Non-wild Type Research Articles

Antimicrobial Resistance in Lactococcus spp. Isolated from Native Brazilian Fish Species: A Growing Challenge for Aquaculture

Lactococcus spp. has emerged as a pathogen that is affecting global aquaculture, with L. garvieae, L. petauri, and L. formosensis causing piscine lactococcosis. While antimicrobials are commonly used to treat diseases in aquaculture, reports of antimicrobial resistance in fish isolates are increasing. However, little is known about the susceptibility patterns of Lactococcus spp. strains isolated from native fish species in Brazil. This study aimed to assess the antimicrobial susceptibility of these strains and establish a provisional epidemiological cutoff value for L. garvieae using the normalized resistance interpretation approach. A total of 47 isolates were tested: 17 L. garvieae, 24 L. petauri, and 6 L. formosensis. The isolates were classified as wild-type (WT) or non-wild-type (NWT) based on inhibition zone diameters. Isolates classified as NWT for three or more antimicrobial classes were considered multidrug-resistant, and the multiple antibiotic resistance (MAR) index was calculated. The results revealed heterogeneity in antimicrobial resistance profiles, with higher resistance to trimethoprim/sulfamethoxazole and norfloxacin. Resistance to other antimicrobials, including florfenicol and oxytetracycline (approved for use in Brazil), varied according to the bacterial species. Lactococcus petauri (87.5%) and L. formosensis (66.7%) showed the highest multidrug resistance, compared to L. garvieae (11.7%), along with higher MAR index values. These findings suggest that multidrug-resistant strains could pose future challenges in the production of native species, underscoring the need for ongoing monitoring of antimicrobial resistance and responsible use of antimicrobials in aquaculture.

Association of multilocus sequence typing, MSH2 gene mutations, and antifungal resistance in Candida glabrata: implications for clinical outcomes in Chinese hospitals

BackgroundCandida glabrata is the second most common cause of invasive candidiasis worldwide. In this study, we determined the clinical characteristics and drug sensitivity of C. glabrata isolates and investigated the associations between MSH2 gene mutations, sequence types (ST), and drug resistance.MethodsA total of 154 C. glabrata isolates were collected from patients being treated in three hospitals in China. The antifungal sensitivity of the strains was assessed using the broth microdilution method. Multilocus sequence typing (MLST) was also performed, followed by MSH2 sequencing. The clinical features and outcomes of C. glabrata infection were analysed for a total of 49 strains, which were collected from patients with invasive Candida infection at Longhua Hospital.ResultsAll 154 isolates were found to be susceptible to amphotericin, 5-fluorocytosine, anidulafungin, caspofungin, and micafungin, whereas 11.7% were fluconazole-resistant, 18.8% were itraconazole non-wild type, and 35.7% were voriconazole non-wild type. ST7 (62.34%) was the most common ST genotype, followed by ST10 (16.88%) and ST15 (7.79%). The total azole resistance rates for all isolates, ST7, ST10, and other STs were 36.4, 42.7, 34.6, and 18.8%, respectively. The ST7 and ST10 isolates were characterised by a higher drug resistance rate than the other minor ST isolates. Moreover, 59.09% of isolates had one or more MSH2 non-synonymous mutations, with V239L being the most commonly detected mutation. The frequency of MSH2 mutations was significantly higher in azole-resistant isolates than in other isolates, whereas P6L or L87P mutations were associated with the highest azole resistance rates of up to 87.5% and 80%, respectively. Our results indicated that ST7 and ST15 are independent predictors of mortality caused by C. glabrata infection and revealed a higher 30-day mortality in patients infected with these strains than in those infected with other ST isolates.ConclusionsOur findings revealed the relationships between MLST, MSH2 gene mutations, and drug resistance in the common pathogenic fungus C. glabrata, and thereby enabled us to identify strains that are associated with higher rates of mortality. These findings will contribute to enhancing our understanding of the pathogenesis of C. glabrata infection.

Multi-Locus Sequence Typing-Based Genetic Analysis, Antifungal Resistance, and Clinical Prognosis of Cryptococcus neoformans Infections in HIV-Infected Patients in Northern China.

This study aimed to investigate the molecular epidemiological characteristics and drug sensitivity of Cryptococcus from HIV-infected patients and their relationship with patients' prognosis. Seventy-six strains were collected and identified to the species level by matrix-assisted laser desorption ionization-time of flight mass spectrometry, confirmed by internal transcribed spacer sequencing. Multi-locus sequence typing was used for the typing of Cryptococcus, and its antifungal susceptibility was tested using FUNGUS 3. The clinical outcomes of the patients were reviewed at 3-, 6-, 9-, and 12-month follow-ups. All strains were Cryptococcus neoformans var. grubii classified into seven sequence types (STs) dominated by ST5, ST31, and a new ST702 strain. The 6- and 9-month survival rates were highest for patients infected with ST31, ST32, and ST174. The antifungal resistant rates were 13.2%, 2.6%, and 1.4% for fluconazole, amphotericin B, and 5-fluorocytosine. Except itraconazole, the minimum inhibitory concentration (MIC) values and wild type (WT)/non-wild type (NWT) of Cryptococcus for antifungal drugs were not related to the clinical prognosis of HIV-infected patients with cryptococcal infection. ST5 was the main ST type, and the new ST702 type was found in a patient who died in a short period of time. Cryptococcus neoformans var. grubii had a relatively high antifungal drug resistance rate to fluconazole. The WT strain accounted for the highest proportions for 5-fluorocytosine, amphotericin B, fluconazole, voriconazole, and itraconazole. The MIC values of Cryptococcus for first-line antifungal drugs showed no relationship with clinical prognosis, implying that MIC values cannot be used to predict the clinical outcome of these patients.

Epidemiology of antimicrobial resistance in commensal E. coli from healthy dairy cattle on a Mediterranean pasture-based system of Australia: A cross-sectional study

This study aimed to determine the prevalence of antimicrobial resistance (AMR) in commensal E. coli from healthy lactating cows and calves in the Mediterranean pasture-based feeding dairy system of Western Australia (WA). Fecal samples were collected from healthy adult lactating cows and healthy calves from dairy farms in WA. Presumptive commensal E. coli was isolated from these samples and confirmed using matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Broth microdilution was used to assess the prevalence and the phenotypic AMR profiles of the E. coli isolates to 8 antimicrobial agents of dairy industry and human importance. The minimum inhibitory concentration (MIC) for each isolate was interpreted using the Epidemiologic Cutoff (ECOFF) and Clinical and Laboratory Standards Institute (CLSI) breakpoints. Genomic characterization provided multi-locus sequence types and AMR genes for a selection of isolates categorised as non-wild type (NWT) by ECOFF values for the combination of ampicillin, trimethoprim-sulfamethoxazole, and tetracycline. From a total of 1,117 fecal samples (633 adult, 484 calf) collected across 26 randomly selected farms, 891 commensal E. coli isolates were recovered (541 adult, 350 calf). Commensal E. coli classified as NWT was highest for ampicillin for both adult (68.8%; 95% CI = 64.7 - 72.7) and calf feces (67.1%; 95% CI = 62.0 - 72.0). A large proportion of tetracycline NWT and trimethoprim-sulfamethoxazole NWT organisms were also identified from calf feces, being 44.0% (95% CI = 38.7 - 49.4) and 24.6% (95% CI = 20.2 - 29.4) respectively. Clinical resistance prevalence was low, being higher for calves than for adult feces (ampicillin (adult: 7.8% (95% CI = 5.7 - 10.3); calf: 30.0% (95% CI = 25.2 - 35.1), tetracycline (adult: 6.3% (95% CI = 4.4 - 8.7); calf: 40.3% (95% CI = 35.1 - 45.6), and trimethoprim-sulfamethoxazole (adult: 2.6% (95% CI = 1.4 - 4.3); calf: 22.0% (95% CI = 17.7 - 26.7)). Commensal E. coli originating from calf feces was significantly higher in NWT prevalence compared with adult feces for ciprofloxacin (P = 0.023), gentamicin (P = 0.02), tetracycline (P < 0.001), and trimethoprim-sulfamethoxazole (P < 0.001). The overall number of antimicrobials an isolate was classified as NWT toward varied among farms and was significantly higher for isolates originating from calf than adult feces (P < 0.001). The strain type and sampling source of the commensal E. coli investigated were both associated with the commonality of the resultant resistance genome. Clinical resistance and NWT classification were highest for ampicillin, tetracycline, and trimethoprim-sulfamethoxazole, all antimicrobials commonly used in the treatment of dairy cattle in Australia. Though highly variable across farms, commensal E. coli isolated from healthy dairy calf feces had significantly higher NWT and multidrug resistance (MDR) prevalence compared with feces from healthy adult lactating dairy cows. The resistant genome identified in MDR isolates, though not always consistent with the phenotype, included QnrS1 and genes encoding AmpC β-lactamase and aminoglycoside phosphotransferase.

Investigating the resistome of haemolytic bacteria in Arctic soils.

Microorganisms inhabiting hostile Arctic environments express a variety of functional phenotypes, some of clinical interest, such as haemolytic ability and antimicrobial resistance. We studied haemolytic bacterial isolates from Arctic habitats, assessing their minimum inhibitory concentration (MIC) against antimicrobials. We then performed whole genome sequencing and analysed them for features conferring antimicrobial resistance. MIC data showed that Micromonospora spp. belong to 33% non-wild type (NWT) for erythromycin and penicillin and 22% NWT for tetracycline. Both Pseudomonas spp. belong to 43% NWT for nalidixic acid and streptomycin and 29% NWT for colistin. Finally, the Pedobacter isolate was in 80% NWT for antimicrobials tested. Whole-genome sequencing analyses revealed that fluoroquinolones, tetracyclines, macrolides and penams were the most frequent drug classes against which genotypic resistance was found. Additionally, resistance genes to heavy metals and disinfectants were identified. Our research demonstrates the presence of antimicrobial resistance in bacteria from Arctic habitats and highlights the importance of conservation efforts in these environments, where anthropogenic influence is becoming more evident. Furthermore, our data suggest the possible presence of novel resistance mechanisms, which could pose a threat if the responsible genes are transferable between species or become widespread due to environmental stress and alterations brought about by climate change.

Genetic and phenotypic diversity of Flavobacterium psychrophilum isolates from Czech salmonid fish farms

BackgroundThe salmonid pathogen Flavobacterium psychrophilum poses a significant economic threat to global aquaculture, yet our understanding of its genetic and phenotypic diversity remains incomplete across much of its geographic range. In this study, we characterise the genetic and phenotypic diversity of 70 isolates collected from rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta m. fario) from fish farms in the Czech Republic between 2012 and 2019 to compare their genomic content with all draft or complete genomes present in the NCBI database (n = 187).ResultsThe Czech isolates underwent comprehensive evaluation, including multiplex PCR-based serotyping, genetic analysis, antimicrobial resistance testing, and assessment of selected virulence factors. Multiplex PCR serotyping revealed 43 isolates as Type 1, 23 as Type 2, with sporadic cases of Types 3 and 4. Multi-locus sequence typing unveiled 12 sequence types (ST), including seven newly described ones. Notably, 24 isolates were identified as ST329, a novel sequence type, while 22 were classified as the globally-distributed ST2. Phylogenetic analysis demonstrated clonal distribution of ST329 in the Czech Republic, with these isolates lacking a phage sequence in their genomes. Antimicrobial susceptibility testing revealed a high proportion of isolates classified as non-wild type with reduced susceptibility to oxolinic acid, oxytetracycline, flumequine, and enrofloxacin, while most isolates were classified as wild type for florfenicol, sulfamethoxazole-trimethoprim, and erythromycin. However, 31 isolates classified as wild type for florfenicol exhibited minimum inhibitory concentrations at the susceptibility breakpoint.ConclusionThe prevalence of the Czech F. psychrophilum serotypes has evolved over time, likely influenced by the introduction of new isolates through international trade. Thus, it is crucial to monitor F. psychrophilum clones within and across countries using advanced methods such as MLST, serotyping, and genome sequencing. Given the open nature of the pan-genome, further sequencing of strains promises exciting discoveries in F. psychrophilum genomics.

An Unusual High Prevalence of Cryptococcus (Naganishia) diffluens Colonization in Neonates Hospitalized in a Referral Neonatal Intensive Care Unit.

Although the Candida species continue to be the most frequent colonizer of neonatal skin, a clear increase of colonization due to rare yeast-like fungi has been reported. In this study, we report an unusual high prevalence of Cryptococcus diffluens colonization in neonates admitted to the neonatal intensive care unit (NICU) over a 1-month period. From January 2020 to June 2021, the study included all neonates who were admitted to the NICU of Bu Ali Sina Hospital at least 28 days old. Skin swabs from different anatomical areas were collected. Sampling was done 3 times/week. Each sample was inoculated into Sabouraud Dextrose Agar containing chloramphenicol and CHROMagar Candida, separately. The plates were incubated at 30 °C and 35 °C, respectively. Identification of the isolates was molecularly confirmed. In vitro antifungal susceptibility testing of the isolates was performed against different antifungal agents using the Clinical Laboratory Standards Institute protocol. Among 1026 samples collected from 78 neonates, 213 yeast isolates were recovered, of which the Candida species were the most common (77.5%), followed by C. diffluens (16.9%). During the study, 55 isolated yeasts were collected from December 26, 2020, to January 26, 2021, of which 65.5% were C. diffluens, while Candida spp. constituted 100% and 98.3% of the isolates before and after this period, respectively. The most frequent sources of C. diffluens were genital regions (27.8%). Of 36 C. diffluens isolates, 13.9%, 22.2%, 52.8%, and 83.3% were non-wild type to fluconazole, amphotericin B, itraconazole and 5-flucytosine, respectively. We reported for the first time an unusual high prevalence of C. diffluens colonization in neonates hospitalized in NICU. Our findings also showed the high minimum inhibitory concentration of amphotericin B and 5-flucytosine against C. diffluens.

Genetic diversity and antifungal susceptibilities of environmental Cryptococcus neoformans and Cryptococcus gattii species complexes

Cryptococcosis is an opportunistic systemic mycosis caused by Cryptococcus neoformans and C. gattii species complexes and is of increasing global importance. Maintaining continued surveillance of the antifungal susceptibility of environmental C. neoformans and C. gattii isolates is desirable for better managing cryptococcosis by identifying resistant isolates and revealing the emergence of intrinsically resistant species. Relevant research data from Egypt are scarce. Thus, this study aimed to report the genetic diversity of C. neoformans and C. gattii species complexes originating from different environmental sources in Egypt, antifungal susceptibility profiles, antifungal combinations, and correlations of susceptibility with genotypes. A total of 400 environmental samples were collected, 220 from birds and 180 from trees. Cryptococcus spp. were found in 58 (14.5%) of the samples, 44 (75.9%) of the isolates were recovered from birds and 14 (24.1%) from trees. These isolates were genotyped using M13 polymerase chain reaction-fingerprinting and URA5 gene restriction fragment length polymorphism analysis. Of the 31 C. neoformans isolates, 24 (77.4%), 6 (19.4%) and one (4.4%) belonged to VNI, VNII, and VNIII genotypes, respectively. The 27 C. gattii isolates belonged to VGI (70.4%), VGII (18.5%), and VGIII (11.1%) genotypes. Non-wild type C. neoformans and C. gattii isolates that may have acquired resistance to azoles, amphotericin B (AMB), and terbinafine (TRB) were observed. C. gattii VGIII was less susceptible to fluconazole (FCZ) and itraconazole (ITZ) than VGI and VGII. C. neoformans isolates showed higher minimum inhibitory concentrations (MICs) to FCZ, ITZ, and voriconazole (VRZ) than those of C. gattii VGI and VGII. Significant (P < 0.001) correlations were found between the MICs of VRZ and ITZ (r = 0.64) in both C. neoformans and C. gattii isolates, FCZ and TRB in C. neoformans isolates, and FCZ and TRB (r = 0.52) in C. gattii isolates.There is no significant differences in the MICs of TRB in combination with FCZ (P = 0.064) or in combination with AMB (P = 0.543) and that of TRB alone against C. gattii genotypes. By calculating the fractional inhibitory concentration (FIC) index, the combination of FCZ + AMB was synergistic against all tested genotypes. These findings expand our knowledge of ecological niches, genetic diversity, and resistance traits of C. neoformans and C. gattii genotypes in Egypt. Further investigations into how they are related to clinical isolates in the region are warranted.

Harnessing Machine Learning to Uncover Hidden Patterns in Azole-Resistant CYP51/ERG11 Proteins.

Fungal resistance is a public health concern due to the limited availability of antifungal resources and the complexities associated with treating persistent fungal infections. Azoles are thus far the primary line of defense against fungi. Specifically, azoles inhibit the conversion of lanosterol to ergosterol, producing defective sterols and impairing fluidity in fungal plasmatic membranes. Studies on azole resistance have emphasized specific point mutations in CYP51/ERG11 proteins linked to resistance. Although very insightful, the traditional approach to studying azole resistance is time-consuming and prone to errors during meticulous alignment evaluation. It relies on a reference-based method using a specific protein sequence obtained from a wild-type (WT) phenotype. Therefore, this study introduces a machine learning (ML)-based approach utilizing molecular descriptors representing the physiochemical attributes of CYP51/ERG11 protein isoforms. This approach aims to unravel hidden patterns associated with azole resistance. The results highlight that descriptors related to amino acid composition and their combination of hydrophobicity and hydrophilicity effectively explain the slight differences between the resistant non-wild-type (NWT) and WT (nonresistant) protein sequences. This study underscores the potential of ML to unravel nuanced patterns in CYP51/ERG11 sequences, providing valuable molecular signatures that could inform future endeavors in drug development and computational screening of resistant and nonresistant fungal lineages.

Epidemiological cutoff values and genetic antimicrobial resistance of Lactococcus garvieae and L. petauri

Lactococcus garvieae and L. petauri are etiological agents of lactococcosis, which cause significant losses in trout aquaculture at elevated water temperatures. Optimizing antimicrobial dosage regimens and identifying the minimum inhibitory concentration (MIC) are important prerequisites for determining clinical breakpoints and effective antimicrobial use. In the present study, epidemiological cutoff values (ECV) were established for 14 antimicrobials using L. garvieae (n = 32) and L. petauri (n = 78), isolated from rainbow trout farms in Türkiye, Spain, Italy, and Greece. The provisional ECVs were calculated using the ECOFFinder and normalized resistance interpretation (NRI) approaches. Furthermore, gene cassettes, capsule gene clusters, and the presence of fourteen antibiotic resistance genes (ARGs) that encode resistance against seven antimicrobial agents, were evaluated. The calculated ECVs of antimicrobials ranged from 1.25 μg mL−1 for amoxicillin to 80.25 μg mL−1 for streptomycin. The percentages of wild type (WT) and non-wild type (NWT) isolates differed based on the approach, and ECVs calculated based on ECOFFinder approach were generally higher. Among the ARGs, those encoding resistance against tetracycline (tetA), β-lactamase (blaSHV, blaTEM, and blaOXA), amphenicol (floR), and macrolide (ermA) were detected in <10% of the isolates. The genes encoding resistance against quinolones (gyrA and qnrA) and aminoglycosides (strB) were the most detected genes. While 40.9% of the isolates contained Class 1 integron cassette regions, 33.6% of the isolates contained Class 2 integron cassette regions, and 9.1% of the isolates contained both cassette regions. None of the screened isolates were positive for the capsule gene cluster. Significant but weak correlations were found among and between ARGs and MIC distribution of antibiotics. The results of this study could be useful for identifying WT and non-WT isolates, as well as for susceptibility surveillance.

Prediction models for COVID-19 disease outcomes

ABSTRACT SARS-CoV-2 has caused over 6.9 million deaths and continues to produce lasting health consequences. COVID-19 manifests broadly from no symptoms to death. In a retrospective cross-sectional study, we developed personalized risk assessment models that predict clinical outcomes for individuals with COVID-19 and inform targeted interventions. We sequenced viruses from SARS-CoV-2-positive nasopharyngeal swab samples between July 2020 and July 2022 from 4450 individuals in Missouri and retrieved associated disease courses, clinical history, and urban-rural classification. We integrated this data to develop machine learning-based predictive models to predict hospitalization, ICU admission, and long COVID. The mean age was 38.3 years (standard deviation = 21.4) with 55.2% (N = 2453) females and 44.8% (N = 1994) males (not reported, N = 4). Our analyses revealed a comprehensive set of predictors for each outcome, encompassing human, environment, and virus genome-wide genetic markers. Immunosuppression, cardiovascular disease, older age, cardiac, gastrointestinal, and constitutional symptoms, rural residence, and specific amino acid substitutions were associated with hospitalization. ICU admission was associated with acute respiratory distress syndrome, ventilation, bacterial co-infection, rural residence, and non-wild type SARS-CoV-2 variants. Finally, long COVID was associated with hospital admission, ventilation, and female sex. Overall, we developed risk assessment models that offer the capability to identify patients with COVID-19 necessitating enhanced monitoring or early interventions. Of importance, we demonstrate the value of including key elements of virus, host, and environmental factors to predict patient outcomes, serving as a valuable platform in the field of personalized medicine with the potential for adaptation to other infectious diseases.

Multidrug-resistant Stenotrophomonas maltophilia in residential aged care facilities: An emerging threat.

Stenotrophomonas maltophilia is a multidrug-resistant (MDR), Gram-negative bacterium intrinsically resistant to beta-lactams, including last-resort carbapenems. As an opportunistic pathogen, it can cause serious healthcare-related infections. This study assesses the prevalence, resistance profiles, and genetic diversity of S. maltophilia isolated from residential aged care facilities (RACFs). RACFs are known for their overuse and often inappropriate use of antibiotics, creating a strong selective environment that favors the development of bacterial resistance. The study was conducted on 73 S. maltophilia isolates recovered from wastewater and facility swab samples obtained from three RACFs and a retirement village. Phenotypic and genotypic assessments of the isolates revealed high carbapenem resistance, exemplifying their intrinsic beta-lactam resistance. Alarmingly, 49.3% (36/73) of the isolates were non-wild type for colistin, with minimum inhibitory concentration values of > 4 mg/L, and 11.0% (8/73) were resistant to trimethoprim-sulfamethoxazole. No resistance mechanisms were detected for either antimicrobial. Genotypic assessment of known lineages revealed isolates clustering with Sm17 and Sm18, lineages not previously reported in Australia, suggesting the potential ongoing spread of MDR S. maltophilia. Lastly, although only a few isolates were biocide tolerant (2.7%, 2/73), their ability to grow in high concentrations (64 mg/L) of triclosan is concerning, as it may be selecting for their survival and continued dissemination.

Commercial Methods for Antifungal Susceptibility Testing of Saprophytic Molds: Can They Be Used to Detect Resistance?

Commercial tests are often employed in clinical microbiology laboratories for antifungal susceptibility testing of filamentous fungi. Method-dependent epidemiological cutoff values (ECVs) have been defined in order to detect non-wild-type (NWT) isolates harboring resistance mechanisms. We reviewed the literature in order to find studies where commercial methods were used to evaluate for in vitro susceptibility of filamentous fungi and assess their ability to detect NWT isolates according to the available ECVs. Data were found for the gradient concentration strips Etest and MIC Test Strips (MTS), broth microdilution Sensititre YeastOne (SYO), Micronaut-AM and the agar dilution VIPcheck assays. Applying itraconazole, voriconazole and posaconazole Etest ECVs for A. fumigatus, Etest was able to detect 90.3% (84/93), 61.2% (90/147) and 86% (31/36) of isolates with known cyp51A mutations, respectively. Moreover, Etest also was able to detect 3/3 fks mutants using caspofungin ECVs and 2/3 micafungin mutant isolates. Applying the voriconazole and posaconazole SYO ECVs, 57.7% (67/116) and 100% (47/47) of mutants with known cyp51A substitutions were classified as NWT, respectively. VIPcheck detected 90.3% (159/176), 80.1% (141/176) and 66% (141/176)of mutants via itraconazole, voriconazole and posaconazole, respectively, whereas Micronaut-AM detected 88% (22/25). In conclusion, Etest posaconazole and itraconazole, as well as micafungin and caspofungin ECVs, detected A. fumigatus mutants. On the other hand, while the posaconazole SYO ECV was able to detect cyp51A mutants, similar data were not observed with the SYO voriconazole ECV.

Fluconazole-resistant Candida parapsilosis genotypes from hospitals located in five Spanish cities and one in Italy: Description of azole-resistance profiles associated with the Y132F ERG11p substitution.

Fluconazole-resistant Candida parapsilosis is a matter of concern. To describe fluconazole-resistant C. parapsilosis genotypes circulating across hospitals in Spain and Rome and to study their azole-resistance profile associated with ERG11p substitutions. We selected fluconazole-resistant C. parapsilosis isolates (n = 528 from 2019 to 2023; MIC ≥8 mg/L according to EUCAST) from patients admitted to 13 hospitals located in five Spanish cities and Rome. Additionally, we tested voriconazole, posaconazole, isavuconazole, amphotericin B, micafungin, anidulafungin and ibrexafungerp susceptibility. Of the 53 genotypes found, 49 harboured the Y132F substitution, five of which were dominating city-specific genotypes involving almost half the isolates. Another genotype involved isolates harbouring the G458S substitution. Finally, we found two genotypes with the wild-type ERG11 gene sequence and one with the R398I substitution. All isolates were fully susceptible/wild-type to amphotericin B, anidulafungin, micafungin and ibrexafungerp. The azole-resistance patterns found were: voriconazole-resistant (74.1%) or voriconazole-intermediate (25.2%), posaconazole-resistant (10%) and isavuconazole non-wild-type (47.5%). Fluconazole-resistant and voriconazole non-wild-type isolates were likely to harbour substitution Y132F if posaconazole was wild type; however, if posaconazole was non-wild type, substitution G458S was indicated if isavuconazole MIC was >0.125 mg/L or substitution Y132F if isavuconazole MIC was ≤0.125 mg/L. We detected a recent clonal spread of fluconazole-resistant C. parapsilosis across some cities in Spain, mostly driven by dominating city-specific genotypes, which involved a large number of isolates harbouring the Y132F ERG11p substitution. Isolates harbouring substitution Y132F can be suspected because they are non-susceptible to voriconazole and rarely posaconazole-resistant.

AMR Threat Perception Assessment of Heterotrophic Bacteria From Shrimp Aquaculture Through Epidemiological Cut off Values.

Emergence and dissemination of antibiotic resistance is one of the major risks associated with the rampant usage of antibiotics in food-producing animals including aquaculture. To determine Epidemiological Cut-OFF (ECOFF) values of heterotrophic bacterial populations from shrimp culture environments against five different antibiotics. In this present study, bacterial samples were isolated from Penaeus vannamei culture environment in different locations of Andhra Pradesh, which is the aquaculture hub of India. The bacterial isolates were assessed for antibiotic resistance towards five antibiotics belonging to different classes (oxytetracycline, chloramphenicol, erythromycin, ciprofloxacin, and co-trimoxazole) by the disc diffusion method. Determination of Epidemiological Cut-OFF (ECOFF) values and analysis by employing normalized resistance interpretation (NRI) was carried out. The most dominant bacterial populations from shrimp culture were Vibrio spp. (pathogenic bacteria) followed by Bacillus spp. (probiotic bacteria). The bacterial isolates showed highest resistance towards oxytetracycline (overall 23.38%) and in location L6 (59.4%) followed by co-trimoxazole (31.1%). ECOFF values calculated by employing NRI showed that the disc diffusion data were distributed in a normalized manner. The maximum ECOFF value was obtained for ciprofloxacin (23.32 mm), while the minimum value was observed for oxytetracycline (9.05 mm). The antibiotic resistant phenotypes showed that the majority of the heterotrophic bacterial isolates (>60%) belonged to the non-wild type phenotype and primarily towards oxytetracycline (90%). The presence of non-wild antibiotic-resistant phenotypes of heterotrophic bacterial populations (which include not only pathogenic bacteria but also probiotic bacteria) indicates that shrimp culture ponds may be a reservoir for drug-resistant bacteria and there is a greater risk associated with transmission of resistant genes across bacterial flora. NRI analysis of antibiotic disc diffusion data of heterotrophic bacterial populations in shrimp aquaculture environments revealed that majority of them belonged to non-wild type (90%) paticularly to oxytetracycline in comparison to other studied antibiotics (chloramphenicol, erythromycin, ciprofloxacin and co-trimoxazole).

Estimation of epidemiological cut-off values for eight antibiotics used for treatment of bovine mastitis caused by Streptococcus uberis and Streptococcus dysgalactiae subsp. dysgalactiae

Interpretive criteria for antimicrobial susceptibility testing are lacking for most antimicrobials used for bovine streptococcal mastitis. The objectives of this study were to determine (tentative) epidemiological cut-off ((T)ECOFF) values for clinically relevant antibiotics used for treatment of bovine mastitis, and to estimate the proportion of acquired resistance (non-wild-types) in Streptococcus dysgalactiae subsp. dysgalactiae and Streptococcus uberis. A total of 255 S. uberis and 231 S. dysgalactiae subsp. dysgalactiae isolates were obtained in Denmark and Norway from bovine mastitis. The isolates were tested for susceptibility to 10 antibiotics using broth microdilution. In accordance with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) standard operating procedure, additional published MIC distributions were included for the estimation of ECOFFs for cloxacillin, cephapirin, lincomycin and tylosin, and TECOFFs for amoxicillin, benzylpenicillin, cephapirin and oxytetracycline. The proportion of non-wild-type (NWT) isolates for the beta-lactams was significantly higher in the Danish S. uberis (45–55%) compared to the Norwegian isolates (10–13%). For oxytetracycline, the proportion of NWT was significantly higher in the Danish isolates, both for S. uberis (28% vs. 3%) and S. dysgalactiae (22% vs. 0%). A bridging study testing in parallel MICs in a subset of isolates (n = 83) with the CLSI-specified and the EUCAST-specified broths showed excellent correlation between the MICs obtained with the two methods. The new ECOFFs and TECOFFs proposed in this study can be used for surveillance of antimicrobial resistance, and - for antimicrobials licensed for streptococcal bovine mastitis - as surrogate clinical breakpoints for predicting their clinical efficacy for this indication.

Surveillance of antimicrobial resistance in Escherichia coli, Salmonella, and Campylobacter recovered from laying hens, their environment and products in Canada indicated a stable level of resistance to critically important antimicrobials, in varying time periods between 2007 and 2021

The study objective was to determine the occurrence and antimicrobial resistance (AMR) attributes of select foodborne bacteria recovered from egg-producing (layer) chickens between 2007 and 2021 using different sample matrices (Study 1: liquid whole eggs, Study 2: spent hen cecal samples, Study 3: environmental sponge swabs and fecal samples from layer chicken barns, and Study 4: fecal samples from layer chicken barns). Samples from each study were submitted for the culture of Escherichia coli and Salmonella. In addition, samples from layer chicken barns were submitted for the culture of Campylobacter. Isolates were tested by microbroth dilution and interpreted using both clinical breakpoints and epidemiological cut-offs (ECOFFs). The ECOFFs were applied to detect non-wild type (NWT) strains. The proportion of resistant, NWT, and distribution of minimum inhibitory concentrations (MIC) were assessed. Ceftriaxone resistance was detected at a low-level in E. coli (< 2 %, all studies) and Salmonella (4.3 %, Study 2). Very low-level ciprofloxacin resistance was detected in E. coli (<1 %, Study 1) but a slightly elevated ciprofloxacin NWT E. coli (1 % to 6 %) observed. Only the farm fecal samples in Studies 2 and 3 were tested for Campylobacter as part of its study design, and moderate level ciprofloxacin resistance (<15 %) was observed. The MIC distribution patterns were similar across the organisms tested/studies and no substantial shifts in the distributions were detected. This analysis demonstrated that resistance to very important antimicrobials in bacteria from layers in Canada remains low, however, the detection of ciprofloxacin-resistant Campylobacter and the implications of this observation to the safety of egg products, and the role of laying flocks (i.e., as reservoir for resistant organisms) needs to be investigated.

Susceptibility Profile and Epidemiological Cut-Off Values Are Influenced by Serotype in Fish Pathogenic Streptococcus agalactiae.

Streptococcus agalactiae is a major health concern in tilapia farming worldwide. In contrast to the availability of susceptibility profile results, interpretative criteria for disk diffusion assays and the influence of serotypes on resistance profiles are not available. To address this, sixty isolates (thirty of each serotype, Ib and III) were evaluated using the disk diffusion assay against six antibiotics, and the epidemiological cut-off value (ECV) was calculated. All the isolates were classified as non-wild type (NWT) for sulfamethoxazole (SUT) and norfloxacin (NOR). The inhibition zones for oxytetracycline (OXY) and doxycycline (DOX) were largely distinct; all serotype Ib and III isolates were classified as wild-type (WT) and NWT, respectively. The results for serotype III of fish group B Streptococcus (GBS) were comparable to the NWT tetracycline profile of human GBS available in EUCAST, suggesting the presence of resistance mechanisms in these fish isolates. The calculation of the cut-off wild type (COWT) values for OXY and DOX was appropriate for both serotypes. Differences between the distribution of florfenicol (FLO) and amoxicillin (AMO) were found, and we attribute this to the faster growth rate of serotype III, which promotes smaller inhibition zones. Therefore, using separate COWT for each serotype is necessary. In conclusion, the serotype of fish GBS affects its susceptibility profile, and it is recommended to use serotype-specific COWT values as interpretative criteria for disk diffusion assays against FLO and AMO.

1947. Activity of Rezafungin against Echinocandin–Non-wild type C. glabrata Clinical Isolates from the Rezafungin Surveillance Program (2014–2021)

Abstract Background Fluconazole (FLC) resistance (R) is common in C. glabrata (CGLA). Echinocandins (ECHs) are often used as first-line therapy. R to ECHs has been associated with FKS1 and FKS2 gene alterations. Rezafungin (RZF), a new ECH approved by the US FDA to treat candidemia and invasive candidiasis, was evaluated against a collection of ECH–non-wild type (NWT) CGLA isolates. Methods A total of 1045 CGLA collected (1/patient) in 2014–2021 from 58 medical centers located in North America (NA; n=459; 20 centers), Europe (EU; n=396; 23 centers), Asia-Pacific (AP; n=130; 10 centers), and Latin America (LA, n=60; 5 centers) were identified by MALDI-TOF and/or sequencing and tested by CLSI broth microdilution. CLSI breakpoints (BP) and epidemiological cut-off values were applied. ECH NWT isolates were submitted to FKS analysis by whole genome sequencing. Results RZF showed similar activity to other ECHs against CGLA (Table), inhibiting 98.5% at ≤0.5 mg/L. Anidulafungin (ANF), caspofungin (CSF), and micafungin (MCF) susceptibility (S) rates were 96.2%, 97.5%, and 97.3%, respectively. ECH-NWT CGLA were detected in 42 isolates (4.0% overall): NA showed the highest rate of ECH NWT isolates (29; 6.3%), followed by EU (10; 2.5%), AP (2; 1.5%), and LA (1; 1.7%). The RZF S rate was 61.9% of ECH-NWT CGLA, while the S rate to ANF, CSF, and MCF was 21.4%, 40.5%, and 33.3%, respectively. Alteration in FKS genes were observed in 31 CGLA (73.8% of ECH NWT; 3.0% overall). RZF was active against &amp;gt;50% of these isolates. RFZ was also active against &amp;gt;90% of ECH-NWT CGLA carrying wildtype FKS genes. Equivalent activity was noted to other ECHs against ECH-NWT isolates. However, ECHs were 4 to 8-fold more active against ECH-NWT CGLA that did not display alterations in FKS genes than those with FKS alterations. FKS2 hot spot(HS)-1 alterations were observed in 22 isolates (12 displayed S663F), while FKS1-HS1 alterations were noted in 9 isolates (7 displayed S629P). FLC-R was observed in 6.9% of CGLA overall and 16.7% of the ECH-NWT isolates. Conclusion RZF demonstrated potent in vitro activity against CGLA and remained active against most isolates of CGLA displaying an ECH-NWT phenotype with or without FKS alterations. Disclosures Cecilia G. Carvalhaes, MD, PhD, AbbVie: Grant/Research Support|bioMerieux: Grant/Research Support|Cipla: Grant/Research Support|CorMedix: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support Paul Rhomberg, BS, MT(ASCP), bioMerieux: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support Abby Klauer, BS, Melinta: Grant/Research Support Lalitagauri M. Deshpande, PhD, Melinta: Grant/Research Support|Paratek: Grant/Research Support Mariana Castanheira, PhD, AbbVie: Grant/Research Support|Basilea: Grant/Research Support|bioMerieux: Grant/Research Support|Cipla: Grant/Research Support|CorMedix: Grant/Research Support|Entasis: Grant/Research Support|Melinta: Grant/Research Support|Paratek: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support

Screening of alkaloids and withanolides isolated from Solanaceae plants for antifungal properties against non-wild type Sporothrix brasiliensis

Antifungal resistance has often been found in animal sporotrichosis in Southern Brazil. The biological potential of compounds from plants of the Solanaceae family against infectious diseases is known, however, it is still unknown against Sporothrix brasiliensis. This study evaluated the anti-Sporothrix brasiliensis activity, synergism, cytotoxicity, and action mechanism of steroidal lactones (withanolides) and alkaloids isolated from these plants. Pure compounds of withanolide D (WNOD), physalin F (PHYF), withanicandin (WNIC), nicandin B (NICB), solasonine (SSON), and solamargine (SMAR) were tested against 12 Sporothrix brasiliensis isolated from cats (n = 11) and dogs (n = 2) through M38-A2 CLSI. For the compounds with the best activity, a checkerboard assay for synergism, sorbitol protection, and ergosterol effect for action mechanism; and MTT test for cytotoxicity were performed. The withanolides WNOD, PHYF, WNIC, and NICB were not antifungal, but SSON (MIC 0.125–1 mg/mL) and SMAR (MIC 0.5–1 mg/mL) were both fungistatic and fungicidal (MFC 0.5–1 mg/mL for both) against wild-type (WT) and non-WT isolates. The activity of SSON and SMAR was indifferent when combined with itraconazole. In the mechanism of action, cell wall and plasma membrane by complexation with ergosterol seemed to be two target structures of SSON and SMAR. SSON was selected for cytotoxicity, whose cell viability in MDBK cells ranged from 28.85 % to 101.75 %, and was higher than 87.49 % at concentrations ≤0.0015 mg/ml. Only the steroidal alkaloids SSON and SMAR were active against non-WT isolates, being promising antifungal candidates for the treatment of feline and canine sporotrichosis with low susceptibility to itraconazole.