Extensively drug-resistant (XDR) Neisseria gonorrhoeae strains threaten the current empirical gonorrhoea treatment. We designed and analytically validated SensiTyper, a rule-based framework that predicts antimicrobial susceptibility from whole-genome sequencing data and generates susceptibility-guided individualized treatment recommendations. SensiTyper was applied to 13,701 gonococcal genomes from 68 countries in 1928-2024, including cultured isolates and clinical molecular samples, and benchmarked predictions against previous genomic and phenotypic data. Susceptibility predictions for ceftriaxone, ciprofloxacin, spectinomycin, and zoliflodacin had 98.9-100% sensitivity and specificity. Azithromycin susceptibility predictions had 92.5% sensitivity and 97.1% specificity, and gepotidacin susceptibility prediction 100% sensitivity, but 0% specificity, illustrating that low-level phenotypic gepotidacin resistance is not caused by the only known resistance determinants (GyrA A92T plus ParC D86N). The SensiTreat module integrates antimicrobial susceptibility profiles into sequential treatment logic, providing susceptibility-guided individualized treatment recommendations while also enabling alternative stewardship strategies prioritizing, e.g., ciprofloxacin. SensiTyper flags XDR isolates for expert follow-up, test-of-cure and comprehensive contact tracing. In conclusion, SensiTyper is a robust and adaptable approach for gonococcal antimicrobial susceptibility prediction, individualized gonorrhoea treatment and antimicrobial stewardship.

Streptococcus suis is an important swine pathogen and an emerging zoonotic bacterium, with multidrug-resistant (MDR) strains representing an increasing concern for both animal and public health. This report describes the pathological findings and antimicrobial susceptibility profiles of MDR S. suis isolates recovered from the central nervous system of two pigs from a commercial swine farm in south-central Chile. Brain and spinal cord samples were examined by histopathology and microbiological culture, followed by bacterial identification and antimicrobial susceptibility testing using an antibiogram and minimum inhibitory concentration (MIC) analysis. Histopathological examination revealed non-suppurative inflammatory lesions characterized by mononuclear perivascular cuffing, consistent with meningoencephalomyelitis and myelitis. S. suis was isolated from the brain and/or spinal cord of both pigs. MIC testing demonstrated resistance to spectinomycin, ampicillin/amoxicillin, ceftiofur, penicillin, florfenicol, enrofloxacin, clindamycin, tilmicosin, tulathromycin, tylosin tartrate, tiamulin, sulfadimethoxine, trimethoprim/sulfamethoxazole, oxytetracycline, and chlortetracycline, indicating an MDR phenotype. Both isolates remained susceptible to gentamicin, while variable susceptibility was observed for danofloxacin, neomycin, tulathromycin, tiamulin, and trimethoprim/sulfamethoxazole. To the authors' knowledge, this is the first report of MDR S. suis associated with neurological disease in pigs from Chile. These findings provide additional information on the antimicrobial susceptibility of S. suis in Chile and support the need for broader antimicrobial resistance surveillance under a One Health framework, as well as the prudent use of antimicrobials in swine production.

The Australian Gonococcal Surveillance Programme (AGSP) has continuously monitored antimicrobial resistance in Neisseria gonorrhoeae (NG) for more than 45 years through the system of jurisdictional Neisseria reference laboratories, the National Neisseria Network (NNN). In 2025, a total of 10,204 isolates across Australia, from public and private sectors, were tested for in vitro antimicrobial susceptibility by standardised methods. In 2025, the AGSP captured antimicrobial susceptibility data for 23.9% of all gonococcal infection notifications nationally. The current treatment recommendation for gonorrhoea, for the majority of Australia, continues to be dual therapy with ceftriaxone and azithromycin. In 2025, of NG isolates tested, 0.73% (74/10,204) met the World Health Organization (WHO) criterion for decreased susceptibility (DS) to ceftriaxone, defined as a minimum inhibitory concentration (MIC) ≥ 0.125 mg/L. This proportion of isolates meeting the ceftriaxone DS criterion was greater than that reported in 2024 (0.51%), with the majority from New South Wales and Victoria. Genomic analysis indicated that 64.9% of these isolates (48/74) possessed the mosaic penA 60.001 allele, the key target associated with ceftriaxone resistance. Resistance to azithromycin was reported in 7.1% of NG isolates nationally, increasing from being proportionally stable (3.9% to 4.7%) since 2019. Of NG isolates tested, 0.20% (20/10,204) exhibited high-level resistance to azithromycin (MIC value ≥ 256 mg/L), with cases reported across Australia, predominantly in New South Wales, Victoria and non-remote Western Australia. There were ten isolates detected in 2025 that had an extensively drug-resistant (XDR) phenotype: i.e., displaying both high-level resistance to azithromycin and decreased susceptibility to ceftriaxone (MIC ≥ 0.125 mg/L). When added to the 14 XDR isolates reported during 2022-2024, this brings the total to 24 XDR NG isolates reported in Australia since 2022. Genomic analyses of the ten XDR isolates reported in 2025 indicated all harboured the mosaic penA 60.001 allele, with several differing sequence types (ST) identified. By contrast, during 2022 and 2024, XDR NG sequence types were limited to ST-16406, consistent with reports from Europe and England. Travel information, where available, indicates infections in 2025 were associated with travel to Southeast Asia but also arose where no travel was involved. In 2025, penicillin resistance was found in 23.4% of gonococcal isolates, and ciprofloxacin resistance in 59.3% of isolates where tested, although there was variation by jurisdiction particularly in remote settings, where acquisition of cultures for antimicrobial susceptibility testing is low. Tetracycline resistance has been included in surveillance in the AGSP in recent years, coincident with increasing use of doxycycline post-exposure prophylaxis for syphilis and chlamydia. Nationally 36.2% of NG isolates in 2025 were tetracycline resistant, with variation by jurisdiction. No isolates were resistant to spectinomycin; data for gentamicin, whilst no breakpoints are defined, are reported by the AGSP as these data are reportable to the WHO Global Antimicrobial Resistance and Use Surveillance System (GLASS). The emergence of antimicrobial resistant NG in Australia has largely occurred through importation, spread and local transmission. In 2025, in Australia and overseas, increased detection of drug-resistant NG isolates is of significant concern as the threat for the future of the current recommended therapeutic agents is increasingly evident, and no ideal alternate therapeutic agent has been identified. Disease prevention strategies under investigation for the future have included the MenB-4C vaccine, designed to protect against meningococcal disease. Despite early promise, recent results from a large Australian trial indicate the MenB-4C vaccine is not effective at preventing gonorrhoea in men who are at high risk of infection. The AGSP continues to monitor antimicrobial resistance and to identify emerging resistant clones by enhanced surveillance approaches, to inform strategies for disease management and treatment of gonorrhoea in Australia.

Background/Objectives: Despite its hostile physicochemical environment characterized by high osmotic pressure, acidic pH, and low water activity, honey serves as a unique ecological niche for resilient microorganisms. This study aimed to recover bacterial isolates from honeys of different botanical origins and to characterize the probiotic-associated properties of a honey-derived Bacillus licheniformis isolate. Methods: A total of 20 bacterial isolates were recovered from four honey samples, including two blossoms and two honeydew honeys. 16S rRNA gene sequence analysis showed that the recovered isolates had taxonomic assignments consistent with Bacillus licheniformis. Because 16S rRNA sequencing does not provide sufficient resolution to establish strain-level diversity or clonality, one isolate, designated Blhoney, was selected for subsequent molecular and functional characterization. Results: In vitro assays revealed that Blhoney exhibits robust tolerance to simulated gastrointestinal stress, including extreme acidity (pH 1-3), bile salts (0.3-1%), pepsin, and pancreatin, maintaining viability for up to 24 h. Phenotypic antimicrobial testing demonstrated measurable inhibition zones for erythromycin, kanamycin, chloramphenicol, ampicillin, tetracycline, gentamicin, streptomycin, and spectinomycin, whereas no measurable inhibition zones were observed for penicillin G, rifampicin, ciprofloxacin, or ceftazidime. However, MIC determination and standardized breakpoint interpretation were not performed; therefore, these findings were interpreted solely as preliminary phenotypic inhibition-zone observations. Although the isolate did not display direct antagonistic activity against the tested bacterial pathogens under experimental conditions, its persistence under the tested gastrointestinal stress conditions supports further investigation of its probiotic-associated characteristics. Conclusions: Overall, these findings indicate that the honey-derived Blhoney isolate exhibits preliminary probiotic-associated characteristics under the tested in vitro conditions. However, further strain-level genomic and safety characterization is required before its potential probiotic application can be established.

We describe in detail an experimental strategy, DOPE (Dependence On Plasmid-mediated Expression), to enable disruption of an essential chlamydial gene. A wild-type Chlamydia trachomatis strain is first transformed with a β-lactamase-selectable plasmid carrying a copy of the gene under the inducible control of an anhydrotetracycline (ATC)-responsive promoter. Transformants are selected under permissive conditions in the absence of ATC to establish stable plasmid maintenance. The resulting chlamydiae are subsequently transformed with a suicide plasmid encoding a group II intron engineered to target the chromosomal copy of the gene. Secondary transformants with the intron inserted into the chromosomal locus are then selected with spectinomycin in the presence of ATC to induce expression of the plasmid-encoded allele. This protocol thus produces a conditional knockout strain in which expression of the essential gene is inducibly controlled by ATC so that its function in chlamydial growth and developmental progression can be delineated.

Genetic manipulation of Chlamydia trachomatis can elucidate the determinants involved in pathogenesis but is difficult due to this bacterium's obligate intracellular lifestyle and comparatively low transformation rate. Deletion mutants can be generated in Chlamydia by allelic exchange via homologous recombination, but require conditionally replicating vectors that have to be maintained and propagated through several rounds of the intracellular developmental cycle. An additional challenge is that Chlamydia and E. coli cannot stably maintain each other's endogenous plasmids. As a solution, the ori (origin of replication) region from C. trachomatis endogenous plasmid pL2 was cloned into the E. coli pUC19 plasmid together with genes for fluorescent reporting and spectinomycin resistance to generate the pKW shuttle vector. pKW can be successfully propagated in both Chlamydia and E. coli, but is rapidly lost by chlamydial transformants upon removal of antibiotic selection pressure. In this chapter, we describe how to prepare the chlamydial stock and generate a pKW-based allelic exchange shuttle vector, with chromosomal sequences upstream and downstream of a target gene, and transform it into C. trachomatis. We provide step-by-step instructions for generating a targeted deletion mutant by growing the transformants under antibiotic selection to allow homologous recombination with the target gene and then removing the antibiotic to induce loss of the suicide vector.

Background and Objectives: Antimicrobial resistance in Neisseria gonorrheae threatens the effectiveness of empirical gonorrhoea treatment. Although global surveillance evidence is increasingly available, resistance data are not consistently reported separately for men with urethral or urogenital infection. This review evaluated antibiotic-specific resistance in N. gonorrhoeae isolates from this clinically defined population and examined the geographic and reporting limitations of the available evidence. Materials and Methods: PubMed, Scopus, ScienceDirect, Google Scholar, and relevant WHO GASP/EGASP resources were searched from inception to [updated search date]. Eligible observational or surveillance studies reported categorical resistance classifications or extractable antibiotic-specific resistant counts and denominators for male urethral or urogenital isolates. Antibiotic-specific proportions were synthesized using random-effects meta-analysis where pooling was considered appropriate. Sparse-event outcomes were additionally evaluated using study-specific estimates and aggregate resistant/tested counts. Certainty of evidence was assessed using the GRADE framework adapted for prevalence outcomes. Results: Six studies met the eligibility criteria, including the 2025 multinational EGASP study. Cefixime resistance was reported in 748 of 13,154 isolates, corresponding to an aggregate proportion of 5.69%; however, the random-effects estimate was unstable because three studies reported no resistant isolates and events were concentrated in two large datasets. Ceftriaxone resistance varied from 0% to 5.17%, with a pooled estimate of 0.32% (95% CI 0.05-2.15; I[2] = 97.8%). Resistance was high but heterogeneous for ciprofloxacin (71.84%, 95% CI 7.53-98.77; I[2] = 97.3%) and penicillin (62.47%, 95% CI 36.87-82.58; I[2] = 98.1%). Spectinomycin resistance was 2.57% (95% CI 1.58-4.16; I[2] = 0%), while azithromycin resistance was 6.38% (95% CI 4.38-9.21; I[2] = 94.9%). Certainty of evidence was very low for all outcomes. Conclusions: Evidence specifically reporting resistance in male urethral or urogenital N. gonorrhoeae isolates remains limited and geographically uneven. Contemporary EGASP data demonstrate that ceftriaxone resistance is not uniformly low across surveillance settings. The pooled estimates should be interpreted as exploratory summaries rather than current global prevalence values or an independent basis for treatment selection. Standardized reporting of sex, anatomical site, breakpoints, MIC distributions, and resistant/tested counts is needed to improve future surveillance synthesis.

The prime editing (PE) system is a precise genome editing technology that works efficiently in monocots; however, its application is limited by low editing efficiency in dicots, particularly Cucurbitaceae and Solanaceae plants. Here, we first significantly improved the transformation efficiency by introducing spectinomycin in cucurbits, then used the tomato elongation factor 1-alpha (SlEF1α) promoter to enhance PE protein expression, and incorporated the Csy4 ribonuclease to process pegRNAs, collectively addressing multiple constraints limiting PE efficiency in cucurbits. The optimized PE systems, particularly Csy4-PE6d, achieved an average desired editing frequency of 80.83% at targeted loci in cucumber via stable genetic transformation, with frequencies reaching up to 100% at certain sites. Moreover, Csy4-PE6d generated homozygous edits in 36.43% of transgenic lines and demonstrated robust editing activity in melon, pumpkin, and potato. Using the Csy4-PE6d tool, we generated heritable edited cucumber lines with dual resistance to bacterial angular leaf spot and downy mildew by targeting the CsSGR gene. Collectively, this optimized system substantially enhances PE efficiency in Cucurbit crops, providing an effective solution to common challenges such as low editing efficiency and limited heritability in these species.

Antimicrobial resistance in Neisseria gonorrhoeae is a global concern. Spectinomycin is unusual in that resistance can emerge rapidly during localized outbreaks yet often disappears from clinical populations after drug withdrawal, suggesting an associated growth cost. To investigate evolutionary routes to spectinomycin resistance, we performed in vitro selection on two N. gonorrhoeae strains and two commensal Neisseria species. Derived cell lines were characterized by minimum inhibitory concentration (MIC) determination, whole-genome sequencing, growth-kinetics analysis, and molecular modelling of the RpsE (ribosomal protein S5) interface with the ribosome. All high-level resistant isolates (MIC > 2048 mg/L) acquired substitutions or deletions in loop 2 of RpsE. Modelling showed that these mutations perturb the conserved network of stabilizing contacts between RpsE residues Lys25 (Lys23 in E. coli numbering) and Lys28 (Lys26), as well as helix 34 nucleotides G922, A923, and C1069 of 16S rRNA, potentially altering the architecture of the spectinomycin-binding site. These mutations were associated with high-level resistance but reduced growth rates, with the resulting growth costs depending on the specific pattern of contact rearrangements. Convergent evolution towards loop 2 mutations supports the existence of a constrained mutational pathway to high-level spectinomycin resistance in the strains and species examined here. This constraint may help explain the rapid decline of resistant variants in the absence of drug pressure and underscores the importance of genomic surveillance.

Arizonosis is a bacterial septicemic disease caused by Salmonella enterica subspecies arizonae. This case report describes S. arizonae septicemia in commercial chukar partridge (Alectoris chukar) chicks that resulted in a 7-day flock mortality of 30% (2100/7000), with mortality rapidly progressing to 99.3% (6950/7000) by 3 wk of age. To our knowledge, this is the first documented case of S. arizonae acute septicemia in chukars. Gross and histopathologic findings in the chukars included hepatosplenomegaly, omphalitis, and multiorgan inflammation. Salmonella arizonae was isolated from the liver, spleen, and yolk sac cultures, and the isolate was serotyped as Salmonella arizonae serovar 18:z4,z23. Minimum inhibitory concentration antimicrobial susceptibility testing performed on S. arizonae classified the isolate as resistant to erythromycin, gentamicin, neomycin, and sulfadimethoxine and not susceptible to amoxicillin and penicillin; the isolate was classified as susceptible to oxytetracycline, tetracycline, ceftiofur, and spectinomycin. In addition to the arizonosis case report, a retrospective analysis of poultry arizonosis cases diagnosed at the California Animal Health and Food Safety laboratories from January 2005 to December 2024 identified 79 poultry cases of S. arizonae, predominantly in turkeys (69/79). A sharp decline in annual S. arizonae poultry cases was noted after 2009. The most common site of isolation was the yolk sac (45/79 cases), suggesting that vertical transmission plays a significant role in the dissemination of the disease. This case report highlights the susceptibility of chukar partridges to S. arizonae infection and disease and underscores the importance of early diagnosis, antimicrobial susceptibility testing, and stringent biosecurity practices to mitigate transmission and economic loss in commercial game bird operations.

Multidrug-resistant Salmonella enterica subsp. enterica serovar Infantis clone, harbouring the pESI megaplasmid, first described in Israel in 2014, is consistently reported in poultry and humans worldwide. This study aimed to investigate the genomic epidemiology of S. Infantis collected by nine European Public Health Institutions from samples of different origins (human, food and animal sources) and understand the evolutionary dynamics of pESI-like in Europe. The resolved pESI-like sequences have also been compared with complete publicly available pESI-like sequences from other countries, in a One Health context. The circulation of a S. Infantis clone in Europe carrying the mosaic megaplasmid pESI-like has been associated with resistance to sulphonamides (sul), tetracycline (tet), streptomycin and spectinomycin (aadA1). In recent years, bla CTX-M-1-positive pESI-like plasmids have been increasingly detected in the extended-spectrum beta-lactamase-producing S. Infantis clone. Using a combined short- and long-read sequencing approach, two main types of pESI variants have been identified, differing in the accessory gene content, including the bla CTX-M variant, indicating a certain stability of pESI variants detected in different geographical regions and sources over time (2011-2021). Moreover, the differences were related to the acquisition of resistance, virulence or fitness-enhancing genes that would potentially benefit the Salmonella host.

Many studies have identified antibiotic resistant (ABR) Escherichia coli on meat. Appropriate hand hygiene and cooking practices should minimize the risk of gastrointestinal colonisation with ABR E. coli found on meat, and the subsequent chance of causing resistant opportunistic extraintestinal infection. There are large gaps in our understanding of the prevalence, origins and zoonotic potential of ABR E. coli found on meat, however, and particularly for meat reared in extensive farming systems. Wales is a devolved nation within the United Kingdom having large populations of extensively-reared sheep and beef cattle. To help address knowledge gaps around ABR E. coli on extensively reared meat, therefore, beef mince and lamb loin/leg steaks/chops were purchased from 50 (beef) and 46 (lamb) independent butchers across Wales. Following enrichment culture, 200 g meat samples were found to be positive for E. coli resistant to amoxicillin (31% positivity), streptomycin (28%), spectinomycin (29%), amoxicillin-clavulanate (11%), 3rd generation cephalosporins (2%) and fluoroquinolones (5%). Phylogenetic analysis confirmed that Welsh lamb meat ABR E. coli isolates (n = 78) are more closely related to those found in faecal samples collected around sheep (n = 352) than around beef cattle (n = 361) on Welsh farms. This suggests that faecal contamination at or around slaughter is their primary origin. We found no closely related meat/infection clones (<20 SNPs distant and the same antibiotic resistance genes) when comparing ABR E. coli from Welsh meat (n = 91) and those causing extraintestinal infections in people (n = 2387) in an English region bordering Wales. We conclude, therefore, that the wider zoonotic implications of finding ABR E coli on beef and lamb meat sold at independent butchers in Wales are small, though a more geographically and temporarily contemporaneous survey is warranted to address this further.