Evidence of pediatric sepsis caused by a drug resistant Lactococcus garvieae contaminated platelet concentrate

Figures & data

Table 1. Pediatric patients’ characteristics.

	Patient 1	Patient 2	Patient 3	Patient 4
Sex	Female	Male	Male	Male
Age, years	8	9	4	9
Nationality	Italian	Italian	Italian	Italian
Disease	ALL	ALL	ALL	Metastatic neuroblastoma
PCs transfusion (day/month/year; hour:minute)	01/09/2020; 16:53	02/09/2020; 14:04	02/09/2020; 14:32	02/09/2020; 16:36
At PCs transfusion:
Immunosuppression	Yes	Yes	Yes	Yes
White blood cells, cells/µL	100.0	800.0	7600.0	700.0
Red blood cells, 10^6cells/µL	2.5	2.3	2.8	3.5
Hb, g/dL	8.0	7.3	8.3	9.6
Ht, %	24.3	21.8	23.3	27.3
MCV, fL	96.5	96.0	84.1	77.1
MCH, pg	31.7	32.0	30.0	27.2
MCHC, g/dL	32.9	33.4	35.6	35.3
RDW, %	18.3	22.2	13.3	13.1
HDW, g/dL	3.4	4.1	3.3	3.1
Platelets, number/µL	20000.0	135000.0	38000.0	29000.0
Neutrophils, %	31.8	36.6	92.2	62.3
Lymphocytes, %	61.2	33.2	2.9	24.8
Monocytes, %	1.2	12.3	3.7	5.9
Eosinophils, %	2.4	1.0	0.3	4.0
Basophils, %	0.0	0.6	0.0	0.3
Leukocytes, %	3.5	11.7	0.8	2.7
Lactococcus garvieae isolation from blood culture (day/month/year; hour:minute)	–	03/09/2020; 20:29	03/09/2020; 11:14	03/09/2020; 09:28
Symptoms after positive blood culture:
Fever, celsius	39.2	39.7	40.0	38.2
Chills	Yes	Yes	Yes	Yes
Heart rates, bpm	110	140	160	150.00
Respiratory rate, breaths/min	22	20	30	22
Arterial blood pressure, mmHg	99/64	117/74	80/40	110/70
Procalcitonin, ng/Ml	18.5	121.6	61.9	356.0
CRP, mg/dL	2.8	15.3	5.9	11.8
Empiric antimicrobial therapy	INN-Tigecycline; Amikacin; β-lactams	INN-Tigecycline; Amikacin; INN-ceftazidime/avibactam	INN-Tigecycline; Amikacin; β-lactams; Quinolones; Metronidazole;	INN-Tigecycline; Amikacin*; Quinolones

ALL: Acute lymphocytic leukemia; PCs: Platelet concentrates; Hb: Hemoglobin; Ht: Hematocrit; MCV: Mean Corpuscular Volume; MCH: Mean Corpuscular Hemoglobin; MCHC: Mean Corpuscular Hemoglobin Concentration; RDW: Red Cell Distribution Width; HDW: Hemoglobin Distribution Width; CRP: C-reactive Protein; INN: International Non-proprietary Name. *Replaced by Gentamycin one day later.

Figure 1. Procalcitonin (Panel A) and C-reactive protein (Panel B) kinetics in the four paediatric patients receiving the platelet concentrates. All patients received platelet concentrates on 2 September 2020 except patient 1 who received the concentrates on 1 September 2020. Isolation of L. garvieae occurred for all patients (patients 1, 2, and 3) on 3 September 2020. Cut-off: normal value. Procalcitonin normal value <0.05 ng/mL; C-reactive protein normal value <0.50 mg/dL.

Figure 2. Estimated maximum likelihood (A) and Bayesian (B) phylogenies based on coreSNP genome of the three L. garvieae strains isolated from pediatric patients at Bambino Gesù Pediatric Hospital, IRCCS. Representative 20 L. garvieae and 4 L. lactis strains retrieved by public databases were also included to obtain an alignment of 27 coreSNP genomes 62,990 nucleotides long. The L. lactis and L. garvieae strains were annotated according to collection source (Orange: Dairy products, Blue: Fishes, Brown: Cow, Red: Humans; Other: Purple). BioSample accession numbers defined the strains. Panel (A) reported the ML tree, inferred using iqTree2 under the nucleotide substitution GTR+I+G4 model and 1000 bootstrap replicates. Panel (B) reported the Bayesian tree, performed by BEAST v.1.10.4 setting a chain length of 100 million states under a strict molecular clock model and the GTR+I+G4 substitution model. Bootstrap values and posterior probabilities are reported along the branches of the trees.

Table 2. Putative virulence factors and resistance genes identified in the three L. garvieae strains isolated from pediatric patients at Bambino Gesù Pediatric Hospital, IRCCS.

Gene product	Accession number and locus	Identity (%)	Length (nt)	E-value*	BitScore**
Chromosomal content
Hemolysin III related protein	NC_017490 region:331642-352295	98.61	217	<1E-100	424
U32 family peptidase [Collagenase]	NC_017490 region: 1699976-1701265	99.53	429	<1E-100	887
MucBP domain-containing protein LCGL 1005	AFCC01000046 region:18820-20355	97.65	680	<1E-100	1321
WxL surface protein	AFCD01000027 region:17404-18222	89.35	629	<1E-100	1019
LPxTG surface protein (Cna B-domain) (orf 25)	HM852551 WP_019291416.1	99.81	2,115	<1E-100	4031
Sortase A	NC_017490 region: 13005315-1306046	96.29	243	<1E-100	443
Adhesins gene cluster - WxL surface protein	NC_017490 region: 878477-872476	96.55	203	<1E-100	389
Adhesins gene cluster - Cell surface putative DUF916 and DUF3324 domain containing protein	DUF916 and DUF3324	93.17	337	<1E-100	629
Adhesins gene cluster - Putative surface protein (COG4713 domain)	HM852557 AFCC01000021 region: 19917-21412	98.95	190	<1E-100	382
Pili specific sortase SpaH/EbpB family LPxTG-anchored major pilin	WP 100222480.1 in AFCD01000058 region: 28828-35485	98.61	502	<1E-100	914
ABC transporter ATP-binding protein LCGL 1621	NC_017490 region: 1594232-1596948	92.64	258	<1E-100	460
ABC transporter substrate-binding protein LCGL 1622		92.62	339	<1E-100	565
ABC transporter substrate-binding protein LCGL 1623		92.01	313	<1E-100	588
Iron ABC transporter permease LCGL 0527	NC_017490 region: 544393-548003	98.85	260	<1E-100	495
Iron ABC transporter permease LCGL 0528		93.98	316	<1E-100	429
Iron ABC transporter permease LCGL 0529		94.90	294	<1E-100	516
Iron-hydroxamate ABC transporter LCGL 0530		98.40	313	<1E-100	605
Ferrous iron transport protein B LCGL RS10540	NC_017490 region: 106587-109166	97.11	693	<1E-100	1321
Phospho-sugar mutase (Phosphoglucomutase) LCGL 1596	NC_017490 region: 1558243-1559955	98.95	570	<1E-100	1158
Enolase [phosphopyruvate hydratase]	NC_017490 region: 1489407-149070	100.00	429	<1E-100	867
Glyceraldehyde-3-phosphate dehydrogenase LCGL RS19790	NC_017490 region: 1928003-1929013	100.00	336	<1E-100	671
Superoxide dismutase LCGL 0285	NC_017490 region: 289210-289818	99.01	202	<1E-100	385
FAD-dependent oxidoreductase LCGL 0664	NC_017490 region: 678241-679578	99.10	445	<1E-100	907
Glycoside hydrolase family 1 protein	NZ_CP065637 region: 716031-717467	99.8	478	<1E-100	487
Antibiotic resistance genes
Lsa(D)^a	MH473150.1	90.76	1493	<1E-100	1993
mdt(A)^a	WP_176490218.1	88.87	1258	<1E-100	1546
vanZ-like domain	NZ_CP065637 region: 571963-572424	93.49	1045	<1E-100	1554
penicillin-binding protein 1A^b	GFO52038.1	97.12	694	<1E-100	1316
penicillin-binding protein 2A^b	GFO51196.1	98.29	762	<1E-100	1453
putative penicillin-binding protein PbpX^b	CEF50305.1	99.68	314	<1E-100	619
penicillin-binding protein 2B^b	GFO51136.1	97.35	718	<1E-100	1329
penicillin-binding protein 1B^b	GFO51088.1	97.65	807	<1E-100	1483
Plasmid content (pUC11C)
Sortase C (n2)	ARE12311	99.58	1179	<1E-100	2150
IS6 family transposase	ARE12304	100	96	6.69E-46	178
mobilization/filimentation protein Fic	ARE12315	98.34	603	<1E-100	1059
Resolvase: Exogenous DNA integration	ARE12303	90.99	555	<1E-100	749
SpaH/EbpB family LPXTG-anchored major pilin	WP_082225092.1	98.6	501	<1E-100	911
VWA domain c protein	WP_082225091.1	98.29	1341	<1E-100	2289
Insertion sequences
IS6 family – IS1216E^c	IS1216E	98.0	132	<1E-100	238
IS6 family – ISS1N^c	ISS1N	98.0	46	<1E-100	61.9

Putative virulence factors were annotated using BLASTN e BLASTX. Identity was estimated against the reference genome for each gene product. Only hits with BIT score >300, coverage >65%, and identity >80% were considered. ^aLsa(D) is a specific resistance factor providing tolerance to lincosamides, while mdt(A) is a drug antiporter responsible for aspecific resistance to tetracyclines and macolides; both of them belong to chromosomal genetic content. ^bPenicillin binding protein (PBP) are chromosomal genes that are normally involved in cell wall biosynthesis; their full resistance mechanism is achieved by the actions of auxiliary genes mainly found in Staphylococcus pathogenic strains (holding chromosomal mec cassette); they provide only partial resistance in other bacterial genera. ^cInsertion Sequences 6 family have been characterized as important factors determining bacterial genome shaping. In fact, they are responsible for exogenous genetic content integration via IS6 family transposase action (found to be part of pUC11C plasmid). *E-value index returns the probability to get a match by chance. **BIT score is the match reliability index.