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Abstract Tritrichomonas foetus is a parasitic protist responsible for bovine trichomonosis, a reproductive disease associated with significant economic burden to the livestock industry throughout the world. Here, we present a chromosome-level reference genome of T. foetus -KV-1 (ATCC 30924) using short-read (Illumina Miseq), long-read (Oxford Nanopore) and chromatin-linked (Hi-C) sequencing. This is the first chromosome-level genome of a parasitic protist of the order Tritrichomonadida and the second within the Parabasalia lineage, after Trichomonas vaginalis, the human-associated causative agent of the sexually transmitted infection in humans. Our constructed genome is 148 Mb in size, with a N50 length of the scaffolds of 22.9 Mb. The contigs are anchored in five super-scaffolds, corresponding to the expected five chromosomes of the species and covering 78% of the genome assembly. We predict 41,341 protein-coding genes, of which 95.10% have been functionally annotated. This high-quality genome assembly serves as a valuable reference genome for T. foetus to support future studies in functional genomics, genetic conservation and taxonomy.

Abstract Q fever, a worldwide-occurring zoonotic disease, can cause economic losses for public and veterinary health systems. Vaccines are not yet available worldwide and currently under development. In this regard, it is important to produce a whole cell antigen, with preserved structural and antigenic properties and free of chemical modifications. Thus, inactivation of Coxiella burnetii with ultraviolet light C (UVC) was evaluated. C. burnetii Nine Mile phase I (NMI) and phase II (NMII) were exposed to decreasing intensities in a time-dependent manner and viability was tested by rescue cultivation in axenic medium or cell culture. Effects on the cell structure were visualized by transmission electron microscopy and antigenicity of UVC-treated NMI was studied by immunization of rabbits. NMI and NMII were inactivated at UVC intensities of 250 µW/cm2 for 5 min or 100 µW/cm2 for 20 min. Reactivation by DNA repair was considered to be unlikely. No morphological changes were observed directly after UVC inactivation by transmission electron microscopy, but severe swelling and membrane degradation of bacteria with increasing severity occurred after 24 and 48 h. Immunization of rabbits resulted in a pronounced antibody response. UVC inactivation of C. burnetii resulted in a structural preserved, safe whole cell antigen and might be useful as antigen for diagnostic purposes or as vaccine candidate.

Klebsiella pneumoniae is a ubiquitous nosocomial pathogen associated with various types of infections in hospitalized patients and different animal species. In the current study, 49 Klebsiella strains isolated from humans, dogs, and cats were investigated using NGS technology. MALDI-TOF failed to identify newly discovered K. variicola and K. quasipneumoniae isolates correctly. MLST analysis revealed different sequence types among K. pneumoniae isolates, and the most frequent STs were ST29, ST219, and ST37. Three ST23 that are generally known as hypervirulent type were identified but they lacked major discriminatory determinants for hypervirulent K. pneumoniae (hvKp). K. pneumoniae isolates showed high diversity, and several isolates from humans and animals were assigned to the same ST and were almost identical. Isolates from humans exhibited more pronounced resistance patterns compared to the animal isolates. High levels of resistance were observed for piperacillin, trimethoprim/sulfamethoxazole, and cephalosporins, and resistance to carbapenem compounds was only found in isolates of human origin. Three strains of human origin were extensively drug-resistant (XDR). A diverse range of resistance genes primarily confer resistance to beta-lactams., phenicol/quinolone, aminoglycoside, macrolide, sulfonamides, and fosfomycin were identified in silico. However, there were inconsistencies between the phenotypic characterization of isolates and the set of resistance genes detected in silico in this set of Klebsiella isolates. Further research using a larger number of isolates from various sources is necessary to fully comprehend the relationship between the presence of antimicrobial resistance determinants and phenotypic data. It is also necessary to monitor the spread of K. pneumoniae from a One Health perspective in Egypt.

BackgroundHuman brucellosis is a neglected disease transmitted to humans from animals such as cattle, goats, dogs, and swine. The causative agents are bacteria of the genus Brucella, intracellular pathogens usually confined to the reproductive organs of their animal hosts causing sterility and abortions. The objective of the study was to determine the seroprevalence of brucellosis among women with spontaneous abortions (SAW) and compare this seroprevalence with that of healthy pregnant women (HPW).MethodsThe case–control study was designed to determine the seroprevalence and molecular detection of brucellosis in women who suffered from spontaneous abortion and healthy pregnant women of the Haripur District of Pakistan. A total of 770 blood samples (n = 385 for each group) were collected from 9 public and 11 private hospitals in Haripur District from December 2021–March 2023. Data on demographic features, epidemiological variables, and risk factors were collected from each participant by structured questionnaires. Initial screening for brucellosis was performed by Rose Bengal Plate Test followed by qRT-PCR for molecular detection of the genus-specific BCSP-31 gene of Brucella.ResultsThe study showed that anti-Brucella antibodies were more found in SAW 23.63% (91/385) than in HPW 1.29% (5/385). Brucella specific DNA was amplified in 89.01% (81/91) seropositive samples of SAW. Demographic features and risk factors such as age, urbanicity, socioeconomic status, education, occupation, and animal contact were found significantly associated with brucellosis (p ≤ 0.05). Consumption of unpasteurized raw milk (OR = 18.28, 95%CI: 8.16–40.94) was found highly concomitant with seroprevalence.ConclusionThis study reports the first evidence of involvement of brucellosis in spontaneous abortions in women of Pakistan. The study can be used to develop strategies for risk management during pregnancy, to raise awareness for brucellosis, and develop control programs.

Abstract 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.

Abstract Background Salmonella infections remain an important public health issue worldwide. Some serovars of non-typhoidal Salmonella (NTS) have been associated with bloodstream infections and gastroenteritis, especially in children in Sub-Saharan Africa with circulating S. enterica serovars with drug resistance and virulence genes. This study identified and verified the clonal relationship of Nigerian NTS strains isolated from humans, animals, and the environment. Methods In total, 2,522 samples were collected from patients, animals (cattle and poultry), and environmental sources between December 2017 and May 2019. The samples were subjected to a standard microbiological investigation. All the isolates were identified using Microbact 24E, and MALDI-TOF MS. The isolates were serotyped using the Kauffmann-White scheme. Antibiotic susceptibility testing was conducted using the disc diffusion method and the Vitek 2 compact system. Virulence and antimicrobial resistance genes, sequence type, and cluster analysis were investigated using WGS data. Results Forty-eight (48) NTS isolates (1.9%) were obtained. The prevalence of NTS from clinical sources was 0.9%, while 4% was recorded for animal sources. The serovars identified were S. Cotham (n = 17), S. Give (n = 16), S. Mokola (n = 6), S. Abony (n = 4), S. Typhimurium (n = 4), and S. Senftenberg (n = 1). All 48 Salmonella isolates carried intrinsic and acquired resistant genes such as aac.6…Iaa, mdf(A), qnrB, qnrB19 genes and golT, golS, pcoA, and silP, mediated by plasmid Col440I_1, incFIB.B and incFII. Between 100 and 118 virulence gene markers distributed across several Salmonella pathogenicity islands (SPIs), clusters, prophages, and plasmid operons were found in each isolate. WGS revealed that strains of each Salmonella serovar could be assigned to a single 7-gene MLST cluster, and strains within the clusters were identical strains and closely related as defined by the 0 and 10 cgSNPs and likely shared a common ancestor. The dominant sequence types were S. Give ST516 and S. Cotham ST617. Conclusion We found identical Salmonella sequence types in human, animal, and environmental samples in the same locality, which demonstrates the great potential of the applied tools to trace back outbreak strains. Strategies to control and prevent the spread of NTS in the context of one’s health are essential to prevent possible outbreaks.

BackgroundBovine mastitis is a common disease of dairy cattle causing major economic losses due to reduced yield and poor quality of milk worldwide. The current investigation aimed to gain insight into the genetic diversity, antimicrobial resistance profiles and virulence associated factors of Staphylococcus (S.) aureus isolated from clinical bovine mastitis in dairy farms in Thuringia, Germany.MethodsForty Staphylococcus aureus isolates collected from clinical bovine mastitis cases from 17 Thuringian dairy farms were phenotyped and genetically characterized using whole genome sequencing.ResultsOut of 40 S. aureus, 30 (75%) were confirmed as methicillin resistant isolates. The isolates showed elevated antimicrobial resistance against penicillin, tetracycline and oxacillin, i.e., 77.5, 77.5, and 75%, respectively. Lower resistance rates were found against moxifloxacin, ciprofloxacin, gentamicin and trimethoprim/sulfamethoxazole, i.e., 35, 35, 30, and 22.5%, respectively. While resistance against clindamycin and erythromycin was rarely found (5 and 2.5%, respectively). All isolates were susceptible to linezolid, teicoplanin, vancomycin, tigecycline, fosfomycin, fusidic acid and rifampicin. These isolates were further allocated into five different sequence types: ST398 (n = 31), ST1074 (n = 4), ST504 (n = 3), ST582 (CC15) (n = 1) and ST479 (n = 1). These isolates were also assigned to seven clusters with up to 100 SNP which has facilitated geographical mapping and epidemiological distribution in Thuringia. Strains belonging to ST398 were classified into clusters 1, 2, 3, 4 and 7. The isolates of ST504 were of cluster 5, those of ST1074 were belonging to cluster 6. Resistance genes blaZ, blaI and blaR associated with penicillin resistance were found in 32 (80%) strains, all except one were belonging to ST398. Methicillin resistance associated mecA was identified in 30 (96.8%) isolates of ST398. All tetracycline and erythromycin resistant isolates were of ST398, and all harbored both tetM and ermA. About 90.3% of tetracycline resistant isolates assigned to ST398 were also carrying tetK gene. The point mutations parC_S80F, gyrA_S84L and parC_S80Y in gyrA and parC associated with quinolone resistance were found in all phenotypically resistant isolates to ciprofloxacin and moxifloxacin (n = 14). Sixty-eight virulence genes were identified among isolates. Both lukD/E and lukM/F-PV-P83 were identified in 22.5% of isolates, all were non-ST398.ConclusionIn this study, ST398 had the highest potential to cause disease and had a massive prevalence in bovine mastitis cases. Five different sequence types and seven clusters were identified in the federal state of Thuringia. The circulation of some clusters in the same region over several years shows the persistence of cluster-associated infection despite the intensive veterinary care. On the other hand, some regions had different clusters at the same year or in different consecutive years. Different sequence types and associated different clusters of S. aureus were geographically widely distributed among dairy farms in Thuringia. The findings of this study show that various clusters have the potential to spread over a large geographical scale. The detection of LA-MRSA on dairy farms, which is known for cabapility to widely spread among different groups of animals, humans and their environment urges for the implementation of national wide strategic programs. The identification of CA-MRSA among the isolates such as ST398 poses a significant risk for the transmission of such strains between animals and humans on dairy farms.

Abstract 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. Graphical Abstract

Abortion is one of the leading causes of economic losses in the livestock industry worldwide. Chlamydia abortus, Coxiella burnetii, and Brucella spp. are the leading cause of late fetal loss in small ruminants. This study determined the seroprevalence of these agents in small ruminants in district Jhang. A total of 385 serum samples were taken from the sheep and goats from different flocks with a history of abortion and subjected to i-ELISA. Further, samples were analysed for liver enzymes and total serum protein using a semi-automated chemistry analyzer. The result of indirect commercial ELISA showed 13.0, 4.2 and 11.2% prevalence for Coxiella burnetii, Chlamydia abortus, and Brucella spp., respectively. Values of different serum parameters (ALT, AST, and total protein) of seropositive animals were also determined. There was a significant rise in AST and ALT values of infected animals (p ≤ 0.05). Total protein decreased for all three infections, but a significant drop was noted in Brucella positive sheep serum samples. Various risk factors were studied. Binary logistic regression proved a significant role of ticks for coxiellosis and brucellosis. Age, parity, and species did not impact the prevalence of diseases studied.

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.

Contagious bovine genital campylobacteriosis (BGC), also known as bovine venereal campylobacteriosis, is a disease relevant to international trade listed by the World Organization for Animal Health (WOAH). It is caused by Campylobacter fetus subsp. venerealis (Cfv), one of three subspecies of Campylobacter fetus. Bulls are the reservoir but BGC may also be spread by artificial insemination (AI). BGC is characterized by severe reproductive losses such as infertility, early embryonic death and abortion with considerable economic losses. This significant economic impact has prompted several countries to adopt stringent eradication and surveillance measures to contain the disease. While there are commercial and autologous vaccines available, scientific evidence for the effectiveness of vaccination is still lacking. In Germany, BCG was already found to be endemic in the 1920s, shortly after the agent and the disease had been described for the first time. It can be assumed that BCG had already circulated uncontrolled for a long time in the predecessor states of Germany, influenced only by the political situation and trading networks of the time. After WW II, BCG was eradicated in the German Democratic Republic due to industrialized cattle production based on AI but it was still endemic at low levels in the Federal Republic of Germany with its diverse cattle production. There has been a steady decline in BGC incidence in re-unified Germany over the past 28 years. A single genetic Cfv lineage was identified which probably emerged in the 19th century and diversified over time. Interestingly, no recurrent cross-border introduction became evident. This review gives insight into the history of bovine genital campylobacteriosis considering the structural change in cattle farming in Germany and reflecting on the political background of the time.

Abstract Background Brucella suis is a zoonotic pathogen with a serious impact on public health and the pig industry worldwide. Information regarding B. suis in pigs in Egypt is scarce. This study aimed to investigate the prevalence of B. suis in slaughtered domestic pigs at El-Basatin abattoir in Cairo, Egypt. A total of 1,116 domestic pigs slaughtered in 2020 were sampled for Brucella isolation and identification. Identified Brucella isolates were molecularly confirmed at species, and biovar levels using Bruce ladder PCR and Suis ladder multiplex PCR. Additionally, high-risk practices of 16 abattoir workers (4 veterinarians, 10 butchering and evisceration workers, and 2 scalding workers) were investigated using a pre-piloted structured questionnaire. Results Brucella isolates were recovered from 1.3% of examined pigs (n = 14) at consistently low rates (1.1—2.9%) across the year of sampling from February to December 2020. All isolates were confirmed as B. suis biovar (bv) 2. Remarkably, 92.9% (13/14) of isolates showed atypical ability to produce H2S and hence were considered as B. suis bv2 atypical phenotype. The prevalence was higher in males (1.8%) than in females (0.9). However, this difference was not significant (Odds ratio = 1.9; CI 95% 0.7 – 5.7; P = 0.2). No detectable pathological lesions were associated with B. suis bv2 infection in examined pigs. All strains were isolated from cervical lymph nodes, highlighting a potential oral transmission. High-risk practices were recorded among swine abattoir workers in this study: 75% do not wear gloves or disinfect their knives daily, and 18.8% were willing to work with open wound injuries. Conclusions To the best of our knowledge, this is the first isolation of B. suis bv2 in Egypt. Detection of H2S producing B. suis bv2 atypical phenotype is alarming as it may result in misinterpretation of these isolates as highly human pathogenic B. suis bv1 in Egypt and possibly elsewhere. Further epidemiological tracing studies are crucial for the detection of the origin of this biovar. Including pigs in the national surveillance program of brucellosis, and an education program for swine abattoir workers about occupational risk of B. suis is a need in Egypt.

Campylobacter (C.) jejuni is a zoonotic bacterium of public health significance. The present investigation was designed to assess the epidemiology and genetic heterogeneity of C. jejuni recovered from commercial turkey farms in Germany using whole-genome sequencing. The Illumina MiSeq® technology was used to sequence 66 C. jejuni isolates obtained between 2010 and 2011 from commercial meat turkey flocks located in ten German federal states. Phenotypic antimicrobial resistance was determined. Phylogeny, resistome, plasmidome and virulome profiles were analyzed using whole-genome sequencing data. Genetic resistance markers were identified with bioinformatics tools (AMRFinder, ResFinder, NCBI and ABRicate) and compared with the phenotypic antimicrobial resistance. The isolates were assigned to 28 different sequence types and 11 clonal complexes. The average pairwise single nucleotide-polymorphisms distance of 14,585 SNPs (range: 0–26,540 SNPs) revealed a high genetic distinction between the isolates. Thirteen virulence-associated genes were identified in C. jejuni isolates. Most of the isolates harbored the genes flaA (83.3%) and flaB (78.8%). The wlaN gene associated with the Guillain–Barré syndrome was detected in nine (13.6%) isolates. The genes for resistance to ampicillin (blaOXA), tetracycline [tet(O)], neomycin [aph(3')-IIIa], streptomycin (aadE) and streptothricin (sat4) were detected in isolated C. jejuni using WGS. A gene cluster comprising the genes sat4, aph(3′)-IIIa and aadE was present in six isolates. The single point mutation T86I in the housekeeping gene gyrA conferring resistance to quinolones was retrieved in 93.6% of phenotypically fluoroquinolone-resistant isolates. Five phenotypically erythromycin-susceptible isolates carried the mutation A103V in the gene for the ribosomal protein L22 inferring macrolide resistance. An assortment of 13 β-lactam resistance genes (blaOXA variants) was detected in 58 C. jejuni isolates. Out of 66 sequenced isolates, 28 (42.4%) carried plasmid-borne contigs. Six isolates harbored a pTet-like plasmid-borne contig which carries the tet(O) gene. This study emphasized the potential of whole-genome sequencing to ameliorate the routine surveillance of C. jejuni. Whole-genome sequencing can predict antimicrobial resistance with a high degree of accuracy. However, resistance gene databases need curation and updates to revoke inaccuracy when using WGS-based analysis pipelines for AMR detection.

Campylobacter fetus subsp. venerealis (Cfv) causes bovine genital campylobacteriosis (BGC), a World Organization for Animal Health (WOAH)-listed trade-relevant disease characterized by severe reproductive losses, such as infertility, early embryonic death and abortion in cattle. BGC has significant economic implications that have prompted several countries to adopt stringent eradication and surveillance measures to contain the disease. In Germany, there has been a low incidence of BGC cases over the past 28 years. This study aimed to illustrate the genomic diversity of German Cfv strains isolated from different federal states in Germany. This study analyzed 63 Cfv strains, collected between 1985 and 2015, by whole-genome sequencing and compared them with genome data of 91 international Cfv isolates. The phylogenetic analysis showed that the Cfv population is genetically conserved and has geographic clusters. In Germany, one phylogenetic lineage comprising all strains was identified. This German lineage was part of a subclade that probably emerged in the nineteenth century and diversified over time. The results of this study point to a non-recurrent cross-border introduction of Cfv in Germany. The BGC control interventions in Germany can be considered successful as no outbreaks were reported since 2015.

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.

Anthrax is an acute infectious zoonotic disease caused by Bacillus anthracis that mostly affects grazing livestock and wildlife. Furthermore, B. anthracis is considered one of the most important biological agents of bioterrorism that could also be potentially misused in biological weapons. The distribution of anthrax in domestic animals and wildlife in Europe with a particular focus on Ukraine as a country of war was analyzed. Between 2005 and 2022, 267 anthrax cases were registered at the World Organization of Animal Health (WOAH) in animals in Europe, including 251 cases in domestic animals and 16 in wildlife. The highest numbers of cases were recorded in 2005 and 2016 followed by 2008, and the highest numbers of registered cases were reported from Albania, Russia, and Italy. In Ukraine, anthrax is currently a sporadic infection. Since 2007, 28 notifications were registered, with isolates mainly from soil samples. The highest number of confirmed anthrax cases was registered in 2018, and Odesa, which is close to Moldova, had the highest number of cases, followed by the Cherkasy region. The presence of thousands of biothermal pits and burial grounds of fallen cattle nationwide favors the re-emergence of new foci. Most confirmed cases were in cattle; however, single cases were confirmed in dogs, horses, and pigs. Further investigation of the disease in wildlife and in environmental samples is needed. The genetic analysis of isolates, investigation of susceptibility to antimicrobial compounds, and determination of virulence and pathogenicity factors are required in this volatile region of the world for awareness raising and preparedness.

The increasing incidence of extended-spectrum β-lactamase (ESBL)-producing Escherichia (E.) coli in backyard chicken farming in Pakistan is of serious concern. This study aimed to assess the prevalence, antimicrobial resistance patterns and risk factors associated with ESBL avian pathogenic E. coli (APEC) isolated from backyard chickens in the Jhang district, Punjab, Pakistan. In total, 320 cloacal swabs were collected from four breeds of backyard chicken (Aseel, Golden, Misri and Necked Neck). ESBL E. coli were phenotypically identified using double disc synergy test (DDST) and corresponding genes were confirmed by multiplex polymerase chain reaction (mPCR). Out of the 320 samples, 164 (51.3%) were confirmed as E. coli, while 74 (45.1%) were characterized as ESBL E. coli. The frequency of isolation of ESBL E. coli was highest in Aseel chickens (35.1%). Of the 164 confirmed E. coli, 95.1%, 78.6%, 76.8%, 71.3%, 70.1%, 68.9%, 60.4% and 57.3% were resistant against tylosin, doxycycline, cefotaxime, enrofloxacin, colistin, trimethoprim/sulfamethoxazole, chloramphenicol and gentamicin, respectively. The ESBL gene types detected and their corresponding proportions were blaCTX-M (54.1 %, 40/74), blaTEM, (12.2%, 9/74) and co-existence (blaCTX-M and blaTEM) were shown in 33.8% (25/74). The blaCTX-M gene sequence showed homology to blaCTX-M-15 from clinical isolates. The mean multiple antibiotic resistance index (MARI) was found to be higher among ESBL E. coli (0.25) when compared to non-ESBL E. coli (0.17). Both free-range husbandry management system (p = 0.02, OR: 30.00, 95% CI = 1.47–611.79) and high antimicrobial usage in the last 6 months (p = 0.01, OR: 25.17, 95% CI = 1.81–348.71) were found significantly associated with isolation of ESBL-producing E. coli in the tested samples using binary logistic regression analysis. This study confirmed the potential of backyard chickens as a reservoir for ESBL E. coli in the Jhang district, Punjab, Pakistan.

Abstract Background Acinetobacter baumannii ability to develop and acquire resistance makes it one of the most critical nosocomial pathogens globally. Whole-genome sequencing (WGS) was applied to identify the acquired or mutational variants of antimicrobial resistance (AMR) genes in 85 German A. baumannii strains utilizing Illumina technology. Additionally, the whole genome of 104 German isolates deposited in the NCBI database was investigated. Results In-silico analysis of WGS data revealed wide varieties of acquired AMR genes mediating resistance mostly to aminoglycosides, cephalosporins, carbapenems, sulfonamides, tetracyclines and macrolides. In the 189 analyzed genomes, the ant (3″)-IIa conferring resistance to aminoglycosides was the most frequent (55%), followed by bla ADC.25 (38.6%) conferring resistance to cephalosporin, bla OXA-23 (29%) and the bla OXA-66 variant of the intrinsic bla OXA-51-likes (26.5%) conferring resistance to carbapenems, the sul2 (26%) conferring resistance to sulfonamides, the tet. B (19.5%) conferring resistance to tetracycline, and mph. E and msr. E (19%) conferring resistance to macrolides. bla TEM variants conferring resistance to cephalosporins were found in 12% of genomes. Thirteen variants of the intrinsic bla OXA-51 carbapenemase gene, bla OXA-510 and bla ADC-25 genes were found in isolates obtained from dried milk samples. Conclusion The presence of strains harboring acquired AMR genes in dried milk raises safety concerns and highlights the need for changes in producing dried milk. Acquired resistance genes and chromosomal gene mutation are successful routes for disseminating AMR determinants among A. baumannii. Identification of chromosomal and plasmid-encoded AMR in the genome of A. baumannii may help understand the mechanism behind the genetic mobilization and spread of AMR genes.

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.

Coxiellosis is a zoonosis in animals caused by Coxiella burnetii. A cross-sectional study was conducted on 920 (591 female and 329 male) randomly selected camels (Camelus dromedarius) of different age groups from 13 districts representative of the three different ecological zones in the Province Punjab, Pakistan to determine the prevalence and associated risk factors of coxiellosis. The blood samples were collected and tested for anti-C. burnetti antibodies using indirect multispecies ELISA. Real-time PCR was used for the detection of C. burnetii DNA to determine the prevalence in heparinized blood pools. Out of 920 investigated camels, anti-C. burnetii antibodies were detected in 288 samples (31.3%) (95% CI: 28.3–34.4%). The highest (78.6%) and lowest (1.8%) seroprevalence were detected in Rahimyar Khan (southern Punjab) and in Jhang (central Punjab), respectively. Potential risk factors associated with seropositivity of the Q fever in camels included desert area (42.5%; OR = 2.78, 95% CI 1.12–3.21) summer season (35.7%; OR = 2.3, 95% CI: 1.31–3.2), sex (female) (39.1; OR = 2.35, 95% CI: 1.34–2.98), tick infestation (51.3%;OR = 2.81, 95% CI: 1.34–3.02), age (>10 years; 46.4%; OR = 1.56, 95% CI: 0.33–2.05) and herd size (38.5%; OR = 1.21, 95% CI: 0.76–1.54). Coxiella burnetii DNA was amplified in 12 (20%) and 1 (10%) of 60 ELISA-negative and 10 suspected camels, respectively. DNA could not be detected in ELISA positive blood pools. This study emphasizes the seroprevalence and associated risk factors of coxiellosis as well as its potential to spill over to animals and humans in contact with these camel herds.

Abstract Clostridium perfringens causes a plethora of devastating infections, with toxin production being the underlying mechanism of pathogenicity in various hosts. Genomic analyses of 206 public-available C. perfringens strains´ sequence data identified a substantial degree of genomic variability in respect to episome content, chromosome size and mobile elements. However, the position and order of the local collinear blocks on the chromosome showed a considerable degree of preservation. The strains were divided into five stable phylogroups (I–V). Phylogroup I contained human food poisoning strains with chromosomal enterotoxin (cpe) and a Darmbrand strain characterized by a high frequency of mobile elements, a relatively small genome size and a marked loss of chromosomal genes, including loss of genes encoding virulence traits. These features might correspond to the adaptation of these strains to a particular habitat, causing human foodborne illnesses. This contrasts strains that belong to phylogroup II where the genome size points to the acquisition of genetic material. Most strains of phylogroup II have been isolated from enteric lesions in horses and dogs. Phylogroups III, IV and V are heterogeneous groups containing a variety of different strains, with phylogroup III being the most abundant (65.5%). In conclusion, C. perfringens displays five stable phylogroups reflecting different disease involvements, prompting further studies on the evolution of this highly important pathogen.

Abstract Glyphosate-based herbicides are among the most used non-selective herbicides worldwide and inhibit synthesis of aromatic amino acids in plants, bacteria, and fungi. Given the broad usage, controversies concerning potential effects of glyphosate on health and especially on gut microbiomes arose. For cattle, it has been proposed based on in vitro data that glyphosate has detrimental effects on the ruminal microbiome, which manifest as a specific inhibition of bacteria involved in fiber degradation and as an enrichment of specific pathogens. In the present study, glyphosate effects on the ruminal microbiome were analyzed in vivo using glyphosate contaminated feedstuffs with strong differences in dietary fiber and dietary energy content in order to reproduce the proposed detrimental glyphosate effects on the rumen microbiome. While significant impact of dietary factors on the ruminal microbiome and its products are pointed out, no adverse glyphosate effects on ruminal microbiome composition, diversity, and microbial metabolites are observed.

Brucellosis is one of the most notorious zoonoses worldwide. The disease is common and endemic in humans and animals of Pakistan, but lack of awareness and lack of research have resulted in an increased incidence in the human population. The present study aimed to determine the seroprevalence and at molecular detection of brucellosis in patients with clinical symptoms in six different hospitals from Lahore, which is the capital city of Punjab province. A total of 218 blood samples were collected from hospitalized patients. The samples were initially screened by the Rose Bengal Plate Test (RBPT), and then quantitative real-time PCR (qRT-PCR) was applied. An overall seroprevalence of 17% (37/218) was found. The highest prevalence was found at the Lady Health center (36.53%), which was followed by the Lady Willingdon Hospital (28.6%). Female patients showed a higher seroprevalence than males and peaked at 34% (n = 32) for women who suffered from abortion. In total, 16.8% of patients younger than 30 years showed seropositive reactions, while the prevalence was 19% in patients between 31 and 50. Thirty-three DNA samples from 24 seropositive and nine seronegative patients tested positive, 32 samples were found positive for B. abortus DNA, and one sample failed to be identified at the species level. Almost all positive cases had direct contact with animals and consumed unpasteurized dairy products. Research on human brucellosis is still scarce in Pakistan. For the diagnosis of brucellosis, serology and molecular tools should be combined if isolation by culture is not possible. Nationwide control activities and increasing awareness for zoonotic brucellosis are needed.

Abstract Klebsiella (K.) pneumoniae as a multi-drug resistant (MDR) pathogen is an emerging challenge for clinicians worldwide. Virulence factors are capsular antigens, adherence factors, the O-lipopolysaccharide, and siderophores promoting infectivity. Mechanisms of antimicrobial resistance are inactivation of compounds via enzymes, change of membrane permeability, and alteration of the target site of the antimicrobial compound. In addition to environmental resistance, K. pneumoniae can survive increasing concentrations of disinfectants, if exposed. This review describes the temporal and spatial distribution of K. pneumoniae in the past decades in Germany, with emphases on the development of resistance in the non-human columns of the One-Health concept. In general, K. pneumoniae is a neglected pathogen in veterinary and environmental health, and the risk of human infection concerning animal contact and food consumption is barely investigated. Few reports exist (n = 26) on antibiotic resistance of isolates from non-human origin. Multi-drug resistance and extended-spectrum β-lactamase (MDR-ESBL) strains also resistant to carbapenems and antibiotics of the ß-lactam group harbor blaCTX-M, blaOXA, blaTEM, blaSHV, blaCMY, and PMQR have been found in animals, foods, and the environment. Colistin resistant strains carrying the mcr-1 gene were detected in wastewater. The blaCTX-M-15 and blaOXA-48 genes are the most frequently identified AMR genes in isolates of humans and were also the most predominant ESBL-genes in samples collected from animal hosts. Several aspects of the molecular epidemiology and resistance development of K. pneumoniae in farm animal populations, wildlife, and foods need intensive research. Environmental health has to be integrated into national research plans, as a lack of data is apparent. Increasing awareness of the fact that non-human sources can act as a reservoir for this pathogen has to be raised.

The Gram-negative, obligate intracellular bacterium Coxiella burnetii is the causative organism of the zoonosis Q fever and is known for its resistance toward various intra- and extracellular stressors. Infected ruminants such as cattle, sheep, and goats can shed the pathogen in their milk. Pasteurization of raw milk was introduced for the inactivation of C. burnetii and other milk-borne pathogens. Legal regulations for the pasteurization of milk are mostly based on recommendations of the Codex Alimentarius. As described there, C. burnetii is considered as the most heat-resistant non-spore-forming bacterial pathogen in milk and has to be reduced by at least 5 log10-steps during the pasteurization process. However, the corresponding inactivation data for C. burnetii originate from experiments performed more than 60 years ago. Recent scientific findings and the technological progress of modern pasteurization equipment indicate that C. burnetii is potentially more effectively inactivated during pasteurization than demanded in the Codex Alimentarius. In the present study, ultra-high heat-treated milk was inoculated with different C. burnetii field isolates and subsequently heat-treated in a pilot-plant pasteurizer. Kinetic inactivation data in terms of D- and z-values were determined and used for the calculation of heat-dependent log reduction. With regard to the mandatory 5 log10-step reduction of the pathogen, the efficacy of the established heat treatment regime was confirmed, and, in addition, a reduction of the pasteurization temperature seems feasible.

Brucellosis is a neglected zoonotic disease of ruminants. It causes severe health problems in humans and significant economic loss. Only a limited number of studies have been conducted in Pakistan to determine the prevalence of human brucellosis and related risk factors. The objectives of the current cross-sectional study were to determine the prevalence of anti-Brucella antibodies in sera collected from symptomatic patients at three hospitals of Abbottabad using a commercial slide agglutination test (SAT) and to determine risk factors for brucellosis for these patients. Five hundred blood samples were collected. A questionnaire was filled in for each patient to obtain information on age, gender, living area, brucellosis associated symptoms, associated risk factors, pregnancy and abortion history. A total of 13.6% (n = 68) patients were found to be SAT positive and in 83.3% (n = 57) of these samples Brucella DNA was detected by genus specific RT-PCR for BCSP-31 gene. Statistical analysis was performed to determine odd ratios, risk ratios, 95% confidence intervals, and p-values. The prevalence of brucellosis by SAT was reported to be higher in women (14.6%, n = 44) than in men (12.1%, n = 24). The age group 25–50 years was found to be at higher risk for brucellosis (14.5%, n = 50) “animal contact” was reported as the main risk factor followed by “consumption of raw animal products.” Out of 131 pregnant women and 21 patients had abortion, the seropositivity of Brucellosis was 9.9% and 23.8%, respectively. The present study reports a striking prevalence of brucellosis among patients including pregnant women at three hospitals of Abbottabad. These findings may foster strategies for controlling human brucellosis at household level, raising of awareness about brucellosis in hospital and family doctors, and finally in setting up an eradication program in the dairy industry.

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 β-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.

Little is known about zoonotic pathogens and their antimicrobial resistance in South American camelids (SAC) in Germany including Clostridioides (C.) difficile. The aim of this study was to investigate prevalence, molecular characteristics and antimicrobial resistance of C. difficile in SAC. Composite SAC faecal samples were collected in 43 husbandries in Central Germany and cultured for C. difficile. Toxinotyping and ribotyping was done by PCR. Whole genome sequencing was performed with Illumina® Miseq™. The genomes were screened for antimicrobial resistance determinants. Genetic relatedness of the isolates was investigated using core genome multi locus sequence typing (cgMLST) and single nucleotide polymorphism analysis. Antimicrobial susceptibility testing was done using the Etest® method. Eight C. difficile isolates were recovered from seven farms. The isolates belonged to different PCR ribotypes. All isolates were toxinogenic. cgMLST revealed a cluster containing isolates recovered from different farms. Seven isolates showed similar resistance gene patterns. Different phenotypic resistance patterns were found. Agreement between phenotypic and genotypic resistance was identified only in some cases. Consequently, SAC may act as a reservoir for C. difficile. Thus, SAC may pose a risk regarding zoonotic transmission of toxinogenic, potentially human-pathogenic and resistant C. difficile isolates.

Clostridium septicum is a Gram-positive, toxin-producing, and spore-forming bacterium that is recognized, together with C. perfringens, as the most important etiologic agent of progressive gas gangrene. Clostridium septicum infections are almost always fatal in humans and animals. Despite its clinical and agricultural relevance, there is currently limited knowledge of the diversity and genome structure of C. septicum. This study presents the complete genome sequence of C. septicum DSM 7534T type strain as well as the first comparative analysis of five C. septicum genomes. The taxonomy of C. septicum, as revealed by 16S rRNA analysis as well as by genomic wide indices such as protein-based phylogeny, average nucleotide identity, and digital DNA–DNA hybridization indicates a stable clade. The composition and presence of prophages, CRISPR elements and accessory genetic material was variable in the investigated genomes. This is in contrast to the limited genetic variability described for the phylogenetically and phenotypically related species Clostridium chauvoei. The restriction-modification (RM) systems between two C. septicum genomes were heterogeneous for the RM types they encoded. C. septicum has an open pangenome with 2,311 genes representing the core genes and 1,429 accessory genes. The core genome SNP divergence between genome pairs varied up to 4,886 pairwise SNPs. A vast arsenal of potential virulence genes was detected in the genomes studied. Sequence analysis of these genes revealed that sialidase, hemolysin, and collagenase genes are conserved compared to the α-toxin and hyaluronidase genes. In addition, a conserved gene found in all C. septicum genomes was predicted to encode a leucocidin homolog (beta-channel forming cytolysin) similar (71.10% protein identity) to Clostridium chauvoei toxin A (CctA), which is a potent toxin. In conclusion, our results provide first, valuable insights into strain relatedness and genomic plasticity of C. septicum and contribute to our understanding of the virulence mechanisms of this important human and animal pathogen.

Brucellosis is a highly contagious and incapacitating disease of humans, livestock and wildlife species globally. Treatment of brucellosis in animals is not recommended, and in humans, combinations of antibiotics recommended by the World Health Organization are used. However, sporadic antimicrobial-resistant (AMR) isolates and relapse cases have been reported from different endemic regions. In the current study, molecular characterization and antibiotic susceptibility testing using the microdilution method for 35 B. abortus and B. melitensis strains isolated from humans, milk and animal were carried out. Additionally, Next-Generation-Sequencing (NGS) technology was applied to confirm Brucella at the species level and investigate AMR and pathogenicity-associated determinants. MALDI-TOF seemed to be a rapid and reliable tool for routine identification of brucellae to the genus level; however, DNA-based identification is indispensable for accurate species identification. Brucella abortus strains were isolated from two human cases and a sheep. Such infections are uncommon in Egypt. Egyptian Brucella strains are still in-vitro susceptible to doxycycline, tetracyclines, gentamicin, ciprofloxacin, levofloxacin, chloramphenicol, streptomycin, trimethoprim/sulfamethoxazole and tigecycline. Probable (no CLSI/EUCAST breakpoints have been defined yet) in-vitro resistance to rifampicin and azithromycin was observed. WGS failed to determine classical AMR genes, and no difference in the distribution of virulence-associated genes in all isolates was found. Isolates of human and non-human origins were still susceptible to the majority of antibiotics used for treatment in humans. The absence of classical AMR genes in genomes of “resistant” Brucella strains may reflect a lack of information in databases, or resistance might not be encoded by single resistance genes. The One Health approach is necessary for tackling brucellosis. Continuous susceptibility testing, updating of breakpoints, assessing mutations that lead to resistance are needed.

Yersinia ruckeri is the causative agent of enteric redmouth disease (ERM), a serious infection that affects global aquaculture with high economic impact. The present study used whole genome sequences to perform a comparative analysis on 10 Y. ruckeri strains and to explore their genetic relatedness to other members of the genus. Y. ruckeri, Yersinia entomophaga, and Yersinia nurmii formed a species complex that constitutes the most basal lineage of the genus. The results showed that the taxonomy of Y. ruckeri strains is better defined by using a core genome alignment and phylogenetic analysis. The distribution of accessory genes in all Yersinia species revealed the presence of 303 distinctive genes in Y. ruckeri. Of these, 169 genes were distributed in 17 genomic islands potentially involved in the pathogenesis of ERM via (1) encoding virulence factors such as Afp18, Yrp1, phage proteins and (2) improving the metabolic capabilities by enhancing utilization and metabolism of iron, amino acids (specifically, arginine and histidine), and carbohydrates. The genome of Y. ruckeri is highly conserved regarding gene structure, gene layout and functional categorization of genes. It contains various components of mobile genetic elements but lacks the CRISPR-Cas system and possesses a stable set of virulence genes possibly playing a critical role in pathogenicity. Distinct virulence plasmids were exclusively restricted to a specific clonal group of Y. ruckeri (CG4), possibly indicating a selective advantage. Phylogenetic analysis of Y. ruckeri genomes revealed the co-presence of multiple genetically distant lineages of Y. ruckeri strains circulating in Germany. Our results also suggest a possible dissemination of a specific group of strains in the United States, Peru, Germany, and Denmark. In conclusion, this study provides new insights into the taxonomy and evolution of Y. ruckeri and contributes to a better understanding of the pathogenicity of ERM in aquaculture. The genomic analysis presented here offers a framework for the development of more efficient control strategies for this pathogen.

ABSTRACT Clostridium perfringens is a spore-forming anaerobic pathogen responsible for a variety of histotoxic and intestinal infections in humans and animals. High-resolution genotyping aiming to identify bacteria at strain level has become increasingly important in modern microbiology to understand pathogen transmission pathways and to tackle infection sources. This study aimed at establishing a publicly available genome-wide multilocus sequence-typing (MLST) scheme for C. perfringens. A total of 1,431 highly conserved core genes (1.34 megabases; 50% of the reference genome genes) were indexed for a core genome-based MLST (cgMLST) scheme for C. perfringens. The scheme was applied to 282 ecologically and geographically diverse genomes, showing that the genotyping results of cgMLST were highly congruent with the core genome-based single-nucleotide-polymorphism typing in terms of resolution and tree topology. In addition, the cgMLST provided a greater discrimination than classical MLST methods for C. perfringens. The usability of the scheme for outbreak analysis was confirmed by reinvestigating published outbreaks of C. perfringens-associated infections in the United States and the United Kingdom. In summary, a publicly available scheme and an allele nomenclature database for genomic typing of C. perfringens have been established and can be used for broad-based and standardized epidemiological studies. IMPORTANCE Global epidemiological surveillance of bacterial pathogens is enhanced by the availability of standard tools and sharing of typing data. The use of whole-genome sequencing has opened the possibility for high-resolution characterization of bacterial strains down to the clonal and subclonal levels. Core genome multilocus sequence typing is a robust system that uses highly conserved core genes for deep genotyping. The method has been successfully and widely used to describe the epidemiology of various bacterial species. Nevertheless, a cgMLST typing scheme for Clostridium perfringens is currently not publicly available. In this study, we (i) developed a cgMLST typing scheme for C. perfringens, (ii) evaluated the performance of the scheme on different sets of C. perfringens genomes from different hosts and geographic regions as well as from different outbreak situations, and, finally, (iii) made this scheme publicly available supported by an allele nomenclature database for global and standard genomic typing.

Black quarter caused by Clostridium (C.) chauvoei is an important bacterial disease that affects cattle and sheep with high mortality. A comparative genomics analysis of 64 C. chauvoei strains, most of European origin and a few of non-European and unknown origin, was performed. The pangenome analysis showed limited new gene acquisition for the species. The accessory genome involved prophages and genomic islands, with variations in gene composition observed in a few strains. This limited accessory genome may indicate that the species replicates only in the host or that an active CRISPR/Cas system provides immunity to foreign genetic elements. All strains contained a CRISPR type I-B system and it was confirmed that the unique spacer sequences therein can be used to differentiate strains. Homologous recombination events, which may have contributed to the evolution of this pathogen, were less frequent compared to other related species from the genus. Pangenome single nucleotide polymorphism (SNP) based phylogeny and clustering indicate diverse clusters related to geographical origin. Interestingly the identified SNPs were mostly non-synonymous. The study demonstrates the possibility of the existence of polymorphic populations in one host, based on strain variability observed for strains from the same animal and strains from different animals of one outbreak. The study also demonstrates that new outbreak strains are mostly related to earlier outbreak strains from the same farm/region. This indicates the last common ancestor strain from one farm can be crucial to understand the genetic changes and epidemiology occurring at farm level. Known virulence factors for the species were highly conserved among the strains. Genetic elements involved in Nicotinamide adenine dinucleotide (NAD) precursor synthesis (via nadA, nadB, and nadC metabolic pathway) which are known as potential anti-virulence loci are completely absent in C. chauvoei compared to the partial inactivation in C. septicum. A novel core-genome MLST based typing method was compared to sequence typing based on CRISPR spacers to evaluate the usefulness of the methods for outbreak investigations.

Brucellosis is an important bacterial zoonosis of domestic and wildlife species. This disease has a significant public health concern and is characterized by reproductive failure resulting in economic losses in the livestock industry. Among thirteen known species, B. abortus, B. melitensis, B. suis, and B. canis are human pathogens. Brucellosis has been extensively investigated in humans and domestic animals. However, the situation in wildlife is still not completely reported and studied. Therefore, a systematic literature search and screening were done to clarify the situation of brucellosis in wildlife in Europe. Sixty-five articles from a total of 13,424 reports published between 1991 and 2021 were selected, applying defined inclusion criteria. Wild boars and brown hares were the most often studied terrestrial wildlife species, whereas seals and porpoises were the most often investigated marine wildlife. Poland, Croatia, and Belgium showed the highest seroprevalences of wild boars caused by B. suis biovar 2. In marine wildlife, brucellosis was mainly caused by B. ceti and B. pinnipedialis. Most samples were from carcasses. Thus, sera could not be collected. It is worrisome that B.abortus and B. melitensis were reported from both terrestrial and marine wild animals, posing a zoonotic threat to people exposed to wild animals. Currently, there is no approved vaccine available for wild animals. The main challenges are the development of specific diagnostics and their validation for use in wildlife.

Poultry is one of the most important reservoirs for zoonotic multidrug-resistant pathogens. The indiscriminate use of antimicrobials in poultry production is a leading factor for development and dissemination of antimicrobial resistance. This study aimed to describe the prevalence and antimicrobial resistance of E. coli isolated from healthy turkey flocks of different ages in Nile delta region, Egypt. In the current investigation, 250 cloacal swabs were collected from 12 turkey farms in five governorates in the northern Egypt. Collected samples were cultivated on BrillianceTM ESBL agar media supplemented with cefotaxime (100 mg/L). The E. coli isolates were identified using MALDI-TOF-MS and confirmed by a conventional PCR assay targeting 16S rRNA-DNA. The phenotypic antibiogram against 14 antimicrobial agents was determined using the broth micro-dilution method. DNA-microarray-based assay was applied for genotyping and determination of both, virulence and resistance-associated gene markers. Multiplex real-time PCR was additionally applied for all isolates for detection of the actual most relevant Carbapenemase genes. The phenotypic identification of colistin resistance was carried out using E-test. A total of 26 E. coli isolates were recovered from the cloacal samples. All isolates were defined as multidrug-resistant. Interestingly, two different E. coli strains were isolated from one sample. Both strains had different phenotypic and genotypic profiles. All isolates were phenotypically susceptible to imipenem, while resistant to penicillin, rifampicin, streptomycin, and erythromycin. None of the examined carbapenem resistance genes was detected among isolates. At least one beta-lactamase gene was identified in most of isolates, where blaTEM was the most commonly identified determinant (80.8%), in addition to blaCTX-M9 (23.1%), blaSHV (19.2%) and blaOXA-10 (15.4%). Genes associated with chloramphenicol resistance were floR (65.4%) and cmlA1 (46.2%). Tetracycline- and quinolone-resistance-associated genes tetA and qnrS were detected in (57.7%) and (50.0%) of isolates, respectively. The aminoglycoside resistance associated genes aadA1 (65.4%), aadA2 (53.8%), aphA (50.0%), strA (69.2%), and strB (65.4%), were detected among isolates. Macrolide resistance associated genes mph and mrx were also detected in (53.8%) and (34.6%). Moreover, colistin resistance associated gene mcr-9 was identified in one isolate (3.8%). The class 1 integron integrase intI1 (84.6%), transposase for the transposon tnpISEcp1 (34.6%) and OqxB -integral membrane and component of RND-type multidrug efflux pump oqxB (7.7%) were identified among the isolates. The existing high incidence of ESBL/colistin-producing E. coli identified in healthy turkeys is a major concern that demands prompt control; otherwise, such strains and their resistance determinants could be transmitted to other bacteria and, eventually, to people via the food chain.

Brucellosis is a bacterial zoonotic disease that affects many animal species and can be transmitted to humans via direct contact or via contaminated food. Although brucellosis is a serious health hazard, its public health concern has been neglected in many countries. In some developing countries, such as Pakistan, where brucellosis is endemic, this disease continues to be of importance. A literature search for the past 11 years (2011–2021) provided a comprehensive insight into brucellosis in Pakistan. In this review, particular emphasis was placed on occurrence, diagnostic tests used, and prevention, treatment, and control in the context of the “One Health” approach.

Q fever remains a neglected zoonosis in many developing countries including Pakistan. The causing agent Coxiella (C.) burnetii is resistant to environmental factors (such as drying, heat and many disinfectants), resulting in a long-lasting infection risk for both human and animals. As the infection is usually asymptomatic, it mostly remains undiagnosed in animals until and unless adverse pregnancy outcomes occur in a herd. In humans, the infection leads to severe endocarditis and vascular infection in chronic cases. Limited data are available on molecular epidemiology and evolution of this pathogen, especially in ruminants. Genomic studies will help speculating outbreak relationships in this scenario. Likewise, pathogenesis of C. burnetii needs to be explored by molecular studies. Awareness programs and ensuring pasteurization of the dairy milk before human consumption would help preventing Q fever zoonosis.

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.

The aim of this study was to investigate the seroprevalence of Q fever in sheep and goats in Kasur, Okara, and Pakpattan in the Punjab of Pakistan. Q fever is a widely reported zoonotic disease caused by Coxiella (C.) burnetii. The main reservoirs are small ruminants that excrete the bacteria in birth by-products in high numbers. Thus, the bacteria can also be detected in the air and the dust of livestock farms. The infection is often asymptomatic in ruminants, but it can lead to reproductive disorders. This cross-sectional study found that a significant number (n = 43; 11.3%) of 300 randomly selected small ruminants of nine tehsils were seropositive using a commercially available ELISA. Seroprevalence was significantly higher in goats (17.1%) than in sheep (4.9%). Binary logistic regression analysis proved that species, age, and breed have a significant effect on the prevalence of Q fever. Tick infestation, contact with animal fomites, contact with other animals, production system, and health status of an animal had a significant impact on the prevalence of Q fever. These findings on Q fever in animals can be used to improve the visibility of this zoonotic disease. These findings will help local health authorities to focus on the origin of the problem and facilitate applying preventive measures to the affected livestock farms.

Brucellae are Gram-negative, aerobic, non-motile coccobacilli causing brucellosis in man and animals. The disease is one of the most significant yet neglected global zoonoses. Especially in developing countries, brucellosis is causing public health problems and economic losses to private animal owners and national revenues. Composed of oligonucleotides, aptamers are chemical analogues of antibodies that are promising components for developing aptamer-based rapid, sensitive, and specific tests to identify the Brucella group of bacteria. For this purpose, aptamers were generated and selected by an enhanced protocol of cell systematic evolution of ligands by exponential enrichment (cell-SELEX). This enhanced cell-SELEX procedure involved the combination of both conventional and toggle cell-SELEX to boost the specificity and binding affinity to whole Brucella cells. This procedure, combined with high-throughput sequencing of the resulting aptamer pools, comprehensive bioinformatics analysis, and wet lab validation assays, led to the selection of a highly sensitive and specific aptamer for those Brucella species known to circulate in Egypt. The isolated candidate aptamer showed dissociation constant (KD) values of 43.5 ± 11, 61.5 ± 8, and 56 ± 10.8 nM for B. melitensis, B. abortus, and B. suis, respectively. This is the first development of a Brucella-specific aptamer using an enhanced combination of conventional and toggle cell-SELEX to the authors’ best knowledge.

Glanders, caused by Burkholderia (B.) mallei is a notifiable zoonotic disease in equidae. For international trade and movement of equids, certificates of negative serological test results for antibodies against B. mallei are required. To date, the complement fixation test (CFT) is the mandatory test to issue these health certificates. The CFT is difficult to standardize and, due to its poor specificity, often leads to false-positive reactions resulting in trade restrictions with considerable financial consequences. In the present study, the new ID Screen Glanders Double Antigen Multispecies ELISA (GLANDA- ELISA) (IDvet, Grabels, France) was evaluated using 400 negative and 370 glanders positive field samples of equidae. The GLANDA-ELISA was significantly more specific (99.8%) than the CFT (97.0%). Considering the comparable sensitivities of CFT (96.5%) and ELISA (98.1%), this new GLANDA-ELISA test appears a suitable confirmatory test and a realistic alternative for serological testing of horses for trade or movement.

Bovine brucellosis is a global zoonosis of public health importance. It is an endemic disease in many developing countries including Pakistan. This study aimed to estimate the seroprevalence and molecular detection of bovine brucellosis and to assess the association of potential risk factors with test results. A total of 176 milk and 402 serum samples were collected from cattle and buffaloes in three districts of upper Punjab, Pakistan. Milk samples were investigated using milk ring test (MRT), while sera were tested by Rose–Bengal plate agglutination test (RBPT) and indirect enzyme-linked immunosorbent assay (i-ELISA). Real-time PCR was used for detection of Brucella DNA in investigated samples. Anti-Brucella antibodies were detected in 37 (21.02%) bovine milk samples using MRT and in 66 (16.4%) and 71 (17.7%) bovine sera using RBPT and i-ELISA, respectively. Real-time PCR detected Brucella DNA in 31 (7.71%) from a total of 402 bovine sera and identified as Brucella abortus. Seroprevalence and molecular identification of bovine brucellosis varied in some regions in Pakistan. With the use of machine learning, the association of test results with risk factors including age, animal species/type, herd size, history of abortion, pregnancy status, lactation status, and geographical location was analyzed. Machine learning confirmed a real observation that lactation status was found to be the highest significant factor, while abortion, age, and pregnancy came second in terms of significance. To the authors' best knowledge, this is the first time to use machine learning to assess brucellosis in Pakistan; this is a model that can be applied for other developing countries in the future. The development of control strategies for bovine brucellosis through the implementation of uninterrupted surveillance and interactive extension programs in Pakistan is highly recommended.

The emergence of Klebsiella pneumoniae (K. pneumoniae) in German healthcare is worrying. It is not well-investigated in the veterinary world and food chains. In the current study, antibiotic susceptibility profiles of 24 K. pneumoniae strains isolated from powdered milk samples produced in Germany were investigated by a microdilution test. Next-generation sequencing (NGS) was applied to identify genomic determinants for antimicrobial resistance (AMR), virulence-associated genes and plasmids replicons. All isolates were susceptible to the majority (14/18) of tested antibiotics. Resistance to colistin, fosfomycin, chloramphenicol and piperacillin was found. The ambler class A ß-lactamase, blaSHV variants were identified in all isolates, of which blaSHV-187 was most prevalent and found in 50% of isolates. Single-nucleotide-variants of oqxA and oqxB conferring resistance to phenicol/quinolone were found in all isolates, and the oqxB17 was the most prevalent found in 46% of isolates. 67% of isolates harbored fosA genes; however, only one was fosfomycin-resistant. Two isolates harbored genes conferring resistance to colistin, despite being susceptible. The majority of identified virulome genes were iron uptake siderophores. Two enterobactins (entB, fepC), six adherence-related genes belonging to E. coli common pilus (ECP) and one secretion system (ompA gene) were found in all isolates. In contrast, yersiniabactin was found in two isolates. One ST23 strain was susceptible to all tested antibiotics, and harbored determinants discriminatory for hypervirulent strains, e.g., aerobactin, salmochelin, yersiniabactin, enterobactin and regulator of mucoid phenotype A genes that are highly associated with hypervirulent K. pneumoniae. The IncF plasmid family was found in all strains, while almost half of the isolates harbored Col440I-type plasmids and nine isolates harbored various Inc-type plasmids. The presence of K. pneumoniae carrying different resistomes and major virulent specific virulomes in powdered milk samples is alarming. This could threaten public health, particularly of neonates and infants consuming dried milk.

Brucellosis is reportedly endemic in ruminants in Pakistan. Both Brucella abortus and B. melitensis infections have been decumented in domestic animals and humans in the country. This study aimed to identify the burden of anti-Brucella antibodies in small ruminants as well as associated potential risk factors with its occurrence at nine institutional livestock farms in Punjab, Pakistan. The sera collected from equal number of sheep and goats (500 from each species) were screened by indirect-ELISA for anti-smooth-Brucella antibodies followed by a serial detection by real-time PCR. Overall, 5.1% (51/1000) seropositivity was registered corresponding to 5% (25/500) prevalence in goats and 5.2% (26/500) in sheep. Brucella-DNA could not be detected in any of the tested sera by real-time PCR. Multiple logistic regression model indicated that farm location (OR 34.05), >4 years of age (OR 2.88), with history of reproductive disorders (OR 2.69), and with BCS of ≤ 3 (OR 12.37) were more likely to test positive for brucellosis at these farms. A routine screening, stringent biosecurity, and quarantine measures are warranted for monitoring and eradication of the infection. Similarly, isolation and molecular investigation of the etiologic agent(s) are needed to understand the relationship of epidemiology and out-breaks of brucellosis in the country.

Abstract Background Poultry remains one of the most important reservoir for zoonotic multidrug resistant pathogens. The global rise of antimicrobial resistance in Gram-negative bacteria is of reasonable concern and demands intensified surveillance. Methods In 2016, 576 cloacal swabs were collected from 48 broiler farms located in five governorates in northern Egypt. Isolates of Enterobacteriaceae could be cultivated on different media and were identified by MALDI-TOF MS and PCR. Escherichia coli isolates were genotyped by DNA-microarray-based assays. The antimicrobial susceptibility to 14 antibiotics was determined and resistance-associated genes were detected. The VITEK-2 system was applied for phenotypical confirmation of extended-spectrum β-lactamase-producing isolates. The determination of colistin resistance was carried out phenotypically using E-test and genotypically using PCR for detection of the mcr-1 gene. Results Out of 576 samples, 72 representatives of Enterobacteriaceae were isolated and identified as 63 E. coli (87.5%), 5 Enterobacter cloacae (6.9%), 2 Klebsiella pneumoniae (2.8%) and 2 Citrobacter spp. (2.8%). Seven out of 56 cultivated E. coli (12.5%) were confirmed as ESBL-producing E. coli and one isolate (1.8%) as ESBL/carbapenemase-producing E. coli. Five out of 63 E. coli isolates (7.9%) recovered from different poultry flocks were phenotypically resistant to colistin and harboured mcr-1 gene. Conclusions This is the first study reporting colistin resistance and emergence of multidrug resistance in Enterobacteriaceae isolated from healthy broilers in the Nile Delta region, Egypt. Colistin-resistant E. coli in poultry is of public health significance. The global rise of ESBL- and carbapenemase-producing Gram-negative bacteria demands intensified surveillance. ESBL-producing E. coli in poultry farms in Egypt are of major concern that emphasizes the possibility of spread of such strains to humans. The results also reinforce the need to develop strategies and to implement specific control procedures to reduce the use of antibiotics.

Abstract Objective The antimicrobial resistance of staphylococci rose worldwide. In total, 96 Staphylococcus isolates from food and clinical samples were collected from two provinces in Algeria. The antimicrobial susceptibility testing was performed and resistance-associated genes were detected. Results Fifty-one strains were isolated from food samples and differentiated into 33 Staphylococcus aureus and 18 coagulase-negative staphylococci. Forty-five staphylococci were collected from hospital and community-acquired infection cases. All S. aureus isolated from food were resistant to penicillin and 45.5% were resistant to tetracycline. The resistance rates of 45 clinical Staphylococcus isolates were 86.7%, 48.9%, 37.8% and 20.0% to penicillin, tetracycline, erythromycin and kanamycin, respectively. Nine isolates were confirmed as MRSA from food and clinical isolates. One S. aureus originated from food was confirmed as vancomycin-resistant. Multidrug-resistance was observed among 25.5% and 53.3% of food and clinical staphylococci, respectively. The tetM/K, blaZ, aacA-aphD, ermC and mecA genes were detected in food and clinical isolates. ermA gene was not found. This study provided insight into the status of antimicrobial resistance of staphylococci isolated from food and clinical samples in Algeria. Further investigations and surveillance programmes are mandatory.

Abstract Background Gallibacterium anatis is an opportunistic pathogen of intensively reared poultry causing oophoritis, salpingitis, peritonitis and enteritis. Gallibacterium anatis infection often remains undiagnosed. Recently multi-drug resistant isolates have been described. Methods A newly developed PCR restriction fragment length polymorphism assay targeting the 16S rRNA gene was used to identify and differentiate Gallibacterium isolates from chicken, turkey and partridge samples originating from 18 different geographical locations in Thuringia, Germany. Antimicrobial susceptibility to 19 compounds of different classes was assessed. Results Nineteen Gallibacterium isolates were investigated. In 9 birds (47.4%) Gallibacterium species were isolated exclusively while in 10 birds (52.6%) other bacterial or viral agents could be detected in addition. In one chicken a mixed infection of Gallibacterium anatis and Gallibacterium genomospecies was identified. All isolates were susceptible to apramycin, florfenicol and neomycin and resistant to clindamycin, sulfathiazole and penicillin. Resistance to sulfamethoxim, spectinomycin, tylosin and oxytetracycline was observed in 93.3%, 93.3%, 86.7% and 80.0% of the field strains, respectively. Conclusions The PCR-RFLP assay allows specific detection and differentiation of Gallibacterium spp. from poultry. Antimicrobial resistance of Gallibacterium spp. is highly significant in Thuringian field isolates.

Staphylococcus aureus is a commensal resident of the skin and nasal cavities of humans and can cause various infections. Some toxigenic strains can contaminate food matrices and cause foodborne intoxications. The present study aimed to provide relevant information (clonal complex lineages, agr types, virulence and antimicrobial resistance-associated genes) based on DNA microarray analyses as well as the origins and dissemination of several circulating clones of 60 Staphylococcus aureus isolated from food matrices (n = 24), clinical samples (n = 20), and nasal carriers (n = 16) in northern Algeria. Staphylococcus aureus were genotyped into 14 different clonal complexes. Out of 60 S. aureus, 13 and 10 isolates belonged to CC1-MSSA and CC97-MSSA, respectively. The CC 80-MRSA-IV was the predominant S. aureus strain in clinical isolates. The accessory gene regulator allele agr group III was mainly found among clinical isolates (70.4%). Panton–Valentine leukocidin genes lukF/lukS-PV were detected in 13.3% of isolates that all belonged to CC80-MRSA. The lukF/S-hlg, hlgA, and hla genes encoding for hemolysins and leucocidin components were detected in all Staphylococcusaureus isolates. Clinical and food isolates harbored more often the antibiotic resistance genes markers. Seventeen (28.3%) methicillin-resistant Staphylococcus aureus carrying the mecA gene localized on a SCCmec type IV element were identified. The penicillinase operon (blaZ/I/R) was found in 71.7% (43/60) of isolates. Food isolates belonging to CC97-MSSA carried several antibiotic resistance genes (blaZ, ermB, aphA3, sat, tetM, and tetK). The results of this study showed that all clones were found in their typical host, but interestingly, some nasal carriers had isolates assigned to CC705 thought to be absent in humans. The detection of MRSA strains among food isolates should be considered as a potential public health risk. Therefore, controlling the antibiotics prescription for a rational use in human and animal infections is mandatory.