Allofrancisella inopinata gen. nov., sp. nov. and

International Journal of Systematic and Evolutionary Microbiology (2016), 66, 4832–4838

DOI 10.1099/ijsem.0.001437

Allofrancisella inopinata gen. nov., sp. nov. and Allofrancisella frigidaquae sp. nov., isolated from water-cooling systems, and transfer of Francisella guangzhouensis Qu et al. 2013 to the new genus as Allofrancisella guangzhouensis comb. nov. Ping-Hua Qu,1 Ying Li,1,2 Nimaichand Salam,3 Shou-Yi Chen,4 Lan Liu,3 Quan Gu,5 Bao-Zhu Fang,3 Min Xiao,3 Mo Li,1 Cha Chen1 and Wen-Jun Li3 1

Department of Clinical Laboratory, Guangdong Provincial Hospital of Traditional Chinese Medicine, Guangzhou 510006, PR China

[email protected]

2

Wen-Jun Li

School of Laboratory Medicine, Tianjin Medical University, Tianjin 300070, PR China

3

State Key Laboratory of Biocontrol and Guangdong Provincial Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, PR China

4

Department of Microbiological Laboratory, Guangzhou Center for Diseases Control and Prevention, Guangzhou 510440, PR China

5

Department of Clinical Laboratory, Tangshan People’s Hospital, Tangshan 063000, PR China

Correspondence Cha Chen

[email protected]

Five bacterial strains (SYSU YG23T, SYSU 10HL1970T, 10HP82-10, 10HL1938, 10HP457) isolated from water reservoirs of cooling systems were characterized using a polyphasic taxonomic approach. The isolates were Gram-stain-negative, strictly aerobic and non-motile. Growth was enhanced in the presence of L-cysteine. The major fatty acids (>5 %) for the five strains were C10 : 0, C16 : 0, C16 : 0 3-OH, C18 : 0 3-OH and C18 : 1!9c. Ubiquinone-8 was detected as the respiratory quinone while the polar lipid profile consisted of phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, three unidentified phospholipids, two unidentified aminophospholipids and three unidentified glycolipids. The strains shared 16S rRNA gene sequence similarities of 99.0–99.2 % with Francisella guangzhouensis 08HL01032T but less than 95.2 % with other members of the family Francisellaceae. The phylogenetic dendrogram based on 16S rRNA gene sequences showed that these strains form a separate cluster along with Francisella guangzhouensis. This cluster was also confirmed from multilocus-sequence typing based on sequences of the mdhA, rpoB and sdhA genes. Matrixassisted laser desorption ionization time-of-flight MS analyses of the strains along with closely and distantly related Francisella strains also showed a distinct cluster for these strains. Based on the findings from the polyphasic taxonomy studies, the strains were considered to represent two novel species of a new genus for which the names Allofrancisella inopinata gen. nov., sp. nov. (type strain SYSU YG23T=KCTC 42968T=DSM 101834T) and Allofrancisella frigidaquae sp. nov. (type strain SYSU 10HL1970T=KCTC 42969T=DSM 101835T) are proposed. In addition, Francisella guangzhouensis Qu et al. 2013 is proposed to be transferred to this new genus as Allofrancisella guangzhouensis comb. nov.

Abbreviation: MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA, mdhA, rpoB and sdhA gene sequences are given in Table S1. Six supplementary figures and two supplementary tables are available with the online Supplementary Material.

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Allofrancisella inopinata gen. nov., sp. nov. and A. frigidaquae sp. nov.

The primary source for Legionnaires’ disease outbreaks is often cooling towers (Dondero et al., 1980; Klaucke et al., 1984; Brown et al., 1999; Greig et al. 2004). It is therefore essential to routinely monitor cooling towers for the presence of Legionella (Nguyen et al., 2006). In 2008, routine investigations to detect Legionella strains from water reservoirs of cooling towers in Guangzhou, China, resulted in the isolation of Francisella guangzhouensis (Qu et al., 2013). In another investigation, more than 300 water samples from cooling towers installed in metro stations and hotels in Guangzhou were collected. This paper reports on the polyphasic characterization of five bacterial strains (SYSU YG23T, SYSU 10HL1970T, 10HP82-10, 10HL1938, 10HP457) isolated from among the samples collected during the investigation. Water samples were collected from cooling towers of air conditioning systems in metro stations and hotels in Guangzhou. Enrichment of the water samples was performed as reported elsewhere (Gu et al., 2015). The pretreated samples were plated on Legionella BCYEa agar or BCYEa-GVPC (BCYEa supplemented with 3 g glycine l 1, 80 000 IU polymyxin B sulphate l 1, 1 mg vancomycin l 1 and 80 mg cycloheximide l 1) at 35 C under a 5 % CO2 atmosphere (Qu et al., 2009, 2013). Colonies obtained were subcultured and purified on BCYEa agar under similar conditions. Growth characteristics were examined on cysteine heart agar (supplemented with 9 % sheep red blood cells; CHAB), Haemophilus chocolate agar, BCYEa-GVPC agar, Thayer-Martin agar, sheep blood agar, Kligler iron agar (KIA) MacConkey agar and Mueller-Hinton (MH) agar (supplemented with X and V factor discs). Anaerobic growth was examined using an anaerobic incubation system (Forma 1025 anaerobic chamber; Thermo Scientific). Optimum growth temperature was determined after 3 days on sheep blood agar incubated at 4, 18, 25, 28, 30, 32, 35, 37 and 42 C. Cell morphology was examined under an Olympus light microscope. Rapid enzymatic tests such as catalase, oxidase and b-lactamase were assayed using bioMerieux reagents. Other biochemical and enzymatic activities were tested using RapidID 32A, API NH and API ZYM kits (bioMerieux). Antibiotic susceptibility assays were performed on CHAB media containing 1.5 % agar by using E-test strips (Oxoid). Susceptibility criteria were interpreted with reference to the MIC breakpoint of Francisella tularensis as listed in the CLSI M45-A2 guidelines for Potential Bacterial Agents of Bioterrorism. All five strains were found to be strictly aerobic. Cells were Gram-stain-negative, with a cocco-bacillus shape (Fig. S1, available in the online Supplementary Material). All strains were found to be catalase-positive, oxidase-negative, nonmotile and non-spore forming. Growth was observed on CHAB, and several media common to clinical laboratories, including Haemophilus chocolate agar, BCYEa-GVPC agar, Thayer-Martin agar and sheep blood agar. Media supplemented with cysteine enhanced the colony size, indicating the requirement of cysteine in growth media (Fig. S2). However, no growth was detected on KIA, MacConkey agar http://ijs.microbiologyresearch.org

or MH agar (supplemented with X and V factors). All five strains were positive for b-lactamase activity, and were further confirmed by the intrinsic resistance to cephalosporins in antibiotic susceptibility assays. However, these strains were sensitive to chloramphenicol, ciprofloxacin, doxycycline, gentamicin, levofloxacin and tetracycline (Table S1). Differential characteristics between the novel strains and related type strains are listed in Table 1 while detailed characteristics are given in the species descriptions. The 16S rRNA gene sequences of the strains were amplified using the universal bacterial primers 27f (5¢-

Table 1. Physiological and biochemical characteristics of the novel strains and related strains of members of the genus Francisella Strains: 1, SYSU YG23T; 2, SYSU 10HL1970T, 10HL1938, 10HP457, 10HP82-10; 3, F. guangzhouensis 08HL01032T; 4, F. philomiragia ATCC 25015T; 5, F. noatunensis subsp. noatunensis 2005/50/F2926CT; 6, F. noatunensis subsp. orientalis Ehime-1T; 7, F. halioticida Shimane-1T. +, Positive; , negative; W, weakly positive; ND, no data available. All strains were positive for enhancement of growth by Lcysteine and activities of catalase, esterase (C4), esterase lipase (C8), acid phosphatase and naphthol-AS-BI-phosphohydrolase. All strains were negative for lipase (C14), valine arylamidase, cystine arylamidase, trypsin arylamidase, a-chymotrypsin, a-mannosidase, a-fucosidase, urease, arginine dihydrolase, a-galactosidase, b-galactosidase 6phosphate, a-glucosidase, b-glucosidase, a-arabinosidase, b-glucuronidase, raffinose fermentation, aesculin hydrolysis and nitrate reduction. Data in columns 5–7 were taken from Mikalsen et al. (2007), Ottem et al. (2009) and Brevik et al. (2011). Characteristic

1

2

3

4

5

6

7

Optimal growth temperature ( C) Oxidase, b-galactosidase, glutamic acid decarboxylase, glutamyl glutamic acid arylamidase, H2S production Indole production Leucyl glycine arylamidase Glucose fermentation Sucrose fermentation Leucine arylamidase Glycine arylamidase Arginine arylamidase Proline arylamidase Pyroglutamic acid arylamidase Tyrosine arylamidase Alanine arylamidase Phenylalanine arylamidase Histidine arylamidase Serine arylamidase

32

32

25– 28

37

22

22

20

+

+

+

W

W

Downloaded from www.microbiologyresearch.org by IP: 113.108.133.54 On: Tue, 13 Dec 2016 14:15:44

+

W

+

+

+ W

+ +

+

+

+ + + W

+

+ +

+ + + + + + + + + + + +

+ +

+ + + +

+

+ +

W W W

ND

+

+

+

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AGAGTTTGATCMTGGCTCAG-3¢) and 1492r (5¢-CGGYTACCTTGTTACGACTT-3¢), corresponding to positions 8– 27 and 1512–1492 of the Escherichia coli 16S rRNA gene, respectively (Frank et al., 2008). Amplification of housekeeping genes was performed as previously described for the detection of the malate dehydrogenase (mdhA) gene (Ottem et al., 2007), RNA polymerase beta subunit (rpoB) gene (Ko et al., 2002) and succinate dehydrogenase (sdhA) gene (Barns et al., 2005). The primer pair used for amplification of the sdhA gene (Barns et al., 2005) failed to amplify this for strains SYSU 10HL1970T, 10HP82-10, 10HL1938 and 10HP457. Therefore, a new primer pair was designed using the PRIMERBLAST tool from NCBI (http://www.ncbi.nlm.nih.gov/tools/ primer-blast/). Sequences retrieved for the related genus Francisella from GenBank were used as the template for designing the new primer pairs sdhA F-286 (5¢-GAGCATGCGCCACAATCAAT-3¢) and sdhA R-897 (5¢-GGCACGCGATACAACATCAC-3¢). Amplification was performed at 95 C for 5 min, 40 cycles of 94 C for 30 s, 54 C for 45 s and 72 C for 45 s, followed by final extension at 72 C for 10 min and a short storage at 4 C. The amplified products for all the four genes were purified and cloned into Trans1-T1 chemically competent cells using pEASY-T1 vector. Sequencing of the clones was performed with an Applied Biosystems automatic sequencer (ABI 3730XL) at Invitrogen. Comparative sequence analysis was performed using the BLAST program (http://blast. ncbi.nlm.nih.gov/Blast.cgi). Phylogenetic dendrograms were generated using MEGA 5.1 (Tamura et al., 2011). To differentiate the proposed novel species from the Francisella strains, matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) fingerprints of these five strains were analysed along with selected Francisella strains listed in Table S1, F. philomiragia ATCC 25015T and Legionella pneumophila ATCC 33823T using the VITEK MS RUO system (bioMerieux). The strains were processed with a standard protocol of ethanol-formic acid protein extraction as previously described (Angeletti et al., 2015). The dendrogram based on the protein fingerprints of the isolates was reconstructed by using SARAMIS software (VITEK MS; bioMerieux), while the characteristics of mass spectra were analysed by Launchpad 2.9 software (VITEK MS; bioMerieux). DNA–DNA re-association studies with closely related strains were performed by the method of fluorometric DNA–DNA hybridization in microdilution wells as previously described (Ezaki et al., 1989). Levels of sequence similarity based on the 16S rRNA gene sequences for strains SYSU 10HL1970T (1440 bp, GenBank accession no. JN620409), 10HP82-10 (1440 bp, JN620410), 10HL1938 (1432 bp, JN620406), 10HP457 (1481 bp, JN620411) and SYSU YG23T (1409 bp, JN620412) with the closely related strains are listed in Table 2. Strains SYSU YG23T and SYSU 10HL1970T shared 99.2 and 99.1 % sequence similarity with F. guangzhouensis 08HL01032T, respectively, but less than 95.1 % similarity with other species of the genus Francisella. The neighbour-joining tree based on the 16S rRNA gene sequence of all members within the family Francisellaceae showed a well-separated 4834

cluster for the five strains along with F. guangzhouensis (Fig. 1). This topology was also supported in the dendrograms generated with the maximum-likelihood and maximum-parsimony methods (Figs S3 and S4). The neighbour-joining tree based on concatenated sequences of the mdhA, rpoB and sdhA genes showed the clustering of the five isolates into two groups (Fig. 2) with close relatedness to F. guangzhouensis. The results of MALDI-TOF MS analysis showed a reliable, robust and effective interspecies differentiation among the selected Francisella strains. F. philomiragia ATCC 25015T presented a nearly identical protein fingerprint to that of previously described F. philomiragia ATCC 25016 (Seibold et al., 2010). While all F. philomiragia strains in this study have a characteristic peak at 5180 (m/z) (similar to that reported for F. tularensis and F. novicida; Seibold et al., 2010), a different characteristic peak at 5110 (m/z) was found for the five strains and the related F. guangzhouensis strains (Fig. S5). The dendrogram based on whole-cell MALDI-TOF mass spectra demonstrated a clear separation of the five strains along with F. guangzhouensis strains from F. philomiragia strains (Fig. 3). The five strains were also grouped into two clusters, represented by strain SYSU YG23T and strains SYSU 10HL1970T, 10HP82-10, 10HL1938 and 10HP457, respectively (Fig. 3). Distinct taxonomic positions of the five strains were also indicated by the DNA–DNA hybridization experiments (Table 2). Strain SYSU 10HL1970T showed DNA–DNA relatedness values of above 70 % with strains 10HP82-10, 10HL1938 and 10HP457, while having relatedness values below 70 % for the other type strains. Strain SYSU YG23T shared a relatedness value of 33.6±6.6 and 33.9±3.8 % with F. guangzhouensis 08HL01032T and F. philomiragia ATCC 25015T, respectively. These results confirmed that strains SYSU 10HL1970T, 10HP82-10, 10HL1938 and 10HP457 belong to the same species. However, strain SYSU 10HL1970T could be differentiated from strain SYSU YG23T and related type strains by the moderately low DNA–DNA relatedness values, which were below the 70 % limit for species delineation (Stackebrandt et al., 2002). Cellular fatty acid methyl ester profiles were determined by GC according to the standard protocol of the MIDI Sherlock MIS system (MIDI version 5.0). Cells were harvested under the same conditions of 32 C and 5 % CO2 atmosphere, using CHAB agar as the cultivation medium. F. philomiragia ATCC 25015T and F. guangzhouensis 08HL01032T were used as controls. Quinones and polar lipids were extracted and analysed as previously described (Goodfellow & Minnikin, 1985). The DNA G+C contents were determined by reversed-phase HPLC as previously described (Tamaoka & Komagata, 1984) under the same conditions. The major fatty acids (>5 %) for strains SYSU YG23T, SYSU 10HL1970T, 10HP82-10, 10HL1938 and 10HP457 consisted of C18 : 1 3-OH (18.2–22.2 %), C10 : 0 (14.3– 21.5 %), C16 : 0 (6.8–17.8 %), C18 : 1!9c (9.1–10.9 %), C16 : 0 3-OH (8.5–9.7 %), C14 : 0 (6.6–7.7 %) and C10 : 0 2-OH (5.1– 7.9 %) (Table S2). Ubiquinone-8 was detected as the respiratory quinone. The polar lipid profiles for strains SYSU

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100*

Francisella noatunensis subsp. orientalis Ehime-1T (EU683030)

88

Francisella asiatica PQ1104T (EU597812)

Francisella noatunensis subsp. noatunensis 2005/50/F292-6CT (DQ295795) 64 100* Francisella pesticida GM2212T (DQ309246) 79 Francisella philomiragia subsp. philomiragia ATCC 25015T (ABYY01000004)

0.02

Francisella halioticida LMG26022T (AB449247) 100*

T 86* Francisella tularensis subsp. tularensis ATCC 6223 (Z21931) 67* Francisella tularensis subsp. holarctica OSU18 (CP000437) 97* Francisella tularensis subsp. mediasiatica GIEM 543T (CP000915)

99* 100*

Francisella tularensis subsp. novicida U112T (CP000439) Francisella hispaniensis FhSp1T (FN252413)

96*

Francisella persica ATCC VR-331T (M21292) Allofrancisella guangzhouensis 08HL01032T (CP010427) 100* 97*

Allofrancisella inopinata SYSU YG23T (JN620412) Allofrancisella frigidaquae 10HP82-10 (JN620410)

Allofrancisella frigidaquae 10HP457 (JN620411) 98* Allofrancisella frigidaquae SYSU 10HL1970T (JN620409) Allofrancisella frigidaquae 10HL1938 (JN620406) Caedibacter taeniospiralis 51T (AY102612)

100*

Fangia hongkongensis UST040201-002T (AB176554) Legionella pneumophila subsp. pneumophila Philadelphia 1T (AE017354)

Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, showing the relationship between strains SYSU YG23T, SYSU 10HL1970T, 10HL1938, 10HP457 and 10HP82-10, and all members of the family Francisellaceae. Asterisks indicate branches of the tree that were also recovered in the maximum-likelihood and maximum-parsimony trees. Numbers at branch points indicate bootstrap percentages (based on 1000 replicates); only values >50 % are indicated. Bar, 0.02 substitutions per nucleotide position.

YG23T and SYSU 10HL1970T were nearly identical, and consisted of phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, two unidentified aminophospholipids, three unidentified phospholipids and three unidentified glycolipids (Fig. S6). The DNA G+C content of strains SYSU YG23T and SYSU 10HL1970T was 32.6 and 30.0 mol%, respectively. The new genus Allofrancisella gen. nov. proposed to accommodate the novel strains is differentiated from F. tularensis subsp. tularensis, F. hispanensis and F. philomiragia by the presence of one unidentified phospholipid (PL4), and by the absence of one unidentified phospholipid (PL1), one unidentified aminophospholipid (APL1), two aminophosphoglycolipids (APGL1, APGL2) and an aminolipid (AL) (Huber et al., 2010). The new genus Allofrancisella is also differentiated from the closely related taxa by the phylogenetic differences as represented in Figs 1, 2 and 3 and DNA–DNA relatedness values. Other phenotypic differences are listed in Table 1. Based on the differential characteristics, strains SYSU YG23T and SYSU 10HL1970T merit recognition as representatives of two novel species of a novel genus, for which the names http://ijs.microbiologyresearch.org

Allofrancisella inopinata gen. nov., sp. nov. and Allofrancisella frigidaquae sp. nov. are proposed, respectively. In addition, F. guangzhouensis 08HL01032T was found to differ considerably from all recognized Francisella species based on both sequence comparison and phylogenetic analysis, while it shares many common traits with the proposed new genus in terms of both phylogenetic comparisons based on the 16S rRNA gene, multi-locus sequence typing and MALDI-TOF MS analysis. It was therefore considered that F. guangzhouensis 08HL01032T should be reclassified as a member of a separate taxon within the newly proposed genus, for which the name Allofrancisella guangzhouensis comb. nov. is proposed.

Description of Allofrancisella gen. nov. Allofrancisella (Al.lo.fran.ci.sel¢la. Gr. adj. allos another, the other; N.L. fem. n. Francisella a bacterial generic name; N.L. fem. n. Allofrancisella the other Francisella referring to the fact that the genus is phylogenetically related to Francisella). Aerobic, Gram-stain-negative coccobacilli. Cells are catalase-positive, non-motile and non-spore forming.


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Francisella tularensis subsp. holarctica FSC200 (CP003862) 52 Francisella tularensis subsp. holarctica OR96-0246 (CP011488) 66 Francisella tularensis subsp. tularensis WY96-3418 (CP000608) 99 Francisella tularensis subsp. tularensis SCHU S4 (CP010290) 100 Francisella tularensis subsp. mediasiatica FSC147T (CP000915)

0.02

Francisella tularensis subsp. novicida U112T (CP009633)

99

59

Francisella tularensis subsp. novicida D9876 (CP009607)

Francisella persica ATCC VR-331T (CP012505) Francisella philomiragia subsp. philomiragia ATCC 25015T (CP010019) Allofrancisella guangzhouensis 08HL01032T (CP010427) Allofrancisella inopinata SYSU YG23T Allofrancisella frigidaquae 10HP82-10

100 100

T 100 Allofrancisella frigidaquae SYSU 10HL1970 56 Allofrancisella frigidaquae 10HL1938 56 Allofrancisella frigidaquae 10HP457

Fig. 2. Neighbour-joining tree of concatenated genes (rpoB, mdhA and sdhA, totalling 1197 nt). Bootstrap values are based on 1000 replicates; only values >50 % are indicated. Bar, 0.02 substitutions per nucleotide position.

Growth is enhanced by supplementation of cysteine. No production of H2S on culture in cysteine-containing media. Produce b-lactamase. Resistant to cephalosporin but sensitive to chloramphenicol, ciprofloxacin, doxycycline, gentamicin, levofloxacin and tetracycline. The respiratory quinone is ubiquinone-8. The polar lipid profile consists of phosphatidylethanolamine,

0

10

20

30

40

50

a

60

70

diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, two unidentified aminophospholipids, three unidentified phospholipids and three unidentified glycolipids. The major fatty acids are C18 : 0 3-OH, C10 : 0, C18 : 1!9c and C16 : 0. Phylogenetically, the genus is affiliated to the family Francisellaceae. The type species of the genus is Allofrancisella inopinata.

80

90 % Allofrancisella frigidaquae 10HP457 Allofrancisella frigidaquae 10HP82-10 Allofrancisella frigidaquae 10HL1938 Allofrancisella frigidaquae SYSU 10HL1970T Allofrancisella guangzhouensis 08HL01032T

b

Allofrancisella guangzhouensis 10HL1960 Allofrancisella guangzhouensis 10HP82-6

c

Allofrancisella guangzhouensis 09HG997 Allofrancisella inopinata SYSU YG23T Francisella philomiragia QH-12 Francisella philomiragia QH-3 Francisella philomiragia QH-2 Francisella philomiragia QH-15

d e

Francisella philomiragia QH-5 Francisella philomiragia QH-4 Francisella philomiragia 10HL1958 Francisella philomiragia ATCC 25015T Legionella pneumophilia ATCC 33823T

Fig. 3. Dendrogram based on the whole-cell MALDI-TOF mass spectra of strains SYSU YG23T, SYSU 10HL1970T, 10HL1938, 10HP457 and 10HP82-10, and related strains of other species of the genus Francisella. The dendrogram was reconstructed using the correlation distance measured with the average linkage algorithm (UPGMA). (a–e) represent the different clusters in the dendrogram. 4836



Table 2. 16S rRNA gene sequence similarities and DNA–DNA hybridization values of strains SYSU YG23T and SYSU 10HL1970T with closely related species 16S rRNA gene sequence similarities (%, upper right) are the results of BLAST analysis. DNA–DNA hybridization values (%, lower left) are expressed as means of the experimental values obtained from four replicates.

SYSU 10HL1970T 10HP82-10* 10HL1938* 10HP457* SYSU YG23T 08HL01032T ATCC 25015T

SYSU 10HL1970T

10HP82-10

10HL1938

10HP457

SYSU YG23T

08HL01032T

ATCC 25015T

– 88.1 90.7 94.0 58.6 32.9 32.7

100 – – – – – –

100 100 – – – – –

100 100 100 – – – –

99.6 99.6 99.6 99.6 – 33.6 33.9

99.1 99.1 99.1 99.0 99.2 – –

95.1 95.1 95.1 95.0 95.3 95.4 –

*DNA–DNA hybridization experiments were not performed among strains 10HP82-10, 10HL1938 and 10HP457.

Description of Allofrancisella inopinata sp. nov.

to the isolation of the type strain from a water-cooling system).

Allofrancisella inopinata (in.o.pi.na¢ta. N.L. fem. adj. inopinata unexpected, referring to the unexpected isolation of the type strain from a water-cooling system in routine investigations to detect Legionella).

Grows well on CHAB, and several media common to clinical laboratories, including selective Haemophilus chocolate agar, selective BCYEa-GVPC agar, ThayerMartin agar and sheep blood agar. No growth on KIA, MacConkey agar or MH agar (supplemented with X and V factors). Growth occurs at 18–37 C, with optimum growth at 32 C. Positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, phenylalanine arylamidase, tyrosine arylamidase, alanine arylamidase, histidine arylamidase and ornithine decarboxylase. Negative for oxidase, lipase (C14), valine arylamidase, cystine arylamidase, trypsin arylamidase, a-chymotrypsin, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase, a-fucosidase, urease, arginine dihydrolase, b-galactosidase 6-phosphate, a-arabinosidase, pyroglutamic acid arylamidase, glycine arylamidase and glutamyl glutamic acid arylamidase. Positive for fermentation of D-glucose, Dfructose and D-mannose but not maltose, sucrose or raffinose. Negative for nitrate reduction, Voges–Proskauer, indole production and gelatin and aesculin hydrolysis tests.

Grows well on CHAB, and several media common to clinical laboratories, including Haemophilus chocolate agar, BCYEa-GVPC agar, Thayer-Martin agar and sheep blood agar. No growth on KIA, MacConkey agar or MH agar (supplemented with X and V factors). Growth occurs at 18– 37 C, with optimum growth at 32 C. Positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, phenylalanine arylamidase and ornithine decarboxylase tests but negative for oxidase, lipase (C14), valine arylamidase, cystine arylamidase, trypsin arylamidase, a-chymotrypsin, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, N-acetyl-b-glucosaminidase, amannosidase, a-fucosidase, urease, arginine dihydrolase, b-galactosidase 6-phosphate, a-arabinosidase, pyroglutamic acid arylamidase, tyrosine arylamidase, alanine arylamidase, glycine arylamidase, histidine arylamidase, glutamyl glutamic acid arylamidase and serine arylamidase. Positive for fermentation of D-glucose, D-fructose and D-mannose but not maltose, sucrose or raffinose. Negative for nitrate reduction, Voges–Proskauer, indole production and gelatin and aesculin hydrolysis tests. The type strain, SYSU YG23T (=KCTC 42968T=DSM 101834T), was isolated from water of a cooling tower in Guangzhou city, China. The G+C content of the type strain is 32.6 mol%.

Description of Allofrancisella frigidaquae sp. nov. Allofrancisella frigidaquae (L.adj. frigidus cold; L.n. aqua water; N.L. gen. n. frigidaquae from/of cold water, referring http://ijs.microbiologyresearch.org

The type strain, SYSU 10HL1970T (=KCTC 42969T=DSM 101835T), was isolated from water of a cooling tower in Guangzhou city, China. The G+C content of the type strain is 30.0 mol%.

Description of Allofrancisella guangzhouensis comb. nov. Allofrancisella guangzhouensis (guang.zhou.en¢sis. N.L. fem. adj. guangzhouensis of or pertaining to Guangzhou, the city in Guangdong province, China, where the first strain was isolated). Basonym: Francisella guangzhouensis Qu et al. (2013).


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The description of the species is as originally described by Qu et al. (2013). The type strain, 08HL01032T (=CCUG 60119T=NCTC 13503T=ATCC BAA-2361T), was isolated from water of a cooling tower in Guangzhou city, China. The DNA G+C content of the type strain is 32.5 mol%.

Acknowledgements This research was supported by the Natural Science Foundation of China (Nos. 3130004 and 3150004), and Research Fund for Outstanding Young in Higher Education Institutions of Guangdong Province (2013LYM_0015). W.-J.L. was also supported by a Guangdong Province Higher Vocational Colleges and Schools Pearl River Scholar Funded Scheme (2014).

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