Effects of Tylosin, a Direct-Fed Microbial and Feedlot Pen Environment on Phenotypic Resistance among Enterococci Isolated from Beef Cattle Feces

In two sequential replicates (n = 90 and n = 96 feedlot finisher cattle, respectively) we measured the impact of an Enterococcus faecium-based probiotic (DFM) and an altered feedlot pen environment on antimicrobial resistance among fecal enterococci in cattle fed (or, not fed) the macrolide tylosin. Diluted fecal samples were spiral-plated on plain and antibiotic-supplemented m-Enterococcus agar. In the first replicate, tylosin significantly (p < 0.05) increased the relative quantity of erythromycin-resistant enterococci. This effect was diminished in cattle fed the DFM in conjunction with tylosin, indicating a macrolide susceptible probiotic may help mitigate resistance. A similar observed effect was not statistically significant (p > 0.05) in the second replicate. Isolates were speciated and resistance phenotypes were obtained for E. faecium and E. hirae. Susceptible strains of bacteria fed as DFM may prove useful for mitigating the selective effects of antibiotic use; however, the longer-term sustainability of such an approach remains unclear.

Impact of host factors on susceptibility to antifungal agents

An obstacle to drug development, particularly in this era of multiple drug resistance, is the under-appreciation for the role the host environment plays in microbial response to drugs. With the rise in fungal infection and drug resistance, particularly in individuals with co-morbidities, the influence serum and its components have on antimicrobial susceptibility requires assessment. This study examined the impact of physiologically relevant glucose and insulin levels in the presence and absence of 50 % human plasma on MICs for clinical isolates of Candida lusitaniae, Candida parapsilosis, Candida albicans, Candida tropicalis, Candida glabrata, Candida krusei and Cryptococcus neoformans. The addition of insulin or glucose at physiologic levels in RPMI medium alone altered the MIC in either a positive or negative fashion, depending on the organisms and drug tested, with C. glabrata most significantly altered with a 40, >32- and 46-fold increase in MIC for amphotericin B, itraconazole and miconazole, respectively. The addition of candida-antibody negative plasma also affected MIC, with the addition of glucose and insulin having a tandem effect on MIC. These findings indicate that phenotypic resistance of Candida and Cryptococcus can vary depending on the presence of insulin with glucose and plasma. This modulation of resistance may help explain treatment failures in the diabetic population and facilitate the development of stable drug-resistant strains. Furthermore, these findings indicate the need for a precision approach in the choice of drug treatment and drug development.

Resistance Patterns, mcr-4 and OXA-48 Genes, and Virulence Factors of Escherichia coli from Apennine Chamois Living in Sympatry with Domestic Species, Italy

The aim of this study was to determine and characterize potential resistance mechanisms against selected Critically Important Antibiotics in Escherichia coli isolates collected from wild and domestic ruminants living in the Maiella National Park, in Central Italy. A total of 38 isolates were obtained from red deer, Apennine chamois, cattle, sheep, and goats grazing in lands with different levels of anthropic pressure. Antimicrobial susceptibility was determined by Minimal Inhibitory Concentration testing, showing phenotypic resistance to colistin, meropenem, or ceftazidime in 9 isolates along with one bacterial strain being resistant to three of the tested antibiotics. In addition, the biomolecular assays allowed the amplification of the genes conferring the colistin (mcr-4), the carbapenems (OXA-48), penicillins and cephalosporins (TEM, SHV, CMY-1, CMY-2) resistance. In order to describe the potential pathogenicity of isolates under study, virulence genes related to Shiga toxin-producing (STEC) and enteropathogenic (EPEC) pathovars were identified. This study is the first report of mcr-4 and OXA-48 genes in resistant E. coli harboring virulence genes in Italian wildlife, with special regard to Apennine chamois and red deer species. The multidisciplinary approach used in this study can improve the early detection of emerging antibiotic resistance determinants in human-animal-environment interfaces by means of wildlife monitoring.

Identification of bacterial blight resistance genes in rice landraces from Yunnan Province, China

Bacterial blight (BB), a serious bacterial disease caused by pathogen Xanthomonas oryzae pv. oryzae (Xoo) affects rice growth and yield. Yunnan Province is regarded as a center of rice diversity in China and indeed the world, and has abundant rice landrace resources, which may offer prospective candidate donors in rice improvement and breeding. In this study, a set of 200 rice landraces were evaluated to determine their resistance to 10 pathogenic Xoo strains resistance by the leaf-clipping method. The results indicated that the tested rice landraces had different resistance levels against different Xoo strains. Multiple comparisons showed that the Xoo strain PXO99 was virulent to the tested rice landraces. Sixty-six rice landraces conferred resistance against at least one Xoo strain. These resistant rice landraces screened were then performed the presence of 14 cloned BB resistance genes by closely linked molecular markers and designed specific primers. The results showed that none of these resistant accessions contained xa13, Xa21, Xa27, and Xa45(t) homologous fragments, while 9, 24, 4, 7, 9, 15, 1, 5, 4 and 27 accessions contained Xa1, Xa2/Xa31(t), Xa14, Xa3/Xa26, Xa4, xa5, Xa7, Xa10, Xa23 and xa25 homologous fragments, respectively. Sequence analysis further revealed that nucleotide variations around functional nucleotide polymorphisms region were observed within these accessions containing the Xa1, Xa2/Xa31(t), Xa14, Xa3/Xa26, Xa4, xa5, Xa10, Xa23 and xa25 homologous fragments. These results along with phenotypic resistance spectrum supported that these accessions carried nine resistance homologous genes. Only one accession (Qishanggu_Wenshan) carried the Xa7 resistance gene. We also found that some resistant rice landraces, especially Xilandigu_Baoshan, and Laoyaling_Lincang without the above resistance genes, which mediated broad spectrum resistance to multiple Xoo strains, were identified as potential sources for breeding rice lines resistance to BB.

Phenotypic and Genotypic Characterization of Methicillin, Vancomycin, and Erythromycin-resistant Staphylococcus aureus Isolated from Milk and Dairy Products

Detailed information on the resistance patterns of Staphylococcus aureus (S. aureus) in milk and cheese is strongly required to facilitate risk assessment analysis in case of food poisoning context and to improve therapeutic approaches used in dairy farms. The present study aimed to perform phenotypic and genotypic antimicrobial characterizations of methicillin, vancomycin, and erythromycin-resistant S. aureus isolated from milk and dairy products through screening mecA, vanA, and ermC using molecular PCR amplification technology. Moreover, the association between each genotypic and its related antibiotic resistance phenotypic features within the isolated S. aureus strains were analyzed. Moreover, the current study aimed to study MRSA's ability to form biofilms. Out of 226 milk and dairy product samples collected from different retailers in Giza Governorate, 69.5% of the samples were positive for the presence of S. aureus. The isolation rate of S. aureus strains from cattle milk, sheep milk, white cheese, flamenco, and mesh samples were 79.7%, 76.5%, 56.0%, 40.0%, and 94.7%, respectively. Multidrug-resistant S. aureus (MDR) was detected in 51% of all isolated S. aureus strains. All tested S. aureus strains were sensitive to trimethoprim-sulfamethoxazole, linezolid, ciprofloxacin, and gentamycin. However, their resistance rates against penicillin, oxacillin, vancomycin, erythromycin, tetracycline, clindamycin and chloramphenicol were 62.4%, 65.0%, 44.6%, 45.9%, 21.0%, 14.0%, and 2.5%, respectively. Of the isolated S. aureus strains, 72.6%, 40.1%, and 48.4% were carriers for mecA, vanA, and ermC genes and the amplified products were at 310, 1030, and 295 bp, respectively. Methicillin-resistant S. aureus isolates were detected in 47.1% of all isolated S. aureus strains. The results indicated that 35.0% of the tested S. aureus strains were genotypic vanA gene carriers and phenotypic resistant to vancomycin representing vancomycin-resistant S. aureus strains. Moreover, 42.7% of all isolated S. aureus strains were carriers for ermC gene and were phenotypic resistant to erythromycin representing erythromycin-resistant S. aureus. The presence of mecA, vanA, and ermC genes in S. aureus was statistically associated with their related phenotypic resistance patterns against both penicillin and oxacillin, vancomycin, and erythromycin, respectively. Moreover, along with an increase in the frequency of mecA, vanA, and ermC genes, their phenotypic antibiotic resistance patterns sharply increased with an odd ratio >1. Of MRSA isolates, 6.8% indicated weak biofilm-formation ability, while 93.2% exhibit no biofilm-forming ability.

Genomic diversity of antimicrobial resistance in non-typhoidal Salmonella in Victoria, Australia

Non-typhoidal Salmonella (NTS) is the second most common cause of foodborne bacterial gastroenteritis in Australia with antimicrobial resistance (AMR) increasing in recent years. Whole-genome sequencing (WGS) provides opportunities for in silico detection of AMR determinants. The objectives of this study were two-fold: (1) establish the utility of WGS analyses for inferring phenotypic resistance in NTS, and (2) explore clinically relevant genotypic AMR profiles to third generation cephalosporins (3GC) in NTS lineages. The concordance of 2490 NTS isolates with matched WGS and phenotypic susceptibility data against 13 clinically relevant antimicrobials was explored. In silico serovar prediction and typing was performed on assembled reads and interrogated for known AMR determinants. The surrounding genomic context, plasmid determinants and co-occurring AMR patterns were further investigated for multidrug resistant serovars harbouring bla CMY-2, bla CTX-M-55 or bla CTX-M-65. Our data demonstrated a high correlation between WGS and phenotypic susceptibility testing. Phenotypic-genotypic concordance was observed between 2440/2490 (98.0 %) isolates, with overall sensitivity and specificity rates >98 % and positive and negative predictive values >97 %. The most common AMR determinants were bla TEM-1, sul2, tet(A), strA-strB and floR. Phenotypic resistance to cefotaxime and azithromycin was low and observed in 6.2 % (151/2486) and 0.9 % (16/1834) of the isolates, respectively. Several multi-drug resistant NTS lineages were resistant to 3GC due to different genetic mechanisms including bla CMY-2, bla CTX-M-55 or bla CTX-M-65. This study shows WGS can enhance existing AMR surveillance in NTS datasets routinely produced in public health laboratories to identify emerging AMR in NTS. These approaches will be critical for developing capacity to detect emerging public health threats such as resistance to 3GC.

Evaluation and Pre-selection of New Grapevine Genotypes Resistant to Downy and Powdery Mildew, Obtained by Cross-Breeding Programs in Spain

The need to develop an environmentally friendly, sustainable viticulture model has led to numerous grapevine improvement programmes aiming to increase resistance to downy and powdery mildew. The success of such programmes relies on the availability of protocols that can quantify the resistance/susceptibility of new genotypes, and on the existence of molecular markers of resistance loci that can aid in the selection process. The present work assesses the degree of phenotypic resistance/susceptibility to downy and powdery mildew of 28 new genotypes obtained from crosses between “Monastrell” and “Regent.” Three genotypes showed strong combined resistance, making them good candidates for future crosses with other sources of resistance to these diseases (pyramiding). In general, laboratory and glasshouse assessments of resistance at the phenotype level agreed with the resistance expected from the presence of resistance-associated alleles of simple sequence repeat (SSR) markers for the loci Rpv3 and Ren3 (inherited from “Regent”), confirming their usefulness as indicators of likely resistance to downy and powdery mildew, respectively, particularly so for downy mildew.

Insecticide susceptibility status and knockdown resistance (kdr) mutation in Aedes albopictus in China

Background Aedes (Stegomyia) albopictus (Skuse, 1894) is the main vector of dengue virus in China. The resistance to insecticides is a huge obstacle for the control of this species, and determining its resistance status and mechanisms in China is essential for the implementation of vector management strategies. Methods We have investigated the larval and adult resistance status of Ae. albopictus to deltamethrin in eight field populations in China. Mutations at the voltage-gated sodium channel gene, related to the knockdown resistance (kdr) effect, were detected by sequencing of PCR products. The eight field populations were examined for pyrethroid resistance using the World Health Organization standard bioassays, and the association between the mutations and phenotypic resistance was tested. Results The eight field populations of larvae of Ae. albopictus in China exhibited high resistance to deltamethrin; the RR50 values ranged from 12 (ZJ) to 44 (GZ). Adult bioassay revealed that Ae. albopictus populations were resistant to deltamethrin (mortality rate < 90%), except ZJ population (probably resistant, mortality rate = 93.5%). Long knockdown time in the field populations was consistent with low mortality rates in adult bioassay. F1534S mutation showed increased protection against deltamethrin in all populations except BJ and SJZ populations, whereas I1532T mutation showed increased protection against deltamethrin in only BJ population. Conclusion There were different degrees of resistance to deltamethrin in field Ae. albopictus populations in China. The longest knockdown time and lowest mortality rate observed in Ae. albopictus population in Guangzhou indicate the severity of high resistance to deltamethrin. The patchy distribution of deltamethrin resistance and kdr mutations in Ae. albopictus mosquitoes suggests the necessity for resistance management and developing counter measures to mitigate the spread of resistance. Graphical Abstract

Effects of a Direct-Fed Microbial and Feedlot Pen Environmental Change on Phenotypic Resistance among Enterococci Isolated from Beef Cattle Feces

In two sequential replicates (n=90 and n=96 feedlot finisher cattle, respectively) we measured the impact of an Enterococcus faecium-based probiotic (DFM) and an altered feedlot pen environment on antimicrobial resistance among fecal enterococci in cattle fed (or, not fed) the macrolide tylosin. Diluted fecal samples were spiral-plated on plain and antibiotic-supplemented m-Enterococcus agar. In the first replicate, tylosin significantly (p&amp;lt;0.05) increased the relative quantity of erythromycin-resistant enterococci. This effect was diminished in cattle fed the DFM in conjunction with tylosin. A similar observed effect was not statistically significant (P &amp;gt; 0.05) in the second replicate. Isolates were speciated and resistance phenotypes were obtained for E. faecium and E. hirae. E. faecium isolates were whole-genome sequenced, which yielded sequence types (ST), resistance genes and phylogeny. Samples of the DFM were sequenced and found to contain E. faecium ST296, which was not present on Day 0 of either replicate. This DFM sequence type was found in fecal samples after Day 0, the majority of which were isolated from cattle in one of the DFM-fed pens. Increased prevalence of ST296 occurred with a concomitant decrease in ST240; of importance, the latter typically harbored both ermB and tet(M) genes.

Third-Generation Cephalosporin Resistance in Intrinsic Colistin-Resistant Enterobacterales Isolated from Retail Meat

Consumption of retail meat contaminated with antimicrobial-resistant (AMR) bacteria is a common route for transmitting clinically relevant resistant bacteria to humans. Here, we investigated the genotypic and phenotypic resistance profiles of intrinsic colistin-resistant (ICR) Enterobacterales isolated from retail meats. ICR Enterobacterales were isolated from 103 samples of chicken, 103 samples of pork, and 104 samples of beef purchased from retail shops in Japan, using colistin-containing media, and their antimicrobial susceptibility was examined. Serratia spp. (440 isolates) showed resistance to cefotaxime (19 isolates, 4.3%), tetracycline (15 isolates, 3.4%), and other antimicrobials (<1%). Hafnia spp. (136) showed resistance to cefotaxime (12 isolates, 8.6%), ceftazidime (four isolates, 2.9%), and tetracycline (two isolates, 1.4%). Proteus spp. (39) showed resistance to chloramphenicol (four isolates, 10.3%), sulfamethoxazole-trimethoprim (four isolates, 10.3%), cefotaxime (two isolates, 5.1%), kanamycin (two isolates, 5.1%), and gentamicin (one isolate, 2.6%). Cedecea spp. (22) were resistant to tetracycline (two isolates, 9.1%) whereas Morganella spp. (11) were resistant to tetracycline (four isolates, 36.4%) and chloramphenicol (one isolate, 9.2%). The resistance genes blafonA, blaACC, and blaDHA were detected in cefotaxime-resistant Serratia spp., Hafnia spp., and Morganella spp. isolates, respectively. This emergence of antimicrobial resistance in ICR Enterobacterales may pose a public health risk.