Yujie Fang

SXT/R391 integrative and conjugative elements in Proteus species reveal abundant genetic diversity and multidrug resistance.

SXT/R391 integrative and conjugative elements (ICEs) are self-transmissible mobile genetic elements that are found in most members of Enterobacteriaceae. Here, we determined fifteen SXT/R391 ICEs carried by Proteus isolates from food (4.2%) and diarrhoea patients (17.3%). BLASTn searches against GenBank showed that the fifteen SXT/R391 ICEs were closely related to that from different Enterobacteriaceae species, including Proteus mirabilis. Using core gene phylogenetic analysis, the fifteen SXT/R391 ICEs were grouped into six distinct clusters, including a dominant cluster and three clusters that have not been previously reported in Proteus isolates. The SXT/R391 ICEs shared a common structure with a set of conserved genes, five hotspots and two variable regions, which contained more foreign genes, including drug-resistance genes. Notably, a class A β-lactamase gene was identified in nine SXT/R391 ICEs. Collectively, the ICE-carrying isolates carried resistance genes for 20 tested drugs. Six isolates were resistant to chloramphenicol, kanamycin, streptomycin, trimethoprim-sulfamethoxazole, sulfisoxazole and tetracycline, which are drug resistances commonly encoded by ICEs. Our results demonstrate abundant genetic diversity and multidrug resistance of the SXT/R391 ICEs carried by Proteus isolates, which may have significance for public health. It is therefore necessary to continuously monitor the antimicrobial resistance and related mobile elements among Proteus isolates.

High Prevalence of Macrolide-resistance and Molecular Characterization of Streptococcus pyogenes Isolates Circulating in China from 2009 to 2016

Streptococcus pyogenes, or group A Streptococcus, is a pathogen responsible for a wide range of clinical manifestations, from mild skin and soft tissue infections and pharyngitis to severe diseases. Its epidemiological characteristics should be comprehensively under surveillance for regulating the national prevention and treatment practice. Herein, a total of 140 S. pyogenes, including 38 invasive and 102 noninvasive isolates, were collected from infected patients in 10 tertiary general hospitals from 7 cities/provinces in China during the years 2009–2016. All strains were characterized by classical and molecular techniques for its emm types/subtypes, virulent factors and antibiotic resistance profiling. Of 140 isolates, 15 distinct emm types and 31 subtypes were detected, dominated by emm12 (60 isolates, 42.9%), emm1(43, 30.7%), and emm89 (10, 7.1%), and 8 new emm variant subtypes were identified. All strains, invasive or not, harbored the superantigenic genes, speB and slo. The other virulence genes, smeZ, speF, and speC accounted for 96.4, 91.4, and 87.1% of collected isolates, respectively. Further multilocus sequence typing (MLST) placed all strains into 22 individual sequence types (STs), including 4 newly-identified STs (11, 7.9%). All isolates were phenotypically susceptible to penicillin, ampicillin, cefotaxime, and vancomycin, whereas 131(93.5%), 132(94.2%), and 121(86.4%) were resistant to erythromycin, clindamycin, and tetracycline, respectively. Our study highlights high genotypic diversity and high prevalence of macrolide resistance of S. pyogenes among clinical isolates circulating in China.

Molecular characterization and antibiotic resistance of clinical Streptococcus dysgalactiae subsp. equisimilis in Beijing, China.

Streptococcus dysgalactiae subsp. equisimilis (SDSE) is presently considered as a human pathogen associated with clinical infection. We characterized 56 SDSE isolates collected from two tertiary hospitals in Beijing, China. Sixteen distinct emm types/subtypes were detected, dominated by stG245.0 (32.1%), stG652.0 (10.7%), stG6.1 (10.7%) and stG485.0 (10.7%), and a novel stG840.0 variant type was identified. All isolates possessed virulence genes of sagA and scpA, and most carried slo (98.2%), ska (98.2%) and speG(dys) (35.7%). By multilocus sequence typing (MLST) analysis, 17 individual sequence types (STs) were distinguished, including 7 newly-identified STs (26.8% of isolates), of which ST127 (30.4%), ST7 (12.5%) and ST44 (10.7%) dominated. Meanwhile, pulsed-field gel electrophoresis (PFGE) analysis revealed 33 pattern types (PTs), which were further combined into 16 pattern clusters (PCs), and 59.3% of isolates were distributed into 2 dominant PCs. Notably, emm types had both close relationship and consistency with STs and PFGE PCs. Furthermore, of 56 SDSE isolates, the predominant antibiotic resistances were erythromycin (71.4%), clindamycin (71.4%) and tetracycline (60.7%). Correspondingly, the prevalent resistance genes of macrolide and tetracycline were erm(B) (78.6%) and tet(M) (73.2%). In addition, multiple point mutations of parC, one of fluoroquinolone resistance genes, were observed (accounting for 75%), and were divided into 12 types, with parC 07 as the predominant type. Our data suggested the wide molecular diversity and distinctive regional features of SDSE from clinical infection in Beijing, China.

First molecular evidence of intrauterine and surgical-site infections caused by Streptococcus dysgalactiae subsp. equisimilis

S. dysgalactiae subsp. equisimilis (SDSE) is infrequently associated with maternal infections during delivery in pregnant women. A rare case is presented of a woman with intrauterine infection and surgical-site infection due to SDSE after cesarean section, which had colonized her genital tract and, via the ascending pathway, reached her intact fetal membrane. All isolates were identified as Streptococcus Lancefield group G, and their emm genes that coded M protein belonged to stG6.1. The isolates tested negative for a series of streptococcal superantigen virulence genes but positive for nonsuperantigenic virulence genes. In particular, molecular typing using pulsed-field gel electrophoresis analysis disclosed that the three isolates from the different infection sites had identical profiles. Furthermore, multilocus sequence typing indicated that the three isolates belonged to a new sequence typing. Our results indicated that SDSE is potentially pathogenic for pregnant women and newborns if colonized.

Microbiological and clinical characteristics of Group B Streptococcus isolates causing materno-neonatal infections: high prevalence of CC17/PI-1 and PI-2b sublineage in neonatal infections

Purpose. Group B Streptococcus (GBS) is one of the major pathogens in severe materno‐neonatal infections. We aimed to describe the clinical and molecular characteristics of GBS isolates causing infections in 45 maternal and 50 neonatal subjects, collected from eight healthcare centres in mainland China over the period 2010‐ 2017. Methodology. The phenotypic and genotypic features of the GBS isolates, including capsular polysaccharide (cps) serotypes, pilus island (PI) genes and antibiotic resistance profiles and genes, were characterized by both conventional and molecular methods. The clonal relationship between these strains was investigated using multilocus sequence typing (MLST). Results. Of the 95 isolates, the predominant serotypes were III (51, 53.7 %), V (13, 13.7 %) and Ib (13, 13.7 %). All GBS strains carried at least one pilus island, with 32.6 % carrying PI‐2b and 67.4 % PI‐2a, singly or in combination. The most frequently‐detected pilus island pattern was the combination of PI‐1 and PI‐2a, accounting for 56.8 % (54 isolates), followed by PI‐1 combined with PI‐2b (28, 29.5 %), PI‐2a (10, 10.5 %) and PI‐2b (3, 3.2 %). The strains were classified into 17 individual sequence types, and further clustered into six clonal complexes (CCs). A high prevalence of CC17/PI‐1 and PI‐2b (17, 34.0 %) was detected in 50 GBS isolates causing neonatal infections. No strain was resistant to penicillin, ampicillin, ceftriaxone or vancomycin, whereas 78.9, 76.8 and 81.5 % were resistant to erythromycin, clindamycin and tetracycline, respectively. Conclusion. Our study highlights the high genotypic diversity of GBS strains causing materno‐neonatal infections, and the CC17/PI‐1 and PI‐2b sublineages should be noted in neonatal infections.

Vibrio fujianensis sp. nov., isolated from aquaculture water

A Gram-stain-negative, facultatively anaerobic strain, designated FJ201301T, was isolated from aquaculture water collected from Fujian province, China. Phylogenetic analysis of 16S rRNA gene sequences indicated that strain FJ201301T belonged to the genus Vibrio, formed a distinct cluster with Vibriocincinnatiensis ATCC 35912T and shared the highest similarity with Vibriosalilacus CGMCC 1.12427T. A 15 bp insertion found in the 16S rRNA gene was a significant marker that distinguished strain FJ201301T from several phylogenetic neighbours (e.g. V. cincinnatiensis). Multilocus sequence analysis of eight genes (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA and topA; concatenated 4135 bp sequence) showed that, forming a long and independent phylogenetic branch, strain FJ201301T clustered with V. cincinnatiensis ATCC 35912T, Vibrioinjenensis KCTC 32233T and Vibriometschnikovii CIP 69.14T clearly separated from V. salilacus CGMCC 1.12427T. Furthermore, the highest in silico DNA-DNA hybridization and average nucleotide identity values between strain FJ201301T and the closest related species were 26.3 and 83.1 % with V. cincinnatiensis ATCC 35912T, less than the proposed cutoff levels for species delineation, i.e. 70 and 95 %, respectively. Biochemical, sequence and genomic analysis suggested the designation of strain FJ201301T representing a novel species of the genus Vibrio, for which the name Vibrio fujianensis sp. nov. is proposed. The type strain is FJ201301T (=DSM 104687T=CGMCC 1.16099T).

Distribution and Genetic Characteristics of SXT/R391 Integrative Conjugative Elements in Shewanella spp. From China

The genus Shewanella consists of facultatively anaerobic Gram-negative bacteria, which are regarded as potential agents of food contamination and opportunistic human pathogens. Information about the distribution and genetic characteristics of SXT/R391 integrative conjugative elements (ICEs) in Shewanella species is limited. Here, 91 Shewanella strains collected from diverse samples in China were studied for the presence of SXT/R391 ICEs. Three positive strains, classified as Shewanella upenei, were obtained from patients and water from a local mill. In light of their close clonal relationships and high sequence similarity, a representative ICE was selected and designated ICESupCHN110003. The BLASTn searches against GenBank showed that ICEVchBan5 was most closely related to ICESupCHN110003, with the coverage of 76% and identity of 99%. The phylogenetic tree of concatenated core genes demonstrated that ICESupCHN110003 formed a distinct branch outside the cluster comprising ICEValA056-1, ICEPmiCHN2410, and ICEPmiChn1. Comparison of the genetic structures revealed that ICESupCHN110003 encoded uncommon genes in hotspots, such as specific type III restriction-modification system, conferring adaptive functions to the host. Based on the low coverage in the sequence analysis, independent clade in the phylogenetic tree, and unique inserted fragments in hotspots, ICESupCHN110003 represented a novel SXT/R391 element, which widened the list of ICEs. Furthermore, the antibiotic resistance genes floR, strA, strB, and sul2 in ICESupCHN110003 and resistance to multiple drugs of the positive isolates were detected. A cross-species transfer capability of the SXT/R391 ICEs was also discovered. In summary, it is necessary to reinforce continuous surveillance of SXT/R391 ICEs in the genus Shewanella.

Rare Shewanella spp . associated with pulmonary and bloodstream infections of cancer patients, China: a case report

BackgroundMembers of Shewanella species are opportunistic pathogens that are found in marine environments. Currently more than sixty species have been identified, whereas the most commonly clinical cases associated with Shewanella species have involved only two species, i.e., S. algae and S. putrefaciens. We present two cases of pulmonary and bloodstream infections caused by two rare Shewanella spp. strains from patients of gastrointestinal cancer.Case presentationTwo male patients with a history of gastrointestinal cancer presented to hospital with pulmonary and bloodstream infections, respectively. The infective pathogens of both cases were primarily isolated and identified as Shewanella algae (case I) and Shewanella putrefaciens (case II) by phenotypic features and VITEK 2 system, but they were further confirmed as Shewanella haliotis and Shewanella upenei by 16S rRNA gene sequence analysis. The major bacterial composition of the bronchoalveolar lavage in case I was also identified as Shewanella by 16S rRNA amplicon sequencing analysis. Antimicrobial susceptibility testing showed that the two strains had broad susceptibility, but S. haliotis in the case I was resistant to ciprofloxacin and levofloxacin and S. upenei in the case II was intermediate to imipenem, piperacillin/tazobactam and ciprofloxacin.ConclusionsTo the best of our knowledge, this is the first cases of the pulmonary and bloodstream infections caused by Shewanella spp. from clinical patients in mainland China. Shewanella as a potential pathogen in China should not be ignored.

Shewanella carassii sp. nov., isolated from surface swabs of crucian carp and faeces of a diarrhoea patient

Two strains, 08MAS2251T and LZ2016-166, were isolated from diverse samples in China collected from the surface of crucian carp and the faeces of a diarrhoea patient, respectively. Both strains were pink-orange coloured, Gram-negative, oxidase- and catalase-positive, facultative anaerobic and motile bacteria, produced H2S and reduced nitrates to nitrites. Growth occurred in the presence of 0-9 % (w/v) NaCl and at 10-42 °C. The optimum conditions were with 1 % (w/v) NaCl and at 35 °C. The phylogenetic tree of 16S rRNA gene demonstrated that strains 08MAS2251T and LZ2016-166 clustered in a distinctive clade next to the species Shewanella chilikensis JC5T within the genus Shewanella. Meanwhile, gyrB gene sequence analysis indicated that the two strains formed an independent branch that was clearly separate from all the other Shewanella species with sequence similarities from 68.49 to 95.74 %. The DNA G+C content of strain 08MAS2251T was 52.68 mol%. Genomic relatedness of in silico DNA-DNA hybridization between strain 08MAS2251T and phylogenetic neighbours ranged from 50.5-51.8 %, below the cutoff of 70 %. In addition, corresponding average nucleotide identity values were between 93.01 to 93.49%, which were lower than 95 % threshold. The major fatty acids of strain 08MAS2251T were C17 : 1ω8c (27.2 %), iso-C15 : 0 (22.5 %), summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c; 8.7 %), C16 : 0 (6.2 %), iso-C13 : 0 (5.6 %) and C17 : 0 (4.5 %). Based on phenotypic and genetic analysis, strains 08MAS2251T and LZ2016-166 are identified as a novel species of the genus Shewanella, for which the name Shewanellacarassii sp. nov. is proposed. The type strain is 08MAS2251T (=DSM 104682T=CGMCC 1.16033T).