eight SNPs were observed for isolates from the same location but several years apart. Full article

The genotyping of the multidrug-resistant Klebsiella pneumoniae species complex is essential to identify outbreaks and to track their source and spread. The aim of this study was to improve and extend the typeability, availability, cost and time efficiency of an existing multi-locus VNTR analysis (MLVA). A modified scheme (MLVA8+) was adopted and validated for strain-level differentiation of the three Klebsiella species involved in human pathology. A diverse set of 465 K. pneumoniae clinical isolates from 22 hospitals and 3 outpatient laboratories in Bulgaria were studied, where 315 were carbapenem-resistant. The MLVA8+ typeability was significantly improved and the typing data were validated against 158 isolates which were previously typed by WGS. The MLVA8+ results were highly concordant with the classic 7-locus MLST and the novel K. variicola MLST, but had greater congruency coefficients (adjusted Wallace). A major advantage was the differentiation of the hybrid cluster ST258 into its corresponding clades. Furthermore, the applicability of MLVA8+ was demonstrated by conducting a retrospective investigation of the intra-hospital spread of blaKPC-, blaNDM- and blaOXA-48-like producers. The MLVA8+ has improved utility and extended typing scope to K. variicola and K. quasipneumoniae, while its cost and time-to-result were reduced. Full article