Phenotypic and genotypic characterization of resistance and virulence in Pseudomonas aeruginosa isolated from poultry farms in Egypt using whole genome sequencing.

Pseudomonas aeruginosa (P. aeruginosa) is an ESKAPE pathogen that can quickly develop resistance to most antibiotics. This bacterium is a zoonotic pathogen that can be found in humans, animals, foods, and environmental samples, making it a One-Health concern. P. aeruginosa threatens the poultry industry in Egypt, leading to significant economic losses. However, the investigation of this bacterium using NGS technology is nearly non-existent in Egypt. In this study, 38 isolates obtained from broiler farms of the Delta region were phenotypically investigated, and their genomes were characterized using whole genome sequencing (WGS). The study found that 100% of the isolates were resistant to fosfomycin and harbored the fosA gene. They were also resistant to trimethoprim/sulfamethoxazole, although only one isolate harbored the sul1 gene. Non-susceptibility (resistant, susceptible with increased dose) of colistin was observed in all isolates. WGS analysis revealed a high level of diversity between isolates, and MLST analysis allocated the 38 P. aeruginosa isolates into 11 distinct sequence types. The most predominant sequence type was ST267, found in 13 isolates, followed by ST1395 in 8 isolates. The isolates were susceptible to almost all tested antibiotics carrying only few different antimicrobial resistance (AMR) genes. Various AMR genes that confer resistance mainly to ß-lactam, aminoglycoside, sulfonamide, and phenicol compounds were identified. Additionally, several virulence associated genes were found without any significant differences in number and distribution among isolates. The majority of the virulence genes was identified in almost all isolates. The fact that P. aeruginosa, which harbors several AMR and virulence-associated factors, is present in poultry farms is alarming and threatens public health. The misuse of antimicrobial compounds in poultry farms plays a significant role in resistance development. Thus, increasing awareness and implementing strict veterinary regulations to guide the use of veterinary antibiotics is required to reduce health and environmental risks. Further studies from a One-Health perspective using WGS are necessary to trace the potential transmission routes of resistance between animals and humans and clarify resistance mechanisms.

Genotypic peculiarities of a human brucellosis case caused by Brucella suis biovar 5.

Human brucellosis cases are rare in non-endemic countries, such as Germany, where infections are predominantly caused by Brucella melitensis. The German National Reference Laboratory for Bovine, Porcine, Ovine and Caprine Brucellosis received a suspected Brucella sp. isolate from a patient for identification. Bacteriological tests and PCR-based diagnostics showed the isolate to be B. suis, but did not yield cohesive results regarding the biovar. Whole genome sequencing and subsequent genotyping was employed for a detailed characterization of the isolate and elucidating the reason for failure of the diagnostic PCR to correctly identify the biovar. The isolate was found to be B. suis bv. 5, a rare biovar with limited geographical distribution primarily found in the Northern Caucasus. Due to a deletion in one of the target regions of the diagnostic PCR, the isolate could not be correctly typed. Based on in silico genotyping it could be excluded that the isolate was identical to one of the B. suis bv. 5 reference strains. Here, we report a rare case of a B. suis bv. 5 field isolate. Furthermore, by reporting this finding, we want to make practitioners aware of possible misinterpretation of PCR results, as it cannot be excluded that the detected deletion is common among the B. suis bv. 5 community, as there is currently a lack of field isolates.

Brucella suis biovar 1 infection in a dog with orchitis in Germany.

In 2021, a case of canine brucellosis diagnosed in a dog with orchitis was presented to a veterinary practice in Germany. Serological testing excluded Brucella (B.) canis as a causative agent, but molecular analysis revealed the presence of B. suis biovar 1. Since biovar 1 is not endemic in Europe and the dog had no history of travel to endemic areas, a comprehensive epidemiological investigation was conducted using whole genome sequence data to determine the source of infection. We describe the clinical progress of the animal and the potential infection of a veterinary clinic employee. The findings highlight the importance of considering less common Brucella species as possible causes of canine brucellosis. The data also emphasize that it is quite challenging to identify Brucella species in a routine diagnostic laboratory and to conduct epidemiological investigations to unveil possible transmission routes.

Genotype diversity of brucellosis agents isolated from humans and animals in Greece based on whole-genome sequencing.

BACKGROUND: Brucellosis is a zoonotic disease whose causative agent, Brucella spp., is endemic in many countries of the Mediterranean basin, including Greece. Although the occurrence of brucellosis must be reported to the authorities, it is believed that the disease is under-reported in Greece, and knowledge about the genomic diversity of brucellae is lacking. METHODS: Thus, 44 Brucella isolates, primarily B. melitensis, collected between 1999 and 2009 from humans and small ruminants in Greece were subjected to whole genome sequencing using short-read technology. The raw reads and assembled genomes were used for in silico genotyping based on single nucleotide substitutions and alleles. Further, specific genomic regions encoding putative virulence genes were screened for characteristic nucleotide changes, which arose in different genotype lineages. RESULTS: In silico genotyping revealed that the isolates belonged to three of the known sublineages of the East Mediterranean genotype. In addition, a novel subgenotype was identified that was basal to the other East Mediterranean sublineages, comprising two Greek strains. The majority of the isolates can be assumed to be of endemic origin, as they were clustered with strains from the Western Balkans or Turkey, whereas one strain of human origin could be associated with travel to another endemic region, e.g. Portugal. Further, nucleotide substitutions in the housekeeping gene rpoB and virulence-associated genes were detected, which were characteristic of the different subgenotypes. One of the isolates originating from an aborted bovine foetus was identified as B. abortus vaccine strain RB51. CONCLUSION: The results demonstrate the existence of several distinct persistent Brucella sp. foci in Greece. To detect these and for tracing infection chains, extensive sampling initiatives are required.

Acinetobacter baumannii Global Clone-Specific Resistomes Explored in Clinical Isolates Recovered from Egypt.

Acinetobacter baumannii (A. baumannii) is a highly problematic pathogen with an enormous capacity to acquire or upregulate antibiotic drug resistance determinants. The genomic epidemiology and resistome structure of 46 A. baumannii clinical isolates were studied using whole-genome sequencing. The isolates were chosen based on reduced susceptibility to at least three classes of antimicrobial compounds and were initially identified using MALDI-TOF/MS, followed by polymerase chain reaction amplification of blaOXA-51-like genes. The susceptibility profiles were determined using a broth microdilution assay. Multi-, extensive-, and pan-drug resistance was shown by 34.8%, 63.0%, and 2.2% of the isolates, respectively. These were most susceptible to colistin (95.7%), amikacin, and trimethoprim/sulfamethoxazole (32.6% each), while only 26.1% of isolates were susceptible to tigecycline. In silico multi-locus sequence typing revealed 8 Pasteur and 22 Oxford sequence types (STs) including four novel STs (STOxf 2805, 2806, 2807, and 2808). The majority of the isolates belonged to Global Clone (GC) 2 (76.4%), GC5 (19.6%), GC4 (6.5%), GC9 (4.3%), and GC7 (2.2%) lineages. An extensive resistome potentially conferring resistance to the majority of the tested antimicrobials was identified in silico. Of all known carbapenem resistance genes, blaOXA-23 was carried by most of the isolates (69.6%), followed by ISAba1-amplified blaADC (56.5%), blaNDM-1 and blaGES-11 (21.7% each), and blaGES-35 (2.2%) genes. A significant correlation was found between carbapenem resistance and carO mutations, which were evident in 35 (76.0%) isolates. A lower proportion of carbapenem resistance was noted for strains possessing both blaOXA-23- and blaGES-11. Amikacin resistance was most probably mediated by armA, aac(6')-Ib9, and aph(3')-VI, most commonly coexisting in GC2 isolates. No mutations were found in pmrABC or lpxACD operons in the colistin-resistant isolates. Tigecycline resistance was associated with adeS (N268Y) and baeS (A436T) mutations. While the lineage-specific distribution of some genes (e.g., blaADC and blaOXA-51-like alleles) was evident, some resistance genes, such as blaOXA-23 and sul1, were found in all GCs. The data generated here highlight the contribution of five GCs in A. baumannii infections in Egypt and enable the comprehensive analysis of GC-specific resistomes, thus revealing the dissemination of the carbapenem resistance gene blaOXA-23 in isolates encompassing all GCs.

Comparison of Illumina and Oxford Nanopore Technology for genome analysis of Francisella tularensis, Bacillus anthracis, and Brucella suis.

BACKGROUND: Bacterial epidemiology needs to understand the spread and dissemination of strains in a One Health context. This is important for highly pathogenic bacteria such as Bacillus anthracis, Brucella species, and Francisella tularensis. Whole genome sequencing (WGS) has paved the way for genetic marker detection and high-resolution genotyping. While such tasks are established for Illumina short-read sequencing, Oxford Nanopore Technology (ONT) long-read sequencing has yet to be evaluated for such highly pathogenic bacteria with little genomic variations between strains. In this study, three independent sequencing runs were performed using Illumina, ONT flow cell version 9.4.1, and 10.4 for six strains of each of Ba. anthracis, Br. suis and F. tularensis. Data from ONT sequencing alone, Illumina sequencing alone and two hybrid assembly approaches were compared. RESULTS: As previously shown, ONT produces ultra-long reads, while Illumina produces short reads with higher sequencing accuracy. Flow cell version 10.4 improved sequencing accuracy over version 9.4.1. The correct (sub-)species were inferred from all tested technologies, individually. Moreover, the sets of genetic markers for virulence, were almost identical for the respective species. The long reads of ONT allowed to assemble not only chromosomes of all species to near closure, but also virulence plasmids of Ba. anthracis. Assemblies based on nanopore data alone, Illumina data alone, and both hybrid assemblies correctly detected canonical (sub-)clades for Ba. anthracis and F. tularensis as well as multilocus sequence types for Br. suis. For F. tularensis, high-resolution genotyping using core-genome MLST (cgMLST) and core-genome Single-Nucleotide-Polymorphism (cgSNP) typing produced highly comparable results between data from Illumina and both ONT flow cell versions. For Ba. anthracis, only data from flow cell version 10.4 produced similar results to Illumina for both high-resolution typing methods. However, for Br. suis, high-resolution genotyping yielded larger differences comparing Illumina data to data from both ONT flow cell versions. CONCLUSIONS: In summary, combining data from ONT and Illumina for high-resolution genotyping might be feasible for F. tularensis and Ba. anthracis, but not yet for Br. suis. The ongoing improvement of nanopore technology and subsequent data analysis may facilitate high-resolution genotyping for all bacteria with highly stable genomes in future.

Determination of Virulence-Associated Genes and Antimicrobial Resistance Profiles in Brucella Isolates Recovered from Humans and Animals in Iran Using NGS Technology.

Brucellosis is a common zoonotic disease in Iran. Antimicrobial-resistant (AMR) Brucella isolates have been reported from different developing countries, posing an imminent health hazard. The objective of this study was to evaluate AMR and virulence-associated factors in Brucella isolates recovered from humans and animals in different regions of Iran using classical phenotyping and next generation sequencing (NGS) technology. Our findings revealed that B. melitensis is the most common species in bovines, small ruminants and camels. B. abortus was isolated only from one human case. Probable intermediate or resistant phenotype patterns for rifampicin, trimethoprim-sulfamethoxazole, ampicillin-sulbactam and colistin were found. Whole genome sequencing (WGS) identified mprF, bepG, bepF, bepC, bepE, and bepD in all isolates but failed to determine other classical AMR genes. Forty-three genes associated with five virulence factors were identified in the genomes of all Brucella isolates, and no difference in the distribution of virulence-associated genes was found. Of them, 27 genes were associated with lipopolysaccharide (LPS), 12 genes were related to a type IV secretion system (virB1-B12), two were associated with the toll-interleukin-1 receptor (TIR) domain-containing proteins (btpA, btpB), one gene encoded the Rab2 interacting conserved protein A (ricA) and one was associated with the production of cyclic ß-1,2 glucans (cgs). This is the first investigation reporting the molecular-based AMR and virulence factors in brucellae isolated from different animal hosts and humans in Iran. Iranian B. abortus and B. melitensis isolates are still in vitro susceptible to the majority of antibiotics used for the treatment of human brucellosis. WGS failed to determine classical AMR genes and no difference was found in the distribution of virulence-associated genes in all isolates. Still, the absence of classical AMR genes in genomes of resistant strains is puzzling, and investigation of phenotypic resistance mechanisms at the proteomic and transcriptomic levels is needed.

Whole-genome sequencing for genetic diversity analysis of Iranian Brucella spp. isolated from humans and livestock.

Brucellosis is one of the most common zoonoses in the Middle East. It is causing economic losses to the livestock industry and has a great public health concern. Little is known about the genetic diversity and distribution of brucellae in Iran. Therefore, forty Brucella spp. strains (B. abortus and B. melitensis) isolated from animals and humans were analyzed by whole genome sequencing (WGS) technology using single nucleotide polymorphism (SNP) analysis and core genome multilocus sequence typing (cgMLST). Brucella isolates were obtained from lymph nodes (cows and camels), milk (cows, camels and sheep), and aborted foetus samples (sheep and goats), as well as cerebrospinal fluid and blood of humans. The isolates were originating from thirteen provinces of Iran and isolated between 2015 and 2020. According to in-silico MLST, ST8 and ST2 were the most frequent sequence types in B. melitensis and B. abortus, respectively. Based on phylogeographic reconstruction using cgSNP analysis, the investigated Iranian B. melitensis strains belonged to the American and Mediterranean lineages of the B. melitensis phylogeny. Furthermore, cgSNP analysis revealed a similarity between Iranian B. abortus isolates and strains from Iraq and Egypt. Therefore, the origin of the Iranian strains can be suggested to be strains from neighboring and Middle East countries. Moreover, cgMLST analysis showed that the Iranian B. melitensis strains were closely relative to strains recovered from sheep and humans in Iraq, Afghanistan, Syria, Turkmenistan, and Pakistan. In the current panel of strains, cgMLST and cgSNP analysis provided an appropriate and accurate tool for effective traceback analyses for Brucella spp. from Iran. The results of cgSNP and cgMLST helped to understand the geographic distribution and interspecies transmission of Iranian strains and highlight the importance of specific brucellosis control measures in Iran with regard to the One-Health approach.

Sequence-based detection and typing procedures for Burkholderia mallei: Assessment and prospects.

Although glanders has been eradicated in most of the developed world, the disease still persists in various countries such as Brazil, India, Pakistan, Bangladesh, Nepal, Iran, Bahrain, UAE and Turkey. It is one of the notifiable diseases listed by the World Organization for Animal Health. Occurrence of glanders imposes restriction on equestrian events and restricts equine movement, thus causing economic losses to equine industry. The genetic diversity and global distribution of the causing agent, Burkholderia (B.) mallei, have not been assessed in detail and are complicated by the high clonality of this organism. Among the identification and typing methods, PCR-based methods for distinguishing B. mallei from its close relative B. pseudomallei as well as genotyping using tandem repeat regions (MLVA) are established. The advent and continuous advancement of the sequencing techniques and the reconstruction of closed genomes enable the development of genome guided epidemiological tools. For achieving a higher genomic resolution, genotyping methods based on whole genome sequencing data can be employed, like genome-wide single nucleotide polymorphisms. One of the limitations in obtaining complete genomic sequences for further molecular characterization of B. mallei is its high GC content. In this review, we aim to provide an overview of the widely used detection and typing methods for B. mallei and illustrate gaps that still require development. The genomic features of Burkholderia, their high homology and clonality will be first described from a comparative genomics perspective. Then, the commonly used molecular detection (PCR systems) and typing systems (e.g., multilocus sequence typing, variable number of tandem repeat analysis) will be presented and put in perspective with recently developed genomic methods. Also, the increasing availability of B. mallei genomic sequences and evolution of the sequencing methods offers exciting prospects for further refinement of B. mallei typing, that could overcome the difficulties presently encountered with this particular bacterium.

First record of the human infection of Brucella melitensis in Kyrgyzstan: evidence from whole-genome sequencing-based analysis.

BACKGROUND: Brucellosis, a zoonosis mainly transmitted by consumption of unpasteurized dairy products as well as direct contact with infected animals, is endemic in Kyrgyzstan. However, Brucella species in humans have not been investigated and the origin of the disease remains poorly known in wide parts of Сentral Asia. Thus, molecular characterization of the circulating strains is a critical first step in understanding Brucella diversity in the country. METHODS: In this study, isolates were collected from patients with suspected brucellosis from different regions in Kyrgyzstan between 2019 and 2020. The detection and identification of Brucella was carried out by Bruce-ladder PCR. Next generation sequencing was used to sequence the 89 Brucella isolates, which were genotyped by cgSNP and cgMLST to identify epidemiological connection between Brucella isolates as well as placing them in the context of the global Brucella phylogeny. RESULTS: The Brucella strains isolated from all regions of Kyrgyzstan were identified as B. melitensis. Based on cgSNP analysis, 18 sequence types were differentiated. The highest numbers of different sequence types were found in Batken (n = 8), Osh (n = 8) and Jalal-Abad (n = 6) oblasts. According to cgSNP and cgMLST analyses, different B. melitensis lineages circulate in Kyrgyzstan, all of them belonging to the Eastern Mediterranean group of the global Brucella phylogeny with the highest similarity to strains from Turkmenistan, Iran and Turkey. CONCLUSION: In the present study, B. melitensis was identified as a causative agent of human brucellosis in Kyrgyzstan and different lineages could be identified. Since this study focused on isolates of human origin, the identity of Brucella species and lineages circulating among animal populations remains elusive. Implementing culture techniques and use of most recent molecular, bioinformatic and epidemiological tools are needed to set up a One Health approach to combat brucellosis in Kyrgyzstan. Further, other Сentral Asian countries need to take part in this effort as brucellosis is a transboundary disease in these regions.

Brucellosis in Humans and Animals in Kyrgyzstan.

Brucellosis is a globally reemerging and neglected zoonosis causing serious public health problems as well as considerable economic losses due to infection of livestock. Although the epidemiology of brucellosis has been well studied and its various aspects in humans and animals are well understood, it is still one of the most challenging health problems in many developing countries such as Kyrgyzstan. This review describes epidemiological characteristics of brucellosis in humans and animals, its impact on animal production and the role of implemented infection control measures in Kyrgyzstan. Particularly, introduction of mass vaccination in small ruminants evidently contributed to control of brucellosis in Kyrgyzstan, reducing the number of infections in animals as well as humans.

Sequencing-Based Genotyping of Pakistani Burkholderia mallei Strains: A Useful Way for Investigating Glanders Outbreaks.

Burkholderia (B.) mallei is a host-adapted equine pathogen that causes glanders, a re-emerging zoonotic disease, which is endemic in Pakistan and other developing countries and seriously impacts the global equine movement. Due to globalization, the geographical restriction of diseases vanishes and the lack of awareness of and experience with eradicated diseases in industrialized countries also promotes the re-introduction of infections in these regions. Owing to the high equine population, the Pakistani province Punjab is a potential hotspot where several glanders outbreaks have been seen over last two decades. For determining the genomic diversity of B. mallei in this and other equine-populated prefectures, the genomes of 19 B. mallei strains isolated between 1999 and 2020 in different locations were sequenced and their genotypes were determined. Particularly, for genetically highly homogenous pathogens like B. mallei genotyping techniques require a high discriminatory power for enabling differentiation on the strain level. Thus, core-genome single nucleotide polymorphism (cgSNP) analysis was applied for distinguishing the highly similar strains. Furthermore, a whole-genome sequence-based core genome multi locus sequence typing (cgMLST) scheme, specific to B. mallei, was developed and additionally applied to the data. It was found that B. mallei genotypes in Pakistan persisted over time and space and genotype clusters preferred connection with a time point rather than the place of isolation, probably due to frequent equine movement, which promotes the spread of glanders. The cgMLST approach proved to work in accord with SNP typing and may help to investigate future glanders outbreaks.

Extremophile Metal Resistance: Plasmid-Encoded Functions in Streptomyces mirabilis.

The extreme metal tolerance of up to 130 mM NiSO4 in Streptomyces mirabilis P16B-1 was investigated. Genome sequencing revealed the presence of a large linear plasmid, pI. To identify plasmid-encoded determinants of metal resistance, a newly established transformation system was used to characterize the predicted plasmid-encoded loci nreB, hoxN, and copYZ. Reintroduction into the plasmid-cured S. mirabilis ΔpI confirmed that the predicted metal transporter gene nreB constitutes a nickel resistance factor, which was further supported by its heterologous expression in Escherichia coli. In contrast, the predicted nickel exporter gene hoxN decreased nickel tolerance, while copper tolerance was enhanced. The predicted copper-dependent transcriptional regulator gene copY did not induce tolerance toward either metal. Since genes for transfer were identified on the plasmid, its conjugational transfer to the metal-sensitive Streptomyces lividans TK24 was checked. This resulted in acquired tolerance toward 30 mM nickel and additionally increased the tolerance toward copper and cobalt, while oxidative stress tolerance remained unchanged. Intracellular nickel concentrations decreased in the transconjugant strain. The high extracellular nickel concentrations allowed for biomineralization. Plasmid transfer could also be confirmed into the co-occurring actinomycete Kribbella spp. in soil microcosms. IMPORTANCE Living in extremely metal-rich environments requires specific adaptations, and often, specific metal tolerance genes are encoded on a transferable plasmid. Here, Streptomyces mirabilis P16B-1, isolated from a former mining area and able to grow with up to 130 mM NiSO4, was investigated. The bacterial chromosome, as well as a giant plasmid, was sequenced. The plasmid-borne gene nreB was confirmed to confer metal resistance. A newly established transformation system allowed us to construct a plasmid-cured S. mirabilis as well as an nreB-rescued strain in addition to confirming nreB encoding nickel resistance if heterologously expressed in E. coli. The potential of intra- and interspecific plasmid transfer, together with the presence of metal resistance factors on that plasmid, underlines the importance of plasmids for transfer of resistance factors within a bacterial soil community.

Molecular Typing of Ukrainian Bacillus anthracis Strains by Combining Whole-Genome Sequencing Techniques.

Anthrax is a recurrent zoonosis in the Ukraine with outbreaks occurring repeatedly in certain areas. For determining whether several Bacillus anthracis genotypes are circulating in this region, four strains from various sources isolated from different regions of the Ukraine were investigated. By combining long- and short-read next-generation sequencing techniques, highly accurate genomes were reconstructed, enabling detailed in silico genotyping. Thus, the strains could be assigned to the Tsiankovskii subgroup of the "TransEurAsia" clade, which is commonly found in this region. Their high genetic similarity suggests that the four strains are members of the endemic population whose progenitor was once introduced in the Ukraine and bordering regions. This study provides information on B. anthracis strains from a region where there is little knowledge of the local population, thereby adding to the picture of global B. anthracis genotype distribution. We also emphasize the importance of surveillance and prevention methods regarding anthrax outbreaks, as other studies predicted a higher number of cases in the future due to global warming.

Molecular Typing of Burkholderia mallei Isolates from Equids with Glanders, India.

We collected 10 Burkholderia mallei isolates from equids in 9 districts in India during glanders outbreaks in 2013-2016. Multilocus variable-number tandem-repeat analysis showed 7 outbreak area-related genotypes. The study highlights the utility of this analysis for epidemiologically tracing of specific B. mallei isolates during outbreaks.

Aquifer community structure in dependence of lithostratigraphy in groundwater reservoirs.

Groundwater microbiology with respect to different host rocks offers new possibilities to describe and map the habitat harboring approximately half of Earths' biomass. The Thuringian Basin (Germany) contains formations of the Permian (Zechstein) and Triassic (Muschelkalk and Buntsandstein) with outcrops and deeper regions at the border and central part. Hydro(geo)chemistry and bacterial community structure of 11 natural springs and 20 groundwater wells were analyzed to define typical patterns for each formation. Widespread were Gammaproteobacteria, while Bacilli were present in all wells. Halotolerant and halophilic taxa were present in Zechstein. The occurrence of specific taxa allowed a clear separation of communities from all three lithostratigraphic groups. These specific taxa could be used to follow fluid movement, e.g., from the underlying Zechstein or from nearby saline reservoirs into Buntsandstein aquifers. Thus, we developed a new tool to identify the lithostratigraphic origin of sources in mixed waters. This was verified with entry of surface water, as species not present in the underground Zechstein environments were isolated from the water samples. Thus, our tool shows a higher resolution as compared to hydrochemistry, which is prone to undergo fast dilution if water mixes with other aquifers. Furthermore, the bacteria well adapted to their respective environment showed geographic clustering allowing to differentiate regional aquifers.