A Longitudinal Evaluation of the Bacterial Pathogens Colonizing Chronic Non-Healing Wound Sites at a United States Military Treatment Facility in the Pacific Region

Figures & data

Table 1 Demographic Characteristics, Comorbidities and Wound Burden of the Population That Was Evaluated in This Study. A Total of 28 Patients Being Treated in the TAMC Vascular Limb Salvage Clinic Were Enrolled

Total: 28 Patients	No. of Patients	(%)
Age (years)
30–49	4	14.3
50–69	11	39.3
70–89	13	46.4
mean (standard deviation)	64.8 (12.7)
Gender
Female	4	14.3
Male	24	85.7
Obesity
No	12	42.9
Yes	16	57.1
Current smoking
No	25	89.3
Yes	3	10.7
Wound
Diabetic	16	57.1
Venous insufficiency	19	67.9
Arterial insufficiency	2	7.1
Number of wounds
1	16	57.1
2	7	25.0
3	4	14.3
4	0	0.0
5	1	3.6

Table 2 Bacteria Species Recovered at Each Visit from 47 Unique Wound Sites from a Total of 28 Patients

Number of Bacterial Species	Initial Visit		2-Week Follow-Up		4-Week Follow-Up
	n	% of Isolates	n	% of Isolates	n	% of Isolates
Gram-positive	53	49	37	51	38	51
Gram-negative	55	51	35	49	36	49
Aerobic bacteria	94	85	60	81	62	84
Anaerobic bacteria	16	15	14	19	12	16
S. aureus	18	16	7	9	12	16
P. aeruginosa	16	14	15	20	10	13
E. faecalis	8	7	5	7	4	5
E. coli	8	7	6	8	5	7
K. pneumoniae	7	6	6	8	5	7
Citrobacter koseri	4	4	0	0	3	4
Proteus mirabilis	4	4	1	1	1	1

Figure 1 A comparison of the numbers of bacterial species that were recovered from chronic non-healing wounds at the initial visit (Day 0), at a 2-week follow-up (Day 15) and at a 4-week follow-up (Day 30).

Table 3 Evaluation of the Antibacterial Resistance (AMR) Genes That Were Identified in the 123 Unique Bacterial Isolates Collected in This Study. AMR Genes Conferring Resistance to the β-Lactam Class of Antibiotic Were the Most Common, Followed by AMR Genes Conferring Resistance to Aminoglycosides and the Macrolides, Respectively

Number of Isolates with Detectable AMR Genes	Antibiotic Class	Target
46	Aminoglycosides	Protein Synthesis
63	β -lactams	Cell Wall Synthesis
9	Fluoroquinolones	DNA Gyrase
24	Fosfomycin	Cell Wall Synthesis
6	Lincosamides	Protein Synthesis
27	Macrolides	Protein Synthesis
11	Phenicols	Protein Synthesis
15	Phenicols/Quinolones	Protein Synthesis/DNA Gyrase
7	Streptothricin	Protein Synthesis
5	Sulfonamides	Purine Base Synthesis
19	Tetracyclines	Protein Synthesis
8	Trimethoprim	Purine Base Synthesis

Table 4 A Summary of Antibacterial Resistance (AMR) Detections by Bacterial Species and Potential Impact on Antibiotic EffectivenesS. The Members of the Enterobacteriaceae (E. Coli), The Non-Fermenters (P. Aeruginosa), and the S. Aureus Were Found to Harbor the Greatest Numbers of AMR Genes

Organism	AMR Genes Detected	Antibiotic Classes Potentially Impacted
Acinetobacter baumanii	ant(3ʹ’)-IIa, blaADC, blaOXA	Aminoglycosides and β -lactams
C. koseri	blaCKO	β -lactams
C. striatum	Erm(X), tet(W)	Macrolides and Tetracyclines
E. cloacae	blaACT-17, oqxA, oqxB	β -lactams and Quinolones
E. faecalis	aac(6ʹ)-le, ant(6)-Ia, aph(2”)-Ia, aph(3”)-IIIa, dfrE, erm(B), lsa(A), sat4, tet(M)	Aminoglycosides, Trimethoprim, Macrolides, Lincosamides, Streptothricin, and Tetracyclines
E. coli	aac(3)-IId, aadA1, aadA2, aadA5, aph(3ʹ)-Ia, aph(6)-Id, blaEC, blaEC-5, blaTEM-1, cmlA1, dfrA17, dfrA5, mph(A), sul1, sul2, sul3, tet(A)	Aminoglycosides, β-lactams, Trimethoprim, Chloramphenicols, Sulfonamides, and Tetracyclines
F. magna	erm(A), erm(B), tet(M)	Macrolides and Tetracyclines
K. aerogenes	ampC, fosA, oqxA, aac(3)-IId	β -lactams, Fosfomycins, Quinolones and Aminoglycosides
K. pneumonia	blaOKP, blaSHV-108, blaSHV-11, blaSHV-36 fosA, oqxA, oqxB, oqxB19, oqxB25	β -lactams, Fosfomycins, quinolones, and Chloramphenicol,
Peptonipilus spp.	erm(A), tet(M)	Macrolides and Tetracyclines
P. mirabilis	catA, tet(J)	Chloramphenicols and Tetracyclines
P. aeruginosa	aph(3ʹ)-IIb, blaOXA, blaOXA-485, blaOXA-488, blaOXA-50, blaPDC, blaPDC-12, blaPDC-19a, blaPDC-3, blaPDC-34 blaPDC-59, catB7, crpP, fosA	Aminoglycosides, β-lactams, Chloramphenicol, and Fosfomycins
S. aureus	aac(6ʹ)-Ie, ant(6)-Ia ant(9)-Ia, aph(2ʹ’)-Ia aph(3ʹ)-IIIa, blaI, blaR1 blaZ, dfrC, erm(A). erm(C), fosB, mecA. mecI, mecR1, mph(C) msr(A), sat4, tet(38)	Aminoglycosides, β-lactams, Macrolides, Streptothrycin, Fosfomycins, and Tetracyclines
S. simulans	ant(9)-Ia, erm(A)	Aminoglycosides and Macrolides
S. agalactiae	erm(B), tet(M)	Macrolides and Tetracyclines

Table 5 Phenotype Conversion of S. Aureus from Sensitive (S) to Resistant (R) for Clindamycin and Erythromycin Over a Period of 30 Days

S. aureus	CFZ	FOX	CIP	CLI	DAP	ERY	GEN	LVX	LZD	OXA	PEN	SXT	VAN
Day 0	R	R	S	S	S	S	R	S	S	R	R	R	S
Day 15	R	R	S	S	S	R	R	S	S	R	R	R	S
Day 30	R	R	S	R	S	R	R	S	S	R	R	R	S

Abbreviations: CFZ, ceftazidime; FOX, cefoxitin; CIP, ciprofloxacin; CLI, clindamycin; DAP, daptomycin; ERY, erythromycin; GEN, gentamicin; LVX, levofloxacin; LZD, linezolid; OXA, oxacillin; PEN, penicillin; SXT, trimethoprim/sulfamethoxazole; VAN, vancomycin.

Table 6 Single Nucleotide Polymorphisms (SNPs) Detected in Three Isolates of S. Aureus Recovered from Wound Sites on a Single Patient on Days 0, 15 and 30. All SNPs Were Localized to Genes That Have Been Predicted to Code for Proteins Involved in the Metabolic Process of the Pathogen and No SNPs Were Detected in Any of the Genes Known to Be Involved in the Development of Antibiotic Resistance

Target Gene	Change	Predicted Effect	Day 0	Day 15	Day 30
Allophanate hydrolase 2 subunit 2	Asparagine to Isoleucine	Substitution	+
Molybdenum cofactor biosynthesis protein MoaC	Synonymous	No Change	+
Aspartate 1-decarboxylase	Synonymous	No Change		+	+
S1 RNA binding domain	Arginine to Lysine	Substitution	+	+	+
Acyl esterase	Serine to Glycine	Substitution	+
Flavin-utilizing monoxygenase	Methionine to Thymine	Substitution		+	+
Na(+)H(+) antiporter subunit A/B	Glutamine to Lysine	Substitution	+
S1 RNA binding domain	Glutamic Acid to Lysine	Substitution	+	+	+
MutT protein	Aspartic Acid to Glycine	Substitution		+	+
Phosphate ABC transporter PstS	Glycine to Arginine	Substitution		+
Dihydroxy-acid dehydratase	Synonymous	No Change	+
Ferrichrome-binding periplasmic protein	Valine to Methionine	Substitution	+
Autolysis histidine Kinase LytS	Synonymous	No Change	+
Hypothetical protein	Tyrosine to Aspartic Acid	Substitution		+
Na(+)H(+) antiporter subunit E	Synonymous	No Change	+