Ribaxamase, an Orally Administered β-Lactamase, Diminishes Changes to Acquired Antimicrobial Resistance of the Gut Resistome in Patients Treated with Ceftriaxone

Figures & data

Table 1 Primer Sequences Used for qPCR Analysis

cfxA	Forward	5ʹ –AGGAAATGTCGGCTGACCAT- 3’
	Reverse	5ʹ –GCAGCACCAAGAGGAGATGT- 3’
vanRd	Forward	5ʹ –CGTTTAATCCGCTGGAAGTG- 3’
	Reverse	5ʹ –CCGCATGTACTGCCTTAGCT- 3’

Figure 1 LEfSe Analysis of Change in Number of Hits from T0 to T1. Each column represents a different AMR gene that demonstrated a significant change (change in linear determinant analysis [LDA] of ≥2) in the number of hits from the T0 collection point (screening) to the T1 collection point (post-antibiotics). Top figure, placebo group and bottom figure, ribaxamase group. Red bars decreased significantly between these collection points while green bars increased significantly. Specific gene classes of interest are indicated by colors on the figure, blue β-lactamases, green vancomycin resistance genes and red efflux pump genes. Individual gene notations correspond to the Comprehensive Antimicrobial Resistance Database.

Figure 2 Comparing Number of Hits per Sample vs Copy Number per Sample. The graph compares data quartiles for the number of hits per sample with the copy number of the gene for each sample for, (A) cfxA and (B) vanRD. The data were separated into quartiles for the 100 samples that were subjected to qPCR analysis and compares the number of hits per sample, as determined by database alignment, vs the copy number per sample, as determined by qPCR analysis. The quartiles were as follows, cfxA Hits, Q1: 0–2658, Q2: 2659–52,776, Q3: 52,777–175,384, Q4: >175,384; Copy Number, Q1: 0–62, Q2: 63–410, Q3: 411–1244, Q4: >1244, vanRD Hits, Q1 & Q2 = 0, Q3: 1–33, Q4: >33, Copy Number, Q1 & Q2 = 0, Q3: 1–4, Q4: >4. Quartiles 1 and 2 were combined for both parameters on the figure for vanRD due to the abundance of samples with zero values in the sample set. The size of each sphere represents the number of samples, based on the copy number, which corresponded to the indicated quartile for hits. The red lines are added for emphasis.

Table 2 Comparison of the Frequency of Antibiotic Resistance Genes at T0 vs T1 in the Ribaxamase vs Placebo-Treated Patients

Gene^a	P-value^b	Resistance Phenotype	Total Hits^c	Data Distribution^d
vanRD^e	0.0053	Vancomycin	3387	≥25%
vanSD^e	0.0130	Vancomycin	539	≥25%
cfxA5^e	0.0153	β-lactam	177,247	≥95%
cfxA^e	0.0201	β-lactam	158,315	≥95%
cfxA4^e	0.0217	β-lactam	171,730	≥95%
cfxA2^e	0.0248	β-lactam	199,467	≥95%
cfxA3^e	0.0296	β-lactam	229,261	≥95%
vanD	0.0849	Vancomycin	1114	≥25%
cblA-1	0.1758	β-lactam	78,578	≥90%
cfxA6	0.4533	β-lactam	535,181	≥90%
vanXYG	0.5832	Vancomycin	866	≥25%
vanWG	0.6375	Vancomycin	1446	≥25%
mefA	0.6929	Macrolide	91,609	≥90%
vanTG	0.7378	Vancomycin	2333	≥25%
vanG	0.7624	Vancomycin	863	≥25%
mel	0.8011	Macrolide	70,445	≥75%
cepA	0.8253	β-lactam	6721	≥25%

Notes: ^aβ-lactamase, vancomycin resistance or macrolide resistance genes selected based on Phase 2b clinical study. ^bChange in hits from T0 to T1, P-value is based on the Mann Whitney test. ^cTotal hits across sample set. Only those with >500 total hits were included in the analysis. ^dPercentage of samples in the 349 sample set which included at least one hit for the gene of interest. ^eAlso identified by linear discriminant analysis effect size (LEfSe) analyses

Table 3 Change in Mean Copy Number Over Time for cfxA and vanRD in Placebo vs Ribaxamase-Treated Patients

Treatment Group	T0	T1	T2
cfxA
Placebo	169,373^a ±24,137^b	279,822 ±58,886	237,738 ±70,114
Ribaxamase	237,012 ±40,149	118,367 ±35,227	150,350 ±46,667
vanRD
Placebo	51 ±34	152 ±63	66 ±31
Ribaxamase	69 ±49	25 ±45	95 ±82

Notes: ^aMean copy number. ^bStandard error.

Table 4 β-Lactamase Gene Variants Demonstrating a Significant Increase in Placebo vs Ribaxamase-Treated Patients from T0 to T1

β-Lactamase Gene Variant	Placebo		Ribaxamase		P-value^b
	T0 (54)^a	T1 (52)	T0 (65)	T1 (66)
cfxA	42,067^c (779^d, 0–4951^e)	113,364 (2,180, 0–20,881)	53,202 (818, 0–9161)	62,854 (952, 0–6063)	0.0241
cfxA2	53,205 (985, 0–6591)	140,885 (2709, 0–23,657)	66,670 (1025, 0–11,581)	79,553 (1205, 0–8166)	0.0324
cfxA3	47,742 (864, 0–5075)	147,920 (2845, 0–17,942)	88,257 (1357, 0–22,534)	79,059 (1197, 0–6188)	0.0525^f
cfxA4	44,115 (816, 0–6372)	119,014 (2289, 0–18,212)	56,108 (863, 0–11,261)	64,788 (981, 0–5605)	0.0279
cfxA5	47,325 (876, 0–5678)	131,480 (2528, 0–26,382)	60,249 (926, 0–10,968)	69,401 (1051, 0–6983)	0.0234

Notes: ^aNumber of samples analyzed (≥95% of the samples had at least one hit each of the five genes). ^bP-value is from linear regression model. ^cTotal hits, ie, the total number of matches for this gene or variant in the sample set. ^dAverage hits. ^eRange of hits per sample. ^fWas above statistical significance by this analysis, but was identified in other analyses.

Table 5 β-Lactamase Gene Variants Demonstrating a Significant Increase in Hits with PPI-Use or Non-Use from T0 to T1

β-Lactamase Gene Variant	Correlated with	PPI Used^a		No PPI Used^b		P-value^c
		T0 (21)^d	T1 (19)	T0 (98)	T1 (99)
cfxA	PPI use	456^e, 295^f (0–2193)^g	1171, 599 (0–5169)	874, 391 (0–5834)	1555, 713 (0–20,881)	0.0003
cfxA2	PPI use	576, 409 (0–11,581)	1590, 787 (3–6677)	1100, 525 (0–3006)	1922, 938 (0–23,657)	0.0018
cfxA3	PPI use	572, 314 (0–2829)	1199, 598 (0–4049)	1265, 507 (0–22,534)	2063, 974 (0–16,908)	0.0165
cfxA4	PPI use	419, 297 (0–1779)	1047, 618 (0–3984)	933, 399 (0–11,261)	1655, 650 (0–18,112)	0.0066
cfxA5	PPI use	432, 256 (0–1761)	1035, 602 (7–4161)	1005, 417 (0–10,968)	1830, 665 (0–26,382)	0.0116
cblA-1	PPI use	187, 101 (2–905)	379, 134 (0–1627)	307, 116 (0–2568)	393, 222 (0–2220)	0.0393
cfxA6	No-PPI	924, 90 (0–5642)	923, 15 (0–6072)	1385, 77 (0–15,844)	1987, 98 (0–21,682)	0.0306

Notes: ^aThe patient received proton pump inhibitors (PPI) between T0 and T1 in the ribaxamase clinical study. ^bThe patient did not receive any PPI in the ribaxamase clinical study. ^cP-value is from linear regression model. ^dNumber of samples analyzed in the group. ^eAverage number of hits, ie, the number of matches for this gene or variant, per group. ^fMedian hits per group. ^gRange of hits per sample.

Table 6 Vancomycin Resistance Genes Demonstrating a Significant Increase in Placebo vs Ribaxamase-Treated Patients from T0 to T1

Gene	Placebo		Ribaxamase		P-value^b
	T0 (54)^a	T1 (52)	T0 (65)	T1 (66)
vanD	382^c (15)^d (25^e, 2–158^f)	1079 (24) (44, 2–298)	1268 (16) (79, 1–539)	693 (21) (33, 1–142)	0.0354
vanRD^g	172 (14) (12, 1–50)	545 (27) (21, 1–107)	506 (24) (21, 1–223)	321 (22) (15, 1–58)	0.0135
vanSD^g	200 (19) (11, 1–76)	543 (31) (18, 1–120)	513 (18) (29, 1–205)	350 (22) (16, 1–81)	0.0112

Notes: ^aNumber of samples analyzed. ^bP-value is from linear regression model. ^cTotal hits, ie, the total number of matches for this gene or variant in the sample set. ^dNumber of samples in set that had at least one hit for the gene. ^eAverage hits in samples that had at least one hit. ^fRange of hits per sample in samples that had at least one hit. ^gWas also identified as being significantly different by linear discriminant analysis effect size (LEfSe) analyses.