Simkania negevensis, an insight into the biology and clinical importance of a novel member of the Chlamydiales order

Figures & data

Figure 1. Evolutionary relationships of Simkaniaceae. The evolutionary history was inferred using the Neighbor-Joining method (Saitou & Nei, 1987) based on 1548 nucleotides from 15 16S rRNA sequences. Sequences were retrieved from the NCBI database using “Simkania*” as a research word. The percentage of replicate trees, in which the associated taxa clustered together in the bootstrap test (100 replicates) are shown next to the branches (Efron et al., 1996). The evolutionary distances were computed using the Tamura 3-parameter method (Tamura, 1992) and are shown as number of base substitutions per site. The rate variation among sites was modeled with a gamma distribution. Evolutionary analyses were conducted in MEGA5 after sequence analysis on Geneious 7.1.7 (Kearse et al., 2012).

Table 1. General features of Simkania negevensis compared to other Chlamydiales members.

	S. negevensis Z*	C. trachomatisD/UW-3/CX†	W. chondrophila WSU86-1044‡	P. acanthamoebaeUV-7¶
Genetic features§
Chromosome size (nt)	2 496 337	1 042 519	2 141 577	3 072 383
Plasmid size (nt)	132 038	7493	15 593ǁ	No
rRNA encoding genes
16S	1	2	2	3
23S	1	2	2	3
Outer membrane structure#
Major membrane protein	MOMP-like	MOMP	MOMP-like	Hypothetical protein (puv)
Cysteine-rich proteins (membrane stabilization)	Absent	OmcA/OmcB	OmcA/OmcB/wcw_0272	OmcB/puv_11160
Porins	MOMP-like	MOMP/PorB/OprB	MOMP-like	Puv_27500/Puv_07550**
MOMP (ompA)	2	1	no	No
MOMP like	35††	n.a.	11	1‡‡
Pmp (auto transporter/cell adhesion)	Yes (PmpB)	Yes	Yes	?¶¶
PG-like structure	Not detected	Yes	Yes	?
NIpD (PG remodeling enzyme)	Yes	Yes	Yes	Yes
AmiA (PG remodeling enzyme)	Yes	Yes	Yes	Yes
Host pathogens interactions§§
CPAF (CMH expression, apoptosis, immune response)	No	Yes	Yes	Yes
Catalase	No	No	Yes	Yes
T3SS	Yes	Yes	Yes	Yes
T4SS	Yes	No	No	Yes
Metabolic pathwaysǁǁ
Glucokinase	Yes	No	Yes	Yes
Citric acid cycle	Complete	Incomplete	Complete	Complete
Nucleotides transporters	4	2	5	5
Tryptophan operon	Complete	Incomplete	Absent	Absent

Table 1 highlights various biological similarities and differences of S. negevensis and other Chlamydiales.

CPAF: Chlamydia protease-like activity factor; MOMP: major outer membrane protein; n.a.: not applicable; nt: nucleotides; PG: peptidoglycan; Pmp: polymorphic membrane protein; T3SS: type III secretion system; T4SS: type IV secretion system.

* (Collingro et al., 2011).

† (Carlson et al., 2008; Stephens et al., 1998).

‡ (Bertelli et al., 2010).

¶ (Horn et al., 2004).

§ (Bertelli et al., 2010; Carlson et al., 2008; Collingro et al., 2011; Horn et al., 2004; Stephens et al., 1998).

ǁ Strain 2032/99 does not have a plasmid.

# (Aistleitner, 2015; Birkelund et al., 2009; Frandi et al., 2014; Jacquier et al., 2014; Kebbi-Beghdadi et al., 2015; Liechti et al., 2014; Pilhofer et al., 2014, 2013).

** Putative Pom encoded in the genome, but not isolated at the membrane.

†† Two of them are considered as homologous of OmpA.

‡‡ One homologue encoded in the genome, but not isolated at the membrane.

¶¶ Putative Pmp encoded in the genome, but not isolated at the membrane.

§§ (Collingro et al., 2011; Rusconi & Greub, 2013).

ǁǁ (Bertelli et al., 2010; Carlson et al., 2006; Collingro et al., 2011; Horn et al., 2004; Knab et al., 2011; Omsland et al., 2014).

Table 2. Prevalence of an acute Simkania negevensis infection in patients suffering from bronchiolitis.

Country, patients	Diagnostic method	Patients (%)		Controls (%)		p values	Co-infections (%)		Reference
Israel, 239	PCR Immunoperoxidase	60/239 14/92	(25) (15)	0/78 1/78	(0) (1)	<0.001<0.001	22/60	(36)	Kahane et al., 1998
Inult Canada, 22	PCR	14/22	(63)				12/14	(85)	Greenberg et al., 2003
USA, 66	PCR	9/55	(16)	2/28	(7)	NS			Kumar et al., 2005
UK, 222	Nested PCR	100/222	(45)						Friedman et al., 2006

PCR: polymerase chain reaction; NS: not significant.

Table 3. Prevalence of an acute Simkania infection in patients suffering from pneumonia.

Population, country, patients	Diagnostic method	Patients (%)		Controls (%)	p values	Simkaniaas onlypathogen	Reference
Adults, Israel, 308	ELISA*	8/308	(3)			4	Lieberman et al., 1997
Children, USA, 25	PCR ELISA*	5/22	(23)	12/31 (39)	NS		Kumar et al., 2005
Children, Israel, 34	Nested PCR MIA	31/34 17/34	(91) (50)	34/35 (97)	NS	20	Kahane et al., 2007b
Children, Finland, 174	MIF†	18/174‡	(10)			6¶	Heiskanen-Kosma et al., 2008
Children, Italy, 101	MIF†	5/101	(5)			2	Fasoli et al., 2008
Children, Brazil, 183	MIF†	3/184	(2)			1	Nascimento-Carvalho et al., 2009
Children, Switzerland, 265	PCR pan-Chlamydiales§	2/265	(1)				Lienard et al., 2011a,b
Adults, Italy, 102	MIF^ǁ	13/102	(13)	2/104 (2)	<0.05		Donati et al., 2013

ELISA: enzyme-linked immunosorbent assay; MIA: membrane immune-assay; MIF: microimmunofluorescence; NS: not significant; PCR: polymerase chain reaction.

* High signal for IgA (>1.2 OD) or an increase of 0.5 OD units between paired sera levels for IgA (1:30) or IgG (1:100).

† IgM ≥1:10 or a four-fold increase between paired sera.

‡ When adjusted for recommended IgM cut-off titers ≥1:32, results are 12/174.

¶ After adjustment 4.

§ Pan-Chlamydiales PCR results are only specific at the family-level lineage. These cases should therefore be considered as Simkaniaceae infections.

ǁ IgA ≥1:8.

Table 4. PCR used to detect Simkania in clinical and environmental samples.

Gene	Primers and probes set	Ampliconlength	References
Simkania-specific
16S	Zpf 5′-AAAGGTAACGAATAATTCCCT-3′	398	Kahane et al., 1998
	ZpR 5′-GCACAGTCGGGGTTGAGACCGACT-3′
	16F2 5′-CAA GAA AAG GTA ACG AAT AAT TGCC-3′	171	Niemi et al., 2011
	16R2 5′-GAG CTC CGG AAT TTC ACA TCT-6′
	16S2 5′-FAM-AAG GGC GCG TAG GCG GGT AAG C-BHQ1-3′
23S	AF 5′-CACAGGTAGGCATGATGA-3′	1099	Everett et al., 1999a,b
	BR 5′-CTAGCTGCGGGTAAACG-3′
	INTF 5′-TTAGATGCACAATGGATAGTTGGA-3′	338	Everett et al., 1999a,b
	INTR 5′-CCATCAGCGCTCATGTGCTCA-3′
Pan-Chlamydiales
16S	ccF 5′-CTT CGG GTT GTA AAG CAC TTT CGC-3′	512	Kahane et al., 2004
	ccR 5′-CCC CGT CAA TTC TTT TGA GTT T-3′
	panChl6F2 5′-CCGCCAACACTGGGACT-3′	207–215*	Lienard et al., 2011a,b
	panChl6R2 5′-GGAGTTAGCCGGTGCTTCTTTAC-3′
	panChl6S 5′-FAM-CTACGGGAGGCTGCAGTCGAGAATC-BHQ1-3′

* Amplicon length varies according to Chlamydiales species.

Table 5. Patients with Simkania-caused pneumonia.

	Patient 1	Patient 2	Patient 3	Patient 4	Patient 5	Patient 6	Patient 7
Age	31 y.o.	32 y.o.	32 y.o.	28 y.o.	57 m.o.	52 m.o.	58 m.o.
Sex	F	M	M	M	M	M	F
Medical history	None	None	FMF Colchicine treatment	None	id.	id.	None
Country				Italy	Italy	Italy	Brazil
Smoking	5 pack-year	20 pack-year	15 pack-year	id.	id.	id.	None
Clinical
Fever	38.5 °C	36.8 °C	39.2 °C	39.4 °C	id.	id.	37.5 °C
Symptoms	Unproductive cough,abdominal pain,diarrhea	Unproductive cough,chest pain, diarrhea,vomiting	Unproductive cough,chest pain	Unproductive cough,chest pain	id.	id.	Cough, dyspnoea
WBC* (cells/ml)	7400	10 800	15 000	6300	14 850	11 870	29 200
CRP^† (mg/l)	<5	<5	<5	<5	id.	id.	id.
PCT^‡ (μg/l)	id.	id.	id.	id.	0.34	0.21	id.
Treatment
Hospitalization	Inpatient 4 days	Inpatient 5 days	Inpatient 3 days	Inpatient 4 days	Outpatient	Outpatient	Inpatient
Antibiotic	Erythromycin	Penicillin after a trial	Erythromycin	Erythromycin	id.	id.	Penicillin
Regimen		erythromycin as an outpatient
Reference	Lieberman et al., 1997	Lieberman et al., 1997	Lieberman et al., 1997	Lieberman et al., 1997	Fasoli et al., 2008	Fasoli et al., 2008	Nascimento-Carvalhoet al., 2009

CRP: C-reactive protein; id.: indeterminate; FMF: familial Mediterranean fever; d.: days; m.o.: months old; PCT: procalcitonin; WBC: white blood cells; y.o.: years old.

* WBC >11 000 is considered significant for a serious bacterial infection.

† CRP >0.5 mg/l is considered significant for a serious bacterial infection.

‡ PCT >0.5 μg/l is considered significant for a serious bacterial infection.

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