Spread of Xanthomonas vasicola pv. musacearum within banana mats: implications for Xanthomonas wilt management

Xanthomonas wilt (XW) of banana caused by Xanthomonas vasicola pv. musacearum (Xvm) does not spread to all plants physically interconnected through the rhizome when one or a few are diseased. However, the factors behind this incomplete systemic spread of Xvm are not fully known yet could inform XW management. This study explored the effect of Xvm inoculum amounts, number and size of suckers, sucker positioning on mother plant corms and other mother plant corm attributes on sucker colonization. A shorter (p <0.05) incubation period (17.9 vs 21.1 days) and higher (p<.001) cumulative number of symptomatic leaves (5.2 vs 1.6 leaves) was observed when all (high inoculum) compared to two leaves (low inoculum) were inoculated. Xvm was recovered in corms at 29 days post inoculation (dpi) in both treatments with no differences (p >0.05) in proportions of corms with Xvm between the treatments. However, Xvm was recovered earlier and at a higher frequency in suckers when all leaves were inoculated. Lower Xvm recoveries occurred in the lower corm sections to which most suckers were attached relative to the middle and upper corm sections. Xvm incidence in corms increased with the number of attached maiden suckers, and the dpi while it declined with increasing mother plant and corm height. Thus, Xvm spread within mats is influenced by the amount of inoculum and the physiological stage (e.g., height) of the plant and attached suckers. The position of suckers, predominantly at the bottom of corms also protects them from infection. Measures that reduce Xvm inoculum build-up in mats are thus crucial for minimizing within mat XW spread.

199. Polymorphisms in Key Regulatory Regions of the bla operon Correlate with the Cefazolin Inoculum Effect in Methicillin-Susceptible Staphylococcus aureus (MSSA)

Background The cefazolin inoculum effect (CzIE), defined as Cz minimum inhibitory concentration ≥ 16 µg/ml at high inoculum (HI-MIC), has been associated with poor clinical outcomes in patients with MSSA bacteremia or osteomyelitis. The CzIE is correlated with the presence of the blaZ gene, one of the components of the bla operon encoding the BlaZ β-lactamase (type A, B, C or D). Other portions of the bla operon include blaR and blaI (encoding the antibiotic sensor and transcriptional repressor, respectively) and the intergenic region with operator and promoter sequences (Figure 1). In BlaR, residue 293 mediates signal transduction, and the Z and R dyads in the intergenic region are the DNA-binding sites for BlaI (Figure 2). Previous experiments have shown that the regulatory portions of the bla operon play a key role in the CzIE. Here, we investigated the association between the CzIE and specific variations in the regulatory sequences of the bla operon. Figure 1. Functioning of the bla operon and the production of the staphylococcal β-lactamase BlaZ. Figure 2. Structure and key regions of the intergenic region of the bla operon, incluiding the promoter and the BlaI DNA-binding regions (Z and R dyads). Methods A total of 437 MSSA containing blaZ were evaluated for the CzIE using broth microdilution at high inoculum. Using whole genome sequencing, the sequences of the bla operons were classified into cassettes based on unique changes in predicted amino acid sequences of BlaZ, BlaR and BlaI paired with specific nucleotide alterations in the intergenic region. The bla operon sequence of S. aureus ATCC29213 was used as reference (cassette 0). Results Among 437 MSSA isolates, 46% exhibited the CzIE. We identified 55 unique bla cassettes. The bla cassettes were phylogenetically grouped in 7 clusters (Figure 3) which grouped cassettes with different BlaZ types and variations in the Z dyad, the -35 box, residue 293 of BlaR, and the blaI ribosomal binding site. Each cluster had an association to the CzIE and distinct Cz HI-MICs. The combination of: a BlaZ type A, C or D, an adenine in the position -66 of blaZ (-35 box of blaZ), a cytosine in the position -22 of blaZ(Z dyad), and either an arginine or a serine in position 293 of BlaR was a very strong predictor of the CzIE (Figure 4). Figure 3. Phylogenetical organization of bla operon cassettes into clusters, their association with polymorphisms in key regulatory regions and the CzIE. GM Cz-MIC: Geometric mean of the Cefazolin MIC at high Inoculum. Figure 4. Variations of the bla operon, their association with the CzIE, their GM (Geometric Mean) of the Cefazolin MIC and the MIC distribution of the strains with each specific combination of polymorphisms. Conclusion Specific variations in regulatory portions of the bla operon, which are likely to influence BlaZ expression, are highly associated with the CzIE, supporting the notion that regulation of blaZ is the key factor responsible for the CzIE in MSSA. Disclosures Lorena Diaz, PhD , Nothing to disclose William R. Miller, MD , Entasis Therapeutics (Scientific Research Study Investigator)Merck (Grant/Research Support) William R. Miller, MD , Entasis (Individual(s) Involved: Self): Scientific Research Study Investigator; Merck (Individual(s) Involved: Self): Grant/Research Support Cesar A. Arias, M.D., MSc, Ph.D., FIDSA, Entasis Therapeutics (Grant/Research Support)MeMed Diagnostics (Grant/Research Support)Merk (Grant/Research Support)

1248. A Machine-Learning Approach to Predict the Cefazolin Inoculum Effect in Methicillin-Susceptible Staphylococcus aureus

Background The cefazolin (Cz) inoculum effect (CzIE), defined as an increase in the Cz MIC to ≥16 µg/mL at high inoculum (107 CFU/mL), has been associated with poor outcomes in MSSA bacteremia and osteomyelitis. The CzIE is associated with the BlaZ β-lactamase, encoded by blaZ and regulated by BlaR (antibiotic sensor) and BlaI (transcriptional repressor). Here, we aimed to obtain a machine-learning (ML) model to predict the presence of the CzIE based on the nucleotide sequence of the entire bla operon and its regulatory components. Methods Using whole genome sequencing, we analyzed the nucleotide sequences of the entire bla operon in 436 MSSA isolates recovered from blood, soft-tissue infections or pneumonia in adults (training-testing cohort, prevalence of the CzIE: 46%). Also, 32 MSSA recovered from pediatric patients with osteomyelitis with the CzIE were included as validation cohort. The CzIE was determined by broth microdilution at high inoculum. K-mer counts were obtained from the bla operon sequences of the isolates from the testing-training cohort, and then used in a ML pipeline which i) discards uninformative K-mers, ii) identifies optimal hyper-parameters and, iii) performs training of the model using 70% of the sequences as training set and 30% as testing set. The pipeline tested 11 different K-mer sizes and 2 models: Logistic Regression (LR) and Support Vector Machine (SVM). Finally, the model with best predictive ability was applied to the sequences of the MSSA osteomyelitis isolates (validation cohort). Results The ML approach had high specificity ( &gt;90%), accuracy ( &gt;80%) and ROC-AUC values ( &gt;0.7) for detecting the CzIE in the testing set of isolates (Figure 1), independently of the type of model or the K-mer size used. The best predictive ability was with LR using K-mers of 17 nucleotides, with an accuracy of 84%, specificity of 96%, and sensitivity of 70% in the testing set (Figure 2). In the validation cohort, the model was capable to correctly identify all the strains exhibiting the CzIE (100% sensitivity). Figure 1. Prediction metrics of the ML pipeline for the detection of the CzIE in MSSA isolates from the training-test cohort. Predictions are shown accordingly to the model and K-mer sizes tested. Figure 2. ROC of best predictive model (Logistic Regression, K-mer size 17) for the detection of the CzIE in MSSA isolates. Conclusion The ML approach is a promising genomic application to detect the CzIE in MSSA isolates of a variety of sources, bypassing phenotypic testing. Further validation is needed to evaluate its possible utility in clinical settings. Disclosures Jonathon C. McNeil, MD, Agency for Healthcare Research and Quality (Research Grant or Support)Allergan (Grant/Research Support)Nabriva (Grant/Research Support, Other Financial or Material Support, Site PI for a multicenter trial) Anthony R. Flores, MD, MPH, PhD, Nothing to disclose Sheldon L. Kaplan, MD, Pfizer (Research Grant or Support) Cesar A. Arias, M.D., MSc, Ph.D., FIDSA, Entasis Therapeutics (Grant/Research Support)MeMed Diagnostics (Grant/Research Support)Merk (Grant/Research Support) Lorena Diaz, PhD , Nothing to disclose

Effects of Growth Medium and Inoculum Size on Pharmacodynamics Activity of Marbofloxacin against Staphylococcus aureus Isolated from Caprine Clinical Mastitis

Staphylococcus aureus (S. aureus) is an important pathogen that causes clinical mastitis in goats and produces infections difficult to cure. Different antimicrobials as fluoroquinolones have been used against S. aureus. However, the studies developed to evaluate the bacterial drug interaction only have used the MIC as a single reference point with artificial growth media. The aims of this study were to describe the effect of marbofloxacin on S. aureus isolated from mastitis goats’ milk by different approaches as the minimum inhibitory and bactericidal concentrations (MIC and MBC) in cation adjusted Mueller–Hinton broth (CAMHB), serum and milk of goats at two inoculum sizes of 105 and 108 CFU/mL, the determination and analysis of the time kill curves (TKC) by non-linear mixed effect models in each growth medium and inoculum size, as well as the estimation of their pharmacokinetics/pharmacodynamics (PK/PD) cutoff values. The results obtained indicate that MIC values were higher and increases 2,4-fold in serum and 3,6-fold in milk at high inoculum, as well as the EC50 values determined by each pharmacodynamics model. Finally, the PK/PD cutoff values defined as fAUC24/MIC ratios to achieve clinical efficacy were highly dependent on inoculum and growth medium, with median values of 60–180, especially at high inoculum in milk, suggesting that further studies are necessary to evaluate and optimize the best therapeutic strategies for treating S. auerus in lactating goats.

A Test For The Rapid Detection of the Cefazolin Inoculum Effect in Methicillin-Susceptible Staphylococcus aureus

The cefazolin inoculum effect (CzIE) has been associated with therapeutic failures and mortality in invasive methicillin-susceptible Staphylococcus aureus (MSSA) infections. A diagnostic test to detect the CzIE is not currently available. We developed a rapid (∼3 h) CzIE colorimetric test to detect staphylococcal-β-lactamase (BlaZ) activity in supernatants after ampicillin induction. The test was validated using 689 bloodstream MSSA isolates recovered from Latin-America and US. Cefazolin MIC determination at high inoculum (107 CFU/mL) was used as reference standard (cut-off of ≥16 μg/mL). All isolates underwent genome sequencing. A total of 257 (37.3%) MSSA exhibited the CzIE by the reference standard method. The overall sensitivity and specificity of the colorimetric test was 82.5% and 88.9%, respectively. Sensitivity in MSSA isolates harboring type A BlaZ (most efficient enzyme against cefazolin) was 92.7% with a specificity of 87.8%. The performance of the test was lower against type B and C enzymes (sensitivities of 53.3% and 72.3%, respectively). When the reference value was set to ≥32 μg/mL the sensitivity for isolates carrying type A enzymes was 98.2%. Specificity was 100% for MSSA lacking blaZ. The overall negative predictive value ranged from 81.4% to 95.6% in Latin-American countries using published prevalence rates of the CzIE. MSSA from US were genetically diverse, with no distinguishing genomic differences from Latin-American MSSA, distributed among 18 sequence types. A novel test can readily identify most MSSA isolates exhibiting the CzIE, particularly those carrying type A BlaZ. In contrast to the MIC determination using high-inoculum, the rapid test is inexpensive, feasible and easy to perform. After minor validation steps, it could be incorporated into the routine clinical laboratory workflow.

In Vitro Activities and Inoculum Effects of Ceftazidime-Avibactam and Aztreonam-Avibactam against Carbapenem-Resistant Enterobacterales Isolates from South Korea

Ceftazidime-avibactam (CAZ-AVI) and aztreonam-avibactam (AZT-AVI) are novel antibiotic combinations active against multidrug-resistant Gram-negative pathogens. This study aimed to evaluate their in vitro activities and inoculum effects in carbapenem-resistant Enterobacterales (CRE), including carbapenemase-producing (CP)-CRE and non-CP-CRE. A total of 81 independent clinical isolates of carbapenem-resistant Escherichia coli and Klebsiella pneumoniae were collected. CAZ-AVI and AZT-AVI minimal inhibitory concentrations (MICs) were evaluated by broth microdilution using standard and high inocula. The inoculum effect was defined as an ≥8-fold increase in MIC with high inoculum. Phenotypic determination of β-lactam resistance mechanism and PCR for carbapenemase genes were performed. Of the 81 CRE isolates, 35 (43%) were CP-CRE. Overall, 73% of the isolates were susceptible to CAZ-AVI, and 95% had low AZT-AVI MICs (≤8 µg/mL). The MIC50/MIC90s of CAZ-AVI and AZT-AVI were 4/≥512 µg/mL and 0.5/4 µg/mL, respectively. CAZ-AVI was more active against non-CP-CRE than against CP-CRE (susceptibility 80% vs. 63%, p = 0.08; MIC50/MIC90, 2/16 μg/mL vs. 4/≥512 μg/mL), whereas AZT-AVI was more active against CP-CRE (MIC50/MIC90, 0.25/1 μg/mL vs. 0.5/8 μg/mL). All four isolates with high AZT-AVI MIC (≥16 μg/mL) were resistant to CAZ-AVI, but only 18% (4/22) of CAZ-AVI-resistant isolates had high AZT-AVI MIC. The rates of the inoculum effect for CAZ-AVI and AZT-AVI were 18% and 47%, respectively (p < 0.001). Interestingly, the frequency of the AZT-AVI inoculum effect was higher in K. pneumoniae than E. coli (64% vs. 8%, p < 0.001). AZT-AVI is more active against CRE than CAZ-AVI, even in CP-CRE and CAZ-AVI-resistant isolates. The presence of a substantial inoculum effect may contribute to clinical failure in high-inoculum infections treated with AZT-AVI.

674. Variations in Agreement and Epidemiological Cutoff Value (ECV) between Fosfomycin (FOF) Agar Dilution and Broth Microdilution Using Standard- and High-Inoculum Protocols for Klebsiella pneumoniae (KP)

Background FOF has been used in the treatment of multidrug-resistant (MDR) KP infections despite established susceptibility breakpoints. At present, agar dilution (AD) is considered the reference method for FOF while broth microdilution (BMD) is specifically recommended against despite its convenience over AD. We therefore sought to assess FOF activity against KP, along with essential and categorical agreement between AD and BMD methods to determine if BMD could be used as a reliable testing method. Methods Minimal inhibitory concentration (MIC) values were determined for a convenience collection of 69 KP isolates (59.4% MDR) from three US institutions. MIC testing was conducted in duplicate on separate days using AD and BMD methods; essential and categorical agreement were calculated using AD as the reference method. Fourteen isolates were also analyzed using high-inoculum AD (105.3-5.9 CFU/mL) similar to the BMD method. MIC values were categorized using Clinical and Laboratory Standards Institute (CLSI) interpretive criteria for Escherichia coli (≤ 64 mg/L, susceptible). ECVs were determined according to CLSI methodology. Results MIC values varied between methods, withMIC50/MIC90 values being 32/256 mg/L for AD and 128/256 mg/L for BMD. Using E. coli criteria, susceptible/intermediate/resistant rates were 82.6/2.9/14.5% (AD) and 44.9/21.7/33.3% (BMD). Essential agreement was 44.9% and categorical agreement was 60.8%. When using high-inoculum AD, MIC values were on average three-fold higher compared to standard-inoculum AD, with 10 of the 14 (71.4%) isolates brought into essential agreement with BMD. Calculated ECVs were 128 mg/L for standard-inoculum AD and 1024 mg/L for BMD. Conclusion Our collection of KP displayed high MIC values to FOF, in addition to substantial discrepancies between AD and BMD methods. Essential agreement increased with the use of high-inoculum AD testing, which better correlated with BMD results. ECV for BMD was three dilutions higher than that for standard-AD ECV. Based on these results, we recommend further investigation of BMD for FOF testing using a larger isolate collection, along with optimization of currently recommended testing methods. In light of these results, KP-specific breakpoints should also be examined. Disclosures Elizabeth B. Hirsch, PharmD, Merck (Grant/Research Support)Nabriva Therapeutics (Advisor or Review Panel member)

28. Regulation of the Staphylococcal β-lactamase Plays a Major Role in the cefazolin Inoculum Effect

Background Cefazolin (Cz) is commonly used to treat methicillin-sensitive Staphylococcus aureus (MSSA) bacteremia. Yet, some MSSA isolates producing the staphylococcal β-lactamase (BlaZ) exhibit the Cz inoculum effect (CzIE), defined as an increase in the minimum inhibitory concentration (MIC) to ≥ 16 µg/mL at high inoculum (107 CFU/mL, HI-MIC). Retrospective clinical data linked the CzIE to increased 30-day mortality and Cz treatment failure in patients with MSSA bacteremia, yet the mechanistic bases of this phenomenon are unknown. We aimed to explore the contribution of blaZ regulation, via BlaR (antibiotic sensor) and BlaI (transcriptional repressor) (Fig 1) to the CzIE by i) in trans expression assays and ii) analysis of their sequences in a set of isolates Figure 1. Structure of the Staphylococcal bla Operon Methods The blaZ genes (with putative promoters) of strains exhibiting and lacking the CzIE (TX0117 and ATCC29213, respectively) were expressed in trans in RN4220 (blaZ neg) using the promotor-less vector pWM401 (Figure 2). We subsequently cloned the blaR and blaI genes of each TX0117 and ATCC29213 upstream of each blaZ allele (Figure 3). The presence of the CzIE was assessed in transformants using broth microdilution at standard (105 CFU/mL, SI-MIC) and high inoculum. We also performed whole-genome sequencing (WGS) in 104 MSSA isolates exhibiting and lacking the CzIE to compare the sequences of BlaZ, BlaR, and BlaI and classified them by allotypes (unique amino acid sequences) using ATCC29213 as reference. Figure 2. In trans expression of blaZ genes from a CzIE+ strain (TX0117) and a CzIE- strain (ATCC29213) in RN4220 Figure 3. In trans expression of the bla Operons from a CzIE+ strain (TX0117) and a CzIE- strain (ATCC29213) in RN4220 Results Expression of blaZTX0117 and blaZATCC29213 with their native promoters in RN4220 resulted in the CzIE with Cz HI-MICs ≥ 64 µg/mL regardless of the origin of the allele (Table 1). Inclusion of the regulatory elements blaR and blaI from TX0117 (CzIE+) did not change the phenotype. In contrast, addition of blaR and blaI from ATCC29213 (CzIE-) led to a marked decrease in the Cz HI-MIC (Table 1). Sequence analyses of 104 MSSA isolates revealed 10, 17 and 6 BlaZ, BlaR and BlaI allotypes, respectively (Table 2). BlaZ-2 and BlaR-4 were linked to the CzIE in 90% of isolates. Table 1. MIC values of transformans after In trans expression of blaZ genes and bla Operons from a S. aureus CzIE+ strain(TX0117) and a CzIE- strain (ATCC29213) in RN4220 Table 2. BlaZ allotypes of 104 Staphylococcus aureus isolates and their association with the CzIE Conclusion Our results suggest that overexpression of blaZ can lead to the CzIE in any MSSA strain. Thus, the regulation of blaZ expression via BlaR and BlaI seem to play a major role in the CzIE. Identification of specific BlaR and BlaI allotypes could predict the presence of the CzIE. Disclosures Cesar A. Arias, M.D., MSc, Ph.D., FIDSA, Entasis Therapeutics (Scientific Research Study Investigator)MeMed (Scientific Research Study Investigator)Merck (Grant/Research Support)